The Interactive Fly
Genes involved in tissue and organ development
Mesoderm Development
Please note: c, s, or v = expression in cardiac, somatic or visceral musculature; h = expression in head mesoderm, d = expression in dorsal medial cells, f = expression in fat body, h = expression in hemocytes
Migration is a complex, dynamic process that has largely been studied using qualitative or static approaches. As technology has improved, it is now possible to take quantitative approaches towards understanding cell migration using in vivo imaging and tracking analyses. In this manner, a four-step model of mesoderm migration during Drosophila gastrulation was establised: (I) mesodermal tube formation, (II) collapse of the mesoderm, (III) dorsal migration and spreading and (IV) monolayer formation. The data provide evidence that these steps are temporally distinct and that each might require different chemical inputs. To support this, the role was analyzed of fibroblast growth factor (FGF) signaling, in particular the function of two Drosophila FGF ligands, Pyramus and Thisbe, during mesoderm migration. It was determined that FGF signaling through both ligands controls movements in the radial direction. Thisbe is required for the initial collapse of the mesoderm onto the ectoderm, whereas both Pyramus and Thisbe are required for monolayer formation. In addition, it was uncovered that the GTPase Rap1 regulates radial movement of cells and localization of the beta-integrin subunit, Myospheroid, which is also required for monolayer formation. These analyses suggest that distinct signals influence particular movements, since it was found that FGF signaling is involved in controlling collapse and monolayer formation but not dorsal movement, whereas integrins are required to support monolayer formation only and not earlier movements. This work demonstrates that complex cell migration is not necessarily a fluid process, but suggests instead that different types of movements are directed by distinct inputs in a stepwise manner (McMahon, 2010).
Mesoderm migration was found to be a combination of complex three-dimensional movements involving many molecular components. live imaging, coupled with quantitative analyses, is important for studying complex cell movements, as it allowed migration to be decomposed into different movement types and thus has allowed description of subtle phenotypes. First, analysis of the directional movements of mesoderm cells within wild-type embryos was extended, focusing on the temporal sequences of events. Cells were found follow a sequential and distinct set of trajectories: movement in the radial direction (tube collapse: -5 to 15 minutes, 0=onset of germband elongation), followed by movement in the angular direction (dorsal migration: 15 to 75 minutes) and ending with small intercalation movements in the radial direction (monolayer formation: 75 to 110 minutes). These movements appear temporally distinct (i.e. stepwise), and thus molecular signals controlling each process were sought (McMahon, 2010).
Which mesoderm movements were FGF-dependent were investigated and, in particular, either Ths- or Pyr-dependent. The interaction between Htl and its two ligands provides a simpler system relative to vertebrates (which exhibit over 120 receptor-ligand interactions) in which to study how and why multiple FGF ligands interact with the same receptor. Previously, it was found that FGF signaling via the Htl FGFR controls collapse of the mesodermal tube but not dorsal-directed spreading (McMahon, 2008). This study demonstrated that FGF signaling is also required for monolayer formation. In addition, distinct, non-redundant roles were defined for the FGF ligands: Ths (but not Pyr) is required for collapse of the mesodermal tube, whereas both Pyr and Ths are required for proper intercalation of mesoderm cells after dorsal spreading (McMahon, 2010).
This analysis raises questions about ligand choice during collapse and monolayer formation. Within the mesodermal tube, cells at the top require a long-range signal in order to orient towards the ectoderm during tube collapse, whereas the signals controlling intercalation during monolayer formation can be of shorter range. It is suggested that the ligands have different activities that are appropriately tuned for these processes. In fact, recent studies of the functional domains of these proteins suggest that Ths has a longer range of action than Pyr, in agreement with the analysis that Pyr does not support tube collapse, but does have a hand in monolayer formation (McMahon, 2010).
This study has demonstrated that Rap1 mutants have a similar mesoderm phenotype to the FGFR htl mutant, with defects in collapse and monolayer formation. It was not possible to establish whether Rap1 acts downstream of FGF signaling, as the complete loss of Mys in Rap1 mutants is more severe than the patchy expression of Mys seen in htl mutants. Therefore, Rap1 could be working in parallel to or downstream of FGF signaling during mesoderm migration. Rap1 has been implicated in several morphogenetic events during Drosophila gastrulation and probably interacts with many different signaling pathways. Further study of Rap1, along with other GTPases, will shed light onto their role during mesoderm migration, how they interact with one another and what signaling pathways control them (McMahon, 2010).
Focus was placed on the more specific phenotype of mys mutants, as its localization is affected in htl mutants and it exhibits a monolayer defect that is similar to pyr and ths mutants. Integrins are important for cell adhesion, so it is not surprising that cells fail to make stable contact with the ectoderm through intercalation in mys mutants. However, some cells do contribute to monolayer formation in the absence of Mys, implying that other adhesion molecules are involved in maintaining contact between the mesoderm and ectoderm. These other adhesion molecules might be activated downstream of FGF signaling as the htl mutant monolayer phenotype is more severe than the mys mutant. Discovering the downstream targets of Htl, which might regulate cell adhesion properties, will help to shed light on the mechanisms supporting collapse of the mesodermal tube (which is not dependent on Mys) and monolayer formation (which is Mys-dependent) (McMahon, 2010).
Cell protrusions, such as filopodia, are important for sensing chemoattractants and polarizing movement during migration. Previous studies have focused on protrusive activity at the leading edge during mesoderm migration in Drosophila and shown that these protrusions are FGF-dependent. In this study, it was found that protrusions exist in all mesoderm cells, not just the leading edge, and that these protrusions also extend into the ectoderm (McMahon, 2010).
The study demonstrates that FGF signaling, as well as integrin activity, is required to support protrusive activity into the ectoderm; this is a potential mechanism by which FGF signaling and Mys could control movement toward the ectoderm during monolayer formation. The function of protrusions at the leading edge remains unclear, as they appear to be reduced in pyr and mys mutants, but migration in the dorsal direction still occurs in both mutant backgrounds. One interpretation is that FGF and Mys are important for generalized protrusive activity and that extensive protrusions are required for intercalation but not dorsal migration (McMahon, 2010).
Based on this study, it is proposed that mesoderm migration is a stepwise process, with each event requiring different molecular cues to achieve collective migration. Invagination of the mesoderm is the first step in this process and is dependent on Snail, Twist, Concertina, Fog and several other genes. Next, collapse of the mesoderm tube onto the ectoderm requires Htl activation via Ths. Rap1 might be involved in this process as well but the phenotype of Rap1 mutants is complex and it is unclear which phenotypes are primary defects (McMahon, 2010).
Following collapse, mesoderm cells spread dorsally by an unknown mechanism. Dorsal migration is unaffected in pyr and ths mutants and occurs in all cells that contact the ectoderm in htl mutants, implying that FGF signaling is, at most, indirectly involved in this step owing to the earlier tube collapse defect (McMahon, 2008). Whether dorsal migration requires chemoattractive signals or whether the cells simply move in this direction because it is the area of least resistance remains unclear (McMahon, 2010).
Finally, after dorsal spreading is complete, any remaining cells not contacting the ectoderm intercalate to form a monolayer. This process is controlled by a combination of both Pyr and Ths interacting through Htl and also by Rap1 and Mys. In other systems, intercalation can lead to changes in the properties of the cell collective, for instance, lengthening of a body plan. However, this study has shown that dorsal migration and spreading are not a result of intercalation, as intercalation occurs after spreading has finished (McMahon, 2010).
Coordination of these signals to control collective migration enables the mesoderm to form a symmetrical structure, which is essential for embryo survival. This model begins to address the question of how hundreds of cells move in concerted fashion and is relevant for a generalized understanding of embryogenesis and organogenesis. It was found that mesoderm migration is accomplished through sequential movements in different directions, implying that collective migration might be best achieved by distinct phases of movement (McMahon, 2010).
The transcription factor Twist initiates Drosophila mesoderm development, resulting in the formation of heart, somatic muscle, and other cell types. Using a Drosophila embryo sorter, enough homozygous twist mutant embryos were isolated to perform DNA microarray experiments. Transcription profiles of twist loss-of-function embryos, embryos with ubiquitous twist expression, and wild-type embryos were compared at different developmental stages. The results implicate hundreds of genes, many with vertebrate homologs, in stage-specific processes in mesoderm development. One such gene, gleeful/lame duck, related to the vertebrate Gli genes, is essential for somatic muscle development and is sufficient to cause neural cells to express a muscle marker (Furlong, 2001).
Formation of muscles during embryonic development is a complex process that requires coordinate actions of many genes. Somatic, visceral, and heart muscle are all derived from mesoderm progenitor cells. The Drosophila twist gene, which encodes a bHLH transcription factor, is essential for multiple steps of mesoderm development: invagination of mesoderm precursors during gastrulation, segmentation, and specification of muscle types. The role of twist in mesoderm development has been conserved during evolution, perhaps because it controls conserved regulatory mesoderm genes. For example, tinman and dMef2 are regulated by Twist in flies and are highly conserved in sequence and function in vertebrates (Furlong, 2001).
In Drosophila, somatic muscle forms from progenitor cells that divide to become muscle founder cells. Founder cells acquire unique identities controlled by transcription factors including Krüppel, S59, vestigial, and apterous. Each of the 30 body wall muscles in an abdominal hemisegment is initiated by a single founder cell and has unique attachments and innervations. To further clarify mechanisms underlying founder cell specification, myoblast fusion, and muscle patterning, Drosophila mutants together with microarrays of cDNA clones were used (Furlong, 2001).
twist mutant embryos develop no mesoderm. A population of mRNA species isolated from twist homozygous embryos was compared with that of stage-matched wild-type embryos. Drosophila lethal mutations are maintained as heterozygotes, in trans to balancer chromosomes. A twist mutation was established in trans to a balancer chromosome carrying a transgene encoding green fluorescent protein (GFP). Embryos were collected from wild-type and twist/GFP-balancer fly stocks at specific developmental stages. The twist/GFP-balancer collections contain a mixed population of embryos: one-quarter twist homozygotes lacking GFP, half heterozygotes with one copy of GFP, and one-quarter homozygous for the balancer chromosome with two copies of GFP. Homozygous twist mutant embryos were separated from their siblings using an embryo sorter. Putative homozygous twist embryos were assessed by immunostaining with an antibody to dMef2. More than 99% of the selected embryos had the twist phenotype (Furlong, 2001).
Three different periods of mesoderm development were analyzed: stages 9-10, 11, and 11-12 . During stage 9-10, the earliest time GFP is detectable in the balancer embryos, mesoderm cells migrate dorsally and become specified as somatic, visceral, cardiac, and fat body mesoderm. twist and its direct targets tinman and dMef2 are expressed throughout stage 9-10 mesoderm. The middle period contains stage 11 embryos and is a transition between the first period (stage 9-10) and the third period (late stage 11-12). During late stage 11 and stage 12, myoblast fusion begins and twist expression remains prominent in only a subset of the somatic muscle cells (Furlong, 2001).
For each developmental period, three independent embryo collections, embryo sortings, and microarray hybridizations were conducted. The microarrays used for the analysis contained over 8500 cDNAs corresponding to 5081 unique genes plus a variety of controls. Each embryonic RNA sample was compared with a reference sample, containing RNA made from all stages of the Drosophila life cycle to allow direct comparisons among all the experiments (Furlong, 2001).
To determine how transcription was affected by the twist mutation, SAM (significance analysis of microarrays) analysis was used. Genes that are normally highly expressed in mesoderm should have lower transcript levels in twist homozygotes. Genes in other tissues whose expression depends on signals from the mesoderm might also have reduced expression. Transcripts of 130 genes, the 'Twist-low' group, were significantly lower in twist mutants than in wild type. Conversely, cells that would have formed mesoderm may take on other fates in the absence of twist, such as neuroectoderm; therefore, many transcript levels could increase in twist mutants. Genes whose transcription is repressed by signals from the mesoderm would also be enriched in twist mutants. One hundred fifty genes, called the 'Twist-high' group, have increased levels of RNA in twist mutant embryos (Furlong, 2001).
In total, 280 of ~5000 genes had significant changes in transcript levels, with 10 false positives. The genes on the array include 15 previously characterized mesoderm-specific genes, all of which are significantly reduced in twist mutant embryos. The arrays also contain genes known to be transcribed in both mesoderm and other cell types. Significant changes in expression were detected for many of these genes (Furlong, 2001).
The 130 Twist-low genes were divided into three groups with similar trends of expression by a self-organizing map (SOM) clustering program. The 24 group A genes, which include tinman, dMef2, and bagpipe, have reduced transcript levels in twist mutants at all developmental stages assayed. Most of the Twist-low genes fall into the B and C groups. The 62 group B 'early genes' encode transcripts with reduced levels of expression in twist mutants only during stages 9-10, not later. One member of group B, stumps (dof/hbr) is essential for mesoderm cell migration. stumps RNA is abundant in the mesoderm at stages 9-10 and is strongly reduced by stage 11. At stage 11, stumps RNA accumulates in trachea, which is largely unaffected in twist mutants (Furlong, 2001).
The 44 group C genes have reduced transcript levels in twist mutant embryos only during late stage 11 and stage 12. These 'lategenes' include blown fuse, a gene essential for myoblast fusion; delilah, a gene required for somatic muscle attachment, and genes such as kettin, which is required to form contractile muscle. Given the predominantly early expression of twist, the early genes in groups A and B are the best candidates for direct transcription targets of Twist, though some indirectly activated genes may be present within these groups. Group C late genes are likely to be regulated by products of genes that are activated by Twist (Furlong, 2001).
In situ hybridizations were done using a previously uncharacterized representative of each Twist-low group. In each case, the hybridization pattern was consistent with the predicted time of transcription. A group A gene, CG15015 (GH16741), is transcribed in somatic muscle throughout stages 9-12. A group B gene, CG12177 (GH22706), is transcribed during early mesoderm development, but not later. CG14848 (GH21860), a group C gene, is expressed in the stomodeum but not the mesoderm during stages 9-10. Its mesoderm expression initiates during stage 11, the latest period of the twist experiment. Thus, combining loss-of-function mutant embryo analysis with staged embryo collections provides gene expression information for both tissue specificity and temporal expression (Furlong, 2001).
The mis-expression of twist in the ectoderm is sufficient to convert both neuronal and epidermal tissues to a myogenic cell fate. RNA from embryos with ubiquitous twist expression was used to evaluate the ability of Twist to initiate mesoderm-like gene expression in cells that would normally form other tissue types. Genes whose transcript levels decrease in twist loss-of-function embryos and increase when twist is ubiquitous are excellent candidates for regulators of mesoderm development or differentiation (Furlong, 2001).
To ectopically express twist, a dominant gain-of-function mutation of the maternal gene Toll (Toll10B) was used. Activated Toll induces the expression of twist and snail in early embryos and of immune response genes in older embryos. Thus, the effects of Toll10B on gene expression reflect the activities of Twist as well as Snail and Dorsal, or their combined actions. Toll10B embryos are essentially bags of mesoderm; epidermal structures are absent or greatly reduced, and they have been used successfully in subtractive hybridization screens to identify mesoderm genes. The gene transcription profile of Toll10B embryos was compared with that of wild-type embryos during four periods of development, using the reference sample to normalize experiments. The earliest period, stage 5, is when twist is initially expressed in presumptive mesoderm. The other three periods analyzed were those used in the twist mutant analysis: stages 9-10, 11, and 11-12 (Furlong, 2001).
In Toll10B embryos, 447 genes had significant changes in RNA levels compared with stage-matched wild-type embryos, 16 of which are predicted to be false positives. Transcripts from 166 genes were reduced in Toll10B embryos compared with wild type. These genes may be involved in neuroectoderm events that are blocked when cells are turned into mesoderm. Transcripts of 281 'Toll-high' genes were increased in Toll10B embryos. Of the 21 previously characterized mesoderm-specific genes on the arrays, 18 have significantly increased transcript levels in Toll10B embryos. The remainder may require activators other than Toll, such as signals from the severely altered ectoderm (Furlong, 2001).
Genes with altered transcription in twist and Toll10B mutants were analyzed with a hierarchical clustering program to identify similar transcription profiles. The genes were divided into putative 'mesoderm' and 'non-mesoderm' groups. Non-mesoderm genes were defined as having increased transcript levels in mesoderm-deficient (twist) embryos and/or decreased expression in mesoderm-enriched (Toll10B) embryos. Mesoderm genes were defined as having decreased transcript levels in twist mutants and/or increased transcripts in Toll10B mutants. The mesoderm genes group would also contain genes expressed in other tissues in a mesoderm-dependent manner (Furlong, 2001).
Non-mesoderm genes in clusters B and C are repressed in Toll10B mutants. Cluster B genes have increased RNA levels in twist mutant embryos, whereas cluster C genes do not change significantly. The overexpression of twist in the presumptive ectoderm in Toll10B embryos results in a conversion of ectodermal cell fate into mesoderm. snail and dorsal are ectopically expressed in Toll10B embryos and transcriptionally repress the expression of neuroectoderm and ectoderm genes. The conversion of ectoderm to mesoderm due to twist misexpression, and the ability of Snail and Dorsal to repress ectoderm genes, suggests that the B and C clusters should contain primarily neuroectodermal genes. Indeed, the non-mesoderm genes include 31 previously characterized neuroectodermal genes. One previously unknown cluster B gene that encodes a putative cell adhesion protein is transcribed in the ventral nerve cord. Another previously unknown gene within cluster C is transcribed within the developing brain (Furlong, 2001).
The Twist-low and Toll-high genes have in common 51 genes that are highly likely to be involved mesoderm development. For example, transcription of the genes in cluster D is reduced in twist mutants and increased in Toll10B mutants during most or all time periods (Furlong, 2001).
A complete overlap between Twist-low and Toll-high gene sets is not expected for three reasons: (1) development of dorsal mesoderm, and of muscle founder cells marked by apterous and connectin, requires the Decapentaplegic signal and perhaps others. Changed characteristics of cells that form ectoderm in Toll10B embryos interfere with these signaling events. A significant reduction is observed in dpp RNA levels in Toll10B embryos. (2) During midgut development, endoderm cells migrate along the mesoderm. Midgut endoderm development is affected in twist mutants. Some Twist-low genes with unchanged expression in Toll10B embryos are transcribed in the midgut. (3) Ectopic Twist inhibits visceral mesoderm and heart development and promotes excess somatic muscle development. Toll10B embryos produce high levels of Twist throughout the embryo, so genes that have reduced RNA levels in both twist and Toll10B mutant embryos are likely to be visceral muscle and heart genes. Indeed, bagpipe and connectin, genes expressed in visceral mesoderm, are among the 79 Twist-low genes not induced by ectopic Twist (Furlong, 2001).
Of the 281 Toll-high genes, 230 are unaffected in twist mutants. Some of the 230 are normally expressed late in embryogenesis in wild-type embryos but are expressed prematurely in Toll10B embryos due to ectopic Twist. These include Myo61F, MSP-300, and Paramyosin, genes normally active in terminally differentiated muscle (stage 16). Ectopic Snail and Dorsal in Toll10B embryos may activate genes that are unaffected in twist mutants. Snail can repress neuroectodermal genes and may also activate mesoderm genes. Dorsal activates immune response genes later in development. relish, drosomycin, and metchnikowin genes -- all immuneresponse genes -- have higher transcript levels in Toll10B embryos (Furlong, 2001).
Data from loss- and gain-of-function experiments, combined with careful staging, yield a useful picture of genes that are likely to be required for mesoderm specification and muscle differentiation. Of 360 identified mesoderm genes, 273 have not been the focus of developmental studies. The predicted proteins encode transcription factors, signal transduction molecules, kinases, and pioneer proteins. The stage at which each gene is active is one criterion for assigning possible functions. Another key criterion will be finding a mutant phenotype. As a pilot, this additional step was undertaken for the gene CG4677 (LD47926). Changes in CG4677 transcript levels were also observed in a Toll10B subtractive hybridization screen (Furlong, 2001).
CG4677 is transcribed in the visceral mesoderm at stages 10-13 and the somatic mesoderm during stages 11-13. This gene encodes a C2H2 zinc finger transcription factor with high sequence similarity to vertebrate Gli proteins: the gene has been named gleeful (gfl). Mammalian Gli proteins act downstream of Hedgehog signaling proteins to control target gene transcription (Furlong, 2001).
The role of gfl in mesoderm development was assessed by disrupting its function using RNA interference. Injection of a double-stranded RNA (dsRNA) control sequence had no effect on mesoderm development. In contrast, gfl dsRNA injection causes severe loss and disorganization of somatic muscle cells, whereas heart and visceral muscle are unaffected. A similar phenotype is seen in Df(3R)hh homozygous embryos: the deficiency removes gfl but not the nearby hedgehog gene (Furlong, 2001).
To determine whether gfl can induce muscle cell development, a UAS-gfl transgenic fly strain was generated. Ectopic expression of gfl using an en-GAL4 driver results in lethality and induction of ectopic dMEF2 expression in the ventral nerve cord. Remarkably, Gfl is sufficient to induce expression of a muscle gene in neuronal cells. Previous studies have shown an essential role for Sonic hedgehog signaling in the formation of slow muscle in avian and zebrafish embryos. gfl may be performing a similar role in Drosophila somatic muscle development (Furlong, 2001).
To delineate the combinatorial relationships between Twist and other TFs, an initial transcriptional network was generated for early mesoderm development. The temporal binding map for Twist was integrated with in vivo binding data for Mef2, Dorsal, and Tinman. A previous study of Mef2-bound enhancers offers the largest collection of regulatory regions bound at this stage of development to date. As it is difficult to visualize all 494 Twist target genes, focus was placed on TFs whose CRMs are cobound by two or more regulators during these stages of development. Therefore, all links in this network represent direct connections to the same CRM at the same stages of development (Sandmann, 2007).
The resulting core network of 51 TFs is already relatively complex, with nine genes [nau, E(spl), eve, bap, Ubx, lbe, odd, hth, and Ptx1] being targeted by three out of the four examined regulators. The topology of the network provides several insights into how Twist functions to regulate multiple aspects of early mesoderm development. Extensive combinatorial binding and feed-forward regulation are abundant features. Dorsal activates twist, which in turn coregulates the majority of known direct Dorsal targets. This network motif is even more prominent within the mesoderm: Twist regulates the expression of Mef2 and tinman, and cobinds with these TFs to many of their targets' enhancers. In fact, Twist co-occupies 42% of all Mef2-bound enhancers during early mesoderm development. Depending on the logical inputs from the two upstream regulators (transcriptional repression or activation), feed-forward loops can aid in cellular decision making by filtering out noisy regulatory inputs or control the timing of a transcriptional response. For example, early gene expression in the mesoderm (e.g., activation of tin) depends on Twist alone, while transcription of other genes initiated at a later stage may require the input from both Twist and Tinman proteins (Sandmann, 2007).
Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, phylogenetic profiling was combined with a DNA-based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. A small number of enhancers were assembled that are active in Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, the number of enhancers was increased by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. A classifier using this enhancer set was then built and trained, and additional related enhancers were identified based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, extensive diversity was found in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type-specific developmental gene expression patterns (Busser, 2012).
There are three main approaches for the prediction of tissue-specific regulatory elements that are based on high-throughput sequencing coupled with chromatin immunoprecipitation (ChIP-Seq), DNA sequence pattern analysis, or hybrid methods that combine both of these strategies. ChIP-Seq for p300 using mouse embryonic tissue has proven to be an accurate means for identifying enhancers and their associated activities, with in vivo validation rates varying from 62% to 88%. Computational analysis of whole-genome histone modification profiles using hidden Markov models and machine learning techniques has also been highly successful at linking chromatin signatures with regulatory elements. Finally, computational models that identify tissue-specific enhancers relying on sequence motifs and linear regression and support vector machines have been similarly effective, with in vivo validation rates of de novo predictions ranging from 62% for heart enhancers to 91% for brain enhancers. Although experimental techniques are often preferred for identifying enhancers on a genome-wide scale, ChIP-Seq has several limitations. For example, ChIP-Seq experiments are typically carried out in only one species and for individual cell types, and are currently not sufficiently precise for low-quality genome sequences. Thus, de novo prediction of regulatory elements based on ChIP-Seq data critically depends on the availability of relevant data for the species, cell type and genomic regions of interest. Currently, computational analysis of DNA sequence patterns shared by a set of regulatory elements with the same or similar biological activity remains a highly effective method for the de novo discovery of tissue-specific enhancers, and the simultaneous elucidation of cell type-specific regulatory codes. The method presented in this study further extends the usefulness of computational sequence analysis by exploring phylogenetic information that can be used to improve the classification accuracy, a strategy that promises to be advantageous in the large number of cases where comparative genomics data are available (Busser, 2012).
Computational approaches for predicting cis-regulatory modules are commonly based on machine learning of arrangements of TFBSs in enhancers that have common functions. These methods rely heavily on a training set of related enhancers to detect over-represented TFBS combinations. Unfortunately, in the vast majority of cases -- including the present study of Drosophila muscle FC enhancers -- the size of the training set is limited by the lack of experimentally validated tissue- and cell type-specific enhancers, which results in overfitting of computational models and poor accuracy of predictions. To overcome this problem, and to provide a generalizable approach for increasing the size of the training set, a phylogenetic profiling strategy was developed based on a search for diverged orthologous counterparts of available enhancers from distantly related species. Twenty-four Drosophila orthologs were identified using this approach, which more than doubled the size of the training set. The ability to accurately distinguish FC enhancers was assessed in a cross-validation framework using the extended training set, and it was determined that the classifier accuracy is 89% as assessed by the AUC approach. This classifier was then developed to scan the entire genome of D. melanogaster for novel FC enhancers, retrieving 5,500 high-scoring predictions at a FPR of 5%. These predictions were significantly associated with genes expressed in FCs, demonstrating that the model was able to capture essential features of FC gene co-regulation. A similar machine learning approach could be applied to a diverse array of datasets, including experimentally-verified regulatory elements from co-expressed targets at either a germ layer, organ, tissue or cellular level from invertebrate and vertebrate databases. Alternatively, a similar approach could be coupled to a training set of predicted regulatory elements derived from genome-wide analyses of chromatin marks or DNAse hypersensitive sites in active enhancers associated with a co-expressed gene set (Busser, 2012).
Evolutionary constraint of functional sequences is routinely employed as an effective filter to improve the prediction of regulatory elements. Furthermore, cross-species comparisons have been successfully exploited to obtain evidence for functional TFBSs. For example, Rouault (2010) used twelve Drosophila species to identify over-represented motifs in the regulatory elements of genes expressed in neural progenitor cells, with sequence orthologs used to enrich the training set and to give prominence to conserved motifs. However, this method extends this approach by including suitably diverged orthologous enhancers from other Drosophila species in the dataset used to train the classifier. The purpose in designing this strategy was two-fold. First, it was desired to enrich for relevant sequence motifs in the training data, allowing for a level of variation that would improve the generalization of the model. Second, a potentially wider variety of TFBS arrangements would be provided that characterize the architecture of authentic FC enhancers. In essence, the addition of orthologous sequences boosts the statistical power of the significance tests, revealing patterns of TFBSs that otherwise could have been neglected (Busser, 2012).
Of note, when 5 of these orthologous sequences were tested in transgenic reporter assays in D. melanogaster, the overall expression pattern generated was similar to the D. melanogaster counterpart despite extensive evolutionary shuffling of known TFBSs. Similar binding site reorganization has been documented for the enhancers that regulate both the segmentation and mesodermal patterns of eve expression. Numerous other studies have shown that the order and spacing of TFBSs is critical for enhancer function. These results suggest that regulatory elements can direct similar expression patterns provided that the overall composition and order of collaborating TFs is maintained. The finding that enhancer function is preserved in the orthologous sequences examined in this study establishes the validity of the sequence conservation thresholds chosen for the present studies, and suggests that the incorporation of orthologous sequences to increase a training set without over-fitting the data will be a generally applicable approach (Busser, 2012).
To assess the accuracy of this method, 12 predicted FC enhancers were selected and their in vivo functions were tested. Seventy-five percent of the putative enhancers were experimentally validated as having transcriptional activity, demonstrating the effectiveness of this approach to identify regulatory sequences. However, of the sequences showing regulatory functions, only 4 of 9 were active in the mesoderm—including 2 in FCs—and 3 of 9 had nervous system activity. These data suggest that the model has been able to reliably recognize general properties of tissue-specific enhancers without specifically distinguishing an overall muscle FC code, even though numerous individual FC-specific motifs were identified. The former finding is similar to the results of Sinha and colleagues (Kantorovitz, 2009) who found that the majority of their classifier predictions were active enhancers, but only a minority were expressed in the predicted pattern. A number of confounding factors can explain this outcome (Busser, 2012).
First, most members of the enhancer training set are active in both FCs and other cell types, including additional mesodermal cells such as the cardiac and visceral mesoderm, as well as some cells of the nervous system. For example, the enhancer responsible for the FC activity of the hunchback gene is also active in the longitudinal visceral mesoderm, and enhancers directing the FC expression of the vestigial, big brain and king-tubby genes are also active in the peripheral nervous system. These results suggest that the regulatory networks specifying the somatic and visceral mesoderm share common features, which is consistent with both the available genetic and genomic evidence for the diverse developmental functions of key mesodermal transcription factors. Second, different members of a given TF family bind to similar motifs but have distinct tissue-specific expression patterns and developmental activities. Thus, combinations of motifs involved in the specification of muscle FCs and the nervous system may overlap. For example, this situation occurs with E-box and NK-homeodomain motifs. Third, some TFs are expressed and functional in the derivatives of more than one germ layer. Fourth, the sequence features characteristic of cell type-specific enhancers, such as those active in muscle FCs, are expected to be under-represented in available training sets owing to the diversity of combinatorial TF models required to specify such a heterogeneous cell type. Identification of many examples of a particular cell-specific signature is a major challenge since each of the approximately 30 FCs in each Drosophila hemisegement expresses a unique combination of cell-specific muscle identity TFs and downstream target genes. Thus, 30 distinct cell states exist, each governed by a different but partially overlapping set of regulatory TFs. In contrast to the difficulties involved in dissecting regulatory codes at single cell resolution, shared features that direct activity to the general level of tissues and organs have been more readily identified using a machine learning approach, as was found in this study for enhancers having mesodermal, although not necessarily FC, activity. This likely reflects the dominant role that some TFs play in the regulatory network specifying the identities of numerous tissues. Fifth, since there appears to be a regulatory signature for enhancers, it is likely that these aspects of enhancer structure will be more significantly over-represented than those features that specify individual FC activity patterns. Sixth, the use of phylogenetic profiling might have expanded the biological function of the training dataset by introducing additional enhancer functions acquired by the orthologs of the original D. melanogaster sequences during their evolution. While this study has been able to show that the phylogenetic profiling approach improves the accuracy of the classifier, one drawback of its use might be that the final classifier recognizes a broader biological domain than the function of the original training set of sequences derived from the reference species. Finally, classifier predictions may represent cis-regulatory elements other than enhancers, for example, silencers and insulators, which would not be detected by the transgenic reporter assays (Busser, 2012).
In summary, a number of confounding factors influenced the ability to identify an enhancer signature that is specific for individual muscle FCs. However, despite these challenges, the successful identification of novel TF binding motifs responsible for the cell type-specific activity of FC enhancers is encouraging the idea that this is a tractable problem that can be solved by an iterative approach to the computational analysis of this and other complex developmental systems. Thus, future studies must focus on obtaining a larger training set of sequences in which enhancers are categorized based on their activities at single cell resolution, combined with the appropriate weighting of newly validated motifs that contribute to the expression pattern of interest. In this manner, each experimental round would improve the accuracy of the classifier (Busser, 2012).
The motifs ranked by the classifier used in this study as having the highest discriminatory power are part of a large regulatory network that is known to be critical for mesoderm specification and myogenesis. These motifs include binding sites for JAK/STAT, Ets, bHLH, Wingless/Tcf, homeodomain and forkhead proteins. Furthermore, it has previously been suggested that Ets is part of a transcriptional code regulating the C1 subset of FC genes, which was validated in this study using site-directed mutational analysis of the Ndg enhancer, a previously characterized regulatory element associated with a C1 FC gene (Busser, 2012).
To extend the components of the myogenic regulatory network beyond these known TFs and motifs, the function was examined of the classifier-defined sequence motifs recognized by POU homeodomain and Myb proteins, transcription factors having no previously known role in Drosophila myogenesis. Mutagenesis of POUHD motifs attenuated the activity of the Ndg enhancer in many mesodermal cells. However, a zygotic loss-of-function mutation in acj6, the only POUHD that was found to be expressed in the mesoderm, had no effect on Ndg gene expression . Given the strong maternal contribution to this gene, RNAi was used to knock down both maternal and zygotic acj6 transcripts, but this manipulation had no effect on Ndg-GFP reporter activity. These findings leave unresolved the identity of the TF that binds to the motif in question. The future characterization of this TF, including exploring the possibility that it is not a POUHD protein, will require searching functional motifs against larger TF databases, combined with analysis of the embryonic expression and function of any new candidates that emerge (Busser, 2012).
Inactivating mutations of the Myb binding sites in the Ndg enhancer led to extensive de-repression of the reporter in other mesodermal cells. Myb is a ubiquitously-expressed DNA binding protein which plays a critical role in controlling regulatory decisions during proliferation and differentiation of progenitor cells. Identifying a putative role for Myb in myogenesis documents the power of this approach, since functional studies tend to focus on genes with restricted expression patterns. However, a definitive assessment requires examining the effect of loss-of-function mutations in Myb. In any event, as myogenesis in Drosophila occurs through a series of asymmetric and symmetric cell divisions, a role for Myb in regulating FC gene expression is entirely consistent with a transcriptional regulator acting at the interface between replication and transcription. Alternatively, Myb may cooperate with other TFs to activate cell or tissue-specific gene expression (Busser, 2012).
Interestingly, T-box motifs scored well in the classification, yet no role for T-box TFs has previously been described in Drosophila somatic muscle development, despite widespread functions of this TF class in mesoderm specification and myogenesis in vertebrates, as well as cardiogenesis in Drosophila and vertebrates. This study shows using both cis and trans tests of TF function, along with gene co-expression, that Optomotor-blind-related-gene-1 (Org-1) is a muscle identity TF. In particular, the cis effects of Org-1 were documented in the FC enhancers associated with two known muscle identity TFs, Slou and Lbl, and org-1 expression localizes to the SBM and VT1, muscles in which the lb genes and slou, respectively, are the only previously described determinants of muscle identity. Slou function is critical for the proper development of muscles LO1 and VT1 and is further required to repress the lb genes in these cells, suggesting a co-regulatory relationship between slou and lb. It is likely that org-1 acts upstream of slou and lb in this regulatory hierarchy since org-1 expression precedes slou and lb, and the ectopic expression of org-1 causes increased expression of slou and lb. In addition, the essential role of org-1 in this regulatory network is revealed by the effects of org-1 overexpression and RNAi knockdown on development of lb- and slou-expressing muscles. Interestingly, the mouse orthologs of org-1 and lb genes, Tbx1 and Lbx1, respectively, have been suggested to regulate myogenic differentiation in the limb. Given the high degree of sequence similarity, and the close correspondence of expression patterns and functions in Drosophila and mouse, the collaborative roles of these two TFs in myogenesis appear to have been conserved through evolution (Busser, 2012).
Computational prediction of regulatory elements requires a thorough understanding of the TFs and motifs that orchestrate gene co-expression patterns. In prior studies, it was established that 5-way and 3-way 'AND' combinations of 3 signal-activated (Tcf, Mad and Pnt) plus 2 tissue-restricted (Twi and Tin) TFs constitute distinct regulatory models for different FC enhancers. The present study significantly extend these prior combinatorial codes for FC gene regulation by identifying four additional classes of TFBSs that are critical for accurate FC enhancer activity, namely POUHD, Myb, Fkh and T-box motifs. Moreover, these findings provided an opportunity to examine the complete spectrum of regulatory motif usage across a collection of regulatory elements that are active in different muscle FCs, which led to the identification of 18 unique combinations of 11 TFBSs for the entire set of 18 known FC enhancers. Thus, unlike other cases that have been studied, a single enhancer archetype does not appear to exist for this subpopulation of myoblasts. This finding likely reflects the fact that although these elements all display FC activity, with some overlap at the level of individual cells, no two FC gene expression patterns directed by this enhancer set are identical (Busser, 2012).
The marked heterogeneity of FC enhancer architecture uncovered in this study reflects not only distinct combinations of various TF classes (including signal-activated, ubiquitous and both tissue- and cell type-specific TFs), but also diversity at other biological levels, including the unique identities of the thirty muscle FCs and their differentiated derivatives in each abdominal hemisegment, and the different gene expression patterns exhibited by those particular cells. Thus, TFBS combinatorics provide a plausible molecular explanation for the functional complexity of enhancers having related but non-identical activites at the resolution of individual cells in the context of the developing embryo (Busser, 2012).
This study has investigated the transcriptional regulatory network specifying individual muscle FCs using an integrated genomics approach that includes identification of orthologous enhancers, de novo motif discovery, classification of enhancer sequence features, empirical testing of candidate enhancers, and cis-trans tests of target gene regulation. It was also established that a small set of training sequences can be expanded with orthologous sequences. Moreover, motifs learned by the classifier were empirically found to be critical for the appropriate spatio-temporal activities of FC enhancers, and suggested new candidate TFs in the myogenic regulatory network. Using this approach, one such candidate TF, Org-1, was identified as a novel muscle identity TF, and further found that no two enhancers with related activities contain the same combination of TFBSs. The tools and strategy used in this study can be readily applied to other cell types to identify the motifs and trans-acting factors regulating a set of co-expressed genes. Finally, it is anticipated that an iterative application of this approach, which could include training on datasets of different epigenetic marks associated with active enhancers or previous ChIP studies of known mesodermally-relevant TFs , will lead to further refinements in the determination of cell type-specific transcriptional codes (Busser, 2012).
Transcription factors (TFs) influence gene expression by binding to DNA, yet experimental data suggests that they also frequently bind regulatory DNA indirectly by interacting with other DNA-bound proteins. This study used a data modeling approach to test if such indirect binding by TFs plays a significant role in gene regulation. Regulatory function of indirectly bound TFs was incorporated into a thermodynamics-based model for predicting enhancer-driven expression from its sequence. The new model was fit to a rich data set comprising hundreds of enhancers and their regulatory activities during mesoderm specification in Drosophila embryogenesis and showed that the newly incorporated mechanism results in significantly better agreement with data. In the process, the first sequence-level model was derived of this extensively characterized regulatory program. It was further shown that allowing indirect binding of a TF explains its localization at enhancers more accurately than with direct binding only. This model also provided a simple explanation of how a TF may switch between activating and repressive roles depending on context (Bhogale, 2022).
Drosophila muscles originate from the fusion of two types of
myoblasts -- founder cells (FCs) and fusion-competent myoblasts (FCMs). To
better understand muscle diversity and morphogenesis, a
large-scale gene expression analysis was performed to identify genes differentially
expressed in FCs and FCMs. Embryos derived from
Toll10b mutants were employed to obtain primarily muscle-forming
mesoderm, and activated forms of Ras or Notch were expressed to induce FC or FCM
fate, respectively. The transcripts present in embryos of each genotype were
compared by hybridization to cDNA microarrays. Among the 83 genes
differentially expressed, genes known to be enriched in FCs or FCMs,
such as heartless or hibris, previously characterized genes
with unknown roles in muscle development, and predicted genes of unknown
function, were found. These studies of newly identified genes revealed new patterns of gene
expression restricted to one of the two types of myoblasts, and also striking
muscle phenotypes. Whereas genes such as phyllopod play a crucial
role during specification of particular muscles, others such as
tartan are necessary for normal muscle morphogenesis (Artero, 2003).
The Toll10b mutation gives rise to embryos
composed primarily of somatic mesoderm. In these embryos FCs and FCMs are
readily detected, and they respond to the Ras and Notch signaling pathways
in the same way as their wild-type counterparts. Advantage was taken of this
fact to enrich Toll10b mutant embryos for FCs or FCMs,
which allowed a concentration on the transcription in these two specific
cell types within the context of the entire embryo. Genes known to be
expressed and regulated in FCs or FCMs emerged from the screen in the proper
categories. Not all known FC/FCM genes were detected in the screen for several
reasons: the high stringency set for interpretation of the array data; the
presence of only about one-third of the genome on the arrays; the loss of Dpp
in the Toll10b background, and the specific window of
myogenesis (5- to 9-hours) that was the focus of this investigation. However, a plethora
of potential new muscle regulators were uncovered, including known genes with
no previously recognized function in the mesoderm (such as phyl and
asteroid), and genes predicted from the Drosophila genome
sequence but not previously analyzed (Artero, 2003).
Various tests were applied to ascertain the validity of the results.
Available databases were analyzed to find evidence
that the known and predicted genes were expressed at the correct time and place. In
addition, Northern analysis with eleven genes tested the reliability of the
microarray detection and selection criteria; the results from all
genes tested agreed with the array data (Artero, 2003).
A Toll10b sample on the Northern blots allowed
ascertainment of why a gene is enriched in a particular condition. For example, in
the case of FC enriched genes, the signal in the Ras and
Notch lanes can be compared with Toll10b alone to
determine whether the Ras/Notch ratio for a gene is due to activation by Ras
or repression by Notch. Those genes that are 'enriched
under Notch conditions', for example, could reflect a variety of transcription
mechanisms that would result in a ratio of less than 0.6. By Northern
analysis, many of the 'Notch-regulated' genes, and hence the predicted
FCM genes, were found to be repressed by Ras signaling and slightly activated by Notch. As
a case in point, hibris is induced by Notch (2-fold)
and repressed by Ras (10-fold), both by Northern analysis and by in situ
hybridization in embryos (Artero, 2003).
A combination of in situ hybridization, immunostaining and confocal
microscopy was used to verify that the differential expression changes that were observed
in these overexpression embryos reflected true differential expression in the
wild-type situation. The expression of nine genes from different
functional categories was analyzed. For
seven of these, expression was detected in the predicted type of myoblast. For
two, asteroid (ast) and cadmus, no specific staining in embryos was detected by in situ hybridization. For
those genes that fell into the category of 'specific role in muscle
development uncertain', in situ hybridization of several (28%) showed
expression in tissues other than somatic mesoderm that are present in the
Toll10b background. These genes changed their expression
levels in response to Ras or Notch, and may be Ras and Notch targets in
non-mesodermal tissues (Artero, 2003).
The most stringent test, mutational analysis, was applied to a set of genes
for which mutations are available. Preliminary analyses of another four FCM-enriched genes was carried out: Elongation factor Tu mitochondrial (EfTuM), Glutamine synthetase 1, cadmus and parcas. All
four mutants have muscle defects, including muscle losses and aberrant muscle
morphologies. Thus all the genes tested show some muscle defect, supporting the usefulness of the genetic and genomic approach (Artero, 2003).
Taken together, these data suggest that the majority of genes detected play important roles in FCs or FCMs during muscle development. Some of these genes might not have been found in traditional forward genetic screens because of partial or
complete genetic redundancy. The data complement traditional forward genetic
approaches for finding genes crucial for muscle morphogenesis (Artero, 2003).
Each of the thirty FCs per abdominal hemisegment is hypothesized to produce
its own unique combination of transcriptional regulators, though the evidence
for this is limited. In turn the combination of regulators would control the
morphology of the final muscle. Although several transcriptional regulators
have been linked to FC identity, the molecular description is far from
complete. This screen contributed two more FC-specific genes. Previously known
markers, such as slouch or eve, once induced in the muscle
FC, are maintained throughout the remainder of development. Ubx,
which emerged from this screen, is a similarly simple case, as its transcripts
are steadily present in most FCs. By contrast, more
complex patterns of gene expression have been identified in FCs, such as the transient transcription of asense in a subset of FCs. The subsequent
transcriptional inactivation of asense may underlie temporal changes in
cell properties (Artero, 2003).
Even less is known about transcriptional regulators controlling FCM
differentiation. Only one gene, lame duck, has been shown to have a
role in FCMs. This screen has uncovered three more potential players: delilah,
E(spl)mß and CG4136, confirming that FCMs follow their own,
distinct, myogenic program. Discovering what aspects of FCM biology are
controlled by these transcriptional regulators awaits analysis of the
loss-of-function phenotypes (Artero, 2003).
Notch and Ras signaling pathways interact during muscle progenitor
segregation. The results suggest that phyl and
polychaetoid (pyd) may be additional links between the two
signaling pathways in FCs. phyl and pyd both interact
genetically with Notch and Delta. The transcription of phyl, which promotes neural differentiation, is negatively regulated by Notch signaling during
specification of SOPs and their progeny. This study
shows a similar regulation in muscle cells, where Notch signaling represses
phyl expression and Ras signaling increases phyl expression. Likewise, in the
nervous system, the segregation of SOPs requires pyd, a Ras target
gene, to negatively regulate ac-sc complex expression. Similarly, Pyd
may restrict the muscle progenitor fate to a single cell, perhaps by
regulating lethal of scute transcription. Thus, Pyd would collaborate
with Notch signaling to restrict muscle progenitor fate to one cell (Artero, 2003).
FCMs appear to integrate Ras and Notch signaling differently. Two genes
whose transcripts were enriched under activated Notch conditions, parcas and asteroid (ast), have been implicated in Ras signaling in other tissues, directly (ast) or indirectly (parcas). These data are suggestive of a role for Ras signaling in the FCMs, in addition to its role in FC specification. In addition, Notch signaling to FCMs may prime cells for subsequent Ras signaling during muscle morphogenesis, much as occurs in FCs where Ras signaling primes the cell for subsequent Notch signaling during asymmetric division of the muscle progenitor (Artero, 2003).
Embryos that lack or ectopically express phyl have morphological
defects in specific muscles, for example, in LL1 and DO4 in response to
diminished phyl function, and in DT1 and LT4 in response to increased
phyl function. The morphological defects in the loss-of-function
embryos appear to be due to a failure to specify particular FCs, a conclusion
that is based upon missing or abnormal production of the FC marker Kr. In eye
development and SOP specification, Phyl directs degradation of the
transcriptional repressor Tramtrack. In a subset
of the primordial muscle cells, Phyl may work similarly, targeting Tramtrack
for degradation. The presence of Tramtrack would contribute to the specific
identity program of the muscle. Since Tramtrack is expressed in the mesoderm,
this possibility is likely. Alternatively, Phyl may be required for targeted
degradation of some other protein in a subset of FCs. The molecular partner
for Phyl during muscle differentiation is unknown, although preliminary data
suggest that sina is also expressed in somatic mesoderm and thus may
be its partner. These studies have
identified a new role for Phyl in muscle progenitor specification and suggest
the importance of targeted ubiquitination for proper muscle patterning (Artero, 2003).
A role for ubiquitination in muscle differentiation is further reinforced
by the identification of the RING finger-containing protein Goliath (Gol), induced by
activated Notch conditions, and CG17492, induced by activated Ras conditions.
Several RING-containing proteins function as E3 ubiquitin ligases, with the
ligase activity mapping to the RING motif itself. Ligase function has been experimentally confirmed for the Gol ortholog GREUL1
in Xenopus. Thus, targeted protein degradation during muscle morphogenesis could serve a host of crucial functions, such as protein turnover, vesicle sorting, transcription factor activation and signal degradation (Artero, 2003).
The simplest view of the 'founder cell' hypothesis is that each FC contains
all the information for the development of a particular muscle. By contrast,
FCMs have been seen as a naïve group of myoblasts, entrained to a
particular muscle program upon fusion to the FC. This work indicates that these
two groups of myoblasts have distinct transcriptional profiles. These data
raise the possibility of a greater role for FCMs in determining the final
morphology of the muscle and emphasize a need to characterize fully those FCM
genes. For
example, this screen identified a protein kinase of the SR splice site selector
factors (SRPK) whose transcripts are enriched in FCMs, suggesting that
regulation of the splicing machinery is important for muscle morphogenesis.
The Mhc gene undergoes spatially and temporally regulated alternative
splicing in body wall muscles conferring different physiological properties on
these muscles. This FCM-specific expression of SRPK may indicate that the
production of a particular Mhc isoform is regulated by the FCMs that
contribute to that muscle, rather than by the particular FC that seeds the
muscle. In addition, a number of observations suggest that FCMs may be a
diverse population of myoblasts, with different subsets having different
potential to contribute to the final muscle pattern. For example, hbs
expression suggests that only a subset of FCMs express the gene, and
twist expression in lame duck mutant embryos persists in a
subset of FCMs. This study provides additional genes
for exploring whether FCMs are a heterogeneous population of myoblasts as well
as determining the nature of FCM contribution to the final muscle (Artero, 2003).
The molecular events underlying complex morphological changes, such as
migration, cell fusion, cell shape changes or changes in the physiology of a
cell, require a rich and dynamic program of transcription changes. This study has described approximately one-third of this transcriptional profile. The FC- or
FCM-specific transcription of seven genes, and the mutant phenotype of four
selected genes, allowed the definition of new muscle mutations that
specifically affect the morphological traits of a subset of muscles (Artero, 2003).
Embryogenesis is remarkably robust to segregating mutations and environmental variation; under a range of conditions, embryos of a given species develop into stereotypically patterned organisms. Such robustness is thought to be conferred, in part, through elements within regulatory networks that perform similar, redundant tasks. Redundant enhancers (or "shadow" enhancers), for example, can confer precision and robustness to gene expression, at least at individual, well-studied loci. However, the extent to which enhancer redundancy exists and can thereby have a major impact on developmental robustness remains unknown. This study systematically assessed and identifies over 1,000 predicted shadow enhancers during Drosophila mesoderm development. The activity of 23 elements, associated with five genes, was examined in transgenic embryos, while natural structural variation among individuals was used to assess their ability to buffer against genetic variation. Data reveal three clear properties of enhancer redundancy within developmental systems. First, it is much more pervasive than previously anticipated, with 64% of loci examined having shadow enhancers. Their spatial redundancy is often partial in nature, while the non-overlapping function may explain why these enhancers are maintained within a population. Second, over 70% of loci do not follow the simple situation of having only two shadow enhancers-often there are three (rols), four (CadN and ade5), or five (Traf1), at least one of which can be deleted with no obvious phenotypic effects. Third, although shadow enhancers can buffer variation, patterns of segregating variation suggest that they play a more complex role in development than generally considered (Cannavò, 2015). The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. The formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, was found to be essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, it was demonstrated that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together these data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease (Deng, 2015).
This study provides the first evidence that Dia is essential for Drosophila myoblast fusion. Dia is expressed in all myoblasts and is recruited to the myoblast fusion site. The spatial and temporal distribution of Dia at the fusion site parallels that of the F-actin focus, which forms the core of an invasive podosome. This actin rich podosome is critical for FCM invasion of the FC/Myotube during fusion. In keeping with its expression pattern, Dia is essential for myoblast fusion progression: both loss and gain of Dia function lead to a fusion block. Under both conditions, the integrity of the F-actin focus and hence the invasive podosome is compromised; myoblasts expressing DiaDN fail to form the focus, whereas myoblasts expressing DiaCA have many filopodia and have a diffuse organization of F-actin, both of which contribute to a failure in invasive podosome formation and fusion. Dia activity is required after FC/Myotube and FCM recognition and adhesion but upstream of Arp2/3 activity. It is required, in parallel with PI(4,5)P2 signaling, to build a functional F-actin focus at the fusion site. These experiments further indicate that Dia activity is critical for actin dynamics at the fusion site, which, in turn, regulate fusion progression. Moreover, the aberrant F-actin organization at the fusion site in both loss and gain of function is also due to altered localization of the Arp2/3 regulators, SCAR and WASp. Taken together, these data support a role for the formin Dia in a critical first step of actin polymerization at the fusion site, downstream of cell-cell recognition and adhesion, and link its activity to the formation of F-actin foci, required for myoblast fusion (Deng, 2015).
Actin remodeling is critical for myoblast fusion, but Arp2/3 was the only known actin polymerization factor that was shown to be necessary for myoblast fusion. This study now shows that the formin Dia is also required during myoblast fusion. Whereas Arp2/3 preferably binds to pre-existing actin filaments and generates uncapped F-actin, formins nucleate F-actin both de novo and from the barbed ends of pre-existing actin filaments. Thus, Dia can generate actin filaments de novo, which Arp2/3 can bind or elongate (Deng, 2015).
This study also shows that the level of Dia activity is critical for myoblast fusion. Too much actin polymerization leads to too many filopodia and absence of an invasive podosome with its characteristic F-actin core. Too little polymerization leads no actin focus and no podosome formation. FRAP data with DiaCA also hint at whether a limited pool of actin is available for the actin polymerization factors during myoblast fusion. Despite the high rates of actin turnover with expression of DiaCA, the final fluorescence levels of actin returns to the same value as in controls. Additional actin monomers are not recruited to the site, even with high levels of polymerization activity. Interestingly, the rate of actin turnover has also been measured in mutants that affect Arp2/3 activity: specifically, mutations in blow, which regulates the Arp2/3 NPF WASp, show lower rates of actin exchange than in controls, due to a reduced exchange rate for WASp on the barbed ends of actin at the fusion site. Together these data suggest future experiments aimed at examination of whether rates of actin polymerization regulated by both Dia and Arp2/3 are optimized for the available actin pool and tightly controlled for myoblast fusion to properly occur (Deng, 2015).
Both cooperative and antagonistic functions between Dia and Arp2/3 have been reported. This study demonstrates that the coordinated and cooperative activities of these two actin polymerization factors leads to the formation of the F-actin focus. With the exception of sltr/Dwip/vrp mutants that form a focus of wild-type size, single mutants in the Arp2/3 NPF pathways, WASp and SCAR, lead to enlarged foci; however, double mutants in WASp and SCAR pathways do not form foci. This is the same phenotype that is seen in myoblasts expressing the DiaDN. The data support Dia activity being upstream of WASp and SCAR activation of Arp2/3 at the fusion site. This suggests that, at the fusion site, Dia initially provides the necessary context upon which Arp2/3 can act and not vice versa, as has been suggested in other contexts in which linear actin filaments emerge from Arp2/3 based structures. Nevertheless, both sets of actin regulators are necessary for F-actin focus formation that provides the core of the invasive podosome. Neither Dia nor Arp2/3 alone are sufficient (Deng, 2015).
The interplay between Dia and Arp2/3 at the fusion site is also reflected by the localization studies. Too little or too much Dia activity resulted in improper localization and, by extension, improper activity of Arp2/3 NPFs. How could Dia regulate this localization? One possibility is that Dia indirectly regulates Arp2/3 localization. Dia could nucleate linear actin filaments, which then would provide the necessary substrate for recruitment, maintenance and /or activation of Arp2/3 and its regulators, such as the WASp-WIP complex. Another possibility is that Dia, through its interactions with members of the SCAR/WAVE complex such as Abi, may directly localize and/or maintain the localization of Arp2/3 regulators, which are then activated at the fusion site. Abi has been reported to bind directly with Dia in vitro, and this interaction is required for the formation and stabilization of cell-cell junctions. Dia likely changes the localization and integrity of the SCAR/WAVE complex by competitively binding to the N-terminal part of Abi, dissociating Kette/Nap1 from the complex, and thus changing the stability and localization of SCAR/WAVE. It has also been established that the recognition and adhesion receptor, Sns, is capable of recruiting the Arp2/3 NPFs, such as WASp, to the fusion site. While Sns is still clustered at the fusion site in DiaDN and DiaCA, its recruitment activity appears not sufficient for focus formation capable of supporting an invasive podosome (Deng, 2015).
This study has shown that localization of Arp2/3 NPFs is affected in Dia loss and gain of function. In addition to this spatial control, another important way of controlling Arp2/3 activity is through activation of the NPFs via small GTPases. SCAR is activated through Rac-dependent dissociation from SCAR inhibitory complex. WASp is activated by binding to Cdc42, which releases it from auto-inhibited state. This study did not examine the localization of these activated GTPases. However, previous work has shown that PI(4,5)P2 signaling is required for proper localization of activated Rac at the fusion site. How the localization and activity of small GTPases at the fusion site contribute to the spatial and temporal interplay between Dia and Arp2/3 regulation of actin polymerization requires further investigation (Deng, 2015).
It remains unresolved how Dia itself is recruited to the fusion site. The data suggest that the recognition and adhesion receptors Duf and Sns would be involved either directly or indirectly in recruiting Dia to the fusion site, as embryos that fail to express either of these adhesion receptors fail to recruit Dia to the fusion site. In addition, recent data from Drosophila epithelial tubes indicate that PI(4,5)P2 serves as a localization cue for Dia. Previous work has shown that PI(4,5)P2 accumulates at the fusion site after FC-FCM recognition and adhesion; sequestering of PI(4,5)P2 results in a significant fusion block. Therefore, whether PI(4,5)P2 regulates Dia localization at the fusion site was tested. Dia was found to be recruited to the fusion site in the PI(4,5)P2 sequestered myoblasts, suggesting that, in this context, PI(4,5)P2 signaling is not required for Dia localization. These data provide possible explanations for why in PI(4,5)P2 sequestering embryos, smaller actin foci are detected: the localized Dia may be sufficient to recruit low levels of Arp2/3 and its NPFs, which, upon activation, lead to the formation of small F-actin foci. Nevertheless, in the absence of PI(4,5)P2 signaling, Dia that is recruited to the fusion site is not sufficient to produce functional actin focus, capable of directing a fusion event. Recent work also indicates that charged residues in the N- and C-termini of mDia1 are sufficient both for mDia's clustering of PI(4,5)P2 and its own membrane anchorage. This interaction between mDia1 and PI(4,5)P2, in turn, regulates mDia1 activity. Whether such a mechanism is in play at the myoblast fusion site needs to be further investigated (Deng, 2015).
A working model is proposed for the interplay between the actin regulators during myoblast fusion. Dia is recruited to the fusion site upon engagement of the recognition and adhesion receptors by a yet-to-be determined mechanism. It is proposed that PI(4,5)P2 signaling at the fusion site regulates the localization and activation of downstream targets such as Rho-family of small GTPases. These small GTPases lead to the activation of Dia. Activated Dia, in turn, polymerizes linear actin filaments and, in combination with the recognition and adhesion receptors and PI(4,5)P2, recruits the Arp2/3 NPFs, SCAR and WASp. Activation of these Arp2/3 NPFs at the fusion site would be accomplished by small GTPases such as Rac. These, in turn, would activate Arp2/3, leading to branched actin and formation of the F-actin focus and the invasive podosome. Whether the Arp2/3 NPFs such as SCAR/WAVE would negatively regulate Dia to downregulate linear actin polymerization, as suggested for mDia2 in cell culture, or whether Dia competes with WASp for barbed end binding remains to be investigated. However, these mechanisms would underscore a switch from linear F-actin filopodium formation to the linear and branched F-actin invasive podosome-like structure that is necessary for fusion (Deng, 2015).
The actin focus formed at the fusion site is an F-actin rich, invasive podosome-like structure that has been suggested to provide a mechanical force for FCMs to invade the FC/Myotube. Similar invasive actin structures named invadosomes have been seen in different cell types, such as podosomes in macrophages and invadopodia in cancer cell. Arp2/3 is known to play a key role in invadosome formation, and recent studies have revealed the involvement of formins in developing invadosomes. The current data indicate that specific temporal and spatial interactions between the formin Dia and Arp2/3 are required for the actin focus and invasive podosome formation. The data thus provide new mechanistic insights for the interplay of Arp2/3 and Formins during invadosome formation in these contexts (Deng, 2015).
Motoneurons directly influence the differentiation of muscle fibers, regulating features such as muscle fiber type and receptor development. Less well understood is whether motoneurons direct earlier events, such as the patterning of the musculature. In Drosophila, the denervation of indirect flight muscles results in a diminished myoblast population and smaller or missing muscle fibers. Whether the neuron-dependent control of myoblast number is due to regulation of cell division, motoneuron-dependent apoptosis, or nerve-dependent localization and migration of myoblasts, was examined. Denervation results in a reduced rate of cell division, as revealed by BrDU incorporation. There is no change in the frequency of apoptotic myoblasts following denervation. Using time lapse imaging of GFP-expressing myoblasts in vivo in pupae, it was observed that despite denervation, the migration and localization of myoblasts remains unchanged. In addition to reducing myoblast proliferation, denervation also alters the segregation of myoblasts into the de novo arising dorso-ventral muscles (DVMs). To address this effect on muscle patterning, the expression of the founder-cell marker Dumbfounded/Kirre (Duf) in imaginal pioneer cells was examined. There is a strong correspondence between cells that express Dumbfounded/Kirre and the number of DVM fibers, consistent with a role for these cells in establishing adult muscles. In the absence of innervation the Duf-positive cells are no longer detected, and muscle patterning is severely disrupted. These results support a model where specialized founder cells prefigure the adult muscle fibers under the control of the nervous system (Fernandes, 2005).
The motoneuron exerts a mitogenic influence on IFM myoblasts. Following unilateral denervation, the BrDU birthdating experiments revealed a significant decline in the rate of proliferation. This decline is likely sufficient to account for the reduced myoblast population observed in denervated hemisegments (previously quantified by morphometry; Fernandes, 1998). The smaller muscles that result are thus likely due to the smaller number of available myoblasts, and possibly to the absence of neuromuscular excitation following denervation (Fernandes, 2005).
Two alternate mechanisms were ruled out: there was no evidence for a change in the rate of myoblast cell death following denervation, indicating that the motoneuron does not provide an essential survival factor for the cells. Also, no significant change was observed in the migratory behavior of myoblasts following unilateral denervation, when examined at either the single cell or population level. This indicates that the reduced population size was not the result of myoblast emigration from denervated sites (Fernandes, 2005).
The second major effect of denervation was the gross disruption of normal muscle formation in the de novo arising DVMs. Normally, myoblasts coalesced into discrete primordia that prefigure the three sets of DVM muscle fibers. When denervated, the DVM myoblasts remain unpatterned, and the muscles fail to form. This may be due to a direct effect of the motoneuron on the myoblasts. However, denervation also disrupts the behavior of a potential intermediary player, the imaginal pioneer cells, that are thought to prefigure the DVM fibers as myoblast fusion targets (Fernandes, 2005).
BrDU birthdating experiments reveal a significant rise in the rate of DLM myoblast proliferation that normally occurs at 18-24 APF. In denervated regions, myoblast proliferation remains unchanged, holding steady at the earlier, basal level seen prior to the onset of myoblast fusion (at 12-16 h APF). Thus, DLM myoblast proliferation involves two components: a basal and nerve-independent phase (at 12-16 h APF) and a later incremental nerve-dependent phase (at 18-24 h APF). It is proposed that the nerve-dependent increase in myoblast proliferation regulates the number of myoblasts available for fusion, and thus is a way for motoneurons to control muscle size (Fernandes, 2005).
The nerve-dependent rise in DLM myoblast proliferation correlates with the expansion of motoneuronal terminal arbors on the muscle fiber surface. While it is possible that the expansion of motoneuron terminals and the change in myoblast proliferation are independent responses to exogenous hormone signals, the data argue that motoneurons strongly influence myoblast cell division, since proliferation is reduced following denervation. It is proposed that the growing motoneuron terminal either releases a factor that influences myoblast cell division, or alternatively potentiates myoblast responsiveness to available growth factors and mitogens. In either case, the nerve-dependent control of myoblast proliferation would in turn influence the growth of the muscle fiber (Fernandes, 2005).
The rise in myoblast proliferation observed during the nerve-dependent phase of DLM development resembles a feature seen during photoreceptor development in the Drosophila eye. During differentiation of the neuroepithelium, there is a rise in cell proliferation, referred to as the second mitotic wave (SMW), which produces additional precursors that are recruited to eventually form a complete ommatidium. It is likely that the rise in DLM myoblast proliferation similarly serves to maintain the size of the myoblast pool, so that cells can be continuously drawn from the pool until the desired muscle size is achieved (Fernandes, 2005).
That the two phases of DLM myogenesis differ in their nerve-dependence
resembles events associated with vertebrate myogenesis. Mammalian skeletal muscles form in two waves: primary myotubes form first, and serve as scaffolds for secondary myotube formation. Primary myogenesis is independent of innervation, while secondary myogenesis is nerve dependent. This is due to the presence of a nerve-dependent population of myoblasts essential for secondary myotube formation (Fernandes, 2005).
The DVMs develop from the de novo fusion of myoblasts, and critically depend on the motoneuron for muscle fiber formation (Fernandes, 1998 and Fernandes, 1999). Denervation also results in a failure of myoblasts to segregate into distinct DVM primordia. Myoblast patterning and fiber development for the DVMs have been proposed to depend on specialized imaginal pioneer cells. The 'imaginal pioneer' (IP) cells lie in close association with motoneuron arbors, as demonstrated by EM analysis, and are thus potentially dependent on neurons for their normal function or survival. The IP cells are thought to serve as myoblast fusion targets, and thus to prefigure the mature muscle fibers. There have been, however, no reported molecular markers for these cells (Fernandes, 2005).
In the Drosophila embryo the mesodermal cells that generate the somatic muscles are critically dependent on specialized founder cells. Each embryonic founder cell is the precursor of a specific muscle fiber, and is the target of fusion-competent myoblasts. The founder cells each express Dumbfounded/Kirre, a key component of the cell fusion machinery. In loss of function duf mutations myoblast fusion is disrupted (Fernandes, 2005).
Intriguingly, it was found that there are Duf-positive cells within the DVM myoblast pool. Their location, number, and size indicate that they are likely to be the IP cells previously described. The Duf-positive cells are present in the DVM I and II primordia in direct correspondence to the final number of DVM fibers, as is the case for the IP cells. Duf-positive cells have also been reported for other pupal muscles, and a correlation exists between Duf-positive cells and the numbers of both IFM and abdominal muscle fibers. Significantly, it was found that denervation affects the Duf cells of the DVM primordia. Although Duf-positive cells are initially present in the denervated hemisegments in the normal pattern and number (at 12 h APF), following denervation they are no longer reliably observed by 18-20 h APF. By 24 h APF, when control hemisegments possess well patterned Duf-positive DVMs, Duf-positive cells on the denervated side are rarely observed (Fernandes, 2005).
These observations support a model where Duf-positive IP cells in the pupa serve as fusion targets of myoblasts, as is the case for the Duf-positive founder cells in the embryo. Since the Duf molecule is an essential component of the cell fusion machinery, its disappearance following denervation suggests that fusion events are severely disrupted and may explain the associated muscle patterning defects. It cannot as yet be determine whether the loss of Duf labeling is due to a loss of Duf expression in the IP cells, or due to apoptosis. Distinguishing between these scenarios will require in situ time lapse imaging of vitally labeled IP cells in denervated hemisegments (Fernandes, 2005).
DLM fibers arise from larval muscles that persist into the pupal stage. Like all embryonically established somatic muscle fibers, the persistent larval fibers also do not depend on the motoneuron for their formation or maintenance. When denervated, the larval fibers persist and DLM fibers still form, albeit at a slower rate (Fernandes, 1998; Fernandes. 1999). Duf expression is also detected in the persistent larval fibers, consistent with the fact that they function as myoblast fusion targets. However, unlike the DVMs, denervation does not result in a loss of Duf expression in the developing DLMs. The reason for this independence remains uncharacterized, but likely reflects the distinct origin of these cells from larval precursor muscles (Fernandes, 2005).
A dependence on motoneurons for the regulation of muscle size and patterning has been observed for several insect systems. When abdominal Drosophila muscles are denervated, the adult fibers are significantly reduced in mass. The most prominent effect involves the male-specific muscle (MSM) of the fifth abdominal segment of the adult. This muscle is larger than other body wall muscle fibers, a difference attributed to the enhanced recruitment of myoblasts from a common myoblast pool. When the abdominal myoblast pool is reduced experimentally through hydroxyurea treatment, a smaller muscle is present at the MSM location in segment A5. A BrDU labeling analysis remains to be performed to confirm the role of myoblast proliferation on MSM development (Fernandes, 2005).
Denervation studies in Manduca have similarly shown that proliferation of myonuclei is reduced in leg, abdominal, and DLM muscles. At the onset of metamorphosis, muscle precursors appear in the region of the future adult muscles and become associated with tendons (leg muscles) or persistent larval muscles (DLM). This accumulation is then followed by the appearance of proliferating 'myonuclei' within the developing primordia. By contrast, in the case of Drosophila DLMs, BrDU incorporation is restricted to myoblasts present outside the primordia, and there is no evidence of nuclear division within the muscle fibers (Fernandes, 2005).
In conclusion, it is proposed that the motoneuron critically influences the size of the myoblast pool through a direct effect on myoblast cell division, and that this helps regulate the final size of adult muscle fibers. The motoneuron has a second role in regulating the development of de novo forming fibers, where it is essential for the partitioning of myoblasts into muscle primordia. Moreover, continued Duf labeling within the primordia depends on the motoneuron's presence. Thus, the motoneuron influences both the number of cells available for fusion, as well as potentially regulates the fusion events themselves. This is an elegant mechanism for controlling muscle fiber differentiation during myogenesis, and may have evolved as a way to ensure that muscle primordia develop into muscles that meet the diverse demands placed on them by the nervous system (Fernandes, 2005).
Coordinated locomotion of an organism relies on the development of proper musculoskeletal connections. In Drosophila, the Slit-Robo signaling pathway guides muscles to tendons. This study shows that the Slit receptor Roundabout 2 (Robo2) plays a non-cell-autonomous role in directing muscles to their corresponding tendons. Robo2 is expressed by tendons, and its non signaling activity in these cells promotes Slit cleavage producing a cleaved Slit-N-terminal guiding signal, which provides short-range signaling into muscles. Consistently, robo2 mutant embryos exhibited a muscle phenotype similar to that of slit, which could not be rescued by a muscle-specific Robo2 expression but rather by an ectodermally derived Robo2. Alternatively this muscle phenotype could be induced by tendon-specific robo2 RNAi. It was further shown that membrane immobilization of Slit, or its N-terminal cleaved form on tendons bypasses the functional requirement for Robo2 in tendons, verifying that the major role of Robo2 is to promote the association of Slit with the tendon cell membrane. Cleaved Slit (Slit-N) tends to oligomerize whereas full-length uncleavable Slit does not. It is therefore proposed that Slit-N oligomers produced at the tendon membrane by Robo2 signal to the approaching muscle by combined Robo;Robo3 activity. These findings establish a Robo2-mediated mechanism, independent of signaling essential to limiting Slit distribution, which might be relevant to the regulation of Slit-mediated short-range signaling in additional systems (Ordan, 2015).
To determine the individual contribution of Robo family receptors to the embryonic muscle pattern, the orientation of the three lateral transverse muscles (LT1-3) and of the dorsal acute muscle 3 (DA3) was analyzed in Drosophila embryos at stage 16 lacking distinct Robo receptors, and it was compared to that of slit. In slit mutant embryos the LT muscles are closer to the posterior segment border and are not spaced correctly, and the DA3 muscle does not extend its leading edge at orthogonal fashion. The orientation of the LT and the DA3 muscles in robo or in robo3 mutants, exhibited almost normal positioning relative to their wild-type (WT) counterparts. Quantification of the ratio between the distance of LT3 muscle and the posterior segment border, divided to overall segmental width (DLT3/Ds) revealed no significant difference between robo and WT, or robo3 and WT. However, in the double robo;robo3 mutant embryos the orientation of these muscles resembled that of slit. robo2 mutants exhibited a phenotype that was comparable to that of slit mutants; the LT muscles were not equally spaced and were closer to the posterior segmental border, a phenotype observed in 85% of the segments. Quantification of DLT3/Ds revealed a smaller value in robo2 mutant. In addition, in 33% of the robo2 mutant segments, the DA3 muscle lost its diagonal orientation and often migrated in a straight ventral-dorsal orientation (Ordan, 2015).
To reveal the dynamic nature of the process of muscle elongation in robo2 mutants, individual muscles were followed in live robo2 mutant and control embryos using specific GFP-marked LT, or DA3 muscles in stage 13-16 throughout muscle elongation. This analysis showed that the polarity of the mutant muscles was similar to that of WT control muscles; however, the LT muscles tended to elongate closer to the segment border and were not evenly spaced. The DA3 muscle often did not turn towards the posterior segment, and both muscles reached their target tendons more slowly. These results suggested differential contributions of the Robo receptors to the directed elongation of LT and DA3 muscles (Ordan, 2015).
To reveal the relative distribution and expression levels of the Robo receptors in muscles, advantage was taken of flies carrying HA tag that was knocked-in within the genomic region of either of the Robo receptors, and embryos were labeled with anti-HA. Robo-HA accumulated at the muscle-tendon junction, and was observed at low levels on the muscle surfaces. Robo2 levels were prominent along ectodermal stripes, but not on muscles, and Robo3 labeling was indistinguishable from background. Double labeling with anti-Robo2 and Stripe or with CD8-GFP driven by the stripe-GAL4 driver verified the ectodermal expression of Robo2 at the surfaces of the tendon cells. The relatively high ectodermal expression of Robo2-HA and its lack of staining in the affected LT and DA3 muscles were consistent with a muscle non-autonomous function of Robo2 in mediating LT and DA3 muscle-patterning (Ordan, 2015).
To address whether undetectable Robo2 protein functioning in the muscles, the ability of Robo2 to rescue the robo2 mutant muscle phenotype was tested when expressed in muscles. Driving Robo2 expression in muscles of robo2 mutant embryos using the mef2-GAL4 driver did not rescue the muscle phenotype, but rather worsen the muscle pattern in all embryos tested compared to wild type embryos overexpressing Robo2. In contrast, knockdown of Robo2 in tendons of robo2 -/+ heterozygous embryos (using sr-GAL4>robo2 RNAi) led to a phenotype similar to that of robo2 mutant; the LT muscles were mis-patterned in 84% of the segments, and the DA3 muscle was aberrant in 31% of the segments. Importantly, partial rescue of the LT muscle pattern was achieved by expressing
Robo2 in the ectoderm using the 69-GAL4 driver. Moreover, embryos in which Robo has been inserted into the robo2 locus, rescued the robo2 muscle phenotype, supporting the critical contribution of Robo2 unique ectodermal distribution rather than its signaling activity. In summary, the ectodermal expression of Robo2, its knockdown in tendons, and the ectodermal rescue imply that Robo2 functions in the ectoderm to induce the muscle pattern (Ordan, 2015).
Next, the requirement was addressed for Slit in the ectodermal activity of Robo2, associated with guiding the LT and DA3 muscles. Strong genetic interaction was found between robo2 and slit in inducing the LT and DA3 muscle pattern. Trans-heterozygous embryos for both robo2 and slit exhibited an aberrant pattern of the LT muscles in 82% of the segments relative to 0 or 10% in robo2 /+ or slit/+ embryos, respectively. In the DA3 muscle, 10% of the segments of the trans-heterozygous slit/robo2 embryos exhibited aberrant muscle orientation, relative to 0% in either of the single heterozygous mutants. These results imply that Robo2 activity is mediated through its ligand Slit (Ordan, 2015).
Next, whether Robo2 affects Slit cleavage, which is critical to short-range Slit signaling, was tested. To this end, Robo2 was overexpressed in the entire embryonic ectoderm using the 69B-GAL4 driver and followed endogenous Slit cleavage by western blotting using anti-Slit antibody, reactive with the C-terminal of Slit; this antibody recognizes both full-length Slit (~170kDa) and the C-terminal cleaved polypeptide, Slit-C, (~80kDa). Strikingly, overexpression of Robo2 in the ectoderm led to an average 9.7-fold increase in the ratio of Slit-C relative to Slit-FL levels, implicating Robo2 in promoting Slit cleavage in the ectoderm. In contrast, overexpression of Robo2 in the muscles (using the mef2-GAL4 driver) did not have this effect. Remarkably, similar enhanced Slit cleavage was observed following overexpression of truncated Robo2 lacking the cytoplasmic domain (Evans and Bashaw, 2010). Notably, overexpression of Robo in the ectoderm similarly enhanced Slit cleavage (data not shown). These results implicated a non-signaling function for Robo2 in promoting Slit cleavage (Ordan, 2015).
It was reasoned that covalently bound Slit oligomers, shown to form by previous crystallographic studies, would enable Slit to bind both juxtaposing Robo receptors, on the muscle side (Robo and/or Robo3) and on the tendon side (Robo2). Therefore Slit oligomerization was tested for in embryos. Overexpression of either GFP-tagged cleaved Slit-N (Slit-N-GFP), or uncleavable full length Slit-UC-myc, was induced in the embryonic ectoderm with 69B-GAL4 driver. The embryo extracts were then boiled under reducing (with β- mercaptoethanol, BME) or non-reducing conditions (without BME), separated on SDS-PAGE, and further reacted by western, either with anti-GFP, or anti-Myc. Under non-reducing conditions, Slit-N-GFP exhibited slower electrophoretic mobility relative to its mobility in reduced conditions (expected to be 124kDa), supporting the formation of Slit-N-GFP oligomers by disulfide bonds. In contrast, uncleavable Slit (Slit-UC-myc) did not exhibit differential electrophoretic mobility in natured versus denatured conditions. This result is consistent with the unique ability of Slit-N to oligomerize (Ordan, 2015).
Taken together, these results suggest that Robo2 in tendons enhances Slit cleavage, producing Slit-N oligomers at the tendon-cell membrane that are potentially capable of binding both Robo2 in cis, and Robo/Robo3 in trans, leading to spatially restricted Slit signaling to the elongating muscle.
Knocked-in membrane-bound full length Slit bypasses the requirement for Robo2. It was reasoned that if the primary function of Robo2 is to promote localized Slit-N oligomers, the knocked-in membrane-bound uncleavable Slit, (Slit- uncleavable(UC)-CD8), would rescue the phenotype of robo2. Knocked-in Slit-UC-CD8 was recombined with robo2 and the phenotype of the DA3 and LT muscles was analyzed in homozygous embryos. Whereas in robo2 mutants, the orientation of the LT muscles was defective in 85% of the segments, the addition of knocked-in Slit-UC-CD8 showed only 37% defective segments. Likewise the DLT3/Ds ratio was rescued in the robo2;Slit-UC-CD8 embryos. Given that Slit-UC-CD8 by itself produced a moderate muscle phenotype, it is concluded that Slit-UC- CD8 is capable of rescuing the robo2 phenotype of the LT muscles. For the DA3 muscle, whereas 32% of the segments showed a phenotype in robo2 mutants, robo2 mutant embryos recombined with Slit-UC-CD8 showed a muscle phenotype in only 13% of the segments, and Slit-UC-CD8 mutants showed only 5% defective segments (Ordan, 2015).
Moreover, consistent with a major function of Robo2 in promoting Slit cleavage, overexpression of the active cleaved Slit-N-GFP in tendon cells rescued the robo2 mutant LT muscle phenotype in 73% of the segments, whereas expression of Slit-UC-myc alone did not. These results are consistent with a major function for Robo2 in promoting Slit-N accumulation at the tendon cell- membrane, presumably by retaining Slit-FL and enhancing its availability proteolytic cleavage. Slit-N oligomers potentially bind to both Robo2 at the tendon side as well as to Robo and/or Robo3 at the muscle side to promote unidirectional short-range Slit-repulsive signal in muscles. In this context Robo2 cytoplasmic signaling domain is dispensable, consistent with Robo2 function in chordotonal organs and with Robo2 non autonomous function in the CNS (Ordan, 2015).
In conclusion, the findings of this study demonstrate a novel, non-signaling role for Robo2 in tendons in regulating the local distribution of active cleaved Slit oligomers at the tendon cell-membrane. This signal is critical in promoting Slit short-range signaling in muscles essential for directional muscle elongation. Such mechanism might be significant in other setups where Slit promotes signaling between neighboring cells (Ordan, 2015).
The type IIa family of receptor protein tyrosine phosphatases (RPTPs), including Lar, RPTPσ and RPTPδ, are well-studied in coordinating actin cytoskeletal rearrangements during axon guidance and synaptogenesis. To determine whether this regulation is conserved in other tissues, interdisciplinary approaches were utilized to study Lar-RPTPs in the Drosophila musculature. This study found that the single fly ortholog, Drosophila Lar (Dlar), is localized to the muscle costamere and that a decrease in Dlar causes aberrant sarcomeric patterning, deficits in larval locomotion, and integrin mislocalization. Sequence analysis uncovered an evolutionarily conserved Lys-Gly-Asp (KGD) signature in the extracellular region of Dlar. Since this tripeptide sequence is similar to the integrin-binding Arg-Gly-Asp (RGD) motif, the hypothesis was tested that Dlar directly interacts with integrin proteins. However, structural analyses of the fibronectin type III domains of Dlar and two vertebrate orthologs that include this conserved motif indicate that this KGD tripeptide is not accessible and thus unlikely to mediate physical interactions with integrins. These results, together with the proteomics identification of basement membrane (BM) proteins as potential ligands for type IIa RPTPs, suggest a complex network of protein interactions in the extracellular space that may mediate Lar function and/or signaling in muscle tissue (Kawakami, 2022).
The somatic muscles, the heart, the fat body, the somatic part of the gonad and most of the
visceral muscles are derived from a series of segmentally repeated primordia in the
Drosophila mesoderm. This work describes the early development of the fat body and its
relationship to the gonadal mesoderm, as well as the genetic control of the development of
these tissues. The first sign of fat body development is the expression of serpent in segmentally repeated clusters within the trunk mesoderm in parasegments 4-9. Segmentation and dorsoventral patterning genes define three regions in each parasegment
in which fat body precursors can develop. The primary and secondary dorsolateral fat body primordia are formed ventral to the visceral muscle primoridium in each parasegment. The ventral secondary cluster forms more ventrally in the posterior portion of each parasegment. Fat body progenitors in these regions are
specified by different genetic pathways. Two dorsolateral regions require engrailed and hedgehog (within the even-skipped domain) for
their development while the ventral secondary cluster is controlled by wingless. Ubiquitous mesodermal en expression leads to an expansion of the primary clusters into the sloppy-paired domain, resulting in a continuous band of serpent-expressing cells in parasegments 4-9. The observed effect of en on fat body development is seen not only on mesodermal overexpression but also when en is overexpressed in the ectoderm. Loss of wingless leads to an expansion of the dorsolateral fat body primordium. decapentaplegic and one or more
unknown genes determine the dorsoventral extent of these regions. High levels of Dpp repress serpent, resulting in the formation of visceral musculature, an alternative cell fate (Reichmann, 1998).
In each of
parasegments 10-12 one of these primary dorsolateral regions generates somatic gonadal precursors instead of fat body. The balance between fat body and somatic gonadal fate in these serially
homologous cell clusters is controlled by at least five genes. A model is suggested in which
tinman, engrailed and wingless are necessary to permit somatic gonadal develoment, while
serpent counteracts the effects of these genes and promotes fat body development. In wg mutant embryos, all dorsolateral mesodermal cells, including those in parasegments 10-12, acquire fat body fate. This phenotype can be interpreted as the combined effects of two separate functions of wg: (1) wg is necessary to repress fat body development in the dorsolateral mesoderm underlying the wg domain in all parasegments; (2) wg is required in the primary cluster to permit somatic gonadal precursor instead of fat body development in parasegments 10-12. Loss of engrailed results in the absence of demonstrable somatic gonadal precursors, similar to the situation in tinman mutants. Ubiquitous mesodermal en expression leads to the formation of additional somatic gonadal precursor cells in parasegments 10-12. The
homeotic gene abdominalA limits the region of serpent activity by interfering in a mutually
repressive feed back loop between gonadal and fat body development. It is unlikely that abdA represses srp directly, since srp can be expressed in cells in which abdA is active. abdA might prevent srp from inhibition of a somatic gonadal precursor competence factor (Riechmann, 1998).
Organisms modulate their growth according to nutrient availability. Although individual cells in a multicellular animal may respond directly to nutrient levels, growth of the entire organism needs to be coordinated. This study provides evidence that in Drosophila, coordination of organismal growth originates from the fat body, an insect organ that retains endocrine and storage functions of the vertebrate liver. A genetic screen for growth modifiers discovered slimfast, a gene that encodes an amino acid transporter. Remarkably, downregulation of slimfast specifically within the fat body causes a global growth defect similar to that seen in Drosophila raised under poor nutritional conditions. This involves TSC/TOR signaling in the fat body, and a remote inhibition of organismal growth via local repression of PI3-kinase signaling in peripheral tissues. These results demonstrate that the fat body functions as a nutrient sensor that restricts global growth through a humoral mechanism (Colombani, 2003).
In multicellular organisms, the control of growth depends on the integration of various genetic and environmental cues. Nutrient availability is one of the major environmental signals influencing growth and, as such, has dictated adaptative responses during evolution toward multicellularity. In particular, complex humoral responses ensure that growth and development are properly coordinated with nutritional conditions (Colombani, 2003).
In isolated cells, amino acid withdrawal leads to an immediate suppression of protein synthesis, suggesting that cells are protected by active sensing mechanims that block translation prior to depletion of internal amino acid stores. In many mammalian cell types, changes in amino acid diet affect the binding of the translation repressor 4EBP1 to initiation factor eIF4E and the activity of ribosomal protein S6 kinase (S6K). These two signaling events require the activity of TOR (target of rapamycin), a conserved kinase recently shown to participate in a nutrient-sensitive complex both in mammalian cells and in yeast. Mutations in the Drosophila TOR homolog (dTOR) results in cellular and physiological responses characteristic of amino acid deprivation and establish that TOR is cell autonomously required for growth in a multicellular organism. Furthermore, the TSC (tuberous sclerosis complex) tumor suppressor, consisting of a TSC1 and TSC2 heterodimer (TSC1/2), as well as the small GTPase Rheb participate to the regulation of TOR function. Overall, these data suggest that TSC, Rheb, TOR, and S6K participate in a conserved pathway that coordinates growth with nutrition in a cell-intrinsic manner (Colombani, 2003).
In multicellular organisms, humoral controls are believed to buffer variations in nutrient levels. However, little is known about how growth of individual cells is coordinated. In vertebrates, growth-promoting action of the growth hormone (GH) is mostly relayed to peripheral tissues through the production of IGF-I. Binding of IGF-I to its cognate receptor tyrosine kinase (IGF-IR) induces phosphorylation of insulin receptor substrates (IRS), which in turn activate a cascade of downstream effectors. These include phospho-inositide 3-kinase (PI3K), which generates the second messenger phosphatidylinositol-3,4,5-P3 (PIP3), and thereby activates the AKT/PKB kinase. Genetic manipulation of IGF-I, IGF-IR, PI3K, and AKT in mice modulates tissue growth in vivo thus demonstrating a requirement of the IGF pathway for growth. In Drosophila, both loss- and gain-of function studies have also exemplified the role of a conserved insulin/IGF signaling pathway in the control of growth. Ligands for the unique insulin receptor (Inr) constitute a family of seven peptides related to insulin, the Drosophila insulin-like peptides (Dilps). Remarkably, three dilp genes (dilp2, dilp3, and dilp5) are expressed in a cluster of seven median neurosecretory cells (m-NSCs) in the larval brain, suggesting that they have an endocrine function. Indeed, ablation of the seven dilp-expressing mNSCs in larvae induces a systemic growth defect (Colombani, 2003).
Both in flies and mice, mutations in IRS provoke growth retardation as well as female sterility similar to what is observed in starved animals. Moreover, PI3K activity in Drosophila larvae depends on the availability of proteins in the food. Overall, this supports the notion that the insulin/IGF pathway might coordinate tissue growth with nutritional conditions. However, upon amino acid withdrawal, neither PI3K nor AKT/PKB activities are downregulated in mammalian or insect cells in culture, suggesting that this pathway does not directly respond to nutrient shortage. Hence, an intermediate sensor mechanism must link nutrient availability to insulin/IGF signaling (Colombani, 2003).
An intriguing possibility is that specific organs could function as nutrient sensors and induce a nonautonomous modulation of insulin/IGF growth signaling in response to changes in nutrient levels. This study used a genetic approach in Drosophila to assess both the cellular and humoral responses to amino acid deprivation in the context of a developing organism. The insect fat body (FB) has important storage and humoral functions associated with nutrition, comparable to vertebrate liver and adipose tissue. During larval stages, the FB accumulates large stores of proteins, lipids, and carbohydrates, which are normally degraded by autophagy during metamorphosis in order to supply the developing tissues but can also be remobilized during larval life to compensate transitory nutrient shortage. In addition to its storage function, the FB also has endocrine activity and supports growth of imaginal disc explants and DNA replication of larval brains in coculture. This study demonstrates that the FB operates as a sensor for variations in nutrient levels and coordinates growth of peripheral tissues accordingly via a humoral mechanism (Colombani, 2003).
In the course of a P[UAS]-based overexpression screen for growth modifiers, a P[UAS]-insertion line (UY681) was found to cause growth retardation upon ectopic activation. Sequence analysis revealed that P(UY)681 is inserted in a predicted gene (CG11128) that encodes a putative protein showing strong homology with amino acid permeases of the cationic amino acid transporter (CAT) family. The P[UAS] element is inserted in the first intron of the CG11128 gene, potentially driving transcription of an antisense RNA in a GAL4-dependent manner. To assess the function of this transporter, 3H-arginine uptake was measured in S2 cells. Results indicate that amino acid uptake is either enhanced by transfection of a CG11128 cDNA or suppressed by RNAi, indicating that the encoded protein presents CAT activity. In situ hybridization revealed basal levels of CG11128 expression in most larval tissues but much higher levels in the FB and the gut, two tissues involved in amino acid processing (Colombani, 2003).
By P element remobilization, an imprecise excision was obtained that deletes the sequences encoding the N-terminal half of the protein. 87% of homozygous mutant animals die during larval stages. The few viable adults emerged after a 2 day delay and were smaller and markedly slimmer than control animals. The associated gene was named slimfast (slif) and the excision allele slif1. Weight measurement indicated that homozygous slif1 adult males displayed a 16% mass reduction compared to control. Accordingly, adult wing size was reduced by 8% due to a reduction of both cell size and cell number. When the slif1 allele was in trans to Df(3L)Δ1AK, a deficiency covering the locus, larval lethality was slightly enhanced, suggesting that slif1 corresponds to a strong hypomorphic allele. The amino acid transporter function of slif, as well as the phenotypes observed upon reduction of slif function suggest that slif mutant animals might suffer amino acid deprivation. A major consequence of amino acid deprivation in larvae is the remobilization of nutrient stores in the FB, which typically results in aggregation of storage vesicles. Consistently, fusion of storage vesicles was observed in the FB of slif1 larvae and was indistinguishable from that observed in animals fed on protein-free media (Colombani, 2003).
GAL4 induction of P(UY)681 resulted in a growth-deficient phenotype similar to that of slif1 loss of function. The antisense orientation of P(UY)681 suggested that the growth defect following GAL4 induction was due to an RNAi effect. Indeed, Northern blot analysis revealed that ubiquitous GAL4-dependent activation of P(UY)681 using the daughterless-GAL4 (da-GAL4) driver strongly reduced slif mRNA levels. Only two of the three alternative first exons are potentially affected by the antisense RNA, possibly explaining the residual accumulation of slif mRNAs in da-GAL4; P(UY)681 animals. Most of these animals died at larval stage, similar to what was observed for slif1 mutants. Specific induction of P(UY)681in the wing disc using the MS1096-GAL4 driver provoked a reduction of the adult wing size, which could be either rescued by coactivation of a UAS-slif transgene or enhanced by reducing slif gene dosage with the heterozygous Df(3L)Δ1AK deficiency. Thus, GAL4-dependent activation of P(UY)681 reduces slif function and defines a conditional loss-of-function allele hereafter termed slifAnti (Colombani, 2003).
As expected, loss of slif function using the slifAnti allele also mimicked amino acid deprivation. Accordingly, ubiquitous slifAnti induction in growing larvae resulted in storage vesicle aggregation and strong reduction of global S6 kinase activity, similar to what was reported in animals raised on protein-free diet. Additionally, an increase in PEPCK1 gene transcription was observed, similar to the effect of amino acid withdrawal. In summary, this study has identified two loss-of-function alleles of the slif gene whose defects mimic physiological aspects of amino acid deprivation. Importantly, the conditional slifAnti allele provides a unique tool to mimic an amino acid deprivation in a tissue-specific manner (Colombani, 2003).
This study established that the FB is a sensor tissue for amino acid levels, as downregulation of the Slif amino acid transporter within the FB is sufficient to induce a general reduction in the rate of larval growth. In contrast, specific disruption of slif in imaginal discs, larval gut, or salivary glands did not induce a nonautonomous growth response, suggesting that these tissues do not participate in the systemic control of growth. The dilp-expressing median neurosecretory cells (m-NSCs) also affect growth control, since selective ablation of these cells in the larval brain induces an overall reduction of animal size. In response to complete sugar and protein starvation, the m-NSCs stop expressing dilp3 and dilp5 genes, suggesting that these neurons also sense nutrient levels. This study shows that the selective reduction of slif function in these cells has no obvious effect on tissue growth and animal development. This indicates that the seven dilp-expressing m-NSCs do not constitute a general amino acid sensor. In contrast, the role of m-NSCs in carbohydrate homeostasis and the observation that they stop expressing certain dilp genes when larvae are deprived of sugar rather suggests that these cells have a role in sensing carbohydrate levels (Colombani, 2003 and references therein).
This analysis also provides a framework in which to understand the phenotype of minidisc, a mutation in an amino acid transporter gene that exhibits nonautonomous growth defects in imaginal discs (Colombani, 2003).
In a number of model systems, both PI3K and TOR have been implicated in linking growth to nutritional status and, until recently, were considered as intermediates of a common regulatory pathway. In yeast, the TOR kinase is part of a cell-autonomous nutrient sensor, which controls protein synthesis, ribosome biogenesis, nutrient import, and autophagy. Genetic analysis in Drosophila indicates that dTOR is required for cell-intrinsic growth control. The results obtained using the slifAnti allele in the wing disc indicate that individual tissues have indeed the potential to respond to amino acid deprivation in a cell-autonomous manner. Nonetheless, this study also demonstrates that the TOR nutritional checkpoint participates in a systemic control of larval growth emanating from the FB. Within a developing organism, each cell may integrate these two distinct inputs regarding nutritional status, one originating from a systemically-acting FB sensor, and the other from TOR-dependent signaling in individual cells. One can further speculate that depending on the strength and duration of starvation, different in vivo nutritional checkpoints will be hierarchically recruited to protect the animal and that the systemic control might, in most physiological situations, override the cell-autonomous control. Indeed, as the data demonstrate, the FB sensor is sufficient to induce a general and coordinated response to starvation without calling individual cell-autonomous mechanisms into play (Colombani, 2003).
Several lines of evidence indicate that the PI3K pathway is not part of the sensor mechanism in FB cells. First, a sensor for PI3K activity in the FB is only marginally affected by amino acid deprivation in that tissue, indicating that the cell-autonomous response to amino acid starvation does not directly influence PI3K signaling. This is reminiscent of previous observations in mammalian cultured cells, showing that PI3K activity does not respond to variations in amino acid levels. Moreover, inhibition of PI3K signaling by dPTEN expression in the FB is not sufficient to trigger the sensing mechanism. Although, dPTEN overexpression causes a complete disappearance of the PI3K sensor accompanied by growth suppression of FB cells, the FB maintains a critical mass that allows for normal larval growth. In contrast, the regulatory subunit p60 whose overexpression potently inhibits PI3-kinase in flies has been shown to induce a systemic effect on larval growth when overexpressed in the FB using an Adh-Gal4 driver. This study found that a pumpless ppl-GAL4-directed expression of p60 also provokes a strong suppression of larval growth and a dramatic inhibition of FB development in young larvae. Thus, the systemic effect on growth observed upon p60 overexpression most likely results from a drastic reduction of FB mass, which then fails to support normal larval growth (Colombani, 2003).
These results further indicate that PI3K signaling is a remote target of the humoral message that originates from the FB in response to amino acid deprivation. This is in agreement with previous data showing that PI3K activity is downregulated by dietary amino acid deprivation and explains why global PI3-kinase inhibition mimics cellular and organismal effects of starvation. The existence of a humoral relay reconciles these in vivo studies with the absence of direct PI3K responsiveness to amino acid levels (Colombani, 2003).
The relative resistance of imaginal disc growth to the systemic control exerted by the FB correlates with maintenance of PI3K activity in these tissues. This is in agreement with previous observations that cells in the larval brain and in imaginal discs maintain a slow rate of proliferation under protein starvation, while larval endoreduplicating tissues (ERTs) arrest. This difference might be attributed to the basal levels of dilp2 expression observed in imaginal discs, allowing a moderate growth rate of these tissues through an autocrine/paracrine mechanism. It was recently shown that clonal induction of PI3K potently induces cell-autonomous growth response even in fasting larvae, indicating that some nutrients are still accessible to support cell growth within a fasted larva. The main function of a general sensor could be to preserve these limited nutrients for use by high priority tissues. In this context, local PI3K activation through an autocrine loop in imaginal tissues could favor the growth of prospective adult structures in adverse food conditions. Thus, the FB would have an active role in controlling the allocation of resources depending on nutritional status. In this respect, it is noteworthy that FB cells are relatively resistant to the FB-derived humoral signal, since the PI3K sensor is not drastically affected in the FB of ppl>slifAnti animals. Thereby, essential regulatory functions of the FB could be preserved even in severely restricted nutritional conditions (Colombani, 2003).
How does the FB signal to other tissues? This study suggests that a humoral signal relays information from the FB amino acid sensor and systemically inhibits PI3K signaling. In addition, this downregulation is not due to a direct inhibition of dilp expression by neurosecretory cells in the brain. Nevertheless, it cannot be ruled out that the secretion of these molecules is subjected to regulation in the mNSCs. Both in vivo and in insect cell culture, several imaginal discs growth factors (IDGF) secreted by the FB have been proposed to function synergistically with Dilp signaling to promote growth. However, this study did not find any modification of IDGF expression in the FB of larvae raised on water- or sugar-only diet, or upon FB induction of slifanti. In vertebrates, the different functions of the circulating IGF-I are modulated through its association with IGF-BPs and acid labile subunit (ALS). In particular, the formation of a ternary complex with ALS leads to a considerable extension of IGF-I half-life. The finding that a Drosophila ALS ortholog is expressed within the FB in an amino acid-dependent manner provides a new avenue to study the molecular mechanisms of nonautonomous growth control mediated by the FB (Colombani, 2003).
This study highlights the contribution that genetics can provide to unravel the mechanisms of physiological control. Using a genetic tool to mimic amino acid deprivation, it was demonstrated that nutrition systemically controls body size through an amino acid sensor operating in the FB. It is proposed that (1) in metazoans, a systemic nutritional sensor modulates the conserved TOR-signaling pathway, and (2) the response to sensor activation is relayed by a hormonal mechanism, which triggers an Inr/PI3K-dependent response in peripheral tissues (Colombani, 2003).
In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation. Autophagy is known to be inhibited by TOR signaling, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body. In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation. Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells. Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival. Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor (Scott, 2004).
Autophagy likely evolved in single-cell eukaryotes to provide an energy and nutrient source allowing temporary survival of starvation. In yeast, Tor1 and Tor2 act as direct links between nutrient conditions and cell metabolism. These proteins sense nutritional status by an unknown mechanism, and effect a variety of starvation responses including changes in transcriptional and translational programs, nutrient import, protein and mRNA stability, cell cycle arrest, and induction of autophagy. Autophagy thus occurs in the context of a comprehensive reorganization of cellular activities aimed at surviving low nutrient levels (Scott, 2004).
In multicellular organisms, TOR is thought to have retained its role as a nutrient sensor but has also adopted new functions in regulating and responding to growth factor signaling pathways and developmental programs. Thus in a variety of signaling, developmental, and disease contexts, TOR activity can be regulated independently of nutritional conditions. In these cases, autophagy may be induced in response to downregulation of TOR despite the presence of abundant nutrients and may potentially play an important role in suppressing cell growth rather than promoting survival. Identification of the tumor suppressors PTEN, and TSC1 and TSC2 as positive regulators of autophagy provides correlative evidence supporting such a role for autophagy in growth control. Alternatively, since TOR activity is required for proper expression and localization of a number of nutrient transporters, inactivation of TOR may lead to reduced intracellular nutrient levels, and autophagy may therefore be required under these conditions to provide the nutrients and energy necessary for normal cell metabolism and survival (Scott, 2004).
The results presented here provide genetic evidence that under conditions of low TOR signaling, autophagy functions primarily to promote normal cell function and survival, rather than to suppress cell growth. This conclusion is based on the finding that genetic disruption of autophagy does not restore growth to cells lacking TOR, but instead exacerbates multiple TOR mutant phenotypes. It is important to note that mutations in TOR do not disrupt larval feeding, and thus disruption of autophagy is detrimental in TOR mutants despite the presence of ample extracellular nutrients. The finding that autophagy is critical in cells lacking TOR further supports earlier studies suggesting that inactivation of TOR causes defects in nutrient import, resulting in an intracellular state of pseudo-starvation (Scott, 2004).
Can the further reduction in growth of TOR mutant cells upon disruption of autophagy be reconciled with the potential catabolic effects of autophagy? TOR regulates the bidirectional flow of nutrients between protein synthesis and degradation through effects on nutrient import, autophagy, and ribosome biogenesis. When TOR is inactivated, rates of nutrient import and protein synthesis decrease, resulting in a commensurate reduction in mass accumulation and cell growth. In addition, autophagy is induced to maintain intracellular nutrient and energy levels sufficient for normal cell metabolism. When autophagy is experimentally inhibited in cells lacking TOR, this reserve source of nutrients is blocked, leading to a further decrease in energy levels, protein synthesis, and growth. It is noted that autophagy may have additional functions in cells with depressed TOR signaling, including recycling of organelles damaged by the absence of TOR activity, or selective degradation of cell growth regulators, analogous to the regulatory roles of ubiquitin-mediated degradation (Scott, 2004).
Autophagy is required for normal developmental responses to inactivation of insulin/PI3K signaling in the nematode C. elegans. In response to starvation or disruption of insulin/PI3K signaling, C. elegans larvae enter a dormant state called the dauer. Autophagy has been observed in C. elegans larvae undergoing dauer formation: disruption of a number of ATG homologs interfers with normal dauer morphogenesis. Importantly, simultaneous disruption of insulin/PI3K signaling and autophagy genes results in lethality, similar to the results presented in this study. Thus despite significant differences in developmental strategies for surviving nutrient deprivation, autophagy plays an essential role in the starvation responses of yeast, flies, and worms (Scott, 2004).
The prevailing view that S6K acts to suppress autophagy was founded on correlations between induction of autophagy and dephosphorylation of rpS6 in response to amino acid deprivation or rapamycin treatment. However, the genetic data presented in this study argue strongly against a role for S6K in suppressing autophagy: unlike other positive components of the TOR pathway, null mutations in S6K do not induce autophagy in fed animals. It is suggested that the observed correlation between S6K activity and suppression of autophagy is due to common but independent regulation of S6K and autophagy by TOR. Thus, autophagy suppression and S6K-dependent functions such as ribosome biogenesis represent distinct outputs of TOR signaling (Scott, 2004).
How might TOR signal to the autophagic machinery, if not through S6K? In yeast, this is accomplished in part through regulation of Atg1 kinase activity and Atg8a gene expression (Kamada, 2000 and Kirisako, 1999). The demonstration of a role for Drosophila ATG1 and ATG8 homologs [see TG8a (CG32672) and ATG8b (CG12334)] in starvation-induced autophagy, and the genetic interaction observed between ATG1 and TOR, are consistent with a related mode of regulation in higher eukaryotes. However, it is noted that other components of the yeast Atg1 complex such as Atg17 and Atg13, whose phosphorylation state is rapamycin sensitive, do not have clear homologs in metazoans, indicating that differences in regulation of autophagy by TOR are likely (Scott, 2004).
In addition to excluding a role for S6K in suppression of autophagy, these results reveal a positive role for S6K in induction of autophagy. S6K may promote autophagy directly, through activation of the autophagy machinery, or indirectly through its effects on protein synthesis. The latter possibility is consistent with previous reports that protein synthesis is required for expansion and maturation of autophagosomes. Interestingly, despite being required for autophagy, S6K is downregulated under conditions that induce it, including chronic starvation and TOR inactivation. Consistent with this, it was found that lysotracker staining is significantly weaker in chronically starved animals or in TOR mutants than in wild-type animals starved 3-4 hr. Furthermore, expression of constitutively activated S6K has no effect in wild-type, but restores lysotracker staining in TOR mutants to levels similar to those of acutely starved wild-type animals. It is suggested that downregulation of S6K may limit rates of autophagy under conditions of extended starvation or TOR inactivation and that this may protect cells from the potentially damaging effects of unrestrained autophagy (Scott, 2004).
Co-culture and conditioned media experiments have shown that the Drosophila fat body is a source of diffusible mitogens. The fat body has also been shown to act as a nutrient sensor through a TOR-dependent mechanism and to regulate organismal growth through effects on insulin/PI3K signaling. The results in this study extend these findings by showing that this endocrine response is accompanied by the regulated release of nutrients through autophagic degradation of fat body cytoplasm. Preventing this reallocation of resources, either through constitutive activation of PI3K or through inactivation of ATG genes, results in profound nutrient sensitivity. Thus, in response to nutrient limitation, the fat body is capable of simultaneously restricting growth of peripheral tissues through downregulation of insulin/PI3K signaling and providing these tissues with a buffering source of nutrients necessary for survival through autophagy (Scott, 2004).
As in many other organisms, the blood of Drosophila consists of several types of hemocytes, which originate from the mesoderm. By lineage analyses of transplanted cells, two separate anlagen have been defined that give rise to different populations of hemocytes: embryonic hemocytes and lymph gland hemocytes. The anlage of the embryonic hemocytes is restricted to a region within the head mesoderm between 70% and 80% egg length. In contrast to all other mesodermal cells, the cells of this anlage are already determined as hemocytes at the blastoderm stage. Unexpectedly, these hemocytes do not degenerate during late larval stages, but have the capacity to persist through metamorphosis and are still detectable in the adult fly. A second anlage, which gives rise to additional hemocytes at the onset of metamorphosis, is located within the thoracic mesoderm at 50% to 53% egg length. After transplantation within this region, clones were detected in the larval lymph glands. Labeled hemocytes are released by the lymph glands not before the late third larval instar. The anlage of these lymph gland-derived hemocytes is not determined at the blastoderm stage, as indicated by the overlap of clones with other tissues. These analyses reveal that the hemocytes of pupae and adult flies consist of a mixture of embryonic hemocytes and lymph gland-derived hemocytes, originating from two distinct anlagen that are determined at different stages of development (Holz, 2003).
The origin of the embryonic hemocytes (EH) can be traced back to the head mesoderm of late stage 11 embryos by morphological criteria. Owing to the fact that srp is expressed in a narrow stripe within the cephalic mesoderm at the blastoderm stage and that a loss of srp function leads to a complete loss of embryonic hemocytes, this domain is considered to be the primordium of the EH. By
homotopic single-cell transplantations it was possible to restrict the anlage to a sharply delimitated region located at 70% to 80% EL within the mesoderm, exactly corresponding to the cephalic expression domain of srp. The fact that none of the EH clones overlapped with other tissues indicates that the hemocytes are already determined at the blastoderm stage. This was confirmed by heterotopic transplantations from the EH anlage into the abdominal mesoderm; these transplanted cells give rise to hemocytes. Since mesodermal cells transplanted into the EH anlage
do not transform into embryonic hemocytes, the determining factor is not able to induce a
hemocyte fate within these cells and seems to function cell-autonomously. A
good candidate for such a factor is Srp. However, since srp is also expressed in many other tissues that do not give rise to hemocytes, there must be additional genes that lead to a determination of the EH at the blastoderm stage. The early determination of the EH is quite unusual, since all other mesodermal tissues analyzed to date -- including the anlage of the lymph gland-derived hemocytes -- are not restricted to a tissue-specific fate prior to the second postblastodermal mitoses. This might be a developmental adaptation of the EH, which at stage 12 are already differentiated into functional macrophages and are responsible for the removal of apoptotic cells within developing tissues (Holz, 2003).
It is commonly believed that in Drosophila during larval
development the EH population is entirely replaced by hemocytes that have been released by the larval lymph glands. However, it is possible to trace hemocytes originating from the head mesoderm through all stages of development until 14-day-old adult flies. The number of hemocytes progressively rises during larval life, from less
than 200 to more than 5000 per individual. Cell lineage analyses
unambiguously demonstrate that this increase is due to postembryonic
proliferation of the EH. The contribution of the lymph
glands to the hemocyte population was determined by means of cell lineage analyses. These studies reveal that the lymph glands do
not release blood cells into the hemocoel during all larval stages but
exclusively at the end of the third larval instar (Holz, 2003).
With the onset of metamorphosis, additional hemocytes are released from the lymph glands. Although the lymph glands do not persist through metamorphosis, the marked
hemocytes released by the labeled lymph glands are still detectable in adult
flies. Hence, all hemocytes found throughout larval life originate solely
from the EH anlage, whereas the pupal and imaginal blood is made up of two
different populations: EH and LGH (Holz, 2003).
The two populations of hemocytes share many functional, morphological and genetic similarities. In both cases, the determination of hemocytes depends on srp, while the specification towards the distinct blood cell
types is induced by the expression of lozenge (lz)
glia cells missing (gcm) and the gcm homolog gcm2. Both EH and LGH differentiate into podocytes, crystal cells and plasmatocytes. Hemocytes of both populations have the capability to adopt macrophage characteristics. However, despite all similarities, the history of the two populations is quite different, since they originate from two different mesodermal regions and are determined at different developmental stages. In view of the fact that the lymph glands do not release hemocytes before the onset of metamorphosis under nonimmune conditions, all hemocytes found in the larval hemocoel represent EH (Holz, 2003).
The many similarities between EG and LGH raise the question why there are
two populations at all. A
massive release of hemocytes by the lymph glands is seen just at the onset of
pupation. The lymph glands additionally have the
capacity to differentiate and release a special type of hemocytes, the
lamellocytes, under immune conditions even before the onset of metamorphosis.
Thus, because under nonimmune conditions the lymph glands do not release any
cells before the onset of pupation, it might be their primary role to provide
a reservoir of immune defensive hemocytes. The massive apoptosis and
accumulation of cell debris might be a secondary trigger to stimulate
proliferation and release of the lymph gland hemocytes (Holz, 2003).
Drosophila hematopoiesis occurs in a specialized organ called the lymph gland.
In this systematic analysis of lymph gland structure and gene expression, the developmental steps in the maturation of blood cells (hemocytes) from
their precursors are defined. In particular, distinct zones of hemocyte maturation,
signaling and proliferation in the lymph gland during hematopoietic progression
are described. Different stages of hemocyte development have been classified
according to marker expression and placed within developmental niches: a
medullary zone for quiescent prohemocytes, a cortical zone for maturing
hemocytes and a zone called the posterior signaling center for specialized
signaling hemocytes. This establishes a framework for the identification of
Drosophila blood cells, at various stages of maturation, and provides a genetic
basis for spatial and temporal events that govern hemocyte development. The
cellular events identified in this analysis further establish Drosophila as a
model system for hematopoiesis (Jung, 2005).
In the late embryo, the lymph gland consists of a single pair of lobes
containing ~20 cells each. These express the transcription factors Srp and
Odd skipped (Odd),
and each cluster of hemocyte precursors is followed by a string of
Odd-expressing pericardial cells that are proposed to have nephrocyte
function. These lymph gland lobes are arranged bilaterally such that they
flank the dorsal vessel, the simple aorta/heart tube of the open circulatory
system, at the midline. By the second larval instar, lymph gland morphology is
distinctly different in that two or three new pairs of posterior lobes have
formed and the primary lobes have increased in size approximately tenfold (to
~200 cells. By the late third instar, the lymph gland has grown significantly in size
(approximately another tenfold) but the arrangement of the lobes and
pericardial cells has remained the same. The cells of the third instar lymph
gland continue to express Srp (Jung, 2005).
The third instar lymph gland also exhibits a strong, branching network of
extracellular matrix (ECM) throughout the primary lobe. This network was
visualized using several GFP-trap lines in which GFP is fused to endogenous
proteins. For
example, line G454 represents an insertion into the viking
locus, which encodes a Collagen IV component of the extracellular matrix.
The hemocytes in the primary lobes of G454
(expressing Viking-GFP) appear to be clustered into small populations within
pockets or chambers bounded by GFP-labeled branches of various sizes. Other lines, such as
the uncharacterized GFP-trap line ZCL2867, also highlight this
branching pattern. What role this intricate ECM network plays in
hematopoiesis, as well as why multiple cells cluster within these ECM
chambers, remains to be determined (Jung, 2005).
Careful examination of dissected, late third-instar lymph glands by
differential interference contrast (DIC) microscopy revealed the presence of
two structurally distinct regions within the primary lymph gland lobes that
have not been previously described. The periphery of the primary lobe generally exhibits a
granular appearance, whereas the medial region looks smooth and compact. These
characteristics were examined further with confocal microscopy using a
GFP-trap line G147, in which GFP is fused to a microtubule-associated
protein. The G147 line is expressed throughout the lymph gland but, in
contrast to nuclear markers such as Srp and Odd, distinguishes morphological
differences among cells because the GFP-fusion protein is expressed in the
cytoplasm in association with the microtubule network. Cells in the
periphery of the lymph gland make relatively few cell-cell contacts, thereby
giving rise to gaps and voids among the cells within this region. This
cellular individualization is consistent with the granularity of the
peripheral region observed by DIC microscopy. By contrast, cells
in the medial region were relatively compact with minimal intercellular space,
which is also consistent with the smoother appearance of this region by DIC
microscopy. Thus, in the late third instar, the lymph gland primary lobes
consist of two physically distinct regions: a medial region consisting of
compactly arranged cells, which was termed the medullary zone; and a peripheral
region of loosely arranged cells, termed the cortical zone (Jung, 2005).
Mature hemocytes have been shown to express several markers, including
collagens, Hemolectin, Lozenge, Peroxidasin and P1 antigen. The expression of the reporter
Collagen-gal4 (Cg-gal4), which is
expressed by both plasmatocytes and crystal cells, is restricted to the
periphery of the primary lymph gland lobe. Comparison of
Cg-gal4 expression in G147 lymph glands, in which the
medullary zone and cortical zone can be distinguished, reveals that maturing
hemocytes are restricted to the cortical zone. In fact,
the expression of each of the maturation markers mentioned above is found to
be restricted to the cortical zone. The reporter hml-gal4 and Pxn,
which are expressed by the plasmatocyte and crystal cell lineages, are
extensively expressed in this region. Likewise,
the expression of the crystal cell lineage marker
Lozenge is restricted in this manner. The spatial restriction of maturing crystal cells to the
cortical zone was verified by several means, including the distribution of
melanized lymph gland crystal cells in the Black cells background and analysis of the
terminal marker ProPOA1. The cortical zone is also the site of P1
antigen expression, a
marker of the plasmatocyte lineage. The uncharacterized GFP fusion line
ZCL2826 also exhibits preferential expression in the cortical zone. Last, it was found that
the homeobox transcription factor Cut is
preferentially expressed in the cortical zone of the primary lobe. Although the role of
Cut in Drosophila hematopoiesis is currently unknown, homologs of Cut
are known to be regulators of the myeloid hematopoietic lineage in both mice
and humans. Cells of the rare third cell type, lamellocytes, are also
restricted to the cortical zone, based upon cell morphology and the expression of a
msn-lacZ reporter (msn06946). In summary, based
on the expression patterns of several genetic markers that identify the three
major blood cell lineages, it is proposed that the cortical zone is a specific
site for hemocyte maturation (Jung, 2005).
The medullary zone was initially defined by structural characteristics and subsequently by
the lack of expression of mature hemocyte markers. However, several markers have been
identified that are exclusively expressed in the medullary
zone at high levels but not the cortical zone. Consistent with the compact
arrangement of cells in the medullary zone, it was found that Drosophila
E-cadherin (DE-cadherin or Shotgun) is highly expressed in this region. No significant
expression of DE-cadherin was observed among maturing cells in the cortical
zone. E-cadherin, in both vertebrates and Drosophila, is a
Ca2+-dependent, homotypic adhesion molecule often expressed by
epithelial cells and is a crucial component of adherens junctions.
Attempts to study DE-cadherin mutant clones in the medullary zone
where the protein is expressed were unsuccessful since no clones were
recoverable. The reporter lines domeless-gal4 and
unpaired3-gal4 are preferentially expressed in the medullary zone. The gene
domeless (dome) encodes a receptor molecule known to mediate
the activation of the JAK/STAT pathway upon binding of the ligand Unpaired. The
unpaired3 (upd3) gene encodes a protein with homology to
Unpaired and has been associated with innate immune function. These
gal4 lines are in this study only as markers that correlate with the
medullary zone and, at the present time, there is no evidence that their
associated proteins have a role in lymph gland hematopoiesis. Other markers of
interest with preferential expression in the medullary zone include the
molecularly uncharacterized GFP-trap line ZCL2897 and
actin5C-GFP. Cells expressing hemocyte maturation markers are not seen in the medullary
zone. It is therefore reasonable to propose that this zone is largely
populated by prohemocytes that will later mature in the cortical zone.
Prohemocytes are characterized by their lack of maturation markers, as well as
their expression of several markers described as expressed in the medullary zone (Jung, 2005).
The posterior signaling center (PSC), a small cluster of cells at the posterior
tip of each of the primary (anterior-most) lymph gland lobes,
is defined by its expression of the Notch ligand Serrate and
the transcription factor Collier.
During this analysis, several additional markers were identified that exhibit
specific or preferential expression in the PSC region. For example, it was found
that the reporter Dorothy-gal4 is
strongly expressed in this zone. The Dorothy gene encodes a UDP-glycosyltransferase, which
belongs to a class of enzymes that function in the detoxification of
metabolites. The upd3-gal4 reporter, which has preferential
expression in the medullary zone, is also strongly expressed among cells of
the PSC. Last, three uncharacterized GFP-gene trap lines, ZCL2375, ZCL2856 and
ZCL0611 were found, that are preferentially expressed in the PSC. This analysis has
made it clear that the PSC is a distinct zone of cells that can be defined by
the expression of multiple gene products (Jung, 2005).
The PSC can be defined just as definitively by the
characteristic absence of several markers. For example, the RTK receptor Pvr,
which is expressed throughout the lymph gland, is notably absent from the PSC. Likewise,
dome-gal4 is not expressed in the PSC, further suggesting
that this population of cells is biased toward the production of ligands
rather than receptor proteins. Maturation markers such as Cg-gal4,
which are expressed throughout the cortical zone, are not
expressed by PSC cells. Additionally, the expression levels of the
hemocyte marker Hemese and the Friend-of-GATA protein U-shaped are
dramatically reduced in the PSC when compared with other hemocytes of the
lymph gland. Taken
together, both the expression and lack of expression of a number of genetic
markers defines the cells of the PSC as a unique hemocyte population (Jung, 2005).
In contrast to primary lobes of the third instar, maturing hemocytes are
generally not seen in the secondary lobes. Correspondingly, secondary lobes
often have a smooth and compact appearance, much like the
medullary zone of the primary lobe. Consistent with this appearance, secondary
lymph gland lobes also express high levels of DE-cadherin. The size of the
secondary lobe, however, varies from animal to animal and this correlates with
the presence or absence of maturation markers. Smaller secondary lobes contain
a few or no cells expressing maturation markers, whereas larger secondary
lobes usually exhibit groups of differentiating cells. Direct comparison of
DE-cadherin expression in secondary lobes with that of Cg-gal4,
hml-gal4 or Lz revealed that the expression of these maturation markers
occurs only in areas in which DE-cadherin is downregulated. Therefore,
although there is no apparent distinction between cortical and medullary zones
in differentiating secondary lobes, there is a significant correlation between
the expression of maturation markers and the downregulation of DE-cadherin, as
is observed in primary lobes (Jung, 2005).
The relatively late 'snapshot' of lymph gland development in the third
larval instar establishes the existence of spatial zones within the lymph
gland that are characterized by differences in structure as well as gene
expression. In order
to understand how these zones form over time, lymph glands of second instar
larvae, the earliest time at which it was possible to dissect and stain, were
examined for the expression of hematopoietic markers. As expected, Srp and Odd
are expressed throughout the lymph gland during the second instar since they are in the
late embryo and third instar lymph gland. Likewise, the
hemocyte-specific marker Hemese is expressed throughout the lymph gland at
this stage, although it is not present in the embryonic lymph gland (Jung, 2005).
To determine whether the cortical zone is already formed or forming in
second instar lymph glands, the expression of various maturation
markers were examined in a pair-wise manner to establish their temporal order. Of the
markers examined, hml-gal4 and Pxn are the earliest to
be expressed. The majority of maturing cells were found to be double-positive
for hml-gal4 and Pxn expression, although a few cells were found to
express either hml-gal4 or Pxn alone. This indicates that
the expression of these markers is initiated at approximately the same time,
although probably independently, during lymph gland development. The marker
Cg-gal4 is next to be expressed since it was found among a subpopulation
of Pxn-expressing cells. Finally, P1 antigen expression is initiated late, usually in
the early third instar. Interestingly, the early expression of each of these
maturation markers is restricted to the periphery of the primary lymph gland
lobe, indicating that the cortical zone begins to form in this position in the
second instar. Whenever possible, each genetic marker was directly compared
with other pertinent markers in double-labeling experiments, except in cases
such as the comparison of two different gal4 reporter lines or when
available antibodies were generated in the same animal. In such cases, the
relationship between the two markers, for example dome-gal4 and
hml-gal4, was inferred from independent comparison with a third
marker such as Pxn (Jung, 2005).
By studying the temporal sequence of expression of hemocyte-specific
markers, one can describe stages in the maturation of a hemocyte. It should be noted,
however, that not all hemocytes of a particular lineage are identical. For
example, in the late third instar lymph gland, the large majority of mature
plasmatocytes (~80%) expresses both Pxn and hml-gal4, but the
remainder express only Pxn (~15%) or hml-gal4 (~5%) alone.
Thus, while plasmatocytes as a group can be characterized by the expression of
representative markers, populations expressing subsets of these markers indeed exist.
It remains unclear at this time whether this heterogeneity in the hemocyte
population is reflective of specific functional differences (Jung, 2005).
In the third instar, Pxn is a prototypical hemocyte maturation marker,
while immature cells of the medullary zone express dome-gal4.
Comparing the expression of these two markers in the second instar reveals an
interesting developmental progression. A group of cells
along the peripheral edge of these early lymph glands already express Pxn.
These developing hemocytes downregulate the expression of dome-gal4, as they do
in the third instar. Next to these developing hemocytes is a group of cells
that expresses dome-gal4 but not Pxn; these cells are most similar to
medullary zone cells of the third instar and are therefore prohemocytes.
Interestingly, there also exists a group of cells in the second instar that
expresses neither Pxn nor dome-gal4. This population is most easily
seen in the medial parts of the gland, close to the centrally placed dorsal. These
cells resemble earlier precursors in the embryo, except they express the
marker Hemese. These cells are called pre-prohemocytes. Interpretation of the expression data
is that pre-prohemocytes upregulate dome-gal4 to become prohemocytes.
As prohemocytes begin to mature into hemocytes, dome-gal4 expression
is downregulated, while the expression of maturation markers is initiated. The
prohemocyte and hemocyte populations continue to be represented in the third
instar as components of the medullary and cortical zones, respectively (Jung, 2005).
The cells of the PSC are already distinguishable in the late embryo by
their expression of collier. It was found that the canonical
PSC marker Ser-lacZ is not expressed in the
embryonic lymph gland and is only expressed in a small number of
cells in the second instar. This relatively late onset of expression is consistent with
collier acting genetically upstream of Ser.
Another finding was that the earliest expression of upd3-gal4
parallels the expression of Ser-lacZ and is restricted to the PSC
region. Finally, Pvr and dome-gal4 are excluded from the PSC in the second instar,
similar to what is seen in the third instar (Jung, 2005).
To determine whether maturing cortical zone cells are indeed derived from
medullary zone prohemocytes, a lineage-tracing experiment was performed in
which dome-gal4 was used to initiate the permanent marking of all
daughter cell lineages. In this system, the dome-gal4 reporter expresses
both UAS-GFP and UAS-FLP. The FLP recombinase excises an intervening
FRT-flanked 'STOP cassette', allowing constitutive expression of lacZ
under the control of the actin5C promoter. At
any developmental time point, GFP is expressed in cells where
dome-gal4 is active, while lacZ is expressed in all
subsequent daughter cells regardless of whether they continue to express
dome-gal4. In this experiment, cortical zone cells
are permanently marked with ß-galactosidase despite not expressing
dome-gal4 (as assessed by GFP), indicating that these cells are
derived from a dome-gal4-positive precursor. This result is
consistent with and further supports independent marker analysis that
shows that dome-gal4-positive prohemocytes downregulate
dome-gal4 expression as they initiate expression of maturation
markers representative of cortical zone cells. As controls to the above
experiment, the expression patterns of two other gal4
lines, twist-gal4 and Serrate-gal4 were determined. The reporter
twist-gal4 is expressed throughout the embryonic mesoderm from which
the lymph gland is derived. Accordingly, the entire lymph gland is permanently
marked by ß-galactosidase despite a lack of twist-gal4 expression (GFP) in the
third instar lymph gland. Analysis of Ser-gal4 reveals that PSC
cells remain a distinct population of signaling cells that do not contribute
to the cortical zone (Jung, 2005).
Genetic manipulation of Pvr function provides valuable insight into its
involvement in the regulation of temporal events of lymph gland development.
To analyze Pvr function, FLP/FRT-based Pvr-mutant clones were
generated in the lymph gland early in the first instar and then examined
during the third instar for the expression of maturation markers. It was found
that loss of Pvr function abolishes P1 antigen and Pxn expression,
but not Hemese expression. The crystal cell markers Lz and ProPOA1
are also expressed normally in Pvr-mutant clones,
consistent with the observation that mature crystal cells lack or downregulate
Pvr. The fact that Pvr-mutant cells express Hemese and
can differentiate into crystal cells suggests that Pvr specifically controls
plasmatocyte differentiation. Pvr-mutant cells do not become TUNEL
positive but do express the hemocyte marker Hemese and can differentiate into crystal
cells, all suggesting that the observed block in plasmatocyte differentiation
within the mutant clone is not due to cell death. Additionally, Pvr-mutant
clones were large and
not significantly different in size from their wild-type twin spots.
Thus, the primary role of Pvr is not in the control of cell
proliferation. Targeting Pvr by RNA interference (RNAi) revealed the same
phenotypic features, confirming that Pvr controls the transition
of Hemese-positive cells to plasmatocyte fate (Jung, 2005).
Entry into S phase was monitored using BrdU incorporation and
distinct proliferative phases were identified that occur during lymph gland hematopoiesis. In
the second instar, proliferating cells are evenly distributed throughout the
lymph gland. By the
third instar, however, the distribution of proliferating cells is no longer
uniform; S-phase cells are largely restricted to the cortical zone. This is
particularly evident when BrdU-labeled lymph glands are co-stained with Pxn. Medullary
zone cells, which can be identified by the expression of dome-gal4,
rarely incorporate BrdU. Therefore, the rapidly cycling prohemocytes of the second
instar lymph gland quiesce as they populate the medullary zone of the third
instar. As prohemocytes transition into hemocyte fates in the cortical zone,
they once again begin to expand in number. This is supported by the
observation that the medullary zone in white pre-pupae does not appear
diminished in size, suggesting that the primary mechanism for the
expansion of the cortical zone prior to this stage is through cell division
within the zone. Proliferating cells in the secondary lobes continue to be
distributed uniformly in the third instar, suggesting that
secondary-lobe prohemocytes do not reach a state of quiescence as do the cells
of the medullary zone. These results indicate that cells of the lymph gland go
through distinct proliferative phases as hematopoietic development proceeds (Jung, 2005).
This analysis of the lymph gland revealed three key features that arise
during development. The first feature is the presence of three distinct zones
in the primary lymph gland lobe of third instar larvae. Two of these zones,
termed the cortical and medullary zones, exhibit structural
characteristics that make them morphologically distinct. These zones, as well
as the third zone, the PSC, are also distinguishable by the expression of
specific markers. The second key feature is that cells expressing
maturation markers such as Lz, ProPOA1, Pxn, hml-gal4 and
Cg-gal4 are restricted to the cortical zone. The medullary zone is
consistently devoid of maturation marker expression and is therefore defined
as a region composed of immature hemocytes (prohemocytes). The finding of
different developmental populations within the lymph gland (prohemoctyes and
their derived hemocytes) is similar to the situation in vertebrates where it
is known that hematopoietic stem cells and other blood precursors give rise to
various mature cell types. Additionally, Drosophila hemocyte
maturation is akin to the progressive maturation of myeloid and lymphoid
lineages in vertebrate hematopoiesis. The third key feature of lymph gland
hematopoiesis is the dynamic pattern of cellular proliferation observed in the
third instar. At this stage, the vast majority of S-phase cells in the primary
lobe are located in the cortical zone, suggesting a strong correlation between
proliferation and hemocyte differentiation. Compared with earlier
developmental stages, cell proliferation in the medullary zone actually
decreases by the late third instar, suggesting that these cells have entered a
quiescent state. Thus, proliferation in the lymph gland appears to be
regulated such that growth, quiescence and expansion phases are evident
throughout its development (Jung, 2005).
Drosophila blood cell precursors, prohemocytes and maturing
hemocytes each exhibit extensive phases of proliferation. The competence of
these cells to proliferate seems to be a distinct cellular characteristic that
is superimposed upon the intrinsic maturation program. Based on the patterns
of BrdU incorporation in developing primary and secondary lymph gland lobes,
it is possible to envision at least two levels of proliferation control during
hematopoiesis. It is proposed that the widespread cell proliferation observed in
second instar lymph glands and in secondary lobes of third instar lymph glands
occurs in response to a growth requirement that provides a sufficient number
of prohemocytes for subsequent differentiation. The mechanisms promoting
differentiation in the cortical zone also trigger cell proliferation, which
accounts for the observed BrdU incorporation in this zone and serves to expand
the effector hemocyte population. The quiescent cells of the medullary zone
represent a pluripotent precursor population because they, similar to
vertebrate hematopoietic precursors, rarely divide and give rise to multiple
lineages and cell types (Jung, 2005).
Based on this analysis a model is proposed by which
hemocytes mature in the lymph gland. Hematopoietic precursors that populate the early lymph gland
are first distinguishable as Srp+, Odd+ (S+O+) cells. These will
eventually give rise to a primary lymph gland lobe where the steps of hemocyte
maturation are most apparent. During the first or early second instar, these
S+O+ cells begin to express the hemocyte-specific marker
Hemese (He) and the tyrosine kinase receptor Pvr. Such cells can be called
pre-prohemocytes and, in the second instar, cells expressing only these
markers occupy a narrow region near the dorsal vessel. Subsequently, a subset
of these Srp+, Odd+, He+, Pvr+
(S+O+H+Pv+) pre-prohemocytes
initiate the expression of dome-gal4 (dg4),
thereby maturing into prohemocytes. The prohemocyte population
(S+O+H+Pv+dg4+)
can be subdivided into two developmental stages. Stage 1 prohemocytes, which
are abundantly seen in the second instar, are proliferative, whereas stage 2
prohemocytes, exemplified by the cells of the medullary zone, are quiescent.
As development continues, prohemocytes begin to downregulate
dome-gal4 and express maturation markers (M; becoming
S+O+H+Pv+dg4lowM+).
Eventually, dome-gal4 expression is lost entirely in these cells
(becoming
S+O+H+Pv+dg4-M+),
found generally in the cortical zone. Thus, the maturing hemocytes of the
cortical zone are derived from prohemocytes previously belonging to the
medullary zone. This is supported by lineage-tracing experiments that show
cells expressing medullary zone markers can indeed give rise to cells of the
cortical zone. In turn, the medullary zone is derived from the earlier,
pre-prohemocytes. Early cortical zone cells continue to express successive
maturation markers (M) as they proceed towards terminal differentiation. Depending on the
hemocyte type, examples of expressed maturation markers are Pxn, P1, Lz, L1,
msn-lacZ, etc. These studies have shown that differentiation of the
plasmatocyte lineage requires Pvr, while previous work has shown that the
Notch pathway is crucial for the crystal cell fate. Both
the JAK/STAT and Notch pathways have been implicated in lamellocyte production (Jung, 2005).
Previous investigations have demonstrated that similar transcription
factors and signal transduction pathways are used in the specification of
blood lineages in both vertebrates and Drosophila. Given this
relationship, Drosophila represents a powerful system for identifying
genes crucial to the hematopoietic process that are conserved in the
vertebrate system. The work presented here provides an analysis of
hematopoietic development in the Drosophila lymph gland that not only
identifies stage-specific markers, but also reveals developmental mechanisms
underlying hemocyte specification and maturation. The prohemocyte population
in Drosophila becomes mitotically quiescent, much as their
multipotent precursor counterparts in mammalian systems. These conserved
mechanisms further establish Drosophila as an excellent genetic model
for the study of hematopoiesis (Jung, 2005).
This paper defines temporal and spatial subdivisions of the embryonic head mesoderm and describes the fate of the main lineages derived from this tissue. During gastrulation, only a fraction of the head mesoderm (primary head mesoderm; PHM) invaginates as the anterior part of the ventral furrow. The PHM can be subdivided into four linearly arranged domains, based on the expression of different combinations of genetic markers (tinman, heartless, snail, serpent, mef-2, zfh-1). The anterior domain (PHMA) produces a variety of cell types, among them the neuroendocrine gland (corpus cardiacum). PHMB, forming much of the'T-bar' of the ventral furrow, migrates anteriorly and dorsally and gives rise to the dorsal pharyngeal musculature. PHMC is located behind the T-bar and forms part of the anterior endoderm, besides contributing to hemocytes. The most posterior domain, PHMD, belongs to the anterior gnathal segments and gives rise to a few somatic muscles, but also to hemocytes. The procephalic region flanking the ventral furrow also contributes to head mesoderm (secondary head mesoderm, SHM) that segregates from the surface after the ventral furrow has invaginated, indicating that gastrulation in the procephalon is much more protracted than in the trunk. This study distinguishes between an early SHM (eSHM) that is located on either side of the anterior endoderm and is the major source of hemocytes, including crystal cells. The eSHM is followed by the late SHM (lSHM), which consists of an anterior and posterior component (lSHMa, lSHMp). The lSHMa, flanking the stomodeum anteriorly and laterally, produces the visceral musculature of the esophagus, as well as a population of tinman-positive cells that is interpreted as a rudimentary cephalic aorta ('cephalic vascular rudiment'). The lSHM contributes hemocytes, as well as the nephrocytes forming the subesophageal body, also called garland cells (de Velasco, 2005).
The mesoderm is a morphologically distinct cell layer that can be recognized in early embryos of most bilaterian phyla and that gives rise to tissues interposed between ectodermal and endodermal epithelia, including muscle, connective, blood, vascular, and excretory tissue. Besides the differentiative fate of tissues derived from it, the mesoderm shares several common properties in regard to its formation during gastrulation. The anlage of the mesoderm is sandwiched in between the anlage of the endoderm and the neurectoderm. This has been documented in most detail in anamniote vertebrates, where signals from the vegetal blastomeres (the anlage of the endoderm) act on the adjacent marginal zone of the future ectoderm to induce mesoderm. Although gastrulation proceeds quite differently in arthropods from the way it does in chordates, the proximity of the mesodermal anlage to future endoderm and neurectoderm is conserved, and numerous signaling pathways and transcriptional regulators that share similar function and expression patterns in arthropods and chordates have been identified (de Velasco, 2005 and references therein).
Following gastrulation, the mesoderm is subdivided along the dorso-ventral axis into several subdivisions laid out in a distinct dorso-ventral order. In vertebrates, cells located in the dorsal part of the mesoderm anlage give rise to notochord and somites, which in turn produce muscular, skeletal, and connective tissue. Next to the somitic mesoderm is the intermediate mesoderm that will form the excretory and reproductive system. The ventral mesoderm (lateral plate) gives rise to blood, vascular system, visceral musculature, and coelomic cavity. In arthropods, fundamentally similar mesodermal subdivisions can be recognized, and similarities extend to the relative positions these domains obtain relative to each other and relative to the adjacent neurectoderm. For example, precursors of visceral muscles, vascular system, and blood are at the edge of the mesoderm facing away from the neural primordium (ventral in vertebrates, dorsal in arthropods (de Velasco, 2005 and references therein).
The subdivision of the vertebrate mesoderm into distinct longitudinal tissue columns with different fates is seen throughout the trunk and head of the embryo. However, several significant differences between the head and the trunk are immediately apparent. For example, cells derived from the anterior neurectoderm form the neural crest that migrates laterally and gives rise to many of the tissues that are produced by mesoderm in the trunk. As a result, the fates taken over by the head mesoderm are more limited than those of the trunk mesoderm. In contrast, the head mesoderm produces several unique lineages, such as the heart (cardiac mesoderm) and a population of early differentiating macrophages. Moreover, some of the signaling pathways responsible for inducing different mesodermal fates in the trunk appear to operate in a different manner in the head. A recently described example is the Wnt signal that induces somatic musculature in the trunk, but inhibits the same fate in the head (de Velasco, 2005 and references therein).
The head mesoderm of arthropods, like that of vertebrates, also appears to deviate in many ways from the trunk mesoderm. For example, specialized lineages like embryonic blood cells and nephrocytes forming the subesophageal body (also called garland cells) arise exclusively in the head. That being said, very little is known about how the arthropod head mesoderm arises and what types of tissues derive from it. The existing literature mainly uses histology, which severely limits the possibilities of following different cell types forward or backward in time. In this paper, several molecular markers have been used to initiate more detailed studies of the head mesoderm in Drosophila. The goal was to establish temporal and spatial subdivisions of the head mesoderm and, using molecular markers expressed from early stages onward, to follow the fate of the lineages derived from this embryonic tissue. Besides hemocytes and pharyngeal muscles described earlier, the head mesoderm also gives rise to several other lineages, including visceral muscle, putative vascular cells, nephrocytes, and neuroendocrine cells. The development of the head mesoderm is discussed in comparison with the trunk mesoderm and in the broader context of insect embryology (de Velasco, 2005).
The Drosophila head mesoderm, as traditionally defined, includes all mesoderm cells originating anterior to the cephalic furrow. The formation of the head mesoderm is complicated by the fact that (unlike the mesoderm of the trunk) only part of it invaginates with the ventral furrow; by far, the majority of head mesoderm cells, recognizable in a stage 10 or 11 embryo, segregate from the surface epithelium of the head after the ventral furrow has formed. Another complicating factor is that head mesoderm cells derived from different antero-posterior levels adopt very different fates, unlike the situation in the trunk where mesodermal fates within different segments along the AP axis are fairly homogenous, with obvious exceptions such as the gonadal mesoderm that is derived exclusively from a subset of abdominal segments. Using several different markers, this study has followed the origin, migration pathways, and later, fates of head mesoderm cells (de Velasco, 2005).
The anterior part of the ventral furrow, called primary head mesoderm (PHM) in the following, includes cells that will contribute to diverse tissues, including muscle, hemocytes, endoderm, and several ill-defined cell populations closely associated with the brain and neuroendocrine system. For clarification, the anterior ventral furrow will be divided into the following domains:
The anterior lip of the T-bar (PHMA) is the source of the corpus cardiacum, as well as other gt-positive cells that at least in part end up as nerve cells flanking the frontal connective and frontal ganglion. These cells continue the expression of giant throughout late embryonic development; they represent a hitherto unknown class of nonneuroblast-derived neurons (de Velasco, 2005).
The posterior lip of the T-bar (PHMB) can be followed towards later stages by its continued expression of htl. These cells, called the procephalic somatic mesoderm, form a bilateral cluster that moves dorso-anteriorly into the labrum and becomes the dorsal pharyngeal musculature. Htl expression almost disappears in these cells around late stage 11, but is reinitiated at stage 12 and stays strong until stage 14, when the dorsal pharyngeal muscles differentiate. Many of the genes expressed in the somatic musculature of the trunk and its precursors (Dmef2, beta-3-tubulin) are also expressed in the procephalic somatic mesoderm (de Velasco, 2005).
The part of the ventral furrow posteriorly adjacent to the T-bar (PHMC) expresses srp, forkhead (fkh), and other endoderm/hemocyte markers. After the ventral furrow closes in the ventral midline (stage 7/8), these cells form a compact median mass, most of which represents part of the anterior endoderm that gives rise to the midgut epithelium. Starting at around this stage, the lateral part of the hemocyte-forming 'secondary head mesoderm' ingresses in between the endoderm and the surface ectoderm. It is likely that some of the PHMC cells invaginating already with the ventral furrow, along with the cells that form the anterior endoderm, also give rise to hemocytes. Precursors of hemocytes and midgut are difficult to distinguish during and shortly after ventral furrow invagination since both express srp and other markers shared between hemocytes and midgut precursors. At around stage 9, the two populations of precursors disengage. The endoderm remains a compact mesenchyme attached to the invaginating stomodeum; hemocyte precursors move dorsally and take on the shape of expanding vertical plates interposed in between endoderm and ectoderm (de Velasco, 2005).
Domain PHMD, the short portion of the ventral furrow situated posterior to the endoderm, along with a considerable portion of the mesoderm behind the cephalic furrow, forms the mesoderm of the three gnathal segments (mandible, maxilla, labium). The gnathal mesoderm in many ways behaves like the mesoderm of thoracic and abdominal segments. It gives rise to somatic muscle (the lateral pharyngeal muscles), visceral muscle, and fat body. Unlike trunk mesoderm, gnathal mesoderm does not produce cardioblasts and pericardial cells. Instead, a large proportion of gnathal mesoderm cells, joining the anteriorly adjacent secondary procephalic mesoderm, adopt the fate of hemocytes (de Velasco, 2005).
Besides the ventral furrow, other parts of the ventral procephalon produce head mesoderm in a complex succession of delamination and ingression events. The head mesoderm that forms from outside the ventral furrow will be called 'secondary mesoderm' (SHM) in the following. Based on the time of formation and the position relative to the stomodeum, the following phases and domains of secondary head mesoderm development can be distinguished.
Following the obliteration of the ventral furrow at stage 8, the eSHM delaminates from the ventral surface 'meso-ectoderm' (considering that this epithelium still contains mesodermal progenitors!) flanking the endodermal mass. The eSHM forms two monolayered sheets that gradually move dorsally and posteriorly; by stage 9, the eSHM cells line the basal surface of the emerging head neuroblasts. An undefined number of primary head mesoderm cells derived from domain PHMC of the ventral furrow are mingled together with the eSHM cells. The ultimate fate of the eSHM is that of hemocytes: they express srp, followed slightly later by other blood cell markers (e.g., peroxidasin and asrij). A subset of hemocytes, called crystal cells, derive from precursors that form a morphologically conspicuous cluster at the dorsal edge of the eSHM, identifiable from early stage 10 onward by the expression of lz. The mechanism by which at least part of the eSHM delaminates is unique. Thus, it is formed by the vertically oriented division of the surface epithelium, whereby the inner daughters will become eSHMe and the outer ones ectoderm. The focus of vertical mitosis has named the procephalic domain in which it occurs 'mitotic domain #9' (de Velasco, 2005).
From late stage 9 onward, the early SHMs are followed inside the embryo by the closely adjacent posterior late SHMs. One cluster of posterior late secondary head mesoderm (lSHMp) cells delaminates from the surface epithelium flanking the posterior lip of the stomodeum; a second lSHMp cluster appears at the same stage at a slightly more posterior level. The first cluster seems to contribute to the hemocyte population; the posterior cluster gives rise to the nephrocytes forming the subesophageal body (also called garland cells; labeled by CG32094). Garland cell precursors are initially arranged as a paired cluster latero-ventrally of the esophagus primordium; subsequently, the clusters fuse in the midline and form a crescent underneath the esophagus. Garland cells are distinguished from crystal cells by their size, location, and arrangement: crystal cells are large, round cells grouped in an oblong cloud dorso-anterior to the proventriculus. Garland cells are smaller, closely attached to each other, and lie ventral of the esophagus
(de Velasco, 2005).
During stages 10 and 11, cells delaminate beside and anterior to the stomodeum, originating from the anlage of the esophagus and the epipharynx (labrum). These cells, called anterior late secondary head mesoderm cells (lSHMa), can be followed by their expression of tin. Two groups can be distinguished. The tin-positive cells delaminating from the esophageal anlage (es) give rise to the visceral musculature (vm) surrounding the esophagus. These cells lose tin expression soon after their segregation, but can be recognized by other visceral mesoderm markers such as anti-Connectin. More dorsally, in the anlage of the clypeolabrum (cl) delaminate, the dorsal subpopulation of the lSHMas, which rapidly migrates posteriorly on either side and slightly dorsal of the esophagus, can be found. These cells retain expression of tin into the late embryo. They assemble into two longitudinal rows stretching alongside the roof of the esophagus primordium. During late embryogenesis, they move posteriorly along with the esophagus towards a position behind the brain commissure. Many of the tin-positive SHMs apparently undergo apoptosis: initially counting approximately 25 on either side, they decrease to 12-15 at stage 14 to finally form a single, irregular row of about 15 cells total in the late embryo. These cells come into contact with the anterior tip of the dorsal vessel. This formation of previously undescribed cells, for which the term 'procephalic vascular cells', is proposed, is interpreted as a rudiment of the head aorta, which forms a prominent part of the dorsal vessel in many insect groups (de Velasco, 2005).
On the basis of additional molecular markers, the tin-positive procephalic vascular cells are further subdivided into two populations. The first subpopulation expresses the muscle and cardioblast-specific marker Dmef2; the second type is Dmef2-negative. In the dorsal vessel of the trunk, tin-positive cells also fall into a Dmef2-positive and a Dmef2-negative population. Dmef2-positive cells of the trunk represent the cardioblasts, myoendothelial cells lining the lumen of the dorsal vessel. Dmef2-negative/tin-positive cells form a somewhat irregular double row of cells attached to the ventral wall of the dorsal vessel. The ultimate fate of these cells has not been explored yet. However, preliminary data suggest that they develop into a muscle band that runs alongside the larval dorsal vessel. This would correspond to the situation in other insects in which such a ventral cardiac muscle band has been described (de Velasco, 2005).
The role of tinman in the formation of the procephalic vascular rudiment was investigated by assaying tin-mutant embryos for the expression of Dmef2. Similar to the cardioblasts of the trunk, the Dmef2-positive cells of the procephalic vascular rudiment are absent in tin mutants. It is quite likely that the (Dmef2-negative) remainder of the procephalic vascular rudiment is affected as well by loss of tin, but in the absence of appropriate markers (besides tin itself, which is not expressed in the mutant), it was not possible to substantiate this proposal (de Velasco, 2005).
At the time of appearance of the ventral furrow, segmental markers such as hh do not allow the distinction between distinct 'preoral' segments. Thus, hh is expressed in a wide procephalic stripe in front of the regularly sized mandibular stripe. During stage 7, the procephalic hh stripe splits into an anterior, antennal stripe and a posterior, short, intercalary stripe. The anterior lip of the ventral furrow (domain PHMA) coincides with the anterior boundary of the antenno-intercalary stripe. Thus, the primary head mesoderm and endoderm originating from within the anterior ventral furrow can be considered a derivative of the antennal and intercalary segments. This interpretation is supported by the expression of the homeobox gene labial (lab) found in the intercalary segment. The labial domain covers much of the anterior ventral furrow, including domains PHMB-C (de Velasco, 2005).
Morphogenetic movements in the ventral head, associated with the closure of the ventral furrow, the formation of the stomodeal placode, and the subsequent invagination of the stomodeum result in a shift of head segmental boundaries. The antennal segment tilts backward, as can be seen from the orientation of the antennal hh stripe that from stage 8 onward forms an almost horizontal line, connecting the cephalic furrow with the sides of the stomodeal invagination (which falls within the ventral realm of the antennal segment, in Drosophila as well as other insects). Since the expression of hh, like that of engrailed (en), coincides with the posterior boundary of a segment, the territory located ventral to the antennal hh stripe falls within the intercalary segment. This implies that most, if not all, of the posterior late SHM, is intercalary in origin. It is further plausible to consider that the anterior lSHM belongs to the intercalary and antennal segment. The vascular cells of the head, a conspicuous derivative of the anterior lSHM in Drosophila, are derived from the antennal mesoderm in other insects. The labrum, with which much of the anterior lSHM is associated, represents a structure that has always been difficult to integrate in the segmental organization of the head. Most likely the labrum represents part of the intercalary segment; this would help explain some of the unusual characteristics of the head mesoderm (de Velasco, 2005).
In conclusion, several fundamental similarities are found between the mesoderm of the head and that of the trunk regarding the tissues they give rise to, and possibly the signaling pathways deciding over these fates. After an initial phase of structural and molecular homogeneity, the trunk mesoderm becomes subdivided into a dorsal and a ventral domain by a Dpp-signaling event that emanates from the dorsal ectoderm. The dorsal domain, characterized by the Dpp-dependent continued expression of tinman, becomes the source of visceral and cardiogenic mesoderm, among other cell types. A role of Dpp/BMP signaling in cardiogenesis seems to be conserved among insects and vertebrates. Subsequent signaling steps, involving both Wingless and Notch/Delta, separate between these two fates and further subdivide the cardiogenic mesoderm into several distinct lineages, such as cardioblast, pericardial cells, and secondary hemocyte precursors (lymph gland). As a result of these signaling events, Tinman and several other fate-determining transcription factors become restricted to their respective lineages: tin to the cardioblasts, odd to pericardial cells and hemocyte precursors, zfh1 and srp to hemocyte precursors and fat body. Dmef2 and several other transcription factors become restricted to various combinations of muscle types (somatic, visceral, cardiac) (de Velasco, 2005).
In the head mesoderm, the above genes are associated with similar fates. Tin and Dmef2 appear widely in the procephalic ventral furrow and the anterior lSHM before getting restricted to the procephalic vascular rudiment and/or the pharyngeal musculature, respectively. In contrast with the initially ubiquitous expression of Tin and Dmef2 in the trunk mesoderm, those parts of the head mesoderm giving rise to hemocytes (PHMC, posterior lSHM) never express these mesodermal genes. Previous work has shown that the head gap gene buttonhead (btd) is responsible for the early repression of tin in the above mentioned domains of the head mesoderm. The early absence of Tin and Dmef2 in the head mesodermal hemocyte precursors is paralleled by the presence of Srp and Zfh1 in these cells. Interestingly, Srp/Zfh-positive cells of the head produce only hemocytes and no fat body, suggesting that an as-yet-uncharacterized signaling step prevents the formation of fat body in the head. It is tempting to speculate that there exists within the mesoderm a 'blood/fat body equivalence group'. Blood cells and fat body share not only the expression of fate-determining genes such as srp and zfh1, but also, later, functional properties that have to do with immunity. In the trunk, the blood/fat body equivalence group gives rise mostly to fat body, producing only a limited number of hemocyte precursors in the dorsal mesoderm of the thoracic segments. In the head, on the other hand, all cells of the equivalence group become hemocytes (de Velasco, 2005).
Attention is drawn to another mesodermal lineage that produces related, yet not identical, cell types in the trunk and the head: the nephrocytes. Nephrocytes are defined by their characteristic ultrastructure (membrane invaginations sealed off by junctions) that attests to their excretory function. In the trunk, nephrocytes are represented by the pericardial cells that settle beside the cardioblasts; a newly discovered nephrocyte population ('star cells') invading the Malpighian tubules is derived from the mesoderm of the tail segments. In the head, nephrocytes aggregate near the junction between esophagus and proventriculus as the subesophageal body, also called garland cells. The fact that from the early stages of development onward different transcription factors are expressed in garland cells and pericardial cells suggests that these cells perform similar, yet not fully overlapping, functions (de Velasco, 2005).
Genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into human muscle physiology and disease. Studies in Drosophila have been particularly useful for discovering key genes involved in muscle specification, myoblast fusion, and sarcomere organization. The muscles of the Drosophila female reproductive system have received little attention despite extensive work on oogenesis. This study used newly available GFP protein trap lines to characterize of ovarian muscle morphology and sarcomere organization. The muscle cells surrounding the oviducts are multinuclear with highly organized sarcomeres typical of somatic muscles. In contrast, the two muscle layers of the ovary, which are derived from gonadal mesoderm, have a mesh-like morphology similar to gut visceral muscle. Protein traps in the Fasciclin 3 gene produced Fas3::GFP that localized in dots around the periphery of epithelial sheath cells, the muscle surrounding ovarioles. Surprisingly, the epithelial sheath cells each contain a single nucleus, indicating these cells do not undergo myoblast fusion during development. Consistent with this observation, the Flp/FRT system was used to efficiently generate genetic mosaics in the epithelial sheath, suggesting these cells provide a new opportunity for clonal analysis of adult striated muscle (Hudson, 2008).
Two protein trap lines showed a striped pattern in ovarian muscle. The GFP transposon in these lines was inserted into the genes for two uncharacterized proteins, CG30084 and CG6416, both of which contain a PDZ domain and a ZASP motif (ZM). CG30084 also contains four LIM domains in its C-terminus. Both CG30084 and CG6416 proteins show homology to the human Z-disc Alternatively Spliced Protein (ZASP) that localizes to the Z-disc in skeletal muscle. Like the ZASP gene, both CG30084 and CG6416 encode a number of alternative splice forms, almost all of which could be tagged based on the locations of the protein trap insertions. An additional isoform of CG30084 has been annotated that encodes only LIM domains and cannot be tagged. Due to the homology of these genes to ZASP, CG30084 was designated as Zasp52, and CG6416 as Zasp66 based on the cytological location of the genes (Hudson, 2008).
The availability of protein trap lines was especially useful for describing intercellular contacts (e.g., Fas3::GFP, Ilk::GFP inserted into Integrin linked kinase) and for showing the location of 'new' sarcomere components (e.g., Zasp52::GFP, Zasp66::GFP). The muscle cells of the oviducts, which are derived from genital disks, have highly organized sarcomeres and a myotube organization typical of somatic muscles in insects, and likely arise by myoblast fusion during development. In contrast, the visceral muscle layers surrounding ovarioles and ovaries have a mesh-like shape similar to the visceral muscles of the gut. Surprisingly, it was found that the epithelial sheath muscles contain one nucleus rather than two as in gut visceral muscles, making them the only known adult somatic or visceral muscle that does not arise by myoblast fusion. It remains to be determined whether the peritoneal sheath cells are mononuclear as well (Hudson, 2008).
The nature of the epithelial sheath muscle makes it ideally suited for analysis of muscle function. The morphology of the cells can be viewed in great detail with an array of available sarcomere and cellular markers. Importantly, the ability to produce cells with a defined genotype by mitotic clone induction provides a new opportunity for studying the phenotypes in adult muscles caused by homozygous mutations in muscle genes, even if the mutations are lethal during early development. Finally, it is straightforward to do short-term in vitro culturing of ovarioles with the epithelial sheath intact and contracting; thus, the behavior of cells bearing mutations can be studied in live tissue under controlled conditions (Hudson, 2008).
It has been shown that species separated by relatively short evolutionary distances may have extreme variations in egg size and shape. Those variations are expected to modify the polarized morphogenetic gradients that pattern the dorso-ventral axis of embryos. Currently, little is known about the effects of scaling over the embryonic architecture of organisms. This problem was examined by asking if changes in embryo size in closely related species of Drosophila modify all three dorso-ventral germ layers or only particular layers, and whether or not tissue patterning would be affected at later stages. This paper reports that changes in scale affect predominantly the mesodermal layer at early stages, while the neuroectoderm remains constant across the species studied. Next, the fate of somatic myoblast precursor cells that derive from the mesoderm was examined to test whether the assembly of the larval body wall musculature would be affected by the variation in mesoderm specification. The results show that in all four species analyzed, the stereotyped organization of the body wall musculature is not disrupted and remains the same as in D. melanogaster. Instead, the excess or shortage of myoblast precursors is compensated by the formation of individual muscle fibers containing more or less fused myoblasts. These data suggest that changes in embryonic scaling often lead to expansions or retractions of the mesodermal domain across Drosophila species. At later stages, two compensatory cellular mechanisms assure the formation of a highly stereotyped larval somatic musculature: an invariable selection of 30 muscle founder cells per hemisegment, which seed the formation of a complete array of muscle fibers, and a variable rate in myoblast fusion that modifies the number of myoblasts that fuse to individual muscle fibers (Belu, 2011).
The data shows that within evolutionary distances as short as 5 mya of divergence time, there can be extreme variations in mesoderm specification. In most species analyzed, the width of mesodermal domain decreases or increases according to embryo size. However, this is not an absolute rule as D. simulans has a larger mesodermal domain than D. melanogaster, despite the fact that those two species vary less in their DV axis (Belu, 2011).
In contrast to the plasticity seen in the mesoderm specification, the width of the neuroectoderm remains constant across species. This latter result is in agreement with two important findings regarding the development of the ventral nerve cord. First, neuroblast maps are nearly identical in a broad range of insect species, sharing similarities even with the crustacean phylum. Second, experiments of genetic manipulation that altered the width of D/V expression domains within the neuroectoderm resulted in the duplication or elimination of neuroblasts of particular identity. Thus, the stable width in the neuroectoderm appears to be essential for the generation of correct neural lineages and axonal scaffolds within Drosophila species. However, the mechanisms that protect the neuroectoderm from scaling effects remain elusive (Belu, 2011).
Based on the stereotyped arrays of muscle fibers and innervation patterns observed in the different species, the data indicate that the mesodermal alterations can be compensated later in development. These corrections would involve an invariable selection of 30 FCs per hemisegment, and a variable rate of myoblast fusion that allows more cells to be incorporated to each muscle fiber. These two cellular mechanisms cooperate and prevent supernumerary or lack of muscle fibers (Belu, 2011).
What protects the development of the somatic body wall musculature from variations in the mesoderm size? This study highlights some key differences between the myogenesis and neurogenesis that may explain why the assembly of the somatic body wall has more alternate ways to cope with the early variations in mesodermal specification than does the ventral nerve cord (Belu, 2011).
The initial steps of both myogenesis and neurogenesis are similar and rely on the formation of groups of equivalent cells, the promuscular and proneural groups, from which a single progenitor cell is selected through lateral inhibition. In the case of the neural progenitor cell, or neuroblast, its identity is determined once it delaminates from the proneural group, when it initiates stereotyped divisions giving rise to a defined number and types of neurons/glial cells. In contrast, the progenitor of somatic muscles undergoes additional asymmetric cell divisions before it gives rise to FCs and adult muscle progenitor cells. Thus, modifications in the specification of muscle progenitor cells and/or their asymmetric cell divisions could generate an identical outcome of 30 embryonic FCs in the different Drosophila species (Belu, 2011).
Another difference between mesodermal and neural tissue specification is the fact that the entire neuroectodermal domain contributes to the formation of a stereotyped tissue, whereas the mesodermal domain is further subdivided and gives rise to non-stereotyped tissues as well, such as the fat body, hematopoietic system and visceral musculature. Therefore, species with reduced mesodermal domain might still be able to assemble the same numbers of promuscular groups at the expense of other mesodermal precursor cells that form non-stereotyped tissues (Belu, 2011).
Finally, the present study reveals that the myoblast fusion step, which is unique to myogenesis, is an important compensatory mechanism for the formation of the somatic body wall musculature (Belu, 2011).
The data shows that during myogenesis of D. busckii and D. pseudoobscura, fewer myoblasts are fused together to form slender muscle fibers in comparison to D. melanogaster. In contrast, more myoblasts fuse into single fibers in D. simulans and D. sechellia, resulting in fibers of increased size. The differential regulation of fusion events appears to be the only characteristic of FC identity that is unique to each species (Belu, 2011).
One of the main regulators of myoblast fusion is the adhesion molecule Kin of Irre/Dumbfounded (Kirre/Duf), which is expressed exclusively by FCs and functions as an attractant to FCMs. The expression of kirre/duf is down-regulated once the correct number of fused FCMs is achieved for a given muscle fiber. If this down-regulation of kirre/duf is modulated by the number of FCM that are aggregated, then there are two ways of increasing myoblast fusion. One would be if inhibitory signals released from fused FCMs are weaker in strength and the other would be if the sensitivity of kirre/duf to these signals is lower. In either case, more myoblasts would be added to the fiber. Recently, the cis-regulatory region of kirre/duf gene was identified in a group of Drosophila species, including D. pseudoobscura and D. simulans, and was found to have stretches of sequence divergence. These results support the view that modifications in the cis-regulatory sequence of kirre/duf could be responsible for different rates of myoblast fusion observed in these Drosophila species. However, further tests would be needed to determine whether constructs with kirre/duf from D. simulans and D. pseudoobscura inserted in D. melanogaster respond as expected by creating fibers with more or less myoblasts, respectively (Belu, 2011).
Variations in embryo size impose challenges to developing organisms, which must be overcome to ensure viability. In all species investigated in this study, some separated by several million years and others by only several thousand years, it was noted that alterations in mesodermal size were resolved by a common mechanism that increases or decreases the rate of myoblast fusion to generate the same stereotyped array of muscles. Since the variation in mesodermal domain and myoblast fusion rates occurred within very short evolutionary distances, these are fast evolving traits. Consistent with this view, there is evidence from the literature that genes belonging to the Toll and Dorsal/NFkappaB pathway, which participate in both immune response and D/V patterning, are fast evolving within twelve Drosophila species. This finding can be explained as adaptation to new pathogens found in the particular niches these species occupy. However, a recent comparison of the genomes of three melanogaster sister species identified components of the Dorsal/NFkappaB pathway that diverged the most in D. melanogaster, but the least in the pair D. simulans/D. sechellia, despite the fact that the latter two species do occupy completely different niches (i.e. one is cosmopolitan and the other is restricted to the plant Morinda, respectively). These data provide further evidence that D/V patterning itself, and not only immunity, evolves fast and point out to specific candidates in the Dorsal/NFkappaB pathway undergoing those changes (Belu, 2011).
Muscles ensure locomotion behavior of invertebrate and vertebrate organisms. They are highly specialized and form using conserved developmental programs. To identify new players in muscle development, Drosophila and zebrafish gene expression databases were screened for orthologous genes expressed in embryonic muscles. More than 100 candidates were selected. Among them is the glycolysis gene Pglym78/pgam2, the attenuated expression of which results in the formation of thinner muscles in Drosophila embryos. This phenotype is also observed in fast muscle fibers of pgam2 zebrafish morphants, suggesting affected myoblast fusion. Indeed, a detailed analysis of developing muscles in Pglym78 RNAi embryos reveals loss of fusion-associated actin foci and an inefficient Notch decay in fusion competent myoblasts, both known to be required for fusion. In addition to Pglym78, the screen identifies six other genes involved in glycolysis or in pyruvate metabolism (Pfk, Tpi, Gapdh, Pgk, Pyk, and Impl3). They are synchronously activated in embryonic muscles and attenuation of their expression leads to similar muscle phenotypes, which are characterized by fibers with reduced size and the presence of unfused myoblasts. The data also show that the cell size triggering insulin pathway positively regulates glycolysis in developing muscles and that blocking the insulin or target of rapamycin pathways phenocopies the loss of function phenotypes of glycolytic genes, leading to myoblast fusion arrest and reduced muscle size. Collectively, these data suggest that setting metabolism to glycolysis-stimulated biomass production is part of a core myogenic program that operates in both invertebrate and vertebrate embryos and promotes formation of syncytial muscles (Tixier, 2013).
During insect myogenesis, myoblasts are organized into a pre-pattern by specialized organizer cells. In the Drosophila embryo, these cells have been termed founder cells and play important roles in specifying muscle identity and in serving as targets for myoblast fusion. A group of adult muscles, the dorsal longitudinal (flight) muscles, DLMs, is patterned by persistent larval scaffolds; the second set, the dorso-ventral muscles, DVMs is patterned by mono-nucleate founder cells (FCs) that are much larger than the surrounding myoblasts. Both types of organizer cells express Dumbfounded, which is known to regulate fusion during embryonic myogenesis. The role of DVM founder cells as well as the DLM scaffolds was tested in genetic ablation studies using the UAS/Gal4 system of targeted transgene expression. In both cases, removal of organizer cells prior to fusion, causes formation of supernumerary fibers, suggesting that cells in the myoblast pool have the capacity to initiate fiber formation, which is normally inhibited by the organizers. In addition to the large DVM FCs, some (smaller) cells in the myoblast pool also express Dumbfounded. It is proposed that these cells are responsible for seeding supernumerary fibers, when DVM FCs are eliminated prior to fusion. When these cells are also eliminated, myogenesis fails to occur. In the second set of studies, targeted expression of constitutively active RasV12 also resulted in the appearance of supernumerary fibers. In this case, the original DVM FCs are present, suggesting alterations in cell fate. Taken together, these data suggest that DVM myoblasts are able to respond to cues other than the original founder cell, to initiate fusion and fiber formation. Thus, the role of the large DVM founder cells is to generate the correct number of fibers, but they are not required for fiber formation itself. Evidence is also presented that the DVM FCs may arise from the leg imaginal disc (Atreya, 2008).
Founder cells (FCs) play an important role during myogenesis as they usually represent a pre-pattern prior to the onset of myoblast fusion. In the Drosophila embryo, each of the 30 muscle fibers in a hemisegment is seeded by a single founder cell. A group of adult flight muscles in Drosophila, the dorso-ventral muscles (DVMs) is patterned by founder cells which unlike the embryo are much larger than surrounding myoblasts. These cells have been referred to as imaginal pioneers and express the embryonic founder cell marker Dumbfounded, as detected by reporter activity. Duf is known to serve as an attractant for myoblasts and to thereby promote fusion. This study examined a role for the DVM FCs in organizing fiber formation. First the FCs were eliminated prior to the onset of fusion through targeted expression of the cell death gene reaper. Removal of these cells does not abolish fiber formation, but rather, excessive numbers of thinner fibers are formed. This outcome resembles what is observed when larval scaffolds for a related group of muscles, the dorsal longitudinal muscles (DLMs), are ablated. These scaffolds also express Duf, and the outcomes of their genetic ablation served as a useful comparison of muscle patterning in a set of functionally related fibers. In examining DVM myogenesis closely, it was found that in addition to the large Duf-expressing FCs, some (smaller) cells in the myoblast pool also express Duf, and it is proposed that these are the alternate/replacement founder cells that can seed fiber formation in absence of the large FCs. Interestingly, supernumerary fibers do not develop when reaper expression is maintained through the period of myogenesis, indicating that Duf expression is necessary in the alternate/replacement founder cells for fiber formation. In a second set of experiments, the Ras-signaling pathway was constitutively activated in using the rP298-Gal4 driver, and this manipulation also resulted in an increase in fiber number. The increase was not due to proliferation of pre-existing founder cells, and the supernumerary fibers develop in presence of the existing FCs. These outcomes for ablations of DVM organizers suggest that (1) DVM fibers can arise in the absence of the large elongate FCs; (2) a subset of cells in the myoblast pool have the potential to initiate fiber formation; (3) signals from the FC normally suppress this capability, so that the correct pattern of muscle fibers can be generated; and (4) interfering with the signaling results in the formation of supernumerary fibers. Thus, the most critical role of the DVM FCs is to regulate fiber number (Atreya, 2008).
An important consequence of eliminating the DVM FCs is that multiple fibers are formed, suggesting that cells from the myoblast pool are recruited to initiate fusion. Formation of supernumerary fibers is also observed when larval scaffolds that pattern the functionally opposing muscle group, the DLMs are ablated, or eliminated using reaper. Thus, although the two muscle groups develop using different modes of development, the larval scaffolds and the single celled FCs serve a similar function-specification of the number of adult fibers. These organizers serve as a pre-pattern, partitioning myoblasts through attractive cues, and subsequently when fusion begins, the final muscle pattern begins to emerge. The size of the myoblast pool remains unaffected by the manipulation (targeted reaper expression) and this is borne out by the fact that muscle volume in the adult thorax is not altered. These outcomes are similar to what is observed after DLM scaffolds are genetically ablated or laser ablated (Fernandes and Keshishian, 1996).
If a role for the large DVM founder cells is to initiate fusion, how does it occur in their absence? Two possibilities are suggested: that myoblasts randomly fuse with each other, or that 'replacement' founder cells begin to seed fiber formation. If the first scenario were true, many more than the 6-7 supernumerary fibers would be expected. Also, it would be a departure from the well understood separation of 'founder' and 'fusion-competent' fates (reviewed in Taylor, 2003) and the normal tendency of myoblasts to only fuse with a founder/scaffold. The current results do suggest the recruitment of 'alternate/replacement' founder cells. The rP298 promoter is active in a small subset of cells in the myoblast pool as early as 6 h APF, as detected by using the rP298-Gal4/UAS mCD8GFP. These cells are the same size as other cells in the myoblast pool and thus much smaller than the DVM FCs. It is proposed that these smaller Duf-expressing cells are recruited to initiate fusion when FCs are eliminated. They are capable of sustaining fusion, as suggested by multiple EWG-positive nuclei within a supernumerary fiber. It was also observed that fiber formation is disrupted upon prolonged exposure to reaper, which is most likely due to elimination of the smaller Duf-expressing cells as well. Thus, it appears that Duf expression is necessary in these cells to generate supernumerary fibers (Atreya, 2008).
It is useful to compare another manipulation which also eliminates Duf-lacZ expressing FCs, but which has different outcomes with respect to fiber formation. When the mesothorax is denervated at the third larval instar, the expression of Dumbfounded in DVM FCs is gradually abolished during the period of fusion, 12-24 h APF. Although the size of the myoblast pool is unaffected by the denervation, and Duf-expressing FCs are present prior to fusion (0-12 h APF), DVM fibers fail to form. In light of the reaper studies, it is reasonable to propose that during the 0-12 h period, DVM FCs engage in a 'lateral inhibition' process that is sufficient to prevent cells of the myoblast pool from seeding fibers during the subsequent fusion phase. Moreover, just as innervation is needed to maintain Duf expression in the FCs during the fusion phase, it may also be necessary to maintain expression in the smaller 'replacement/alternate' founder cells (Atreya, 2008).
In considering a role for the smaller Duf-positive cells during normal development, it is proposed that a subset of cells in the myoblast pool have the capability to initiate fiber formation, and that interactions with the founder cell prior to fusion are responsible for active suppression of this 'organizer' competency. When the FCs are eliminated, it is conceivable that the suppression is incomplete and they can go on to seed fibers. Thus, the smaller Duf-expressing cells serve as a reserve pool of founder cells to initiate fusion when necessary. The suppression could involve signaling mechanisms such as those that have been described for the embryonic mesoderm, wherein an equivalence group is first defined, from which a single founder cell is then selected, and the potential of the remaining cells is inhibited (Atreya, 2008).
Supernumerary fibers can arise despite the presence of pre-existing rP298-lacZ positive FCs
Overexpression of RasV12 with the rP298-Gal4 driver prior to the onset of fusion also results in the development of supernumerary fibers. Two additional phenotypes are seen compared to what is seen under conditions of reaper expression (1) the large rP298-lacZ expressing FCs are still present and (2) many more rP298-lacZ expressing nuclei are seen in the myoblast pool than in controls. The formation of additional rP298-lacZ expressing cells is considered first. The BrDU studies demonstrate that they do not arise by proliferation. Thus the cells are being generated from the myoblast pool due to a change in cell fate. Two possibilities are suggested: one that activation of the Ras/Map kinase pathway in FCs signals cells in the myoblast pool to turn on rP298, and the second and more intriguing possibility considers the small subset of cells that express the rP298-reporter at low levels. There may be additional cells that are below the level of detection with the lacZ reporter; if the Ras/Map kinase pathway is activated in several of these cells, it would similarly result in activation of the rP298 promoter, giving rise to the increased number of rP298-positive cells. The levels of RasV12 activated in these unfused myoblasts may be just enough to alter cell fate, conferring founder cell like properties. In the Drosophila embryo, when RasV12 is targeted to the embryonic mesoderm an overproduction of progenitor cells is seen, which is brought about as a result of a change in cell fate of mesodermal cells. Although RasV12 is also being targeted to the large DVM FC, it does not alter cell fate there since that cell is already expressing high levels of rP298 and it continues to seed fiber formation, as in controls (Atreya, 2008).
Ras regulates intracellular signaling through the ERK/MAP kinase pathway, and RasV12 is known to increase levels of activated MAPK within cells. A recent report on founder cell specification during adult abdominal myogenesis in Drosophila has shown that restricted activation of the Ras/Map kinase pathway by the FGF receptor Htl, in a few myoblasts reinforces founder cell properties in them, leading to the upregulation of rP298-lacZ levels, and the eventual loss of reporter expression in the other myoblasts. While expression of Htl has not been reported in IFM myoblasts, a similar mechanism that uses the ERK/MAPK pathway might be at play. It is also considered that overexpression of RasV12 causes death of the founder cells. But this is clearly not the case, since TUNEL labeling does not show any cell death, and the original founder cells are present and seed fiber formation (Atreya, 2008).
Expressing RasV12 in DLM scaffolds has two prominent outcomes. First, there is an increase in the number of rows of nuclei within the developing fibers. The increase in number of nuclei is suggestive of rapid fusion, and an outcome of enhanced Ras signaling. Another interesting aspect is in regard to muscle splitting. The six DLM fibers are generated as a collective outcome of the longitudinal splitting of the three larval scaffolds and fusion of myoblasts with these scaffolds. It is thought that as myoblasts fuse with the scaffolds there is an increase in surface area, which is manifested as splitting. Under conditions of RasV12 expression, there is an increase in the number of nuclei within each DLM fiber, indicative of rapid fusion. However, in 45% of animals, splitting does not proceed normally. It follows therefore, that fusion occurs too rapidly, and becomes uncoupled from a sustained incorporation of new membrane, which is then manifested as a lag in splitting. Under conditions of nerve ablation, a lag in muscle splitting is observed, but in that case, a reduced pool of myoblasts and slower fusions are responsible (Atreya, 2008).
Which of the known organizer cells do they resemble — grasshopper pioneer, larval scaffolds, or embryonic FCs? The cells that organize the DVM myoblasts share features that are common to embryonic founder cells and to grasshopper pioneers. Like the embryonic FCs and grasshopper pioneer cells, they serve as fusion targets and prefigure a muscle fiber. They bear a closer resemblance to grasshopper pioneers with respect to their large size including cellular expanse. However, they are unique with respect to the manner in which they interact with surrounding myoblasts, a feature that has been revealed as a result of manipulations carried out in the present study. They are not necessary for fiber formation, per se, as in their absence, a reserve pool of 'alternate/replacement' FCs is recruited. When these cells are removed as well, fiber formation fails and this scenario is the most similar to the outcome of targeting reaper to embryonic founder cells (Atreya, 2008).
A distinction between adult and embryonic myogenesis is exemplified by the manner in which founder cells are selected during abdominal myogenesis. rP298-lacZ expressing founder cells in the adult abdomen are specified by signaling through the FGF receptor, Heartless, which is under positive as well as negative regulation through Sprouty and Heartbroken. This is different from founder cell specification in the embryo which uses Notch-mediated lateral inhibition. This difference between embryonic and adult abdominal muscles is particularly striking since the two muscle groups are more similar to each other with respect to the occurrence of segmentally repeated patterns, a feature that is not present in the adult thorax. The manner in which founder cells of the adult thoracic muscles are specified is not known, but it does not include a Notch-mediated lateral inhibition (Atreya, 2008).
In considering how a myoblast pool is patterned, these studies thus far have shown that an important property of organizer cells that regulate DVM patterning, is the regulation of fiber number. This property is related to the expression of Duf, which is known to serve as an attractant for myoblasts. Thus, the pre-pattern of large, elongate rP298-lacZ positive cells enables myoblast segregation in a manner that the fidelity of fiber number is ensured. There is a scattering of smaller rP298-lacZ expressing adult myoblasts in the pool, and these have the potential to seed fiber formation. The manipulations have shown that the DVM FCs suppress these smaller myoblasts from seeding fibers, and that interfering with this capacity can result in the formation of supernumerary fibers being generated. The interference can take the form of eliminating FCs or by activating the Ras/Map kinase pathway. Interestingly, cues from the epidermis are able to guide the nascent supernumerary fibers to attach appropriately. The organizer cells are contacted by neurons prior to the onset of fusion and this is the basis for another property that is distinct from embryonic founder cells -- the nerve dependence of rP298-lacZ expression. The innervating neurons continue to exert an influence on myogenesis during the stages of fusion and proliferation as well (Atreya, 2008).
Future work will be aimed at examining the mechanisms by which the correct number of FCs is established, the signaling mechanisms underlying founder cell-myoblast interactions, and how supernumerary founder cells are normally prevented from arising in the myoblast pool (Atreya, 2008).
The Hand basic helix-loop-helix transcription factors play an important role in the specification and patterning of various tissues in vertebrates and invertebrates.
This study has investigated the function of Hand in the development of the Drosophila wing hearts which consist of somatic muscle cells as well as a mesodermally derived epithelium. Hand was found to be essential in both tissues for proper organ formation. Loss of Hand leads to a reduced number of cells in the mature organ and loss of wing heart functionality. In wing heart muscles Hand is required for the correct positioning of attachment sites, the parallel alignment of muscle cells, and the proper orientation of myofibrils. At the protein level, α-Spectrin and Dystroglycan are misdistributed suggesting a defect in the costameric network. Hand is also required for proper differentiation of the wing heart epithelium. Additionally, the handC-GFP reporter line is not active in the mutant suggesting an autoregulatory role of Hand in wing hearts. Finally, in a candidate-based RNAi mediated knock-down approach Daughterless and Nautilus were identified as potential dimerization partners of Hand in wing hearts (Togel, 2013).
In hand null mutants, wing hearts are formed but exhibit severe morphological defects resulting in loss of wing heart function. Consequently, almost all individuals display opaque wings and are unable to fly. Moreover, over time many of the mutant flies accumulate hemolymph in their wings. This long term effect occurs also very frequently in flies that completely lack wing hearts. During wing inflation, hemolymph is forced into the wings by elevated hemolymph pressure in the thorax which is effectuated by rhythmic contractions of the abdomen. However, this does not result in uncontrolled hemolymph accumulation as observed in animals lacking wing heart function since the epidermal cells of the wings still interconnect the opposing wing surfaces at this stage. Only after their delamination, more hemolymph may accumulate resulting in balloon-like wings which explains the long term character of this phenotype. However, since a rather large amount of hemolymph may accumulate in the wings some mechanism must exist that prevents backflow into the body cavity. In the tubular connection between wing and wing heart, a back-flow valve exists that prohibits hemolymph flow from the body cavity into the wing and thus is unsuitable to maintain a large amount of hemolymph inside the wing. In the region of the hinge, no valves are present and hemolymph may freely enter or leave the wings. It is therefore assumed that the apoptotic epidermal cells that remain in the wings due to loss of wing heart function form clots in the inflow and outflow tracts and thereby block hemolymph passage. Animals exhibiting these long term effects are probably affected in various ways. Most obviously, flies with filled wings have difficulties moving around and tend to fall during climbing. However, there are probably also physiological effects since the amount of hemolymph trapped in the wings must be lacking in the body cavity and should therefore affect internal hemolymph pressure as well as tissue homeostasis. Thus, it is proposed that the long term effects on wing morphology may contribute to the observed shortened life span of adult hand mutants (Togel, 2013).
Based on handC-GFP reporter activity, wing hearts express hand throughout their entire development and probably also during their mature state. However, the requirement for Hand seems only critical during early pupal stages at the time when the wing hearts are formed. Similarly, hand mutants display a phenotype in the adult only with regard to the heart and the midgut indicating that Hand is likewise required only during metamorphosis in these organs. In the adult heart, loss of hand leads to disorganized myofibrils, a phenotype that was also observed in mature wing heart muscles. Additionally, it was found that the attachment sites are less regular leading to a disruption of the dorso-ventral order of the muscle cells and loss of their parallel alignment. In many cases, muscle cells even form ectopic attachment sites in an area where they never occur in the wild-type. In an attempt to characterize the phenotype at the protein level, it was found that α-Spectrin and Dystroglycan are not properly distributed. Both proteins constitute components of the costameric network and are enriched at the membrane overlying Z-discs in the wild-type. In hand mutants, however, their pattern is altered to a more or less homogenous distribution at the membrane. In knock-downs of α- or β-Spectrin in the postsynaptic neuromuscular junction (NMJ), it was shown that spectrins are required for normal growth of NMJs and normal distribution of Dlg at the junctions. The enlarged NMJs visible in the Dlg staining, support the observation that α-Spectrin is misdistributed in hand mutants. The similar localization of α-Spectrin and Dystroglycan at the membrane raises the question whether their misdistribution in the mutant is somehow interconnected or independent of each other. Spectrins are organized in tetramers, consisting of two α/β-Spectrin heterodimers, which bind actin and are connected to the plasma membrane via Ankyrins. Ankyrins, in turn, have binding sites for Dystroglycan and E-Cadherin and together Ankyrin and the actin/spectrin network are thought to stabilize cell-cell and cell-matrix attachments. A hint that the misdistribution of α-Spectrin and Dystroglycan may be interconnected comes from observations in Dystrophin deficient mdx mice. There, Dystroglycan and β-Spectrin are both irregularly distributed but always co-localize. This let the authors conclude that their organization is coordinated. A possible explanation for the general loss of costamere organization may be that the costameric γ-Actin, although expressed normally, does not form a stable link between Z-discs and the membrane in mxd mice. However, loss of Dystrophin results mostly in the disruption of the linear arrangement of the proteins at the Z- and M-lines and does not lead to their homogenous distribution as observed in hand mutants. Nevertheless, the data obtained in this study and the phenotypic analysis of loss of function studies strongly suggest that the Spectrin and Dystroglycan phenotypes in hand mutants are interconnected caused by a, yet unknown, defect in the costameric network. Moreover, the misdistribution of these two proteins suggests that other proteins might be affected in a similar way including receptors required for directed outgrowth of muscle cells and proper targeting of tendon cells. This would explain why many muscle cells attach at improper positions or are misaligned. However, the cytoskeleton is not affected as a whole since the muscle cells still attain an elongated shape with attachment sites at their ends and a wild-typic βPS-Integrin pattern (Togel, 2013).
In the epithelium, loss of hand results in the failure of cells to integrate into the developing epithelium leading to gaps and the loss of cells. In mature organs, cells are predominantly missing in the area that dorsally extends the muscle cells suggesting that epithelial cells have greater difficulties attaching to their own type than to the muscle cells. On the protein level, it was found that Arm does not localize to the periphery of the cells except for small dot-like areas in the remaining filopodia-like cellular interconnections. Arm (β-Catenin) constitutes an intracellular adapter protein that links the transmembrane receptor E-Cadherin to actin filaments in adherens junctions. Adherens junctions are predominantly found between cells of the same type whereas Integrin based hemiadherens junctions connect to the ECM and additionally form specialized junctions between different cell types (e.g. myotendinous junctions). Based on the correct distribution of βPS-Integrin and the absence of Arm at the cell borders in hand mutants, it could be that the epithelial cells are able to form hemiadherens junctions towards the muscle cells but fail to establish a sufficient number of adherens junctions towards other epithelial cells. However, an alternative explanation would be that the formation of hemiadherens junctions is not affected and the remaining cells are simply too far apart to establish proper cell-cell contacts. Further experiments are needed to clarify this point (Togel, 2013).
It has been suggested that Hand proteins are involved in the inhibition of apoptosis based on the observation that loss of Hand function leads to hypoplasia and that block of apoptosis in the mutant background, at least partially, rescues the hand phenotype. This study observed a similar effect with respect to wing heart cell number. However, live cell imaging showed that also in the wild-type muscle cells are removed by apoptosis suggesting that this is a normal process during regulation of muscle cell number. Consequently, block of apoptosis in the controls led to an increase in muscle cell number. This suggests further that wing hearts in general have the potential to form more functional muscle cells. In hand mutants, the same removal of muscle cells occurs indicating that hand does not in general block apoptosis in wing hearts. Moreover, since the inhibition of apoptosis by P35 also affects the apoptosis involved in regulation of muscle cell number it cannot be excluded that the observed effect is actually induced hyperplasia in the mutant background mimicking a rescue instead of a real rescue of the hand phenotype. Additionally, live cell imaging showed that the cells of the wing heart epithelium forming the dorsal extension arise at their correct position in a sufficient number so that no gaps are visible. Only after they fail to establish proper cell–cell contacts they are removed from the wing hearts. It is therefore proposed that loss of cells by apoptosis in hand mutants is only a secondary effect caused by the inability of cells to integrate into the forming wing hearts (Togel, 2013).
It has been shown that Hand proteins can function as transcriptional activators in vertebrates and invertebrates. However, no direct targets have been identified in Drosophila so far. This study reports that the handC-GFP reporter line shows almost no activity in wing heart progenitors during postembryonic development suggesting that hand itself is a direct target of Hand. Remarkably, in some individuals a few nuclei of the wing hearts still show reporter activity indicating that hand is not the only transcription factor involved in postembryonic activation of the reporter. Moreover, the fact that the hand null mutant is not always a null with respect to reporter activation makes it a variable phenotype. Similarly, the severity of the phenotype observed in individual wing hearts (e.g. left and right side of the same animal) may differ considerably. So, how can a null mutation cause variable phenotypes? The answer may lie in the fact that all bHLH transcription factors need to form homo- or heterodimers for DNA binding. It was therefore proposed that the absence of a bHLH transcription factor not only affects its direct downstream targets but also the entire bHLH factor stoichiometry within the cell suggesting that the pool of bHLH dimers might be dynamically balanced. In the absence of Hand, new and presumably also artificial bHLH dimers are formed which consequently can cause a variety of delicate differentiation defects. The scenario is becoming even more complicated by the observation that the dimerization property of Hand is modulated by its phosphorylation state as well as by the finding that Hand can inhibit the dimerization of other transcription factors by blocking their protein interaction sites (Togel, 2013).
A crucial prerequisite for understanding the bHLH network in wing hearts is therefore the identification of dimerization partners. In a candidate based RNAi approach, two bHLH proteins, Da and Nau, were identified which evoke a phenotype very similar to the hand mutant. In order to verify the indication that these factors are interacting with Hand, Y2H analysis was applied and and an interaction between both Hand and Da as well as Hand and Nau was confirmed at the protein level. Furthermore, in vertebrates it was shown that these proteins are also able to form heterodimers with each other and that Hand is able to compete for heterodimer formation and DNA binding. Thus, based on Y2H interaction as well as phenotype similarity, the potential bHLH network in wing hearts likely includes Hand/Da and Hand/Nau heterodimers which activate different sets of downstream genes. In hand null mutants, the balance may be shifted to Da/Nau heterodimers or even Da/Da or Nau/Nau homodimers which may be able to activate some of Hand's target genes but with lower or higher efficiency. The competition of all these dimers with different transcriptional activation efficiency for the hand targets might explain the variations observed in the hand mutants (Togel, 2013).
Dmca1D was identified as the channel that mediates the Ca2+ inward current in the Drosophila embryonic muscle. Dmca1D has been shown to be the primary Ca2+ channel involved in the generation of voltage-dependent inward current in the larval abdominal muscles (Ren, 1998). This means that Drosophila skeletal muscles express the same α-subunit throughout the embryonic and larval stages. The Dmca1D channel current in the larval muscle as carried by Ba2+ was detected at - 20 mV and more positive potential, with the peak current attained at 0 mV. In the present study, Ca2+ currents appeared at - 20 mV and peaked at + 20 mV with normal [Ca2+]OUT. A similar shift in the voltage at which the inward current reaches the peak was observed when Dmca1D was heterologously expressed in Xenopus oocytes. It might be that the internal milieu of the cell from which currents were recorded had an impact on the voltage dependence of channel activation. The Ba2+ current in the larval muscle slowly declined during a command step of 500 ms in the voltage range between - 20 and 0 mV, indicating that its inactivation is slow and least voltage dependent, similar to the Ca2+ current in the embryonic muscle. The Ca2+ inward current recorded in this study exhibited ultraslow inactivation kinetics with time constants of ∼1000 ms, which is on the same order of magnitude as that for a slow inactivation component of L-type Ca2+ currents in dorsal horn neurons (∼2000 ms1). A study of mammalian Ca2+ channels expressed in Xenopus oocytes demonstrated that substitution of a β1b-subunit with a β2a-subunit in the α1A+α2-δ Ca2+ channel complex converts Ca2+ currents from inactivating into non-inactivating. This finding raises the intriguing possibility that the ultraslow inactivation observed in the current experiments was dependent on β-subunit subtypes recruited to the Ca2+ channel complex in Drosophila embryonic muscles. Another important question to be addressed by a future study is to determine whether or not the Ca2+ entry contributed to the inactivation of Ca2+ current, as was the case in the skeletal muscle fibers of the stick insect. The fact that the inactivation became evident only at the prepulse potentials beyond which the Ca2+ inward current was activated (i.e., ~- 20 mV) opens the possibility that the observed inactivation was, at least in part, dependent on Ca2+ influx. This possibility can be tested using a prepulse to the reversal potential of Ca2+ current so that the Ca2+ entry during the prepulse is minimal. Since the biophysical properties of the Dmca1D Ca2+ channel in the larval muscle have not been fully described, more detailed comparisons of Dmca1D Ca2+ channels in the embryonic and larval muscles are difficult. The larval abdominal muscles have an additional Ca2+ current component, which is sensitive to amiloride and similar to the vertebrate T-type current in kinetics (Gielow, 1995). In fact, some of the Ca2+ current traces in the current experiment had a rapidly decaying component. It remains to be determined whether the amiloride-sensitive transient Ca2+ current is present or not in the embryonic muscle (Hara, 2015).
This study found that the Ca2+ conductance saturates as the [Ca2+]OUT increases. Compared with the saturating Ca2+ conductance reported in other insect muscles, the saturation of the Ca2+ current in Drosophila embryonic muscle becomes evident at much lower [Ca2+]OUT, giving rise to a smaller dissociation constant for a presumptive binding site within the channel pore. This suggests that the interaction of Ca2+ with the channel wall during permeation is several folds stronger in the Dmca1D channel analyzed in this study. In a future study, it would be of interest to examine how other permeable and impermeable cations interact with the Dmca1D channel in penetrating or blocking it (Hara, 2015).
The body wall muscles formed in the embryo are used across the developmental stages. These muscles serve primarily for hatching in embryos and for crawling during the larval stage. Despite this change in their function, the current results and published works together suggest that Dmca1D remains the primary α-subunit type of Ca2+ channels in the body wall muscles across the developmental stages. It is envisaged that evolution preferentially regulated muscle excitability by means other than alteration of the Ca2+ channel subunits during development. In fact, Drosophila muscles have a variety of K+ channels with different compositions of subunits each encoded by different genes, including Shaker, Hyperkinetic, ether-á-gogo, Shab, slowpoke, and others, which confer distinct kinetics, and permeability properties and sensitivities to voltage, Ca2+, and/or cyclic nucleotides on the K+ channels thus formed. It remains to be determined how these K+ channels conform muscle membrane excitability to the requirement for different functions of body wall muscles in different developmental stages and how they cooperate with Dmca1D in modulating muscle contractile properties according to such functional requirements (Hara, 2015).
Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. This study used electron microscopy methods to study this key form of cell-cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. It was found that IFM myoblast-myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Cell adhesion was found to be necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell-cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process (Dhanyasi, 2015).
Striated muscle thick filaments are composed of myosin II and several non-myosin proteins which define the filament length and modify its function. Myosin II has a globular N-terminal motor domain comprising its catalytic and actin-binding activities and a long α-helical, coiled tail that forms the dense filament backbone. Myosin alone polymerizes into filaments of irregular length, but striated muscle thick filaments have defined lengths that, with thin filaments, define the sarcomere structure. The motor domain structure and function are well understood, but the myosin filament backbone is not. This study reports on the structure of the flight muscle thick filaments from Drosophila melanogaster at 4.7 Å resolution, which eliminates previous ambiguities in non-myosin densities. The full proximal S2 region is resolved, as are the connecting densities between the Ig domains of stretchin-klp. The proteins, flightin, and myofilin are resolved in sufficient detail to build an atomic model based on an AlphaFold prediction. The results suggest a method by which flightin and myofilin cooperate to define the structure of the thick filament and explains a key myosin mutation that affects flightin incorporation. Drosophila is a genetic model organism for which These results can define strategies for functional testing (Yeganeh, 2023).
Guanine nucleotide exchange factors (GEF) of the BRAG subfamily activate small Arf GTPases, which are pivotal regulators of intracellular membrane traffic and actin dynamics. Consequently, BRAG proteins have been implicated to regulate the surface levels of adhesive and signaling receptors. However, not much is known about the mechanism leading to the regulation of these surface proteins. This study found that the Drosophila BRAG GEF Schizo interacts physically with the Abl-interactor (Abi). schizo mutants display severe defects in myoblast fusion during syncytial muscle formation and show increased amounts of the cell adhesion protein N-cadherin. The schizo myoblast fusion phenotype can be rescued by the expression of the Schizo GEF (Sec7) and membrane-binding (pleckstrin homology) domain. Furthermore, the expression of the Sec7-PH domain in a wild-type background decreases the amounts of N-cadherin and impairs myoblast fusion. These findings support the notion that the Sec7-PH domain serves as a constitutive-active form of Schizo. Using a yeast-two hybrid assay, it was shown that the SH3 domain of Abi interacts with the N-terminal region of Schizo. This region is also able to bind to the cytodomain of the cell adhesion molecule N-cadherin. To shed light on the function of Schizo and Abi in N-cadherin removal, epistasis experiments were employed in different developmental contexts of Drosophila. These studies point towards a new model for the regulation of Schizo. It is proposed that the binding of Abi to the N-terminal part of Schizo antagonizes Schizo function to inhibit N-cadherin removal (Lubke, 2021).
Contractile tension is critical for musculoskeletal system development and maintenance. In insects, the muscular force is transmitted to the exoskeleton through the tendon cells and tendon apical extracellular matrix (ECM). In Drosophila, tendon cells were found to secrete Dumpy (Dpy), a zona pellucida domain (ZPD) protein, to form the force-resistant filaments in the exuvial space, anchoring the tendon cells to the pupal cuticle. Dpy undergoes filamentous conversion in response to the tension increment during indirect flight muscle development. Another ZPD protein Quasimodo (Qsm) was found to protect the notum epidermis from collapsing under the muscle tension by enhancing the tensile strength of Dpy filaments. Qsm is co-transported with Dpy in the intracellular vesicles and diffuses into the exuvial space after secretion. Tissue-specific qsm expression rescued the qsm mutant phenotypes in distant tissues, suggesting Qsm can function in a long-range, non-cell-autonomous manner. In the cell culture assay, Qsm interacts with Dpy-ZPD and promotes secretion and polymerization of Dpy-ZPD. The roles of Qsm underlies the positive feedback mechanism of force-dependent organization of Dpy filaments, providing new insights into apical ECM remodeling through the unconventional interaction of ZPD proteins (Chu, 2021).
To ensure locomotion and body stability, the active role of muscle contractions relies on a stereotyped muscle pattern set in place during development. This muscle patterning requires a precise assembly of the muscle fibers with the skeleton via a specialized connective tissue, the tendon. Like in vertebrate limbs, Drosophila leg muscles make connections with specific long tendons that extend through different segments. During the leg disc development, cell precursors of long tendons rearrange and collectively migrate to form a tube-shaped structure. A specific developmental program underlies this unique feature of tendon-like cells in the Drosophila model. This study provides for the first time a transcriptomic profile of leg tendon precursors through fluorescence-based cell sorting. From promising candidates, the Kruppel-like factor Dar1 was identified as a critical actor of leg tendon development. Specifically expressed in the leg tendon precursors, loss of dar1 disrupts actin-rich filopodia formation and tendon elongation. These findings show that Dar1 acts downstream of Stripe and is required to set up the correct number of tendon progenitors (Laurichesse, 2021).
Genes controlling muscle size and survival play important roles in muscle wasting diseases. In Drosophila melanogaster metamorphosis, larval abdominal muscles undergo two developmental fates. While a doomed population is eliminated by cell death, another persistent group is remodelled and survives into adulthood. To identify and characterize genes involved in the development of remodelled muscles, a workflow was developed consisting of in vivo imaging, targeted gene perturbation and quantitative image analysis. Inhibition of TOR signalling and activation of autophagy promote developmental muscle atrophy in early, while TOR and yorkie activation are required for muscle growth in late pupation. Changes were discovered in the localization of myonuclei during remodelling that involve anti-polar migration leading to central clustering followed by polar migration resulting in localization along the midline. The Cathepsin L orthologue Cp1 was demonstrated to be required for myonuclear clustering in mid, while autophagy contributes to central positioning of nuclei in late metamorphosis. In conclusion, studying muscle remodelling in metamorphosis can provide new insights into the cell biology of muscle wasting (Kuleesha, 2016).
Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres. Mutations in many genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue. However, the pathways underlying muscle degeneration, and manifestation of myopathy phenotypes are not well understood. This study explored transcriptional alterations that ensue from the absence of the two major muscle proteins - myosin and actin - using the Drosophila indirect flight muscles. The aim of this study was to understand how the muscle tissue responds as a whole to the absence of either of the major scaffold proteins, whether the responses are generic to the tissue; or unique to the thick versus thin filament systems. The results indicated that muscles respond by altering gene transcriptional levels in multiple systems active in muscle remodelling, protein degradation and heat shock responses. However, there were some responses that were filament-specific signatures of muscle degeneration, like immune responses, metabolic alterations and alterations in expression of muscle structural genes and mitochondrial ribosomal genes. These general and filament-specific changes in gene expression may be of relevance to human myopathies (Madan, 2017).
Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. Previously work has shown that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. This study presents a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching; this method was used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. Body posture was also examined and advantage was taken of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching (Chandel, 2019).
A muscle undergoing cyclical contractions requires fast and efficient muscle activation and relaxation to generate high power with relatively low energetic cost. To enhance activation and increase force levels during shortening, some muscle types have evolved stretch activation (SA), a delayed increased in force following rapid muscle lengthening. SA's complementary phenomenon is shortening deactivation (SD), a delayed decrease in force following muscle shortening. SD increases muscle relaxation, which decreases resistance to subsequent muscle lengthening. Although it might be just as important to cyclical power output, SD has received less investigation than SA. To enable mechanistic investigations into SD and quantitatively compare it to SA, this study developed a protocol to elicit SA and SD from Drosophila and Lethocerus indirect flight muscles (IFM) and Drosophila jump muscle. When normalized to isometric tension, Drosophila IFM exhibited a 118% SD tension decrease, Lethocerus IFM dropped by 97%, and Drosophila jump muscle decreased by 37%. The same order was found for normalized SA tension: Drosophila IFM increased by 233%, Lethocerus IFM by 76%, and Drosophila jump muscle by only 11%. SD occurred slightly earlier than SA, relative to the respective length change, for both IFMs; but SD was exceedingly earlier than SA for jump muscle. These results suggest SA and SD evolved to enable highly efficient IFM cyclical power generation and may be caused by the same mechanism. However, jump muscle SA and SD mechanisms are likely different, and may have evolved for a role other than to increase the power output of cyclical contractions (Loya, 2022).
Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4 short (dNedd4S) and long (dNedd4Lo). DNedd4Lo has a unique N-terminus containing a Pro-rich region. While dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, binding partners to the N-terminal unique region of dNedd4Lo were sought in larval lysates. Mass-spectrometry identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains, which regulates muscle transverse tubule (T-tubule) formation in flies. The interaction was validated by coimmunoprecipitation, and direct binding between dAmph-SH3 domain and dNedd4Lo-N-terminus was demonstrated. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and to impaired T-tubule formation, phenocopying amph null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C->A). It is proposed that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function (Safi, 2016).
The Drosophila melanogaster larval body wall muscles are established during embryogenesis beginning with the invagination of the mesoderm, which spreads along the ectoderm and then forms numerous mesodermal derivatives. Somatic mesodermal specification produces three different types of myoblasts. Fusion of muscle founder cells and fusion-competent myoblasts form the syncytial myotube, which develops into the embryonic and larval body wall muscles. After myoblast fusion, nuclei are positioned correctly throughout the myotube and form connections to surrounding tendon cells to establish the myotendinous junction, which is innervated by motorneurons in a process called neuromuscular (NM) synaptogenesis. The contractile apparatus is then assembled, and muscles begin to contract. During larval stages, the essential muscle pattern created in the embryo does not change, except that the muscles continue to expand along with the growth of the larva. In Drosophila larva, a repeated pattern of 30 unique muscle fibers is present in each abdominal hemisegment, which are innervated by 36 motor neurons. Each muscle fiber is distinguishable by size, shape, orientation, number of nuclei, innervation, and tendon attachment sites. Throughout development, internal and external cues guide muscles to adopt specific properties that allow them to perform particular functions (Safi, 2016).
Ubiquitination is the process of conjugating ubiquitin onto proteins, and it plays an important role in controlling protein degradation/stability, as well as in trafficking, sorting, and endocytosis of transmembrane proteins. The ubiquitination cascade involves three enzymes: E1, E2, and E3, with the last responsible for substrate recognition and ubiquitin transfer, either indirectly (e.g., RING E3 ligases) or directly (Safi, 2016).
Although many proteins are involved in the regulation of NM synaptogenesis in flies, the role of the ubiquitin system in this process is less well characterized. Studies have shown that the RING-family ubiquitin ligase complex Highwire inhibits synapse formation and function by inhibiting the kinase Wallenda/DLK1, the activator of JNK , whereas the deubiquitinating enzyme Fat Facet targets Liquid Facet/Epsin and promotes synaptic growth (Safi, 2016 and references therein).
Neuronal precursor cell expressed developmentally down-regulated 4 (Nedd4) family members belong to the HECT family of E3 ligases and contain a common C2-WW(n)-HECT domain architecture. Drosophila contains a single dNedd4 gene, which undergoes alternative splicing to produce several splice isoforms, including two prominent ones: dNedd4-short (dNedd4S) and dNedd4-long (dNedd4Lo). Differences between the two isoforms of dNedd4 include an alternate start codon site, resulting in a longer N-terminal region in dNedd4Lo, and an extra exon inserted between those encoding WW1 and WW2 domains (Zhong, 2011; Safi, 2016 and references therein).
Previous studies have shown that dNedd4S promotes NM synaptogenesis in flies by interacting and ubiquitinating Commissureless (Comm), which leads to endocytosis of Comm from the muscle surface, a step required for NM synaptogenesis. Whereas dNedd4S is essential for proper NM synaptogenesis, dNedd4Lo inhibits it (Zhong, 2011). Of importance, dNedd4Lo also inhibited normal larval locomotion (Zhong, 2011). These adverse effects of dNedd4Lo were caused by unique N-terminal and Middle regions found in dNedd4Lo (and absent from dNedd4S) and required a functional HECT domain (Zhong, 2011). Of interest, during embryonic muscle development, dNedd4Lo expression is dramatically decreased, whereas that of dNedd4S remains relatively high (Zhong, 2011; Safi, 2016 and references therein).
Because it was observed that the muscle and synaptogenesis defects of dNedd4Lo larvae were not caused by altered phosphorylation of dNedd4Lo, dNedd4Lo-mediated inhibition of catalytic activity of dNedd4S, or diminished effects of dNedd4Lo on Comm endocytosis (Zhong, 2011), it was suspected that dNedd4Lo might inhibit muscle development and/or function by interacting with other proteins via its unique regions (Safi, 2016).
This study identified, using mass spectrometry, Drosophila Amphiphysin (dAmph) as a binding partner of the unique N-terminal region of dNedd4Lo. Amphiphysins are members of the BAR-SH3 domain-containing family of proteins. Mammalian amphiphysin Amph I is involved in endocytosis and synaptic vesicle recycling during neurotransmission by interacting with clathrin/dynamin. In contrast, both mammalian Amph IIb (Bin1) and its fly orthologue, dAmph, are postsynaptic, lack binding sites for clathrin/dynamin, and are not involved in endocytosis. Instead, Bin1 and dAmph regulate transverse tubule (T-tubule) biogenesis in muscles (Safi, 2016).
This study demonstrates that the N-terminus of dNedd4Lo directly binds to dAmph-SH3 domain. In accord, dNedd4Lo and dAmph are colocalized postsynaptically at neuromuscular junctions (NMJs) and muscle transverse tubules (T-tubules). The data show that dNedd4Lo regulates the levels of dAmph postsynaptically and in muscles. Moreover, expression of dNedd4Lo in muscles results in impaired T-tubule formation, phenocopying amph-null mutants. These results demonstrate an important role of dNedd4Lo in regulating T-tubule organization of Drosophila muscles (Safi, 2016).
Previous work has shown that unlike dNedd4 (dNedd4S), muscle-specific overexpression of the dNedd4Lo isoform inhibits NM synaptogenesis and leads to impaired larval locomotion and lethality (Zhong, 2011). This effect required the catalytic activity of dNedd4Lo, since a mutant dNedd4Lo(C->A) with an inactivating mutation in the HECT domain (Cys -> Ala) was not inhibitory. This suggested that the same gene (dNedd4) can encode isoforms with opposite functions. In accord with this, it was observed that during stages of embryonic development when synaptogenesis takes place (14-24 h), dNedd4S expression remains relatively high, whereas that of the inhibitory dNedd4Lo is strongly reduced (Zhong, 2011), suggesting tight regulation of expression of these isoforms to promote muscle development at a very precise time. The inhibitory effect of dNedd4Lo is mediated by two regions unique to dNedd4Lo (N-terminus and Middle region), as deletion of these unique regions alleviated the synaptogenesis defects and increased viability. Therefore it is postulated that the unique regions of dNedd4Lo might negatively regulate NM synaptogenesis and muscle development by targeting specific substrate(s) (Safi, 2016).
This study identified dAmph as a binding partner for the unique N-terminal region of dNedd4Lo by mass spectrometry and validated the interaction in vitro and in vivo in flies. Transiently expressed dAmph coimmunoprecipitates with dNedd4Lo but not with dNedd4S in Drosophila S2 cells and demonstrated direct binding between dAmph-SH3 domain and the dNedd4Lo N-terminus. In vivo results showed that dAmph colocalizes with dNedd4Lo postsynaptically at neuromuscular junctions and muscle T-tubules, where their expression overlaps with the postsynaptic/T-tubule marker, Dlg. Of importance, it was demonstrated that dNedd4Lo expression significantly reduced the levels of dAmph in the postsynaptic region and muscles, an effect not observed in larvae expressing dNedd4S or dNedd4Lo(C->A). As expected, due to the disappearance of endogenous dAmph in larvae expressing dNedd4Lo (and the inability to 'treat' live larvae with proteasome inhibitors), it was not possible to detect ubiquitination of dAmph in these larvae. In addition to biochemical interactions, genetic interactions were also shown between dNedd4Lo and dAmph (Safi, 2016).
The reduction in dAmph levels in the dNedd4Lo-expressing muscles correlated with impaired T-tubule formation, mimicking the phenotype of the amph-null flies. These results could help explain (along with previously described NM synaptogenesis defects; Zhong, 2011) the observed locomotion defects in the dNedd4Lo-overexpressing larvae. At present, it is not possible to quantify the contribution of the T-tubule defects versus the NM synaptogenesis defects to the impaired muscle locomotion/function (Safi, 2016).
Interestingly, it was found that flies overexpressing the dAmph(ΔSH3) mutant in muscles showed reduced localization at the postsynaptic region and T-tubules and no longer colocalized with dNedd4. It is known that the SH3 domain of some membrane-associated proteins is important for their targeting to specific subcellular locations. Similarly, it was found that the SH3 domain of dAmph is also important for its location in the postsynaptic region and the muscle, since the ΔSH3 dAmph protein mislocalized to a region near the muscle plasma membrane. It is not known whether this SH3-dependent localization is related to the ability of this domain to bind dNedd4Lo or due to its interaction(s) with other molecules (Safi, 2016).
Amphiphysin has been implicated in T-tubule biogenesis due to its N-terminal amphipathic helix and BAR domain (N-BAR), which promotes membrane curvature. The BAR domain of the isoform of mammalian amphiphysin 2 (Bin1) is known to be associated with T-tubule formation in skeletal and cardiac muscles, where it induces tubular plasma membrane invaginations. Similar to Bin1, dAmph was shown to participate in plasma membrane remodeling during cleavage furrow ingression, which is required for de novo formation of cells in the Drosophila embryo; the BAR domain of dAmph is required for the formation of endocytic tubules that form at the cleavage furrow tips. This study shows that dNedd4Lo expression reduces the levels of dAmph in the muscle and significantly inhibits T-tubule formation. The degradation of dAmph by dNedd4Lo could impair T-tubule biogenesis by the BAR domain of dAmph, which could help to explain the larval locomotion defects that were observed (Safi, 2016).
Cardiac Bin1 has been implicated in calcium channel trafficking and formation of the inner membrane folds of the cardiac T-tubules. Bin1 localizes to cardiac T-tubules with the L-type calcium channel, Cav1.2, by tethering dynamic microtubules to membrane scaffolds, allowing targeted delivery of Cav1.2 to cardiac T-tubules. Knockdown of Bin1 reduces surface Cav1.2 and delays development of the calcium transient. In cardiomyopathy, decrease in Bin1 alters T-tubule morphology and can cause arrhythmia. Mice with cardiac Bin1 deletion show decreased T-tubule folding, which leads to free diffusion of local extracellular ions, prolonging action-potential duration and increasing susceptibility to arrhythmias. Bin1 is also important for maintenance of intact T-tubule structure and Ca2+ homeostasis in adult skeletal muscle. Adult mouse skeletal muscles with Bin1 knockdown display swollen T-tubule structures, alterations to intracellular Ca2+ release, and compromised coupling between the voltage-gated calcium channel, dihydropyridine receptor (DHPR), and the intracellular calcium channel, ryanodine receptor 1 (Safi, 2016).
Similar to Bin1, dAmph is also required for the organization of the excitation-contraction coupling machinery of muscle. Accordingly, dAmph mutant larvae and flies show defects in T-tubule formation, severe locomotor defects, and flight impairments, indicative of defects in muscle function. Therefore degradation of dAmph in the muscle and inhibition of T-tubule biogenesis by dNedd4Lo could have adverse effects on the localization of T-tubule-associated calcium channels and coupling between the DHPR and ryanodine receptor, as a result altering calcium signaling in muscles. Efficient intracellular Ca2+ homeostasis in skeletal muscle requires intact triad junctional complexes comprising T-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Because dNedd4Lo expression significantly reduced T-tubule projections, this would likely impair intracellular Ca2+ homeostasis and result in locomotor defects (Safi, 2016).
Although there is no direct homologue of dNedd4Lo in species other than Drosophila, the mammalian Nedd4 relative Itch has a proline-rich N-terminal region that binds the SH3 domain of Sorting Nexin 9 . In yeast, Rsp5 (the yeast orthologue of Nedd4 proteins) regulates the Amphiphysin homologue Rvs167 by monoubiquitination of lysine in the SH3 domain of Amphiphysin, demonstrating that Nedd4 family members can interact with SH3 domains, including that of amphiphysin, in other species in addition to flies (Safi, 2016).
In addition to dAmph, this study identified several other interacting partners of the N-terminal and Middle regions of dNedd4Lo that could potentially be targeted by dNedd4Lo. Similar to dAmph, two of these proteins, Syndapin (which, like dAmph, bound the unique N-terminus region of dNedd4Lo), and Sorting Nexin 9 (SH3PX1, which bound the unique Middle region of dNedd4Lo), contain BAR and SH3 domains. The SH3 domain-containing protein Cindr/CG31012 (orthologue of the mammalian Cd2ap and Cin85) was also identified as a binding partner to the unique N-terminus of dNedd4Lo. It is not known whether these proteins are bone fide substrates of dNedd4Lo or contribute to the NMJ and T-tubule defects caused by overexpression of dNedd4Lo in the muscle during development (Safi, 2016).
In conclusion, the severely reduced locomotion activity of larvae overexpressing dNedd4Lo in the muscle may be explained by both impaired neuromuscular synaptogenesis, which has been demonstrated previously (Zhong, 2011), and by impaired T-tubule formation as a result of dAmph degradation by dNedd4Lo, which is shown in this study. The defective T-tubule branching would likely impair coupling between the DHPR and ryanodine receptor, possibly by affecting the localization of calcium channels to the T-tubule network. Reduced surface calcium channels on the T-tubule network would alter calcium homeostasis and compromise excitation and contraction coupling, causing larval locomotor defects (Safi, 2016).
Work on genetic model systems such as Drosophila and mouse has shown that the fundamental mechanisms of myogenesis are remarkably similar in vertebrates and invertebrates. Strikingly, however, satellite cells, the adult muscle stem cells that are essential for the regeneration of damaged muscles in vertebrates, have not been reported in invertebrates. This study shows that lineal descendants of muscle stem cells are present in adult muscle of Drosophila as small, unfused cells observed at the surface and in close proximity to the mature muscle fibers. Normally quiescent, following muscle fiber injury, these cells express Zfh1 and engage in Notch-Delta-dependent proliferative activity and generate lineal descendant populations, which fuse with the injured muscle fiber. In view of strikingly similar morphological and functional features, these novel cells are considered to be the Drosophila equivalent of vertebrate muscle satellite cells (Chaturvedi, 2017).
The TET (Ten-eleven translocation) 1, 2 and 3 proteins have been shown to function as DNA hydroxymethylases in vertebrates and their requirements have been documented extensively. Recently, the Tet proteins have been shown to also hydroxylate 5-methylcytosine in RNA. 5-hydroxymethylcytosine (5hmrC) is enriched in messenger RNA but the function of this modification has yet to be elucidated. Because Cytosine methylation in DNA is barely detectable in Drosophila, it serves as an ideal model to study the biological function of 5hmrC. This study characterized the temporal and spatial expression and requirement of Tet throughout Drosophila development. Tet was shown to be essential for viability as Tet complete loss-of-function animals die at the late pupal stage. Tet is highly expressed in neuronal tissues and at more moderate levels in somatic muscle precursors in embryos and larvae. Depletion of Tet in muscle precursors at early embryonic stages leads to defects in larval locomotion and late pupal lethality. Although Tet knock-down in neuronal tissue does not cause lethality, it is essential for neuronal function during development through its affects upon locomotion in larvae and the circadian rhythm of adult flies. Further, the function of Tet in ovarian morphogenesis is reported. Together, these findings provide basic insights into the biological function of Tet in Drosophila, and may illuminate observed neuronal and muscle phenotypes observed in vertebrates (Wang, 2018).
In Drosophila melanogaster, the receptor tyrosine kinase (RTK) Anaplastic lymphoma kinase (Alk) and its ligand Jelly belly (Jeb) are required to specify muscle founder cells in
the visceral mesoderm. This study identified a critical role for the
scaffolding protein Cnk (Connector
enhancer of kinase suppressor of Ras) in this signaling pathway.
Embryos that ectopically expressed the minimal Alk interaction region in
the carboxyl terminus of Cnk or lacked maternal and zygotic cnk
did not generate visceral founder cells or a functional gut musculature,
phenotypes that resemble those of jeb and Alk mutants.
Deletion of the entire Alk-interacting region in the cnk locus
affected the Alk signaling pathway in the visceral mesoderm and not
other RTK signaling pathways in other tissues. In addition, the
Cnk-interacting protein Aveugle (Ave)
was shown to be critical for Alk signaling in the developing visceral mesoderm. Alk
signaling stimulates the MAPK/ERK pathway, but the scaffolding protein
Ksr, which facilitates activation of this pathway, was not required to
promote visceral founder cell specification. Thus, Cnk and Ave represent
critical molecules downstream of Alk, and their loss genocopies the lack
of visceral founder cell specification of Alk and jeb
mutants, indicating their essential roles in Alk signaling (Wolfstetter, 2017).
Receptor tyrosine kinase (RTK) signaling plays an essential role in development by transducing external signals into the nucleus and other cellular compartments, thereby altering gene expression and promoting intracellular responses. The hallmarks of RTK signaling are conserved among eukaryotic organisms and involve ligand-dependent activation of a transmembrane receptor protein tyrosine kinase and the recruitment of canonical intracellular signaling modules and cascades, such as the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Alk activation stimulates this pathway through the guanosine triphosphatase Ras and the serine-threonine kinases Raf [a MAPK kinase kinase (MAPKKK)], MEK (a MAPK kinase), and MAPK/ERK. Other factors that contribute to or modulate the activity of this pathway have been identified, such as the kinase suppressor of Ras (Ksr), which was identified by mutagenesis screens in Ras-sensitized genetic backgrounds in Drosophila melanogaster and Caenorhabditis elegans. Because of inconsistent findings regarding the catalytic activity of its kinase domain, the role of Ksr has remained controversial. Different models have proposed distinct roles for Ksr as an activator of Raf in parallel to or downstream of Ras or as a scaffolding protein for the assembly of Raf-MEK protein complexes. There is no evidence for a direct interaction between Ksr and Ras, but dimerization between Ksr and Raf can stimulate Raf activity in a manner that is independent of the kinase activity of Ksr, suggesting that Ksr may act as a scaffold in the context of RTK signaling (Wolfstetter, 2017).
A genetic modifier screen using ectopic expression of a dominant-negative, chimeric version of Ksr in the fly eye led to the identification of another critical factor for Ras-ERK signaling named Connector enhancer of kinase suppressor of Ras (Cnk). The cnk locus encodes a large protein of 1557 amino acids containing an N-terminal sterile α motif (SAM), followed by a conserved region in Cnk (CRIC), a PDZ domain, proline-rich motifs, and a pleckstrin homology (PH) domain. The protein structure suggests that Cnk acts as a multidomain protein scaffold. Like Ksr, Cnk functions downstream of various RTK signaling events including epidermal growth factor receptor (EGFR)-tribution of Cnk to embryonic Torso signaling, supporting the finding - and EGFR-dependent air sac development in the dorsal thorax. Ectopic expression of the Cnk N-terminal region enhances the effects of activated RasV12 independently of MAPK/ERK activation in the Drosophila eye. The C-terminal region contains a Raf inhibitory region (RIR) that binds to and represses Raf, which is released upon phosphorylation of Cnk by the Src family kinase Src42A. Thus, Cnk functions as a molecular scaffold to support Ksr-mediated Raf activation and to recruit and integrate additional signaling components such as Src42A (Wolfstetter, 2017).
During embryonic development in D. melanogaster, the visceral mesoderm (VM) gives rise to a lattice of midgut muscles that ensheaths the larval midgut. The VM consists of naïve myoblasts that become specified as either founder cells (FCs) or fusion competent myoblasts (FCMs). Subsequently, the FCs fuse one-to-one with FCMs and eventually form the binucleate visceral myotubes. Specification of VM cells requires the Drosophila ortholog of the receptor anaplastic lymphoma kinase (ALK), initially identified as part of a chimeric protein created by the 2;5 (p23:q35) translocation in human anaplastic large cell lymphoma cell lines. Drosophila Alk is expressed in the segmental clusters of the embryo that segregate from the dorsal trunk mesoderm to form the VM. Alk protein can be detected at the membrane of all VM cells, but only the distal arch within each cluster comes into direct contact with a secreted, small low-density lipoprotein domain ligand named Jelly belly (Jeb). Binding of Jeb to the extracellular part of Alk activates a downstream signaling cascade that results in ERK phosphorylation and triggers expression of an FC-specific subset of genes including Hand, optomotor-blind-related-gene-1 (org-1), and kin of irre (kirre; also referred to as dumbfounded or duf). Jeb-Alk signaling is crucial for visceral myoblasts to commit to the FC fate. In the absence of either ligand or receptor, neither ERK phosphorylation nor the expression of FC-specific marker genes in the VM occurs. Moreover, visceral cells fail to undergo myoblast fusion, and the VM subsequently disintegrates in jeb and Alk mutant embryos (Wolfstetter, 2017).
This study has identified the multidomain scaffolding protein Cnk as a potential Alk binding partner and essential component in the Alk signaling pathway. Cnk bound to the intracellular part of Alk by its C-terminal region. Loss of cnk function or expression of dominant-negative cnk constructs in Drosophila interfered with Alk signaling in multiple developmental contexts. Moreover, germline clone-derived embryos lacking maternal and zygotic Cnk failed to specify visceral FCs and did not develop a functional midgut. In agreement with its proposed function, epistasis experiments revealed that Cnk operates between Ras and Raf in the Alk signaling pathway. Further targeted deletion of a minimal Alk interaction region (AIR) in Cnk resulted in a specific decrease of Jeb-Alk-induced ERK phosphorylation within the visceral FC row. Deletion of the larger Alk interacting region blocked specification of visceral FCs in response to Alk activation. Although the SAM domain containing Cnk binding partner Aveugle (Ave) was essential for Alk signaling, it was found that Ksr is not essentially required to drive Alk signaling in the developing VM. Thus, Cnk and its binding partner Ave serve as critical components for Alk signaling in Drosophila (Wolfstetter, 2017).
This study uncovered an essential function for the protein scaffold Cnk in Alk signaling. The identification of multiple Cnk preys as AlkICD interactors in the Y2H analysis revealed a region in Cnk that likely mediates this interaction and allowed definition of a minimal AIR that was sufficient to bind Alk. The importance of Cnk in Alk signaling was supported by the loss of FCs in the VM of germline clone-derived cnk mutants [cnk (m-/z-)], which genocopied the embryonic Alk loss-of-function phenotype and the dominant-negative effect of ectopic CnkAIR expression on visceral FC specification. The tissue-specific decrease of ERK phosphorylation in the visceral FC row of cnkΔAIR mutants and the loss of visceral FCs upon deletion of the entire Alk interacting region identified by the Y2H approach further support a direct interaction between Cnk and Alk (Wolfstetter, 2017).
Various interactions between Cnk and membrane-associated factors (although not other RTKs) have been reported. In cultured mammalian cells, CNK1 promotes insulin signaling by binding to and localizing cytohesins at the plasma membrane, and binding of CNK1 to the transmembrane ligand EphrinB1 links fibronectin-mediated cell adhesion to EphrinB-associated JNK signaling. Moreover, mammalian CNK2 (also called MAGUIN), the mammalian CNK homolog most similar to Drosophila Cnk, binds to various members of the membrane-associated MAGUK family proteins and Densin-180. It will be interesting to determine whether the RTK binding capacities of Cnk are limited to Alk (Wolfstetter, 2017).
Cnk localizes close to the plasma membrane. Although the Alk-Cnk interaction appears to be important for ERK activation and visceral FC specification in the VM, Alk does not appear to be required for the subcellular localization of Cnk. Whether the Alk-Cnk interaction depends on Alk activity, potentially resulting in posttranslational modification of Cnk, will be interesting to pursue in further studies. Notably, Drosophila Cnk is tyrosine-phosphorylated upon coexpression with the activated form of the RTK Sevenless (SEVS11) in S2 cells, and activation of the platelet-derived growth factor receptor induces tyrosine phosphorylation of mammalian CNK1, leading to changes in CNK1 subcellular localization (Wolfstetter, 2017).
Cnk is generally abundant in the Drosophila embryo where its function is required in various RTK signaling pathways. However, the CnkAIR appears to be critical only for Alk signaling. Morphological analysis further reveals a minor contribution of Cnk to embryonic Torso signaling, supporting the finding that Torso signals are processed by three or more parallel branches. This finding also agrees with earlier analyses of embryonic Torso signaling in avem-/z- mutants, which form terminally derived structures. Notably, the differences in tracheal phenotypes caused by the cnk63F null allele and the strong, Raf-repressing cnksag alleles, which have C-terminal nonsense mutations and abrogate Alk signaling in the VM, indicate that distinct Cnk domains are involved in specific RTK-signaling pathways. This notion is also supported by the observation that the strong, Heartless-related phenotypes exhibited by cnkm-/z- null mutants were barely apparent in cnkΔY2H m-/z- embryos, which, on the other hand, displayed a complete loss of Alk-driven FC specification in the VM (Wolfstetter, 2017).
Selectivity for a requirement of CNK by different RTKs has also been observed in mammalian cells because CNK2 appears to be required for nerve growth factor, but not EGF-induced ERK activation in PC12 cells. Therefore, Cnk contributes to multiple signaling events, but its importance to different RTKs varies, perhaps reflecting differential wiring of downstream signaling in different developmental processes, an aspect that will be interesting to explore in future studies.
Cnk has been described as a protein scaffold that facilitates Ras-Raf-MAPK signaling at the plasma membrane, allowing signal integration to enhance Raf and MAPK activation. The epistatic analysis presented in this study shows that Cnk is required downstream of activated Alk and RasV12 but upstream of activated Raf in the VM, which agrees with previous studies in Drosophila. Thus, activated Ras seems to require Cnk to transmit signaling to Raf in the VM (Wolfstetter, 2017).
Alk activation at the membrane of a prospective visceral FC by the ligand Jeb, which is secreted from the neighboring somatic mesoderm, induces the Raf/MAPK/ERK signaling cascade, eventually leading to the transcriptional activation of downstream targets including kirre, org-1, and Hand. Cnk and Ave are core components of the Alk signaling pathway that are required downstream of the receptor and upstream of Raf to mediate visceral FC specification. Although not essential for Jeb-Alk signaling, Ksr appears to be required for full activation of ERK (Wolfstetter, 2017).
Ave directly binds to Cnk through an interface formed by their SAM domains. This interaction is thought to be necessary to recruit Ksr to a complex that in turn promotes Raf activation in the presence of activated Ras. Although this study identified Ave as a critical component for Cnk function downstream of Alk, the single Ksr in Drosophila was not required for Alk-mediated FC specification. Cnk was originally identified in a Ksr-dependent genetic screen in Drosophila, and its function has been proposed to mediate the association between Ksr and Raf, suggesting that Ksr should also play an important role in Alk signaling. However, the role of Ksr is unclear, with early reports suggesting an inhibitory function rather than an activating potential in RTK signaling). Ksr requires the presence of additional factors such as 14-3-3 proteins or activated Ras , and loss of Ksr-1 suppresses RasE13-induced but not wild-type signaling during C. elegans vulva formation, suggesting altered affinities of Ksr for different variants of Ras. Because this analyses revealed a function for ksr in driving robust ERK phosphorylation, it is plausible that, although nonessential, Ksr might enhance Alk signaling by integrating signals from activated Ras to Cnk-associated Raf (Wolfstetter, 2017).
The importance of ERK activation in RTK-mediated signaling in the Drosophila embryo is difficult to address directly because the rolled locus (encoding the only MAPK/ERK1-2 ortholog in Drosophila) is located close to the centromere and therefore not accessible for standard germline clone analysis. The removal of the 42-amino acid AIR from Cnk by genomic editing of the cnk locus suggests that ERK phosphorylation, even at decreased amounts, is sufficient to promote Alk-induced specification of visceral FCs in vivo. Whereas the AIR is sufficient to mediate the Alk-Cnk interaction in vitro, the ability of the cnkΔAIR mutant to support Alk-driven FC specification in the developing embryo highlights additional requirements. The Y2H analysis supports a direct interaction between Alk and Cnk mediated by a more extensive binding interface within the Alk-Y2H binding region of Cnk. Accordingly, deletion of this region in the cnk locus (cnkΔY2H) blocks VM specification in vivo. However, the possibility cannot be excluded that the Y2H region of Cnk may form interactions with additional Alk binding proteins, which could mediate the Alk-Cnk interaction in an indirect manner (Wolfstetter, 2017).
In summary, this study has identified an interaction between Alk and Cnk mediated by an Alk binding region in Cnk. This region was specifically required for the activation of ERK and formation of FCs downstream of the activated Alk receptor in the Drosophila VM. Together with Ave, Cnk represents an important signaling module that is required for Alk-mediated signaling during embryogenesis. Cnk and Ave represent molecules identified downstream of Alk, whose loss genocopies the lack of visceral FC specification of Alk and jeb mutants. Further work should allow a better understanding of the importance of Cnk in Alk signaling and whether this is conserved in mammalian systems (Wolfstetter, 2017).
Development of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. This study used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). This study confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, this paper reports a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterizes the role of Kah in the regulation of embryonic midgut morphogenesis (Mendoza-Garcia, 2021).
Specification of FCs in the VM depends on Alk signaling in response to Jeb secretion from the somatic mesoderm Signaling via Alk activates the Ras/MAPK pathway, translocating the FCM fate-promoting TF Lameduck (Lmd) from the nucleus to the cytoplasm. A similar mechanism has been suggested for a still unknown FC-fate repressor triggering the FC-specific transcriptional program in the VM. This transcriptional program remains relatively unexplored with only a few identified targets reported, such as Hand, org-1, kirre, dpp and Alk itself. Although ChIP has been the predominant approach for mapping protein-chromatin interactions, it requires significant amounts of starting material and specific antibodies. RNA-seq has also been intensely employed for transcriptomic analyses and, although straightforward for cell culture studies, isolation of the VM would be required for its use in identifying Alk transcriptional targets in Drosophila . Therefore, in efforts to identify novel Alk transcriptional targets in the VM, TaDa was used, allowing genome-wide RNA PolII occupancy to be investigated in the specific tissue of choice. TaDa requires less starting material and provides cell-type specificity, although resolution can be less accurate compared with RNA-seq and ChIP-seq due to its dependency on frequency of GATC sites in the genome (Mendoza-Garcia, 2021).
The experimental design used in this study was based on either activating or inhibiting Alk signaling throughout the VM, followed by TaDa analysis. Comparison of the TaDa dataset with previously published RNA-seq data from cells isolated from the mesoderm suggest the data recapitulated endogenous binding of RNA PolII. The dataset also agreed with current understanding of Alk signaling and induction of cell fate specification in the trunk VM, including observed differential expression of previously identified Alk transcriptional targets, such as Hand, org-1, kirre and dpp . However, these FC-enriched targets were not the most significantly expressed genes within the TaDa dataset, perhaps reflecting lower levels or smaller temporal windows of active transcription that may additionally be complicated by differentially stable mRNA or protein products. Taken together, a combination of different analyses supported this approach as replicating transcriptional events in the VM and led to validation of TaDa-identified genes in the VM as targets of Alk-driven signaling events (Mendoza-Garcia, 2021).
A number of differentially expressed genes were validated by in situ hybridization during embryogenesis. mRNA was indeed visualized in the visceral musculature, and VM expression was confirmed in the HandC-GFP scRNA-seq dataset for fax, Sumo and CG11658. Expression of fax was observed in the VM and CNS. Interestingly, Fax has been identified in a screen for diet-regulated proteins in the Drosophila ovary. Insulin signaling in response to diet promotes activation of the Ribosomal protein S6 Kinase (S6K), which drives fax expression. Notably, Alk modulates insulin signaling in the brain during nutrient restriction, making Fax interesting for further study. Another interesting candidate is Sumo, which encodes the Drosophila SUMO-1 homologue. Sumo is not exclusively expressed in the VM, but is rather expressed maternally and ubiquitously throughout the embryo. Functional studies have identified a role for Sumo in the post-translational modification of several TFs, as well as in modulation of signaling in the fly. Overall, the TaDa analysis identified numerous uncharacterized genes, and further investigation will be crucial to decipher their role in the developing visceral muscles (Mendoza-Garcia, 2021).
One interesting uncharacterized target was Kah, which encodes a Snail family TF. Kah overexpression in the thorax has been reported to block development of thoracic bristles, revealing a potential to drive changes in cell identity (Singari, 2014). It was possible to validate Kah as an Alk target locus in the embryonic VM, with differences in Kah expression when Alk signaling was either blocked or activated. However, Alk-independent Kah transcription in the early VM was noted in addition to the Alk-modulated transcription that is reminiscent of the VM expression reported for org-1. Currently, the TFs downstream of Alk that regulate Kah transcription are unknown, although this will be interesting to study in the future (Mendoza-Garcia, 2021).
Alk signaling in the VM drives FC specification via Ras/MAPK pathway activation, leading to the transcription of FC-specific genes such as Hand, org-1, kirre, dpp and also Kah. Loss of Hand, org-1 and dpp does not alter FC specification in the VM, suggesting a complex temporal regulation that assures FC specification and eventually formation of visceral muscles. Characterization of Kah mutant alleles suggest that, similar to Hand and org-1, Kah is dispensable for VM FC specification, although formally Kah could be responsible for FC-specific transcriptional changes of yet unidentified targets (Mendoza-Garcia, 2021).
Kah mutants exhibit defects in midgut constriction formation. A number of players are implicated in this event, such as Wg, Dpp, Ultrabithorax (Ubx), Pnt, Extra macrochaetae (Emc) and Org-1. Interestingly, Alk signaling activity is important for VM Dpp expression and maintenance of Org-1 in FCs. Although it is difficult to define a role for Alk in VM events post FC specification, this study shows that Alk may indeed play a role in later midgut development. It is also noted that inappropriate activation of Alk signaling via Jeb expression results in a range of later gut defects. Thus, perturbation of Alk signaling appears to disrupt later events in midgut constriction. However, whether this is a consequence of earlier, Alk-dependent, visceral FC specification or refers to an independent role of Alk signaling in the VM is unclear. This study was particularly interested in Pnt, as this ETS domain TF has been reported by the FlyBi-project to bind Kah in high-throughput Y2H, and pnt mutant embryos also exhibit a midgut constriction phenotype. Like Kah, Pnt is not required for VM FC specification. The observation of low penetrance midgut constriction defects in transheterozygous KahΔATG/pntΔ88 embryos suggest that Kah and Pnt may function together in this process, which is further supported by live imaging, through which stronger midgut constriction phenotypes were observed in Kah, pntΔ88 double mutants than in pntΔ88 alone. Employing publicly available Kah-ChIP datasets, it was possible to define a Kah-binding motif similar to that of Snail. Analysis of publicly available Pnt-ChIP datasets identified numerous targets of both Kah and Pnt, including Kah itself, nej, put, Mad and Ras85D. Interestingly, although several components of the Dpp signaling appear to be misregulated in Kah mutants, wg and dpp expression appear normal, and robust pMAD activity is observed in both KahΔATG and KahΔZnF mutants. Earlier work reported that wg and dpp expression are also normal in the VM of pnt mutant embryos. It is likely that as yet unidentified players function downstream of Kah in this process, and it was not yet possible to identify a key component downstream of Kah that could explain the underlying molecular mechanisms. It is also important to note that, although this work focused on a role in the VM, Kah is also expressed in the embryonic SM and CNS. This analysis of Kah mutant RNA-seq together with Kah-ChIP and Pnt-ChIP datasets identifies candidates to be focused on in future studies (Mendoza-Garcia, 2021).
The TaDa approach successfully allowed identification of transcriptional targets of Alk signaling in the developing mesoderm, including the transcriptional regulator Kahuli. Many of these targets are currently uncharacterized and future studies should allow their function(s) in the VM to be elucidated. The in-depth study of Kah highlights a role for this TF in later visceral musculature development, where it appears to work in concert with other factors, including Pnt, to regulate midgut constriction. Combined ChIP and RNA-seq analyses highlights a group of interesting and largely uncharacterized genes, which should shed further light on the midgut constriction process (Mendoza-Garcia, 2021).
Tissue homeostasis often requires a properly placed niche to support stem cells. Morphogenetic processes that position a niche are just being described. For the Drosophila testis, recent work showed that pro-niche cells, specified at disparate positions during early gonadogenesis, must assemble into one collective at the anterior of the gonad. Slit and FGF signals emanating from adjacent visceral mesoderm regulate assembly. In response to signaling, niche cells express islet, which was found to be also required for niche assembly. Without signaling, niche cells specified furthest from the anterior are unable to migrate, remaining dispersed. The function of such niches is severely disrupted, with niche cells evading cell cycle quiescence, compromised in their ability to signal the incipient stem cell pool, and failing to orient stem cell divisions properly. This work identifies both extrinsic signaling and intrinsic responses required for proper assembly and placement of the testis niche (Anllo, 2022).
Mutations in the human N-glycanase 1 (NGLY1) cause a rare, multisystem congenital disorder with global developmental delay. However, the mechanisms by which NGLY1 and its homologs regulate embryonic development are not known. This study shows that Drosophila Pngl encodes an N-glycanase and exhibits a high degree of functional conservation with human NGLY1. Loss of Pngl results in developmental midgut defects reminiscent of midgut-specific loss of BMP signaling. Pngl mutant larvae also exhibit a severe midgut clearance defect, which cannot be fully explained by impaired BMP signaling. Genetic experiments indicate that Pngl is primarily required in the mesoderm during Drosophila development. Loss of Pngl results in a severe decrease in the level of Dpp homodimers and abolishes BMP autoregulation in the visceral mesoderm mediated by Dpp and Tkv homodimers. Thus, these studies uncover a novel mechanism for the tissue-specific regulation of an evolutionarily conserved signaling pathway by an N-glycanase enzyme (Galeone, 2017).
The broad phenotypes of children affected with NGLY1 deficiency and the semi-lethality of Pngl-/- flies indicate that NGLY1 plays important roles during animal development. However, the N-glycanase function has not been linked to any developmental signaling pathway. This study reports that fly Pngl regulates BMP signaling during embryonic midgut development without affecting BMP signaling in ectodermal and head regions of the embryo. The data indicate that Pngl is not required in the midgut endoderm to receive the BMP signal, but rather is required in the visceral mesoderm (VM) to send the BMP signal. It has previously been shown that BMP signaling uses a paracrine/autocrine loop in the VM to sustain and increase the expression of Dpp in PS3 and PS7 of embryonic VM. This loop is proposed to ensure that the level of BMP ligands in the VM is high enough to induce signaling in the endoderm and to specify gastric caeca, the second midgut constriction and the acid zone. Several lines of evidence indicate that the BMP autoregulation mediated by the para-autocrine loop in the VM is the step which is impaired in Pngl-deficient embryos. First, Pngl is not required for the initial, Ubx-dependent expression of dpp. In fact, even a 50% decrease in the expression of dpp in the visceral mesoderm of dpps2/+ animals does not impair BMP autoactivation and midgut development. Second, despite expressing Dpp at early stages, BMP signaling is not activated in Pngl-/- VM, as evidenced by the lack of pMad staining. Third, overexpression of Dpp-GFP in the mesoderm is able to induce BMP signaling in the endoderm in Pngl-/- embryos. Lastly, bypassing the para-autocrine loop by transgenic expression of a constitutively active BMP receptor in the mesoderm results in restoration of BMP signaling in PS3 and PS7 regions of the endoderm and in partial rescue of lethality in Pngl-/- embryos (Galeone, 2017).
In the BMP para-autocrine loop, VM cells both secrete the BMP ligand and respond to it. Therefore, theoretically, Pngl might play a critical role in sending the BMP signal, receiving the BMP signal, or both. Although the data do not allow exclusion of any of these possibilities, based on the following observations, a scenario is favored in which Pngl is required in VM cells to send the Dpp signal not to receive it: (1) Pngl is not required to receive the BMP signal in the endoderm; (2) Loss of Pngl and Pngl KD result in a dramatic decrease in the level of Dpp homodimers and the Dpp-positive puncta; (3) Expression of a constitutively active form of Tkv in the mesoderm is able to restore midgut pMad staining in embryos and the copper cell region in the adult midgut, and partially rescue the lethality of Pngl-/- animals; (4) Loss of Pngl almost fully suppresses the aberrant BMP signaling caused by mesodermal overexpression of Dpp-GFP (Galeone, 2017).
Whole larval protein extracts from Pngl-deficient animals show an increase in the level of the monomeric forms of Dpp (full-length and a cleavage product) and a simultaneous decrease in the bands corresponding in size to Dpp dimers. Moreover, Pngl-/- embryos show a decrease in Dpp-positive puncta both in the mesoderm, where signaling is impaired, and in the ectoderm, where signaling is not impaired. Together, these observations indicate that the effect of loss of Pngl on the Dpp protein itself is not limited to the mesoderm. Indeed, protein extracts from Pngl-deficient midgut and carcass (without midgut) both show a decrease in Dpp dimer levels. This suggests that either Pngl regulates BMP signaling by affecting Dpp dimer levels in other larval tissues not identified yet, or that Dpp dimers are only important in the midgut and although they are decreased elsewhere, Dpp-Gbb heterodimers compensate for the lack of Dpp dimers in most other tissues. Regardless, it is proposed that loss of BMP signaling in Pngl mutant midguts results from a requirement for Dpp homodimers in the para-autocrine autoregulatory loop present in the visceral mesoderm (Galeone, 2017).
BMP ligands can signal both as homodimers and as heterodimers. In vitro and in vivo studies have shown that in general, BMP heterodimers have stronger bioactivity than their homodimers counterparts. In some cases, the homodimers induce weak to moderate signaling, and in other cases they either do not elicit signaling or even play an antagonistic role. Stronger activity of BMP heterodimers can at least in part be explained by differential affinities of individual BMP ligands for different BMP receptors, combined with stronger signal transduction by heterodimeric type I receptors compared to homodimers of each type I receptor. For example, in Drosophila, Dpp has a higher affinity for Tkv, whereas the other two ligands-Gbb and Scw-have a higher affinity for Sax. A similar receptor-ligand binding preference has been observed among the vertebrate orthologs. In the embryonic dorsal midline and the wing imaginal disc, Dpp/Scw and Dpp/Gbb heterodimers induce high levels of signaling, respectively, through Tkv/Sax heterodimers. Comparison of the gbb mutant phenotypes in the midgut with those caused by Pngl loss and by dpp KD indicates that Dpp homodimers are the only productive form of ligand in PS7. Moreover, mesodermal KD of tkv severely decreases BMP signaling in PS7, but mesodermal KD of sax not only does not decrease pMad staining in PS7, but also results in an expansion of pMad expression domain in the PS7 region, similar to gbb mutant embryos. Together, these observations strongly support the notion that the BMP autoregulatory loop in the VM, which is essential for the activation of BMP signaling in the endoderm, relies solely on Dpp and Tkv homodimers, and therefore is impaired in Pngl mutants due to the severe decrease in the level of Dpp homodimers in these animals (Galeone, 2017).
Vertebrate and invertebrate BMP proteins and other members of the TGFβ superfamily each harbor several N-linked glycosylation sites, which have been shown to be glycosylated in many cases. Various functional roles have been ascribed to N-glycans on these ligands, including enhancing receptor binding of BMP6, keeping the TGFβ1 ligand in a latent state, and promoting inhibin (α
/β) heterodimer formation at the expense of activin (β/β) homodimer formation. Accordingly, given the significant increase in Dpp monomeric forms and the simultaneous decrease in Dpp dimers upon loss of Pngl, it is possible that Pngl removes one or more N-glycans from Dpp and thereby promotes the formation or the stability of Dpp homodimers. Whether the regulation of Dpp by Pngl is direct or mediated via other proteins will remain to be explored (Galeone, 2017).
In agreement with a previous report, the current data suggest that the lethality of Pngl mutants cannot be fully explained by shortening of the gastric caeca and impairment of BMP signaling in midgut development. Pngl KD with mesodermal drivers leads to a higher degree of lethality compared to dpp KD with the same drivers. Moreover, how24B > PnglRNAi animals show ~70% lethality, even though they do not have gastric caeca defects. Finally, restoring BMP signaling in the midgut by expressing tkvCA only recues the lethality in ~30% of Pngl-/- animals. Phenotypic analysis of Pngl mutants combined with rescue and KD experiments suggest that a failure to properly empty the gut before puparium formation contributes to lethality in these animals. The molecular mechanisms for the food accumulation phenotype and other potential Pngl-/- phenotypes contributing to lethality are still under investigation (Galeone, 2017).
In summary, this work indicates that the fly Pngl is an evolutionarily conserved N-glycanase enzyme necessary to sustain BMP autoactivation in the VM mediated by para-autocrine activity of Dpp homodimers through Tkv homodimers. Although it cannot be. excluded that Pngl plays important roles in other cell types as well, the data indicate that Pngl is primarily required in the mesoderm during midgut development and its loss results in Dpp-dependent and Dpp-independent midgut defects. Given the reports on potential para-autocrine functions of mammalian Dpp homologs and prominent human pathologies associated with dysregulated BMP signaling in ophthalmic, gastrointestinal and musculoskeletal systems, tissue-specific alterations in BMP signaling might contribute to some of the NGLY1 deficiency phenotypes including retinal abnormalities, delayed bone age and osteopenia, small feet and hands, and chronic constipation. Finally, understanding the mechanisms underlying the food accumulation phenotype in Pngl-/- larvae might shed light on the pathophysiology of chronic constipation in NGLY1 deficiency patients (Galeone, 2017).
This study investigated the dynamics of chromatin regulatory landscapes during embryogenesis at single-cell resolution. Using single-cell combinatorial indexing assay for transposase accessible chromatin with sequencing (sci-ATAC-seq), chromatin accessibility was profiled in over 20,000 single nuclei from fixed Drosophila embryos spanning three embryonic stages: stage 5 blastoderm nuclei; 6-8 h after egg laying, to capture a midpoint in embryonic development when major lineages in the mesoderm and ectoderm are specified; and 10-12 h after egg laying, when cells are undergoing terminal differentiation. The results show that there is spatial heterogeneity in the accessibility of the regulatory genome before gastrulation, a feature that aligns with future cell fate, and that nuclei can be temporally ordered along developmental trajectories. During mid-embryogenesis, tissue granularity emerges such that individual cell types can be inferred by their chromatin accessibility while maintaining a signature of their germ layer of origin. Analysis of the data reveals overlapping usage of regulatory elements between cells of the endoderm and non-myogenic mesoderm, suggesting a common developmental program that is reminiscent of the mesendoderm lineage in other species. 30,075 distal regulatory elements were identified that exhibit tissue-specific accessibility. The germ-layer specificity of a subset of these predicted enhancers was validated in transgenic embryos, achieving an accuracy of 90%. Overall, these results demonstrate the power of shotgun single-cell profiling of embryos to resolve dynamic changes in the chromatin landscape during development, and to uncover the cis-regulatory programs of metazoan germ layers and cell types (Cusanovich, 2018).
The Drosophila Hsp67Bc gene encodes a protein belonging to the small heat-shock protein (sHSP) family, identified as the nearest functional ortholog of human HSPB8. The most prominent activity of sHSPs is preventing the irreversible aggregation of various non-native polypeptides. Moreover, they are involved in processes such as development, aging, maintenance of the cytoskeletal architecture and autophagy. In larval muscles Hsp67Bc localizes to the Z- and A-bands, which suggests its role as part of the conserved chaperone complex required for Z-disk maintenance. In addition, Hsp67Bc is present at neuromuscular junctions (NMJs), which implies its involvement in the maintenance of NMJ structure. This study reports the effects of muscle-target overexpression of Drosophila Hsp67Bc hot-spot variants Hsp67BcR126E and Hsp67BcR126N mimicking pathogenic variants of human HSPB8. Depending on the substitutions, a different impact was observed on muscle structure and performance. Expression of Hsp67BcR126E affects larval motility, which may be caused by impairment of mitochondrial respiratory function and/or by NMJ abnormalities manifested by a decrease in the number of synaptic boutons. In contrast, Hsp67BcR126N appears to be an aggregate-prone variant, as reflected in excessive accumulation of mutant proteins and the formation of large aggregates with a lesser impact on muscle structure and performance compared to the Hsp67BcR126E variant (Jablonska, 2018).
Sarcomeres are extremely highly ordered macromolecular assemblies where structural organization is intimately linked to their functionality as contractile units. Although the structural basis of actin and Myosin interaction is revealed at a quasiatomic resolution, much less is known about the molecular organization of the I-band and H-zone. This study reports the development of a powerful nanoscopic approach, combined with a structure-averaging algorithm, that allowed determination of the position of 27 sarcomeric proteins in Drosophila melanogaster flight muscles with a quasimolecular, approximately 5- to 10-nm localization precision. With this protein localization atlas and template-based protein structure modeling, refined I-band and H-zone models were assembled with unparalleled scope and resolution. In addition, it was found that actin regulatory proteins of the H-zone are organized into two distinct layers, suggesting that the major place of thin filament assembly is an M-line-centered narrow domain where short actin oligomers can form and subsequently anneal to the pointed end (Szikora, 2020)
Drosophila underreplicate the DNA of thoracic nuclei, stalling during S phase at a point that is proportional to the total genome size in each species. In polytene tissues, such as the Drosophila salivary glands, all of the nuclei initiate multiple rounds of DNA synthesis and underreplicate. Yet, only half of the nuclei isolated from the thorax stall; the other half do not initiate S phase. To address this problem, flow cytometry was used to compare underreplication phenotypes between thoracic tissues. When individual thoracic tissues are dissected and the proportion of stalled DNA synthesis is scored in each tissue type, it was found that underreplication occurs in the indirect flight muscle, with the majority of underreplicated nuclei in the dorsal longitudinal muscles (DLM). Half of the DNA in the DLM nuclei stall at S phase between the unreplicated G0 and fully replicated G1. The dorsal ventral flight muscle provides the other source of underreplication, and yet, there, the replication stall point is earlier (less DNA replicated), and the endocycle is initiated. The differences in underreplication and ploidy in the indirect flight muscles provide a new tool to study heterochromatin, underreplication and endocycle control (Johnston, 2020).
Developmental progression and cellular diversity are largely driven by transcription factors (TFs); yet, characterizing their loss-of-function phenotypes remains challenging and often disconnected from their underlying molecular mechanisms. This study combined single-cell regulatory genomics with loss-of-function mutants to jointly assess both cellular and molecular phenotypes. Performing sci-ATAC-seq at eight overlapping time points during Drosophila mesoderm development could reconstruct the developmental trajectories of all major muscle types and reveal the TFs and enhancers involved. To systematically assess mutant phenotypes, a single-nucleus genotyping strategy was developed to process embryo pools of mixed genotypes. Applying this to four TF mutants could identify and quantify their characterized phenotypes de novo and discover new ones, while simultaneously revealing their regulatory input and mode of action. This approach is a general framework to dissect the functional input of TFs in a systematic, unbiased manner, identifying both cellular and molecular phenotypes at a scale and resolution that has not been feasible before (Secchia, 2022).
The continuous temporal resolution of the time course was exploited to reconstruct regulatory trajectories for three muscle lineages, starting from unspecified mesodermal cells. Ordering cells along pseudotime revealed extensive and dynamic temporal changes in accessibility for both regulatory elements and genes along each lineage's trajectory. The loci of many upstream identity genes change in accessibility (in both directions) as the development of each lineage progresses. This includes lmd, kah, NK7.1, Pdp1, tx, and nau (dMyoD) in the somatic trajectory, in addition to more downstream effector genes required for differentiated muscle function (Mhc, Mlc, and Tropomyosin). Cardiomyocytes are specified by a highly conserved set of TFs from flies to humans, including members of the NKx2.5 (tinman [tin] in Drosophila), GATA (pannier [pnr]), T-box (Dorsocross-3 [Doc3]), and islet 1 (tailup [tup]) TFs. The dynamic usage of all factors and many more are observed along the cardiac lineage. Both the somatic and visceral muscles are formed from two populations of cells-founder cells (FCs), which give the muscle its identity, and fusion-competent myoblasts (FCMs), which fuse to FCs during differentiation to form a multinucleated syncytium. Two trajectories were observed for the visceral-muscle lineage. Starting from common precursors, one lineage branches toward the somatic body wall muscle, while the other has a distinctive lineage that remains separate from the rest of the muscle populations (Secchia, 2022).
To explore the visceral-muscle lineages further, all annotated visceral-muscle cells from the Mef2+ time course were combined with the Bin+ FAC sorted cells, and these these 4,187 visceral-muscle cells were reclustered separately, revealing a more complex multi-branched structure. The visceral muscle represents a collection of muscles with different developmental origins. The FCs and FCMs of the circular trunk visceral muscle (CVM) are specified at stages 10 and 11 (6-8 h), after which they migrate laterally and undergo myoblast fusion (stage 12) to form a continuous muscle that encloses the gut. The longitudinal VM (LVM) is formed from FCs in the caudal mesoderm toward the end of the embryo, which migrate on top of the circular VM, using it as a scaffold. The single-cell trajectory captures these diverse origins and the temporal delay of the LVM development. The main branch consists of both FCs and FCMs, as seen by their enrichment in markers for visceral-muscle FC specification (including Alk, numb, bap, and bin) and FCM specific genes (lmd and sns). A subset of cells progress to a more differentiated state, expressing specific myoblast-fusion genes in the intermediate state before differentiating to contractile muscle, indicated by the expression of sarcomere protein genes such as MHC, Tmod, Tm1, and Tm2. Given the timing of these transitions and the presence of bap (a TF not expressed in LVM), this likely reflects circular VM development. The LVM and hindgut VM (HVM) branches have different developmental origins, as expected, and are enriched in longitudinal (cluster 5) and hindgut (cluster 4) visceral-muscle markers. The precursor population has a second branch, expressing a broader set of myoblast-fusion genes, perhaps representing a more mature or more diverse FCM population. Some FCMs were proposed to remain unfused and wait for the LVM founder cells to migrate over the circular VM and then fuse during late stage 12 and stage 13 (~10-12 h). The results support this and suggest that a proportion of these cells may also fuse to the overlying somatic-muscle FCs, given their expression of Vrp1/wip and wasp, two genes specific to myoblast fusion of the somatic muscle, and their progressive acquisition of a chromatin state similar to the somatic muscle (Secchia, 2022).
To determine the functional impact of TF mutants on both the developmental trajectories of cells and their regulatory programs, sci-ATAC-seq to was applied to loss-of-function mutant embryos of four mesodermal TFs and assessed the cells' behavior by integrating them with the wild-type developmental trajectory (Figure 1D). Myocyte enhancer factor 2 (Mef2) is essential for myoblast fusion and terminal differentiation of all muscle types. Tinman (Nkx2-5) is essential for the subdivision of the dorsal mesoderm into cardiac and visceral muscle, through the activation of bagpipe (NKx3-2), which initiates biniou (FoxF2) expression and the visceral-muscle lineage. Although the occupancy of each TF has been examined in bulk, their contribution to enhancer accessibility and to an individual cell's state, remains unknown (Secchia, 2022).
Recessive lethal mutants in animal models must be maintained as heterozygotes; therefore, only 25% of offspring embryos are homozygous for the mutation of interest. This study first developed an easy and generalizable approach to profile single-cell genomic measurements from embryos of mixed genotypes. Rather than genotyping and hand sorting homozygous mutant embryos, all embryos were retained representing a pool of three genotypes (homozygous loss-of-function mutant, heterozygous, and homozygous nonmutant), and sci-ATAC-seq was performed on their dissociated pooled nuclei. In Drosophila, such heterozygous mutants are maintained over balancer chromosomes. The genetic background of the loss-of-function mutants and the balancer chromosome was sequenced, allowing each nucleus to be genotyped based on informative SNPs in the sci-ATAC reads. In contrast to standard allelic imbalance studies, this requires relatively few informative reads per nucleus, as described below (Secchia, 2022).
The characterized mutations of these TFs were generated over 20 years ago and will have accumulated additional mutations that could impact chromatin accessibility independently of the TFs' function. To circumvent this, CRISPR-Cas9 editing with single-stranded oligo donors (ssODNs) was used to recreate the characterized loss-of-function mutations for each factor in a common isogenic genetic background. These new alleles were sequence verified and recapitulate the expected characterized muscle phenotypes. As essential factors, they are homozygous lethal, and, importantly, the new alleles do not complement the characterized loss-of-function allele when placed in trans, confirming that the lethality is due to the mutation of the TF and not a CRISPR off-target effect (Secchia, 2022).
To phenotype mutants, staged embryos were collected from the heterozygous adults (mutantCRISPR/balancer chromosome), which were formaldehyde fixed and processed for sci-ATAC-seq. The dissociated nuclei thereby come from a pool of F1 embryos which represent 25% homozygous loss-of-function mutant/mutant, 50% heterozygous mutant/balancer, and 25% homozygous balancer/balancer. After sci-ATAC-seq, each nucleus was genotyped de novo based on the fraction of reads mapping to the mutant or balancer chromosomes. The genotype assignment is based on over 450,000 genetic variants between the balancer and the mutants genetic background. With a median of roughly 1,000 variants covered per cell, it was possible to genotype 99.9% of all theoretically assignable nuclei with high confidence (>0.9 posterior probability). The genotype assignments were very robust, being 98% identical when performing the assigments using two sets of variants identified with a stringent or a more lenient filtering threshold (Secchia, 2022).
De novo single-nucleus genotyping approach has a number of advantages for single-cell profiling of mutants. It eliminates the need, and associated experimental time, to hand select embryos of the correct genotype and is therefore faster and more reliable. Profiling nuclei from homozygous and heterozygous siblings in the same experiment has an additional advantage to aid in batch correction. As the heterozygous nuclei are essentially wild type, they can be used to align mutant data from the same batch to the wild-type reference trajectory, avoiding 'over fitting' by batch aligners of biologically real mutant phenotypes (Secchia, 2022).
Mef2 regulates differentiation of all major muscle types. To determine the functional impact of Mef2 on chromatin accessibility and mesodermal cell fate, sci-ATAC-seq was performed on Mef2 mutant embryos (a pool of homozygous and heterozygous) at 10-12 h (mainly stage 13) during the initiation of terminal muscle differentiation in the somatic and visceral muscle. The de novo genotyping strategy assigned the expected proportion of profiled nuclei as homozygous mutant (expected: 25%, observed: 26%) (Secchia, 2022).
As these experiments were performed on whole embryos, a first round of clustering was performed to identify muscle cells. The profiled 12,926 cells cluster by cell type, rather than by genotype, revealing 8 broad cell states, including one large muscle population. Selecting the muscle cells, genotype and cluster-specific peaks were identified and those 2,567 cells were reclustered. This resulted in five cell clusters with distinct chromatin accessibility, three of which could be identified as somatic, cardiac, and visceral muscle. Digitally genotyped Mef2-/- mutant nuclei are almost completely absent from the somatic-muscle cluster and instead are highly enriched in two additional 'muscle clusters,' which appear close to, but distinct from, somatic-muscle cells (Mutant1 and Mutant2 clusters). Mutant1 is composed of 89%, while Mutant2 56%, Mef2-/- cells. This indicates that in the absence of Mef2, mesodermal cells are unable to establish the regulatory landscape to become somatic muscle, and instead form a new altered state. Cells in the Mutant2 cluster have lower (~2.2-fold) coverage than the other clusters. This may represent cells in a more naive state or undergoing apoptosis, although the clustering of these cells might be driven primarily by their lower coverage. The proportion of homozygous mutant cells is also partially reduced in the visceral muscle, while it is largely unaffected in the heart, at these stages. These tissue-specific differences likely reflect differences in the timing of differentiation between muscle lineages. Terminal differentiation of the somatic and visceral muscle begins after myoblast fusion at ~10-12 h (stage 13), while it cannot occur in the heart until later stages, after dorsal closure is complete at ~13 h (stage 15) (Secchia, 2022).
The somatic and visceral muscle were also the two tissues that displayed the highest relationship between Mef2 binding at 10-12 h and open chromatin signatures (chromVAR scores) in wild-type embryos, suggesting that such scores are a good indicator of the tissues and time points that will most likely be affected by the removal of a TF. For example, while Mef2 is also expressed in the cardiac muscle, Mef2-bound regions are generally less accessible compared with the somatic and visceral muscle and, concordantly, the proportion of mutant cells remains close to normal in the cardiac muscle. However, such computational inference from wild-type embryos cannot predict all mutant phenotypes. While the visceral and somatic muscle display similar accessibility (ChromVar scores) at Mef2-bound regions in wild-type embryos, the mutant analyses revealed clear differences in the response of both tissues to loss of Mef2; mutant cells are almost completely absent in the somatic muscle but only partially reduced in visceral, indicating a difference in Mef2 dependency for accessibility between these two tissues, which was not evident in wild-type conditions (Secchia, 2022).
To experimentally test the accuracy of the de novo genotyping strategy, homozygous mutant embryos were hand sorted from the Mef2 mutant based on a GFP-marked balancer chromosome, and sci-ATAC-seq was performed on these 100% Mef2-/- nuclei. The hand-sorted mutant nuclei show the same properties as the genotyped mutant nuclei; they are absent from the somatic muscle and accumulate in two mutant clusters-mainly in Mutant1, where 88% of the homozygous mutant cells reside, similar to the digital genotyping above. Both the genotyped and hand-sorted homozygous mutant nuclei display the same alterations in chromatin accessibility at individual loci, as shown for two muscle contractile proteins, Mlc1 and Msp300. Both genes have multiple Mef2-bound regions overlapping open chromatin in cells from the somatic cluster, which are almost completely closed in both the digitally genotyped and hand-sorted Mutant1 cells. Concordant gains in accessibility were also observed at regulatory regions in Mef2-/- cells, including the enhancer VT30021, which is embryonically active, but normally not in muscle tissues. This proof of principle indicates that the de novo nuclear genotyping strategy correctly assigns homozygous mutant nuclei (Secchia, 2022).
The whole-embryo single-cell data allowed exploration of whether Mef2 mutant cells adopt another cell state, either from within the mesoderm or another germ layer. To assess this, clusterwise accessibility correlations of the mutant clusters against all cell types (both mesodermal and nonmesodermal cell clusters) were computed in embryos at 10-12 h. Both Mutant1 and Mutant2 are most highly correlated to clusters within the myogenic mesoderm, in particular the somatic muscle, and are clearly separated from the nonmyogenic mesoderm, ectoderm, and endoderm lineages, indicating that these cells are specified to become muscle, but appear blocked in their development. To determine if they are stuck in an earlier myogenic state, the mutant cell data was combined (combining the digitally genotyped and hand-sorted mutant cells, given that they appear identical) and the heterozygous cells with all cells in the wild-type reference trajectory and reclustered the data. The heterozygous cells behave indistinguishably from the reference cells, falling within the expected wild-type populations on the trajectory. In contrast, Mef2-/- Mutant1 and Mutant2 cells cluster separately, off the wild-type muscle trajectory, but roughly at the appropriate 'temporal' time point. If these cells were blocked in their developmental progression, they would be expected to cluster on the trajectory at some earlier time point in muscle development, which is not what was observe. To explore this further, clusterwise accessibility correlations were computed of the mutant and muscle clusters for each time point, which confirmed that both Mutant1 and Mutant2 are progressing to the appropriate developmental stage. This indicates that Mef2 mutant cells are not simply immature muscle cells but rather have developed a new abnormal 'muscle-like' state, which is likely defined by the inactivation or decreased expression of late muscle function genes, combined with the inappropriate activation of nonmuscle enhancers and genes. Taken together, this suggests that Mef2 is not only required as a differentiation factor to regulate the expression of muscle contractile genes, but also to prevent muscle cells from undergoing other cell-state changes (Secchia, 2022).
The same approach was applied to three other loss-of-function mutants for TFs involved in the specification of the dorsal mesoderm (tinman) and its derived visceral muscle (bagpipe and biniou) that forms the gut musculature. These TFs have a hierarchical relationship between them, where Tinman regulates Bagpipe expression at stage 10 (6-8 h), which in turn regulates Biniou expression. To examine the function of these TFs, bagpipe and biniou mutants were assessed at 6-8 h of development, which coincides with the initiation of their expression and with the specification of the visceral muscle (stages 10 and 11). As Tinman acts upstream of both bap and bin, = the time window was shifted 1 h earlier (5-7 h; stage 9, 10) to capture these events. For all three mutants, sci-ATAC-seq was performed on a pool of homozygous and heterozygous embryos, as above. Staged bagpipe and biniou mutant embryos were collected at 6-8 h (late stage 10, mainly stage 11) and the mesodermal population isolated by Mef2 FAC sorting, as in the wild-type trajectory, obtaining high-quality profiles for 6,306 and 5,833 mesodermal cells, respectively. Presorting for the mesodermal population was not possible for tinman, as it regulates Mef2 expression. Therefore sci-ATAC-seq was assessed on whole embryos of tinman mutants and a first round of clustering was performed to identify 6,786 high-quality mesodermal cells (Secchia, 2022).
To assess the fate of the mutant cells, their development was directly compared with the wild-type trajectory by co-clustering the combined mutant data, representing 18,925 homozygous and heterozygous cells, together with the wild-type mesoderm time course, correcting for batch-level effects. Reclustering and reannotation of this joint dataset , representing 40,232 cells, revealed a structure that is generally consistent with the wild-type trajectory. Nuclei from heterozygous mutant cells (+/-) for tinman are present in the cardiac and early visceral-muscle clusters. Similarly, bagpipe and biniou heterozygous mutant cells are present in the visceral mesoderm, spanning both branches, and extending to later stages of embryogenesis (Secchia, 2022).
Examining the homozygous mutant nuclei (-/-) revealed that, in contrast to Mef2, the proportion was significantly lower than the expected 25%, representing 15%, 19%, and 17% for tinman, bagpipe, and biniou, respectively. This indicates that a proportion of homozygous mutant cells is not maintained and likely undergoes apoptosis as they cannot progress in their development. The trajectories of the remaining mutant cells (-/-) are very different from their heterozygous siblings; tinman -/- cells are completely absent from the cardiac lineage and late-stage visceral muscle, with few remaining cells in the early visceral-muscle clusters. Moreover, there is a significant reduction of homozygous mutant cells in late mesoderm stages, which likely represents the dorsal mesoderm. Similarly, the bagpipe and biniou homozygous mutant nuclei are absent from visceral-muscle clusters at later stages of development. The early visceral cells are more prominently affected in tinman mutants and to a lesser extent in bagpipe and biniou mutants, reflecting the hierarchical position of these TFs with Tinman acting upstream of both factors. These findings indicate that the circular VM cells are initially specified in bagpipe and biniou mutant embryos but are blocked from further expansion and differentiation, resulting in a loss of the VM at later stages. Interestingly, the hindgut and longitudinal visceral muscles appear largely unaffected in all three mutants, reflecting their different developmental origin (Secchia, 2022).
This molecular data can therefore phenotype all three mutants de novo, identifying the gross phenotypes described by immunostaining of mutant embryos. In addition, the single-cell approach provides more fine-grained quantitative information on the proportion of missing cells at a given stage, in addition to revealing more subtle phenotypes not previously observed, including a gain of mutant cells in other muscle lineages. For example, there is a significant overrepresentation of tinman mutant cells in the early mesoderm and in the somatic lineage. The removal of these TFs thereby not only results in a loss of tissue (one cell fate) but also a more subtle gain of cells dispersed in other tissues from different mesodermal trajectories, highlighting the plasticity of cell fates within the myogenic mesoderm (Secchia, 2022).
Mef2 is required for chromatin accessibility at its high-affinity sites and for gene expression
Applying single-cell ATAC-seq to TF mutants in the context of a developing embryo allowed exploration of the extent to which such single-cell data can discern regulatory properties of the TF or its enhancers. As a proof of principle, focus was placed on the Mef2-/- mutant cell cluster (Mutant1), as it contains the highest number of mutant cells (943 cells). The somatic-muscle cluster is the closet cell type to Mutant 1. Of the 8,725 accessible regions in both cell clusters, 408 have significant differential accessibility (DA) in Mef2-/- mutant cell. The majority of DA sites have reduced accessibility (67% [274/408]) and reduced sites often have a larger fold-change. Mef2-responsive sites are generally more gene distal, compared with unchanged sites and are overrepresented in muscle enhancers: Mef2 -/- DA regions more frequently overlap (1) characterized muscle enhancers, and (2) two large collections of putative muscle enhancers defined by ChIP and DNase-seq of FACS-sorted muscle cells, compared with non-DA regions (Secchia, 2022).
Ninety percent of the 8,725 accessible regions in either the somatic and/or Mutant1 cluster were identified in bulk DNase-seq from FACS-sorted muscle cells purified at different stages of embryogenesis. While this highlights the quality of the sci-ATAC-seq data, having such information at a single-cell resolution goes beyond the detection of regulatory regions, as it reveals enhancer usage along developmental trajectories of specific cell types and enables a detailed analysis of the functional input of TFs to enhancers. In addition, single-cell data have enhanced the sensitivity to identify regions that change in mutant embryos and enhanced the precision to uncover the cellular context that is susceptible to that change. To demonstrate this, the analysis was repeated in by testing for differential accessibility between nonmutant and mutant cells across the whole embryo and across the whole muscle population, in order to mimic samples profiled by bulk ATAC-seq in either whole embryos or FACS-purified muscle, respectively. Of the 408 DA sites in Mef2 mutants, only 7% (28) and 43% (176) are identified as significantly changed compared with whole-embryo and muscle mimic samples, respectively. The single-cell approach therefore has enhanced sensitivity to reveal regulatory changes that are not discoverable by traditional approaches, unless complex and extensive FAC sorting is used (Secchia, 2022).
To explore the 408 DA sites further, they were first catagorized into Mef2-bound and -unbound sites, using bulk Mef2 ChIP data at multiple time points of embryonic development. Almost half of the DA regions (48%, 197/408) are bound by Mef2 at this stage or earlier in embryogenesis. Mef2-bound DA sites almost exclusively lose accessibility, consisting of 66% of all DA sites with reduced accessibility. In contrast, regions that gain accessibility are generally not bound by Mef2 and involve regions less related to muscle function (Secchia, 2022).
Removal of Mef2 affects the accessibility of only a specific subset (15%) of all Mef2-bound regions at 10-12 h. Mef2-bound sites that are sensitive to, or resistant to, Mef2 removal could depend on either their extent of co-occupancy by other factors or on the affinity of Mef2 binding. To distinguish between these two possibilities, the co-occupancy of ten TFs active in mesoderm was examined. Susceptible sites are generally less frequently occupied by these TFs compared to nonsusceptible sites. The fraction of DA sites tends to decrease as sites are bound by an increasing number of mesodermal TFs. Examining the occupancy of a much larger set of TFs, 280 factors from modERN also shows an inverse relationship between differential chromatin accessibility in Mef2-/- cells and the number of bound TFs: the median number of bound TFs is 3 for the DA class and 40 for non-DA sites. Therefore, sites that require Mef2 for their accessibility tend to be bound by Mef2 alone or with a small number of other factors, perhaps cooperatively, suggesting that these regions are very Mef2 dependent. To investigate this further, the quantitative Mef2 ChIP signal was used as a proxy for Mef2 affinity. Susceptible (DA) sites have significantly higher Mef2 ChIP signal compared with nonsusceptible (non-DA) sites. Moreover, the proportion of DA sites steadily increases as the Mef2 ChIP signal increases, going from 5% of DA sites in the lowest to 35% in the highest ChIP quantile. This indicates that sites bound more strongly by Mef2 are more likely to have reduced chromatin accessibility upon Mef2 removal. Although both classes are occupied by Mef2, susceptible sites have a 2.5-fold enrichment (61% in the DA group versus 17% in the non-DA group) in the presence of a Mef2 motif. These results indicate that Mef2 is required to establish and/or maintain chromatin accessibility at a large fraction of its high-affinity sites (Secchia, 2022).
Many of these regions overlap characterized or putative muscle enhancers. The loss of accessibility at these sites may therefore lead to changes in the expression of mesoderm/muscle genes, which most likely contributes to the mutant phenotype. To examine this, bulk RNA-expression data was integrated from Mef2 mutant embryos, and genes were sought with a Mef2-bound site in their vicinity (defined as 5 kb upstream and intronic regions). Using this metric, 1,705 differentially expressed genes are associated with at least one Mef2-bound open chromatin region. Of these, those with significantly downregulated, but not upregulated, expression in Mef2 -/- mutants are highly overrepresented for a loss or reduction in chromatin accessibility in at least one of their Mef2-bound associated peaks. Moreover, genes with reduced Mef2-bound sites have significantly stronger changes in both their chromatin accessibility and gene expression, compared with genes with unchanged Mef2-bound sites. Many known Mef2 target genes are among this set, including Mhc, Mlc1/2, Tm1, Mp20, Mlp60A, and Msp300. In addition, their expression changes become more severe with increasing numbers of associated regulatory regions with reduced accessibility. These findings indicate that Mef2 functions primarily as an activator and as the predominant regulator for the expression of these genes, which in turn likely leads to the muscle defects in Mef2 mutant embryos. It is also a rare example demonstrating that a single TF can affect the regulation of many genes by having a cumulative effect on their expression through the action of multiple dependent enhancers (Secchia, 2022).
This paper presents a general framework to obtain a fine-grained view of TF function at both a cellular and molecular level using a systematic, unbiased approach. Phenotypes of developmental mutants are typically assessed by immunostaining with tissue markers and often described in qualitative and somewhat arbitrary terms. There are many examples where other phenotypes were missed as the tissue was outside the interests or scope of the study, and in some cases, suitable tissue markers were not available because they are downstream of the mutated TF. When they are, translating such coarse-grained tissue defects to the underlying molecular function of the TF remains a challenge, and typically the regulatory input is only assessed by occupancy in wild-type embryos compared with gene-expression changes in the mutant. This study shows how single-cell regulatory trajectories, obtained by a dense time course of developmental stages, provide a new opportunity to map developmental mutants to much more precise cell states, thereby providing more fine-grained insights into mutant phenotypes. In the four mutants studied, this approach not only revealed the loss of the expected cell types but could also quantify the proportion of cells lost, pinpoint the development stages, and reveal more subtle phenotypes, such as a gain of some mutant cells in seemingly normal trajectories of other tissues. This highlights the plasticity of mesodermal cell states and also a high degree of canalization to developmental programming, even upon mutation of these essential TFs. This could increase the overall robustness of embryogenesis, for example, by providing an excess of cells that can partly compensate for the loss of others when defects occur. The data provide a rich resource of regulatory changes associated with each step of mesoderm specification and differentiation into different muscle types, which is provide das easy to search, interactive UMAPs for further exploration. Going forward, this approach could be applied to reassess phenotypes and regulatory programs of 'classic' developmental mutants and also to uncover phenotypes of completely uncharacterized mutants de novo, beckoning a new era for joint cellular and molecular phenotyping (Secchia, 2022).
Although the approach presented in this study can readily identify cellular phenotypes from mutant embryos, it might not be possible to study molecular phenotypes in mutants in cases where specific cell types are not specified or maintained. This limitation could be overcome by using conditional depletion/knockout strategies. Dissection of molecular phenotypes using scATAC-seq could also be masked by the binding of other TFs to the same enhancers. Using single-cell ChIP against H3K27ac and/or nascent RNA-seq in this study to measure eRNA at single-cell resolution would help, although both approaches are still very challenging to apply to embryos (Secchia, 2022).
The pathophysiological effects of a number of metabolic and age-related disorders can be prevented to some extent by exercise and increased physical activity. However, the molecular mechanisms that contribute to the beneficial effects of muscle activity remain poorly explored. Availability of a fast, inexpensive, and genetically tractable model system for muscle activity and exercise will allow the rapid identification and characterization of molecular mechanisms that mediate the beneficial effects of exercise. This study reports the development and characterization of an optogenetically-inducible muscle contraction (OMC) model in Drosophila larvae that was used to study acute exercise-like physiological responses. To characterize muscle-specific transcriptional responses to acute exercise, bulk mRNA-sequencing was performed, revealing striking similarities between acute exercise-induced genes in flies and those previously identified in humans. This larval muscle contraction model opens a path for rapid identification and characterization of exercise-induced factors (Ghosh, 2022).
Spatio-temporal gradients in thermal and oxygen conditions trigger evolutionary and developmental responses in ectotherms' body size and cell size, which are commonly interpreted as adaptive. However, the evidence for cell-size responses is fragmentary, as cell size is typically assessed in single tissues. In a laboratory experiment, genotypes of Drosophila melanogaster were raised at all combinations of two temperatures (16 °C or 25 °C) and two oxygen levels (10% or 22%), and body size and the sizes of cells were measured in different tissues. For each sex,epidermal cells in a wing and a leg and ommatidial cells of an eye were measured. For males, epithelial cells of a Malpighian tubule and muscle cells of a flight muscle were measured. On average, females emerged at a larger body size than did males, having larger cells in all tissues. Flies of either sex emerged at a smaller body size when raised under warm or hypoxic conditions. Development at 25 °C resulted in smaller cells in most tissues. Development under hypoxia resulted in smaller cells in some tissues, especially among females. Altogether, these results show thermal and oxygen conditions trigger shifts in adult size, coupled with the systemic orchestration of cell sizes throughout the body of a fly. The nature of these patterns supports a model in which an ectotherm adjusts its life-history traits and cellular composition to prevent severe hypoxia at the cellular level. However, the results revealed some inconsistencies linked to sex, cell type, and environmental parameters, which suggest caution in translating information obtained for single type of cells to the organism as a whole (Czarnoleski, 2023).
Decline in skeletal muscle cell size (myofiber atrophy) is a key feature of cancer-induced wasting (cachexia). In particular, atrophy of the diaphragm, the major muscle responsible for breathing, is an important determinant of cancer-associated mortality. However, therapeutic options are limited. This study used Drosophila transgenic screening to identify muscle-secreted factors (myokines) that act as paracrine regulators of myofiber growth. Subsequent testing in mouse myotubes revealed that mouse Fibcd1 is an evolutionary-conserved myokine (mouse homolog of Drosophila CG30280 by BLAST search) that preserves myofiber size via ERK signaling. Local administration of recombinant Fibcd1 (rFibcd1) ameliorates cachexia-induced myofiber atrophy in the diaphragm of mice bearing patient-derived melanoma xenografts and LLC carcinomas. Moreover, rFibcd1 impedes cachexia-associated transcriptional changes in the diaphragm. Fibcd1-induced signaling appears to be muscle selective because rFibcd1 increases ERK activity in myotubes but not in several cancer cell lines tested. It is proposed that rFibcd1 may help reinstate myofiber size in the diaphragm of patients with cancer cachexia (Graca, 2022).
Muscle development is a multistep process that involves cell specification, myoblast fusion, myotube migration, and attachment to the tendons. In spite of great efforts trying to understand the basis of these events, little is known about the molecular mechanisms underlying myotube migration. Knowledge of the few molecular cues that guide this migration comes mainly from studies in Drosophila. The migratory process of Drosophila embryonic muscles involves a first phase of migration, where muscle progenitors migrate relative to each other, and a second phase, where myotubes migrate searching for their future attachment sites. During this phase, myotubes form extensive filopodia at their ends oriented preferentially toward their attachment sites. This myotube migration and the subsequent muscle attachment establishment are regulated by cell adhesion receptors, such as the conserved proteoglycan Kon-tiki/Perdido. Laminins have been shown to regulate the migratory behavior of many cell populations, but their role in myotube migration remains largely unexplored. This study showed that laminins, previously implicated in muscle attachment, are indeed required for muscle migration to tendon cells. Furthermore, it was fond that laminins genetically interact with kon-tiki/perdido to control both myotube migration and attachment. All together, these results uncover a new role for the interaction between laminins and Kon-tiki/Perdido during Drosophila myogenesis. The identification of new players and molecular interactions underlying myotube migration broadens understanding of muscle development and disease (Perez-Moreno, 2022).
Tissue homeostasis involves the elimination of abnormal cells to avoid compromised patterning and function. Although quality control through cell competition is well studied in epithelial tissues, it is unknown if and how homeostasis is regulated in mesenchymal collectives. This study demonstrates that collectively migrating Drosophila muscle precursors utilize both fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signaling to promote homeostasis via anoikis, a form of cell death in response to substrate de-adhesion. Cell-cycle-regulated expression of the cell death gene head involution defective is responsible for caudal visceral mesoderm (CVM) anoikis. The secreted BMP ligand drives cell-cycle progression via a visceral mesoderm-specific cdc25/string enhancer to synchronize collective proliferation, as well as apoptosis of cells that have lost access to substrate-derived FGF. Perturbation of BMP-dependent cell-cycle progression is sufficient to confer anoikis resistance to mismigrating cells and thus facilitate invasion of other tissues. This BMP-gated cell-cycle checkpoint defines a quality control mechanism during mesenchymal collective migration (Macabenta, 2022).
Cell fate specification is essential for every major event of embryogenesis, and subsequent cell maturation ensures individual cell types acquire specialized functions. The mechanisms that regulate cell fate specification have been studied exhaustively, and each technological advance in developmental biology ushers in a new era of studies aimed at uncovering the most fundamental processes by which cells acquire unique identities. What is less appreciated is that mechanisms are in place to ensure cell identity is maintained throughout the life of the organism. The body wall musculature in the Drosophila embryo is a well-established model to study cell fate specification, as each hemisegment in the embryo generates and maintains thirty muscles with distinct identities. Once specified, the thirty body wall muscles fuse with mononucleate muscle precursors that lack a specific identity to form multinucleate striated muscles. Multinucleate body wall muscles do not fuse with each other, which maintains a diversification of muscle cell identities. This study shows the serine/threonine kinase Back seat driver (Bsd) prevents inappropriate muscle fusion to maintain cell identity. Thus, the regulation of cell fusion is one mechanism that maintains cell identity (Yang, 2023).
During morphogenesis, large-scale changes of tissue primordia are coordinated across an embryo. In Drosophila, several tissue primordia and embryonic regions are bordered or encircled by supracellular actomyosin cables, junctional actomyosin enrichments networked between many neighbouring cells. This study shows that the single Drosophila Alp/Enigma-family protein Zasp52, which is most prominently found in Z-discs of muscles, is a component of many supracellular actomyosin structures during embryogenesis, including the ventral midline and the boundary of the salivary gland placode. This study reveals that Zasp52 contains within its central coiled-coil region a type of actin-binding motif usually found in CapZbeta proteins, and this domain displays actin-binding activity. Using endogenously-tagged lines, it was identified that Zasp52 interacts with junctional components, including APC2, Polychaetoid and Sidekick, and actomyosin regulators. Analysis of zasp52 mutant embryos reveals that the severity of the embryonic defects observed scales inversely with the amount of functional protein left. Large tissue deformations occur where actomyosin cables are found during embryogenesis, and in vivo and in silico analyses suggest a model whereby supracellular Zasp52-containing cables aid to insulate morphogenetic changes from one another (Ashour, 2023).
Sarcomeres are the force-producing units of all striated muscles. Their nanoarchitecture critically depends on the large titinprotein, which in vertebrates spans from the sarcomeric Z-disc to the M-band and hence links actin and myosin filaments stably together. This ensures sarcomeric integrity and determines the length of vertebrate sarcomeres. However, the instructive role of titins for sarcomeric architecture outside of vertebrates is not as well understood. This study used a series of nanobodies, the Drosophila titin nanobody toolbox, recognising specific domains of the two Drosophila titin homologs Sallimus and Projectin to determine their precise location in intact flight muscles. By combining nanobodies with DNA-PAINT super-resolution microscopy, this study found that, similar to vertebrate titin, Sallimus bridges across the flight muscle I-band, whereas Projectin is located at the beginning of the A-band. Interestingly, the ends of both proteins overlap at the I-band/A-band border, revealing a staggered organisation of the two Drosophila titin homologs. This architecture may help to stably anchor Sallimus at the myosin filament and hence ensure efficient force transduction during flight (Schueder, 2023).
Many myofibrillar proteins undergo isoform switching in a spatio-temporal manner during muscle development. The biological significance of the variants of several of these myofibrillar proteins remains elusive. One such myofibrillar protein, the Muscle LIM Protein (MLP), is a vital component of the Z-discs. This paper shows that one of the Drosophila MLP encoding genes, Mlp60A, gives rise to two isoforms: a short (279 bp, 10 kDa) and a long (1461 bp, 54 kDa) one. The short isoform is expressed throughout development, but the long isoform is adult-specific, being the dominant of the two isoforms in the indirect flight muscles (IFMs). A concomitant, muscle-specific knockdown of both isoforms leads to partial developmental lethality, with most of the surviving flies being flight defective. A global loss of both isoforms in a Mlp60A-null background also leads to developmental lethality, with muscle defects in the individuals that survive to the third instar larval stage. This lethality could be rescued partially by a muscle-specific overexpression of the short isoform. Genetic perturbation of only the long isoform, through a P-element insertion in the long isoform-specific coding sequence, leads to defective flight, in around 90% of the flies. This phenotype was completely rescued when the P-element insertion was precisely excised from the locus. Hence, these data show that the two Mlp60A isoforms are functionally specialized: the short isoform being essential for normal embryonic muscle development and the long isoform being necessary for normal adult flight muscle function (Wishard, 2023).
Transcription is initiated at the core promoter, which confers specific functions depending on the unique combination of core promoter elements. The downstream core promoter element (DPE) is found in many genes related to heart and mesodermal development. However, the function of these core promoter elements has thus far been studied primarily in isolated, in vitro or reporter gene settings. tinman (tin) encodes a key transcription factor that regulates the formation of the dorsal musculature and heart. Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, this study showed that a substitution mutation of the functional tin DPE motif within the natural context of the core promoter results in a massive perturbation of Tinman's regulatory network orchestrating dorsal musculature and heart formation. Mutation of endogenous tin DPE reduced the expression of tin and distinct target genes, resulting in significantly reduced viability and an overall decrease in adult heart function. This study has demonstrated the feasibility and importance of characterizing DNA sequence elements in vivo in their natural context, and accentuate the critical impact a single DPE motif has during Drosophila embryogenesis and functional heart formation (Sloutskin, 2023).
Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin filaments. The actin and myosin filaments are organized into repeated units called sarcomeres, which form the myofibrils. Muscle contraction is achieved by the simultaneous shortening of sarcomeres, which requires all sarcomeres to be the same size. Muscles have a variety of ways to ensure sarcomere homogeneity. It has been shown shown that the controlled oligomerization of Zasp proteins sets the diameter of the myofibril. This study looked for Zasp-binding proteins at the Z-disc to identify additional proteins coordinating myofibril growth and assembly. This study found that the E1 subunit of the oxoglutarate dehydrogenase complex localizes to both the Z-disc and the mitochondria, and is recruited to the Z-disc by Zasp52. The three subunits of the oxoglutarate dehydrogenase complex are required for myofibril formation. Using super-resolution microscopy, it was revealed the overall organization of the complex at the Z-disc. Metabolomics identified an amino acid imbalance affecting protein synthesis as a possible cause of myofibril defects, which is supported by OGDH-dependent localization of ribosomes at the Z-disc (Morales, 2023).
Phosphorylation reactions performed by protein kinases are one of the most studied post-translational modifications within cells. Much is understood about conserved residues within protein kinase domains that perform catalysis of the phosphotransfer reaction, yet the identity of the target substrates and downstream biological effects vary widely among cells, tissues, and organisms. This study characterize key residues essential for NUAK kinase activity in Drosophila melanogaster myogenesis and homeostasis. Creation of a NUAK kinase-dead mutation using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 results in lethality at the embryo to larval transition, while loss of NUAK catalytic function later in development produces aggregation of the chaperone protein αB-crystallin/CryAB in muscle tissue. Yeast 2-hybrid assays demonstrate a physical interaction between NUAK and CryAB. It was further shown that a phospho-mimetic version of NUAK promotes the phosphorylation of CryAB and this post-translational modification occurs at 2 previously unidentified phosphosites that are conserved in the primary sequence of human CryAB. Mutation of these serine residues in D. melanogaster NUAK abolishes CryAB phosphorylation, thus, proving their necessity at the biochemical level. These studies together highlight the importance of kinase activity regulation and provide a platform to further explore muscle tissue proteostasis (Zhao, 2023).
Development of the visceral musculature of the Drosophila midgut encompasses a closely coordinated sequence of migration events of cells from the trunk and caudal visceral mesoderm that underlies the formation of the stereotypic orthogonal pattern of circular and longitudinal midgut muscles. This study focuses on the last step of migration and morphogenesis of longitudinal visceral muscle precursors and shows that these multinucleated precursors utilize dynamic filopodial extensions to migrate in dorsal and ventral directions over the forming midgut tube. The establishment of maximal dorsoventral distances from one another, and anteroposterior alignments, lead to the equidistant coverage of the midgut with longitudinal muscle fibers. Teyrha-Meyhra (Tey), a tissue-specific nuclear factor related to the RNF220 domain protein family, as a crucial regulator of this process of muscle migration and morphogenesis that is further required for proper differentiation of longitudinal visceral muscles. In addition, Tey is expressed in a single somatic muscle founder cell in each hemisegment, regulates the migration of this founder cell, and is required for proper pathfinding of its developing myotube to specific myotendinous attachment sites (Frasch, 2023).
To investigate the pathophysiology and the molecular mechanisms underlying DM1, several DM1 models, both mouse, have been created. The reduction in MBNL1 and stabilization of CELF1 are thought to be involved in most DM1 phenotypes. Indeed, Mbnl1 knockout mice develop muscle myotonia, weakness/wasting, and cardiac defects including dilated cardiomyopathy and heart conduction block. Mice overexpressing CELF1 in the heart show conduction abnormalities and dilated cardiomyopathy thus confirming the contribution of MBNL1 sequestration and CELF1 up-regulation to DM1 pathogenesis. Overall, the mouse models reproduced multiple DM1 features including RNA foci formation and various alternative splice defects (Souidi, 2023).
A series of inducible Drosophila DM1 lines was generated bearing UAS-iCTG constructs with 240, 480, 600, and 960 CTGs. These lines were used to model DM1 in larval somatic muscles showing not only nuclear foci formation and Mbl sequestration but also muscle hypercontraction, splitting of muscle fibers, reduced fiber size, and myoblast fusion defects leading to impaired larva mobility (Picchio, 2013). The severity of phenotypes in these Drosophila models could be correlated with repeat size (Picchio, 2013), as also observed in DM1 patients. Finally, the overexpression of Drosophila CELF1 ortholog Bru3 and attenuation of MBNL1 counterpart mbl offer further valuable models for identifying gene deregulations underlying DM1 (Souidi, 2023).
Among molecular mechanisms associated with DM1, the deregulation of miRNAs and in particular reduced levels of evolutionarily conserved muscle- and heart-specific miRNA, miR-1, has been reported in DM1 patients and in DM1 models including mouse and Drosophila (Fernandez-Costa, 2013). However, the impact of miR-1 down-regulation on DM1-associated phenotypes has not yet been analyzed (Souidi, 2023).
This study made use of Drosophila DM1 models to explore miR-1 involvement in cardiac dysfunction in DM1. It was observed that dmiR-1 level was reduced in the cardiac cells of DM1 flies and that its down-regulation in the heart led to DCM, thus suggesting that reduced dmiR-1 levels contribute to DM1-associated DCM. Among potential dmiR-1 regulated genes from in silico screening, this study identified Multiplexin (Mp)/Collagen15A1 (Col15A1) The reduction in mammalian MBNL1 and stabilization of mammalian CELF1 are thought to be involved in most DM1 phenotypes. Indeed, Mbnl1 knockout mice develop muscle myotonia, weakness/wasting, and cardiac defects including dilated cardiomyopathy and heart conduction block. Mice overexpressing CELF1 in the heart show conduction abnormalities and dilated cardiomyopathy thus confirming the contribution of MBNL1 sequestration and CELF1 up-regulation to DM1 pathogenesis. Overall, the mouse models reproduced multiple DM1 features including RNA foci formation and various alternative splice defects (Souidi, 2023).
Myotonic dystrophy type 1 is the most common muscular dystrophy in adults. Cardiac repercussions including DCM are among the main causes of death in DM1. However, the underlying mechanisms remain poorly understood, impeding the development of adapted treatments. As was previously demonstrated, Drosophila DM1 models recapitulate all the cardiac phenotypes observed in DM1 patients and so could help gain insight into gene deregulations underlying DM1-associated DCM (Souidi, 2023).
In humans, DCM is characterized by left ventricular dilation and systolic dysfunction defined by a depressed ejection fraction. Similarly, in DCM-developing flies, the cardiac tube is enlarged and shows an increased diastolic and systolic diameter with reduced contractility. The loss of cardiac miRNAs and in particular miR-1 has already been correlated to DCM and heart failure in mice. miR-1 sequence is highly conserved between Drosophila and Human, and it is well known that it regulates genes involved in cardiac development and function including Nkx2.5, SRF, and components of WNT and FGF signaling pathways (Kura et al, 2020) and that its level is reduced in the pathological context of DM1. However, it was not known whether the low miR-1 level caused DM1-associated DCM, nor what were the downstream miR-1 targets. This study shows that two heart-targeting Drosophila DM1 models, Hand > mblRNAi and Hand > Bru3 mimicking sequestration of MBNL1 and stabilization of CELF1, respectively, developed DCM and showed a reduced expression of dmiR-1 in cardiac cells including cardiomyocytes and pericardial cells. Regarding the influence of Hand-Gal4 driven expression in pericardial cells on the DM1 heart phenotypes, previously work tested all the DM1 models using cardioblast-specific Tin-GAL4 driver. DM1 cardiac phenotypes such as conduction defects observed in the Hand > Bru3 model and DCM observed in Hand > mblRNAi and Hand > Bru3 models are observed when using Tin-Gal4 driver. These results suggest that the cardiac phenotypes observed in the DM1 Drosophila heart, including DCM, are mainly due to gene deregulations within the cardiomyocytes. Because the overexpression of CELF1 and the loss of MBNL1 also result in DCM in mice, Drosophila appears well-suited to assessing the impact of reduced miR-1 in DM1-associated DCM. One mechanism explaining why miR-1 levels fall in the DM1 context is the sequestration of MBNL1, which can no longer play its physiological role in pre-miR-1 processing into mature miR-1. This study observed reduced dmiR-1 also upon the cardiac overexpression of CELF1 ortholog Bru3. How CELF1/Bru3 impinges on miR-1 levels is not fully understood, but it was demonstrated that CELF1 could bind UG-rich miRNAs (such as miR-1) and mediate their de-adenylation and degradation by recruiting poly(A)-specific ribonuclease (PARN). Given that Drosophila DM1 models developing DCM showed markedly reduced dmiR-1 in cardiac cells, this study sought to determine whether heart-targeted attenuation of dmiR-1 was sufficient to induce DCM: dmiR-1 knockdown in the heart mimics DM1-associated DCM (Souidi, 2023).
To identify candidate dmiR-1 target genes involved in DCM in silico screening was performed for dmiR-1 seed sites in the 3'UTR regions of genes up-regulated in cardiac cells at 5 weeks of age in DM1 models developing DCM. Among 1,189 3'UTR sequences tested, 162 bore potential dmiR-1 seed sites, including the 3'UTR of Multiplexin (Mp). Mp codes for extracellular matrix protein belonging to a conserved collagen XV/XVIII family. Mp was top-ranked because of its known role in setting the size of the cardiac lumen. The embryos overexpressing Mp display an enlarged cardiac tube and conversely, Mp-/- embryos were found to present a narrower lumen with reduced contractility of the heart tube. In parallel, the mouse mutants of Mp ortholog, Col15A1, showed age-related muscular and cardiac deterioration linked to a degraded organization of the collagen matrix. This prompted an examination Mp expression in the adult fly heart and the effect of its overexpression. Using Mp specific antibody Mp was detected on the surface of the cardiac cells and found that Mp accumulated to a high level in both Hand > mblRNAi and Hand >> Bru3 DM1 lines. Whether the in silico identified dmiR-1 seed site was required for the regulation of Mp expression was examined and confirmed that Mp is a direct in vivo target of dmiR-1 in cardiac cells. As the potential binding site for human dmiR-1 is present also in 3'UTR of Col15A1 transcript it was hypothesize that Mp/Col15A1 are evolutionarily conserved dmiR-1 targets. Consistent with its role downstream of dmiR-1, Mp overexpression in the heart mimicked the dmiR-1 knockdown phenotype, leading to a significantly enlarged heart with reduced contractility. Moreover, heart-specific attenuation of Mp expression in the Hand > Bru3 DM1 context reduced heart dilation and rescued DCM phenotype in aged flies, thus demonstrating that increased Mp levels contribute to DCM observed in DM1 flies. Previous reports revealed increased expression levels of different collagens associated with DCM in both animal models and patients. This study reports evidence that Col15A1 is specifically up-regulated at both transcript and protein levels in cardiac samples from DM1 patients and in particular in those with DCM, with down-regulation of miR-1. Altogether, the observations that Col15A1 expression level is abnormally elevated in DCM-developing DM1 patients and that attenuation of its Drosophila ortholog Mp could ameliorate the DCM phenotype suggest that Col15A1 could be a novel therapeutic target in DM1 (Souidi, 2023).
A large number of genes have so far been implicated in DCM, attesting to the complex molecular origin of this cardiac condition. For example, in Drosophila, DCM was observed in mutants of genes encoding contractile and structural muscle proteins such as Troponin I (TpnI), Tropomyosin 2 (Tm2), δ-sarcoglycan and Dystrophin but also associated with deregulations of EGF, Notch, Cdc42 and CCR4-Not signaling pathway components. In humans, DCM-causing mutations were also identified in a large number of genes including those encoding cytoskeletal proteins such as FLNC, nuclear membrane protein LMNA or involved in sarcomere stability (Titin, TNNT2, MYH7, TPM1) but also RNA-binding protein RBM20 (Souidi, 2023).
This study focused on DCM associated with DM1. A previous study on a mouse model overexpressing CELF1 and developing DCM, identified down-regulation of Transcription factor A mitochondrial (Tfam), Apelin (Apln), and Long-chain fatty acid-CoA ligase 1 (Acsl1) as potentially associated with DCM. It was suggested that CELF1 might regulate their mRNA stability by binding to their 3'UTR regions and causing destabilization and degradation of their transcripts. In this DCM-developing mouse DM1 model, Col15a transcripts were elevated, but the role of Col15a in DCM was not analyzed. Using Drosophila DM1 models with a DCM phenotype, this study identified up-regulation of Col15A1 ortholog Mp as a molecular determinant of DM1-associated DCM. Reduced miR-1 levels were detected in DCM-developing DM1 cardiac cells to the up-regulation of Mp, establishing that Mp is an in vivo target of dmiR-1 (Souidi, 2023).
Importantly, these findings show that in DM1 patients, Collagen 15A1 is up-regulated in the hearts of patients with DCM. In DM1 patients, the DCM phenotype appears several years after onset and is less common than the conduction system defects and arrhythmias. However, DCM is frequently associated with poor prognosis and indication for heart transplant (Souidi, 2023).
In summary, this study report2 evidence for the importance of miR-1-dependent gene deregulations in DM1, and Mp was identified as a new miR-1 target involved specifically in DM1-associated DCM. Mbl depletion and Bru3 up-regulation in the heart were shown to have overlapping impacts on DM1 pathogenesis, both leading to reduced miR-1, up-regulation of Mp, and so to DCM (Souidi, 2023).
The conclusion is that in a physiological context, Mp level is moderately triggered by Mbl-dependent regulation of dmiR-1 processing and Bru3-dependent regulation of dmiR-1 stability. However, in the DM1 context, Mbl is sequestered in nuclear foci while Bru3 levels increase, leading to a reduced dmiR-1 and the up-regulation of its target gene Mp. Considering the known role of Mp as a positive regulator of cardiac lumen size, Mp accumulation in the adult heart would also be expected to promote heart tube enlargement, leading to the DCM phenotype. Whether like in embryos this Mp function involves the Slit/Robo signaling pathway remains to be investigated, but the finding that Robo2 is among identified miR-1 targets up-regulated in DCM-developing DM1 flies upports this possibility. Finally, the fact that Mp ortholog Col15A1 is highly elevated in cardiac samples from DM1 patients with DCM and that reducing Mp rescues the DCM phenotype in DM1 fly model suggests that Mp/Col15A1 could be an attractive diagnostic and/or therapeutic target for DM1-associated DCM (Souidi, 2023).
Cell growth and proliferation must be balanced during development to attain a final adult size with the appropriate proportions of internal organs to maximize fitness and reproduction. While multiple signaling pathways coordinate Drosophila development, it is unclear how multi-organ communication within and between tissues converge to regulate systemic growth. One such growth pathway, mediated by insulin-like peptides that bind to and activate the insulin receptor in multiple target tissues, is a primary mediator of organismal size. This study uncover a signaling role for the NUAK serine/threonine kinase in muscle tissue that impinges upon insulin pathway activity to limit overall body size, including a reduction in the growth of individual organs. In skeletal muscle tissue, manipulation of NUAK or insulin pathway components influences sarcomere number concomitant with modulation of thin and thick filament lengths, possibly by modulating the localization of Lasp, a nebulin repeat protein known to set thin filament length. This mode of sarcomere remodeling does not occur in other mutants that also exhibit smaller muscles, suggesting that a sensing mechanism exists in muscle tissue to regulate sarcomere growth that is independent of tissue size control (Brooks, 2022).
The troponin complex regulates the Ca2+-activation of myofilaments during striated muscle contraction and relaxation. Troponin genes emerged 500-700 million years ago during early animal evolution. Troponin T (TnT) is the thin filament-anchoring subunit of troponin. Vertebrate and invertebrate TnTs have conserved core structures, reflecting conserved functions in regulating muscle contraction, and also contain significantly diverged structures, reflecting muscle type- and species-specific adaptations. TnT in insects contains a highly diverged structure consisting of a long glutamic acid-rich C-terminal extension of ~70 residues with unknown function. C-terminally truncated Drosophila TnT (TpnT-CD70) retains binding of tropomyosin, troponin I, and troponin C, indicating a preserved core structure of TnT. However, the mutant TpnTCD70 gene residing on the X chromosome resulted in lethality in male flies. This X-linked mutation produces dominant-negative phenotypes, including decreased flying and climbing abilities, in heterozygous female flies. Immunoblot quantification with a TpnT-specific monoclonal antibody indicated expression of TpnT-CD70 in vivo and normal stoichiometry of total TnT in myofilaments of heterozygous female flies. Light and electron microscopy examinations revealed primarily normal sarcomere structures in female heterozygous animals, whereas Z-band streaming could be observed in the jump muscle of these flies. Although TpnT-CD70-expressing flies exhibited lower resistance to cardiac stress, their hearts were significantly more tolerant to Ca2+ overloading induced by high-frequency electrical pacing. These findings suggest that the Glu-rich long C-terminal extension of insect TnT functions as a myofilament Ca2+buffer/reservoir, potentially critical to the high-frequency asynchronous contraction of flight muscles (Cao, 2020).
A major challenge in biology is to understand how complex gene expression patterns are encoded in the genome. While transcriptional enhancers have been studied extensively, few transcriptional silencers have been identified, and they remain poorly understood. This study used a novel strategy to screen hundreds of sequences for tissue-specific silencer activity in whole Drosophila embryos. Almost all of the transcriptional silencers that were identified were also active enhancers in other cellular contexts. These elements are bound by more transcription factors than non-silencers. A subset of these silencers forms long-range contacts with promoters. Deletion of a silencer caused derepression of its target gene. These results challenge the common practice of treating enhancers and silencers as separate classes of regulatory elements and suggest the possibility that thousands or more bifunctional CRMs remain to be discovered in Drosophila and in humans (Gisselbrecht, 2019).
This study has adapted an enhancer-fluorescence-activated cell sorting-sequencing (FACS-seq) technology for highly parallel screening of elements for enhancer activity in Drosophila embryos (Gisselbrecht, 2013) into silencer-FACS-seq (sFS) technology, which enriches for elements that tissue specifically silence reporter gene expression. Briefly, this study generated a reporter vector, pSFSdist, which drives GFP expression under the control of an element from a library of candidate silencers, positioned at least 100 bp upstream of a strong, ubiquitous enhancer (ChIPCRM2078; Gisselbrecht, 2013). This vector contains a target sequence for a site-specific recombinase, permitting assaying all of the tested elements in the same genomically integrated context. Flies carrying single insertions from the reporter library are crossed to a strain in which the expression of the exogenous marker protein rat CD2 is driven in a tissue or cell type of interest, and the resulting informative embryos are dissociated to produce a single-cell suspension. By sorting for CD2+ cells in which GFP expression is reduced from the level driven by the strong ubiquitous enhancer in the absence of silencing activity, cells were enriched for that contained silencers active in the cell type of interest, which were then recover and identify by high-throughput sequencing. Insertion of an element with known mesodermal silencing activity into this vector consistently yielded a larger fraction of CD2+GFPreduced cells) than were observed when a negative control sequence (derived from Escherichia coli genomic DNA) was used (Gisselbrecht, 2019).
A library of 591 genomic elements, chosen to represent a variety of chromatin states or enhancer activity patterns, was designed to test for silencer activity in the embryonic mesoderm at stages 11-12 (5.5-7.5 h after embryo deposition). Since general features of silencers are unknown, this library was designed to test 3 main hypotheses about what kinds of sequences act as silencers in this developmental context (Gisselbrecht, 2019).
First, it was noted that 2 bifunctional CRMs had been identified previously in Drosophila that function as enhancers in one context and as silencers in other contexts. As this phenomenon is known to occur in multiple eukaryotic systems from a small number of examples and could be important in understanding the architecture of regulatory DNA, it was desirable to assess the generality of this phenomenon. Therefore, CRMs were selected from the REDfly and CAD2 databases that exhibited no or highly restricted mesodermal expression at embryonic stage 11. Elements associated with genes that show widespread mesodermal expression at this stage were filtered out. Second, a potential mechanistic signature of transcriptional silencers is the binding of well-characterized transcriptional corepressors, by analogy to the prediction of enhancers by binding of the coactivator CBP. Therefore genomic elements identified by ChIP as binding sites for the co-repressors Groucho or CtBP were selected. As Groucho has canonically been associated with long-range repression and CtBP has been associated with short-range repression, it was predicted that Groucho binding sites would be a richer source of silencer activity in this assay, in which candidate silencers were placed >100 bp upstream of the enhancer driving reporter gene activity (Gisselbrecht, 2019).
Third, genomic regions were selected that were associated with the markers of both enhancers and repressed chromatin structure in whole-mesoderm or whole-embryo experiments. It was reasoned that silencers are active regulatory elements, distinct from the silenced chromatin that results from their activity, yet must recruit factors that exert repressive functions, and therefore may show association with both classes of chromatin marks. Moreover, these 'bivalent' chromatin states may represent sequences of the above-mentioned type, that act as enhancers in one cell type and as silencers in another. In this class, two sets of sequences were included: (1) DNase I hypersensitive sites (DHSs) that colocalize with ChIP signal for the well-studied repressive chromatin mark histone H3 trimethyllysine 27 (H3K27me3) in sorted mesoderm, and (2) coincident mesodermal peaks for H3K27me3, the canonical enhancer mark histone H3 monomethyllysine 4 (H3K4me1), and histone H3 acetyllysine 27 (H3K27ac), which has been associated with active enhancers and promoters. All of the sequences identified from genome-wide ChIP methods were filtered for the absence of widespread mesodermal expression of associated nearby genes (Gisselbrecht, 2019).
Finally, 15 sequences were included for which enhancer chromosomal contact sequencing (4C-seq) data for sorted mesodermal cells were available. Three positive control sequences were included that were previously shown to have mesodermal silencing activity and two types of putative negative controls were included: broadly active mesodermal enhancers and length-matched regions of the E. coli genomic sequence (Gisselbrecht, 2019).
The library of genomic elements was screened for silencer activity in the embryonic mesoderm in 2 rounds. Testing of this library yielded a readily detectable population of mesodermal cells in which GFP expression was reduced; the elements enriched in these cells are referred to as sFS+ elements. Of the 591 sequence elements chosen for inclusion in this library, 501 were genomically integrated into transgenic flies after injection of the pooled library (Gisselbrecht, 2019).
Overlap with transcription start sites (TSSs) were identified as a highly enriched feature of sFS+ elements, which likely reveals the presence of promoter competition. Competition among promoters for association with active enhancers is one mechanism that has been proposed to account for the specificity with which enhancers target genes for activation and has been shown to restrict enhancer-driven activation of gene expression in reporter assays. Overall, the initial set of 41 'hits' that overlapped promoter regions was significantly enriched for mapped instances of the TATA box. While these are technical positives in the silencer screen, since the goal of this study was to analyze CRMs that silence gene expression by other means, any sequences that overlapped promoter regions were omitted from subsequent analyses. Moreover, many of the library elements tested overlapped other library elements; these were mergend for downstream analysis. After filtering to remove elements that overlapped promoters and collapsing overlapping genomic regions, 29 of a total of 352 genomic regions tested for mesodermal silencer activity were positive in the sFS screen (Gisselbrecht, 2019).
To validate the results from the sFS screen, pure transgenic lines were generated from a subset of library elements, and then their silencer activity was assayed by FACS analysis of embryos resulting from these individual reporter strains. Next, whether the silencers detected by the sFS screen could also silence the activity of enhancers other than the strong, ubiquitous enhancer used in the sFS screen was investigated. Thus, silencing by several sFS+ elements was assessed visually by placing these elements upstream of the following mesoderm-specific enhancers and imaging the resulting GFP expression. ChIPCRM2613 is an intronic enhancer of the pan-mesodermal gene heartless, and it drives reporter gene expression throughout the presumptive mesoderm from the beginning of gastrulation (Gisselbrecht, 2013). The Mef2 I-ED5 enhancer drives expression in the fusion-competent myoblasts beginning in late stage 11. In every case examined (15 of 15), at least one of these additional enhancers showed reduced activity in the mesoderm in the context of the tested element. These results not only verify the silencing activity of these sFS+ elements but they also demonstrate that silencers are not specific for a particular enhancer (Gisselbrecht, 2019).
The resulting set of mesodermal silencers was analyzed to determine which genomic features that were explicitly sampled in the design of the element library were predictive of silencer activity. Despite the inclusion in the sFS library of ChIP peaks for transcriptional co-repressors and for a repressive chromatin mark, the only screened element types that were significantly enriched among the active mesodermal silencers were positive controls and non-mesodermal enhancers. In fact, 22 of 29 regions containing mesodermal silencers had been previously reported to have enhancer activity. Testing of the remaining 7 silencers for enhancer activity revealed that 6 of 7 also function as enhancers in the embryo: 5 of 6 were entirely non-mesodermal, while 1 showed restricted mesodermal expression. In total, 28 of 29 of the elements were found to act as mesodermal silencers also exhibited enhancer activity in a different cellular context. Overall, >10% (26 of 200) of the previously known enhancers tested in the assay exhibited mesodermal silencer activity. This constitutes more bifunctional transcriptional regulatory elements than were previously known across all biological systems (Gisselbrecht, 2019).
A different class of bifunctional CRMs was reported recently (Erceg, 2017), in which developmental enhancers have an additional function as Polycomb response elements (PREs). PREs provide genomic binding sites for sequence-specific DNA binding proteins that recruit protein subunits of Polycomb repressive complexes and could, in principle, play a role in silencing target genes. Therefore, this study tested the hypothesis that the silencer activity of enhancers discovered in the current assay resulted from PRE activity. Only 4 of 29 regions displaying mesodermal silencer activity overlapped PREs as defined by Erceg (2017) on the basis of ChIP for the PRE-binding factors Pho and dSfmbt, versus 24 of 323 mesodermal non-silencers, indicating that PREs are not a major source of mesodermal silencers (Gisselbrecht, 2019).
The results suggest a view of enhancers as CRMs with distinct spatiotemporal patterns of both activation and repression. To further assess the generality of this phenomenon, the effects of a subset of the newly discovered mesodermal silencers was visualized on enhancers that are active broadly in the mesoderm at different developmental stages. This enabled simultaneous evaluation of a variety of spatiotemporal domains of silencer and enhancer activity (Gisselbrecht, 2019).
Several elements exhibited apparently uniform silencing activity across the mesoderm and at different stages. The most commonly observed temporal pattern was a lack of silencing activity at gastrulation and strong silencing during the later stages at which sFS was performed. An element, hkb_0.6kbRIRV, was also observed that silenced much more strongly earlier than later in embryonic development; this element simultaneously acted as an enhancer in its previously characterized pattern in the midgut primordia. One element, the oc otd186 enhancer, which was observed to drive expression in the head, exhibited spatially patterned silencing within the mesoderm during gastrulation but not later in development; silencing was moderate in the anterior portion of the germband, but much stronger in the posterior portion, as seen in the context of 2 different early pan-mesodermal enhancers. Two different later-acting mesodermal enhancers showed moderate, uniform silencing across the anteroposterior extent of the germband. Finally, a tested element exhibited enhancer-specific silencing activity. The lz crystal cell enhancer was a moderately weak silencer when tested on different late-acting mesodermal enhancers and a strong silencer at gastrulation in the context of ChIPCRM2613, yet it completely failed to silence activity driven during gastrulation by ChIPCRM7759. These results highlight that silencers exhibit a similarly diverse range of spatiotemporal regulatory patterns as those of enhancers (Gisselbrecht, 2019).
To investigate whether the silencer activity observed in reporter assays reflects the activity of the putative silencer in its native chromosomal context, the average mesodermal transcription was profiled in the genomic neighborhood of silencers or other functional elements. Using published RNA-seq data from sorted mesodermal cells, reads were aggregated within 500-bp windows over a 25-kb region centered on each element, representing the typical size of chromatin state domains observed in a high-resolution Drosophila Hi-C experiment. Then the results were averaged over all of the elements in a class to create a meta-profile of transcript levels surrounding each class of cis-regulatory elements. As expected, transcription near a previously published set of mesodermal enhancers (Gisselbrecht, 2013) is elevated. In contrast, transcription near silencers is below the baseline level of transcription observed near a negative control set of genomic regions. Both effects decay to background levels in the meta-profiles over a scale of ~5 kb, suggesting that silencers act within approximately the same distance range as transcriptional enhancers. The bifunctional PREs reported by Erceg (2017) are also associated with strongly elevated transcription, but this effect appears to spread more broadly on the chromatin domain scale, suggesting that bifunctional PREs may act by a mechanism that is distinct from that of the silencers identified in the sFS screen (Gisselbrecht, 2019).
Next, to further demonstrate the functional importance of the silencers that were identified in the sFS screen, CRISPR-Cas9 genome editing was used to generate a Drosophila strain containing a deletion of the hkb_0.6RIRV element. This element was originally reported as an enhancer driving the expression of the gap gene huckebein (hkb) at the termini of the blastoderm embryo, and this study identified it as a mesodermal silencer in the screen. Mesodermal cells from embryos homozygous for this deletion and from wild-type control embryos were screened and hkb RNA was found to be significantly upregulated in the homozygous mutant mesoderm, which supports a role for this element in silencing its endogenous target gene during normal embryonic development (Gisselbrecht, 2019).
Various types of epigenomic features, including chromatin accessibility, post-translational modifications of histones, and occupancy by TFs and chromatin-modifying enzymes, have been associated with different categories of functional elements in the genome, such as active promoters and enhancers. However, relatively little is known about the chromatin features of active silencers. Therefore the epigenomic environment at the set of 29 mesodermal silencers was explored by assessing the enrichment or depletion of signal from various published epigenomic datasets, as compared to elements that did not display silencer activity in the sFS screen (Gisselbrecht, 2019).
It was hypothesized that since bifunctional elements are more functionally complex than CRMs that act only as enhancers, they may exhibit a more complex suite of TF interactions across various tissues. This study observed that validated silencers are strongly enriched for overlap with highly occupied target (HOT) regions as exceeding a TF complexity score threshold of ~10 overlapping bound factors. Since silencing activity is likely mediated through the effects of bound sequence-specific transcriptional repressors, the set of 29 mesodermal silencers was searched for enriched combinations of evolutionarily conserved DNA binding site motif occurrences for TFs annotated as repressors. The only motif combination that was significantly enriched among silencers was a 3-way combination of the motifs for the TFs Snail, Dorsal, and Tramtrack-PF, which were found together in 12 of 29 mesodermal silencers (Gisselbrecht, 2019).
Snail is a well-known repressor of non-mesodermal genes in the developing mesoderm. Dorsal and Tramtrack have also been shown to play roles in mesodermal gene repression. Analysis of ChIP data for Snail revealed significant enrichment for Snail occupancy at silencers. To validate that this enrichment reflects Snail activity at silencers, predicted Snail binding sites were mutated in 4 silencer elements with high levels of Snail ChIP signal, and the silencer activity of the mutant was compared to wild-type sequences within whole embryos in the FACS-based reporter assay. All 4 elements showed significantly reduced silencer activity. Mutating sites for an unrelated TF, as a negative control, caused no significant reduction in silencer activity. While Snail has been well characterized as a short-range repressor acting within 150 bp, all of the Snail binding sites that were found to be required for full silencer activity are >400 bp away from the silenced enhancer in the reporter construct, indicating that Snail can act as a repressor at distances longer than those described for short-range repression (Gisselbrecht, 2019).
Finally, the evidence was examined for the direct action of Snail binding to silencers on the expression of the silencers' endogenous target genes. Since the majority of elements exhibiting silencer activity in this study were originally reported as enhancers, it was possible to identify published target genes of these bifunctional elements and examine the effect of loss of snail function on their expression. Target genes of 12 elements bound by Snail in ChIP-seq data showed significant derepression in sna mutant embryos. In contrast, no target of any of the 14 Snail-unbound elements was significantly derepressed. Therefore, it is concluded that the known role of Snail in mesodermal repression explains the activity of a large minority (41%) of the observed silencers, while the transcriptional regulators mediating silencing activity through the majority of the silencers remain to be determined (Gisselbrecht, 2019).
In an attempt to identify a 'silencer signature' that is analogous to the previously described chromatin signatures of enhancers and promoters, published ChIP data was assembled from whole embryos or from sorted mesoderm, where available, for several chromatin marks previously associated with active or repressed chromatin states, and hierarchical clustering was performed of all 352 tested genomic regions according to these histone modification chromatin profiles. As expected, clusters of elements with greater signal for the repressive marks H3K27me3 and H3K9me3 are enriched for silencers, while other clusters are depleted of silencers. It was also observed, however, that many non-silencers belonged to these clusters, and that some silencers belonged to other clusters that were instead enriched for non-silencers, suggesting that these commonly profiled chromatin marks do not constitute a general chromatin signature of silencers. Similarly, neither the Groucho nor the CtBP co-repressors were significantly enriched at silencers (Gisselbrecht, 2019).
For a subset of silencers (18 of 29), the individual FACS validation data provided a measure of the strength of silencer activity, in terms of the percentage of cells in the GFPreduced population. Using these quantitative estimates of silencer activity, it was found that H3K27me3 and H3K9ac, a mark that previously had been associated with bivalent promoters and active enhancers, are significantly correlated with silencer strength across these 18 elements, possibly reflecting the fact that mesodermal silencers are active enhancers in other cellular contexts. No single mark or combination of marks that was tested from among the publicly available histone modification profiles accurately discriminates active silencers as a whole from other types of cis-elements (Gisselbrecht, 2019).
It has been well established that enhancers can act directly on their target promoters by looping to create direct, 3-dimensional (3D) physical contacts between genomic elements widely separated in sequence space. Such contacts have also been shown to play a role in repression by heterochromatin and at PREs. Silencers could, in principle, act directly to recruit repressive activities to regulated promoters, or alternatively by sequestering enhancers that would otherwise interact with promoters, or by other mechanisms that do not involve focal contacts to regulated elements, such as nucleating a repressive chromatin state that spreads along the chromosome (Gisselbrecht, 2019).
Therefore data was examined from assays of genomic contacts based on proximity ligation to attempt to distinguish among these hypotheses. Mesodermal silencer activity was observed in a CRM previously characterized by circular chromosome conformation capture (4C). This element makes mesodermally enriched contacts with 2 regions that overlap the promoters of genes that are not expressed in the mesoderm, suggesting the possibility that silencing may be mediated by direct silencer-promoter looping (Gisselbrecht, 2019).
To test the generality of this potential mechanism, Hi-C data was generated from sorted mesodermal and non-mesodermal cells at embryonic stages 11-12, the same developmental stages that was assayed by sFS. Mesodermally enriched contacts were observed at 1-kb resolution in each of 2 paired replicates using the chromoR package and compared the results to the mesodermally enriched 4C contacts previously reported. The frequency of random contacts observed in Hi-C assays is greater with closer genomic distance. Therefore, to control for such nonspecific interactions in this analysis, negative control sets of 'contact regions' were generated by reflecting each observed contact around the viewpoint region. Each Hi-C replicate showed significantly greater overlap with 4C contacts than with negative control regions, indicating that the Hi-C contacts agree with published genomic contacts (Gisselbrecht, 2019).
The features of these mesodermally enriched silencer contacts were examined, as these are potential targets of silencing activity. A list was created of these potential targets by filtering for contacts that were observed in both of the Hi-C replicates and that overlap sFS-tested elements, and then the (epi)genomic features of these regions were examined. Since the TF Snail (Sna) has previously been associated with short-range repression and 'antilooping', the features of regions contacted by Sna-bound mesodermal silencers, Sna-unbound mesodermal silencers, and elements that did not act as silencers in mesoderm were compared. Regions that made mesoderm-specific contacts to Sna-unbound mesodermal silencers are significantly enriched for overlapping TSSs, as compared to those contacting either Sna-bound silencers or non-silencers, indicating that the Sna-unbound mesodermal silencers contact promoters. This suggests that Snail-unbound silencers are targeted primarily by long-range repressors, whereas Snail-bound silencers show almost no promoter contacts, which is consistent with previous reports of short-range repression and antilooping associated with this TF (Gisselbrecht, 2019).
Next, whether the expression levels of silencer-contacted genes were consistent with silencer activity was inspected. For genes whose promoters made contact with any of the 352 tested library elements, RNA-seq data from sorted mesodermal cells was compared with the elements' histone marks that showed significant correlation with silencer activity. The level of H3K27me3 found at a library element was significantly anticorrelated with the mesodermal expression of genes contacted by that element, supporting the model of contact-based repression by silencers (Gisselbrecht, 2019).
Attempts were made to test the alternate model that silencers directly contact enhancers that would otherwise be active. Thus, the contacted regions were examined for overlap with CRMs that have been reported to act as enhancers, according to the REDfly database. Each of the 3 sets of library elements (Sna-bound silencers, Sna-unbound silencers, and non-silencers) were separately tested, and no significant enrichment or depletion of CRM contact was observed in any of the sets. It was further reasoned that direct action by silencers on enhancers would result in enrichment of the enhancer mark H3K4me1 in regions that contact silencers. In this scenario, this enrichment should be apparent in histone mark data from whole embryos and across a broad range of time points, reflecting their enhancer activity in non-mesodermal tissues and/or other developmental stages. Instead a significant depletion of H3K4me1 was observed at Sna-unbound silencer contacts versus non-silencer contacts, which does not support the model of silencers interacting directly with enhancers. The results support models in which distinct classes of transcriptional silencers act by antilooping or by acting directly on the promoters of repressed genes. While the existence of silencers that may sequester enhancers from contacting promoters cannot be ruled out, the results do not support this alternate mechanism (Gisselbrecht, 2019).
This study has developed a highly parallel reporter assay carried out in whole, developing animals to identify a set of transcriptional silencers on the basis of their tissue-specific function. Analysis of RNA-seq data indicated that genes located near these silencers in their endogenous context are expressed at lower levels. Deletion of 1 of these elements at its native genomic locus by CRISPR-Cas9 genome editing demonstrated the importance of that element for the proper expression level of its target gene. This study also integrated a wide variety of data types from previously published datasets, including ChIP of histone modifications and specific factors, with newly generated tissue-specific 3D chromosomal physical interaction data to assess enriched features of the set of tissue-specific silencers and has explored potential mechanisms (Gisselbrecht, 2019).
Many enhancers were found in fact to be bifunctional elements, capable of up- and downregulating gene expression in different cellular contexts. While this phenomenon has been observed previously in studies of individual regulatory elements, the extent of CRM bifunctionality had not been appreciated. It is important to note that many CRMs that failed to show silencing activity in the screen are known enhancers that are not active in the tissue tested. Silencers are therefore an identifiable set of active elements, distinct from 'quenched' or inactive enhancers that neither activate nor repress gene expression (Gisselbrecht, 2019).
While prior studies have found histone modifications associated with enhancer activity, this study suggests that despite the extensive genome-scale ChIP profiling studies by numerous investigators and consortia, the available chromatin profiling data are not sufficient to identify silencers. This is possibly explained by the existence of various silencer classes. Alternatively, there are dozens of chromatin marks that have not been characterized extensively that may mark silencers. Expanded efforts in profiling larger sets of tissue-specific chromatin marks may reveal a signature of active silencers. Similarly, it was surprising that co-repressor occupancy was not a good predictor of silencers. One potential explanation is that many of these elements may be silencers in other cell types or at other developmental stages than were assayed in this study, since co-repressor ChIP data were generated in whole embryos across a broad range of ages. Another possibility is that different subclasses of CRMs with silencer activity may be endowed with subclass-specific chromatin and/or TF signatures. The list of 29 silencers discovered by the sFS assay provides a training set that can be used for the further study of regulatory features that govern silencing (Gisselbrecht, 2019).
Enriched Snail binding was observed at a subset of mesodermal silencers. Snail is a well-characterized short-range repressor protein acting in the mesoderm, and it has been proposed to act by preventing regulated elements from looping to promoters. The current results are consistent with this general model; however, the effects of Snail repression spread over hundreds of base pairs and into neighboring regulatory elements in the reporter assay, in contrast to previously reported limits of short-range repression. Thus, the results indicate that Snail can act by different modes of repression, which had not been observed previously (Gisselbrecht, 2019).
This study provides evidence supporting a model of silencer activity in which a subset of silencers makes direct 3D contacts with the promoters of regulated genes. These physical interactions are important to consider when interpreting genome-wide maps of chromosome conformation. Not all promoter-interacting regions will act as enhancers, and it will be necessary to develop approaches that integrate a wide range of genomic data types to identify and functionally characterize cis-regulatory elements, including distinguishing those acting as enhancers versus silencers (Gisselbrecht, 2019).
It has recently been shown that many developmental enhancers also act as PREs. Despite some common features, including evidence for looping to target promoters, this set of bifunctional enhancer elements is nearly distinct from the elements this study has characterized that act as both enhancers and silencers, and appears to act by distinct mechanisms. It was previously reported that a Drosophila insulator element has a second role in mediating long-range enhancer-promoter interactions. It is suggested that a taxonomy of regulatory elements as enhancers, silencers, insulators, and so forth is likely an oversimplification, and that it is more useful to think generally of CRMs, which can activate and repress, recruit chromatin modifiers and remodelers, and/or structure the 3D genome in a context-sensitive fashion (Gisselbrecht, 2019).
It has been estimated that there may be >50,000 enhancers in the D. melanogaster genome. This study has detected mesodermal silencer activity in >10% of tested non-mesodermal enhancers. If these elements are representative of the broader enhancer population, then this result suggests that there may be thousands of such bifunctional elements across a range of tissues in Drosophila; since many of the elements were tested could be silencers in a cell type that was not examined or at a later developmental stage, these numbers are likely even higher. The sFS approach could be adapted in future studies to screen for bifunctional elements in mammals (Gisselbrecht, 2019).
These results suggest that most, if not all, silencers are also enhancers in a different cell type. CRM bifunctionality complicates the understanding of how gene regulation is specified in the genome and how it is read out in different cell types. The observation that the vast majority of complex trait- and disease-associated variants identified from genome-wide association studies (GWASs) map to noncoding sequences, most of which occur within DNase I hypersensitive sites, emphasizes the importance of understanding these elements. The characterization of bifunctional elements will help in elucidating how precise gene expression patterns are encoded in the genome and aid in the interpretation of cis-regulatory variation (Gisselbrecht, 2019).
Drosophila obscurin (Unc-89) is a titin-like protein in the M-line of the muscle sarcomere. Obscurin has two kinase domains near the C-terminus, both of which are predicted to be inactive. This study has identified proteins binding to the kinase domains. Kinase domain 1 bound Ballchen (Ball, an active kinase), and both kinase domains 1 and 2 bound MASK (a 400-kDa protein with ankyrin repeats). Ball was present in the Z-disc and M-line of the indirect flight muscle (IFM) and was diffusely distributed in the sarcomere. MASK was present in both the M-line and the Z-disc. Reducing expression of Ball or MASK by siRNA resulted in abnormalities in the IFM, including missing M-lines and multiple Z-discs. Obscurin was still present, suggesting that the kinase domains act as a scaffold binding Ball and MASK. Unlike obscurin in vertebrate skeletal muscle, Drosophila obscurin is necessary for the correct assembly of the IFM sarcomere. Ball and MASK act downstream of obscurin, and both are needed for development of a well defined M-line and Z-disc. The proteins have not previously been identified in Drosophila muscle (Katzemich, 2015).
A stable lattice of thick and thin filaments in striated muscle is needed to maintain the optimum register of the filaments as the fibres contract. Thin filaments from neighbouring sarcomeres are anchored in the Z-disc by α-actinin and other cross-linking proteins, and thick filaments are held in position by cross-links at the M-line in the middle of the sarcomere. The register of thick filaments is also maintained by elastic links between the ends of the filaments and the Z-disc. Large modular proteins of the titin family, associated with thick filaments, contribute to both the stability and the stiffness of the sarcomere. These proteins are made up of tandem immunoglobulin (Ig) and fibronectin-like (Fn3) domains and can have one, or sometimes two, kinase domains near the C-terminus, and there can also be signalling domains (Katzemich, 2015).
The M-line protein, obscurin, has a similar modular structure in invertebrates and vertebrates, although the number of modules in different isoforms and the position of the signalling domains vary. Both Unc-89 (the obscurin homologue in Caenorhabditis elegans; note that this protein is also known as Unc-89 in Drosophila) and obscurin in Drosophila have SH3 and Rho-GEF signalling domains near the N-terminus and two kinase domains near the C-terminus. In vertebrate obscurin, the signalling domains are near the C-terminus; the isoform obscurin A has an ankyrin-binding domain instead of the two C-terminal kinase domains in obscurin B. Both these isoforms are at the periphery of myofibrils in the M-line region of mature skeletal fibres. Binding of obscurin A to ankyrins creates a link between the sarcoplasmic reticulum (SR) and the myofibril. By contrast, Drosophila obscurin is found throughout the M-line and there is no ankyrin-binding domain, so direct binding to the SR is unlikely. However, in the nematode, loss-of-function mutations in unc89 result in displaced ryanodine receptor and SERCA, as well as abnormal Ca2+ signalling. This suggests that there is a function for Unc-89 in Ca2+ regulation involving the SR. So far, five large isoforms of obscurin have been identified in Drosophila muscles: one expressed in the larva, and four expressed in the pupa and adult. All these isoforms have Ig domains in the tandem Ig region, and at least the first of the kinase domains (denoted Kin1). The indirect flight muscle (IFM) has two isoforms: a major isoform of 475 kDa and a minor isoform that is somewhat smaller. The two remaining isoforms are in other thoracic muscles. Drosophila obscurin is essential for the formation of an M-line, and for the correct assembly of thick and thin filaments in the sarcomere: lack of obscurin in the IFM results in asymmetrical thick filaments and thin filaments of abnormal length and polarity. Paradoxically, vertebrate obscurin is not necessary for normal sarcomere structure, given that obscurin knockout in the mouse had no serious effect on sarcomere assembly or maintenance (Katzemich, 2015).
The kinase domains of titin-like proteins often function as scaffolds binding other proteins, and might or might not be active kinases. In C. elegans, the Unc-89 kinase 1 domain (PK1) is predicted to be inactive because the ATP-binding site lacks essential residues. The Unc-89 kinase 2 domain (PK2) might be active, although a motif contributing to ATP-binding is atypical. Both Unc-89 kinase domains interact with the protein small, C-terminal domain, phosphatase-like 1 (SCPL-1), which is thought to be involved in muscle-specific signalling. Unc-89 PK1 also interacts with the LIM-domain protein, LIM-9; the complex of PK1, SCPL-1 and LIM-9 links Unc-89 to integrin adhesion sites at the cell surface. In Drosophila, both Obscurin kinase domains (denoted Kin1 and Kin2) are predicted to be inactive because the catalytic site lacks the catalytic aspartate and other crucial residues. Both kinase domains in vertebrate obscurin B are predicted to be catalytically active, and can apparently be auto-phosphorylated. The kinase domains are reported to interact with membrane associated proteins: kinase domain 1 (SK1) with the extracellular domain of a Na+/K+-ATPase at adherens junctions, which is not a substrate, and kinase domain 2 (SK2) with the cell-adhesion molecule, N-cadherin, which is an in vitro substrate (Katzemich, 2015).
The kinase domains in titin-like proteins have sequences at the C-terminus that sterically block the active site (the C-terminal regulatory domain). This sequence can inhibit an active kinase, or regulate ligand binding; it can also be part of the structure of the kinase domain, and necessary to maintain the stability of the domain. Titin-like kinases are linked to stretch-activated signalling pathways in muscle. Mechano-sensing by the kinase can result in changes in the C-terminal regulatory domain and transient binding of ligands to the kinase scaffold. The precise mechanism of regulation varies in different species (Katzemich, 2015).
The aim of this study was to identify proteins binding to the two kinase domains in Drosophila obscurin, and to determine the effect of the proteins on the assembly of an ordered sarcomere in IFM. Ball (a protein kinase) was shown to bind to Kin1, and MASK (an ankyrin repeat protein) binds to both Kin1 and Kin2. The kinase ligands are essential for the formation of an intact M-line and Z-disc in the IFM sarcomere (Katzemich, 2015).
The kinase domains of Drosophila Obscurin differ from those of the C. elegans homologue, Unc89, and the vertebrate protein. Both domains are predicted to be inactive as kinases. However, some pseudokinases can become catalytically active by replacing missing residues with residues from neighbouring domains, or from associated ligands. Pseudokinases commonly act as scaffolds for binding proteins involved in signal transduction, and are often tethered to other domains, including Ig and Fn3 domains, which contribute to the binding site. Titin kinase in the M-line region of vertebrate skeletal muscle forms part of a binding site for the autophagy and kinase scaffold proteins, Nbr1 and SQSTM1, and the ubiquitin ligase, MuRF1 (also known as TRIM63). In the case of MuRF1, the site includes the preceding Ig and Fn3 domains, which will also bind MuRF1 without the kinase domain (Katzemich, 2015).
This study has found that Kin1 in Drosophila Obscurin binds Ball, which has the hallmarks of an active serine-threonine kinase. Ball differs from other kinase molecules in having a long extension C-terminal to the kinase sequence. This extension binds to Kin1 of Obscurin, with or without the flanking Ig and regulatory domains. Given that Ball also binds to the Ig domain alone, it is likely the molecule spans a region of Obscurin that includes the Ig domain as well as the kinase. Binding to Kin1 with the regulatory domain was unexpected. However, it is not clear how much of the sequence downstream of the kinase is included in a possible regulatory domain. In pseudokinases that are part of a larger molecule, the association of the regulatory domain with a ligand-binding site can be altered by force applied to the whole molecule. The regulatory domain of Kin1, taken out of its usual context, might not associate with the active site in the same way as it would in vivo. Alternatively, the regulatory domain might be required to stabilise the kinase structure when the muscle is stretched, as suggested for twitchin kinase (Katzemich, 2015).
Ball is found in the Z-disc of IFM, as well as being diffusely distributed in the sarcomere and in some samples, Ball is also detected at the M-line. Ball might migrate to bind to Kin1 in the M-line when the kinase activity of Ball is needed. There are other examples of protein migration in the muscle sarcomere. A transient translocation from the M-line to the Z-disc and cytoplasm has been observed for titin kinase ligands in cardiac muscle. In zebrafish, the myosin chaperone, Unc-45, is associated with myosin during myofibrillogenesis; in the adult, Unc-45 is in the Z-disc in normal fibres and it migrates to the A-band under conditions of stress, where it transiently associates with myosin again. Similarly, Ball might migrate from the Z-disc to the M-line under some conditions. Although this study has shown that Ball is capable of binding to Kin1 in vivo, the conditions necessary for the association are not yet known. Ball is still present in the IFM of obscurin-knockdown flies, though with a less-ordered distribution in the sarcomere, which suggests there are likely to be other binding partners for Ball (Katzemich, 2015).
Kin2 binds MASK, which has two regions with ankyrin repeats, and a relatively long sequence C-terminal to a KH domain. A peptide near the end of the molecule binds to Kin2 with or without the Fn3 domain preceding the kinase. MASK does not bind to the Fn3 domain alone, nor does it bind to Kin2 with sequence C-terminal to the kinase. It is not clear at present whether the C-terminal sequence acts as a regulatory domain (Katzemich, 2015).
The dual position of MASK in both the Z-disc and the M-line of IFM is unlikely to be due to migration of a protein of 400 kDa. The RNA coding for ankyrin-repeat proteins undergoes extensive alternative splicing, which can alter the binding sites of the different ankyrin isoforms. The independence of the binding sites for MASK in the M-line and Z-disc is confirmed by the finding that in obscurin-knockdown flies, MASK is almost eliminated from the M-line but still present in the Z-disc. Thus, Obscurin is needed for MASK to bind in the M-line, but not to the Z-disc. The presence of Obscurin in the M-line of MASK-knockdown flies is consistent with an Obscurin scaffold that binds MASK (Katzemich, 2015).
In addition to MASK, Tropomyosin-1 was identified as a ligand associated with Kin2 expressed in vivo. As tropomyosin is a thin filament protein, the significance of an association with Obscurin, which is at the midline of the thick filament, is not clear. Smaller isoforms of Obscurin have been detected in IFM, and it is possible that Tropomyosin-1 could bind, outside the M-line, to a small isoform containing a Kin2 domain (Katzemich, 2015).
The effect of downregulating Ball or MASK on the structure of the M-line and Z-disc in IFM shows the importance of these proteins in the development of a regular filament lattice. In the IFM of Ball-knockdown flies, the shifted position of the H-zone and M-line is associated with fragmented Z-discs; where the Z-disc is normal within a sarcomere, the H-zone and M-line are at the midline. The aggregates of multiple Z-discs in the IFM of MASK-knockdown flies dominate the sarcomere and there is no regular H-zone. This phenotype differs from the effect of reducing the expression of obscurin in IFM, where the H-zone and M-line are often shifted from the midline, without a corresponding anomaly in the Z-disc. The difference might be due to the presence of Ball and MASK in the Z-disc, whereas Obscurin is solely in the M-line. The high mortality rate at the larval and pupal stages of flies when either Ball or MASK is reduced differs from the survival of flies with reduced obscurin, which is unaffected in RNAi lines. Evidently, the crucial function of Ball and MASK is in binding to the Z-disc. The association of the proteins with Obscurin in the M-line is not necessary for the performance of most muscles, although it is essential for the development of the precise filament lattice that is needed for the performance of the IFM. The function of Obscurin in the assembly of the IFM sarcomere is not seen in vertebrates. Knockouts of obscurin in the mouse have no effect on the assembly of myosin filaments, Z-disc or M-line, but they do impair the assembly of the SR. Whether or not invertebrate and vertebrate obscurin-like proteins are true homologues (with the same functions, rather than just having a similar patterns of domains) is at present uncertain (Katzemich, 2015).
Ball and MASK are reported to be involved in cell proliferation, growth and differentiation in Drosophila. Ball regulates the proliferation and differentiation of germline stem cells and neuroblasts (Herzig, 2014; Yakulov, 2014). However, it is not clear whether this has any relevance to the function of Ball in Drosophila muscle. The VRK family of kinases (of which Ball is a member) phosphorylate the barrier-to-autointegration factor (BAF), which is necessary for the correct assembly of chromatin. Comparison of domains in the sequences of Drosophila Ball (also called NHK-1) and VRK in human, mouse, Xenopus and C. elegans, shows that C. elegans and Drosophila are the only homologues with a long C-terminal sequence extension; the other species have a relatively short stretch of sequence following the kinase domain. This suggests that Ball has a different function in the invertebrates; in Drosophila, the C-terminal sequence binds to Kin1 (Katzemich, 2015).
MASK was first identified in the developing eye of Drosophila, where it is required for proliferation and differentiation of photoreceptors. MASK genetically interacts with Corkscrew (CSW), a protein phosphatase that acts downstream of the epidermal growth factor receptor (EGFR) in a signalling pathway involved in myogenesis in Drosophila. Through these interactions, Obscurin can potentially be linked to a receptor tyrosine kinase (RTK) pathway involved in myogenesis. Obscurin and Kettin are present at an early stage in the development of pupal IFM. Both are large titin-like proteins with tandem Ig domains, which have a dual function in myogenesis and in the mature muscle. Obscurin kinase domains appear to be scaffolds for binding MASK. Ankyrin repeats act as adaptor modules, binding cytoskeletal proteins and signalling molecules. The repeats stabilise protein networks, often together with large structural proteins. Therefore, an interaction between obscurin kinases and MASK could provide a platform for the assembly of signalling proteins, and this could be affected by force on the obscurin molecule. Ankyrin-repeat proteins in vertebrate skeletal muscle (MARPs) interact with the N2A elastic region of titin; in the case of Ankrd2 (also known as Arpp), expression is induced by stretch, and MARPs are thought to be involved in stretch-induced signalling pathways. There are two smaller homologues of MASK in human cells: MASK1 (Ankhd1) and MASK2 (Ankrd17), which have the same domain structure as Drosophila MASK. The Drosophila protein (~400 kDa) is larger than the human proteins (~280 kDa), mainly due to a longer stretch of sequence between the ankyrin repeat domains. MASK is a cofactor of Drosophila Yorkie (Yki) and mammalian Yes-associated protein (YAP) in the Hippo signalling pathway, which controls tissue growth. The signalling function in this pathway is similar for Drosophila and human MASK; however, MASK isoforms have not been found it human muscle. There is a sequence in the C. elegans genome that codes for a protein with homology to Drosophila, mouse and human MASK. The protein, ankyrin repeat and KH domain-containing protein is predicted to be 287 kDa, so similar in size to the human protein. The function is unknown (Katzemich, 2015).
The presence of Ball and MASK in mature IFM suggests the proteins have a signalling function in the adult fly. There is turnover of contractile proteins in Drosophila muscles, including the IFM, throughout the life of a fly. The function of obscurin kinase domains as scaffolds for the assembly of signalling proteins is likely to be important in the continual remodelling of the muscle. During contraction, the M-line experiences shearing stress, due to unbalanced forces in the two half sarcomeres, and the M-line is thought to act as a strain sensor. Ball and MASK might be recruited to the M-line in response to mechanical stress sensed by obscurin. Importantly, mutations in human titin kinase lead to a phenotype (Z-disc streaming) similar to that of Ball and MASK knockdowns, possibly by disrupting protein turnover, which supports the finding that Z-disc abnormalities can be an indirect consequence of mutations in proteins associated with the M-line (Katzemich, 2015).
In summary, this study has identified two proteins, Ball and MASK, that are essential for the assembly of an ordered IFM. The pseudokinase domains of obscurin act as scaffolds binding the proteins. This raises the possibility of investigating the regulation of signalling pathways involved in assembly and maintenance of IFM through interaction with obscurin (Katzemich, 2015).
In sarcomeres, α-actinin crosslinks thin filaments and anchors them at the Z-disc. Drosophila melanogaster Zasp52 also localizes at Z-discs and interacts with α-actinin via its extended PDZ domain, thereby contributing to myofibril assembly and maintenance, yet the detailed mechanism of Zasp52 function is unknown. This study shows a strong genetic interaction between actin and Zasp52 during indirect flight muscle assembly, indicating that this interaction plays a critical role during myofibril assembly. The results suggest that Zasp52 contains an actin-binding site, which includes the extended PDZ domain and the ZM region. Zasp52 binds with micromolar affinity to monomeric actin. A co-sedimentation assay indicates that Zasp52 can also bind to F-actin. Finally, in vivo rescue assays of myofibril assembly were used to show that the α-actinin-binding domain of Zasp52 is not sufficient for a full rescue of Zasp52 mutants suggesting additional contributions of Zasp52 actin-binding to myofibril assembly (Liao, 2020).
Myosin is an essential motor protein, which in muscle is comprised of two molecules each of myosin heavy-chain (MHC), the essential or alkali myosin light-chain 1 (MLC1), and the regulatory myosin light-chain 2 (MLC2). It has been shown previously that MLC2 phosphorylation at two canonical serine residues is essential for proper flight muscle function in Drosophila; however, MLC2 is also phosphorylated at additional residues for which the mechanism and functional significance is not known. This study found that a hypomorphic allele of Pkcδ causes a flightless phenotype; therefore, it was hypothesized that PKCδ phosphorylates MLC2. Flight disability was rescued by duplication of the wild-type Pkcδ gene. Moreover, MLC2 is hypophosphorylated in Pkcδ mutant flies, but it is phosphorylated in rescued animals. Myosin isolated from Pkcδ mutant flies shows a reduced actin-activated ATPase activity, and MLC2 in these myosin preparations can be phosphorylated directly by recombinant human PKCδ. The flightless phenotype is characterized by a shortened and disorganized sarcomere phenotype that becomes apparent following eclosion. It is concluded that MLC2 is a direct target of phosphorylation by PKCδ, and that this modification is necessary for flight muscle maturation and function (Vaziri, 2020).
The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. This study shows that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) (Cheerio) and CryAB. NUAK biochemically and genetically interacts with the cochaperone Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. It was further demonstrated that Fil/Cheerio is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), ref(2)p/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, these results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases (Brooks, 2020).
Proteins must fold into an intrinsic three dimensional structure to perform distinct cellular functions. Denatured or misfolded proteins can be refolded by chaperones or are subject to degradation by the ubiquitin-proteasome system (UPS) and/or the autophagosome-lysosome pathway (ALP). The accumulation of misfolded proteins upon genetic mutation or decreased chaperone function causes protein aggregates that are not effectively cleared by the UPS or the ALP. Environmental insults or aging may exacerbate this accumulation of misfolded proteins, resulting in disease and eventual cell death (Brooks, 2020).
A specialized autophagy pathway, termed chaperone-assisted selective autophagy (CASA), has been verified in both Drosophila and mammalian systems. The CASA complex includes BAG3 in concert with the chaperones HSC70/HSPA8 (HSP70 family), HSPB8 (small HSP family), and the ubiquitin (Ub) ligase CHIP/STUB1. CASA regulates the removal and degradation of Fil from the Z-disc in striated muscle or actin stress fibers in non-muscle cells. The N-terminal actin-binding domain (ABD) in Fil is followed by multiple immunoglobulin (Ig)-like repeats which bind numerous proteins to link the internal cytoskeleton to the sarcolemma. Tension exerted by contractile muscle tissue requires continuous folding and refolding of individual Ig-like domains in Fil, eventually damaging the ability of the protein to sense and transmit mechanical strain. The BAG3-HSC70 protein complex binds to the mechanosensor region (MSR) of Fil and upon detection of protein damage, CHIP ensures the addition of polyubiquitin (polyUb) moieties. Rather than promoting delivery to the proteasome, these Ub chains instead recruit the autophagic Ub adapter protein p62/SQSTM1. p62 interacts with Atg8a/LC3 to induce autophagophore formation and the subsequent clearance of Fil through lysosomal degradation. Fil aggregates and a block in autophagosome-lysosome fusion are present in lysosomal associated membrane protein 2 (LAMP2)-deficient muscles, thus linking impaired autophagy to abnormal protein deposits (Brooks, 2020).
Drosophila NUAK encodes for a conserved serine/threonine kinase that is homologous to the mammalian kinases NUAK1/ARK5 and NUAK2/SNARK. These proteins comprise a family of twelve AMP-activated protein kinase (AMPK)-related kinases (NUAK1 and 2, BRSK 1 and 2, QIK, QSK, SIK, MARK 1-4, and MELK) that share a conserved N-terminal kinase domain activated by the upstream liver kinase B1 (LKB1). NUAK1 and NUAK2 proteins are broadly expressed, but enriched in cardiac and skeletal muscle. Muscle contraction and LKB1 phosphorylation can activate both NUAK proteins. NUAK2 activity is additionally stimulated by oxidative stress, AMP, and glucose deprivation in various cell types. Interestingly, NUAK2 expression increases during muscle differentiation and in response to stress or in aging muscle tissue, whereas dominant-negative (DN)-NUAK2 induces atrophy. Homozygous NUAK1 KO mice are embryonic lethal and <10% of NUAK2 homozygotes survive, precluding analysis of post-embryonic contributions. Because of this embryonic lethality, conditional NUAK1 KO mice were generated to examine muscle function . However, no change was observed in muscle mass or fiber size between control or muscle-specific NUAK1 KO mice, likely due to functional redundancy (Brooks, 2020).
The presence of single NUAK orthologs in worms (Unc-82) or flies (NUAK/CG43143) allows for the study of NUAK protein function without compensation from additional family members that may mask cellular roles. unc-82 associates with Paramyosin and likely Myosin B to promote proper myofilament assembly in C. elegans. The kinase domain in Drosophila NUAK shares 61% identity and 80% similarity to human NUAK1 and NUAK2. In flies, RNAi knockdown of NUAK phenocopies weak Lkb1 defects in regulating cell polarity during ommatidial formation and actin cone formation in spermatogenesis. NUAK kinase targets or additional functions in other tissues have not been reported (Brooks, 2020).
This study identified Drosophila NUAK as a key regulator of autophagic protein clearance in muscle tissue. NUAK physically interacts with and phosphorylates Fil [encoded by Drosophila cheerio (cher)]. NUAK also genetically and biochemically interacts with the Stv-Hsc-70-4 complex and Stv overexpression is sufficient to rescue NUAK-mediated muscle deterioration. The identification of Fil as a cargo protein that abnormally accumulates in muscle tissue deficient for NUAK, Stv, Hsc70-4, and Atg8a links protein aggregation to defects in autophagic disposal (Brooks, 2020).
Prior to this study, few substrates of NUAK kinase activity had been uncovered. One of these is Myosin phosphatase targeting-1 (MYPT1), a regulatory subunit of myosin light-chain phosphatase. Two Drosophila regulatory subunits, MYPT75D and Myosin binding subunit (Mbs), were tested in Stv NUAK sensitized genetic assay and no protein aggregation and/or muscle degeneration was observed. While negative, this data nevertheless argues that this family of phosphatases likely does not function with NUAK in muscle tissue. Since the mammalian NUAK1-MYPT1 interaction was identified in vitro and further validated in HEK293 cells, NUAK likely has cell and tissue-specific targets that regulate diverse biological outputs (Brooks, 2020).
Based upon the discovery of Fil as a novel NUAK substrate, two scenarios are envisioned that are not mutually exclusive to explain the molecular function of NUAK in preventing protein aggregation. First, the increase in sarcomere number upon muscle-specific NUAK RNAi suggests that at least one role of NUAK may be to negatively regulate the addition of proteins (such as Fil) into sarcomeres. This data is consistent with studies that show C. elegans unc-82 regulates myofilament assembly. Notably, one key feature of the misincorporated proteins in unc-82 mutants is their inclusion into aggregate-like structures, similar to the accumulation of Fil and CryAB in NUAK-/- muscles. An additional, or alternative possibility, is that NUAK phosphorylates unfolded or 'damaged' Fil for removal from the sarcomere, thereby triggering the Stv-Hsc70-4 complex to promote autophagic turnover. Thus, proteins such as Fil that fail to get incorporated into sarcomeres and/or sustain damage due to repeated rounds of tension-induced muscle contraction, may destabilize myofilament architecture and trigger abnormal protein (Brooks, 2020).
In both contractile muscle tissue and in adherent cells subjected to mechanical force, BAG3 acts as a hub to coordinate Fil-induced tension-sensing and autophagosome formation. The MSR of Fil is comprised of Ig repeats whose conformational transitions between open and closed states dictate differential protein-protein interactions and biological outputs. While the chaperones Hsc70/HSPA8 and HSPB8 weakly bind to the MSR of Fil, this biochemical interaction is greatly enhanced in the presence of BAG3. Interestingly, BAG3 interacts with Ig repeats 19-21 in the MSR, while the selected interaction domain of NUAK with Fil comprises Ig repeats 15-18. These data suggest that NUAK and Stv each bind to a separate region of the MSR in Fil (Brooks, 2020).
It remains to be determined if NUAK-mediated phosphorylation is a prerequisite for the removal of damaged Fil protein by BAG3. The rescue results suggest that this phosphorylation event is not required as Stv overexpression alleviates protein aggregation and muscle degeneration upon a loss of NUAK. An alternative possibility is that this excess Stv protein is present in sufficient amounts to interact with Fil and overcome the necessity for phosphorylation by NUAK. The inability of NUAK overexpression to restore muscle defects due to knockdown of Stv, Hsc70-4, or Atg8a suggests that NUAK functions upstream or parallel to this pathway. It seems likely that NUAK has additional target substrates for kinase activity that may regulate autophagic protein clearance in muscle tissue (Brooks, 2020).
Recent studies demonstrate that increased autophagic degradation of Fil by BAG3 also induces fil transcription as a compensatory mechanism to ensure steady-state Fil levels. Thus, whether loss of NUAK or Stv alters gene expression upon a block in protein clearance was investigated. While the mRNA levels of cher, CryAB, Hsc70-4, or Atg8a were not altered in NUAK or stv mutants, there was a large increase in p62 transcripts. Thus, this increase in p62 mRNA synthesis may contribute to the elevated p62 protein levels observed upon loss of NUAK or Stv as multiple stress conditions increase p62 transcription, including proteasome inhibition, starvation and atrophic muscle conditions. Data that support a role for an autophagic block include the localization of p62 and Atg8a to regions of protein aggregation (Brooks, 2020).
A model for NUAK is proposed that incorporates these new findings with existing roles for BAG3. Fil and CryAB are physically associated at the Z-disc in Drosophila larval muscle. The phosphorylation of Fil by NUAK may control the incorporation of Fil into the Z-disc during myofibril assembly and/or may be required for the disposal of damaged Fil protein. BAG3 and chaperones such as Hsc70/HSPA8 are thought to monitor the MSR of Fil to detect force-induced damage and to promote the addition of K63-linked polyUb chains. Recruitment of the ubiquitin autophagic adapter p62/SQSTM1 induces autophagosome initiation through the accumulation of Atg8a. Eventual fusion of these autophagosomes with lysosomes promotes protein client complex destruction (Brooks, 2020).
Upon loss of NUAK, excess Fil protein that fails to be incorporated into the Z-disc and/or is damaged due to tension-induced muscle contraction begins to accumulate near the Z-disc. The presence of CryAB in Fil-like aggregates may be due to the normal association of CryAB with Fil at the Z-disc, either to monitor Fil protein damage, or to prevent protein aggregation. It is interesting that while both Fil and CryAB contain actin-binding domains, these associations are lost in NUAK-/- muscle tissue as F-actin is displaced from regions of Fil-CryAB accumulation. At this point it cannot be determined if NUAK preferentially binds to the short (~90kD) and/or long (~240 kD) Fil isoforms since the mapped interaction domains (Ig domains 15-18) are present in both isoforms (Brooks, 2020).
In the initial stages of aggregate formation, nearly all Fil puncta are decorated with Ub. It is hypothesized that the observed decrease in Ub-Fil colocalization in large regions of aggregate formation may be due to intrinsic properties of aggregation-prone proteins whereby protein misfolding triggers aggregation of Fil with itself and other proteins. The accumulation of p62 and circular structures that stain positive for Atg8a in regions of Fil accumulation demonstrate that the autophagosome machinery is recruited to BAG3-client complexes. The absence of lysosomes in these aggregate regions suggest that either fusion and/or transport to sites of degradation are compromised (Brooks, 2020).
CASA-mediated autophagy via the BAG3-client complex includes Hsc70-4/HSPA8, HSPB8, and the E3 ligase CHIP/STUB1, the latter of which ubiquitinates Fil for the subsequent recruitment of p62 to initiate autophagosome formation. However, fibroblasts deficient for CHIP are not defective in autophagy and mice or flies lacking CHIP/STUB1 are viable. A failure to enhance protein aggregation defects upon CHIP RNAi knockdown in the sensitized NUAK+/- or stv+/- backgrounds suggests that additional Ub ligases cooperate with the Stv/BAG3 complex to remove damaged proteins. Future studies will also determine which Drosophila protein is the equivalent of HSPB8 since no genetic interactions were observed with putative CG14207 or Hsp67Bc RNAi lines. This negative data does not rule out the possibility that protein levels are not reduced enough to see phenotypes upon RNAi induction or possible functional redundancy exists between CG14207 and Hsp67Bc (Brooks, 2020).
An interesting hallmark of protein aggregate diseases is the accumulation of specific proteins in affected cells or tissues. Thus, proteins susceptible to aggregation in vivo may possess specific structural characteristics or shared biological functions. This latter feature is evident in a group of protein aggregate diseases termed myofibrillar myopathies (MFM). Laser microdissection of aggregates from normal or affected muscles reveal specificity in the types of proteins that accumulate in patients afflicted with MFMs. Common proteins present in these aggregates include Filamin C (FILC), αB-crystallin (CRYAB), BAG3, and Desmin (DES), among others. The inability of MFM patients to clear these aggregates results in myofibrillar degeneration and a decline in muscle function. Interestingly, mutations in Drosophila NUAK phenocopy both structural and functional deficits observed in MFM patients, including Fil and CryAB accumulation, muscle degeneration, and locomotor defects. The discovery of cellular degeneration and protein aggregation in muscle tissue upon loss of the single fly NUAK ortholog highlights the power of Drosophila as a model. Future studies will focus on identifying kinase targets of NUAK and defining additional proteins that function in NUAK and stv-mediated autophagy for the eventual development of therapeutic targets to treat MFMs and other protein aggregate diseases (Brooks, 2020).
Nuclei are actively positioned and anchored to the cytoskeleton via the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex. This study identified mutations in the Parkin-like E3 ubiquitin ligase Ariadne-1 (Ari-1) that affect the localization and distribution of LINC complex members in Drosophila. ari-1 mutants exhibit nuclear clustering and morphology defects in larval muscles. Ari-1 mono-ubiquitinates the core LINC complex member Koi. Surprisingly, functional redundancy between Parkin and Ari-1 was discovered: increasing Parkin expression rescues ari-1 mutant phenotypes and vice versa. It was further shown that rare variants in the human homolog of ari-1 (ARIH1) are associated with thoracic aortic aneurysms and dissections, conditions resulting from smooth muscle cell (SMC) dysfunction. Human ARIH1 rescues fly ari-1 mutant phenotypes, whereas human variants found in patients fail to do so. In addition, smooth muscle cells (SMCs) obtained from patients display aberrant nuclear morphology. Hence, ARIH1 is critical in anchoring myonuclei to the cytoskeleton (Tan, 2018).
The localization of a nucleus in a specific subcellular location is a highly regulated and dynamic process. Depending on the cell type, nuclear localization can affect cell division and differentiation, or cellular function. In humans, loss of proper nuclear positioning has been implicated in muscular dystrophy, lissencephaly, cerebellar ataxia, and deafness. The LINC complex is required for proper nuclear localization and consists of various proteins (Lamins, SUN [Sad1 and UNc84 homology proteins] and KASH [Klarsicht, ANC-1, and Syne Homology] proteins) that cross the nuclear envelope and project into the cytoplasm to connect the nucleoskeleton to the cytoskeletal network. Lamins, the main component of the nuclear lamina, interact with SUN proteins that cross the inner nuclear membrane into the perinuclear space. Within the perinuclear space, the SUN domain interacts with the C terminus of KASH proteins that in turn span the outer nuclear membrane and interact with various members of the cytoskeleton. Apart from placing a nucleus in its appropriate position in the cell, the LINC complex also endows a cell with the ability to respond to changes in its micro-environment, such as changes in cell shape or migration. This process was recently dubbed nuclear mechanosensing. External stimuli that alter cell shape introduce mechanical tension on the plasma membrane, which is transferred through the cytoskeleton and the LINC complex to alter nuclear morphology and/or transcription. This suggests that the LINC complex is required for mechanosensing, an important feature for cells, in particular muscle cells that have to respond to constant changes in mechanical forces that are applied to them (Tan, 2018).
Some of the LINC complex components have been shown to be the subject of few posttranslational modifications. However, a role for ubiquitination in the regulation of the LINC complex has not been documented so far. The human genome encodes ∼700 E3 ligases, 14 of which are RING-between-RING (RBR) ligases. The best-known member of the RBR family in Drosophila is encoded by the parkin locus, because mutations in human PARK2 (parkin) cause a recessive form of early-onset Parkinson's disease. Parkin plays a role in clearing dysfunctional mitochondria based on fly mutants and mammalian cell culture experiments, but numerous mouse knockouts of Park2 have revealed little about the protein's in vivo function in mice. Much less is known about the other members of the RBR family of E3 ligases. The human homolog of Ari-1 (ARIH1) was recently shown to function together with Cullins of the RING family of E3 ligases to regulate several cell cycle regulators. In this setting, ARIH1 transfers the first ubiquitin molecule to the Cullin substrate, upon which Cullin lengthens the poly-ubiquitin chain. In flies, Ari-1 was shown to be an essential protein that can dimerize and can regulate the insect hormonal ecdysone signaling pathway (Tan, 2018).
Interestingly, although Parkin and Ari-1 are thought to have unique biological functions, the two proteins are similar at the structural level and Ari-1 interacts with a subset of established Parkin substrates. In addition, both proteins are activated by the same E2 conjugating enzyme, UbcD10 (fly)/UBCH7 (human) (Tan, 2018).
This study identified lethal mutations in ari-1 from an X chromosome mutagenesis screen in Drosophila. Phenotypic characterization of the mutants revealed a role for Ari-1 in positioning nuclei in muscles by regulating the LINC complex. This study shows that Ari-1 mono-ubiquitinates Koi. In the absence of Ari-1, Parkin can partially compensate for the loss of Ari-1 and vice versa. In contrast, ari-1 missense mutations act in a dominant negative fashion and the mutant forms of Ari-1 affect Parkin, inhibiting its function in nuclear positioning. Furthermore, three variants of ARIH1 were identified in patients with aortic or cerebrovascular aneurysms. Aortic tissues from these patients reveal that vascular smooth muscle cell (vSMC) nuclear morphology is severely affected. Further testing of the identified ARIH1 variants in Drosophila show that the mutant forms of ARIH1 are less functional or non-functional. In sum, this study has identified an evolutionarily conserved role of Ari-1/ARIH1 with Parkin in regulating myonucleo-cytoskeletal anchorage and shape, and variants in ARIH1 are associated with developing aortic aneurysms in humans (Tan, 2018).
This study reveals that the RBR E3 ubiquitin ligase Ari-1 regulates the subcellular localization and morphology of muscle nuclei in flies. Loss of ari-1 affects the protein levels and/or distribution of members of the LINC complex. Interestingly, Ari-1 and Parkin, a second RBR E3 ligase family member, exhibit redundant functions. Indeed, overexpressing parkin in ari-1 mutants suppresses the observed phenotypes and vice versa. Koi, a subunit of the LINC complex, can be ubiquitinated by both Ari-1 and Parkin. Finally, rare dominant variants were identified in the human homolog of ari-1, ARIH1. These variants are associated with cerebrovascular and aortic aneurysms and this study documented that patient samples have altered nuclear morphology (Tan, 2018).
Loss of ari-1 leads to clustering of nuclei in Drosophila striatal skeletal muscles. The protein distribution of several LINC complex members is affected in ari-1 mutants. The data show that Koi is the target of Ari-1. First, Koi protein levels are upregulated in ari-1 mutants, whereas Koi mRNA levels remain unchanged. Second, in human vSMCs, the protein level of the Koi homolog, SUN2, is elevated upon ARIH1 knockdown. Third, overexpressing Koi phenocopies ari-1 mutants. Fourth, Koi is the only LINC complex member that physically interacts with Ari-1 when tested in cell culture. Fifth, Ari-1 mono-ubiquitinates Koi in in vitro experiments. It is proposed that Ari-1's mono-ubiquitination of Koi primes Koi for subsequent poly-ubiquitination and degradation by another E3 ligase. This other E3 ligase could be Cullin-1, as, in human cells, the Cullin-1 ubiquitination complex regulates the turnover of SUN2. Furthermore, a cooperative ubiquitin-tagging activity has been shown for vertebrate ARIH1 and Cullin-1 (Tan, 2018).
How do elevated levels of Koi impair the LINC complex? One possibility is that excessive amounts of Koi proteins disrupt the stoichiometry of the functional LINC complex, resulting in imbalanced mechanical tensions applied across the nuclear envelope. Also, excessive Koi at the nuclear envelope may redistribute Koi to other compartments continuous to the nuclear envelope, including the endoplasmic reticulum (ER). In fact, overexpressed SUN1 was found to enter the ER-Golgi secretory pathway, and retention of SUN in the Golgi is associated with the pathology of laminopathies, supporting observations that elevated Koi/SUN levels can lead to cellular and physiological defects (Tan, 2018).
The observation that a missense mutation induces a stronger phenotype than its corresponding null mutation was recently dubbed 'recessive antimorph'. This genetic phenomenon reveals the presence of a second, functionally redundant protein that is inhibited by a missense mutant but can execute its function in the absence of the protein. The current data suggest that the missense mutations function as recessive antimorphs because (1) the mutations cause recessive lethality with a dominant muscle nuclear phenotype; (2) mutant phenotypes are rescued by introducing wild-type copies of the gene; (3) crossing ari-1 missense mutant alleles to a deficiency diminishes the nuclear positioning phenotype compared with homozygous/hemizygous missense mutants; and (4) the missense mutant alleles can be rescued in a dose-dependent fashion. These findings strongly support the view that the three identified missense mutations behave as dominant negative alleles (Tan, 2018).
Ari-1 and Parkin are members of the same E3 ligase subfamily and are evolutionarily related. An evolutionary tree of the RBR domains of all Ariadne and Parkin RBR proteins reveals that ariadne genes are the ancestral genes as they, in contrast to parkin, are present in all unicellular eukaryotic organisms tested. Parkin has an established role in mitochondrial quality control. However, loss of PARK2 homologs have been associated with significant phenotypic discrepancies across species and even among cell types in the same organism. These differences have challenged the ability to unravel the in vivo role of Parkin and have questioned the cellular requirements of Parkin. The data argue that there is some functional redundancy between Ari-1 and Parkin. First, the double homozygous mutants are first instar lethal, whereas the single null mutants are viable (parkin) or late pupal lethal with escapers (ari-1). Second, double heterozygous mutants display a much stronger myonuclear clustering phenotype than either heterozygous mutant. Third, overexpressing Parkin partially rescues ari-1 mutant phenotypes. Fourth, overexpressing ARIH1 in parkin mutants ameliorates phenotypes associated with the loss of parkin. Moreover, Ari-1 and Parkin can form homodimers or homomultimers. In addition, Ari-1 can be found in a complex with Parkin. These data are therefore most consistent with the following model: it is proposed that there are three complexes (Ari-1/Ari-1, Parkin/Parkin, Ari-1/Parkin), each of which can perform some functions related to nuclear and mitochondrial phenotypes, but they differ in efficiency. Under normal conditions, Ari-1 mainly regulates nuclear positioning and morphology but is aided by Parkin. In the absence of Ari-1, Parkin can still regulate nuclear positioning but with lower efficiency leading to a mild nuclear clustering phenotype. Although the requirement of Ari-1 is clearly more prominent for nuclear phenotypes than the requirement of Parkin, the reverse is true for mitochondrial quality control. In the latter case, Parkin plays the major role and no mitochondrial morphology phenotype is observed in Ari-1 mutants, but Ari-1 can clearly suppress some of the mitochondrial phenotypes associated with loss of parkin. Finally, in an ari-1 missense mutant background, mutant Ari-1 sequesters Parkin in a non-functional complex, causing a stronger nuclear positioning phenotype (Tan, 2018).
Is this functional redundancy observed among all RBR family members? Although the data show that Ari-1, Parkin, and Ari-2 can form pairwise complexes such as Ari-1/Ari-2, Parkin/Ari-1, and Parkin/Ari-2, overexpressing Ari-2 does not rescue nuclear clustering phenotypes in ari-1 mutants, suggesting no redundancy exists between these two Ari members for this function. It is, however, possible that, in different cellular contexts, one or more RBR protein(s) may be able to compensate for the loss of another RBR protein, and different species may have evolved to express these proteins at different levels in various tissues. Hence, the observed phenotypic discrepancies between parkin mutants in different species and cells may be attributable to redundancy/dependency issues between the Ari-1 and Parkin isoforms (Tan, 2018).
This study has identified three rare variants in ARIH1 in patients with aortic and cerebrovascular aneurysms. All three variants likely represent loss-of-function alleles because they, in contrast with wild-type ARIH1, fail to rescue nuclear clustering, eclosion, and longevity of ari-1 mutant flies. Similarly, the nuclei of patients' vSMCs display aberrant nuclear morphology, and reducing ARIH1 expression in vSMCs induces nuclear morphological changes, as observed in the patients (Tan, 2018).
How could loss of ARIH1 lead to aneurysms? To date, the genes associated with aortic aneurysms can be divided into several groups based on the cellular process that is affected, one of which is cellular mechanotransduction. Mechanotransduction is important for muscles to adapt and respond to dynamic mechanical forces in their environment. In vascular smooth muscle cells (vSMCs), proper contraction and mechanosensing depends on functional contractile units and their connections to the extracellular matrix through focal adhesions. Genes that encode major structural components of the contractile unit, ACTA2 (encodes SMC-specific α-actin), MYH11 (SMC-specific myosin heavy chain), MYLK (myosin light-chain kinase), PRKG1 (type I cyclic glutathione S-transferase [GMP]-dependent protein kinase), and the extracellular matrix component FBN1 (encodes fibrillin-1) have been shown to be mutated in aortic aneurysm patients. It has been proposed that, in these cases, mechanotransduction between vSMCs and the extracellular matrix is impaired, affecting mechanosensing of the vSMCs and thereby altering molecular pathways leading to aortic disease. Given that contractile units in vSMCs are connected to both the focal adhesions and the nuclear membrane, and that proper mechanosensing is important in preventing aneurysms, the observation that ARIH1 disease-associated variants disrupt the nuclear envelope's structure in vSMCs implicates a defect in mechanosensing that may be responsible for the aortic and cerebrovascular diseases (Tan, 2018).
Interestingly, a growing body of evidence implicates the LINC complex in mechanosensing. Integrin adhesion complexes relay extracellular signals via the cytoskeleton and the LINC complex onto the nuclear envelope to permit mechano-regulation of gene expression. Mutations in the LINC complex members have been associated with striatal and cardiac muscle diseases as well as vascular diseases. For example, mutations in Lamin (LMNA), and SUN genes (SUN1 and 2) are associated with muscular dystrophies. Interestingly, missense mutations in SYNE1 (the human Msp-300 homolog) are associated with dominant muscular dystrophies, while mutations that encode truncated SYNE1 cause recessive cerebellar ataxias with no overt muscular phenotype. However, all of the above mutations affect nuclear positioning or nuclear envelope integrity (Tan, 2018).
Given that Ari-1 regulates the LINC complex in flies, it is highly likely that it assumes a similar role in human vSMCs. Loss of ARIH1 in aortic vSMCs would thereby affect mechanosensing, weaken the aortic wall muscles to induce aneurysms and dissections. It is intriguing to note that patients with ARIH1 mutations have no reported skeletal muscle abnormalities, even though mutations in members of the LINC complex are associated with muscular dystrophies. The current data suggest that functional redundancies of RBR proteins in skeletal muscles may underlie the lack of phenotype. Interestingly, patients with Parkinson's disease are about two times more likely to develop heart failure, suggesting that parkin may be a risk factor for heart disease (Tan, 2018).
In summary, Ari-1 regulates nuclear morphology in Drosophila skeletal muscles and human vSMCs. Loss of ARIH1 is associated with aortic and cerebrovascular aneurysms, likely due to the disruption of mechanosensing in vSMCs. This study also found that Ari-1 and Parkin are to some extent functionally redundant as either gene can partially suppress some of the phenotypes associated with the loss of the other gene. Hence, Ari-1 and Parkin regulate nuclear positioning and mitochondrial quality control respectively but have each retained the ability to perform both functions to some extent (Tan, 2018).
Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. This study reports that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type-specific gene and splice isoform expression, notably loss of an indirect flight muscle-specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. It was further shown that Rbfox1 directly binds the 3'-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type-specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type-specific splicing and expression dynamics of identity genes and structural proteins (Nikonova, 2022).
Tankyrase (Tnks) transfers poly(ADP-ribose) on substrates. Whereas studies have highlighted the pivotal roles of Tnks in cancer, cherubism, systemic sclerosis, and viral infection, the requirement for Tnks under physiological contexts remains unclear. This study report that the loss of Tnks or its muscle-specific knockdown impairs lifespan, stress tolerance, and energy homeostasis in adult Drosophila. Tnks is a positive regulator in the JNK signaling pathway, and modest alterations in the activity of JNK signaling can strengthen or suppress the Tnks mutant phenotypes. JNK was identified as a direct substrate of Tnks. Although Tnks-dependent poly-ADP-ribosylation is tightly coupled to proteolysis in the proteasome, it was demonstrated that Tnks initiates degradation-independent ubiquitination on two lysine residues of JNK to promote its kinase activity and in vivo functions. This study uncovers a type of posttranslational modification of Tnks substrates and provides insights into Tnks-mediated physiological roles (Li, 2018).
Tankyrase (Tnks) belongs to the poly(ADP-ribose) polymerase (PARP) superfamily, which consists of 17 members in human. PARP-1 is the founding member of the family and has a critical role in the repair of DNA damage. The PARPs are characterized by a structurally similar PARP catalytic domain that successively transfers ADP-ribose from NAD+ onto substrate proteins. This post-translational modification is referred to as poly-ADP-ribosylation, or PARsylation. It has been shown that some PARPs actually catalyze mono-ADP-ribosylation rather than the polymerization of poly(ADP-ribose) chains. Because of this reason, the PARP family members have been renamed as ADP-ribosyltransferases diphtheria toxin-like (ARTDs). Tnks was identified as a telomere-associated protein that binds to the telomere-specific DNA binding protein TRF1. In addition to the PARP domain, Tnks contains two unique domains that distinguish it from other PARPs, including an Ankyrin repeat domain that is involved in the recruitment of substrate and a sterile alpha motif (SAM) that mediates oligomerization. Tnks is evolutionarily conserved in human, mouse, rat, chicken, C. elegans, and Drosophila. The human and mouse genome encodes two Tnks proteins (Tnk1/PARP5A/ARTD5 and Tnk2/PARP5B/ARTD6), whereas there is only one Tnks homolog in Drosophila (Li, 2018).
Tnks catalyzes PARsylation on its substrates and is involved in a variety of cellular processes, such as telomere homeostasis, cell-cycle progression, Wnt/β-catenin signaling, PI3K signaling, Hippo signaling, glucose metabolism, stress granule formation, and proteasome regulation. Various amino acid residues including lysine, arginine, aspartate, glutamate, asparagine, cysteine, phospho-serine, and diphthamide may serve as acceptors for PARsylation. In many cases, proteins modified by Tnks are subsequently poly-ubiquitinated and targeted for proteasomal degradation. For example, Tnks promotes telomere elongation by mediating the degradation of TRF1, a negative regulator of telomere length maintenance. Tnks PARsylates the β-catenin destruction complex component Axin, triggers its degradation, and thereby activates Wnt signaling. Tnks also regulates the stability of centrosomal P4.1-associated protein CPAP to limit centriole elongation during mitosis. On the other hand, the effects of PARsylation on some Tnks substrates, such as the nuclear mitotic apparatus protein NUMA, have not been elucidated, implying that alternative regulation following PARsylation may exist (Li, 2018).
Aberrant Tnks expression or activity has been implicated in a diversity of diseases including cancer, systemic sclerosis, cherubism, Herpes simplex, and Epstein-Barr viral infections. Particularly, the pro-oncogenic role of Tnks in many types of cancer strongly suggests a therapeutic benefit of Tnks inhibition. Whereas a substantial amount of studies have highlighted the important functions of Tnks under pathological conditions, the requirement for Tnks under physiological contexts is largely unexplored (Li, 2018).
This study investigate the physiological functions of Tnks during the adult stage in Drosophila with its mutant alleles and RNAi strains. The loss of Tnks was shown to impair lifespan, stress tolerance, and energy storage in adult flies. Ubiquitous or muscle-specific knockdown of Tnks causes defects similar to those observed in the mutant alleles. It was further shown that Tnks is specifically required for the activity of the c-Jun N-terminal kinase (JNK) and positively regulates the outputs of JNK signaling during organ development. In addition, mild reduction and slight increase in the activity of JNK signaling via genetic manipulations can strengthen and suppress the phenotypes of the Tnks mutants, respectively. Last, the results reveal for the first time that Tnks mediates degradation-independent ubiquitination on its substrate. Drosophila JNK was identified as a direct substrate protein of Tnks. The PARsylation by Tnks triggers K63-linked poly-ubiquitination on JNK, enhancing its kinase activity and maintaining its in vivo functions. Together, this study uncovers that Tnks is a positive regulator of JNK activity, mediates a novel type of post-translational modification, and functions through JNK signaling to affect lifespan, stress resistance, and energy homeostasis in Drosophila (Li, 2018).
PARsylation by Tnks appears to be tightly coupled to poly-ubiquitination and subsequent proteasome-dependent degradation, as observed for known Tnks substrates including Axin, TRF1, PTEN, 3BP2, CPAP, and AMOT family proteins. This study found that Tnks mediated PARsylation and poly-ubiquitination of Drosophila JNK (Bsk), however, without affecting its protein levels. This observation prompted investigation og the form of poly-ubiquitin chain assembled on Bsk in the presence of Tnks. Although all the seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) and the N-terminal methionine residue in the ubiquitin can serve as the attachment site to generate poly-ubiquitin chain, K48-linked and K63-linked poly-ubiquitination are two predominant forms in cells. It is well-established that K48-linked poly-ubiquitination targets substrates to the 26S proteasome and promotes protein degradation, while K63-linked poly-ubiquitin chain performs non-proteolytic functions. Consistently, this study observed that Tnks promoted the assembly of K63-linked poly-ubiquitin chain on Bsk and increased its kinase activity. In contrast, Tnks did not affect K48-linked poly-ubiquitination of Bsk. Although the possibility cannot be completely that these modifications occur on a Bsk partner that tightly binds to Bsk even after stringent washing, this study reveals for the first time that Tnks mediates degradation-independent ubiquitination of its substrate, which may represent a novel type of modification induced by PARsylation. Indeed, it has been reported that PARsylation of some Tnks substrates such as the proteasome regulator PI31 and the mitotic spindle-pole protein NuMA may affect the activities of these proteins. It will be interesting to investigate whether similar modification occurs during these processes (Li, 2018).
JNK is an evolutionarily conserved stress-activated protein kinase (SAPK) and is one of the most versatile stress sensors in metazoans. The JNK signaling pathway adapts growth and metabolism to environmental conditions, and mediates stress tolerance, damage repair, and apoptosis. Therefore, JNK signaling has pivotal roles in regulating homeostasis, longevity, and organ development. This study reports an essential role of Tnks in regulating JNK signaling. As an initial hint, adult Tnks mutants are more sensitive to oxidative stress and to starvation, and live much shorter than the wild-type controls. Although several signaling pathways are known to coordinate these functions in Drosophila, this study observed that the loss of Tnks specifically decreased the activity of Bsk without affecting that of ERK, p38, Akt, and Nrf2 signaling. It was further found that Tnks was required for the outputs of JNK signaling during the development of eye, wing, and thorax. The knockdown of Tnks in the thorax strengthened the thoracic cleft phenotype caused by bsk knockdown. The loss of Tnks, or reducing its gene dosage, suppressed ectopic JNK signaling-induced phenotypes during the formation of compound eye and wing vein. In addition, Tnks overexpression in the wing disc activated puc expression in a bsk-dependent manner. Last, it was observed that bsk with mutations in Tnks-induced ubiquitination sites lacked the ability to rescue the lethality of the bsk1/2 mutant and displayed impaired activity in regulating stress tolerance, climbing, energy storage, and organ development compared to wild-type bsk. This work thus reveals Tnks as a positive regulator of the JNK signaling pathway (Li, 2018).
Aberrant Tnks expression or activity has been implicated in a variety of disease states including cancer, cherubism, systemic sclerosis, and viral infection. The proposed roles of Tnks in telomere homeostasis, mitosis, and Wnt signaling have made it an attractive drug target in many types of cancer. Tnks is implied in a diversity of additional cellular processes, such as glucose metabolism, stress granule assembly, and proteasome regulation. However, the requirement for Tnks under physiological contexts remains poorly understood. While a double knockout of Tnks1 and Tnk2 is embryonically lethal in mice, Tnks mutant flies do not display any noticeable developmental defects. Although a previous study reported that Tnks-deficient mice exhibited substantially reduced adiposity, the underlying molecular mechanism is unclear. This work has focused on the physiological changes in Tnks mutant flies during the adult stage. It is reported that the loss of Tnks or its knockdown shortens lifespan, decreases climbing ability, reduces resistance to oxidative stress and starvation, and impairs energy storage in adult flies. Through tissue-specific knockdown, it is concluded that the above functions of Tnks are mainly mediated via its activity in the muscle. These physiological functions of Tnks are attributed to its regulation of JNK signaling activity, which is supported by several lines of evidence. First, the loss of Tnks specifically weakens Bsk activity and JNK signaling. Second, mild reduction in JNK signaling activity by removing one copy of bsk gene aggravates the phenotypes in Tnks mutants, whereas slightly elevated JNK signaling largely rescues these phenotypes. Third, this study shows that Tnks triggers PARsylation and K63-linked poly-ubiquitination of Bsk and enhances the kinase activity and in vivo functions of Bsk (Li, 2018).
Adherens junctions (AJ) are involved in cancer, infections and neurodegeneration. Still, their composition has not been completely disclosed. Poly(ADP-ribose) polymerases (PARPs) catalyze the synthesis of poly(ADP-ribose) (PAR) as a posttranslational modification. Four PARPs synthesize PAR, namely PARP-1/2 and Tankyrase-1/2 (TNKS). In the epithelial belt, AJ are accompanied by a PAR belt and a subcortical F-actin ring. F-actin depolymerization alters the AJ and PAR belts while PARP inhibitors prevent the assembly of the AJ belt and cortical actin. It was asked wondered which PARP synthesizes the belt and which is the PARylation target protein. Vinculin (VCL) participates in the anchorage of F-actin to the AJ, regulating its functions, and colocalized with the PAR belt. TNKS has been formerly involved in the assembly of epithelial cell junctions. TNKS poly(ADP-ribosylates) (PARylates) epithelial belt VCL, affecting its functions in AJ, including cell shape maintenance. In this work, some of the hypothesis predictions have been tested. It was demonstrated that: (1) VCL TBMs were conserved in vertebrate evolution while absent in C. elegans; (2) TNKS inhibitors disrupted the PAR belt synthesis, while PAR and an endogenous TNKS pool were associated to the plasma membrane; (3) a VCL pool was covalently PARylated; (4) transfection of MCF-7 cells leading to overexpression of Gg-VCL/*TBM induced mesenchymal-like cell shape changes. This last point deserves further investigation, bypassing the limits of the transient transfection and overexpression system. In fact, a 5th testable prediction would be that a single point mutation in VCL TBM-II under endogenous expression control would induce an epithelial to mesenchymal transition (EMT). To check this, a CRISPR/Cas9 substitution approach followed by migration, invasion, gene expression and chemo-resistance assays should be performed (Vilchez Larrea, 2021).
Reactive oxygen species, generated as by-products of mitochondrial electron transport, can induce damage to mitochondrial DNA (mtDNA) and proteins. This study investigated whether the moderate accumulation of mtDNA damage in adult muscles resulted in accelerated aging-related phenotypes in Drosophila. DNA polymerase γ (Polγ) is the sole mitochondrial DNA polymerase. The muscle-specific silencing of the genes encoding the polymerase subunits resulted in the partial accumulation of mtDNA with oxidative damage and a reduction in the mtDNA copy number. This subsequently resulted in the production of abnormal mitochondria with reduced membrane potential and, consequently, a partially reduced ATP quantity in the adult muscle. Immunostaining indicated a moderate increase in autophagy and mitophagy in adults with RNA interference of Polγ (PolγRNAi) muscle cells with abnormal mitochondria. In adult muscles showing continuous silencing of Polγ, malformation of both myofibrils and mitochondria was frequently observed. This was associated with the partially enhanced activation of pro-apoptotic caspases in the muscle. Adults with muscle-specific PolγRNAi exhibited a shortened lifespan, accelerated age-dependent impairment of locomotor activity, and disturbed circadian rhythms. These findings in this Drosophila model contribute to understanding how the accumulation of mtDNA damage results in impaired mitochondrial activity and how this contributes to muscle aging (Ozaki, 2022).
The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex transduces nuclear mechanical inputs suggested to control chromatin organization and gene expression; however, the underlying mechanism is currently unclear. This study has shown that the LINC complex is needed to minimize chromatin repression in muscle tissue, where the nuclei are exposed to significant mechanical inputs during muscle contraction. To this end, the genomic binding profiles of Polycomb, Heterochromatin Protein1 (HP1a) repressors, and of RNA-Pol II were studied in Drosophila larval muscles lacking functional LINC complex. A significant increase in the binding of Polycomb and parallel reduction of RNA-Pol-II binding to a set of muscle genes was observed. Consistently, enhanced tri-methylated H3K9 and H3K27 repressive modifications and reduced chromatin activation by H3K9 acetylation were found. Furthermore, larger tri-methylated H3K27me3 repressive clusters, and chromatin redistribution from the nuclear periphery towards nuclear center, were detected in live LINC mutant larval muscles. Computer simulation indicated that the observed dissociation of the chromatin from the nuclear envelope promotes growth of tri-methylated H3K27 repressive clusters. Thus, it is suggested that by promoting chromatin-nuclear envelope binding, the LINC complex restricts the size of repressive H3K27 tri-methylated clusters, thereby limiting the binding of Polycomb transcription repressor, directing robust transcription in muscle fibers (Pavlov, 2023).
Metabolic flexibility of muscle tissue describes the adaptive capacity to use different energy substrates according to their availability. The disruption of this ability associates with metabolic disease. Using a Drosophila model of systemic metabolic dysfunction triggered by yorkie-induced gut tumors, this study showed that the transcription factor REPTOR is an important regulator of energy metabolism in muscles. Evidence is presented that REPTOR is activated in muscles of adult flies with gut yorkie-tumors, where it modulates glucose metabolism. Further, in vivo studies indicate that sustained activity of REPTOR is sufficient in wildtype muscles to repress glycolysis and increase tricarboxylic acid (TCA) cycle metabolites. Consistent with the fly studies, higher levels of CREBRF, the mammalian ortholog of REPTOR, reduce glycolysis in mouse myotubes while promoting oxidative metabolism. Altogether, these results define a conserved function for REPTOR and CREBRF as key regulators of muscle energy metabolism (Saavedra, 2023).
The biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. In this study, temporal muscle inactivation in Drosophila larvae carrying a temperature-sensitive mutation in the shibire (shi1) gene was induced to mimic sedentary behavior during early life and study its transcriptional outcome. The findings indicated a significant change in the epigenetic profile, as well as the genomic profile, of RNA Pol II binding in the inactive muscles relative to control, within a relatively short time period. Whole-genome analysis of RNA-Pol II binding to DNA by muscle-specific targeted DamID (TaDa) protocol revealed that muscle inactivity altered Pol II binding in 121 out of 2010 genes (6%), with a three-fold enrichment of genes coding for lncRNAs. The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. Moreover, inducing muscle inactivation exerted a multi-level impact upon chromatin modifications, triggering an altered epigenetic balance of active versus inactive marks. The downregulated genes in the inactive muscles included genes essential for muscle structure and function, carbohydrate metabolism, longevity, and others. Given the multiple analogous genes in Drosophila for many human genes, extrapolating these findings to humans may hold promise for establishing a molecular link between sedentary behavior and metabolic diseases (Brener, 2023).
Nuclear movement is a fundamental process of eukaryotic cell biology. Skeletal muscle presents an intriguing model to study nuclear movement because its development requires the precise positioning of multiple nuclei within a single cytoplasm. Furthermore, there is a high correlation between aberrant nuclear positioning and poor muscle function. Although many genes that regulate nuclear movement have been identified, the mechanisms by which these genes act is not known. Using Drosophila melanogaster muscle development as a model system, and a combination of live-embryo microscopy and laser ablation of nuclei, studies have found that clustered nuclei encompass at least two phenotypes that are caused by distinct mechanisms. Specifically, Ensconsin is necessary for productive force production to drive any movement of nuclei whereas Bocksbeutel and Klarsicht are necessary to form distinct populations of nuclei that move to different cellular locations. Mechanisitcally, Ensconsin regulates the number of growing microtubules that are used to move nuclei whereas Bocksbeutel and Klarsicht regulate interactions between nuclei (Collins, 2021).
The mitochondrial contact site and cristae organizing system (MICOS) is a multi-protein interaction hub that helps define mitochondrial ultrastructure. While the functional importance of MICOS is mostly characterized in yeast and mammalian cells in culture, the contributions of MICOS to tissue homeostasis in vivo remain further elucidation. This study examined how knocking down expression of Drosophila MICOS genes affects mitochondrial function and muscle tissue homeostasis. CG5903/MIC26-MIC27 was found to colocalize and function with Mitofilin/MIC60 and QIL1/MIC13 as a Drosophila MICOS component; knocking down expression of any of these three genes predictably altered mitochondrial morphology, causing loss of cristae junctions, and disruption of cristae packing. Furthermore, the knockdown flies exhibited low mitochondrial membrane potential, fusion/fission imbalances, increased mitophagy, and limited cell death. Reductions in climbing ability indicated deficits in muscle function. Knocking down MICOS genes also caused reduced mtDNA content and fragmented mitochondrial nucleoid structure in Drosophila. Together, these data demonstrate an essential role of Drosophila MICOS in maintaining proper homeostasis of mitochondrial structure and function to promote the function of muscle tissue (Wang, 2020).
Skeletal muscles play a central role in human movement through forces transmitted by contraction of the sarcomere. It has been recently showed that mammalian sarcomeres are connected through frequent branches forming a singular, mesh-like myofibrillar matrix. However, the extent to which myofibrillar connectivity is evolutionarily conserved as well as mechanisms which regulate the specific architecture of sarcomere branching remain unclear. This study demonstrates the presence of a myofibrillar matrix in the tubular, but not indirect flight (IF) muscles within Drosophila melanogaster. Moreover, it was found that loss of transcription factor H15 increases sarcomere branching frequency in the tubular jump muscles, and it was shown that sarcomere branching can be turned on in IF muscles by salm-mediated conversion to tubular muscles. Finally, it was demonstrated that neurochondrin misexpression results in myofibrillar connectivity in IF muscles without conversion to tubular muscles. These data indicate an evolutionarily conserved myofibrillar matrix regulated by both cell-type dependent and independent mechanisms (Ajayi, 2022).
Organ growth and size are finely tuned by intrinsic and extrinsic signaling molecules. In Drosophila, the BMP family member Dpp is produced in a limited set of imaginal disc cells and functions as a classic morphogen to regulate pattern and growth by diffusing throughout imaginal discs. However, the role of TGFβ/Activin-like ligands in disc growth control remains ill-defined. This study demonstrates that Myoglianin (Myo), an Activin family member, and a close homolog of mammalian Myostatin (Mstn), is a muscle-derived extrinsic factor that uses canonical dSmad2-mediated signaling to regulate wing size. It is proposed that Myo is a myokine that helps mediate an allometric relationship between muscles and their associated appendages. Although Babo/dSmad2 signaling has been previously implicated in imaginal disc growth control, the ligand(s) responsible and their production sites(s) have not been identified. Previous in situ hybridization and RNAi knockdown experiments suggested that all three Activin-like ligands contribute to control of wing size. However, no expression of these Activin-like ligands was found in imaginal discs, with the exception of Actβ which is expressed in differentiating photoreceptors of the eye imaginal disc. It is concluded that the small wing phenotypes caused by RNAi knockdown of Actβ or daw are likely the result of off-target effects and that Myo is the only Activin-type ligand that regulates imaginal disc growth (Upadhyay, 2020).
Although Babo/dSmad2 signaling has been previously implicated in imaginal disc growth control, the ligand(s) responsible and their production sites(s) have not been identified. Previous in situ hybridization and RNAi knockdown experiments suggested that all three Activin-like ligands (Myoglianin, Activinβ, and Dawdle) contribute to control of wing size. However, this study found no expression of these Activin-like ligands in imaginal discs, with the exception of Actβ which is expressed in differentiating photoreceptors of the eye imaginal disc. More importantly, using genetic null mutants, this study showed that only loss of myo affects imaginal disc size. The discrepancy in phenotypes between tissue-specific knockdown results and the genetic nulls is often noted and not fully understood. In addition to simple off-target effects within the wing disc itself, one possible explanation is that many GAL4 drivers are expressed in tissues other than those reported, potentially resulting in deleterious effects for the animal that indirectly affect imaginal disc size. Another possibility is that in Actβ and daw genetic null backgrounds a non-autonomous compensatory signal is generated by another tissue and this signal is not activated in the case of tissue-specific knockdown. Both of these explanations are thought unlikely in this instance since it was demonstrated that only the Babo-A receptor isoform is expressed and required in discs. Since it was previously shown that Daw only signals through isoform Babo-C, it is unclear why knockdown of daw in the wing disc would result in a small wing phenotype as previously reported. It is concluded that the small wing phenotypes caused by RNAi knockdown of Actβ or daw are likely the result of off-target effects and that Myo is the only Activin-type ligand that regulates imaginal disc growth (Upadhyay, 2020).
The signaling ability of TGFβ ligands is modulated by the specific combinations of receptors and co-receptors to which they bind. In Drosophila, the receptor requirements for effective signaling through dSmad2 likely vary for each ligand and tissue. This study found that Myo signaling in the wing disc requires Punt as the type II receptor and Babo-A as the type I receptor. Furthermore, it was establish that Myo is the exclusive Activin-like ligand signaling to the discs since loss of Myo eliminated detectable phosphorylation of dSmad2 in the wing imaginal disc. Since Babo-A is the only isoform expressed in wing discs, it is also concluded that Myo is able to signal through this isoform in the absence of other isoforms. Whether Myo can also signal through Babo-B or C is not yet clear, but in the context of mushroom body remodeling Babo-A also appears to be the major receptor isoform utilized. The co-receptor Plum (see Plum, an Immunoglobulin Superfamily Protein, Regulates Axon Pruning by Facilitating TGF-β Signaling) is also required for mushroom body remodeling, suggesting that Plum and Babo-A are both necessary for efficient Myo signaling. However, it is noteworthy that Plum null mutants are viable while Myo null mutants are not. This observation suggests that Plum is not required for all Myo signaling during development. Further studies will be required to evaluate whether Plum is essential to mediate Myo signaling in imaginal discs (Upadhyay, 2020).
The requirement of Punt as a type II receptor for production of an efficient signaling complex with Myo may be context dependent. In the mushroom body, indirect genetic evidence suggests that the two Type II receptors function redundantly. Although both punt and wit are expressed in imaginal discs, only loss of punt produces a phenotype in the brk reporter assay. To date, clear signaling has not been seen in S2 cells expressing Punt and Babo-A when Myo is added. It is also notable that a previous attempt to study Myo signaling in a heterotypic cell culture model also failed. In that study, Myo was found to form a complex with Wit and Babo-A in COS-1 cells but no phosphorylation of dSmad2 was reported. One explanation is that effective signaling by Myo requires Punt, and Babo-A, and perhaps another unknown co-receptor that substitutes for Plum. Despite this caveat, the current results provide in vivo functional evidence for a Myo signaling complex that requires Babo-A and Punt to phosphorylate dSmad2 for regulation of imaginal disc growth (Upadhyay, 2020).
Final tissue size is determined by several factors including cell size, proliferation, death rates, and duration of the growth period. While cell size changes were observed upon manipulation of Myo signaling, the direction of change depended on the genotype. In myo mutants, estimation of cell size via apical surface area indicates that the cells are ~20% smaller than wild-type. Although this measurement does not indicate the actual volume of the cells, it gives an indication of cell density in the epithelial sheet of the wing pouch, which is analogous to counting cells in the adult wing. RNAi knock down of babo-a in the entire disc produced smaller adult wings with larger (less dense) cells. This result differs from the myo mutant, but is similar to the reported adult wing phenotypes of babo mutants and larval disc phenotypes of dSmad2 mutants. When babo-a is knocked down in one compartment, that compartment is reduced in size with smaller cells. It is concluded that tissue size reduction is the consistent phenotype upon loss of Myo signaling, but cell size changes depend on the specific type of manipulation (Upadhyay, 2020).
While cell size effects may be context dependent, it is notable that neither reduction in size of imaginal discs nor adult wing surface area can be explained solely by a cell size defect. Since no apoptotic increase was seen in myo mutant discs, and because dSmad2 knockdown also fails to alter apoptotic rate, the mostly likely cause is an altered proliferation rate. Consistent with this view is that the large disc phenotype exhibited by dSmad2 protein null mutants is clearly dependent on Myo and it has been previously shown that this is the result of enhanced proliferation. Similarly earlier studies also showed that expression of activated Babo or activated dSmad2 in wing discs also leads to larger wings with slightly smaller cells which is most easily explained by an enhanced proliferation rate. It is worth noting that this proposed enhanced proliferation rate is difficult to detect since cell division is random with regard to space and time during development. Thus a ~ 20% reduction in adult wing size caused by a proliferation defect translates into about 1/5th of disc cells dividing on average one less time throughout the entire time course of larval development. Therefore, without prolonged live imaging, this small reduction in proliferation rate will not be detectable using assays that provide only static snapshots of cell division. It is worth noting, however, that previous clonal studies also concluded that dSmad2 or Babo loss in wing disc clones resulted in a reduced proliferation rate (Upadhyay, 2020).
One attempt to shed light on the transcriptional output of TGFβ signaling responsible for wing disc size employed microarray mRNA profiling of wild-type versus dSmad2 gain- and loss-of-function wing discs. However, this study did not reveal a clear effect on any class of genes including cell cycle components, and it was concluded that the size defect is the result of small expression changes of many genes. Consistent with this view are dSmad2 Chromatin Immunoprecipitation experiments in Kc cells which revealed that dSmad2 is associated with many genomic sites and thus may regulate a myriad of genes (Upadhyay, 2020).
Insect myoglianin is a clear homolog of vertebrate Myostatin (Mstn/GDF8), a TGFβ family member notable for its role in regulating skeletal muscle mass. Mstn loss-of-function mutants lead to enlarged skeletal muscles. Mstn is thought to affect muscle size through autocrine signaling that limits muscle stem cell proliferation, as well as perturbing protein homeostasis via the Insulin/mTOR signaling pathways. Similarly, Gdf11, a Mstn paralog, also regulates size and proliferation of muscles and adipocytes, and may promote healthy aging. Mstn and Gdf11 differ in where they are expressed and function. Mstn is highly expressed in muscles during development while Gdf11 is weakly expressed in many tissues. Both molecules are found to circulate in the blood as latent complexes in which their N-terminal prodomains remain associated with the ligand domain. Activation requires additional proteolysis of the N-terminal fragment by Tolloid-like metalloproteases to release the mature ligand for binding to its receptors. Interestingly in Drosophila, the Myoglianin N-terminal domain was also found to be processed by Tolloid-like factors, but whether this is a prerequisite for signaling has not yet been established. In terms of functional conservation in muscle size control, the results of both null mutants and RNAi depletion indicates that it has little effect on muscle size. This contradicts a previous study in which muscle-specific RNAi knockdown of myo was reported to produce larger muscles similar to the vertebrate observation. The discrepancy between the tissue-specific RNAi knockdown and previous studies is not clear, but the current null mutant analysis strongly argues that Drosophila Myo does not play a role in muscle size control. Intriguingly however, this study found that loss of Actβ, another ligand that signals through Babo and dSmad2, results in a smaller muscles) contrary to that produced by loss of vertebrate Mstn and various other vertebrate Activin family members. Recent data has shown that Drosophila Actβ is the only Activin-like ligand that affects muscle growth, and it does so, in part, by regulating Insulin/Tor signaling in the opposite direction compared to vertebrates. Thus, in Drosophila the Myo/Activin pathway promotes muscle growth while in vertebrates it inhibits muscle growth (Upadhyay, 2020).
The most intriguing finding of this study is that muscle-derived Myo acts non-autonomously to regulate imaginal disc growth. This is in stark contrast to the two BMP ligands, Dpp and Gbb, which are produced by disc cells and act autonomously within the disc itself to regulate both growth and pattern. The fact that a TGFβ ligand can act in an endocrine-like manner is not particularly novel since many vertebrate members of the TGFβ family, including Myostatin, the closest homolog to Drosophila Myoglianin, are found in the blood. Even the disc intrinsic molecule Dpp has been recently shown to be secreted into the hemolymph where it circulates and signals to the prothoracic gland to regulate a larval nutritional checkpoint. Several additional reports indicate that ligands from the Drosophila Activin-like subfamily also circulate in the hemolymph and function as inter-organ signals. For example, muscle-derived Actβ and Myo signal to the fat body to regulate mitochondrial function and ribosomal biogenesis, respectively. In addition, Daw produced from many tissue sources can signal to the Insulin producing cells and the midgut to stimulate Insulin secretion and repress expression of sugar metabolizing genes, respectively. Thus, many TGFβ type factors act as both paracrine and endocrine signals depending on the tissue and process involved (Upadhyay, 2020).
The phenotype of the myo mutant animal supports the claim that endogenous Myo contributes to imaginal disc growth. The ectopic expression assay produced various wing disc sizes when Myo was expressed in different tissues, indicating that the growth response likely depends on the level of Myo being produced in the distal tissue. Loss of glial derived Myo is not sufficient to suppress overgrowth of dSmad2 mutant discs, but overexpression of Myo in glia did partially rescue size of myo null wing discs, likely because the repo-Gal4 driven overexpression produces more ligand than endogenous glia. Likewise, expression from a large tissue such as muscle or fat body likely produces more Myo than glia leading to normal disc growth or even overgrowth. It is also possible that Myo signaling activity is modified depending on the tissue source. Like other TGFβ family members, Myo requires cleavage by a furin protease at its maturation site to separate the C-terminal ligand from the prodomain. Myo may also require a second cleavage by a Tolloid protease family member to achieve full dissociation of the prodomain from the ligand to ensure complete activation. Either of these cleavage reactions, or any other step impacting the bioavailability of active Myo ligand, may vary with tissue or may be modulated by environmental conditions (Upadhyay, 2020).
What is the rationale for larval muscle regulating imaginal discs size? A possible reason is that for proper appendage function, the muscle and the structure (leg, wing, and haltere) that it controls should be appropriately matched to ensure optimal organismal fitness for the environmental niche the adult occupies. For example, a large muscle powering a small wing might result in diminished fine motor control. Conversely, a small muscle may not be able to power a large wing to support flight. However, the multi-staged nature of muscle and appendage development complicates this picture. Larval muscles are histolysed during metamorphosis and do not contribute to the adult muscle. However, remnants of larval muscles in the thoracic segment are preserved as fibers that act as scaffolds upon which the larval myoblasts infiltrate and fuse to become the adult indirect flight muscles. Thus, at least for the indirect flight muscles, the size of the larval muscle scaffold might contribute to the building of a bigger adult muscle. Another possibility invokes a signal relay system. Wg and Serrate/Notch signaling from the wing disc epithelial cells control myoblast proliferation during larval development. Thus it may be that Myo signaling from the larval muscles stimulates proliferation of the disc epithelial layer which in turn enhances Wnt and Serrate/Notch signaling to myoblasts to increase their number thereby coordinating the adult appendage size with muscle size. A final scenario is that, since muscle is a major metabolic and endocrine organ, Myo production may be regulated by the general metabolic state of the larva. If healthy, high levels of Myo, in concert with other growth signals such as insulin, leads to a bigger fly with large wings, and if the metabolic state is poor then lower Myo levels leads to diminished proliferation and a smaller cell size resulting in a smaller fly with small wings (Upadhyay, 2020).
Regardless of the precise mechanism, the ability of the muscle to control appendage size has interesting implications in terms of evolutionary plasticity. The proportionality of insect wing size to body size can vary over a large range, but the mechanism responsible for determining this particular allometric relationship for a given species is not understood. It was recently demonstrated that in Drosophila, motor neuron derived Actβ, another TGFβ superfamily member, can dramatically affect muscle/body size (Moss-Taylor, 2019). Therefore, it is tempting to speculate that evolutionary forces might modulate the activity of these two genes to produce an appropriate body-wing allometry that is optimal for that species' ecological niche (Upadhyay, 2020).
Lysosomes are compartments for the degradation of both endocytic and autophagic cargoes. The shape of lysosomes changes with cellular degradative demands; however, there is limited knowledge about the mechanisms or significance that underlies distinct lysosomal morphologies. This study found an extensive tubular autolysosomal network in Drosophila abdominal muscle remodeling during metamorphosis. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein Syntaxin17 and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. Among ATG-deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, this study revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling (Murakawa, 2020)
The mRNA export pathway is responsible for the transport of mRNAs from the nucleus to the cytoplasm, and thus is essential for protein production and normal cellular functions. A partial loss of function allele of the mRNA export factor Nxt1 in Drosophila shows reduced viability and sterility. A previous study has shown that the male fertility defect is due to a defect in transcription and RNA stability, indicating the potential for this pathway to be implicated in processes beyond the known mRNA transport function. This study investigated the reduced viability of Nxt1 partial loss of function mutants and describes a defect in growth and maintenance of the larval muscles, leading to muscle degeneration. RNA-seq revealed reduced expression of a set of mRNAs, particularly from genes with long introns in Nxt1 mutant carcass. Differential expression was detected of circRNA, and significantly fewer distinct circRNAs expressed in the mutants. Despite the widespread defects in gene expression, muscle degeneration was rescued by increased expression of the costamere component tn (abba) in muscles. This is the first report of a role for the RNA export pathway gene Nxt1 in the maintenance of muscle integrity. These data also links the mRNA export pathway to a specific role in the expression of mRNA and circRNA from common precursor genes, in vivo (van der Graaf, 2021).
Proper muscle function is heavily dependent on highly ordered protein complexes. UNC45 is a USC (named since this region is shared by three proteins UNC45/CRO1/She4P) chaperone that is necessary for myosin incorporation into the thick filaments. UNC45 is expressed throughout the entire Drosophila life cycle and it has been shown to be important during late embryogenesis when initial muscle development occurs. However, the effects of UNC45 manipulation at later developmental times, after muscle development, have not yet been studied. UNC45 was knocked down with RNAi in a manner that permitted survival to the pupal stage, allowing for characterization of sarcomere organization in the well-studied third instar larvae. Second harmonic generation (SHG) microscopy revealed changes in the striated pattern of body wall muscles as well as a reduction of signal intensity. This observation was confirmed with immunofluorescence and electron microscopy imaging, showing diminished UNC45 signal and disorganization of myosin and z-disk proteins. Concomitant alterations in both synaptic physiology and locomotor function were also found. Both nerve-stimulated response and spontaneous vesicle release were negatively affected, while larval movement was impaired. This study highlights the dependency of normal sarcomere structure on UNC45 expression. The known role of UNC45 for myosin localization was confirmed, and it was further shown the I-Z-I complex is also disrupted. This suggests a broad need for UNC45 to maintain sarcomere integrity. Newly discovered changes in synaptic physiology reveal the likely presence of a homeostatic response to partially maintain synaptic strength and muscle function (Karunendiran, 2021).
Myosin dimers arranged in layers and interspersed with non-myosin densities have been described by cryo-EM 3D reconstruction of the thick filament in Lethocerus (Hemiptera) at 5.5 Å resolution. One of the non-myosin densities, denoted the 'red density', is hypothesized to be flightin, an LMM-binding protein essential to the structure and function of Drosophila indirect flight muscle (IFM). Each independent red density winds its way through the myosin dimers, such that it links four dimers in a layer and one dimer in a neighboring layer. This area in which three distinct interfaces within the myosin rod are contacted at once and the red density extends to the thick filament core is designated the "multiface". Present within the multiface is a contact area inclusive of E1563 and R1568. Mutations in the corresponding Drosophila residues interfere with flightin accumulation and phosphorylation. This study examined the LMM area in direct apposition to the red density and identified potential binding residues spanning up to ten helical turns. The red density is associated within an expanse of the myosin coiled-coil that is unwound by the third skip residue and the coiled-coil is re-oriented while in contact with the red density. These findings suggest a mechanism by which Flightin induces ordered assembly of myosin dimers through its contacts with multiple myosin dimers and brings about reinforcement on the level of a single myosin dimer by stabilization of the myosin coiled-coil (Menard, 2021).
Transgelins are a conserved family of actin-binding proteins involved in cytoskeletal remodeling, cell contractility, and cell shape. In both mammals and Drosophila, three genes encode transgelin proteins. Transgelins exhibit a broad and overlapping expression pattern, which has obscured the precise identification of their role in development. This study reports the first systematic developmental analysis of all Drosophila transgelin proteins, namely, Mp20, CG5023, and Chd64 in the living organism. Drosophila transgelins display overall higher sequence identity with mammalian TAGLN-3 and TAGLN-2 than with TAGLN. Detailed examination in different developmental stages revealed that Mp20 and CG5023 are predominantly expressed in mesodermal tissues with the onset of myogenesis and accumulate in the cytoplasm of all somatic muscles and heart in the late embryo. Notably, at postembryonic developmental stages, Mp20 and CG5023 are detected in the gut's circumferential muscles with distinct subcellular localization: Z-lines for Mp20 and sarcomere and nucleus for CG5023. Only CG5023 is strongly detected in the adult fly in the abdominal, leg, and synchronous thoracic muscles. Chd64 protein is primarily expressed in endodermal and ectodermal tissues and has a dual subcellular localization in the cytoplasm and the nucleus. During the larval-pupae transition, Chd64 is expressed in the brain, eye, legs, halteres, and wings. In contrast, in the adult fly, Chd64 is expressed in epithelia, including the alimentary tract and genitalia. Based on the non-overlapping tissue expression, it is predicted that Mp20 and CG5023 mostly cooperate to modulate muscle function, whereas Chd64 has distinct roles in epithelial, neuronal, and endodermal tissues (Vakaloglou, 2021).
he folding of two-dimensional epithelial sheets into specific three-dimensional structures is a fundamental tissue construction mechanism in animal development. A common mechanism that mediates epithelial folding is apical constriction, the active shrinking of cell apices driven by actomyosin contractions. It remains unclear whether cells outside of the constriction domain also contribute to folding. During Drosophila mesoderm invagination, ventrally localized mesoderm epithelium undergoes apical constriction and subsequently folds into a furrow. While the critical role of apical constriction in ventral furrow formation has been well demonstrated, it remains unclear whether, and if so, how the laterally localized ectodermal tissue adjacent to the mesoderm contributes to furrow invagination. This study combined experimental and computational approaches to test the potential function of the ectoderm in mesoderm invagination. Through laser-mediated, targeted disruption of cell formation prior to gastrulation, it was found that the presence of intact lateral ectoderm is important for the effective transition between apical constriction and furrow invagination in the mesoderm. In addition, using a laser-ablation approach widely used for probing tissue tension, this study found that the lateral ectodermal tissues exhibit signatures of tissue compression when ablation was performed shortly before the onset of mesoderm invagination. These observations led to the hypothesis that in-plane compression from the surrounding ectoderm facilitates mesoderm invagination by triggering buckling of the mesoderm epithelium. In support of this notion, it was shown that the dynamics of tissue flow during mesoderm invagination displays characteristic of elastic buckling, and this tissue dynamics can be recapitulated by combining local apical constriction and global compression in a simulated elastic monolayer. It is proposed that Drosophila mesoderm invagination is achieved through epithelial buckling jointly mediated by apical constriction in the mesoderm and compression from the neighboring ectoderm (Guo, 2022).
All growth and/or proliferation may require the reprogramming of metabolic pathways, whereby a switch from oxidative to glycolytic metabolism diverts glycolytic intermediates towards anabolic pathways. This study identified a novel role for TRIM32 in the maintenance of glycolytic flux mediated by biochemical interactions with the glycolytic enzymes Aldolase and Phosphoglycerate mutase. Loss of Drosophila TRIM32, encoded by thin (tn), shows reduced levels of glycolytic intermediates and amino acids. This altered metabolic profile correlates with a reduction in the size of glycolytic larval muscle and brain tissue. Consistent with a role for metabolic intermediates in glycolysis-driven biomass production, dietary amino acid supplementation in tn mutants improves muscle mass. Remarkably, TRIM32 is also required for ectopic growth: loss of TRIM32 in a wing disc-associated tumor model reduces glycolytic metabolism and restricts growth. Overall, these results reveal a novel role for TRIM32 for controlling glycolysis in the context of both normal development and tumor growth (Biwa, 2020a).
The metabolism of all cells must adapt to meet the energetic and biosynthetic needs of growth and homeostasis. For example, tissues composed of non-dividing, differentiated cells must strike a balance between catabolic pathways that provide energy for cellular homeostasis and anabolic pathways that repair the cell and generate cell-type-specific molecules. In contrast, the metabolic requirements of cell growth and proliferation often require a shift toward anabolic pathways that favors the synthesis of macromolecules, such as proteins, lipids, nucleic acids, and complex carbohydrates. Striking this delicate balance between degradative and biosynthetic processes requires the integration of extracellular and intracellular information by complex signaling networks (Biwa, 2020a).
The mechanisms by which cell proliferation and tissue growth rewire metabolism to enhance biosynthesis are diverse and complex. These changes in metabolic flux involve pathways such as the pyrimidine and purine biosynthesis, one carbon metabolism, and the interplay between the citric acid cycle and amino acids pools. However, the pathway most commonly associated with enhanced biosynthesis is glycolysis, where in biological systems ranging from yeast to human T-cells, glycolytic flux is often elevated in the context of cell growth and proliferation. This observation is particularly apparent in the fruit fly Drosophila melanogaster, where the onset of larval development is preceded by a metabolic switch that induces the coordinate upregulation of genes involved in glycolysis, the pentose phosphate pathway, and lactate dehydrogenase (LDH). The resulting metabolic program allows larvae to use dietary carbohydrates for both energy production and biomass accumulation. Moreover, studies of Drosophila larval muscles reveal that this metabolic transition is essential for muscle growth and development, suggesting that glycolysis serves a key role in controlling growth. The mechanisms that control glycolysis specifically in larval muscle, however, remain relatively unexplored. As a result, Drosophila larval development provides an excellent model for understanding how glycolysis and biomass production are regulated in a rapidly growing tissue. Moreover, since larval muscle increases in size without cell divisions, larval muscle provides an unusual opportunity to understand how glycolytic metabolism promotes growth independent of cell division (Biwa, 2020a).
Of the known factors that promote muscle development, TRIM32 is an intriguing candidate for coordinating metabolism with cell growth. This protein is a member of the Tripartite motif (TRIM)-containing family of proteins defined by an N-terminal RING domain, one or two B-boxes, a coiled-coil domain, and a variable C-terminal region. In TRIM32, six Ncl-1, HT2A, Lin-41 (NHL) repeats comprise the C-terminus and are proposed to mediate the diverse functions of TRIM32, including cell proliferation, neuronal differentiation, muscle physiology and regeneration, and tumorigenesis. A single mutation in the B-box region of TRIM32 causes the multisystemic disorder Bardet-Biedl syndrome (BBS), while multiple mutations that cluster in the NHL domains result in the muscle disorders Limb-girdle muscular dystrophy type 2H (LGMD2H) and Sarcotubular Myopathy (STM) (Biwa, 2020a).
A complete understanding of TRIM32 function is confounded by its ubiquitous expression and multitude of potential substrates for E3 ligase activity via the RING domain. Many known TRIM32 target substrates include proteins implicated in muscle physiology, consistent with a role for TRIM32 in LGMD2H. However, additional polyubiquitinated substrates, including p53, Abi2, Piasy, XIAP, and MYCN, are implicated in tumorigenesis. Importantly, TRIM32 protein levels are upregulated in multiple tumor types, suggesting that TRIM32 is a key player in growth regulation. There is precedence for NHL function in controlling cell proliferation as two other Drosophila NHL-containing proteins, Brat and Mei-P26, act as tumor suppressors in the larval brain and female germline, respectively (Biwa, 2020a).
This study provide a novel mechanism for TRIM32 in cell growth. The data show that TRIM32 promotes glucose metabolism through the stabilization of glycolytic enzyme levels. This increased rate of TRIM32-mediated glycolytic flux generates precursors that are utilized for biomass production. Surprisingly, this mechanism operates in both non-dividing muscle cells as well as in proliferating larval brain cells, demonstrating a universal metabolic function for TRIM32 in growth control (Biwa, 2020a).
A unique feature of Drosophila larval development is the inherent glycolytic nature of muscle and brain tissue, which promotes biomass synthesis during this stage of rapid organismal growth. Maintenance of such a high metabolic rate predicts that enzymes are present at sufficient concentrations in the cell to mediate the rapid shunting of intermediates through the pathway. This study shows that TRIM32 directly interacts with and maintains the levels of two glycolytic enzymes. Decreased protein levels of both Ald and Pglym (and possibly other glycolytic enzymes) cripple this rapid flux, effectively blunting the generation of metabolic intermediates that contribute to anabolic synthesis necessary to sustain cell growth (Biwa, 2020a).
It has been recently reported that pathways controlling lactate and glycerol-3-phosphate metabolism function redundantly in larval growth. Removal of LDH and hence lactate production caused an increase in glycerol-3-phosphate, which was sufficient to maintain larval redox balance. Since both LDH and GPDH1 regulate redox balance necessary for maintaining high glycolytic flux to promote biomass accumulation, Ldh, Gpdh1 double mutants exhibit severe growth defects with reduced brain size. The current results show that loss of TRIM32 decreases both lactate and glycerol-3-phosphate levels, thus mimicking the reduced carbohydrate metabolism in Ldh, Gpdh1 double mutants (Biwa, 2020a).
It is not clear how mutations in a ubiquitously expressed protein such as TRIM32 result in tissue-specific diseases. One prediction is that TRIM32 differentially interacts with proteins in diverse cell types to elicit distinct biological outputs. There is strong evidence to support this hypothesis in the context of LGMD2H. TRIM32 is upregulated in proliferating satellite cells and loss of this protein prevents myotube regeneration, partially through the misregulation of NDRG and c-Myc. Muscle-specific targets that contribute to disease progression are less clear. TRIM32-mediated deregulation of key muscle substrates, including actin, α-actinin, tropomyosin, and desmin, contribute to muscle atrophy, but studies have not been performed to directly test this model in the context of LGMD2H. Interestingly, mammalian glycolytic type II fibers are preferentially affected over oxidative type I fibers in muscle atrophy induced by aging/starvation, as well as in Duchenne's and Becker muscular dystrophies (DMD). TRIM32 KO muscles also show a decrease in the glycolytic proteins GAPDH and PyK, just as this study observed a reduction in Ald and Pglym levels in tn-/- muscles, suggesting that TRIM32-mediated regulation of glycolysis may be a general mechanism that underlies some muscular dystrophies (Biwa, 2020a).
The multi-faceted roles exhibited by TRIM32 in muscle physiology and cancer seem quite different on the surface. However, control of glycolytic flux may be a common mode of regulation that has been overlooked. As in muscle tissue, the majority of studies on TRIM32 and cancer have focused on identifying substrates that are subject to poly-ubiquitination and subsequent proteasomal degradation. Piasy, p53 and Abi2 are known targets of TRIM32 E3 activity that regulate the proliferative balance in cancer cells (Albor, 2006; Kano, 2008; Liu, 2014). The proteolytic turnover of these proteins may affect signaling pathways independent of glycolytic TRIM32 regulation or may be a compensatory mechanism in response to metabolic shifts in which normal cells can transiently adopt cancer-like metabolism during periods of rapid proliferation (Biwa, 2020a).
How does this loss of TRIM32 lead to a reduction in glycolytic enzymes? Glycolytic proteins may be substrates for TRIM32 E3 ligase activity. It seems unlikely that TRIM32 polyubiquitinates Ald or Pglym for proteasomal degradation as protein levels are not elevated upon loss of this putative E3 activity. Furthermore, co-immunoprecipitaion of higher molecular weight forms of Ald or Pglym with TRIM32 suggests a yet unidentified post-translational modification. Another possibility, which this study favors, is that the NHL domain of TRIM32 serves as a scaffold for the subcellular localization of glycolytic proteins to limit diffusion of substrates during glycolysis. This does not negate, but rather expands the repertoire of TRIM32 functions (Biwa, 2020a).
Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. Previous work established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. This study shows that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N and 520fs) induce myofibril abnormalities, altered nuclear morphology and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, the protein levels of βPS integrin and Sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant, aberrantly accumulate α- and β-Dystroglycan and α-Sarcoglycan. It is speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration (Biwa, 2020b).
It is unclear how loss of the single TRIM32 protein in muscle tissue initiates or promotes LGMD2H pathology. Interpretation of mouse TRIM32 models created to mimic LGMD2H pathology has proven complex, as both neurogenic and myopathic abnormalities are present in TRIM32KO or TRIM32 D489N knock-in mice. Moreover, loss of TRIM32 in satellite cells limits muscle regeneration, thereby promoting further tissue deterioration. This stud took advantage of the lack of satellite cells in Drosophila larval muscles to separate the muscle-intrinsic function of TRIM32 from its role in mammalian muscle regeneration. It was confirmed that a decrease in neuronal TRIM32 does not contribute to muscle pathology, while the expression of myopathic mutations in larval and adult muscle leads to pathogenic defects with reduced TRIM32 protein levels. Most importantly, this study has substantiated a role for costameric proteins in disease progression (Biwa, 2020b).
TRIM32 is a multidomain protein that consists of RING domains and NHL repeats that are structurally and functionally conserved among different species. At least 12 different molecules, consisting of either RNA or protein, have been shown to interact with NHL-containing proteins. Moreover, mutations and/or deletions in the NHL region of TRIM family members are linked to diverse human diseases. In addition to LGMD2H caused by mutations in TRIM32, axonal neuropathy and gliomas result from mutations in TRIM2 and TRIM3, respectively. However, understanding of how these repeats function in maintaining adequate protein-protein interactions, especially in muscle cytostructure, is limited (Biwa, 2020b).
Despite the moderate amino acid identity between Drosophila and human TRIM32, this study has demonstrated that structural homology of the entire NHL region is remarkably close and serves as an ideal model to investigate the downstream consequences of these myopathic mutations. Previous modeling of the R394H and D487N mutations used the crystal structure of the related NHL-containing protein Drosophila Brat as a template. The curren domain mutation analysis, based upon the structure solved for TRIM32, reveals alterations that impact not only local protein structure. For example, the R394H mutation located in NHL domain 1 causes backbone perturbations and residue energy changes in NHL domains 3 and 4, thus providing a molecular understanding for the destabilization of the R394H mutant relative to WT NHL by DSC. These results provide evidence that mutations exert debilitating effects on NHL structure and reduce protein levels, further contributing to disease progression (Biwa, 2020b).
Similar to LGMD2H patients, the expression of disease-causing alleles (R394H, D487N and 520fs) in Drosophila larval muscle causes muscle degeneration with reduced TRIM32 protein levels and locomotor defects. It has been reported that RNAi-mediated knockdown of TRIM32 disrupts adult myofiber architecture at day 7 and day 11 after tn RNAi induction posteclosion. In contrast, the current genetic assays show that expression of the R394H and 520fs mutations in IFMs cause severe damage to the myofibers by day 1, which continue to degenerate with age. Expression of the D487N mutation also triggered muscle deterioration, but delayed compared with other mutations. It is also clear that TRIM32 protein levels correlate with protein destabilization and muscle degeneration. TRIM32 did not localize in the IFMs of R394H and 520fs mutants, but remained relatively well localized in D487N mutant myofibers until day 3. In all destabilizing mutations, variable accumulation of TRIM32 puncta was observed. The presence of puncta, rather than the complete absence of protein, may indicate that TRIM32 no longer retains its correct sarcomeric association or possibly represents aggregated TRIM32 protein in response to the damage caused by the myopathic mutations. It is believed that the differences in muscle degeneration and TRIM32 expression in different alleles partially explains the variable onset and phenotype severity observed in LGMD2H patients (Biwa, 2020b).
Do components of the dystrophin-glycoprotein components (DGC) and integrin adhesion system play a critical role in LGMD2H disease progression? Mutations in the DGC and integrin adhesion complex are associated with muscular dystrophies and cardiomyopathies. Deficiency of δ-sarcoglycan in mammalian skeletal muscle results in the absence of α-, β-, and γ-sarcoglycan, suggesting that sarcoglycan is a core component of sarcoglycan complex assembly. Similarly, integrin adhesion molecules at the costamere are indispensable for the development and maintenance of sarcomeric architecture. Work performed in mice and flies showed that integrins play an important role in Z-disk formation. Previous work revealed that TRIM32 is essential for costamere integrity, whereby βPS integrin, talin, spectrin, vinculin, and Scgδ accumulate abnormally along the sarcolemma (LaBeau-DiMenna, 2012). The current results further support that loss of costamere stability may be involved in disease pathogenesis, as the expression of TRIM32 mutations also phenocopies the mislocalization of βPS integrin and Scgδ. Similar data were obtained upon expression of a catalytically inactive version of TRIM32 in murine myoblasts, extending in mammals the novel role of TRIM32 in promoting the degradation of Drosophila costamere components. Importantly, although TM protein is mislocalized upon loss of TRIM32, it retains its normal localization when pathogenic alleles are expressed in muscle tissue. These results suggest that TRIM32 can regulate the levels of sarcomeric proteins such as TM, but this accumulation is not sufficient to cause muscle degeneration as observed with costamere proteins (Biwa, 2020b).
Several studies have shown that upregulation of utrophin or integrin α7 partially compensates for the lack of dystrophin in mdx mice or in human DMD patients. Although the integrin adhesion complex (assayed by βPS integrin) and the DGC complex (assayed by Scgδ) are also up-regulated upon loss of TRIM32, this buildup of costameric proteins at the sarcolemma compromises the attachment between the membrane and myofibrils, leading to muscle degeneration. Some or all of these costamere proteins may be substrates for TRIM32 E3 ligase activity. The abnormal accumulation of βPS integrin and Scgδ along the sarcolemma may result from the inability to be ubiquitinated and turned over by the proteasome and/or the inability to maintain its normal localization due to protein damage during muscle contraction (Biwa, 2020b).
There are conflicting data about whether LGMD2H mutations in the NHL domain of TRIM32 inhibit multimer formation or alter ubiquitination activity. It is speculated that disease-causing mutations elevate protein levels of at least a subset of costamere proteins, either by altering protein interactions or by abolishing the catalytic activity of TRIM32. However, additional work is required to characterize the ubiquitination signature by TRIM32 and the resulting fate of substrate proteins. The structural and functional conservation of TRIM32, combined with the muscle-intrinsic Drosophila genetic model, will continue to provide novel insights into LGMD2H initiation and progression not currently available through study in other model organisms (Biwa, 2020b).
In holometabolous insects, developmentally controlled programmed cell death (PCD) is a conserved process that destroys a subset of larval tissues for the eventual creation of new adult structures. This process of histolysis is relatively well studied in salivary gland and midgut tissues, while knowledge concerning larval muscle destruction is limited. This study examined the histolysis of a group of Drosophila larval abdominal muscles called the dorsal external oblique muscles (DEOMs). Previous studies have defined apoptosis as the primary mediator of DEOM breakdown, whose timing is controlled by ecdysone signaling. However, very little is known about other factors that contribute to DEOM destruction. In this paper, the role of thin (tn), which encodes for the Drosophila homolog of mammalian TRIM32, was examined in the regulation of DEOM histolysis. Loss of Tn blocks DEOM degradation independent of ecdysone signaling. Instead, tn genetically functions in a pathway with the death-associated inhibitor of apoptosis (DIAP1), Dronc, and death-associated APAF1-related killer (Dark) to regulate apoptosis. Importantly, blocking Tn results in the absence of active Caspase-3 immunostaining, upregulation of DIAP1 protein levels, and inhibition of Dronc activation. DIAP1 and Dronc mRNA levels are not altered in tn mutants, showing that Tn acts post-transcriptionally on DIAP1 to regulate apoptosis. This study also found that the RING domain of Tn is required for DEOM histolysis as loss of this domain results in higher DIAP1 levels. Together, these results suggest that the direct control of DIAP1 levels, likely through the E3 ubiquitin ligase activity of Tn, provides a mechanism to regulate caspase activity and to facilitate muscle cell death (Vishal, 2018).
PCD is required for the destruction of certain larval tissues during metamorphosis. Previous work established that histolysis of the abdominal muscles is regulated by apoptosis, while blocking autophagy does not affect muscle breakdown. In addition to the ecdysone receptor, only a handful of nuclear proteins are known to function in dorsal exterior oblique muscle (DEOM) histolysis. Loss of East results in a partial block in DEOM degeneration, whereas premature muscle destruction is observed in muscles that lack Chromator. Moreover, the two nuclear receptors, FTZ-F1 and HR39, antagonistically function to regulate the timing of DEOM histolysis. This study has further identified Tn as a novel protein in pupal muscle remodeling. However, loss of Tn does not affect salivary gland and midgut histolysis, highlighting an exclusive muscle role for Tn during Drosophila metamorphosis (Vishal, 2018).
Genetic assays demonstrate that tn functions with core components of the cell death machinery to regulate DEOM destruction. It was surprising that inhibition of apoptotic activity in Dark or Dronc mutants was not sufficient to completely block histolysis by 24 h APF. One explanation is the existence of additional cell death mechanisms other than apoptosis. While histolysing DEOMs contained autophagic vesicles, a reduction in autophagy components did not block or delay muscle degradation at 8 h APF. Tests were performed to see if a decrease in Tn-mediated apoptosis could sensitize muscle cells to initiate autophagy as a compensatory mechanism to assure cell death. However, this does not seem to be the case as RNAi knockdown of Atg1, Atg5, or Atg18 does not further block DEOM histolysis in a tn RNAi background. A second explanation is that the hypomorphic nature of these alleles may not completely abrogate Dark and Dronc function. Alternatively, additional effector caspases, including Dcp-1, Decay, and/or Damm, may be operating in the latter stages of DEOM histolysis since these caspases may function redundantly or act independent of the DIAP1-Drice axis (Vishal, 2018).
More than a partial block in DEOM histolysis was expected upon manipulation of DIAP1 (i.e., DIAP1 OE alone or tn RNAi+DIAP1 OE) at 12 h APF. It is possible that normal or overexpressed DIAP1 levels in the DEOMs are not high enough to block apoptosis, especially using RNAi approaches to reduce Tn levels. However, the use of tn-null alleles clearly shows a complete block in muscle degradation and a corresponding inhibition of active Dronc. Seemingly a delicate balance exists to regulate mRNA and protein expression, as well as protein turnover and proteolytic processing of active caspases. Cells must normally prevent cell death and only activate the apoptotic cascade upon a commitment to die. Thus, threshold levels of caspase activity must be reached for this terminal fate. There is evidence for stage or tissue-specific differential sensitivity to pro-apoptotic factors. Early L3 individuals are resistant to apoptosis, while wandering L3 larvae have elevated levels of Dark, Dronc, and Drice that are sufficient to trigger cell death under the appropriate stimuli. A model is proposed whereby Tn, through its RING domain, normally ubiquitinates DIAP1 for delivery to the proteasome during DEOM histolysis. This degradation of DIAP1 relieves Dronc inhibition, thereby initiating the caspase cascade for the execution of cell death. A general reduction in Tn, or loss of RING domain activity, prevents the addition of ubiquitin moieties and causes an increase in DIAP1 levels, effectively blocking cell death by limiting caspase activity (Vishal, 2018).
Numerous roles have been identified for mammalian TRIM32 in normal and cancerous cells. In muscle, mutations in the NHL repeats result in limb-girdle muscular dystrophy type 2H or sarcotubular myopathy. Several structural muscle proteins are targets of TRIM32 activity, including tropomyosin, desmin, α-actinin, and dysbindin. However, it is not yet clear if regulation of these muscle substrates contributes to normal muscle physiology, is required to prevent atrophy, or plays a critical role in disease pathology. The TRIM32-mediated degradation of additional protein substrates, including p53, Abi2, Piasy, and the X-linked IAP (XIAP), contribute to oncogenic or tumor suppressor activities that either confer resistance or susceptibility to apoptosis. Tumor necrosis factor-α can trigger death receptor-mediated apoptosis through the regulation of XIAP activity. TRIM32 colocalizes and directly interacts with XIAP in human kidney epithelial cells (HEK293). Moreover, TRIM32 induces apoptosis through the direct ubiquitination and subsequent protein turnover of XIAP degradation. This control of apoptotic cell death mirrors the current genetic results, strongly suggesting that this TRIM32-mediated regulation of IAP family members may be a conserved mechanism to regulate apoptosis. It would be interesting to further investigate if Tn and mammalian TRIM32 regulates apoptotic decisions in other contexts of muscle development and/or disease (Vishal, 2018).
Muscle contraction is a fundamental biological process where molecular interactions between the myosin molecular motor and actin filaments result in contraction of a whole muscle, a process spanning size scales differing in eight orders of magnitude. It was hypothesized that a model based on molecular measurements must be modified to include a weakly-bound interaction between myosin and actin in order to fit measurements at the muscle fiber or whole muscle scales. This hypothesis is further supported by the model's need for a weakly-bound state in order to qualitatively reproduce the force response that occurs when a muscle fiber is rapidly stretched a small distance. This hypothesis was tested by measuring steady-state force as a function of shortening velocity, and the force transient caused by a rapid length step in Drosophila jump muscle fibers. Then, by performing global parameter optimization, the predictions of two mathematical models, one lacking a weakly-bound state and one with a weakly-bound state, were quantitatively compared to these measurements. Both models could reproduce the force-velocity measurements, but only the model with a weakly-bound state could reproduce the force transient measurements. However, neither model could concurrently fit both measurements. It was found that only a model that includes weakly-bound cross-bridges with force-dependent detachment and an elastic element in series with the cross-bridges is able to fit both the measurements. This result suggests that the force response after stretch is not a reflection of distinct steps in the cross-bridge cycle, but rather arises from the interaction of cross-bridges with a series elastic element. Additionally, the model suggests that the curvature of the force-velocity relationship arises from a combination of the force-dependence of weakly- and strongly-bound cross-bridges. Overall, this work presents a minimal cross-bridge model that has predictive power at the fiber level (Jarvis, 2021).
The innate immune system is the primary defense response to limit invading pathogens for all invertebrate species. In insects, immune cells are central to both cellular and humoral immune responses, however few genetic resources exist beyond Drosophila to study immune cell function. Therefore, the development of innovative tools that can be widely applied to a variety of insect systems is of importance to advance the study of insect immunity. This study has adapted the use of clodronate liposomes (CLD; a hydrophilic molecule that can be encapsulated within phospholipid bilayers) to deplete phagocytic immune cells in the vinegar fly, Drosophila melanogaster, and the yellow fever mosquito, Aedes aegypti. Through microscopy and molecular techniques, the depletion of phagocytic cell populations was validated in both insect species, and the integral role of phagocytes in combating bacterial pathogens demonstrated. Together, these data demonstrate the wide utility of CLD in insect systems to advance the study of phagocyte function in insect innate immunity (Kumar, 2021).
The endotoxin lipopolysaccharide (LPS) from Gram-negative bacteria exerts a direct and rapid effect on tissues. While most attention is given to the downstream actions of the immune system in response to LPS, this study focuses on the direct actions of LPS on skeletal muscle in Drosophila melanogaster. It was noted in earlier studies that the membrane potential rapidly hyperpolarizes in a dose-dependent manner with exposure to LPS from Pseudomonas aeruginosa and Serratia marcescens. The response is transitory while exposed to LPS, and the effect does not appear to be due to calcium-activated potassium channels, activated nitric oxide synthase (NOS), or the opening of Cl(-) channels. The purpose of this study was to further investigate the mechanism of the hyperpolarization of the larval Drosophila muscle due to exposure of LPS using several different experimental paradigms. It appears this response is unlikely related to activation of the Na-K pump or Ca(2+) influx. The unknown activation of a K(+) efflux could be responsible. This will be an important factor to consider in treatments of bacterial septicemia and cellular energy demands (Potter, 2021).
Recent evidence indicates that the composition of the ribosome is heterogeneous and that multiple types of specialized ribosomes regulate the synthesis of specific protein subsets. In Drosophila, expression of the ribosomal RpS28 protein variants RpS28a and RpS28-like were found to preferentially occurs in the germline, a tissue resistant to aging; it significantly declines in skeletal muscle during aging. Muscle-specific overexpression of RpS28a at levels similar to those seen in the germline decreases early mortality and promotes the synthesis of a subset of proteins with known anti-aging roles, some of which have preferential expression in the germline. These findings indicate a contribution of specialized ribosomal proteins to the regulation of the muscle proteome during aging (Jiao, 2021).
Metabolic adaptations occur with aging but the significance and causal roles of such changes are only partially known. In Drosophila, this study found that skeletal muscle aging is paradoxically characterized by increased readouts of glycolysis (lactate, NADH/NAD+) but reduced expression of most glycolytic enzymes. This conundrum is explained by lactate dehydrogenase (LDH), an enzyme necessary for anaerobic glycolysis and whose expression increases with aging. Experimental Ldh overexpression in skeletal muscle of young flies increases glycolysis and shortens lifespan, suggesting that age-related increases in muscle LDH contribute to mortality. Similar results are also found with overexpression of other glycolytic enzymes (Pfrx/PFKFB, Pgi/GPI). Conversely, hypomorphic mutations in Ldh extend lifespan whereas reduction in PFK, Pglym78/PGAM, Pgi/GPI, and Ald/ALDO levels shorten lifespan to various degrees, indicating that glycolysis needs to be tightly controlled for optimal aging. Altogether, these findings indicate a role for muscle LDH and glycolysis in aging (Hunt, 2021).
Muscle atrophy, or a decline in muscle protein mass, is a significant problem in the aging population and in numerous disease states. Unraveling molecular signals that trigger and promote atrophy may lead to a better understanding of treatment options; however, there is no single cause of atrophy identified to date. To gain insight into this problem, changes in protein profiles during muscle atrophy were investigated in Manduca sexta and Drosophila melanogaster. The use of insect models provides an interesting parallel to probe atrophic mechanisms since these organisms undergo a normal developmental atrophy process during the pupal transition stage. Leveraging the inherent advantages of each model organism, protein signature changes during Manduca intersegmental muscle (ISM) atrophy were defubed and then genetic approaches were used to confirm their functional importance in the Drosophila dorsal internal oblique muscles (DIOMs). The data reveal an upregulation of proteasome and peptidase components and a general downregulation of proteins that regulate actin filament formation. Surprisingly, thick filament proteins that comprise the A band are increased in abundance, providing support for the ordered destruction of myofibrillar components during developmental atrophy. The actin filament regulator Ciboulot (Cib) was uncovered as a novel regulator of muscle atrophy. These insights provide a framework towards a better understanding of global changes that occur during atrophy and may lead to eventual therapeutic targets (Brooks, 2021).
An 'interactome' screen of all Drosophila cell-surface and secreted proteins containing immunoglobulin superfamily (IgSF) domains discovered a network formed by paralogs of Beaten Path (Beat) and Sidestep (Side), a ligand-receptor pair that is central to motor axon guidance. This study describes a new method for interactome screening, the Bio-Plex Interactome Assay (BPIA), which allows identification of many interactions in a single sample. Using the BPIA, four more members of the Beat-Side network were 'deorphanized'. Interactions were confirmed using surface plasmon resonance. The expression patterns of beat and side genes suggest that Beats are neuronal receptors for Sides expressed on peripheral tissues. side-VI is expressed in muscle fibers targeted by the ISNb nerve, as well as at growth cone choice points and synaptic targets for the ISN and TN nerves. beat-V genes, encoding Side-VI receptors, are expressed in ISNb and ISN motor neurons (Li, 2017).
Protein-protein interactions (PPIs) control a vast array of processes in metazoans, ranging from signal transduction and gene regulation within cells to signaling between cells via cell surface and secreted proteins (CSSPs). The strength of PPIs varies widely, from high-affinity interactions that create stable protein complexes to weak transient interactions. Defining global PPI patterns ('interactomes') has been the focus of much recent research. Progress has been made in generating high-throughput protein interaction data for a variety of organisms, including S. cerevisiae, C. elegans and D. melanogaster. Methods used to create interactomes include affinity purification/mass spectrometry (AP-MS) and the yeast two-hybrid assay (Y2H) (Li, 2017).
It is estimated that up to one sixth of human genes encode CSSPs. CSSPs control signaling from the extracellular milieu to cells and the flow of information between cells. Due to their importance and accessibility, CSSPs are often the targets for therapeutic agents, including humanized monoclonal antibody drugs such as checkpoint inhibitors, the non-Hodgkin's lymphoma drug Rituxan, and the breast cancer drug Herceptin. However, the biochemical properties of many CSSP interactions prevent them from being detected by commonly used techniques employed in high-throughput PPI screens, and CSSPs are underrepresented in global interactomes. There are several reasons for this. First, these proteins are usually glycosylated and have disulfide bonds, so they need to be expressed in the extracellular compartment. CSSP interactions between monomers are also often weak, with KDs in the μM range, making them difficult to capture due to their short half-lives. Lastly, insoluble transmembrane domains on cell surface proteins preclude their purification with standard biochemical techniques, which makes them difficult to study using methods such as AP-MS (Li, 2017).
Despite these difficulties, recent advances have been made in the study of global CSSP interaction patterns. Interactions among cell-surface proteins (CSPs) often occur between clusters of proteins on cell surfaces, and avidity effects (stronger binding due to clustering) can make these cell-cell interactions stable even when monomers bind only weakly. To facilitate detection of interactions among CSSP extracellular domains (ECDs) in vitro, several groups have taken advantage of avidity effects by attaching ECDs to protein multimerization domains and expressing ECD fusions as soluble secreted proteins. These methods have been shown to be effective, allowing detection of interactions that otherwise would not have been observable (Li, 2017).
Özkan (2013) scaled up the avidity-based approach, developing a high-throughput ELISA-like screening method, the Extracellular Interactome Assay (ECIA). The ECIA was used to define interactions among 202 Drosophila CSSPs, comprising all CSSPs within three ECD families. These were the immunoglobulin superfamily (IgSF), fibronectin type III (FNIII) and leucine-rich repeat (LRR) families. The ECIA utilized dimers as 'bait' and pentamers as 'prey'. It detected 106 interactions, 83 of which were previously unknown (Li, 2017).
The most striking finding from the ECIA interactome was that a subfamily of 21 2-IgSF domain CSPs, the Dprs, selectively interacts with a subfamily of 9 3-IgSF domain CSPs, the DIPs, forming a network called the 'Dpr-ome' (Özkan, 2013). Each Dpr and DIP that has been examined is expressed by a small and unique subset of neurons at each stage of development. One Dpr-DIP pair is required for normal synaptogenesis and influences neuronal cell fate. In the pupal optic lobe, neurons expressing a Dpr are often presynaptic to neurons expressing a DIP to which that Dpr binds in vitro. The Dpr-ome initially defined by the global interactome contained several 'orphans', proteins with no binding partner (Özkan, 2013). By expressing new versions of Dprs and DIPs, including chimeras, and using these to conduct a 'mini-interactome' analysis of the Dpr-ome, it was possible to find partners for all but one orphan. That protein, Dpr18, has changes to conserved binding interface residues and may lack the capacity to bind to any DIPs (Li, 2017).
The ECIA also identified a second IgSF network, formed among members of the Beaten Path (Beat) and Sidestep (Side) protein subfamilies. Beat-Ia and Side were identified by genetic screens for motor axon defects, and were later shown to have a ligand-receptor relationship. They control the projection of motor axons to muscle targets. Beat-Ia is expressed on motor axons, where it binds to Side, which is expressed on muscles. This binding causes motor axons to decrease their adhesion to each other, allowing them to leave their bundles and turn onto the muscle fibers. beat-Ia and side have strong motor axon phenotypes. In the absence of either protein, motor axons often remain in their fascicles and never leave to arborize on their target muscles (Li, 2017).
The ECIA detected the known Beat-Ia::Side interaction, and also uncovered other interactions between members of the Beat and Side subfamilies. Seven of the 14 Beats were found to bind to four of the eight Sides. The remaining Beats and Sides were still orphans with no binding partners in the other subfamily. The functions of the newly defined interactions between Beats and Sides were unknown. Most beat genes are expressed in embryonic neurons. Some Beats were genetically characterized using deletion mutations and RNAi, but loss of these Beats did not cause strong motor axon phenotypes. None of the other Side subfamily members had been examined (Li, 2017).
This paper describes the development of a new method for interactome screening, which is called the BPIA (Bio-Plex Interactome Assay). This method uses the 'Bio-Plex' system, which employs Luminex xMAP technology. This method detects binding of a prey protein to many bait proteins, each conjugated to a bead of a different color, in each assay well. For the ECIA, the number of assays required for the interactome screen was the square of the number of proteins examined, while with the Bio-Plex the number of assays could be equal to the number of proteins. In principle, then, the Bio-Plex might greatly speed up interactome screening, and might also be more sensitive, since the available signal-to-background ratio is much greater for the Bio-Plex than for the ECIA. As a test of the method, a Bio-Plex 200 was used to do a mini-interactome screen of the Beat-Side network. Based on the the fact that the Dprs and DIPs that were initially orphans were later shown to have binding partners, it was hypothesized that some of the orphan Beats should have Side partners, and vice versa. Consistent with this hypothesis, it was possible to deorphanize two more Beats and two Sides using the BPIA (Li, 2017).
To further understanding of Beat and Side function during embryonic development, this study characterized expression of several Beats and Sides, focusing primarily on Side-VI and the three Beat-Vs, which were the strongest interactors in both the ECIA and BPIA screens. The three beat-Vs exhibit differential expression in identified motor neurons, while side-VI is expressed at motor axon choice points and in a subset of target muscle fibers (Li, 2017).
In principle, the Bio-Plex system can allow 500 unique protein-protein (bait-prey) interaction pairs to be analyzed in a single well. In this method, capture of proteins from media with streptavidin-coupled beads bypasses the purification step for bait proteins. The assay is also compatible with the use of unpurified prey proteins, thereby reducing the workload for multiplexed screenings. The small size of the beads, the ability to probe multiple interactions simultaneously, and the small volume of the binding reactions all help reduce the amount of protein and reagents needed for the assay. It was possible to produce enough bait and prey proteins for the mini-interactome described in this study (a 23 x 23 matrix) with a single 10 cm dish transfection per protein (Li, 2017).
As a test of the system, the BPIA was used to examine interactions between the Drosophila Beat and Side IgSF protein subfamilies. Beat-Ia is a receptor on motor growth cones that recognizes Side expressed on muscles, and in the absence of Beat-Ia or Side motor axons fail to leave their bundles and arborize on their muscle targets. There are 14 Beat subfamily members and 8 Side subfamily members, but all of these proteins except Beat-Ia and Side itself were orphans until the global IgSF interactome revealed interactions between six other Beats and three Sides (Li, 2017).
In the Dpr-ome, the other IgSF network uncovered by the interactome, every Dpr protein likely to be capable of binding has an interaction partner in the DIP subfamily. Based on this, it is predicted that there should be additional interactions to be discovered within the Beat-Side subfamily network. Using the BPIA, three new interactions were found: Beat-VI::Side-II, Beat-Ic::Side-III, and Beat-Ic::Side. These results suggest that the BPIA is more sensitive than the ECIA. Like the ECIA, the BPIA should be able to find new receptor-ligand interactions even if proteins not previously known to have any interactions were tested. Of course, for both assays any candidate receptor-ligand pairs need to be confirmed as genuine using other methods. For the Beat-Side network, all three new interactions found by the BPIA, as well as the interactions between the three Beat-Vs and Side-VI found by the ECIA, were verified by SPR. This study also demonstrated that Beat-Vs and Side-VI interact using cell-based binding assays and binding to live-dissected embryos (Li, 2017).
There are still five Beats and two Sides that remain orphans. Since the structure of Beat-Side complexes is unknown, it cannot be determined whether these Beats and Sides are likely to be able to bind, but it is speculated that at least the three Beat-IIIs are likely to have Side partners. The Beat-II and Beat-V clusters each interact with a single Side partner, and perhaps the Beat-IIIs interact with one of the two orphan Sides. It is possible that changes in methodology, such as using more highly multimerized preys and/or baits, could increase sensitivity and allow detection of additional interactions (Li, 2017).
The expression patterns of side and beat genes were examined in order to obtain insights into their possible functions. Most sides are expressed in cells in the periphery as well as in the CNS, while most beats are expressed only by CNS neurons, including motor neurons. Beat-Ia::Side interactions are required for normal motor axon guidance, and highly penetrant motor axon defects in which muscles remain uninnervated are observed in mutants lacking either protein. By contrast, partial loss of function of beat-Ib, beat-Ic, both beat-IIs, or beat-VI causes motor axon defects with less than 20% penetrance. Genetic redundancy is a common theme in motor axon guidance , so it is not surprising that only low-penetrance defects are observed when Beat paralogs are not expressed. Given that Beat-Ia and Side both interact with other partners, it is perhaps remarkable that beat-1a and side have such strong phenotypes as single mutants (Li, 2017).
Beat-V and Side-VI also have redundant functions in motor axon guidance. side-VI is expressed in motor axon targets, including muscles 12 and 13 and interacts with the three Beat-Vs, at least two of which are expressed in subsets of motor neurons. Beat-V::Side-VI interactions produced the strongest signals in both the ECIA and BPIA. Sow-penetrance (~1/5 of stage 17 embryonic hemisegments affected) muscle 12 innervation defects were observed in side-VI insertion mutants or in deletion mutants lacking all three beat-V genes. There were also low-penetrance ISN guidance defects in both mutants. The fact that most muscle 12 s are innervated normally in beat-V or side-VI mutants indicates that, while Beat-V::Side-VI interactions may contribute to correct targeting of the RP5 axon to muscle 12, other cues must also be involved. Muscles 12 and/or 13 also express Wnt-4 (a repulsive ligand) and the LRR protein Capricious (Caps; probably an adhesion molecule), and low-penetrance RP5 targeting defects are observed in Wnt-4 and caps mutants. Perhaps muscle 12 is distinguished from other nearby muscles by a set of partially redundant cues, so that strong targeting phenotypes are not observed in any single mutant (Li, 2017).
Although Beat and Side paralogs may not be central to motor axon guidance, their expression patterns suggest that they could be important for determining synaptic connections within the CNS. side-VIII, encoding an orphan Side, is expressed in a small subset of embryonic CNS neurons. In the optic lobe of the pupal brain, an RNAseq analysis of two photoreceptors (R7 and R8) and five types of lamina neurons (L1-L5) revealed that beats and sides have highly specific expression patterns. For example, beat-VII is specific to L2, beat-VI to L5, beat-IIa to L3 (with lower levels in L4), and beat-IIIc to R8, being expressed at much higher levels in those cells relative to all other cells. side and side-III are specific to L3, side-II is specific to L1, side-IV is specific to L2, and side-V is specific to L5. Each of the L neuron types as well as R7 and R8 synapse with different sets of neurons in the medulla, a ten-layered structure that processes visual information from the retina and lamina. It has been observed that R and L neurons expressing specific Dprs often form synapses on medulla neurons expressing DIPs to which those Dprs bind in vitro. In a similar manner, perhaps some of the medulla neurons that are postsynaptic to L or R neurons expressing specific Sides or Beats express their in vitro binding partners, and these Beat-Side interactions might be important for synapse formation or maintenance (Li, 2017).
Despite several decades of studies on the neuromuscular system, the relationship between muscle stem cells and motor neurons remains elusive. Using the Drosophila model, evidence is provided that adult muscle precursors (AMPs), the Drosophila muscle stem cells, interact with the motor axons during embryogenesis. AMPs not only hold the capacity to attract the navigating intersegmental (ISN) and segmental a (SNa) nerve branches, but are also mandatory to the innervation of muscles in the lateral field. This so-far-ignored AMP role involves their filopodia-based interactions with nerve growth cones. In parallel, the expression of the guidance molecule-encoding genes sidestep and side IV in AMPs is reported. Altogether, these data support the view that Drosophila muscle stem cells represent spatial landmarks for navigating motor neurons and reveal that their positioning is crucial for the muscles innervation in the lateral region. Furthermore, AMPs and motor axons are interdependent, as the genetic ablation of SNa leads to a specific loss of SNa-associated lateral AMPs (Lavergne, 2020).
During Drosophila embryogenesis, a stereotypical pattern of AMPs per abdominal hemisegment in ventral (V-AMP), lateral (L-AMPs), dorsolateral (DL-AMPs) and dorsal (D-AMPs) positions can be distinguished. This study has investigated the relationship between AMPs and motor axons, and their dynamics, during development using embryos carrying the M6-gapGFP transgene, which allows visualization of the membrane of AMP cells. The intersegmental nerve (ISN) established contacts with the DL-AMPs during the embryonic stage 13 and then navigated toward the D-AMP to contact it at stage 15. Within the lateral field, the segmental nerve a (SNa) is sub-divided into two branches, dorsal (D-SNa), which innervates the lateral transverse muscles (LTs 1-4), and lateral (L-SNa), which targets the segmental border muscle (SBM). The SNa sub-division takes place during stage 15 and it was observed that the L-SNa branch migrated towards the L-AMPs before innervating the SBM. In parallel, the anterior L-AMP underwent shape changes and directional migration towards the L-SNa. In a similar way, one of the DL-AMPs moves dorsally following ISN migration and the D-AMPs appear to extend toward the ISN. However, AMPs survival and behavior are not affected in the absence of motor axons, as shown in the prospero mutant, where motor axons fail to exit the CNS. To better characterize dynamics of AMP-motor axons interactions, the M6-GAL4; UAS-Life-actin GFP reporter line was used that allows in vivo visualization of both the motor axons and the AMPs. The M6-Gal4 and M6-gapGFP lines are both driven by the same regulatory elements; however, the expression in motor axons, which is low and difficult to distinguish in M6-gapGFP embryos, is enhanced by the GAL4/UAS system and is clearly present in the M6>lifeActGFP context. Live imaging revealed that, among the numerous oriented cytoplasmic projections sent out by the AMPs, those contacting the growth cones of motor axons became stabilized. In particular, stabilization of filopodial connections between L-AMPs and SNa coincided with the setting of the SNa branching point and specification of its lateral branch. Oriented filopodial dynamics were found in the dorsal region with the contact between D-AMP projections and ISN growth cone prior to ISN migration toward the D-AMP. As previously demonstrated, muscle founders are needed for terminal defasciculation of the main motor axon branches. In this context, AMP positioning and the fact that they actively engage with the navigating motor axons might also participate in this process by acting as spatial check-points that either induce and or attract targeted defasciculation of ISN and SNa (Lavergne, 2020).
To investigate the impact of L-AMPs on the SNa pathway and branching, the effect of a genetic ablation of the AMP cells was assessed using the M6-GAL4-driven expression of the pro-apoptotic gene reaper. This enabled targeted induction of apoptosis in AMPs, leading to AMP cell loss without strong defects in the ISN and SNa trajectory, despite the expression of M6-GAL4 in the motor neurons. This differential effect could be due to a lower expression level of M6-GAL4 in motor neurons than in AMPs, and/or a stronger resistance of neural cells to the Reaper-induced apoptosis. Importantly, in 86% of hemisegments, complete loss of L-AMPs was associated with absence of the lateral branch of SNa (L-SNa), strongly suggesting that L-AMPs play an instructive role in L-SNa formation and/or stabilization. In contrast, loss of L-SNa in hemisegments where the L-AMPs were still present occurred in 5.6% of analyzed hemisegments. The L-SNa loss in this context was thus higher than the one observed in the M6-GAL4 line with only 1.8% of hemisegments without L-SNa. To explain this difference, an effect of Reaper expression in the motor system cannot be excluded, but this could also be a consequence of early stages of apoptosis in L-AMPs. Thus, M6-GAL4-induced apoptosis created a context in which loss of the L-SNa branch was observed in L-AMP-devoid segments where the L-SNa target muscle (SBM) was still present. This suggests that L-SNa branch formation might not be dependent on its muscle target, and so prompted a test of whether the L-SNa would form or persist in an SBM-devoid context. reaper expression was targeted to the developing SBM using the SBM(lbl)-GAL4 driver. The SBM(lbl)-GAL4-driven apoptosis resulted in a systematic loss of the SBM and only sporadic loss of the L-AMPs (12% hemisegments). In the SBM-devoid context but with L-AMP cells correctly located, the L-SNa branch was still present (73% of hemisegments). Additionally, in a subset of SBM-deficient embryos, L-AMPs shifted toward the navigating SNa, leading to a shortened L-SNa (13% of the hemisegments). These observations thus suggest an instructive role for AMPs in L-SNa establishment, and reveal that SBM might not be needed for this process and is at least dispensable for its stabilization. To further test the role of L-AMPs in lateral defasciculation of the SNa, different genetic contexts were analyzed in which AMP specification is affected. First a perturbation of asymmetric cell divisions was induced. To adversely affect divisions of progenitor cells that give rise to AMPs, the asymmetry determinant Numb was ectopically expressed using the pan-mesodermal driver Twist-GAL4. In the lateral region, this led predominantly to the loss of one of the L-AMPs and a duplication of the SBM with no major impact on L-SNa formation compared with the control Twist-GAL4 line. However, in a small subset of hemisegments, loss of both L-AMPs but not SBM (often duplicated) was observed. In this rare context, the L-SNa was absent in 88% of hemisegments, supporting the view that L-AMPs are required for L-SNa branching. These findings are also consistent with the effects of generalized mesodermal expression of the identity gene Pox meso (Poxm), which can lead to a loss of L-AMPs without affecting SBM. In such a context, the L-SNa formation is impaired in 89% of L-AMP-devoid segments against 37% in random Twi>Poxm hemisegments. Interestingly, pan-mesodermal expression of Pox meso can also induce misplacement of L-AMPs along the SBM, leading to aberrant L-SNa trajectory. Hence, L-AMPs and their spatial positioning appear crucial to achieve the formation and correct pathfinding of the L-SNa (Lavergne, 2020).
The findings described above suggest that L-AMPs are a source of attractive signals that promote lateral sub-branching of the SNa, making it competent to innervate the SBM. Interestingly, the SBM is the only lateral muscle innervated by the Connectin-positive SNa, which does not express this homophilic target recognition molecule. In such a context, L-AMP-mediated lateral sub-branching of SNa offers a way to drive L-SNa to its specific muscle target. As L-AMPs seem not to express Connectin either, in contrast to previous suggestions, their role in attracting SNa and inducing the L-SNa sub-branching might rely on other guidance molecules (Lavergne, 2020).
It has been previously shown that the mutants of sidestep and beat-1a, which encode interacting membrane proteins of the immunoglobulin superfamily, displayed loss of L-SNa, a phenotype similar to the one observed when L-AMPs are missing. However, the mechanisms leading to the loss of the L-SNa in sidestep and beat-1a mutants have not been elucidated. In addition, the embryonic expression pattern of sidestep has been only partially characterized. By using in situ hybridization, this study found that sidestep mRNA is strongly enriched in all the AMPs, suggesting its potential involvement in the dialogue between AMPs and motor axons. It was therefore decided to test Sidestep protein distribution at the time when L-SNa sub-branching is taking place. By examining stage 14 to 15 embryos, a previously unreported faint and transient expression of Sidestep was found specifically in L-AMPs. To confirm this observation, the expression of Sidestep was analyzed in a mutant for beat-1a. It has been reportedthat the contact of Beat-1a-expressing motor axons with Sidestep-expressing cells leads to a negative regulation of the expression of sidestep. If this contact is missing, cells normally expressing sidestep transiently and at a low level will continue to do so, leading to continuous and higher Sidestep level in these cells. Analyses of beat-1a mutants confirmed that the L-AMPs are Sidestep-expressing cells and that Sidestep expression onset coincides with L-SNa sub-branching. The high Sidestep expression resulting from the lack of beat-1a was still detected in late-stage embryos in which it became gradually restricted to the most anterior L-AMPs. This late differential Sidestep expression may point to a leading role for the anterior L-AMPs in the process of interaction with SNa and in its lateral sub-branching. Additionally, an increased Sidestep expression in L-AMPs was also observed in SNa-devoid pros mutants and in the Duf-GAL4; UAS-NetrinB (NetB) context. Importantly, the SBM does not express Sidestep, making the L-AMPs the only Sidestep-expressing cells in the L-SNa pathway. Thus, this newly reported expression pattern suggests that L-AMPs could attract the L-SNa through the temporally and spatially restricted expression of sidestep (Lavergne, 2020).
Interestingly, the sidestep mutants also display a stall phenotype of the ISN suggestive of a potential role of the D-AMPs. Indeed, this study observed that the aberrantly located D-AMPs, after the mesodermal overexpression of the activated form of the Notch receptor (NICD), are able to attract the ISN, suggesting that they are a source of guiding signals. However, because only faint sidestep transcript expression was observed in D-AMPs and Sidestep protein was not detected, it is expected that other guiding cues may be in play. It is important to notice that to visualize the attractive potential of mis-positioned D-AMPs induced pan-mesodermal expression of NICD was observed via a GAL4/UAS system known to be thermo-sensitive. High mesodermal expression of NICD induced at 29°C leads to the loss of majority of muscles but, as is shown in this study, several muscles persist in Twi-GAL4;UAS-NICD embryos incubated at 25°C, thus allowing uncouple effects of delocalized D-AMPs from potential influence of muscles loss on ISN trajectories. However, loss of D-AMPs, observed in this study in a Poxm gain-of-function context, appears to have only a minor effect on the capacity of ISN motor axons to target dorsal muscles, which are correctly innervated by the ISN in 65% of hemisegments without D-AMP. These results highlight differential requirements of AMPs for motor axons defasciculation and navigation with L-AMPs being mandatory for L-SNa branching and D-AMPs acting as guiding cells for the ISN. However, the functional significance underlying the guidance of motor nerves by muscle stem cells remains to be determined (Lavergne, 2020).
It is also important to state that the loss of L-SNa in the sidestep mutants is not fully penetrant (observed in less than 10% of hemisegments), suggesting that sidestep is not the only player in L-SNa sub-branching. This could be due to functional redundancy between several members of Side and Beat families comprising 8 and 14 members, respectively. Expression and function of Side and Beat family members remain largely unexplored, but the fact that Sidestep labels L-AMPs and its paralog, side VI, is expressed in the DL-AMPs suggests there might be a 'Side expression code' that operates in AMPs and makes them competent to interact with navigating motor axons. In support of this hypothesis, this study found that side IV, another member of the Side family, is also expressed in AMPs with a higher transcript levels detected in L-AMPs, suggesting it could contribute to the interactions between L-AMPs and the SNa. To gain insight into AMP functions of sidestep and side IV in setting interactions with motor neurons, the selective AMP-targeting tools need to be developed to generate AMP-specific mutant rescue (Lavergne, 2020).
It has previously been shown that the nervous system is required for the establishment of the adult muscle pattern and that motor axons serve as a support for migration of AMPs during larval and pupal development. More recently, it has also been suggested that the nervous system could be involved in the selection of founder cells from the pool of AMPs. This study took advantage of a previously described genetic context (pan-muscular expression of NetB) to affect the SNa and tested impact of SNa loss on L-AMPs. In stage 16 DUF>NetB embryos, loss of the SNa observed in 84% of the hemisegments does not affect the number of L-AMPs. However, in surviving 3rd instar larvae in hemisegments lacking the SNa, number of L-AMPs is dramatically reduced. Interestingly, a specific depletion was observed of normally associated with SNa anterior L-AMPs (complete loss in 10 out of 26 hemisegments analyzed), while the posterior L-AMPs associated with the transverse nerve (TN) remained unaffected. Thus, this data provides evidence for a cross-talk between AMPs and motor axons, with the AMPs attracting navigating motor axons, which in turn are required for AMP maintenance during larval stages. The loss of anterior L-AMPs in SNa-devoid larva suggests that SNa-derived signals promote survival of associated L-AMPs, but precise underlying mechanisms remain to be elucidated (Lavergne, 2020).
Thus, in Drosophila, the dynamic interactions and close association between AMPs and the motor axon network contribute to setting ISN trajectory and are required for SNa sub-branching and proper innervation of lateral muscles, which is itself needed in larvae for the maintenance of anterior L-AMPs. In vertebrates, it has previously been described that muscle pioneers can impact motor axon pathfinding in zebrafish and more recently in mice that muscle stem cells activate and contribute to neuromuscular junction regeneration in response to denervation, and that depletion of muscle stem cells induced neuromuscular junction degeneration. This study, conducted in Drosophila, represents the first demonstration that, during development of neuromuscular system, muscle stem cells interact with motor neurons and contribute to proper muscle innervation (Lavergne, 2020).
Identifying neural substrates of behavior requires defining actions in terms that map onto brain activity. Brain and muscle activity naturally correlate via the output of motor neurons, but apart from simple movements it has been difficult to define behavior in terms of muscle contractions. By mapping the musculature of the pupal fruit fly and comprehensively imaging muscle activation at single cell resolution, this study describes a multiphasic behavioral sequence in Drosophila. This characterization identifies a previously undescribed behavioral phase and permits extraction of major movements by a convolutional neural network. Movements were deconstructed into a syllabary of co-active muscles and specific syllables were identified that are sensitive to neuromodulatory manipulations. Muscle activity shows considerable variability, with sequential increases in stereotypy dependent upon neuromodulation. This work provides a platform for studying whole-animal behavior, quantifying its variability across multiple spatiotemporal scales, and analyzing its neuromodulatory regulation at cellular resolution (Elliott, 2021).
The behavioral hallmark of pupal development is the Drosophila pupal ecdysis sequence, which is one of a general class of behavioral sequences used by insects to molt. These sequences typically divide into three principal phases during which the animal first loosens and then sheds its old exoskeleton before expanding its newly secreted one. Ecdysis sequences are strongly dependent on the action of hormones for their initiation and progression, and together with vertebrate reproductive behaviors, they have long served as a useful model of hormone-behavior interactions. Neural control of ecdysis behaviors is typically exercised by a conserved complement of hormones including eclosion hormone, ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP), and Bursicon. The sites of action of these hormones within the nervous system have been principally studied in Drosophila, but detailed neuroethological studies have been undertaken in larger insects, such as the locust, Schistocerca gregaria, and cricket, Teleogryllus oceanicus. Motor program organization of the adult ecdysis sequences in these insects is quite stereotyped, suggesting central control of motor execution, but sensory feedback can also modulate behavioral performance. While characterization of central nervous system (CNS) activity underlying ecdysis sequences has remained limited in larger insects, progress has been made in Drosophila where a fictive ecdysis sequence can be elicited in an excised pupal brain by exposure to ETH. Fictive activity imaged using Ca++ indicators grossly correlates with the motor patterns executed during pupal ecdysis, but comprehensively interpreting CNS activity remains a challenge (Elliott, 2021).
The Drosophila pupal ecdysis sequence occurs at the onset of metamorphosis and has been adapted to initiate transformation of the body plan. Although it occurs in the context of the pupal molt and has three principal phases characterized by distinct motor programs, its function in molting is limited to casting off the cuticular linings of the gut and trachea. Its primary function is, instead, to create internal pressure at points along the body to evert adult parts such as the head, legs, and wings. ETH initiates the pupal ecdysis sequence after a long period of behavioral quiescence and targets neurons that express its two receptor isoforms, ETHRA and ETHRB. Neurons expressing ETHRB are largely dispensable during larval life, but are essential for pupal ecdysis. Neurons expressing ETHRA include the neuroendocrine cells that secrete CCAP and Bursicon. These cells are likewise essential at pupal, but not larval, ecdysis. They become active approximately 10 min after the onset of pupal ecdysis, and their activation mediates the transition to the next behavioral phase. Understanding how the nervous system transforms such hormonal signals into temporally ordered behavioral sequences will require a more complete description of the motor neuron activity that dictates the behavioral sequences themselves (Elliott, 2021).
This study use body-wide fluorescence imaging from the dorsal, lateral, and ventral views to characterize the pupal ecdysis sequence at single-cell resolution. Using improved imaging methods-including a new pan-muscle LexA driver that permits dual imaging of muscle and neuron activity-this study identified novel elements of the pupal ecdysis sequence, including previously undescribed movements and a phase of stochastic muscle activity preceding ecdysis. Muscle activity was found to exhibit a high degree of variability, with individual muscles recruited stochastically into repeating small ensembles, termed called syllables. Syllable activity is synchronized over anatomical compartments to form movements, which are sufficiently stereotyped to be learned by a convolutional neural network (CNN). It is possible to prevent synchronization at specific motor program transitions by suppressing neuromodulatory neurons, which is lethal. The suppression of proprioceptive neurons, which blocks initiation of pupal ecdysis, is also unexpectedly lethal. Overall, this analysis at single-cell resolution reveals a dynamic system in which movements are not rigidly specified but form from variable components subject to neuromodulatory reorganization (Elliott, 2021).
Behavior is linked to neural mechanisms by the muscle activity that governs movement. To gain insight into how nervous systems specify behavior, muscle activity was examined during the Drosophila pupal ecdysis sequence at single-cell resolution using genetic Ca++ indicators. The pupal ecdysis sequence consists of multiple motor programs, dependent for their execution on hormonal cues. Hormonal signaling was found to coordinate muscle activity across individual muscle ensembles and anatomical compartments to ensure stereotypy of behavioral execution. Although stereotypy is evident at the level of phases, the recruitment of muscles into movements is not stereotyped and some muscle activity is not correlated with movement. Importantly, a phase of stochastic muscle Ca++ activity precedes the onset of behavior, indicating that prior to the action of ETH, nervous system activity exhibits intrinsic variability. This variability is reduced, but not eliminated, by the action of neuromodulators, which incrementally increase behavioral coherence (Elliott, 2021).
The results significantly extend previous descriptions of pupal ecdysis and illustrate the power of pan-muscle Ca++ imaging. Behavioral fine-mapping at single-cell resolution permits the definition and automated detection of elemental movements, the identification of a syllabary of movement-associated muscles and muscle ensembles, and the analysis of their sensitivity to neuronal manipulations. Importantly, single-cell analysis permits the identification of muscle activity that is not consistently associated with movements. The most salient example of such idiosyncratic activity occurs in P0, a previously undescribed phase of muscle activity lacking coordinated movement. Variability persists in phases that exhibit idiosyncratic muscle activation comingled with stereotyped movement syllables. Furthermore, muscle recruitment into syllables, and recruitment of syllables into movements, exhibits considerable variability both within and across animals. All observations suggest that variability in the order of recruitment of behavioral elements is a pervasive feature of the pupal ecdysis sequence with stereotypy emerging only at higher levels of behavioral description (Elliott, 2021).
Variability in pupal ecdysis behavior may arise from the need to adjust movement to changing forces on the body wall, both from inside and outside. Inside the animal, hydrostatic pressure varies globally in response to local contractions of the body wall. This pressure may need to be countered to maintain control of movement. Outside the animal, the wall of the puparium may form an inhomogeneous substrate as the distribution of molting fluid and air at different places varies. A notable feature of pupal ecdysis is that it is heralded by the appearance of a large air bubble in the abdomen that is expelled into the puparium by the movements executed during P1. After expulsion from the body, air is displaced by the animal's movements, first posteriorly and then anteriorly. In the presence of residual molting fluid, pockets of air likely cause fluctuations in surface tension between the body wall and puparium. Forces exerted both by internal pressure and by substrate interactions within the puparium may thus require that motor output be dynamically adjusted, presumably by sensory cues (Elliott, 2021).
The importance of sensory cues to pupal ecdysis is evident from the finding that animals lacking proprioceptive input die at P0 without initiating the behavioral sequence. The cause of death remains to be determined, but its timing suggests that proprioceptive feedback may signal muscle responsiveness to neural input during the period of muscle reactivation and thus provide a readiness signal for ecdysis initiation. Alternatively, pressure on the body wall due to air bubble growth may trigger pupal ecdysis. Sensory cues have been shown to gate behavioral transitions in the adult ecdysis sequences of crickets and also to adjust motor program execution when extrication from parts of the old cuticle fails (Elliott, 2021).
For the pupal ecdysis sequence, more work will be required to determine the sources of the observed variability. Notably, sources that arise frequently in the context of other behaviors, such as external environmental stimuli (i.e., stimuli outside the puparium) and competing physiological needs, are absent for pupal ecdysis. In addition, proprioceptive cues, while they may tune ecdysis behavior, are not essential for generating it in that a fictive sequence is generated by an excised pupal brain treated with ETH. Finally, the evidence suggests that at least some behavioral variability derives from the operation of the ecdysis neural network itself since stochasticity is clearly evident at P0 and then appears to extend to other phases (Elliott, 2021).
The variability of P0 muscle bouts is reminiscent in some ways of the neurogenic bursts of muscle activity observed in Drosophila embryos prior to hatching. Initial embryonic bursts consist of uncoordinated muscle activity that becomes increasingly organized over several hours as the locomotor networks mature. By the time of hatching, complete peristaltic motor sequences are regularly performed. Similar precocious network activity is also found in a variety of other systems, including pupal moths where the developing circuitry for flight drives low-threshold neuromuscular responses in muscles that fail to elicit contractions. Such activity has also been proposed to support network maturation. P0 motor activity may share this function as patterns of muscle activity become increasingly complex during P0 and recognizable syllables emerge. However, in contrast to other systems, fully coordinated activity does not appear until the phase ends with the first P1 Lift, and even after this muscle and syllable recruitment remain irregular, suggesting continued network variability (Elliott, 2021).
This apparently intrinsic variability may relate to the tradeoffs required of any multifunctional system. Pupal ecdysis, like most behaviors, depends on the integration of signals from CPGs, proprioceptors, neuromodulators, and possibly higher-order command systems-all of which are likely used in the context of larval behavior. For example, circuits that generate waves of activity along the anteroposterior axis are required for both larval locomotion and pupal ecdysis, but while they generate coordinated bilateral activity in locomotion, they generate mostly alternating activity during ecdysis. This degree of flexibility may be bought at the cost of reproducibility of execution. Indeed, precise reproducibility may not be prioritized by nervous systems. This may instead favor solutions that are 'good enough' as has been generally proposed for biological control systems. According to this view, further optimization of pupal ecdysis, in the form of behavioral stereotypy,may incur costs on performance in the execution of other behaviors that rely on the same circuit elements (Elliott, 2021).
Variability in pupal motor output is substantially altered by the neuromodulatory action of the ecdysis hormones ETH, CCAP, and Bursicon. Previous evidence indicates that these hormones induce the motor activity characteristic of P1 and P2, even in an isolated pupal nervous system. Consistent with the ability of neuromodulators to reconfigure motor networks, this induction likely represents reorganization and possibly stabilization of network activity, and also increased network coherence. P1 is distinguished from P0 by the presence of coherent movements and P2 is more coherent than P1 in recruitment of muscles, syllables, and compartmental activity. The reduced stereotypy in P3 may result from waning neuromodulatory action. The ecdysis hormones thus reorganize motor output and increase the stereotypy of its execution. This conclusion is supported by suppression of neuromodulatory signaling, which disrupts muscle activity at multiple levels, as expected from the broad distribution of ecdysis hormone receptors (Elliott, 2021).
What causes the release of Bursicon, CCAP, and ETH at pupal ecdysis is unknown, but the results suggest that ecdysis activity itself may be a factor. Release of ETH occurs only after muscle responsiveness to neural stimulation is ensured, suggesting that the latter may represent a checkpoint for release. Similarly, the aborted swing-like activity occurring in the absence of NCCAP and NBurs activity indicates that entry into P2 has a hormone-independent component that may act as a checkpoint for hormone release, which then sustains P2 network activity. One possible mechanism might be that as the animal pulls back during P1 and creates an anterior space, sensory feedback from the head signals the readiness for P2. Similar checkpoint control mechanisms have been proposed to operate in the adult ecdysis sequence of locusts and crickets (Elliott, 2021).
The granular analysis of muscle activity presented in this study also reveals how neuromodulators may serve to coordinate action across anatomically and functionally distinct compartmental boundaries. ETHRB neuron suppression blocks the Lift, a movement of the posterior compartment, while suppressing CCAP neurons prematurely terminates the first (and only) swing-like movement by blocking its progression into the anterior compartment. Additionally, the distribution of CCAP-R appears to reflect mechanisms for selectively regulating distinct compartments across the D-V axis. CCAP-R is expressed selectively in motor neurons that innervate dorsal and ventral muscles. These motor neurons have dendrites that occupy similar positions on the myotopic map and share similar synaptic inputs. This distinguishes them from motor neurons that innervate the lateral transverse muscles, which do not express CCAP-R. However, a subset of lateral transverse muscles (i.e., M21-23) themselves express CCAP-R, and the pattern of CCAP-R expression may thus represent a mechanism for synchronizing activity across the D-V axis during the swing movements of P2 when CCAP is released. Notably, muscle synchrony across compartments in posterior segments is lost in the one, partially executed Swing when CCAP-expressing neurons are suppressed and no further Swings are executed (Elliott, 2021).
The putative source of CCAP for the muscles of PME2 are the type III terminals on muscle M12, which are selectively retained in the anterior compartment (i.e., segments 1-4). CCAP-R-expressing muscles in the anterior compartment may thus receive CCAP modulation prior to those in the posterior compartment. This may facilitate the delayed appearance of the contralateral brace, which is formed principally by segmental PME2s and which changes the character of the Swing after head eversion. Interestingly, the anatomical asymmetry in the distribution of M12 is one of several observed in pupal muscles across the D-V and A-P body axes. These have only been partially described previously and result from the selective degradation of larval muscles in the ventral and posterior compartments. The asymmetries in muscle distribution correlate with several pupal movements that are physically partitioned across the A-P axis, including the Lift, which occurs only in the posterior compartment during P1, and the AntComp, which is restricted to the anterior compartment during P3. Synaptic mechanisms for controlling movements across the A-P boundary in the larva have been described previously, and similar mechanisms may synergize with anatomical asymmetries and neuromodulatory mechanisms in the pupa to spatially and temporally constrain muscle activity to specific compartments (Elliott, 2021).
It is worth noting that the expression of CCAP-R by the lateral transverse muscles indicates the potential importance of muscle modulation in shaping behavior. Whether modulation of muscle properties also underlies the generalized failure of muscles to respond to synaptic input at the onset of P0 is unclear. Animals stop moving shortly after pupariation and do not resume until pupal ecdysis, but neuromuscular activity may be maintained throughout this period. This study observed neuromuscular activity without muscle responses at the earliest pre-pupal stages examined (approximately stage P2). However, more detailed imaging would be required to determine the extent, continuity, and pattern of the input. Drosophila muscles are targets of a variety of neuropeptides including myoinhibitory peptides, which have also been implicated in pupal ecdysis behavior, and it is possible that these play a role in suppressing muscle responses to neural input. Alternatively, neuromuscular signaling may be progressively potentiated during P0. Such a mechanism has been proposed to operate in pupal moth flight muscles, which respond electrically, but fail to contract, to synaptic input corresponding to flight motor patterns. As animals approach eclosion, rising octopamine levels upregulate neuromuscular efficacy so that flight muscles activate in response to input (Elliott, 2021).
A goal of computational neuroethology is to describe behavior at a level of resolution that permits the identification of its neural determinants. The muscle-level description provided in this study lends itself naturally to this purpose. For example, the activity patterns of the muscles comprising PME2 have likely neural correlates within the synaptic and neuromodulatory networks that govern pupal ecdysis behavior. At the neuromodulatory level, the NCCAP cells that terminate on M12 are likely sources of CCAP modulation of PME2 muscles, perhaps to help reverse their P-to-A activation at P2. At the synaptic level, the regular, intersegmental pattern of activation of PME2 muscles in nearly all pupal movements and behavioral bouts suggests that PME2 motor neurons are driven by CPG neurons that generate anteroposterior rhythms. The results thus provide testable predictions about the patterns of neuromodulatory and synaptic connectivity between muscles, motor neurons, and premotor interneurons of various types (Elliott, 2021).
Testing these predictions will be facilitated by data emerging from reconstruction of the larval CNS at synaptic resolution. Although pupal behaviors differ from those of the larva, broad similarities suggest that at least some neural substrates are shared. The initial P-to-A activity flow of the pupal ecdysis sequence is reminiscent of larval forward peristalsis, and its reversal after alternating bilateral Swing movements resembles the switch to backward peristalsis following sensory stimuli. An important difference, however, is that the basic features of larval locomotion are identifiable in the default activity of the excised larval brain, whereas the pupal nervous system produces patterned activity resembling pupal ecdysis only in response to ETH. Pupal ecdysis will thus permit investigation of the mechanisms by which intrinsic neuronal activity is organized by neuromodulatory control. Analyzing behavior at single-muscle resolution, as demonstrated in this study, will facilitate this investigation and should be applicable to other translucent preparations of neuroscientific interest, such as larval fruit flies, roundworms, and larval zebrafish (Elliott, 2021).
Genesis of syncytial muscles is typically considered as a paradigm for an irreversible developmental process. Notably, transdifferentiation of syncytial muscles is naturally occurring during Drosophila development. The ventral longitudinal heart-associated musculature (VLM) arises by a unique mechanism that revokes differentiation states of so-called alary muscles and comprises at least two distinct steps: syncytial muscle cell fragmentation into single myoblasts and successive reprogramming into founder cells that orchestrate de novo fiber formation of the VLM lineage. This study provides evidence that the mesodermal master regulator twist plays a key role during this reprogramming process. Acting downstream of Drosophila Tbx1 (Org-1), Twist is regulating the activity of the Hippo pathway effector Yorkie and is required for the initiation of syncytial muscle dedifferentiation and fragmentation. Subsequently, fibroblast growth factor receptor (FGFR)-Ras-mitogen-activated protein kinase (MAPK) signaling in resulting mononucleated myoblasts maintains Twist expression, thereby stabilizing nuclear Yorkie activity and inducing their lineage switch into founder cells of the VLM (Rose, 2022).
Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power
of contraction. Little is known about how T-tubules maintain highly
organized structures and contacts throughout the contractile system
despite the ongoing muscle remodeling that occurs with muscle atrophy,
damage and aging. This study uncovered an essential role for autophagy
in T-tubule remodeling with genetic screens of a developmentally regulated
remodeling program in Drosophila abdominal muscles. It was shown
that autophagy is both upregulated with and required for progression
through T-tubule disassembly stages. Along with known mediators of
autophagosome-lysosome fusion, the screens uncover an unexpected shared
role for Rab2
with a broadly conserved function in autophagic clearance. Rab2 localizes
to autophagosomes and binds to HOPS complex members, (Jiang, 2014; Takáts, 2014) suggesting a direct
role in autophagosome tethering/fusion. Together, the high membrane flux
with muscle remodeling permits unprecedented analysis both of T-tubule
dynamics and fundamental trafficking mechanisms (Fujita, 2017).
Differentiated muscle cells, or myofibers, are highly organized in order to coordinate the roles of specialized subcellular structures involved in contraction. Myofibril bundles of sarcomeres provide the contractile force. The power of contraction, however, requires synchronous sarcomere function under control of the 'excitation-contraction coupling' system that includes two membranous organelles, the sarcoplasmic reticulum (SR) and Transverse (T)-tubules (Al-Qusairi, 2011). The T-tubule membrane network is continuous with the muscle cell plasma membrane, with tubulated membranes that invaginate radially inward in a repeated pattern at each sarcomere. With excitation-contraction coupling, neuromuscular action potentials are transmitted along the muscle T-tubule membrane to the SR junction, or dyad/triad, triggering coordinated SR Ca2+ release and synchronous sarcomere contractions (Al-Qusairi, 2011). Formation of organized T-tubule membranes is thus critical for muscle function (Takeshima, 2015). Mechanisms must also remodel the T-tubule membrane network with ongoing myofiber reorganization in response to muscle use, damage, atrophy and aging. However, the extent and mechanisms of T-tubule remodeling remain largely unknown, in part due to challenges with observing T-tubule membrane network dynamics within intact mammalian myofibers (Fujita, 2017).
The T-tubule network includes both transversal and longitudinal tubular membrane elements that form and mature with myofiber differentiation and growth. In mouse skeletal muscle, mostly longitudinal tubular membranes initially present in embryonic muscle are remodeled postnatally with expansion to predominantly transversal tubular elements. In contrast, both longitudinal and transversal T-tubule elements are maintained in adult mammalian cardiac muscle and in insect muscles. Relatively few molecular factors are known to shape the T-tubule network, and perhaps not surprisingly, all of which so far encode for membrane-associated functions (CAV3, DYSF, BIN1/Amph2, MTM1, DNM2). Mutations in each also are associated with human myopathy and/or cardiomyopathy with T-tubule disorganization, pointing to the critical importance of membrane-mediated mechanisms to maintain the T-tubule membrane network (Fujita, 2017).
Drosophila is a powerful system for insights into the functional requirements for T-tubule formation and remodeling. The BIN1 BAR-domain protein has a conserved function involved in membrane tubulation required for T-tubule formation that was first described for the single Drosophila homolog, Amphiphysin. The amph null mutant flies lack transversal T-tubule element membranes in myofibers at all developmental stages, corresponding with both larval and adult mobility defects. In contrast, the myotubularin (mtm) fly homolog of mammalian MTM1/MTMR2/MTMR1 subfamily of phosphatidylinositol 3-phosphate phosphatases is required only at later stages in development for T-tubule remodeling. While mtm loss of function has no obvious effects on larval muscle T-tubule organization or function, mtm-depleted post-larval stage muscles lack transversal T-tubule membranes with adult mobility defects in eclosion and flight. Together, the amph and mtm mutant conditions that both lack transversal T-tubule elements in post-larval stage muscle yet different early development requirements underscores that distinct mechanisms are involved in T-tubule formation (amph-dependent) versus maintenance/remodeling (amph- and mtm-dependent) (Fujita, 2017).
In Drosophila, a set of larval body wall muscles that persist as viable pupal abdominal muscles, called dorsal internal oblique muscles (IOMs), are essential for adult eclosion. During metamorphosis, changes in IOM cell size and myofibril content have been noted. Previous studies have shown that wildtype IOMs undergo dramatic cortical and membrane remodeling with costamere integrin adhesion complex disassembly and reassembly at discrete pupal stages (Ribeiro, 2011). In contrast, the mtm-depleted IOMs exhibited persistent disassembly or a block in reassembly of integrin costameres along with the loss of transversal T-tubule membranes at late pupal stages, but without any precocious cell death (Ribeiro, 2011). A striking feature in the mtm-depleted IOMs was the accumulation of endosomal-like membranes decorated with integrin and T-tubule markers, Amph and Discs large (Dlg1, a PDZ protein). Altogether, these results suggest that T-tubule membranes may undergo disassembly-reassembly with normal myofiber remodeling, including the delivery of disassembled T-tubule membrane into an endomembrane trafficking pathway. The role for a molecular-cellular program in control of T-tubule remodeling that is at least partially distinct from that involved in initial T-tubule formation raises many questions about possible mechanisms, including the regulation of T-tubule organization and dynamics, the membrane fate(s) and source(s) with disassembly-reassembly, respectively, and the specific membrane trafficking routes and effectors involved. Possible hints may come from studies of other specialized dynamic cell membrane invaginations shown to involve endosomal and Golgi membrane trafficking pathways, such as cellularization of Drosophila syncytial embryos and the tubulated demarcation membrane system in megakaryocyte platelet formation (Fujita, 2017).
Membrane trafficking relies on the large family of Rab GTPases, with over sixty Rabs in humans and thirty in flies. The different Rabs are under distinct spatiotemporal regulation for recruitment, activation and functions at specific membrane compartments or domains. Guanine nucleotide exchange factors (GEFs) convert specific inactive GDP-bound Rabs to an active GTP-bound form. Active Rab-GTP then recruits a range of specific effector proteins to the membrane that mediate key trafficking functions, including cargo selection, vesicle budding, transport, tethering and fusion. Subsequently, GTPase-activating proteins (GAPs) deactivate Rabs by promoting GTP hydrolysis. Many membrane compartments have been defined by well-established localized functions of specific Rabs, for example: ER (Rab1), Golgi (Rab1, Rab6), secretory vesicles (Rab8), early endosomes (Rab5, Rab21), recycling endosomes (Rab11, Rab35), late endosomes (Rab7, Rab9), lysosomes (Rab7) and others. Thus, identifying the specific Rabs required for a cellular process can provide clues to potential underlying membrane trafficking mechanisms involved. However, examples exist of Rabs with multiple known sites of function or yet unknown functions, and conversely, certain cellular processes - like T-tubule remodeling - lack defined roles yet for any Rabs (Fujita, 2017).
This study utilized the advantages of Drosophila IOMs to screen for Rab GTPases and related membrane trafficking functions required for T-tubule remodeling in intact muscle. The results show that the entire contractile and excitation-contraction coupling system, including T-tubules, are disassembled and reassembled in IOMs during Drosophila metamorphosis. Autophagy, the membrane trafficking process for degradation of cytoplasmic contents by delivery to lysosomes, is upregulated with IOM remodeling where it plays an indispensable role for progression through T-tubule disassembly to reassembly. Genetic analysis of IOM remodeling also reveals an unexpected and broad role for Rab2 in autophagy in flies and mammals. From these data, it is proposed that Rab2 localizes to autophagosomes where it interacts with the HOPS complex, which in turn, mediates tethering and trans-SNARE complex formation with Rab7-marked lysosomes to promote autophagosome-lysosome fusion. Together, these results show that Drosophila IOM remodeling provides an unprecedented in vivo context for discovery and analysis of T-tubule dynamics with relevance to human myopathy, as well as an ideal system due to high membrane flux to study fundamental trafficking pathways (Fujita, 2017).
This study has characterized a wildtype myofiber remodeling program by confocal and electron microscopy in intact muscles in vivo. In Drosophila IOMs during metamorphosis, the entire contractile and excitation-contraction coupling system, including T-tubules, are disassembled and then reassembled. This process highlights that myofibers harbor distinct programs for initial T-tubule formation versus regulated T-tubule remodeling. This likely includes additional mechanisms for T-tubule membrane disassembly and renovation, features that reflect those seen with mammalian myofiber atrophy and recovery. The Drosophila body wall muscles provide an unprecedented system permitting a combination of powerful visualization and systematic perturbation analysis, including the first genetic screens, of T-tubule dynamics and organization (Fujita, 2017).
Autophagy is upregulated with the onset of IOM remodeling during metamorphosis. Further, disruption of autophagy initiation, autophagosome formation or clearance all induced loss of T-tubules with a block in IOM remodeling at/after T-tubule disassembly. This is the first report of a non-cell death role of autophagy in Drosophila metamorphosis. The role of autophagy in IOMs that persist and redifferentiate during metamorphosis is clearly different from its roles in pupal midgut and salivary gland cells that undergo autophagic forms of cell death. There are multiple speculative direct or indirect role(s) for autophagy specifically in T-tubule membrane remodeling: (1) a direct role in T-tubule membrane recycling, as a means to deliver disassembled T-tubule membrane via autophagosomes to lysosomes or related organelles for intracellular storage, then later redeployed to contribute to T-tubule reassembly; (2) an indirect role in cell renovation, including T-tubule membrane clearance, to permit cell space for redifferentiation; or (3) an indirect role in cell metabolism, to support cell survival and/or the energy cost of redifferentiation with starvation during metamorphosis. Most likely, autophagy serves some combination of these roles in IOM remodeling (Fujita, 2017).
How could autophagy play a direct role in T-tubule remodeling? It was surprising that mCD8:GFP-positive small vesicles accumulated to a similar degree as autophagosome numbers in IOMs when autophagosome-lysosome fusion was blocked. This suggests that mCD8:GFP localizes to autophagosomes during IOM remodeling. It is possible that T-tubule membranes are a source of autophagosomal membrane, at least in part: mCD8:GFP labels the muscle plasma membrane and T-tubules in larval muscle precursor cells of IOMs, and T-tubule disassembly coincides with the upregulation in autophagy early in metamorphosis. Also, disruption of autophagy induction blocked normal progression in disassembly and remodeling of T-tubule-derived mCD8:GFP-marked membranes. In the absence of autophagy initiation, mCD8:GFP-positive stacked membranes were observed, likely retained or partially disassembled T-tubules. It is proposed that T-tubules are remodeled through autophagosomes. It is important to note that T-tubules are not an apparent autophagic cargo, but instead, a possible source of autophagosome membrane. In this scenario, T-tubules are disassembled into autophagosomes and then reassembled from subsequent autolysosome-related structures, both of which successively increased in numbers during wildtype IOM remodeling (Fujita, 2017).
Alternatively or additionally, other roles for autophagy could indirectly impact T-tubule remodeling. Extensive IOM atrophy with nearly complete disassembly of the contractile and excitation-contraction systems by 1d APF is followed by a rapid re-differentiation within hours after 3.5d APF. Autophagy could be required to simply clear away and degrade the old contraction systems in order to make space to rebuild and realign new systems, as well as permit the normal central repositioning of nuclei away from the myofiber cortex. However, the persistent block in early IOM remodeling with autophagy disruption suggests that the remodeling normally proceeds through a progression of interrelated steps rather than independent programs for disassembly and reassembly. Autophagy also has a well-established role in metabolic homeostasis through the recycling of amino acids and turnover of damaged mitochondria in the lysosome. The current data suggest that mitochondria are a major autophagic cargo with IOM remodeling. In conditions that disrupted autophagy initiation (Atg1, Atg18 RNAi), the cytoplasm was abnormally filled with mitochondria in IOMs at 4d APF. Consistent with that, a significant portion of autophagosomes harbored intact mitochondria when autophagosome-lysosome fusion was blocked (Rab2, Rab7 or Stx17 RNAi). This is different from observations in larval muscle, in which mitochondria were notably absent in autophagosomes that accumulated with a block in autophagy. It is possible that mitophagy, a selective form of autophagy for mitochondrial turnover, is upregulated and could play both metabolic and cell renovation roles in IOM remodeling. Interestingly, the autophagy-blocked IOMs remained viable throughout metamorphosis, suggesting that autophagy is not absolutely required for cell survival through the starvation with metamorphosis (Fujita, 2017).
Through a systemic screen of all Drosophila Rab GTPases, an unexpected role was uncovered for Rab2 in autophagy. The striking Rab2 RNAi IOM phenotype was shared with RNAi of other functions known to be specifically required for autophagosome-lysosome fusion. Genetic blockade of autophagosome-lysosome fusion resulted in a dramatic phenotype, with massive accumulations of autophagosomes within IOMs. Previously, autophagosome-lysosome fusion was shown to involve the cooperative functions of Rab7, the HOPS tethering complex, and a trans-SNARE complex between Stx17, SNAP29 and VAMP7/8. Among these tethering and fusion functions, it has been shown that Stx17 (a hairpin SNARE) is recruited to autophagosomal membranes, while Rab7 and VAMP7/8 localize to endolysosomal membranes. Stx17 localizes to autophagosomes as well as to the ER and mitochondria, but the HOPS complex directly associates and colocalizes with Stx17 only at autophagosomes (Jiang et al., 2014; Takáts et al., 2014). This suggests that Stx17 is not a sole determinant for HOPS complex recruitment (Fujita, 2017).
It is proposed that Rab2 is required for the autophagosomal recruitment of the HOPS complex. Rab2 specifically localized to completed autophagosomes, and Rab2 had an affinity with the HOPS complex, as does Stx17. It is envisioned that upon completion of autophagosome biogenesis/maturation, Rab2 and Stx17 are recruited to the outer autophagosomal membrane. Then, the HOPS complex is subsequently recruited to autophagosomes in a Rab2-depedent manner through coincident interactions with both Stx17 and Rab2 (see Hierarchal analysis of Rab2 and factors involved in autophagosome-lysosome fusion). At the same time, the HOPS complex binds Rab7 on lysosomes. In turn, the HOPS complex tethers autophagosomes and lysosomes to promote trans-SNARE complex formation between Stx17, SNAP29 and Vamp7/8 and ultimately autophagosome-lysosome fusion (Fujita, 2017).
Rab2 role in autophagy discovered in fly muscle relates to a broader autophagy requirement in other cell types and across species. The localization of Rab2 on autophagosomes in Drosophila IOMs was conserved for both Rab2A and Rab2B in mouse embryonic fibroblasts (MEFs). As in flies, the Rab2A/2B double knockout led to a delay or block in autophagy clearance as indicated by accumulation of LC3/Atg8. However, the specific Rab2 loss-of-function phenotypes were not identical. While Rab2 was required for autophagosome-lysosome fusion in fly IOMs, the Rab2A/2B double knockouts in MEFs indicated a requirement at a later step in autophagic clearance. Interestingly, this disparity in autophagy phenotypes across species is also seen with Rab7. In flies and yeast, Rab7/Ypt7 is essential for autophagosome-lyososome/vacuole fusion, while mammalian Rab7 knockdowns more clearly indicate a required role in autolysosome maturation. Other examples indicate that the autophagosome-lysosome fusion machinery is not highly evolutionarily conserved. The Stx17-SNAP29-VAMP7/8 trans-SNARE complex is conserved in Drosophila and mammals, but not in yeast, where no autophagosomal SNARE has been reported so far. Moreover, budding yeast do not encode for Rab2 (Fujita, 2017).
Altogether, it is plausible that Rab2 is required for autophagosome-lysosome fusion efficiency, and Rab2-dependency is variable across different tissues or species. Two possible models could explain the different Rab2 autophagy requirements in flies and mouse cells. First, it is suggested that autophagosomes sequentially fuse with endosomes then lysosomes to become amphisomes and autolysosomes, respectively. If either of the steps requires Rab2A/2B, then intermediates with partially degraded contents could accumulate in double knockout MEFs. Alternatively, an autophagosome may normally fuse with multiple lysosomes to ensure full degradation of its contents. In the absence of Rab2A/2B in MEFs, autophagosomes could still fuse but not with a sufficient number of lysosomes, resulting in an accumulation of partially digested autolysosomes (Fujita, 2017).
Rab2 has been previously associated with transport events at the Golgi apparatus, ER-to-Golgi traffic and secretory granule formation, as well as in a C. elegans endocytic/phagocytic pathway. Gillingham et al. systematically explored Rab effectors in Drosophila cultured cells, and found that Rab2 interacts with the HOPS complex besides known Golgi-resident effectors (Gillingham, 2014). The interaction between Rab2 and HOPS complex is also conserved in mammals, and the unexpected Rab2 localization to autophagosomes was found. Thus, it is likely that Rab2 exerts multiple functions through interaction with different effectors at different places. A possible Rab2 function in the endosome-lysosome system that affects autophagic flux cannot be excluded, although no clear lysosomal defects were detected in Rab2A/B knockout MEFs. Several other factors that localize to autophagosomes or late endosomes-lysosomes, including Atg14, PLEKHM1 and EPG5, have been shown to control autophagosome maturation. It is plausible that Rab2 contributes to autophagosome maturation through both a direct role in the fusion mechanism and an indirect role in endo-lysosome maturation, the same as Rab7 and the HOPS complex (Fujita, 2017).
How Rab2 localizes to autophagosomes remains unclear. Localization of Rab2 on autophagosomes in IOMs did not depend on HOPS complex subunits, Vps39 and Vps41, or on Stx17. Further studies will be needed to determine the identities of the Rab2 guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) that regulate Rab2 GTPase activity in autophagosome-lysosome fusion. A conserved TBC domain protein, OATL1/TBC1D25, is a strong candidate for a Rab2 GAP, given OATL1 localization to autophagosomes and involvement in autophagosome-lysosome fusion. Further, it was reported that OATL1 directly bound to and showed GAP activity for Rab2A (Fujita, 2017).
Autophagy is critical for the maintenance of myofiber homeostasis in mammalian skeletal muscle. It is known that several myopathies are associated with excess accumulation of autophagic structures in muscle. Further, loss of autophagy in mouse skeletal muscle shows anomalies, including abnormal mitochondria, disassembled sarcomeres and disorganized triads, as also seen in aged muscle. It is established that autophagy is down-regulated during the course of aging. This evidence points to a possible significance of autophagy in myofiber remodeling and in T-tubule maintenance. Jumpy/MTMR14 PI3-phosphatase and Dynamin-2 (DNM2) GTPase, two causative genes of human centronuclear myopathy, are required for not only T-tubule maintenance but also proper progression of autophagy. Based on these reports and the current findings, it is speculated that their roles in T-tubule maintenance are mediated, at least in part, through autophagy (Fujita, 2017).
Signaling pathways that regulate atrophy and hypertrophy in Drosophila have been identified, however, the mechanisms and direct mediators of muscle remodeling remain largely unknown. IOM remodeling is a good model to study the mechanisms of muscle remodeling, given that the signaling pathways that control muscle remodeling are conserved between Drosophila and mammals. Advantages of the IOM system are not only its genetic tractability, but also its reproducibility and structure. As a relatively giant single cell along the body wall, IOMs enable tracking of a single cell and its subcellular organization during metamorphosis. The results show that studies in IOMs can provide new insights into the mechanisms of muscle remodeling as well as regulation of fundamental membrane trafficking pathways, such as autophagy and endocytosis (Fujita, 2017).
Actin-based thin filament arrays constitute a fundamental core component
of muscle sarcomeres. This study used formation of the Drosophila
indirect flight musculature for
studying the assembly and maturation of thin-filament
arrays in a skeletal muscle model system. Employing GFP-tagged actin
monomer incorporation, several distinct phases in the dynamic construction
of thin-filament arrays were identified. This sequence includes assembly
of nascent arrays after an initial period of intensive microfilament
synthesis, followed by array elongation, primarily from filament
pointed-ends, radial growth of the arrays via recruitment of peripheral
filaments and continuous barbed-end turnover. Using genetic approaches,
the single Drosophila homolog of the FHOD sub-family of formins, Fhos,
was identified as a primary and versatile mediator of IFM thin-filament
organization. Localization of Fhos to the barbed-ends of the arrays,
achieved via a novel N-terminal domain, appears to be a critical aspect of
its sarcomeric roles (Shwartz, 2016).
During metamorphosis in Drosophila, multi-nucleated larval dorsal abdominal muscles either undergo cell death or are remodeled to temporary adult muscles. Muscle remodeling is associated with anti-polar nuclear migration and atrophy during early pupation followed by polar migration and muscle growth during late pupation. Loss of Cathepsin-L has been shown to inhibit anti-polar movements, while knockdown of autophagy-related genes affected nuclear positioning along the medial axis in late metamorphosis. This study has developed new descriptors of myonuclear distribution. Nuclear tracks were used to distinguish between fast moving nuclei associated with fragments of dead muscles (sarcolytes) and slow-moving nuclei inside remodelled muscles. Anti-polar migration leads to a longitudinal nuclear spread decrease. Unexpectedly, the decrease in longitudinal nuclear spread was significantly enhanced by Atg9, Atg5 and Atg18 silencing, indicating that the loss of autophagy promotes the migration and clustering of nuclei. In vivo imaging and quantitative image analysis of Drosophila metamorphosis promise to provide novel insights into the relationship between muscle wasting and myonuclear positioning (Kuleesha, 2017).
The proper differentiation and maintenance of myofibers is fundamental to a functional musculature. Disruption of numerous mostly structural factors leads to perturbations of these processes. Among the limited number of known regulatory factors for these processes is Mind bomb2 (Mib2), a muscle-associated E3 ubiquitin ligase, which was previously established to be required for maintaining the integrity of larval muscles. This study has examined the mechanistic aspects of Mib2 function by performing a detailed functional dissection of the Mib2 protein. The ankyrin repeats, in its entirety, and the hitherto uncharacterized Mib-specific domains (MIB), are shown to be important for the major function of Mib2 in skeletal and visceral muscles in the Drosophila embryo. Furthermore, novel mib2 alleles were identified that have arisen from a forward genetic screen aimed at identifying regulators of myogenesis. Two of these alleles are viable, but flightless hypomorphic mib2 mutants, and harbor missense mutations in the MIB domain and RING finger, respectively. Functional analysis of these new alleles, including in vivo imaging, demonstrates that Mib2 plays an additional important role in the development of adult thorax muscles, particularly in maintaining the larval templates for the dorsal longitudinal indirect flight muscles during metamorphosis (Domsch, 2017).
Cell death can have both cell autonomous and non-autonomous roles in normal development. Previous studies have shown that the central cell death regulators grim and reaper are required for the developmentally important elimination of stem cells and neurons in the developing central nervous system (CNS). This study shows that cell death in the nervous system is also required for normal muscle development. In the absence of grim and reaper, there is an increase in the number of fibers in the ventral abdominal muscles in the Drosophila adult. This phenotype can be partially recapitulated by inhibition of cell death specifically in the CNS, indicating a non-autonomous role for neuronal death in limiting muscle fiber number. It was also shown that FGFs produced in the cell death defective nervous system are required for the increase in muscle fiber number. Cell death in the muscle lineage during pupal stages also plays a role in specifying fiber number. Altogetger, data suggests that FGFs from the CNS act as a survival signal for muscle FCs. Thus, proper muscle fiber specification requires cell death in both the nervous system and in the developing muscle itself (Sarkissian, 2016). The modulation of an animal's
behavior through external sensory stimuli, previous experience and its
internal state is crucial to survive in a constantly changing
environment. In most insects, octopamine (OA) and its precursor tyramine
(TA) modulate a variety of physiological processes and behaviors by
shifting the organism from a relaxed or dormant condition to a
responsive, excited and alerted state. Even though OA/TA neurons of the
central brain are described on single cell level in Drosophila
melanogaster, the periphery was largely omitted from anatomical studies.
Given that OA/TA is involved in behaviors like feeding, flying and
locomotion, which highly depend on a variety of peripheral organs, it is
necessary to study the peripheral connections of these neurons to get a
complete picture of the OA/TA circuitry. This study describes the
anatomy of this aminergic system in relation to peripheral tissues of
the entire fly. OA/TA neurons arborize onto skeletal muscles all over
the body and innervate reproductive organs, the heart, the corpora
allata, and sensory organs in the antennae, legs, wings and halteres
underlining their relevance in modulating complex behaviors (Pauls, 2018).
The adrenergic system of mammals influences various aspects of the animal's life. Its transmitters/hormones, adrenaline and noradrenaline, modulate a variety of physiological processes and behaviors. They are secreted into the bloodstream by the adrenal glands in response to stress. In addition, they are synthesized and released by axonal terminals in the central nervous system (CNS) as well as sympathetic fibers of the autonomic nervous system. Adrenaline and noradrenaline have been described as modulators to shift the organism from a relaxed or dormant state to a responsive, excited and alerted state. Stressful stimuli induce a metabolic and behavioral adaptation, leading to enhanced energy supply, increased muscle performance, increased sensory perception and a matched behavior. This so-called 'fight or flight' response can be seen in vertebrates and invertebrates. In insects, the stress response is mediated -- among others -- by octopamine (OA) and its precursor tyramine (TA). TA is synthesized from tyrosine by the action of a tyrosine decarboxylase enzyme (Tdc) and functions as an independent neurotransmitter/-modulator as well as the intermediate step in OA synthesis. For this, TA is catalyzed by the tyramine-β-hydroxylase (TΒH). Similar to the vertebrate adrenergic system, OA and TA act through specific G-protein coupled receptors. Besides structural similarities between OA/TA and adrenaline/noradrenaline and the corresponding receptors, functional similarities are illustrated by the action of these transmitters/hormones in the regulation of physiological processes and behaviors. OA and TA are known to modulate muscle performance, glycogenolysis, fat metabolism, heart rate, and respiration in insects (Pauls, 2018).
While the role of TA as an independent signaling molecule was underestimated for a long time, OA has been extensively studied and was shown to have effects on almost every organ, sensory modality and behavior in a great variety of insects. The most intensively studied peripheral organs regarding the modulatory role of OA are muscles. Here, OA is thought to not exclusively modulate muscle performance or motor activity. OA rather modulates muscle action according to metabolic and physiological processes, for example by promoting energy mobilization directly from the fat body, or indirectly by promoting the release of adipokinetic homones (AKH) from neuroendocrine cells in the corpora cardiaca (CC, a homolog of the vertebrate anterior pituitary gland and an analog of mammalian pancreatic alpha cells). In addition to the impact of OA/TA on muscles, fat body and AKH cells, OA is shown to modulate the heart, trachea and air sacs, gut, hemocytes, salivary glands, Malpighian tubules and ovaries in insects, mainly to induce a general stress or arousal state. However, in total OA seems to modulate a vast number of behaviors, which are not necessarily coupled to stress responses. The OA/TA system is shown to also act on inter alia (i.a.) learning and memory, sleep, feeding, flight, locomotion, and aggression (Pauls, 2018).
As mentioned above, OA and TA act as neurotransmitters and neuromodulators, allowing them to act in a paracrine, endocrine or autocrine fashion. In the fruit fly Drosophila, huge efforts were made to describe OA/TA neurons (OANs/TANs) in the brain and ventral nervous system (VNS) down to the single cell level. Nevertheless, although knowledge about physiological processes and behaviors modulated by the OA/TA system in the brain is rich, less is known about how OA and TA reach all its target organs and tissues in the periphery (exceptions: reproductive organs and muscles) (Pauls, 2018).
This study used the genetically tractable fruit fly Drosophila melanogaster to describe the arborizations of Tdc2-Gal4-positive, and therefore OANs and TANs in the periphery, as the Drosophila Tdc2 gene is expressed neurally. OANs/TANs were found to be widespread distributed throughout the fly's body with innervations in the skeletal muscles, reproductive organs, corpora allata, antennae, legs, wings, halteres and the heart. This diverse innervation pattern reflects the modulatory role of OA/TA in many different behaviors and physiological processes. These results provide, for the very first time, a complete and comprehensive map of the OA/TA circuitry in the entire insect body. This map allows assumptions about the type of OA/TA signaling (paracrine or endocrine) to a specific organ and, at the same time, it provides a deeper understanding to what extend the OA/TA-dependent activity of peripheral organs is altered, for example by genetically manipulating Tdc2-Gal4-positive neurons in the brain and VNS (Pauls, 2018).
This study used the Tdc2-Gal4 line, allowing Gal4 expression under the control of a regulatory sequence of the tyrosine decarboxylase enzyme. As this enzyme is essential for the synthesis of TA from tyrosine, the Tdc2-Gal4-line labels both TANs and OANs. Within the Drosophila brain, Tdc2-Gal4 labels in total about 137 cells, while additional 39 cells are located in the VNS. The small number of Tdc2Ns lead to arborizations in large parts of the central brain, optic lobes and the thoracic and abdominal ganglion. Based on the profound innervation of Tdc2Ns in the brain and VNS, the variety of behaviors modulated by the OA/TA system including learning and memory, feeding, vision, and sleep, are not surprising. Beyond the brain and VNS, OANs and TANs massively innervate regions within the periphery of the fly. This study described arborizations on most skeletal muscles, the antennae, wings, halteres and reproductive system and parts of the circulatory system and stomodaeal ganglion (Pauls, 2018).
The findings are in line with previous reports focusing on the expression of different OA and TA receptors in the fly. Accordingly, the OA receptor OAMB is expressed in reproductive organs (in both male and female flies) and muscles, which are directly innervated by Tdc2Ns. Additionally, the midgut and trachea contain OA and TA receptors, but do not seem to be innervated by Tdc2Ns, even though axons run in close vicinity to these organs. Likewise, the OA receptor Octβ2R is expressed in the fat body, salivary glands and Malpighian tubules, tissues that seem not to be innervated by Tdc2Ns, while the expression of Octβ1R and Octβ3R is more specific56,57. The three tyramine receptors TyrR, TyrRII and TyrRIII show a broad expression in the periphery, also in tissues not innervated by Tdc2Ns56. The lack of direct innervation of these peripheral tissues might argue for volume transmission or hemolymph released OA/TA from Tdc2Ns. Alternatively, Drosophila TDC1, the product of the non-neurally expressed Tdc gene, is expressed in the gut musculature, rectal papillae, Malpighian tubules and two small clusters in the thoracic nervous system and might be the source for peripheral TA. Interestingly, TyrR seems to be the only receptor expressed in the heart, suggesting that only TA modulates heart function. Contrary, OA has a modulatory effect on the heart of other insect species including honeybees, olive fruit flies and cockroaches. This is also in line with a previous report providing evidence that OA modulates the heart rate of the Drosophila fly and pupa, but not the larva (Pauls, 2018).
OA-dependent modulation of organs and tissues is mainly elicited through muscle action, especially in terms of its impact on the 'fight or flight' response. In line with this, it was observed Tdc2-Gal4-positive arborizations on nearly all skeletal muscles and many visceral muscles. In both Drosophila and desert locusts OA and TA is expressed in type II terminals of skeletal muscles50. OA has an excitatory effect on Drosophila flight muscles, while TA was shown to inhibit excitatory junction potentials, and thereby reduce muscle contractions and locomotion at least in the larva. In addition, flies lacking OA show severe deficits in flight initiation and maintenance. Interestingly, in an antagonistic effect to serotonin, OA reduces crop muscle activity presumably via Octβ1R, suggesting that OA has different effects on muscle activity dependent on the type of muscle. However, the data do not provide any convincing evidence of a direct innervation of Tdc2Ns of the crop, even though many fibers run in close vicinity, suggesting that OA might target the crop by volume transmission (Pauls, 2018).
Furthermore, OA modulates ovulation and fertilization in insects. Flies lacking OA display a severe egg-laying phenotype. Remarkably, within the female reproductive organ two different OA receptors, OAMB and Octβ2R, are necessary. Again, OA has a strong impact on muscle activity within the reproductive system. Octβ2R is expressed in the visceral oviduct muscle and elicits muscle relaxation through an increase of intracellular cAMP levels. Such an OA-dependent modulation appears to be conserved as OA is found in dorsal unpaired median neurons of locusts innervating oviduct muscles through the oviducal nerve. However, the data suggest that OA-positive fibers not only innervate oviduct muscles, but also enter the organs themselves. The OAMB receptor is expressed in epithelial cells inducing fluid secretion through increasing intracellular Ca2+ levels. Thus, OA affects different processes within the female reproductive organ due to the expression of different receptors and their coupled signaling pathways, which may be a general mechanism of the OA/TA system to fulfill an extensive modulatory function (Pauls, 2018).
OA does not exclusively modulate muscle activity, but also sensory neurons of external tissues like the antennae, halteres and wings. OA has also been shown to increase the spontaneous activity of olfactory receptor neurons (ORN). The modulation of ORNs allows OA to modulate the innate response to attractive stimuli like fruit odors or pheromones. Further, this modulation helps nestmate recognition in ants. In addition to Tdc2-Gal4-positive arborizations in the funiculus, Tdc2-Gal4-positive sensory neurons were found in the JO, a chordotonal organ sensitive to mechanosensory stimuli and thus important for hearing in insects. In mosquitos, OA modulates auditory frequency tuning and thereby affects mating behavior. In locusts, OA similarly modulates the response of chordotonal neurons in the legs to encode proprioceptive information. These data suggest that chordotonal neurons in the legs, wings, halteres and thorax are included in the Tdc2-Gal4 line suggesting a conserved modulatory role of OA/TA for insect proprioception (Pauls, 2018).
Taken together, this study suggests that the OA/TA system massively modulates various organs and tissues in the periphery of Drosophila. Through distinct receptors and coupled signaling pathways OANs/TANs mainly induce 'fight or flight' responses by modulating muscle activity, proprioception, and heart rate. As a result, the innervation pattern in the periphery supports the idea that the OA/TA system is crucial for insects to switch from a dormant to an excited state, by a positive modulation of muscle activity, heart rate and energy supply, and a simultaneous negative modulation of physiological processes like sleep (Pauls, 2018).
Myofibrils are huge cytoskeletal assemblies embedded in the cytosol of muscle cells. They consist of arrays of sarcomeres, the smallest contractile unit of muscles. Within a muscle type, myofibril diameter is highly invariant and contributes to its physiological properties, yet little is known about the underlying mechanisms setting myofibril diameter. This study shows that the PDZ and LIM domain protein Zasp, a structural component of Z-discs, mediates Z-disc and thereby myofibril growth through protein oligomerization. Oligomerization is induced by an interaction of its ZM domain with LIM domains. Oligomerization is terminated upon upregulation of shorter Zasp isoforms which lack LIM domains at later developmental stages. The balance between these two isoforms, which are call growing and blocking isoforms sets the stereotyped diameter of myofibrils. If blocking isoforms dominate, myofibrils become smaller. If growing isoforms dominate, myofibrils and Z-discs enlarge, eventually resulting in large pathological aggregates that disrupt muscle function (Gonzalez-Morales, 2019).
Inside cells, proteins are assembled into complex functional units. The correct assembly of these units is crucial for their function. Myofibrils are highly organized assemblies of cytoskeletal proteins forming an array of sarcomeres that are embedded in the cytosol of myotubes and mediate contractility. Sarcomeres are composed of actin-containing thin filaments and myosin-containing thick filaments arranged into antiparallel cables. Thin filaments are anchored to a large multiprotein complex called the Z-disc, and thick filaments are anchored to another large multiprotein complex called the M-line. Anchoring of myofibrils to the exoskeleton provides the mechanical tension that aligns sarcomeres into myofibrils and coordinates their development. Once aligned, sarcomeres grow to their final size. Electron and confocal microscopy studies showed that sarcomeres form initially from small structures called Z-bodies that grow eventually into mature Z-discs to which thin filaments are anchored. The size of the Z-disc therefore sets the diameter of the myofibril. While mechanisms have been proposed that set the length of sarcomeres, Z-disc growth and growth termination is poorly understood (Gonzalez-Morales, 2019b).
A hallmark of genetically caused myopathies is the appearance of large aggregates composed mainly of Z-disc proteins. Interestingly, many myopathy-associated mutations encode Z-disc proteins. Mutations in any of the four α-actinin genes or in Zasp and other Alp/Enigma family genes in humans cause myopathies characterized by the presence of large aggregates. The aggregation phenotype is conserved among animals: fruit flies with mutations in myopathy-related genes also develop Z-disc aggregates (Gonzalez-Morales, 2019b).
α-Actinin and Z-disc Alternatively Spliced Protein (Zasp) are conserved proteins that coordinate Z-disc formation. α-Actinin forms a rod-shaped antiparallel homodimer at the Z-disc, where it crosslinks and serves as an attachment point for actin filaments. Zasp and other members of the Alp/Enigma family of proteins are scaffolding proteins with an α-actinin-binding PDZ domain, an uncharacterized Zasp Motif (ZM; InterPro: IPR031847 and IPR006643) domain, and one to four protein-protein interaction LIM domains (InterPro: IPR001781). Zasp and α-actinin proteins are present at the earliest stages of Z-disc formation, and are required for Z-disc assembly (Gonzalez-Morales, 2019b).
Vertebrates have seven Alp/Enigma genes, each encoding several isoforms. The Drosophila genome has three Zasp genes, Zasp52, Zasp66, and Zasp67, which encode 21, 12, and 4 isoforms, respectively. Zasp66 and Zasp67 are duplications of Zasp52 and resemble the smallest isoforms of Zasp52 (González-Morales, 2019a). The number of isoform variants and genes adds an additional layer of complexity and regulation to sarcomere formation (Gonzalez-Morales, 2019b).
Drosophila indirect flight muscles wee used, and it was asked if the mechanism that controls Z-disc size relates to the pathological aggregation behavior known for Z-disc-related myopathies. This study shows that accumulation of multivalent Zasp growing isoforms (with multiple LIM domains) causes Z-disc growth, whereas upregulation of monovalent blocking isoforms at later developmental time points terminates Z-disc growth. An imbalance of growing and blocking Zasp isoforms results either in aggregate formation, enlarged Z-disc size or reduced Z-disc size. It is proposed that this mechanism has wide implications for diseases caused by aggregate formation (Gonzalez-Morales, 2019b).
These findings indicate that Z-disc formation and growth is driven by multivalent oligomerization of Zasp proteins with multiple LIM domains, and eventually terminated at the proper Z-disc size by the upregulation of blocking isoforms without LIM domains. The process of Z body and Z-disc formation is reminiscent of membraneless organelles with compositions distinct from the surrounding cytosol, which form through a mechanism of phase separation (e.g. Cajal bodies or P bodies) (Boeynaems, 2018; Weber, 2017). Both have sharp boundaries between themselves and the cytoplasm, they form and organize as discrete puncta in the cytosol, and multivalent protein domains are often involved in their formation (Gonzalez-Morales, 2019b).
Sarcomere size is stereotyped in a given muscle type but distinct among different muscles. How can the current model explain differences in sarcomere sizes? The sarcomere grows, while the Zasp growing isoforms dominate the Zasp isoform pool. Different sarcomere sizes can be achieved in two ways. First, the sarcomere growth period (i.e., the window of time in which Zasp growing isoforms dominate) might vary among muscle types. Second, the speed at which new Zasp molecules are recruited to the Z-disc, might be different among muscle types, while the growth period remains constant. Given the diversity of muscle types and therefore sarcomere sizes that exist, it is likely that a combination of these two strategies occur simultaneously (Gonzalez-Morales, 2019b).
Finally, apart from the ZM-LIM mechanism described in this study, additional redundant mechanisms to control Z-disc growth might exist, as evidenced by the observation that Zasp52-PR overexpression makes big Z-discs and aggregates, while the mutant removing Zasp52 LIM domains reduces Z-disc size to a comparatively small degree. Redundant mechanisms might operate through other LIM domain proteins, or the coordination of Z-disc and M-line growth, all of which may provide important buffering functions to ensure proper myofibril size, which is crucial for fully functional muscles (Gonzalez-Morales, 2019b).
The ZM domain is a conserved domain without a clearly defined function. On its own, the ZM domain from two mouse Zasp proteins localizes to the Z-disc (Klaavuniemi, 2004; Klaavuniemi, 2006). The data suggest that Z-disc localization is a conserved feature of ZM domains from vertebrates to insects. ZM-containing proteins are tethered to the Z-disc by the physical interaction with the LIM domains of other Zasp proteins. In sum, the LIM domain serves as a recruitment signal for Zasp proteins and potentially other unidentified ZM-containing proteins to join the Z-disc. In addition, given the appearance of the ZM domain in bilateral animals with canonical Z-discs, it is postulated that a conserved mechanism involving LIM-ZM binding underlies Z-disc growth and growth termination (Gonzalez-Morales, 2019b).
In vertebrates, the Zasp proteins are very diverse and are better known as Alp/Enigma family: ZASP/Cypher/Oracle/LDB3/PDLIM6, ENH/PDLIM5, PDLIM7/ENIGMA/LMP-1, CLP36/PDLIM1/Elfin/hCLIM1, PDLIM2/Mystique/SLIM, ALP/PDLIM3, and RIL/PDLIM4. The ZM/DUF4749 motif occurs in ZASP, CLP36, PDLIM2, ALP and RIL. The LIM domain occurs in all Zasp proteins, either as one domain in Alp family members or as three domains in Enigma family members. Two Zasp genes were identified that encode only blocking isoforms in fruit flies: Zasp66 and Zasp67, and one gene, Zasp52, that encodes blocking and growing isoforms. Although Zasp66 and Zasp67 genes are insect-specific (Gonzalez-Morales, 2019a), vertebrate Alp/Enigma genes also express isoforms without LIM domains that could fulfill a blocking isoform function. In addition, because Zasp52-PK, which only contains one LIM domain, behaves as a blocking isoform, the Alp members with only one LIM domain might also behave as blocking isoforms (Gonzalez-Morales, 2019b).
The function of the growing isoforms of Zasp requires multiple functional LIM domains. As the Enigma family members contain three C-terminal LIM domains, they are the ideal candidates to fulfill the growing role in vertebrates. Three Enigma proteins exist in vertebrates: PDLIM7/Enigma/LMP-1, ENH/PDLIM5 and ZASP/Cypher/Oracle/LDB3/PDLIM6. Functional redundancy between them at the Z-disc is likely common and demonstrated in one case. In Cypher knockout mice sarcomere assembly occurs normally during development, followed by immediate sarcomere failure after postnatal onset of contractility. ENH mutants exhibit cardiac dilation and abnormal Z-disc structure in the heart. Intriguingly, in both Cypher and ENH single mutants, as well as Cypher ENH double mutants, sarcomeres look considerably smaller in diameter in electron microscopy images. Thus, a similar role for Enigma proteins in setting sarcomere diameter in vertebrates appears likely (Gonzalez-Morales, 2019b).
Is the mechanism that controls Z-disc size related to the protein aggregation defects in human myopathies? The Z-disc oligomerization hypothesis agrees well with the observation that many myopathies present aggregates, and several human ZASP mutations have been linked to aggregate-forming myopathies. Many ZASP mutations linked to disease lie within the ZM domain or one of the LIM domains. Protein aggregation in myopathy patients might be a consequence of an imbalance in the mechanism that controls sarcomere size, favoring the growing over the blocking isoforms. If this were the case, the data points to a potential therapeutic avenue: blocking the growing isoforms with short peptides containing a ZM domain (Gonzalez-Morales, 2019b).
In conclusion, it is proposed that a conserved mechanism involving LIM-ZM binding underlies Z-disc growth and therefore myofibril diameter (Gonzalez-Morales, 2019b).
How cells position organelles is a fundamental biological question. During Drosophila embryonic muscle development, multiple nuclei transition from being clustered together, to splitting into two smaller clusters, to spreading along the myotube's length. Perturbations of microtubules and motor proteins disrupt this sequence of events. These perturbations do not allow intuiting which molecular forces govern the nuclear positioning; therefore computational screening was used to reverse engineer and identify these forces. The screen reveals three models: two suggest that the initial clustering is due to the nuclear repulsion from the cell poles, while the third, most robust, model poses that this clustering is due to a short-ranged internuclear attraction. All three models suggest that the nuclear spreading is due to the long-ranged internuclear repulsion. The robust model was quantitatively tested by comparing it to data from perturbed muscle cells. The model was also tested by using agent-based simulations with elastic dynamic microtubules and molecular motors. The model predicts that, in longer mammalian myotubes with a great number of nuclei, the spreading stage would be preceded with segregation of the nuclei into a large number of clusters, proportional to the myotube length, with a small average number of nuclei per cluster (Manhart, 2020).
Muscle myofibrils and sarcomeres present exceptional examples of highly ordered cytoskeletal filament arrays, whose distinct spatial organization is an essential aspect of muscle cell functionality. This study utilized ultra-structural analysis to investigate the assembly of myofibrils and sarcomeres within developing myotubes of the indirect flight musculature of Drosophila. A temporal sequence composed of three major processes was identified: subdivision of the unorganized cytoplasm of nascent, multi-nucleated myotubes into distinct organelle-rich and filament-rich domains; initial organization of the filament-rich domains into myofibrils harboring nascent sarcomeric units; and finally, maturation of the highly-ordered pattern of sarcomeric thick (myosin-based) and thin (microfilament-based) filament arrays in parallel to myofibril radial growth. Significantly, organized microtubule arrays were present throughout these stages and exhibited dynamic changes in their spatial patterns consistent with instructive roles. Genetic manipulations confirm these notions, and imply specific and critical guidance activities of the microtubule-based cytoskeleton, as well as structural interdependence between the myosin- and actin-based filament arrays. These observations highlight a surprisingly significant, behind-the-scenes role for microtubules in establishment of myofibril and sarcomere spatial patterns and size, and provide a detailed account of the interplay between major cytoskeletal elements in generating these essential contractile myogenic units (Dhanyasi, 2020).
Using Drosophila, a role for was uncovered for myocyte enhancer factor 2 (MEF2) in modulating diet-dependent lipid droplet diversification within adult striated muscle, impacting mortality rates. Muscle-specific attenuation of MEF2, whose chronic activation maintains glucose and mitochondrial homeostasis, leads to the accumulation of large, cholesterol ester-enriched intramuscular lipid droplets in response to high calorie, carbohydrate-sufficient diets. The diet-dependent accumulation of these lipid droplets also correlates with both enhanced stress protection in muscle and increases in organismal lifespan. Furthermore, MEF2 attenuation releases an antagonistic regulation of cell cycle gene expression programs, and up-regulation of Cyclin E is required for diet- and MEF2-dependent diversification of intramuscular lipid droplets. The integration of MEF2-regulated gene expression networks with dietary responses thus plays a critical role in shaping muscle metabolism and function, further influencing organismal lifespan. Together, these results highlight a potential protective role for intramuscular lipid droplets during dietary adaptation (Zhao, 2020).
Cancer cells demand excess nutrients to support their proliferation, but how tumours exploit extracellular amino acids during systemic metabolic perturbations remain incompletely understood. This study used a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis to uncover a systemic host-tumour metabolic circuit that supports tumour growth. Coordinate induction is demonstrated of systemic muscle wasting with tumour-autonomous Yorkie-mediated SLC36-family amino acid transporter expression as a proline-scavenging programme to drive tumourigenesis. Indole-3-propionic acid was identified as an optimal amino acid derivative to rationally target the proline-dependency of tumour growth. Insights from this whole-animal Drosophila model provide a powerful approach towards the identification and therapeutic exploitation of the amino acid vulnerabilities of tumourigenesis in the context of a perturbed systemic metabolic network (Newton, 2020).
Cancer cells require a constant supply of metabolic intermediates to support their proliferation. To meet the biosynthetic demands associated with tumourigenesis, cancer cells actively acquire nutrients from the extracellular space. Cancer is a systemic disease that associates with a range of host metabolic abnormalities such as obesity, insulin resistance and cancer-associated cachexia; each of which alters the host systemic nutritional environment. These changes in both nutrient composition and availability may have profound effects on cancer development and progression. However, how cancer cells sense and respond to nutritional changes in the context of organismal metabolic alterations remains an underexplored area in cancer biology (Newton, 2020).
Cancer-associated cachexia is a systemic metabolic syndrome of weight loss associated with progressive skeletal muscle wasting. The multifactorial and heterogeneous condition of cachexia involves a complex multi-organ interplay, which has impeded its comprehensive understanding at the molecular level. A series of recent studies using Drosophila melanogaster have shown that tumour-derived factors modulate host metabolism. In addition, tumour-derived factors promote the release of nutrients from the tumour microenvironment to promote tumour growth. This study has leveraged a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis and demonstrates that HSD-enhanced tumours induce SLC36-family transporter expression as a coordinated mechanism to exploit exogenous proline for tumourigenesis during systemic muscle wasting. Furthermore, these mechanistic insights were used to rationally target the proline dependency of tumours as an approach to inhibit tumour growth (Newton, 2020).
Muscle wasting is observed in chronic muscle wasting diseases due to cancer (cachexia), ageing/senescence (sarcopenia), myopathies and other metabolic diseases, as well as in acute conditions due to burns and sepsis. This study demonstrates that the combination of diet-induced obesity and tumour growth induces systemic muscle wasting, associated with functional locomotion defect. This muscle phenotype was not due to the inability to properly form muscle during development; the muscles are wholly formed at the early stage of larval development, and waste progressively at the later larval stage, as the tumours develop. In addition, it was demonstrated that the muscle phenotype observed in this model is unrelated to a developmentally related degeneration process (Newton, 2020).
This study reveals a systemic amino acid-utilising circuit whereby HSD-enhanced tumours induce muscle wasting as a systemic metabolic network to drive tumourigenesis. A consequence of muscle wasting in ras1G12V;csk-/- animals raised on HSD was increased release of proline into the circulation. Plasma amino acid profiling of patients with sarcopenia - a condition of muscle wasting associated with aging—showed elevated plasma proline levels, indicating that elevated free circulating proline is a common feature of muscle wasting. This study identified a proline vulnerability of HSD-enhanced tumours; SLC36-family amino acid transporter Path is required for tumour growth and exogenous proline promotes tumourigenesis through Path. This study highlights two layers of coordination in tumour metabolic response: (1) at the whole organism level, by promoting muscle wasting and systemic amino acid availability, and (2) at the tumour-autonomous level, by altering amino acid transporter repertoire (Newton, 2020).
Path and CG1139 have different transport characteristics for proline, with Path having a lower transport capacity compared to CG1139. Despite this, labelled proline uptake experiments indicated uptake of extracellular proline in Ras/Src/Path-tumours but not in Ras/Src/CG1139-tumours, suggesting that CG1139 and Path may exhibit different activities in the context of an oncogenic background. Consistent with a previous report (Goberdhan, 2005), the current data support the signalling role of Path through activation of the Tor-S6K pathway. Proline metabolism has been demonstrated to support cancer clonogenicity and metastasis. Furthermore, proline promotes cancer cell survival under nutrient-limited or hypoxic microenvironments. The current data extend these studies to reveal a tumour-promoting role of proline in response to systemic host metabolic changes (Newton, 2020).
Indole-3-propionic acid (IPA) specifically targets the proline dependency of tumour growth in Drosophila. Furthermore, this study demonstrates a functional effect of IPA on human cells. A recent study demonstrated that proline uptake in Ras-driven tumour cells is much higher in spheroids in three-dimensional cultures—which better mimic conditions in vivo—compared to monolayers in two-dimensional cultures, suggestive of a potentially stronger effect in tumours that are dependent on proline for growth in vivo. Proline is a limiting amino acid for protein synthesis in kidney cancers. Therefore, reducing proline uptake with SLC36-inhibitors -- as exemplified here by use of IPA -- may warrant consideration as a therapeutic strategy to break the nutritional circuit between systemic muscle wasting and tumour growth in proline vulnerable cancers (Newton, 2020).
Organ growth and size are finely tuned by intrinsic and extrinsic signaling molecules. In Drosophila, the BMP family member Dpp is produced in a limited set of imaginal disc cells and functions as a classic morphogen to regulate pattern and growth by diffusing throughout imaginal discs. However, the role of TGFβ/Activin-like ligands in disc growth control remains ill-defined. This study demonstrates that Myoglianin (Myo), an Activin family member, and a close homolog of mammalian Myostatin (Mstn), is a muscle-derived extrinsic factor that uses canonical dSmad2-mediated signaling to regulate wing size. It is proposed that Myo is a myokine that helps mediate an allometric relationship between muscles and their associated appendages. Although Babo/dSmad2 signaling has been previously implicated in imaginal disc growth control, the ligand(s) responsible and their production sites(s) have not been identified. Previous in situ hybridization and RNAi knockdown experiments suggested that all three Activin-like ligands contribute to control of wing size. However, no expression of these Activin-like ligands was found in imaginal discs, with the exception of Actβ which is expressed in differentiating photoreceptors of the eye imaginal disc. It is concluded that the small wing phenotypes caused by RNAi knockdown of Actβ or daw are likely the result of off-target effects and that Myo is the only Activin-type ligand that regulates imaginal disc growth (Upadhyay, 2020).
Although Babo/dSmad2 signaling has been previously implicated in imaginal disc growth control, the ligand(s) responsible and their production sites(s) have not been identified. Previous in situ hybridization and RNAi knockdown experiments suggested that all three Activin-like ligands (Myoglianin, Activinβ, and Dawdle) contribute to control of wing size. However, this study found no expression of these Activin-like ligands in imaginal discs, with the exception of Actβ which is expressed in differentiating photoreceptors of the eye imaginal disc. More importantly, using genetic null mutants, this study showed that only loss of myo affects imaginal disc size. The discrepancy in phenotypes between tissue-specific knockdown results and the genetic nulls is often noted and not fully understood. In addition to simple off-target effects within the wing disc itself, one possible explanation is that many GAL4 drivers are expressed in tissues other than those reported, potentially resulting in deleterious effects for the animal that indirectly affect imaginal disc size. Another possibility is that in Actβ and daw genetic null backgrounds a non-autonomous compensatory signal is generated by another tissue and this signal is not activated in the case of tissue-specific knockdown. Both of these explanations are thought unlikely in this instance since it was demonstrated that only the Babo-A receptor isoform is expressed and required in discs. Since it was previously shown that Daw only signals through isoform Babo-C, it is unclear why knockdown of daw in the wing disc would result in a small wing phenotype as previously reported. It is concluded that the small wing phenotypes caused by RNAi knockdown of Actβ or daw are likely the result of off-target effects and that Myo is the only Activin-type ligand that regulates imaginal disc growth (Upadhyay, 2020).
The signaling ability of TGFβ ligands is modulated by the specific combinations of receptors and co-receptors to which they bind. In Drosophila, the receptor requirements for effective signaling through dSmad2 likely vary for each ligand and tissue. This study found that Myo signaling in the wing disc requires Punt as the type II receptor and Babo-A as the type I receptor. Furthermore, it was establish that Myo is the exclusive Activin-like ligand signaling to the discs since loss of Myo eliminated detectable phosphorylation of dSmad2 in the wing imaginal disc. Since Babo-A is the only isoform expressed in wing discs, it is also concluded that Myo is able to signal through this isoform in the absence of other isoforms. Whether Myo can also signal through Babo-B or C is not yet clear, but in the context of mushroom body remodeling Babo-A also appears to be the major receptor isoform utilized. The co-receptor Plum (see Plum, an Immunoglobulin Superfamily Protein, Regulates Axon Pruning by Facilitating TGF-β Signaling) is also required for mushroom body remodeling, suggesting that Plum and Babo-A are both necessary for efficient Myo signaling. However, it is noteworthy that Plum null mutants are viable while Myo null mutants are not. This observation suggests that Plum is not required for all Myo signaling during development. Further studies will be required to evaluate whether Plum is essential to mediate Myo signaling in imaginal discs (Upadhyay, 2020).
The requirement of Punt as a type II receptor for production of an efficient signaling complex with Myo may be context dependent. In the mushroom body, indirect genetic evidence suggests that the two Type II receptors function redundantly. Although both punt and wit are expressed in imaginal discs, only loss of punt produces a phenotype in the brk reporter assay. To date, clear signaling has not been seen in S2 cells expressing Punt and Babo-A when Myo is added. It is also notable that a previous attempt to study Myo signaling in a heterotypic cell culture model also failed. In that study, Myo was found to form a complex with Wit and Babo-A in COS-1 cells but no phosphorylation of dSmad2 was reported. One explanation is that effective signaling by Myo requires Punt, and Babo-A, and perhaps another unknown co-receptor that substitutes for Plum. Despite this caveat, the current results provide in vivo functional evidence for a Myo signaling complex that requires Babo-A and Punt to phosphorylate dSmad2 for regulation of imaginal disc growth (Upadhyay, 2020).
Final tissue size is determined by several factors including cell size, proliferation, death rates, and duration of the growth period. While cell size changes were observed upon manipulation of Myo signaling, the direction of change depended on the genotype. In myo mutants, estimation of cell size via apical surface area indicates that the cells are ~20% smaller than wild-type. Although this measurement does not indicate the actual volume of the cells, it gives an indication of cell density in the epithelial sheet of the wing pouch, which is analogous to counting cells in the adult wing. RNAi knock down of babo-a in the entire disc produced smaller adult wings with larger (less dense) cells. This result differs from the myo mutant, but is similar to the reported adult wing phenotypes of babo mutants and larval disc phenotypes of dSmad2 mutants. When babo-a is knocked down in one compartment, that compartment is reduced in size with smaller cells. It is concluded that tissue size reduction is the consistent phenotype upon loss of Myo signaling, but cell size changes depend on the specific type of manipulation (Upadhyay, 2020).
While cell size effects may be context dependent, it is notable that neither reduction in size of imaginal discs nor adult wing surface area can be explained solely by a cell size defect. Since no apoptotic increase was seen in myo mutant discs, and because dSmad2 knockdown also fails to alter apoptotic rate, the mostly likely cause is an altered proliferation rate. Consistent with this view is that the large disc phenotype exhibited by dSmad2 protein null mutants is clearly dependent on Myo and it has been previously shown that this is the result of enhanced proliferation. Similarly earlier studies also showed that expression of activated Babo or activated dSmad2 in wing discs also leads to larger wings with slightly smaller cells which is most easily explained by an enhanced proliferation rate. It is worth noting that this proposed enhanced proliferation rate is difficult to detect since cell division is random with regard to space and time during development. Thus a ~ 20% reduction in adult wing size caused by a proliferation defect translates into about 1/5th of disc cells dividing on average one less time throughout the entire time course of larval development. Therefore, without prolonged live imaging, this small reduction in proliferation rate will not be detectable using assays that provide only static snapshots of cell division. It is worth noting, however, that previous clonal studies also concluded that dSmad2 or Babo loss in wing disc clones resulted in a reduced proliferation rate (Upadhyay, 2020).
One attempt to shed light on the transcriptional output of TGFβ signaling responsible for wing disc size employed microarray mRNA profiling of wild-type versus dSmad2 gain- and loss-of-function wing discs. However, this study did not reveal a clear effect on any class of genes including cell cycle components, and it was concluded that the size defect is the result of small expression changes of many genes. Consistent with this view are dSmad2 Chromatin Immunoprecipitation experiments in Kc cells which revealed that dSmad2 is associated with many genomic sites and thus may regulate a myriad of genes (Upadhyay, 2020).
Insect myoglianin is a clear homolog of vertebrate Myostatin (Mstn/GDF8), a TGFβ family member notable for its role in regulating skeletal muscle mass. Mstn loss-of-function mutants lead to enlarged skeletal muscles. Mstn is thought to affect muscle size through autocrine signaling that limits muscle stem cell proliferation, as well as perturbing protein homeostasis via the Insulin/mTOR signaling pathways. Similarly, Gdf11, a Mstn paralog, also regulates size and proliferation of muscles and adipocytes, and may promote healthy aging. Mstn and Gdf11 differ in where they are expressed and function. Mstn is highly expressed in muscles during development while Gdf11 is weakly expressed in many tissues. Both molecules are found to circulate in the blood as latent complexes in which their N-terminal prodomains remain associated with the ligand domain. Activation requires additional proteolysis of the N-terminal fragment by Tolloid-like metalloproteases to release the mature ligand for binding to its receptors. Interestingly in Drosophila, the Myoglianin N-terminal domain was also found to be processed by Tolloid-like factors, but whether this is a prerequisite for signaling has not yet been established. In terms of functional conservation in muscle size control, the results of both null mutants and RNAi depletion indicates that it has little effect on muscle size. This contradicts a previous study in which muscle-specific RNAi knockdown of myo was reported to produce larger muscles similar to the vertebrate observation. The discrepancy between the tissue-specific RNAi knockdown and previous studies is not clear, but the current null mutant analysis strongly argues that Drosophila Myo does not play a role in muscle size control. Intriguingly however, this study found that loss of Actβ, another ligand that signals through Babo and dSmad2, results in a smaller muscles) contrary to that produced by loss of vertebrate Mstn and various other vertebrate Activin family members. Recent data has shown that Drosophila Actβ is the only Activin-like ligand that affects muscle growth, and it does so, in part, by regulating Insulin/Tor signaling in the opposite direction compared to vertebrates. Thus, in Drosophila the Myo/Activin pathway promotes muscle growth while in vertebrates it inhibits muscle growth (Upadhyay, 2020).
The most intriguing finding of this study is that muscle-derived Myo acts non-autonomously to regulate imaginal disc growth. This is in stark contrast to the two BMP ligands, Dpp and Gbb, which are produced by disc cells and act autonomously within the disc itself to regulate both growth and pattern. The fact that a TGFβ ligand can act in an endocrine-like manner is not particularly novel since many vertebrate members of the TGFβ family, including Myostatin, the closest homolog to Drosophila Myoglianin, are found in the blood. Even the disc intrinsic molecule Dpp has been recently shown to be secreted into the hemolymph where it circulates and signals to the prothoracic gland to regulate a larval nutritional checkpoint. Several additional reports indicate that ligands from the Drosophila Activin-like subfamily also circulate in the hemolymph and function as inter-organ signals. For example, muscle-derived Actβ and Myo signal to the fat body to regulate mitochondrial function and ribosomal biogenesis, respectively. In addition, Daw produced from many tissue sources can signal to the Insulin producing cells and the midgut to stimulate Insulin secretion and repress expression of sugar metabolizing genes, respectively. Thus, many TGFβ type factors act as both paracrine and endocrine signals depending on the tissue and process involved (Upadhyay, 2020).
The phenotype of the myo mutant animal supports the claim that endogenous Myo contributes to imaginal disc growth. The ectopic expression assay produced various wing disc sizes when Myo was expressed in different tissues, indicating that the growth response likely depends on the level of Myo being produced in the distal tissue. Loss of glial derived Myo is not sufficient to suppress overgrowth of dSmad2 mutant discs, but overexpression of Myo in glia did partially rescue size of myo null wing discs, likely because the repo-Gal4 driven overexpression produces more ligand than endogenous glia. Likewise, expression from a large tissue such as muscle or fat body likely produces more Myo than glia leading to normal disc growth or even overgrowth. It is also possible that Myo signaling activity is modified depending on the tissue source. Like other TGFβ family members, Myo requires cleavage by a furin protease at its maturation site to separate the C-terminal ligand from the prodomain. Myo may also require a second cleavage by a Tolloid protease family member to achieve full dissociation of the prodomain from the ligand to ensure complete activation. Either of these cleavage reactions, or any other step impacting the bioavailability of active Myo ligand, may vary with tissue or may be modulated by environmental conditions (Upadhyay, 2020).
What is the rationale for larval muscle regulating imaginal discs size? A possible reason is that for proper appendage function, the muscle and the structure (leg, wing, and haltere) that it controls should be appropriately matched to ensure optimal organismal fitness for the environmental niche the adult occupies. For example, a large muscle powering a small wing might result in diminished fine motor control. Conversely, a small muscle may not be able to power a large wing to support flight. However, the multi-staged nature of muscle and appendage development complicates this picture. Larval muscles are histolysed during metamorphosis and do not contribute to the adult muscle. However, remnants of larval muscles in the thoracic segment are preserved as fibers that act as scaffolds upon which the larval myoblasts infiltrate and fuse to become the adult indirect flight muscles. Thus, at least for the indirect flight muscles, the size of the larval muscle scaffold might contribute to the building of a bigger adult muscle. Another possibility invokes a signal relay system. Wg and Serrate/Notch signaling from the wing disc epithelial cells control myoblast proliferation during larval development. Thus it may be that Myo signaling from the larval muscles stimulates proliferation of the disc epithelial layer which in turn enhances Wnt and Serrate/Notch signaling to myoblasts to increase their number thereby coordinating the adult appendage size with muscle size. A final scenario is that, since muscle is a major metabolic and endocrine organ, Myo production may be regulated by the general metabolic state of the larva. If healthy, high levels of Myo, in concert with other growth signals such as insulin, leads to a bigger fly with large wings, and if the metabolic state is poor then lower Myo levels leads to diminished proliferation and a smaller cell size resulting in a smaller fly with small wings (Upadhyay, 2020).
Regardless of the precise mechanism, the ability of the muscle to control appendage size has interesting implications in terms of evolutionary plasticity. The proportionality of insect wing size to body size can vary over a large range, but the mechanism responsible for determining this particular allometric relationship for a given species is not understood. It was recently demonstrated that in Drosophila, motor neuron derived Actβ, another TGFβ superfamily member, can dramatically affect muscle/body size (Moss-Taylor, 2019). Therefore, it is tempting to speculate that evolutionary forces might modulate the activity of these two genes to produce an appropriate body-wing allometry that is optimal for that species' ecological niche (Upadhyay, 2020).
Muscle forces are produced by repetitive stereotyped acto-myosin units called sarcomeres. Sarcomeres are chained into linear myofibrils spanning the entire muscle fiber. In mammalian body muscles, myofibrils are aligned laterally resulting in their typical cross-striated morphology. Despite this detailed textbook knowledge about the adult muscle structure, it is still unclear how cross-striated myofibrils are built in vivo. This study investigated the morphogenesis of Drosophila abdominal muscles and establishrf them as in vivo model for cross-striated muscle development. Using live imaging, ong immature myofibrils lacking a periodic acto-myosin pattern were found to be built simultaneously in the entire muscle fiber and then align laterally to mature cross-striated myofibrils. Interestingly, laser micro-lesion experiments demonstrate that mechanical tension precedes the formation of the immature myofibrils. Moreover, these immature myofibrils do generate spontaneous Ca2+ dependent contractions in vivo, which when chemically blocked result in cross-striation defects. Together, these results suggest a myofibrillogenesis model, in which mechanical tension and spontaneous muscle twitchings synchronise the simultaneous self-organisation of different sarcomeric protein complexes to build highly regular cross-striated myofibrils spanning throughout large muscle fibers (Weitkunat, 2017).
A stereotyped array of body wall muscles enables precision and stereotypy of animal movements. In Drosophila, each syncytial muscle forms via fusion of one founder cell (FC) with multiple fusion competent myoblasts (FCMs). The specific morphology of each muscle, i.e. distinctive shape, orientation, size and skeletal attachment sites, reflects the specific combination of identity transcription factors (iTFs), such as Apterous, Even-Skipped and Slouch/S59, expressed by its FC. This study addressed three questions: Are FCM nuclei naive? What is the selectivity and temporal sequence of transcriptional reprogramming of FCMs recruited into growing syncytium? Is transcription of generic myogenic and identity realisation genes coordinated during muscle differentiation? The tracking of nuclei in developing muscles shows that FCM nuclei are competent to be transcriptionally reprogrammed to a given muscle identity, post fusion. In situ hybridisation to nascent transcripts for FCM, FC-generic and iTF genes shows that this reprogramming is progressive, beginning by repression of FCM-specific genes in fused nuclei, with some evidence that FC nuclei retain specific characteristics. Transcription of identity realisation genes is linked to iTF activation and regulated at levels of both transcription initiation rate and period of transcription. The generic muscle differentiation programme is activated independently. It is concluded that transcription reprogramming of fused myoblast nuclei is progressive, such that nuclei within a syncytial fibre at a given time point during muscle development are heterogeneous with regards to specific gene transcription. This comprehensive view of the dynamics of transcriptional (re)programming of post-mitotic nuclei within syncytial cells provides a new framework for understanding the transcriptional control of the lineage diversity of multinucleated cells (Bataille, 2017).
Hemolymph is driven through the antennae of Drosophila melanogaster by the rhythmic contraction of muscle 16 (m16), which runs through the brain. Contraction of m16 results in the expansion of an elastic ampulla, opening ostia and filling the ampulla. Relaxation of the ampullary membrane forces hemolymph through vessels into the antennae. This study shows that m16 is an auto-active rhythmic somatic muscle. The activity of m16 leads to the rapid perfusion of the antenna by hemolymph. In addition, it leads to the rhythmic agitation of the brain, which could be important for clearing the interstitial space (Kay, 2021).
Conservation of genetic toolkits in disparate phyla may help reveal commonalities in organ designs transcending their extreme anatomical disparities. A male accessory sexual organ in mammals, the prostate, for instance, is anatomically disparate from its analogous, phylogenetically distant counterpart - the male accessory gland (MAG) - in insects like Drosophila. It has not been ascertained if the anatomically disparate Drosophila MAG shares developmental parallels with those of the mammalian prostate. This study shows that the development of Drosophila mesoderm-derived MAG entails recruitment of similar genetic toolkits of tubular organs like that seen in endoderm-derived mammalian prostate. For instance, like mammalian prostate, Drosophila MAG morphogenesis is marked by recruitment of fibroblast growth factor receptor (FGFR) - a signalling pathway often seen recruited for tubulogenesis - starting early during its adepithelial genesis. A specialisation of the individual domains of the developing MAG tube, on the other hand, is marked by the expression of a posterior Hox gene transcription factor, Abd-B, while Hh-Dpp signalling marks its growth. Drosophila MAG, therefore, reveals the developmental design of a unitary bud-derived tube that appears to have been co-opted for the development of male accessory sexual organs across distant phylogeny and embryonic lineages (Kumari, 2021).
The nuclei of multinucleated skeletal muscles experience substantial external force during development and muscle contraction. Protection from such forces is partly provided by lamins (see Drosophila Lamin), intermediate filaments that form a scaffold lining the inner nuclear membrane. Lamins play a myriad of roles, including maintenance of nuclear shape and stability, mediation of nuclear mechanoresponses, and nucleo-cytoskeletal coupling. This study investigated how disease-causing mutant lamins alter myonuclear properties in response to mechanical force. This was accomplished via a novel application of a micropipette harpooning assay applied to larval body wall muscles of Drosophila models of lamin-associated muscular dystrophy. The assay enables the measurement of both nuclear deformability and intracellular force transmission between the cytoskeleton and nuclear interior in intact muscle fibers. These studies revealed that specific mutant lamins increase nuclear deformability while other mutant lamins cause nucleo-cytoskeletal coupling defects, which were associated with loss of microtubular nuclear caging. Microtubule caging of the nucleus was found to depended on Msp300, a KASH domain protein that is a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Taken together, these findings identified residues in lamins required for connecting the nucleus to the cytoskeleton and suggest that not all muscle disease-causing mutant lamins produce similar defects in subcellular mechanics (Shaw, 2022).
Nemaline myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 (TPM2), which encodes a skeletal muscle-specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as cap myopathy, congenital fiber type disproportion, and distal arthrogryposis (DA). The in vivo pathomechanisms underlying TPM2-related disorders are unknown, so this study expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found 4 variants significantly affected muscle development and muscle function. Transient overexpression of the 4 variants also disrupted the morphogenesis of mouse myotubes in vitro and negatively affected zebrafish muscle development in vivo. Transient overexpression assays in zebrafish were used to characterize two potentially novel TPM2 variants and one recurring variant that were identified in patients with DA (V129A, E139K, A155T, respectively) and found these variants caused musculoskeletal defects similar to those of known pathogenic variants. The consistency of musculoskeletal phenotypes in these assays correlated with the severity of clinical phenotypes observed in patients with DA, suggesting disrupted myogenesis is a potentially novel pathomechanism of TPM2 disorders and that myogenic assays can predict the clinical severity of TPM2 variants (McAdow, 2022).
Muscles have evolved to power a wide variety of movements. A protein component critical to varying power generation is the myosin isoform present in the muscle. However, how functional variation in muscle arises from myosin structure is not well understood. The influence of the converter, a myosin structural region at the junction of the lever arm and catalytic domain, was studied using Drosophila because its single myosin heavy chain gene expresses five alternative converter versions (11a-e). Five transgenic fly lines were generated, each forced to express one of the converter versions in their indirect flight muscle (IFM) fibers. Electron microscopy showed that the converter exchanges did not alter muscle ultrastructure. The four lines expressing converter versions (11b-e) other than the native IFM 11a converter displayed decreased flight ability. IFM fibers expressing converters normally found in the adult stage muscles generated up to 2.8-fold more power and displayed up to 2.2-fold faster muscle kinetics than fibers with converters found in the embryonic and larval stage muscles. Small changes to stretch-activated force generation only played a minor role in altering power output of IFM. Muscle apparent rate constants, derived from sinusoidal analysis of the chimeric converter fibers, showed a strong positive correlation between optimal muscle oscillation frequency and myosin attachment kinetics to actin, and an inverse correlation with detachment related cross-bridge kinetics. This suggests the myosin converter alters at least two rate constants of the cross-bridge cycle with changes to attachment and power stroke related kinetics having the most influence on setting muscle oscillatory power kinetics (Glasheen, 2018).
The myosin molecular motor interacts with actin filaments in an ATP-dependent manner to yield muscle contraction. Myosin heavy chain residue R369 is located within loop 4 at the actin-tropomyosin interface of myosin's upper 50 kDa subdomain. To probe the importance of R369, this study introduced a histidine mutation of that residue into Drosophila myosin and implemented an integrative approach to determine effects at the biochemical, cellular, and whole organism levels. Substituting the similarly charged but bulkier histidine residue reduces maximal actin binding in vitro without affecting myosin ATPase activity. R369H mutants exhibit impaired flight ability that is dominant in heterozygotes and progressive with age in homozygotes. Indirect flight muscle ultrastructure is normal in mutant homozygotes, suggesting that assembly defects or structural deterioration of myofibrils are not causative of reduced flight. Jump ability is also reduced in homozygotes. In contrast to these skeletal muscle defects, R369H mutants show normal heart ultrastructure and function, suggesting that this residue is differentially sensitive to perturbation in different myosin isoforms or muscle types. Overall, these findings indicate that R369 is an actin binding residue that is critical for myosin function in skeletal muscles, and suggest that more severe perturbations at this residue may cause human myopathies through a similar mechanism (Trujillo, 2022).
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Summary:
100s of Small Open Reading Frames (smORFs) are actively translated and display conserved sequence, structure and evolutionary signatures indicating that the translated peptides could fulfil important biological roles. Some of these smORFs display conserved functions with their mutations being associated with pathogenesis. Thus, investigating smORF roles in Drosophila will not only expand understanding of their functions but it may have an impact in human health. This study describe the function of a novel and essential Drosophila smORF gene named purriato (prto CG9034). prto belongs to an ancient gene family whose members have expanded throughout the Protostomia clade. prto encodes a transmembrane peptide which is localized in endo-lysosomes and perinuclear and plasma membranes. prto is dynamically expressed in mesodermal tissues and imaginal discs. Targeted prto knockdown (KD) in these organs results in changes in nuclear morphology and endo-lysosomal distributions correlating with the loss of sarcomeric homeostasis in muscles and reduction of mitosis in wing discs. Consequently, prto KD mutants display severe reduction of motility, and shorter wings. Finally, genetic interaction experiments show that prto function is closely associated to the CASA pathway, a conserved mechanism involved in turnover of mis-folded proteins and linked to muscle dystrophies and neurodegenerative diseases. Thus, this study shows the relevance of smORFs in regulating important cellular functions and supports the systematic characterisation of this class of genes to understand their functions and evolution (Pueyo, 2023).
Summary:
Cardiomyocyte maturation requires a massive increase in respiratory enzymes and their assembly into long-lived complexes of oxidative phosphorylation (OXPHOS). The molecular mechanisms underlying the maturation of cardiac mitochondria have not been established. To determine whether the mitochondria-specific lipid cardiolipin is involved in cardiac maturation, a cardiomyocyte-restricted knockout (KO) of cardiolipin synthase (Crls1 ) was created in mice, and the postnatal development of the heart was studied. The turnover rates were also measured of proteins and lipids in cardiolipin-deficient flight muscle from Drosophila, a tissue that has mitochondria with high OXPHOS activity like the heart. Crls1KO mice survived the prenatal period but failed to accumulate OXPHOS proteins during postnatal maturation and succumbed to heart failure at the age of 2 weeks. Turnover measurements showed that the exceptionally long half-life of OXPHOS proteins is critically dependent on cardiolipin. Cardiolipin is essential for the postnatal maturation of cardiomyocytes because it allows mitochondrial cristae to accumulate OXPHOS proteins to a high concentration and to shield them from degradation (Ren, 2023).
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