The Interactive Fly
Zygotically transcribed genes
Dorsoventral (DV) patterning of the Drosophila embryo is initiated by a broad Dorsal (Dl) nuclear gradient, which is regulated by a conserved signaling pathway that includes the Toll receptor and Pelle kinase. What are the consequences of expressing a constitutively activated form of the Toll receptor, Toll(10b), in anterior regions of the early embryo? Using the bicoid 3' UTR, localized Toll(10b) products result in the formation of an ectopic, anteroposterior (AP) Dl nuclear gradient along the length of the embryo. The analysis of both authentic Dorsal target genes and defined synthetic promoters suggests that the ectopic gradient is sufficient to generate the full repertory of DV patterning responses along the AP axis of the embryo. For example, mesoderm determinants are activated in the anterior third of the embryo, whereas neurogenic genes are expressed in central regions. These results raise the possibility that Toll signaling components diffuse in the plasma membrane or syncytial cytoplasm of the early embryo (Huang, 1997).
The Huang (1997) paper also clearly summarizes what is known about the regulation of genes involved in dorsal/ventral patterning. There are five distinct thresholds of gene activity in response to the Dorsal nuclear gradient in early embryos. The type I target gene folded gastrulation is activated only in response to peak levels of the Dl gradient, so that expression is restricted to a subdomain of the presumptive mesoderm. The PE enhancer from the twist promoter region exhibits a similar pattern of expression. This enhancer contains a cluster of low-affinity Dl binding sites that restrict expression to the ventral-most regions of early embryos. The type II target gene snail contains a series of low-affinity Dl-binding sites, as well as binding sites for the bHLH activator, Twist. The Dl and Twist proteins appear to make synergistic contact with the basal transcription complex, so that snail is activated throughout the presumptive mesoderm in response to both peak and high levels of the Dl gradient. The ventral midline arises from the mesoderm, which is derived from the ventral-most regions of the neuroectoderm. Mesectoderm differentiation is controlled by the bHLH-PAS gene, sim. Some of the E(spl) complex also exhibit early expression in the presumptive mesectoderm. A synthetic enhancer containing high-affinity Dl-binding sites and Twist binding sites exhibits expression in this region. The type IV target gene rhomboid is expressed in lateral stripes that encompass the ventral half of the presumptive neuroectoderm. These stripes are regulated by a 300-bp enhancer (NEE) that contains high-affinity Dl-binding sites, Twist-binding sites, and "generic" E-box sequences that appear to bind ubiquitously distributed bHLH activators (Daughterless and Scute), which are present in the unfertilized egg. The fifth and final threshold response is defined by the lowest levels of the Dl gradient. The zerknullt target gene is repressed by high and low levels of the gradient, so that expression is restricted to the presumptive dorsal ectoderm. The zen promoter region contains high-affinity Dl-binding sites and closely linked "corepressor" sites. Efficient occupancy of the Dl sites appears to depend on strong, cooperative DNA-binding interactions between Dl and the corepressors. The same low levels of Dl that repress zen also repress sog. The sim, E(spl), rho and sog expression patterns are restricted to the neurogenic ectoderm and excluded from the ventral mesoderm by Snail, which encodes a zinc finger repressor (Huang, 1997).
This study also provides evidence that neurogenic repressors may be important for the establishment of the sharp mesoderm/neuroectoderm boundary in the early embryo. About half of the embryos carrying the Toll anteriorly expressed transgene exhibit a ventral gap in the endogenous ventral expression pattern of snail behind the ectopic anterior staining pattern. Although the identity of the repressor creating this gap is unknown, it is conceivable that members of the E(spl) complex encode putative snail repressors because previous studies have shown that the m7 and m8 genes are expressed in the lateral neuroectoderm of early embryos. Proteins coded for by these genes are known to repressors. These proteins might be regulated by the gene hierarchy responsible for D/V polarity (Huang, 1997).
Developmental patterning relies on morphogen gradients, which generally involve feedback loops to buffer against perturbations caused by fluctuations in gene dosage and expression. Although many gene components involved in such feedback loops have been identified, how they work together to generate a robust pattern remains unclear. The network of extracellular proteins that patterns the dorsal region of the Drosophila embryo by establishing a graded activation of the bone morphogenic protein (BMP) pathway has been studied. The BMP activation gradient itself is robust to changes in gene dosage. Computational search for networks that support robustness shows that transport of the BMP class ligands (Scw and Dpp) into the dorsal midline by the BMP inhibitor Sog is the key event in this patterning process. The mechanism underlying robustness relies on the ability to store an excess of signaling molecules in a restricted spatial domain where Sog is largely absent. It requires extensive diffusion of the BMP-Sog complexes, coupled with restricted diffusion of the free ligands. Dpp is shown experimentally to be widely diffusible in the presence of Sog but tightly localized in its absence, thus validating a central prediction of a theoretical study (Eldar, 2002).
Graded activation of the BMP pathway subdivides the dorsal region of Drosophila embryos into several distinct domains of gene expression. This graded activation is determined by a well-characterized network of extracellular proteins, which may diffuse in the perivitelline fluid that surrounds the embryo. The patterning network is composed of two BMP class ligands (Scw and Dpp), a BMP inhibitor (Sog), a protease that cleaves Sog (Tld) and an accessory protein (Tsg), all of which are highly conserved in evolution and are used also for patterning the dorso-ventral axis of vertebrate embryos. Previous studies have suggested that patterning of the dorsal region is robust to changes in the concentrations of most of the crucial network components. For example, embryos that contain only one functional allele of scw, sog, tld or tsg are viable and do not show any apparent phenotype. Misexpression of scw or of tsg also renders the corresponding null mutants viable (Eldar, 2002).
To check whether robustness is achieved at the initial activation gradient, signaling was monitored directly by using antibodies that recognize specifically an activated, phosphorylated intermediate of the BMP pathway (pMad). Prominent graded activation in the dorsal-most eight cell rows was observed for about 1h, starting roughly 2h after fertilization at 25°C. This activation gradient was quantified in heterozygous mutants that were compromised for one of three of the crucial components of the patterning network, Scw, Sog or Tld. Whereas homozygous null mutants that completely lack the normal gene product have a deleterious effect on signaling, the heterozygotes, which should produce half the amount of the gene product, were indistinguishable from wild type. Similarly, overexpression of the Tld protein uniformly in the embryo did not alter the activation profile. The activation profile at 18°C is the same as that at 25°C. This robustness to temperature variations is marked, considering the wide array of temperature dependencies that are observed in this temperature span. By contrast, the profile of pMad is sensitive to the concentration of Dpp. The dosage sensitivity of Dpp is exceptional among morphogens and is singled out as being haploid-insufficient (Eldar, 2002).
No apparent transcriptional feedback, which might account for the robustness of dorsal patterning, has been identified so far. Robustness should thus be reflected in the design of interactions in the patterning network. To identify the mechanism underlying robustness, a general mathematical model of the dorsal patterning network was formulated. For simplicity, initial analysis was restricted to a single BMP class ligand (Scw or Dpp), a BMP inhibitor (Sog) and the protease (Tld). The general model accounted for the formation of the BMP-Sog complex, allowed for the diffusion of Sog, BMP and BMP-Sog, and allowed for the cleavage of Sog by Tld, both when Sog is free and when Sog is associated with BMP. Each reaction was characterized by a different rate constant (Eldar, 2002).
Extensive simulations were carried out to identify robust networks. At each simulation, a set of parameters (rate constants and protein concentrations) was chosen at random and the steady-state activation profile was calculated by solving three equations numerically. A set of three perturbed networks representing heterozygous situations was then generated by reducing the gene dosages of sog, tld or the BMP class ligand by a factor of two. The steady-state activation profiles defined by those networks were solved numerically and compared with the initial, nonperturbed network. A threshold was defined as a given BMP value (corresponding to the value at a third of the dorsal ectoderm in the nonperturbed network). The extent of network robustness was quantified by measuring the shift in the threshold for all three perturbed networks. Over 66,000 simulations were carried out, with each of the nine parameters allowed to vary over four orders of magnitude (Eldar, 2002).
As expected, in most cases (97.5%) the threshold position in the perturbed networks was shifted by a large extent (>50%). In most of those nonrobust cases, the BMP concentration was roughly uniform throughout the dorsal region. By contrast, Sog was distributed in a concentration gradient with its minimum in the dorsal midline, defining a reciprocal gradient of BMP activation. Thus, the key event in this nonrobust patterning mechanism is the establishment of a concentration gradient of Sog, which was governed by diffusion of Sog from its domain of expression outside the dorsal region, coupled with its cleavage by Tld inside the dorsal region. Although such a gradient has been observed, it is also compatible with other models (Eldar, 2002).
A small class of networks (198 networks, 0.3%) was identified in which a twofold reduction in the amounts of all three genes resulted in a change of less than 10% in the threshold position. Notably, in all of these robust cases, BMP was redistributed in a sharp concentration gradient that peaked in the dorsal midline. In addition, this concentration gradient decreases as a power-low distribution with an exponent n = 2, which indicates the uniqueness of the robust solution. In these cases, Sog was also distributed in a graded manner in the dorsal region. Analysis of the reaction rate constants of the robust networks showed a wide range of possibilities for most parameters. But two restrictions were apparent and defined the robust network design: (1) in the robust networks the cleavage of Sog by Tld was facilitated by the formation of the complex Sog-BMP; (2) the complex BMP-Sog was broadly diffusible, whereas free BMP was restricted (Eldar, 2002).
To identify how robustness is achieved, an idealized network was considered by assuming that free Sog is not cleaved and that free BMP does not diffuse. The steady-state activation profile defined by this network can be solved analytically; the solution reveals two aspects that are crucial for ensuring robustness. First, the BMP-Sog complex has a central role, by coupling the two processes that establish the activation gradient: BMP diffusion and Sog degradation. This coupling leads to a quantitative buffering of perturbations in gene dosage. Second, restricted diffusion of free BMP enables the system to store excess BMP in a confined spatial domain where Sog is largely absent. Changes in the concentration of BMP alter the BMP profile close to the dorsal midline but do not change its distribution in most of the dorsal region (Eldar, 2002).
The complete system, comprising Sog, Tld, Tsg, both Scw and Dpp, and their associated receptors was examined next. Two additional molecular assumptions are required to ensure the robustness of patterning. First, Sog can bind and capture the BMP class ligands even when the latter are associated with their receptors. Second, Dpp can bind Sog only when the latter is bound to Tsg. Indeed, it has been shown that, whereas Sog is sufficient for inhibiting Scw, both Tsg and Sog are required for inhibiting Dpp. This last assumption implies that Tsg functions to decouple the formation of the Scw gradient from the parallel generation of the Dpp gradient, ensuring that Scw and Dpp are transported to the dorsal midline independently by two distinct molecular entities (Eldar, 2002).
The complete model was solved numerically for different choices of rate constants. In particular, the effect of twofold changes in gene dosage was assessed. The steady-state activation profiles can be superimposed, indicating the robustness of the system. In addition, with the exception of Dpp, the expression of all other crucial network components can be altered by at least an order of magnitude before an effect on the position of a given threshold is observed. In the model, the lack of robustness to Dpp stems from its insufficient dosage. Note that the time taken to reach steady state is sensitive to these concentrations of protein. For the wide range of parameters that were used, however, the adjustment time does not exceed the patterning time. Flexible adjustment time thus facilitates the buffering of quantitative perturbations (Eldar, 2002).
This analysis has identified two principle molecular features that are essential for robust network design: first, free Sog is not cleaved efficiently -- an assumption that is supported by the in vitro finding that Sog cleavage by Tld requires BMP; second, the diffusion of free BMP is restricted. This is the central prediction of the theoretical study, namely, that Scw diffusion requires Sog, whereas Dpp diffusion requires both Sog and Tsg. Although several reports suggest that in wild-type embryos both Dpp and Scw are widely diffusible, their ability to diffuse in a sog or tsg mutant background has not been examined as yet (Eldar, 2002).
To monitor the diffusion of Scw or Dpp, the even-skipped (eve) stripe-2 enhancer (st2) was used to misexpress Dpp or Scw in a narrow stripe perpendicular to the normal BMP gradient. In transgenic embryos, dpp or scw RNA was detected in a stripe just posterior to the cephalic furrow. Initially the stripe was about 12 cells wide at early cleavage cycle 14, but refined rapidly to about 6 cells by late cycle 14. The st2-dpp and st2-scw embryos were viable, despite the high expression of these proteins as compared with their endogenous counterparts (Eldar, 2002).
The activation of the BMP pathway was monitored either by staining for pMad or by following dorsal expression of the target gene race, which requires high activation. Scw is a less potent ligand than is Dpp. This experimental setup could not be used to study Scw diffusion properties because expressing st2-scw did not alter the pattern of pMad or race expression in wild-type or sog-/- embryos. By contrast, expression of st2-dpp led to an expansion of both markers in a region that extends far from the st2 expression domain, indicating a wide diffusion of Dpp in a wild-type background. Conversely, on expression of st2-dpp in sog-/- or in tsg-/- embryos, both markers were confined to a narrow stripe in the st2 domain. The width of this stripe was comparable to that of st2-dpp expression, ranging from 6 to 12 cells, indicating that Dpp does not diffuse from its domain of expression in the absence of Sog or Tsg. Taken together, these results show that both Sog and Tsg are required for Dpp diffusion, as predicted by the theoretical analysis (Eldar, 2002).
The computation ability of biochemical networks is striking when one considers that they function in a biological environment where the amounts of the network components fluctuate, the kinetics is stochastic, and sensitive interactions between different computation modules are required. Studies have examined the effect of these properties on cellular computation mechanisms, and robustness has been proposed to be a 'design principle' of biochemical networks. The applicability of this principle to morphogen gradient patterning has been shown during early development. Quantitative analysis can be used to assess rigorously the robustness of different patterning models and to exclude incompatible ones. The remaining, most plausible model points to crucial biological assumptions and serves to postulate the central feedback mechanisms. Applying the same modelling principles to other systems might identify additional 'design principles' that underlie robust patterning by morphogen gradients in development (Eldar, 2002).
Dorsal-ventral (DV) patterning of the Drosophila embryo is initiated by Dorsal, a sequence-specific transcription factor distributed in a broad nuclear gradient in the precellular embryo. Previous studies have identified as many as 70 protein-coding genes and one microRNA (miRNA) gene that are directly or indirectly regulated by this gradient. A gene regulation network, or circuit diagram, including the functional interconnections among 40 Dorsal (Dl) target genes and 20 associated tissue-specific enhancers, has been determined for the initial stages of gastrulation. This study attempts to extend this analysis by identifying additional DV patterning genes using a recently developed whole-genome tiling array. This analysis led to the identification of another 30 protein-coding genes, including the Drosophila homolog of Idax, an inhibitor of Wnt signaling. In addition, remote 5' exons were identified for at least 10 of the ~100 protein-coding genes that were missed in earlier annotations. As many as nine intergenic uncharacterized transcription units (TUs) were identified, including two that contain known microRNAs, miR-1 and -9a. The potential functions of these recently identified genes are discussed and it is suggested that intronic enhancers are a common feature of the DV gene network (Biemar, 2006).
The Dl nuclear gradient differentially regulates a variety of target genes in a concentration-dependent manner. The gradient generates as many as five different thresholds of gene activity, which define distinct cell types within the presumptive mesoderm, neuroectoderm, and dorsal ectoderm. Total RNA was extracted from embryos produced by three different maternal mutants: pipe–/pipe–, Tollrm9/Tollrm10, and Toll10B. pipe–/pipe– mutants completely lack Dl nuclear protein and, as a result, overexpress genes that are normally repressed by Dl and restricted to the dorsal ectoderm. For example, the decapentaplegic (dpp) TU is strongly "lit up" by total RNA extracted from pipe–/pipe– mutant embryos. The intron-exon structure of the transcribed region is clearly delineated by the hybridization signal, most likely because the processed mRNA sequences are more stable than the intronic sequences present in the primary transcript. There is little or no signal detected with RNAs extracted from Tollrm9/Tollrm10 (neuroectoderm) and Toll10B (mesoderm) mutants. Instead, these other mutants overexpress different subsets of the Dl target genes. For example, Tollrm9/Tollrm10 mutants contain low levels of Dl protein in all nuclei in ventral, lateral, and dorsal regions. These low levels are sufficient to activate target genes such as intermediate neuroblasts defective (ind), ventral neuroblasts defective (vnd), rhomboid (rho), and short gastrulation (sog) but insufficient to activate snail (sna). In contrast, Toll10B mutants overexpress genes (e.g., sna) normally activated by peak levels of the Dl gradient in ventral regions constituting the presumptive mesoderm (Biemar, 2006).
To identify potential Dl targets, ranking scores were assigned for the six possible comparisons of the various mutant backgrounds, pipe vs. Tollrm9/Tollrm10, pipe vs. Toll10B, Tollrm9/Tollrm10 vs. Toll10B, Tollrm9/Tollrm10 vs. pipe, Toll10B vs. Tollrm9/Tollrm10, and Toll10B vs. pipe, using the TiMAT software package. As a first approximation, only hits with a median fold difference of 1.5 and above were considered. For further analysis, the top 100 TUs were selected for each of the comparisons, with the exception of Tollrm9/Tollrm10 vs. pipe for which the TiMAT analysis returned only 43 hits that meet the cutoff. To refine the search for TUs specifically expressed in the mesoderm, where levels of nuclear Dl are highest, only those present in the Toll10B vs. Tollrm9/Tollrm10 and Toll10B vs. pipe, but not pipe vs. Tollrm9/Tollrm10 comparisons were selected. For TUs induced by intermediate and low levels of nuclear Dl in the neuroectoderm, those present in both the Tollrm9/Tollrm10 vs. Toll10B and Tollrm9/Tollrm10 vs. pipe, but not pipe vs. Toll10B comparisons were selected. For TUs restricted to the dorsal ectoderm, only those present in the pipe vs. Tollrm9/Tollrm10 and pipe vs. Toll10B, but not Tollrm9/Tollrm10 vs. Toll10B, were selected. Finally, the TUs corresponding to annotated genes already identified in the previous screen were eliminated to focus on annotated genes not previously considered as potential Dorsal targets, as well as transcribed fragments (transfrags) not previously characterized. Using these criteria, 45 previously annotated protein-coding genes were identified, along with 23 uncharacterized transfrags. Of the 45 protein-coding genes, 29 exhibited localized patterns of gene expression across the DV axis, whereas the remaining 16 were not tested (Biemar, 2006).
The previous microarray screen relied on high cutoff values for the identification of authentic DV genes. For example, only genes exhibiting 6-fold up-regulation in pipe–/pipe– mutant embryos were tested by in situ hybridization for localized expression in the dorsal ectoderm. Many other genes displayed >2-fold up-regulation but were not explicitly tested for localized expression. The whole-genome tiling array permitted the use of much lower cutoff values. For example, CG13800, which was identified by conventional microarray screens, falls just below the original cutoff value but displays 5-fold up-regulation in pipe–/pipe– mutants in the analysis. In situ hybridization assays reveal localized expression in the dorsal ectoderm. This pattern is greatly expanded in embryos derived from pipe–/pipe– mutant females, as expected for a gene that is either directly or indirectly repressed by the Dl gradient. Genes exhibiting even lower cutoff values were also found to display localized expression. Among these genes is a Wnt homologue, Wnt2, which is augmented only 2.25-fold in mutant embryos lacking the Dl nuclear gradient (Biemar, 2006).
The 4-fold cutoff value used in the previous screen for candidate protein-coding genes expressed in the neuroectoderm also excluded genes expressed in this tissue. The Trim9 gene exhibits just a 2-fold increase in mutant embryos derived from Tollrm9/Tollrm10 females. Nonetheless, in situ hybridization assays reveal localized expression in the neuroectoderm of WT embryos. As expected, expression is expanded in Tollrm9/Tollrm10 mutant embryos. Another gene, CG9973, displays just 1.8-fold up-regulation but is selectively expressed in the neuroectoderm. CG9973 encodes a putative protein related to Idax, an inhibitor of the Wnt signaling pathway. Idax inhibits signaling by interacting with the PDZ domain of Dishevelled (Dsh), a critical mediator of the pathway. A Wnt2 homologue is selectively expressed in the dorsal ectoderm. Recent studies identified a second Wnt gene, WntD, which is expressed in the mesoderm. Thus, the CG9973/Idax inhibitor might be important for excluding Wnt signaling from the neuroectoderm. Such a function is suggested by the analysis of Idax activity in vertebrate embryos (Biemar, 2006).
Additional genes were also identified that are specifically expressed in the mesoderm. Among these is CG9005, which encodes an unknown protein that is highly conserved in different animals, including frogs, chicks, mice, rats, and humans. It displays <2-fold up-regulation in Toll10B embryos but is selectively expressed in the ventral mesoderm of WT embryos. Expression is expanded in embryos derived from Toll10B mutant females (Biemar, 2006).
Other protein-coding genes were missed in the previous screen because they were not represented on the Drosophila Genome Array used at the time. These include, for instance, CG8147 in the dorsal ectoderm and CG32372 in the mesoderm (Biemar, 2006).
An interesting example of the use of tiling arrays to identify tissue-specific isoforms is seen for the bunched (bun) TU. bun encodes a putative sequence-specific transcription factor related to mammalian TSC-22, which is activated by TGFβ signaling. It was shown to inhibit Notch signaling in the follicular epithelium of the Drosophila egg chamber. Three transcripts are expressed from alternative promoters in bun, but it appears that only the short isoform (bun-RC) is specifically expressed in the dorsal ectoderm. A number of bun exons are ubiquitously transcribed at low levels in the mesoderm, neuroectoderm, and dorsal ectoderm. However, the 3'-most exons are selectively up-regulated in pipe–/pipe– mutants. It is conceivable that Dpp signaling augments the expression of this isoform, which in turn, participates in the patterning of the dorsal ectoderm (Biemar, 2006).
In addition to protein-coding genes, the tiling array also identified uncharacterized TUs not previously annotated. Some of them are associated with ESTs, providing independent evidence for transcriptional activity in these regions. For 14 of these transfrags (61%), visual inspection of neighboring loci using the Integrated Genome Browser suggested coordinate expression of a neighboring protein-coding region (i.e., overexpressed in the same mutant background). The N-Cadherin gene (CadN) has a complex intron-exon structure consisting of ~20 different exons. The strongest hybridization signals are detected within the limits of exons, but an unexpected signal was detected ~10 kb upstream of the 5'-most exon. It is specifically expressed in the mesoderm, suggesting that it represents a previously unidentified 5' exon of the CadN gene. Support for this contention stems from two lines of evidence: (1) in situ hybridization using a probe against the 5' exon detects transcription in the presumptive mesoderm, the initial site of CadN expression; (2) using primers anchored in the 5' transfrag as well as the first exon of CadN, confirmation was obtained by RT-PCR that the recently identified TU is part of the CadN transcript. This recently identified 5' exon appears to contribute to the 5' leader of the CadN mRNA. It is possible that this extended leader sequence influences translational efficiency as seen in yeast. Because there seems to be a considerable lag between the time when CadN is first transcribed and the first appearance of the protein, it is suggested that this extended leader sequence might inhibit translation. An interesting possibility is that it does so through short upstream ORFs, as has been shown for several oncogenes in vertebrates (Biemar, 2006).
A 5' exon was also identified for crossveinless-2 (cv-2), a component of the Dpp bone morphogenetic protein (BMP) signaling pathway. cv-2 binds BMPs and functions as both an activator and inhibitor of BMP signaling. It is specifically required in the developing wing disk to generate peak Dpp signaling in the presumptive crossveins. cv-2 is also expressed in the dorsal ectoderm of early embryos, but its role during embryonic development has not been investigated. The whole-genome tiling array identified a 5' exon located ~10 kb 5' of the transcription start site of the cv-2 TU. Using RT-PCR and in situ hybridization assays, it was confirmed that the exon is part of the cv-2 transcript. It is possible that the exon resides near an embryonic promoter that is inactive in the developing wing discs. Future studies will determine whether this 5' exon influences the timing or levels of Cv-2 protein synthesis (Biemar, 2006).
In addition to the identification of 10 5' exons associated with previously annotated genes such as CadN and cv-2, three other transfrags appear to correspond to 3' exons, and nine of the RNAs seem to arise from autonomous TUs. Three of these represent annotated computational RNA (CR) genes: CR32777, CR31972, and CR32957. CR32777 corresponds to roX1, which is ubiquitously expressed at the blastoderm stage, hence it represents a false positive. The other two potential noncoding RNAs were recently identified independently in two other studies, and although the expression of CR32957 could not be detected by in situ hybridization, CR31972 transcripts are detected in the mesoderm. There is no evidence that these transcripts are processed into miRNAs, but noncoding genes corresponding to known miRNA loci were also identified in the screen. Transfrag 22 corresponds to the miR-9a primary transcript (pri-mir9a) and is detected in both the dorsal- and neuroectoderm. Expression of pri-mir9a is ubiquitous in embryos derived from pipe–/pipe– or Tollrm9/Tollrm10 females. Transfrag 8 corresponds to pri-mir1, which is present in the mesoderm (Biemar, 2006).
A third noncoding transcript (Transfrag 12) maps next to a known miRNA, miR-184. It is selectively expressed in the mesoderm and overexpressed in Toll10B mutants. The mesodermal expression of miR-184 has been reported. It is possible that Transfrag 12 corresponds to pri-mir-184, and that secondary structures in the miRNA region preclude detection on the array. This is seen for several other miRNA precursors expressed at various stages during embryogenesis. Alternatively, Transfrag 12 might represent the fragment resulting from Drosha cleavage of the pri-mir-184 to produce the miR-184 precursor hairpin (pre-miR-184). A similar situation has been observed for the iab4 locus. Like miR-1, miR-184 is selectively expressed in the ventral mesoderm. It will be interesting to determine whether the two miRNAs jointly regulate some of the same target mRNAs (Biemar, 2006).
The identity of the last three transfrags is less clear. Visual inspection using the Integrated Genome Browser suggests expression of Transfrag 10 in the mesoderm, Transfrag 21 in the neuroectoderm, and Transfrag 11 in both the dorsal ectoderm and neuroectoderm. However, in situ hybridization assays confirm the predicted expression pattern only for Transfrag 11. Computational analyses designed to estimate the likelihood of translation suggest a protein-coding potential for Transfrag 10 [Likelihood Ratio Test (LRT) P < 0.001] and possibly Transfrag 11 (LRT P < 0.01), whereas Transfrag 21 could not be analyzed because of lack of conservation in other Drosophila species (Biemar, 2006).
This work has attempted to identify nonprotein coding genes involved in patterning the DV axis of the Drosophila embryo using an unbiased approach to survey the entire genome. This study, along with earlier analyses, identified as many as 100 protein-coding genes and five to seven noncoding genes that are differentially expressed across the DV axis of the early Drosophila embryo. Roughly half of the noncoding RNAs correspond to miRNAs, although <1% of the annotated genes in the Drosophila genome encode miRNAs. Future studies will determine how these RNAs impinge on the DV regulatory network (Biemar, 2006).
Recent studies have identified large numbers of noncoding transcripts in the mouse and human genomes. If the present study is predictive, less than one-fourth of the transcripts correspond to novel noncoding RNAs of unknown function, akin to CR31972 and Transfrag 11 expressed in the mesoderm and ectoderm, respectively. Most of the noncoding transcripts are likely to derive from intronic sequences because of the occurrence of cryptic remote 5' exons as seen for the CadN and cv-2 genes. At least 10% of the DV protein-coding genes were found to contain such exons. As a result, these genes contain large tracts of intronic sequences that might encompass regulatory DNAs such as tissue-specific enhancers. The FGF8-related gene, thisbe (ths), represents such a case. A neurogenic-specific enhancer that was initially thought to reside 5' of the TU actually maps within a large intron because of the occurrence of a remote 5' exon. It is suggested that such exons are responsible for the evolutionary "bundling" of genes and their associated regulatory DNAs. Gene duplication events are more likely to retain this linkage when regulatory DNAs map within the TU. In contrast, enhancers mapping in flanking regions can be uncoupled from their normal target gene by chromosomal rearrangements (Biemar, 2006).
The dorsal-ventral patterning of the Drosophila embryo is controlled by a well-defined gene regulation network. This study addressed how changes in this network produce evolutionary diversity in insect gastrulation. Focus was placed on the dorsal ectoderm in two highly divergent dipterans, the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae. In D. melanogaster, the dorsal midline of the dorsal ectoderm forms a single extra-embryonic membrane, the amnioserosa. In A. gambiae, an expanded domain forms two distinct extra-embryonic tissues, the amnion and serosa. The analysis of approximately 20 different dorsal-ventral patterning genes suggests that the initial specification of the mesoderm and ventral neurogenic ectoderm is highly conserved in flies and mosquitoes. By contrast, there are numerous differences in the expression profiles of genes active in the dorsal ectoderm. Most notably, the subdivision of the extra-embryonic domain into separate amnion and serosa lineages in A. gambiae correlates with novel patterns of gene expression for several segmentation repressors. Moreover, the expanded amnion and serosa anlage correlates with a broader domain of Dpp signaling as compared with the D. melanogaster embryo. Evidence is presented that this expanded signaling is due to altered expression of the sog gene (Goltsev, 2007).
A variety of dorsal patterning genes were examined in A. gambiae
embryos in an effort to determine the basis for the formation of distinct
ectodermal derivatives. For example hindsight (hnt; also
known as peb - Flybase) is expressed along the dorsal midline of D. melanogaster embryos, while tailup (tup) is expressed in a broader pattern that encompasses both the presumptive amnioserosa and dorsolateral ectoderm. The hnt expression pattern seen in A. gambiae is similar to that detected in D. melanogaster, although there is a marked expansion in the dorsal-ventral limits of the presumptive extra-embryonic territory. By contrast, the
tup pattern in A. gambiae is dramatically different from
that seen in D. melanogaster -- it is excluded from the prospective
serosa and restricted to the future amnion (Goltsev, 2007).
The T-box genes Dorsocross1 (Doc1) and Doc2 are
involved in amnioserosa development and expressed along the dorsal midline and
in a transverse stripe near the cephalic furrow of gastrulating D.
melanogaster embryos. The Doc1 and Doc2 orthologues in A. gambiae exhibit restricted expression in the presumptive amnion, similar to the tup pattern. The expression patterns of the two genes are identical but only Doc1 is shown. They
are initially expressed in a broad dorsal domain but come to
be repressed in the serosa. There is also a head stripe of expression
comparable to the D. melanogaster pattern. Additional
dorsal-ventral patterning genes are also expressed in a restricted pattern
within the developing amnion. Overall, the early expression patterns of tup, Doc1 and Doc2 (and additional patterning genes) foreshadow the subdivision of the dorsal ectoderm into separate serosa and amnion lineages in Anopheles (Goltsev, 2007).
In D. melanogaster, the patterning of the dorsal ectoderm depends
on Dpp and Zen, along with a variety of genes encoding Dpp signaling
components, such as the Thickveins (Tkv) receptor. Most of the corresponding
genes are expressed in divergent patterns in A. gambiae embryos. For example,
dpp and tkv are initially expressed throughout the dorsal
ectoderm, but become excluded from the presumptive serosa and
restricted to the amnion. By contrast, both genes have broad, nearly uniform expression patterns in the dorsal ectoderm of D. melanogaster embryos (Goltsev, 2007).
There is an equally dramatic change in the zen expression pattern.
In A. gambiae, expression is restricted to the presumptive serosa
territory, even at the earliest stages of development. By contrast,
zen is initially expressed throughout the dorsal ectoderm of
cellularizing embryos in D. melanogaster, and becomes restricted to the dorsal midline by the onset of gastrulation. Thus, the dpp/tkv and zen expression patterns are essentially complementary in A. gambiae embryos, but extensively overlap in Drosophila (Goltsev, 2007).
The loss of dpp, tkv, Doc1, Doc2 and tup expression in the presumptive serosa of A. gambiae embryos raises the possibility that
zen activates the expression of one or more repressors in the serosa.
It is unlikely that Zen itself is such a repressor since the expression of the
A. gambiae zen gene in transgenic Drosophila embryos does
not alter the normal development of the amnioserosa (Goltsev, 2007).
Different segmentation genes were examined in an effort to identify
putative serosa-specific repressors. For example, the gap gene
hunchback (hb) is initially expressed in the anterior
regions of A. gambiae embryos, in a similar pattern to that seen in
D. melanogaster, but by the onset of gastrulation a novel
pattern arises within the presumptive serosa.
hb expression has also been seen in the developing serosa of other insects, including a primitive fly (Clogmia) and the flour beetle, Tribolium (Goltsev, 2007).
Two additional segmentation genes behave like hb, empty spiracles
(ems) and tramtrack (ttk). ems is
involved in head patterning in D. melanogaster. Its
expression is limited to a single stripe in anterior regions of cellularizing
D. melanogaster embryos. Staining is seen in a comparable anterior region of A.
gambiae embryos, but a second site of expression (not seen in Drosophila) is also detected in the presumptive serosa (Goltsev, 2007).
Ttk is a maternal repressor that helps establish the expression limits of
several pair-rule stripes. It is ubiquitously expressed throughout the early D.
melanogaster embryo, but has a tightly localized expression pattern within the
presumptive serosa of A. gambiae embryos. Thus, novel patterns
of ems and ttk expression are consistent with the
possibility that serosa-specific repressors help subdivide the dorsal ectoderm
into separate serosa and amnion lineages in A. gambiae embryos (Goltsev, 2007).
The analysis of dorsal-ventral patterning genes identified two critical
differences between the pre-gastrular fly and mosquito embryos. First, there
are separate serosa and amnion lineages in A. gambiae, but just a
single amnioserosa in D. melanogaster. Second, there is an expansion
in the limits of the dorsal ectoderm in A. gambiae as compared with
the D. melanogaster embryo. Localized repressors might help explain
the former observation of separate lineages, but do not provide a basis for
the expansion of the dorsal ectoderm (Goltsev, 2007).
In D. melanogaster, the limits of Dpp signaling are established by
the repressor Brinker and the inhibitor Sog. Genetic studies suggest that Sog is the more critical determinant in early embryos. It is related to Chordin, which inhibits BMP signaling in vertebrates, and is expressed in broad lateral stripes encompassing the entire neurogenic ectoderm. The secreted Sog protein directly binds Dpp, and blocks its ability to interact with the Tkv receptor. However, Sog-Dpp complexes are proteolytically processed by the Tolloid (Tld) metalloprotease, which is expressed throughout the dorsal ectoderm of early Drosophila embryos. Tld helps ensure that high levels of the Dpp signal are released at the dorsal midline located far from the restricted source of the inhibitor Sog (Goltsev, 2007).
The expression patterns of the sog and tld genes in
A. gambiae are very different from those seen in D.
melanogaster. sog expression is primarily detected in the ventral mesoderm,
although low levels of sog transcripts might extend into the
ventral-most regions of the neurogenic ectoderm. This pattern is
more restricted across the dorsal-ventral axis than the D. melanogaster
sog pattern. tld expression is restricted to lateral regions of A.
gambiae embryos and is excluded from the dorsal ectoderm, which is the
principal site of expression in Drosophila. These significant
changes in the sog and tld expression patterns might
account, at least in part, for the expanded limits of Dpp signaling in the
dorsal ectoderm of A. gambiae embryos (Goltsev, 2007).
Direct evidence for broader Dpp signaling was obtained using an antibody
that detects phosphorylated Mad (pMad), the activated form of Mad obtained upon induction of the Tkv receptor. In D. melanogaster pMad expression is restricted to the dorsal midline. This is the domain where Sog-Dpp complexes are processed and peak levels of Dpp interact with the receptor Tkv. The spatial limits of the sog expression pattern are
decisive for this restricted domain of pMad activity. Just a twofold reduction
in the levels of Sog (sog/+ heterozygotes) causes a significant
expansion in pMad expression (Goltsev, 2007).
There is a marked expansion of the pMad expression domain in A.
gambiae embryos as compared with Drosophila. The domain
encompasses the entire presumptive serosa and extends into portions of the
presumptive amnion. The dpp and tkv expression patterns are
downregulated in the presumptive serosa, nonetheless, the
pMad staining pattern clearly indicates that this is the site of peak Dpp
signaling activity. The early expression of both dpp and tkv
encompasses the entire dorsal ectoderm. It would appear that peak Dpp
signaling is somehow maintained in the developing serosa even after the
downregulation of dpp and tkv expression in this tissue. A similar scenario is seen in the Drosophila embryo, in that there is downregulation of both dpp and tkv expression along the dorsal midline of gastrulating embryos (Goltsev, 2007).
To determine the basis for expanded Dpp signaling a sog enhancer was identified and characterized in A. gambiae. The D.
melanogaster enhancer is located in the first intron of the sog
transcription unit. It is ~300 bp in length and contains four evenly spaced, optimal Dorsal
binding sites. These sites permit activation of sog expression by
low levels of the Dorsal gradient; however, closely linked Snail repressor
sites inactivate the enhancer in the ventral mesoderm. A putative A.
gambiae enhancer was identified by scanning the sog locus for
potential clusters of Dorsal binding sites. The recently developed
cluster-draw program was used for this purpose since it successfully
identified a sim enhancer in the honeybee, Apis mellifera,
which is even more divergent than Anopheles. The best putative Dorsal binding cluster was identified within the first intron of the A. gambiae sog locus. Several genomic DNA fragments were tested for enhancer activity, but only this cluster was found to activate gene expression in transgenic Drosophila embryos (Goltsev, 2007).
Two different genomic DNA fragments, 3.7 kb and 1.1 kb, that encompass the
intronic binding cluster were tested in transgenic embryos. Both fragments were
attached to a lacZ reporter gene containing the core eve
promoter from D. melanogaster, and both direct lacZ
expression in the presumptive mesoderm. They exhibit the same restricted dorsal-ventral limits of expression as that seen for the endogenous sog gene in A. gambiae, although the smaller fragment produces ventral stripes whereas the larger fragment directs
a more uniform pattern. The change in the dorsal-ventral limits --
broad expression in D. melanogaster and restricted expression in
A. gambiae -- might be due to the quality of individual Dorsal binding
sites in the two enhancers (Goltsev, 2007).
Therefore, s comprehensive analysis of dorsal-ventral patterning genes in the A.
gambiae embryo reveals elements of conservation and divergence in the
gastrulation network of D. melanogaster. There is broad conservation
in the expression of regulatory genes responsible for the patterning of the
mesoderm and neurogenic ectoderm, including sequential expression of sim,
vnd and ind in the developing nerve cord. By contrast, there are
extensive changes in the expression of regulatory genes that pattern the
dorsal ectoderm. These changes foreshadow the subdivision of the dorsal
ectoderm into separate serosa and amnion lineages in A. gambiae (Goltsev, 2007).
The major difference in the early patterning of the mesoderm in flies and
mosquitoes concerns the manner in which mesoderm cells enter the blastocoel of
gastrulating embryos. In D. melanogaster, there is a coherent
invagination of the mesoderm through the ventral furrow, much like the
movement of bottle cells through the blastocoel of Xenopus embryos. By contrast,
there is no invagination of the mesoderm in A. gambiae. Instead, the
mesoderm undergoes progressive ingression during germband elongation. This
type of ingression is seen in D. melanogaster mutants lacking
fog signaling. The A. gambiae genome lacks a clear homologue of
fog, and it is therefore conceivable that fog represents an
innovation of the higher Diptera that was only recently incorporated into the
D. melanogaster dorsal-ventral patterning network (Goltsev, 2007).
D. melanogaster is somewhat unusual in having an amnioserosa,
rather than separate serosa and amnion tissues as seen in most insects. In
certain mosquitoes the serosa secretes an additional proteinaceous membrane
that provides extra protection against desiccation.
The changes in gene expression in the D. melanogaster and A.
gambiae dorsal ectoderm provide a basis for understanding the
evolutionary transition of two dorsal tissues in A. gambiae into a
novel single tissue in higher dipterans (Goltsev, 2007).
The D. melanogaster amnioserosa expresses a variety of regulatory
genes, including Doc1/2 and tup. The expression of most of
these genes is restricted in the presumptive amnion of the A. gambiae
embryo. zen is the only dorsal patterning gene, among those tested,
that exhibits restricted expression in the serosa. Several segmentation genes
have a similar pattern, and one of these, ttk, encodes a known
repressor. Ectopic expression of Ttk causes a variety of patterning defects in
Drosophila embryos, including disruptions in head involution and
germband elongation that might arise from alterations in the amnioserosa. It is
proposed that zen activates ttk in the serosa of A. gambiae embryos. The encoded repressor might subdivide the dorsal ectoderm into separate serosa and amnion tissues by inhibiting the expression of Doc1/2 and tup in the serosa. The loss of this putative zen-ttk regulatory linkage might be sufficient to allow Dpp signaling to activate tup and Doc1/2 throughout the dorsal ectoderm,
thereby transforming separate serosa and amnion tissues into a single
amnioserosa. According to this scenario, the loss of zen binding
sites in ttk regulatory sequences might be responsible for the
evolutionary transition of the amnioserosa (Goltsev, 2007).
The formation of separate amnion and serosa tissues is not the only
distinguishing feature of A. gambiae embryos when compared with
D. melanogaster. There is also a significant expansion in the overall
limits of the dorsal ectoderm. This can be explained, in part, by distinct
patterns of sog expression. The broad expression limits of the Sog inhibitor are responsible for restricting Dpp/pMad signaling to the dorsal midline of the D.
melanogaster embryo. This pattern depends on a highly sensitive response of the sog intronic enhancer to the lowest levels of the Dorsal gradient. The Dorsal binding sites in the sog enhancer are optimal sites, possessing perfect matches to the
idealized position weighted matrix of Dorsal recognition sequences.
By contrast, the A. gambiae intronic sog enhancer
contains low-quality Dorsal binding sites, similar to those seen in the
regulatory sequences of genes activated by peak levels of the Dorsal gradient,
such as twist. The binding sites in the D. melanogaster sog
enhancer have an average score of ~10. By contrast, the best sites in the
A. gambiae sog enhancer have scores in the 6.5-7 range, typical of
enhancers that mediate expression in the mesoderm in response to high levels
of the Dorsal gradient. Although every potential
regulatory sequence in the A. gambiae sog locus was not explicitly tested, none of the putative Dorsal binding clusters in the vicinity of the gene possess the quality
required for activation by low levels of the Dorsal gradient in the neurogenic
ectoderm. Thus, the narrow limits of sog expression in A.
gambiae embryos can be explained by the occurrence of low-quality Dorsal
binding sites, along with the loss of Snail repressor sites (Goltsev, 2007).
The altered sog expression pattern is probably not the sole basis
for the expansion of the dorsal ectoderm. A. gambiae embryos also
exhibit a significant change in the tld expression pattern.
tld is expressed throughout the dorsal ectoderm in D.
melanogaster, but restricted to the neurogenic ectoderm of A.
gambiae. Tld cleaves inactive Tsg-Sog-Dpp complexes to produce peak Dpp
signaling along the dorsal midline of Drosophila embryos. It is proposed that the
altered tld pattern in combination with altered sog leads to
two dorsolateral sources of the active Dpp ligand in mosquito embryos. The sum of these sources might produce a step-like distribution of pMad across dorsal regions of mosquito embryos. This broad plateau of pMad activity might be responsible for the observed expansion of the dorsal ectoderm territory, and the specification of the serosa (Goltsev, 2007).
In Drosophila, tld is regulated by a 5' silencer element
that prevents the gene from being expressed in ventral and lateral regions in
response to high and low levels of the Dorsal gradient. This silencing activity is due to close linkage of Dorsal binding sites and recognition sequences for 'co-repressor' proteins. Preliminary studies suggest that Dorsal activates the A. gambiae tld gene, possibly by the loss of co-repressor binding sites in the 5' enhancer (Goltsev, 2007).
It is proposed that there are at least two distinct threshold readouts of Dpp
signaling in the dorsal ectoderm of A. gambiae embryos. Type 1 target
genes, such as hb, ems, ttk and zen, are activated by high
levels and thereby restricted to the presumptive serosa. Type 2 target genes,
such as tup and Doc1/2, can be activated - in principle - by
both high and low levels of Dpp signaling in the presumptive serosa and
amnion. However, these target enhancers contain binding sites for one or more
type 1 repressors expressed in the serosa. The favorite candidate repressor is
Ttk. Perhaps the type 2 tup enhancer contains optimal pMad activator
sites as well as binding sites for the localized repressor Ttk, which keeps
tup expression off in the serosa and restricted to the amnion. As
discussed earlier, the simple loss of ttk regulation by the Dpp
signaling network might be sufficient to account for the evolutionary conversion of separate serosa and amnion tissues into a single amnioserosa. Localization of this single tissue within a restricted domain along the dorsal midline would arise from concomitant dorsal shifts in the sog and tld expression patterns (Goltsev, 2007).
Bone morphogenetic proteins (BMPs) are of central importance for dorsal-ventral (DV) axis specification. They are core components of a signalling cascade that includes the BMP ligand decapentaplegic (DPP) and its antagonist short gastrulation (SOG) in Drosophila melanogaster. These components are very ancient, with orthologs involved in DV patterning in both protostomes and deuterostomes. Despite such strong conservation, recent comparative work in insects has revealed interesting differences in the way the patterning function of the DV system is achieved in different species. This paper characterised the expression patterns of the principal components of the BMP DV patterning system, as well as its signalling outputs and downstream targets, in the non-cyclorrhaphan moth midge Clogmia albipunctata (Diptera: Psychodidae). Previously work has shown ventral expression patterns of dpp in the pole regions of C. albipunctata blastoderm embryos. Strikingly, ventral sog and posteriorly restricted tkv expression, as well as expanded polar activity of pMad were also found. The results from gene knock-down by embryonic RNA interference were used to propose a mechanism of polar morphogen shuttling in C. albipunctata. These results were compared to available data from other species and discuss scenarios for the evolution of DV signalling in the holometabolan insects. It is concluded that a comparison of gene expression patterns across hemipteran and holometabolan insects reveals that expression of upstream signalling factors in the DV system is very variable, while signalling output is highly conserved. This has two major implications: first, as long as ligand shuttling and other upstream regulatory mechanisms lead to an appropriately localised activation of BMP signalling at the dorsal midline, it is of less importance exactly where the upstream components of the DV system are expressed. This, in turn, explains why the early-acting components of the DV patterning system in insects exhibit extensive amounts of developmental systems drift constrained by highly conserved downstream signalling output (Wotton, 2017).
Drosophila Decapentaplegic (Dpp), a member of the BMP2/4 class of the TGF-betas, is required for organ growth, patterning and differentiation. However, much remains to be understood about the mechanisms acting downstream of these multiple roles. This issue was investigated during the development of the Drosophila eye. viriato (vito) has been identified as a dMyc-target gene encoding a nucleolar protein that is required for proper tissue growth in the developing eye. By carrying out a targeted in vivo double-RNAi screen to identify genes and pathways functioning with Vito during eye development, a strong genetic interaction was found between vito and members of the Dpp signaling pathway including the TGF-beta receptors tkv (type I), put (type II), and the co-Smad medea (med). Analyzing the expression of the Dpp receptor Tkv and the activation pattern of the pathway's transducer, p-Mad, vito was found to be required for a correct signal transduction in Dpp-receiving cells. Overall, this study validated the use of double RNAi to find specific genetic interactions and, in particular, a link between the Dpp pathway and Vito, a nucleolar component, was uncovered. vito would act genetically downstream of Dpp, playing an important role in maintaining a sufficient level of Dpp activity for the promotion of eye disc growth and regulation of photoreceptor differentiation in eye development (Marinho, 2013).
Different genetic relationships are uncovered by the detection of aggravating synthetic interactions. A pair of genes could act in parallel pathways converging on the same biological process ('between-pathway' interaction), or could either act at the same level or different levels of one pathway ('within-pathway' interaction). Ultimately, it is also possible that each gene may act in unrelated processes revealing an indirect interaction, even though the breakdown of the system occurs when both genes are compromised. Within this conceptual framework, this study reports the first in vivo double-RNAi screen to study genetic interactions during Drosophila development, providing evidence for the usefulness of tissue-targeted RNAi screens for the detection of aggravating synthetic genetic interactions. An in vivo double-RNAi was performed screen to uncover genes and pathways functioning with the nucleolar regulator Vito during eye development and 12 interactor genes were identified. Eleven out of the 12 Vito interactor genes identified have been described, or predicted, to be involved in the development of the nervous system. Furthermore, a significant interaction was detected between vito and the retinal determination genes ey, eya, and so. However these interactions are weaker (lower interaction scores) than the interactions between vito and Dpp signaling genes. Dpp and Eya (this latter partnering with So, the Six2 homolog) are both required downstream of Hh for retinogenesis. Both Dpp and Eya/So are then required for further differentiation of the retina and the repression of hth, a transcription factor that maintains the progenitor state. Dpp and Hh are also required redundantly to establish So expression. Thus, the interaction between vito and the retinal determination genes could potentially be a consequence of the significant modulation of Dpp signaling by vito. Interestingly, it was observed that vito interacts with members of the TGF-β signaling pathway, including the Dpp signaling pathway receptors tkv and put, but also with members of the activin signaling branch, such as the R-Smad smox/dSmad2 and the receptor type-I baboon. Recent knowledge about the cross-talk between the activin and Dpp branches is arising as it was shown that Smox (the activin dedicated R-Smad) has a role in wing disc growth that requires the function of Mad. Moreover, it was also reported that Baboon, the type-I activin receptor, is able to phosphorylate the Dpp branch dedicated Mad in a Smox-concentration dependent manner. These reports hint at the complex inter-regulation between both branches of the TGF-β signaling, which complicates the detailed analysis of the exact contribution of vito to the signaling activities of the two branches (Marinho, 2013).
Taken together, the data demonstrate that Vito acts downstream of Dpp, having a dual role during eye development: Vito cooperates with Dpp in growth stimulation during early stages of eye disc development and also in later stages during the process of eye disc patterning. Vito/Dpp interaction does not seem to be based on Vito's requirement for survival in the developing eye because no increase in the number of apoptotic cells was detected when Vito was depleted together with the Dpp receptor tkv. The vito-Dpp interaction specifically takes place in the context of eye development where vito is required for Myc-stimulated growth. To assess a potential interaction between the Dpp pathway and Myc in the developing eye, double-RNAi experiments were carried that revealed a synthetic interaction between dMyc and Med. However, the interaction observed after co-depleting vito and med is even stronger. These results point to a specific and direct interaction of vito with the Dpp signaling pathway that is not simply an indirect effect from the previously described dMyc-vito interaction (Marinho, 2013).
Overall, these data are consistent with a role of Vito in positively regulating Dpp signaling since depletion of Vito partially reverted Dpp overexpression phenotypes, and the phenotype of depleting vito in a Dpp weak RNAi background resembled a strong RNAi for a Dpp pathway component. Remarkably, overexpression of low levels of Vito could rescue the absence of differentiation caused by a strong reduction in Dpp activity by put RNAi. Moreover, vitoRNAi eye discs showed a delay in MF progression and an irregular activation of p-Mad within the furrow, which was accompanied with a reduction in Tkv levels. Whether Vito regulates Dpp signaling by direct modulation of Tkv levels, or whether this down-regulation of Tkv is an indirect effect due to a decreased signaling output from Dpp signaling remains an open question. In conclusion, the genetic data reveal that Vito, a nucleolar putative RNA 5'-3' exonuclease, modulates Dpp signaling during fly eye development. Extensively known for its role in ribosome biogenesis, recent studies suggest that the nucleolar sub-nuclear compartment is also linked to cell-cycle and developmental decisions. As an example, Nucleostemin is a nucleolar GTP-binding protein with both ribosomal and non-ribosomal roles, and was recently shown to maintain self-renewal of embryonic stem cells and to play a role in injured-induced liver regeneration. As a further example, differentiation of primary spermatocytes into mature spermatids was shown to require the nucleolar sequestration of Polycomb repression complex 1 factors. Provocatively, one of the reports by the ENCODE project that surveyed the transcriptome of nuclear subcompartments in the K562 cell line revealed that a small fraction of transcripts with distinct GO-enrichment is unique to the nucleolar compartment. Although the genetic data do not reveal the molecular mechanism underlying the Vito/Dpp interaction, it is interesting to note that alterations of Vito expression levels have profound effects on nucleolar architecture. In the face of the dynamic and potentially important role of the nucleolus in the control of gene expression, the interaction between Dpp and vito could result from altered expression or sub-nuclear localization of yet to be identified regulators of Dpp signaling when vito expression is knocked-down. Thus, further experiments are necessary to elucidate the mechanisms underlying the role of Vito in the modulation of Dpp functions in Drosophila eye development (Marinho, 2013).
Bone morphogenetic proteins (BMPs), a subgroup of the transforming growth factor (TGF)-beta family, transduce their signal through multiple components downstream of their receptors. Even though the components involved in the BMP signaling pathway have been intensely studied, many molecules mediating BMP signaling remain to be addressed. To identify novel components that participate in BMP signaling, RNA interference (RNAi)-based screening was established by detecting phosphorylated Mad (pMad) in Drosophila S2 cells. Ter94, a member of the family of AAA ATPases, was identified as a novel mediator of BMP signaling, which is required for the phosphorylation of Mad in Drosophila S2 cells. Moreover, the mammalian orthlog of Ter94 valosin-containing protein (VCP) plays a critical role in the BMP-Smad1/5/8 signaling pathway in mammalian cells. Genetic evidence suggests that Ter94 is involved in the dorsal-ventral patterning of the Drosophila early embryo through regulating Decapentaplegic (Dpp)/BMP signals. Taken together, these data suggest that Ter94/VCP appears to be an evolutionarily conserved component that regulates BMP-Smad1/5/8 signaling (Zeng, 2014: PubMed).
Enhancers constitute one of the major components of regulatory machinery
of metazoans. Although several genome-wide studies have focused on finding
and locating enhancers in the genomes, the fundamental principles
governing their internal architecture and cis-regulatory grammar remain
elusive. This study describes an extensive, quantitative perturbation
analysis targeting the dorsal-ventral
patterning gene regulatory network (GRN) controlled by Drosophila
NF-κB homolog Dorsal. To
understand transcription factor interactions on enhancers, an ensemble of
mathematical models were employed to test effects of cooperativity,
repression, and factor potency. Models trained on the dataset correctly
predict activity of evolutionarily divergent regulatory regions, providing
insights into spatial relationships between repressor and activator
binding sites. Importantly, the collective predictions of sets of models
are effective at novel enhancer identification and characterization. The
study demonstrates how experimental dataset and modeling can be
effectively combined to provide quantitative insights into cis-regulatory
information on a genome-wide scale (Sayal, 2016).
In the early Drosophila melanogaster embryo, the gene regulatory network controlled by Dpp signaling is involved in the subdivision of dorsal ectoderm into the presumptive dorsal epidermis and amnioserosa. This study identified new Dpp downstream targets involved in dorsal ectoderm patterning. Oligonucleotide Drosophila microarrays were used to identify the set of genes that are differential expressed between wild type embryos and embryos that overexpress Dpp (nos-Gal4>UAS-dpp) during early stages of embryo development. By using this approach, 358 genes were identified whose relative abundance significantly increased in response to Dpp overexpression. Among them, the entire set of known Dpp target genes that function in dorsal ectoderm patterning (zen, doc, hnt, pnr, ush, tup, and others) were identified, in addition to several up-regulated genes of unknown functions. One of the candidate genes, CG13653, which is expressed at the dorsal-most cells of the embryo during a restricted period of time was further analyzed. CG13653 orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa. The enhancer region of CG13653 was characterized, and CG13653 was shown to be directly regulated by Dpp signaling pathway (Dominguez, 2016).
Histone modifications are frequently used as markers for enhancer states, but how to interpret enhancer states in the context of embryonic development is not clear. The poised enhancer signature, involving H3K4me1 and low levels of H3K27ac, has been reported to mark inactive enhancers that are poised for future activation. However, future activation is not always observed, and alternative reasons for the widespread occurrence of this enhancer signature have not been investigated. By analyzing enhancers during dorsal-ventral (DV) axis formation in the Drosophila embryo, it was found that the poised enhancer signature is specifically generated during patterning in the tissue where the enhancers are not induced, including at enhancers that are known to be repressed by a transcriptional repressor. These results suggest that, rather than serving exclusively as an intermediate step before future activation, the poised enhancer state may be a mark for spatial regulation during tissue patterning. The possibility is discussed that the poised enhancer state is more generally the result of repression by transcriptional repressors (Koenecke, 2016).
Epithelial cells are characterized by apical-basal polarity. Intrinsic factors underlying apical-basal polarity are crucial for tissue homeostasis and have often been identified to be tumor suppressors. Patterning and differentiation of epithelia are key processes of epithelial morphogenesis and are frequently regulated by highly conserved extrinsic factors. However, due to the complexity of morphogenesis, the mechanisms of precise interpretation of signal transduction as well as spatiotemporal control of extrinsic cues during dynamic morphogenesis remain poorly understood. Wing posterior crossvein (PCV) formation in Drosophila serves as a unique model to address how epithelial morphogenesis is regulated by secreted growth factors. Decapentaplegic (Dpp), a conserved bone morphogenetic protein (BMP)-type ligand, is directionally trafficked from longitudinal veins (LVs) into the PCV region for patterning and differentiation. These data reveal that the basolateral determinant Scribbled (Scrib) is required for PCV formation through optimizing BMP signaling. Scrib regulates BMP-type I receptor Thickveins (Tkv) localization at the basolateral region of PCV cells and subsequently facilitates Tkv internalization to Rab5 endosomes, where Tkv is active. BMP signaling also up-regulates scrib transcription in the pupal wing to form a positive feedback loop. These data reveal a unique mechanism in which intrinsic polarity genes and extrinsic cues are coupled to promote robust morphogenesis.
This study shows that the Scrib complex, a basolateral determinant, is a novel feedback component that optimizes BMP signaling in the PCV region of the Drosophila pupal wing (Gui, 2016).
During PCV development, limited amounts of Dpp ligands are provided by the Dpp trafficking mechanism. Furthermore, amounts of receptors appear to be limited since tkv transcription is down-regulated in the cells in which the BMP signal is positive, a mechanism that serves to facilitate ligand diffusion and sustain long-range signaling in the larval wing imaginal disc. To provide robust signal under conditions in which both ligands and receptors are limiting, additional molecular mechanisms are needed. Previous studies suggest that two molecules play such roles. Crossveinless-2 (Cv-2), which is highly expressed in the PCV region, serves to promote BMP signaling through facilitating receptor-ligand binding. Additionally, the RhoGAP protein Crossveinless-c (Cv-c) provides an optimal extracellular environment to maintain ligand trafficking from LVs into PCV through down-regulating integrin distribution at the basal side of epithelia. Importantly, both cv-2 and cv-c are transcriptionally regulated by BMP signaling to form a feedback or feed-forward loop for PCV formation (Gui, 2016).
Scrib, a third component, sustains BMP signaling in the PCV region in a different manner. First, to utilize Tkv efficiently, Scrib regulates Tkv localization at the basolateral region in the PCV cells, where ligand trafficking takes place. Regulation of receptor localization could be a means of spatiotemporal regulation of signaling molecules during the dynamic process of morphogenesis. Second, to optimize the signal transduction after receptor-ligand binding, Scrib facilitates Tkv localization in the Rab5 endosomes. Localization of internalized Tkv is abundant at Rab5 endosomes in the PCV region of wild-type, but not scrib RNAi cells. While the physical interaction between Scrib, Tkv and Rab5 in the pupal wing remains to be addressed, the data in S2 cells suggest that physical interactions between these proteins are key for preferential localization of Tkv at the Rab5 endosomes. Recently, Scrib has been implicated in regulating NMDA receptor localization through an internalization-recycling pathway to sustain neural activity. Therefore, Scrib may be involved in receptor trafficking in a context-specific manner, the molecular mechanisms of which, however, remain to be characterized. Third, BMP/Dpp signaling up-regulates scrib transcription in the pupal wing. Moreover, knockdown of scrib leads to loss of BMP signaling in PCV region but not loss of apical-basal polarity. These facts suggest that upregulation of Scrib is key for optimizing BMP signaling by forming a positive feedback loop (Gui, 2016).
Previous studies indicate that cell competition takes place between scrib clones and the surrounding wild-type tissues in the larval wing imaginal disc. Moreover, cell competition has been documented between loss-of-Dpp signal and the surrounding wild-type tissues. It is presumed that the mechanisms proposed in this study are independent of cell competition for the following reasons. First, scrib RNAi and AP-2μ RNAi data reveal that loss of BMP signal in the PCV region is produced without affecting cell polarity. Thus, cell competition is unlikely to occur in this context. Second, BMP signal is intact in scrib mutant clones of the wing imaginal disc, suggesting that cell competition caused by scrib clones is not a direct cause of loss of BMP signaling in scrib mutant cells (Gui, 2016).
Previous studies established that receptor trafficking plays crucial roles in signal transduction of conserved growth factors, including BMP signaling. Several co-factors have been identified as regulators of BMP receptor trafficking. Some of them down-regulate BMP signaling while others help maintain it. It is proposed that the Scrib-Rab5 system is a flexible module for receptor trafficking and can be utilized for optimizing a signal. During larval wing imaginal disc development, BMP ligands are trafficked through extracellular spaces to form a morphogen gradient. Although previous studies indicate that regulation of receptor trafficking impacts BMP signaling in wing imaginal discs, BMP signaling persists in scrib or dlg1 mutant cells in wing discs. Wing disc cells interpret signaling intensities of a morphogen gradient. In this developmental context, an optimizing mechanism might not be beneficial to the system. In contrast, cells in the PCV region use the system to ensure robust BMP signaling (Gui, 2016).
Taken together, these data reveal that a feedback loop through BMP and Scrib promotes Rab5 endosome-based BMP/Dpp signaling during PCV morphogenesis. Since the components (BMP signaling, the Scrib complex, and Rab5 endosomes) discussed in this work are highly conserved, similar mechanisms may be widely utilized throughout Animalia (Gui, 2016).
Tight regulation of signalling activity is crucial for proper tissue patterning and growth. This study investigates the function of Pentagone (Pent/Magu), a secreted protein that acts in a regulatory feedback during establishment and maintenance of BMP/Dpp morphogen signalling during Drosophila wing development. It was shown that Pent internalises the Dpp co-receptors, the glypicans Dally and Dally-like protein (Dlp), and the study proposes that this internalisation is important in the establishment of a long range Dpp gradient. Pent-induced endocytosis and degradation of glypicans requires dynamin- and Rab5, but not clathrin or active BMP signalling. Thus, Pent modifies the ability of cells to trap and transduce BMP by fine-tuning the levels of the BMP reception system at the plasma membrane. In addition, and in accordance with the role of glypicans in multiple signalling pathways, Pent was found to be required for Wg signalling. These data propose a novel mechanism by which morphogen signalling is regulated (Norman, 2016).
Bone morphogenetic protein (BMP) signalling is required in a wide variety of processes across higher organisms, from the establishment of the dorso-ventral (DV) axis in insects to the maintenance of the mammalian gut. Many of the biological functions of BMP signalling require a high degree of spatial regulation; accordingly, multiple mechanisms have evolved that control the movement, stability and activity of BMP ligands (Norman, 2016).
One of the most intensely studied examples of extracellular regulation of BMP comes from Drosophila wing development, a tissue where Dpp (Drosophila BMP2/4) acts as a morphogen to control both patterning and growth. During larval wing development, Dpp is produced in a stripe of cells at the anterior-posterior (AP) boundary and disperses into both compartments, by mechanisms that are still not fully understood, to organize a BMP signalling activity gradient along the AP axis with highest levels in medial and lowest in lateral regions. Dpp, together with a second, uniformly expressed ligand, Glass bottom boat (Gbb), activates membrane bound receptors and induces the phosphorylation of the transcription factor Mad. Phosphorylated Mad (pMad) accumulates in the nucleus with the cofactor Medea, where the activated Smad complex directly regulates BMP-target gene transcription (Norman, 2016).
In addition to the localized production of Dpp, many other determinants impact on proper establishment and maintenance of the activity gradient. Prominent amongst them are membrane-bound BMP-binding proteins, such as Thickveins (Tkv) and Dally, which have dual functions in the establishment of the pMad gradient. Tkv is cell-autonomously required for signalling as it is the main type I BMP receptor in Drosophila. At the same time, Tkv critically affects Dpp tissue distribution through ligand trapping and internalisation, and thus globally shapes the BMP activity gradient. Similarly, Dally, a GPI-anchored heparan sulfate proteoglycan (HSPG), binds and concentrates Dpp at cell surfaces and, together with a second glypican, Dally-like protein (Dlp), is required for both local signal activation and long-range distribution of the ligand. Absence of glypicans- for example in a clone of cells- can result in both a reduction of BMP signalling within the clone and an interruption of Dpp spreading within and beyond the clone. In addition, glypicans can also, by virtue of their ligand-binding capacity, hinder the movement of ligands in several contexts. For example, the diffusion of BMP4 (a vertebrate homolog to Dpp) during early Xenopus embryogenesis has been shown to be restricted through its interactions with HSPGs. Similarly, in the wing disc, increasing the levels of Dally at the source of Dpp causes a local increase of signalling activity and a drastic compaction of the gradient due to ligand trapping. Reflecting the importance of the activity of Tkv and Dally for proper gradient establishment, the levels of both proteins are tightly regulated along the AP axis of the developing disc. Through complex transcriptional regulation, which involves repression by BMP signalling itself, both Tkv and Dally are down-regulated near the ligand source to maintain the proper balance between Dpp signalling and Dpp dispersion (Norman, 2016).
Pentagone (Pent; also known as Magu), is an additional determinant in the establishment, maintenance and scaling of the BMP signalling gradient in the developing wing. The transcription of pent is directly repressed by BMP signalling, hence its production is restricted to the lateral-most cells of the disc. Pent protein is, however, secreted and distributes in a gradient that is inverse to the pMad gradient. Pent mutants have a restricted pMad gradient with abnormally high levels in the centre of the disc and very low levels in lateral regions; consequently, adult wings have growth and patterning defects in lateral regions. The pMad gradient of pent mutants thus resembles the abnormal gradients caused by medial over-expression of Tkv or Dally, suggesting an interaction of the protein with the BMP-reception system. Indeed, past work established that Pent physically associates with Dally on cell membranes, but the consequences of this interaction have remained unclear (Norman, 2016).
This study presents data showing that Pent binds and induces the internalisation of both Drosophila glypicans, resulting in reduction of Dally and Dlp protein levels. Endocytosis of glypicans is dependent on dynamin and Rab5, but does not require clathrin or Dpp signalling. Additionally, it was shown that Pent influences Wg signalling, which also depends on glypicans. It is concluded from these data that Pent modulates glypican levels in order to modify multiple signalling pathways during wing morphogenesis. The data suggest an additional, protein-level feedback mechanism to tightly control levels of signalling, which cooperates with transcriptional regulatory feedback loops to ensure proper morphogen gradient formation and organ development (Norman, 2016).
It has been proposed that the function of Pent in Dpp gradient formation could be to either enhance the ability of Dally to displace Dpp, or to reduce the co-receptor function of Dally. From the data presented in this study, it is proposed that Pent reduces the co-receptor function of glypicans by binding them and inducing their internalisation. It is suggested that Pent may promote spreading of Dpp by reducing Dpp co-receptors and therefore Dpp trapping and signal transduction. Internalisation of glypicans is independent of signalling and Dpp itself, which fits the model that removal of glypicans by Pent enhances spreading. Furthermore, this study has presented data showing that by regulating glypican levels, Pent is also able to modulate Wingless signalling (Norman, 2016).
The work proposes that Pent modulates Dpp signalling via the co-receptors Dally and Dlp. The relative contribution of Dally and Dlp to Dpp signalling is unknown, although both must be removed in order for a reduction in pMad to occur. Data showing that Pent influences the co-receptor but not the receptor itself distinguishes Pent from other BMP signalling modifiers in D. melanogaster, such as Crossveinless-2, Short gastrulation and Twisted gastrulation, which bind either the BMP ligand, the receptor, or both. This could reflect the different roles that BMP signalling must fulfill in D. melanogaster, where it forms a long-range gradient in the larval wing disc but short-range gradients in the embryo and pupal wing (Norman, 2016).
The data show that Pent binds and internalises glypicans. Prior to endocytosis, it is probable that glypicans are clustered at the cell surface by Pent, and this might also inhibit their function without necessarily inducing their internalisation. Glypicans share physical properties, notably a GPI anchor and heparan sulphate side chains, upon which Pent binds. This study has shown that internalisation of glypicans by Pent requires dynamin and Rab5 but not clathrin. Cell culture experiments have shown that GPI proteins are commonly endocytosed via clathrin independent mechanisms, but this has not been demonstrated before in Drosophila. Many clathrin independent mechanisms have been described in cultured cells, but in vivo evidence for many of them is lacking. Lipid-rich microdomains, in some cases marked by flotillin, can be involved, but this study found no requirement for flotillin in the endocytosis of glypicans by Pent (Norman, 2016).
One of the key problems cells must overcome to internalise GPI anchored proteins is that they have no cytoplasmic region to mediate recruitment into endocytic pits. A similar process to that described in this study, the Hh mediated internalisation of GPC3, is thought to utilise LRP1 in order to communicate with the endocytic machinery. This does not seem to be the case with Pent and Dally, as knockdown of the Drosophila homologue of LRP1 does not affect internalisation. It is possible that protein clustering, and the membrane deformations this has been predicted to cause, may be involved in the internalisation of glypicans by Pent. The precise mechanism by which Pent internalises glypicans will be an interesting avenue of future research (Norman, 2016).
While the data implicate glypicans as the direct target of Pent's activity, it cannot be ruled out that the effect on Dpp gradient formation involves the regulation of Tkv levels and/or activity. While Pent does not bind to Tkv directly and Pent over-expression seems not to affect the levels of membrane bound Tkv, a substantial amount of the receptor is found in Pent- and Dally positive endocytic vesicles. This raises the possibility that Pent might target a specific subpopulation of Tkv for degradation, and that this interaction requires glypicans as adaptors (Norman, 2016).
The data show that Pent binds and internalises Dally and Dlp. Glypicans, in particular Dally, have been shown to regulate the spreading of Dpp, in addition to being essential for Dpp signal transduction itself. The molecular basis for these activities is the binding of Dpp to the heparan sulphate side-chains of glypicans, probably a first step that serves to concentrate Dpp at the surface of the disc epithelium. Glypican-bound Dpp molecules can follow multiple routes, as they can be passed to receptors (promoting signalling), to glypicans of neighbouring cells (promoting ligand dispersion), or can persist on glypicans of the same cell resulting in local ligand enrichment. It is probable that the specific outcome at any position along the morphogen field will depend on the relative levels and activities of the involved factors, i.e. the ligand, receptors and glypicans. A similar balance between glypicans, receptors and ligands has been proposed to explain the biphasic activity of Dlp in Wg signalling in the wing imaginal disc. In the case of Dpp, levels of glypicans need to be tightly regulated to allow for the optimal balance between ligand release, trapping and receptor binding. The data suggest that Pent contributes to this balance by fine-tuning the levels of glypicans. It is proposed that in the absence of Pent, glypican levels are too high and this results in excessive ligand trapping and enhanced local signalling. Such local effects would be accompanied with a non-autonomous reduction in ligand spreading and shrinkage of the pMad gradient. An approximation mimicking this situation is artificially elevating levels of Dally in medial regions, which has been shown to locally increase pMad. This study has shown that this increase in signalling by Dally is at the expense of Dpp spreading to the rest of the disc and the formation of the long range pMad gradient. This clearly shows that excess Dally can block spreading of Dpp. Notably, pent mutants display a similarly compacted activity gradient with high medial and low lateral pMad levels. Importantly, ligand-binding properties of HSPGs have been described to impede ligand spreading in multiple physiological contexts, including BMP4 in Xenopus early dorso-ventral patterning and FGF10 in its role in branching morphogenesis (Norman, 2016).
Multiple transcriptional feedback loops are required for the maintenance of the Dpp signalling gradient in the wing. Primary amongst these is the repression of Tkv and Dally transcription by Dpp signalling. This ensures that receptor and co-receptor levels are low near the Dpp producing cells, allowing Dpp to spread out from the centre of the disc. These feedback loops are important for proper establishment of the Dpp signalling gradient. However, such direct feedbacks targeting the production of molecules with ligand-binding properties may have limitations. In response to a reduction in spreading of Dpp, Tkv and Dally levels would increase to locally compensate the reduction in Dpp signalling activity. Such an increase would, however, further enhance trapping and internalisation of the ligand and, at the level of the whole wing disc, would further block Dpp spreading. From the data it is suggested that Pent, a secreted negative regulator of Dpp signalling, fine-tunes the signalling gradient at a different level, by directly adjusting glypican levels and reducing the inbuilt increase in co-receptor and ligand-trapping upon a reduction in the extent of the pMad gradient. This might happen at a critical region of the wing disc, the mediolateral cells, where declining levels of the spreading ligand face increasing levels of the receptor and co-receptor (Tkv and Dally, respectively). Pent, secreted by lateral cells next to this region, could reduce the glypican pool to allow Dpp to overcome excessive ligand trapping and thus promote further spreading. Consistent with such a 'remote' activity, Pent can be detected throughout the wing disc. As Pent is transcriptionally repressed by Dpp signalling and, unlike Tkv and Dally, does not bind Dpp, Pent might be a good candidate for how the system overcomes the inherent limitations of feedback loops involving membrane tethered, Dpp-binding proteins (Norman, 2016).
The key extracellular signalling molecules of the wing disc, Dpp, Wg and Hh, all bind to glypicans. The regulatory proteins Pent, Notum and Shifted also bind glypicans, putting glypicans at the centre of signalling regulation in the wing disc. Consequently, any factor that affects glypican function, such as Pent, is likely to modify multiple signalling pathways. This study has shown that Pent is also able to influence Wg signalling, thus providing a possible link between the Wg and Dpp pathways (Norman, 2016).
The role of glypicans in Wg signalling is well described and complex. Dlp can stimulate Wg signalling, Wg accumulates on cells over-expressing Dlp and fails to accumulate on cells mutant for Dlp. Similarly, the data show that excess Pent internalises glypicans and reduces extracellular Wg. Precise in vitro assays have shown that low levels of Dlp enhance Wg signalling, but too much Dlp reduces signalling. Furthermore, recent evidence shows that deacylation of Wg by Notum, which reduces Wg signalling activity, requires glypicans. It is clear, then, that the level of glypicans must be very finely balanced for Wg signalling to be at the correct level. It is proposed that the elevated glypican levels observed in the absence of Pent push this fine balance towards inhibition of signalling, due to the increased levels of glypicans sequestering Wg away from the receptor and also increasing the platform upon which Notum can deacylate Wg. Consistent with this conclusion, the effects of Notum and Dlp over-expression can be suppressed by increasing the level of Pent protein (Norman, 2016).
Interestingly, inactivation of the BMP-response elements in the regulatory region of the pent gene locus results in prominent expression of pent at the DV boundary, hinting at an input into pent transcription from DV signals. Future studies, including quantitative studies and modelling, should give further insight into pathway interaction and coordination during tissue development by molecules such as Pent (Norman, 2016).
A model is proposed that Pent internalises glypicans to modify multiple signalling pathways. Future work should address the influence of Pent on glypican organisation at the nanoscale, and also the type of membranes at which Tkv and Dally localise, questions that are challenging to answer using current methods. In order to fully understand the role of Pent in establishment of the long range Dpp gradient, first it is important to gain a better understand how glypicans function in Dpp signalling and how Dpp is spread throughout the tissue (Norman, 2016).
In the last few years, accumulated information has indicated that the evolution of an extra-embryonic membrane in dipterans was accompanied by changes in the gene regulatory network controlled by the BMP/Dpp pathway, which is responsible for dorsal patterning in these insects. However, only comparative analysis of gene expression levels between distant species with two extra-embryonic membranes, like A. gambiae or C. albipunctata, and D. melanogaster, has been conducted. Analysis of gene expression in ancestral species, which evolved closer to the amnioserosa origin, could provide new insights into the evolution of dorsoventral patterning in dipterans. This study describes the spatial expression of several key and downstream elements of the Dpp pathway and show the compared patterns of expression between Musca and Drosophila embryos, both dipterans with amnioserosa. Most of the analyzed genes showed a high degree of expression conservation, however, several differences were found in the gene expression pattern of M. domestica orthologs for sog and tolloid. Bioinformatics analysis of the promoter of both genes indicated that the variations could be related to the gain of several binding sites for the transcriptional factor Dorsal in the Md.tld promoter and Snail in the Md.sog enhancer. These altered expressions could explain the unclear formation of the pMad gradient in the M. domestica embryo, compared to the formation of the gradient in D. melanogaster. It is concluded that gene expression changes during the dorsal-ventral patterning in insects contribute to the differentiation of extra-embryonic tissues as a consequence of changes in the gene regulatory network controlled by BMP/Dpp. This work, in early M. domestica embryos, identified the expression pattern of several genes members involved in the dorsoventral specification of the embryo. These data can contribute to understanding the evolution of the BMP/Dpp pathway, the regulation of BMP ligands, and the formation of a Dpp gradient in higher cyclorraphan flies (Hodar, 2018).
G protein-coupled receptors play particularly important roles in many organisms. The novel Drosophila gene anchor is an orthologue of vertebrate GPR155. However, the roles of anchor in molecular functions and biological processes, especially in wing development, remain unknown. Knockdown of anchor resulted in an increased wing size and additional and thickened veins. These abnormal wing phenotypes were similar to those observed in BMP signalling gain-of-function experiments. The BMP signalling indicator p-Mad: was significantly increased in wing discs in which anchor RNAi was induced in larvae and accumulated abnormally in intervein regions in pupae. Furthermore, the expression of target genes of the BMP signalling pathway was examined using a lacZ reporter, and the results indicated that omb and sal were substantially increased in anchor-knockdown wing discs. An investigation of genetic interactions between Anchor and the BMP signalling pathway revealed that the thickened and ectopic vein tissues were rescued by knocking down BMP levels. These results suggested that Anchor functions to negatively regulate BMP signalling during wing development and vein formation (Wang, 2018).
Magnesium transporter subtype 1 (MagT1) is a magnesium membrane transporter with channel like properties. MagT1 (CG7830) has been identified in the Drosophila genome and its protein product characterized by electrophysiological means. This study reports the generation of fly MagT1 mutants and shows that MagT1 is essential for early embryonic development. In wings and primordial wings, by clonal analysis and RNAi knock down of MagT1, this study found that loss of MagT1 results enhanced/ectopic Wingless (Wg, a fly Wnt) signaling and disrupted Decapentaplegic (Dpp) signaling, indicating the crucial role of MagT1 for fly development at later stages. Finally, this study directly demonstrated that magnesium transportations are proportional with the MagT1 expressional levels in Drosophila Kc167cells. Taken together, these findings may suggest that MagT1 is a major magnesium transporter/channel profoundly involved in fly development by affecting developmental signaling pathways, such as Wg and Dpp signaling (Xun, 2018).
Seminal fluid proteins (SFPs) exert potent effects on male and female fitness. In Drosophila, most SFPs are produced in the accessory glands, which are composed of approximately 1,000 fertility-enhancing "main cells" and approximately 40 more functionally cryptic "secondary cells." Inhibition of bone morphogenetic protein (BMP) signaling in secondary cells suppresses secretion, leading to a unique uncoupling of normal female postmating responses to the ejaculate: refractoriness stimulation is impaired, but offspring production is not. Secondary-cell secretions might regulate more global SFP functions and proteome composition. Secondary-cell-specific BMP signaling inhibition compromises sperm storage and increases female sperm use efficiency. It also impacts second male sperm, tending to slow entry into storage and delay ejection. First male paternity is enhanced, which suggests a constraint on ejaculate evolution whereby high female refractoriness and sperm competitiveness are mutually exclusive. Using quantitative proteomics, this study reveals changes to the seminal proteome that surprisingly encompass alterations to main-cell-derived proteins, indicating important cross-talk between classes of SFP-secreting cells. These results demonstrate that ejaculate composition and function emerge from the integrated action of multiple secretory cell types, suggesting that modification to the cellular make-up of seminal-fluid-producing tissues is an important factor in ejaculate evolution (Hopkins, 2019).
Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests that prion-like proteins are prevalent in various species; however, it remains unclear what functional space they occupy in multicellular organisms. This study reports the identification of a prion-like protein, Herzog (CG5830), through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning, likely being involved in TGF-beta/BMP and FGF/EGF signaling pathways. Remarkably, monomeric Herzog, a protein-serine/threonine phosphatase, is enzymatically inactive and becomes active upon amyloid-like assembly. The prion-like domain of Herzog is necessary for both its assembly and membrane targeting. Removal of the prion-like domain impairs activity, while restoring assembly on the membrane using a heterologous prion-like domain and membrane-targeting motif can restore phosphatase activity. This study provides an example of a prion-like domain that allows an enzyme to gain essential functionality via amyloid-like assembly to control animal development (Nil, 2019).
Most proteins adopt one specific conformation in a cell dictated by their primary amino acid sequence. The relationship between a protein's sequence and one structure has in recent years been complemented by an exciting alternative. One group of proteins, called prion-like proteins, can assume distinct conformational states in the same cell, and often one of these states leads to a higher-order state that can vary in physical nature-from liquid to hydrogel to non-amyloid or amyloid-like aggregates. Prion-like proteins often contain a modular domain, called a prion-like domain (PrD), which is necessary and sufficient for higher-order state. Prion-like proteins are important regulators of various physiological processes in diverse species, such as adaptation to changing environmental conditions, immune response, and memory formation. Recent in silico analyses suggest that proteins with prion-like domains are prevalent in higher eukaryotic proteomes and may occupy broader functional space. However, with few exceptions, in most cases it remains unclear what the functional relevance is, if any, of the prion-like domains (Nil, 2019).
The establishment of cell fates during tissue specification and embryonic patterning are achieved by regulated activation and deactivation of various signaling pathways. Protein phosphorylation, a common dynamic post-translational modification (PTM), lies at the heart of developmental signaling pathways. Cells precisely control the state and the amplitude of signaling pathways by adding, stabilizing, or removing phosphate groups through the opposing activity of protein kinases and phosphatases. The complexity and specificity of phosphorylation is generally associated with the diversity of the kinases, and almost 2% of the eukaryotic protein coding genes encode for protein kinases. In contrast to kinases, a small number, ∼0.6% of genes, encode for protein phosphatases, and often in vitro multiple phosphatases act on the same substrate or the same phosphatase can act on multiple substrates, implying a low level of substrate specificity and regulation. Despite this apparent promiscuity and redundancy, loss of phosphatases often leads to very specific phenotypes. Therefore, unlike kinases, it remains unclear how seemingly promiscuous phosphatases achieve substrate specificity or control activity both in time and space (Nil, 2019).
A small-scale systematic screen of the Drosophila melanogaster proteome led to the identification of five potential prion-like proteins. One of these candidates, which was named Herzog (Hzg), is a functionally uncharacterized gene (CG5830), with homology to the haloacid dehalogenase (HAD) subfamily small CTD phosphatases (SCPs). Hzg, a membrane-associated phosphatase, is a maternal effect gene required for proper establishment of segment polarity in the embryos. Hzg is expressed ubiquitously throughout embryonic development but changes from monomers to aggregates with features of amyloids during gastrulation, and this state change leads to a gain of phosphatase activity. The phosphatase activity requires both membrane localization and amyloid-like fibrillization through its N-terminal prion-like domain. Taken together, this study provides a definitive example of how a prion-like domain can control embryonic development by orchestrating phosphatase activity in space and time (Nil, 2019).
In silico sequence-based analysis indicates that proteins with prion-like domains are quite abundant in all branches of life. However, with a few exceptions, the functional relevance of prion-like domains in multicellular eukaryotes remains largely unknown. This stduy reports that Hzg, through its N-terminal prion-like domain, changes from an inactive monomer to an active amyloid-like aggregate on the membrane during gastrulation, likely to control segment polarity. Although detailed structural information is lacking, the fact that substrate recognition does not require the N-terminal domain, but phosphatase activity does, and that the only fibrillar (recombinant) protein has enzymatic activity suggest that the conformational change into an amyloid like aggregate allows Hzg to be catalytically active (Nil, 2019).
Hzg belongs to HAD family of phosphatases, an ancient large class of enzymes, with unknown functions. For most phosphatases, a recognizable regulatory domain apart from their catalytic domains controls enzymatic function by controlling compartmentalization or substrate binding. HAD subfamily of SCPs (to which Hzg belongs), like prokaryotic HADs, contains a single catalytic domain without any of the recognizable structural regulatory domains. They also do not have a cap module in their active site, which is used by other HAD family members for substrate selectivity. Therefore, Hzg likely utilizes the conformational reorganization associated with amyloid-like fold to control phosphatase activity. The use of a prion-like domain to induce a conformational change and thereby catalytic activity adds to the repertoire of regulatory modes of enzymatic activity. Considering that several phosphatases and kinases in Drosophila and in other species harbor prion-like domains, it is possible that the aggregation-based modulation of enzymatic activity may not be rare. Intriguingly, the prion-like domain domain of Hzg is not only important for aggregation but also for its membrane localization. It is uncertain how Hzg prion-like domain targets the protein to the membrane and whether aggregation occurs in the membrane or whether preformed aggregates are recruited to the membrane. Whatever the mechanism, membrane localization and regulated aggregation and dissolution can confine the phosphatase activity in space and time, allowing a phosphatase to act in a substrate-specific manner. A key question for the future is how Hzg aggregation is regulated (Nil, 2019).
The screen was biased toward identifying prion-like proteins that form stable assemblies. Nonetheless, it begs the question as to why an enzyme forms an amyloid-like state, seemingly a stable state, to control dynamic signaling pathways. One possibility is that unlike liquid, gel, or glass-like assemblies, where the proteins are organized randomly, in an amyloid-like state the ordered assembly and as a result the ordered organization of the catalytic domain can offer a more reliable interaction between enzyme and substrate. Mechanistically, it is possible that the catalytic domain is hidden in the monomer and that the amyloid-like assembly reveals that catalytic domain. It is hypothesized that since the substrate binding and catalytic domain (M domain) is outside the N-terminal prion-like domain, the substrate does not need to access the core of the fiber. Conformational changes associated with amyloid-like fibrilization simply orient the substrate binding and catalytic domain to the surface of the fiber. Alternatively, three-dimensional (3D) domain swapping associated with amyloid-like assembly may turn inactive monomers to an enzymatically active oligomer or polymer. Indeed, almost a decade ago, Eisenberg and colleagues elegantly showed that two inactive monomers of bovine ribonuclease A can be arranged into an enzymatically active amyloid-like aggregate. It is speculated that Hzg represents a natural example of turning an inactive enzyme active via amyloid-like assembly (Nil, 2019).
In addition to conformational alteration, what are the consequences of adopting an amyloid-like state with respect to function? Historically, amyloids are thought to be irreversible primarily based on in vitro studies of disease-causing amyloids that originate from misfolded or truncated proteins. However, over the years it has emerged that cellular proteins such as enzymes, hormone peptides, mRNA, and DNA-binding proteins can adopt amyloid-like states and disassemble under normal cellular conditions. Moreover, from recent atomic-level structural analyses of various amyloid-like proteins, it is also emerging that in addition to canonical cross-β sheet, there are also α-helical amyloid-like states and amyloid-like states with charged residues in the core that confer flexibility. Although, the physiological relevance of such diversity and structural flexibility remains unclear, it is speculated that flexibility may allow an enzyme like Hzg to form a stable assembly that nonetheless can be taken apart. Development, which needs to accommodate the changing environment, might utilize such molecular stability and flexibility to tune the time course of development. With respect to heterogeneity in structure, as stated earlier, the number of phosphatases encoded by the genome is relatively small compared to kinases considering that almost one-third of all proteins in the cell are regulated by phosphorylation. The structural flexibility may help with expanding the functional space (Nil, 2019).
Proteomics analysis followed by pairwise interaction assays for Hzg-interacting partners suggest that there could be at least seven putative substrates for Hzg; Babo, Dah, Irk, Pch2, Ras64B, Sax, and Src64B. Multiple substrates implies that the phenotypes of hzg mutants may be pleotropic. Considering the multiple putative membrane-associated substrates, biochemical function, and mobility of Hzg aggregates on the membrane, he following speculative model is proposed: Hzg forms functional amyloidogenic aggregates on the membrane during gastrulation, and these aggregates moves along the membrane, allowing Hzg to act on different targets over time (Nil, 2019).
Mutant embryos expressing only the N-terminal prion-like domain of Hzg show defects in segment polarity, and this phenotype is reminiscent of mutations of wingless (wg) signaling antagonists such as zw3/GSK, D-axin, and D-Apc2. However, in the current proteomics analysis, no interaction of Hzg was seen with canonical Wg-signaling components, suggesting that Hzg may influence wg signaling indirectly. It is possible that the action of Hzg in TGF-β/BMP and EGF/FGF signaling (Ras64B and Src64B) pathways that are known to cross talk with the wg-signaling pathway contributes to the mutant phenotype. Further work would be necessary to obtain detailed mechanistic insight into the role of Hzg in embryonic development (Nil, 2019).
The Toll-Dorsal signaling pathway controls dorsal-ventral (DV) patterning in early Drosophila embryos, which defines specific cell fates along the DV axis and controls morphogenetic behavior of cells during gastrulation and beyond. The extent by which DV patterning information regulates subcellular organization in pre-gastrulation embryos remains unclear. This study found that during Drosophila cleavage, the late endosome marker Rab7 is increasingly recruited to the yolk granules and promotes the formation of dynamic membrane tubules. The biogenesis of yolk granule tubules is positively regulated by active Rab7 and its effector complex HOPS, but negatively regulated by the Rab7 effector retromer. The occurrence of tubules is strongly biased towards the ventral side of the embryo, which shows is controlled by the Toll-Dorsal signaling pathway. This work provides the first evidence for the formation and regulation of yolk granule tubulation in oviparous embryos and elucidates an unexpected role of Toll-Dorsal signaling in regulating this process (Reed, 2021).
High-risk Human Papillomaviruses (HPV) 16 and 18 are responsible for more than 70% of cervical cancers and majority of other HPV-associated cancers world-wide. Recently, a Drosophila model of HPV18E6 plus human E3 ubiquitin ligase (hUBE3A; see Drosophila Ubiquitin protein ligase E3A) was developed, and it was demonstrated that the E6-induced cellular abnormalities are conserved between humans and flies. Subsequently, it was demonstrated that reduced level and activity of IKKβ, a regulator of NF-κB, suppresses the cellular abnormalities induced by E6 oncoprotein and that the interaction of IKKβ and E6 is conserved in human cells. In this study, transcriptomic analysis was performed to identify differentially expressed genes that play a role in IKKβ-mediated suppression of E6-induced defects. Transcriptome analysis identified 215 genes whose expression were altered due to reduced levels of IKKβ. Out of these 215 genes 151 genes showed annotations. These analyses were followed by functional genetic interaction screen using RNAi, overexpression, and mutant fly strains for identified genes. The screen identified several genes including genes involved in Hippo and Toll pathways as well as junctional complexes whose downregulation or upregulation resulted in alterations of E6-induced defects. Subsequently, RT-PCR analysis was performed for validation of altered gene expression level for a few representative genes. These results indicate an involvement for Hippo and Toll pathways in IKKβ-mediated suppression of E6 + hUBE3A-induced cellular abnormalities. Therefore, this study enhances understanding of the mechanisms underlying HPV-induced cancer and can potentially lead to identification of novel drug targets for cancers associated with HPV (Collins, 2023).
CRISPR-Cas has revolutionized genetics and extensive efforts have been made to enhance its editing efficiency by developing increasingly more elaborate tools. This study evaluated the CRISPR-Cas9 system in Drosophila melanogaster to assess its ability to induce stem cell-derived tumors in the intestine. Conditional tissue-specific CRISPR knockouts were generated using different Cas9 expression vectors with guide RNAs targeting the BMP, Notch, and JNK pathways in intestinal progenitors such as stem cells (ISCs) and enteroblasts (EBs). Perturbing Notch and BMP signaling increased the proliferation of ISCs/EBs and resulted in the formation of intestinal tumors, albeit with different efficiencies. By assessing both the anterior and posterior regions of the midgut, regional differences were observed in ISC/EB proliferation and tumor formation upon mutagenesis. Surprisingly, high continuous expression of Cas9 in ISCs/EBs blocked age-dependent increase in ISCs/EBs proliferation and when combined with gRNAs targeting tumor suppressors, it prevented tumorigenesis. However, no such effects were seen when temporal parameters of Cas9 were adjusted to regulate its expression levels or with a genetically modified version, which expresses Cas9 at lower levels, suggesting that fine-tuning Cas9 expression is essential to avoid deleterious effects. These findings suggest that modifications to Cas9 expression results in differences in editing efficiency and careful considerations are required when choosing reagents for CRISPR-Cas9 mutagenesis studies. In summary, Drosophila can serve as a powerful model for context-dependent CRISPR-Cas based perturbations and to test genome-editing systems in vivo (Bahuguna, 2021).
Over the years Ski and Sno have been found to be involved in cancer progression e.g. in oesophageal squamous cell carcinoma, melanoma, oestrogen receptor-positive breast carcinoma, colorectal carcinoma, and leukaemia. Often, their prooncogenic features have been linked to their ability of inhibiting the anti-proliferative action of TGF-β signalling. Recently, not only pro-oncogenic but also anti-oncogenic functions of Ski/Sno proteins have been revealed. Besides Ski and Sno, which are ubiquitously expressed other members of Ski/Sno proteins exist which show highly specific neuronal expression, the SKI Family Transcriptional Corepressors (Skor). Among others Skor1 and Skor2 are involved in the development of Purkinje neurons and a mutation of Skor1 has been found to be associated with restless legs syndrome. But neither Skor1 nor Skor2 have been reported to be involved in cancer progression. Using overexpression studies in the Drosophila eye imaginal disc, this study analysed if the Drosophila Skor homologue Fuss has retained the potential to inhibit differentiation and induce increased proliferation. Fuss expressed in cells posterior to the morphogenetic furrow, impairs photoreceptor axon pathfinding and inhibits differentiation of accessory cells. However, if its expression is induced prior to eye differentiation, Fuss might inhibit the differentiating function of Dpp signalling and might maintain proliferative action of Wg signalling, which is reminiscent of the Ski/Sno protein function in cancer (Rass, 2022).
Negative regulators of the TGF-β signalling pathway are inhibitory Smads (I-Smads), Smurfs and the Ski/Sno protein family. Proteins of the latter group possess two structural domains: the Ski/Sno homology domain and the SMAD4-binding domain. With the help of these domains, Ski/Sno proteins can interact, among others, with R-Smads, N-CoR, Sin3a, SMAD4 and the histone deacetylase HDAC1 and this complex leads to transcriptional repression of target genes. By their expression domains, Ski/Sno proteins can be further subdivided into ubiquitously expressed genes (human Ski and Sno), and mainly neuronally expressed genes, the SKI Family Transcriptional Corepressors (Skor1 and Skor2). The Ski/Sno proteins fulfil a wide range of different physiological functions such as axonal morphogenesis, Purkinje cell development, myogenesis and mammary gland alveogenesis (Rass, 2022).
However, the Ski/Sno proteins were not discovered by their physiological functions but via the transforming capability of the viral ski (v-ski) homologue found in the Sloan-Kettering virus. The first evidence that Ski/Sno proteins possess oncogenic capabilities came from overexpression experiments, where it was shown that the truncation of v-ski is not responsible for the transformation of chicken embryo fibroblasts, but that overexpression of v-ski, Ski or Sno is sufficient for this transformation. Despite this background, their role in carcinogenesis is still not fully understood, if not even contradictory at times. Ski and Sno have been found to be upregulated in different types of cancer e.g. oesophagus squamous cell carcinoma, melanoma, and colorectal cancer. Further evidence for a pro-oncogenic role was found in downregulation analyses of Sno or Ski. This downregulation resulted in decreased tumour growth in breast cancer cells and pancreatic cancer cells. But as stated before, there is some objection that Ski and Sno function purely as oncogenes. Mice, which were heterozygous mutant for Ski or Sno, showed an increased level of tumour induction after carcinogen treatment. In metastatic non-small cell lung cancer, Ski expression is significantly reduced, whereas increased expression of Ski in these cells reduced the invasiveness inhibiting epithelial-mesenchymal transition. Therefore, this could reflect that the outcome of Ski or Sno expression in cancer cells is dependent on the cell type or the actual status of the cancer cells and cancer cells often exploit Ski or Sno to inhibit the anti-proliferative effects of TGF-β signalling. Whereas Ski or Sno have been found to be involved in a lot of different cancer types, there is sparse evidence for deregulation of Skor proteins in cancer cells. Endogenously, Skor proteins have been linked to neurodevelopmental processes. After Skor1 overexpression, genes involved in axonal guidance or post-synapse assembly were differentially expressed. Skor2 is important for cerebellar Purkinje cell differentiation as in Skor2 knockout mice dendrite formation of Purkinje cells was impaired. Pathophysiologically, Skor1 has mainly been linked to restless leg syndrome and localized scleroderma (Rass, 2022).
In Drosophila melanogaster, only one homologue of Ski and Sno, which is designated Snoo, and one homologue of Skor1 and Skor2, which is designated Fuss, exist. It has been recently shown that Fuss is interacting with SMAD4 and HDAC1. In overexpression assays, Fuss can inhibit Dpp signalling and endogenously, the Fuss/HDAC1 complex is required for bitter gustatory neuron differentiation and fuss mutant flies pause more often during walking. However, this study was interested whether the Skor/Fuss proteins retained their ability to inhibit differentiation and induce increased proliferation. For this purpose, Fuss was overexpressed in differentiating cells of the eye imaginal disc, an excellent model tissue to study regulatory gene function in the context of carcinogenesis. This overexpression impaired photoreceptor axon guidance and inhibited the differentiation of accessory cells such as cone cells and primary pigment cells, which are all transformed into a basal pigment cell type. In a second approach, fuss overexpressing clones were generated early during development in the eye imaginal discs, when cells are still proliferating. This resulted in vast outgrowths of undifferentiated tissue of the eye imaginal disc because fuss overexpression most likely inhibited Dpp-signalling, a member of the TGF-β superfamily. This work shows that Fuss retained the ability of Ski/Sno proteins to inhibit the antiproliferative effects of TGF-β signalling by analogous inhibition of Dpp-signalling, allowing proliferation to be sustained (Rass, 2022).
The overexpression of fuss posterior to the morphogenetic furrow with the GMR-Gal4 driver line resulted in a nearly complete loss of all cell types in the adult eye. During development, photoreceptor axons were not able to target the appropriate layers of the optic lobe anymore and cone cells, primary pigment cells and bristle cells were transformed into a basal pigment cell fate. This transformation was caused by the inhibition of sv expression, which is crucial for accessory cell differentiation. Additionally, increased apoptosis during pupal development lead to the removal of photoreceptors and lastly adult eyes only consisted of cells containing pigment granules. This lack of differentiation cannot be explained by the Dpp inhibiting role Fuss exerts, when overexpressed, because inhibiting the Dpp signaling pathway via knockdown of Tkv or Med had no effect. Photoreceptor axon guidance is impaired, if Dpp signaling is disrupted in photoreceptors by the expression of the inhibitory Smad Dad. Thus, the observed photoreceptor axon guidance phenotype, when fuss is overexpressed with GMR, could indeed be a result of Dpp signaling inhibition. However, the loss of nearly all eye cell types is due to other effects (e.g. downregulation of sv and apoptosis) than Dpp signaling repression alone, because loss of Dpp signaling behind the morphogenetic furrow only results in mild patterning defects of the pupal retina. Nonetheless, the inhibition of cell differentiation has already been shown in other cancer models e.g., when two copies of the constitutive active form of the receptor tyrosine kinase dRETMEN2B are expressed with the GMR-Gal4 line, pupal retinas are devoid of any distinguishable cell types. This phenotype is indistinguishable from the phenotype of the pupal retinas generated by the overexpression of fuss via GMR-Gal4. In a screen for novel oncogenes from breast cancer patients, human transgenes have been overexpressed with the GMR-Gal4 driver line. Overexpression of human RPS12, a subunit of the small ribosomal subunit, whose expression is increased in various cancer types, leads also to a glazed eye phenotype. Therefore, different oncogenes can result in different outcomes when expressed with the GMR-Gal4 driver line and are not always leading to massive tissue overgrowth like the Yorkie overexpression. Most importantly, with this approach to overexpress fuss in cells which already were destined for acquiring a cell fate and have left the cell cycle, it was not possible to induce increased proliferation anymore, but could prevent cell differentiation (Rass, 2022).
Consequently, a more pluripotent cell type in the eye imaginal disc was used and fuss overexpressing clones were induced prior to the formation of the morphogenetic furrow. These results allowed the assumption that in this context, fuss overexpressing clones do not react to the antiproliferative effects of the Dpp morphogen anymore. Instead, wg expression and thus, proliferation promotion might be maintained. This leads to outgrowths of clonal tissue from the eye imaginal disc of third instar larvae, which showed an increased number of mitotic events. If these flies survived to adulthood, undifferentiated, extra tissue was visible in the complex eye (Rass, 2022).
An analogous mechanism can be observed in tumors which overexpress Ski or Sno. The TGF-β signaling pathway is also anti-proliferative, but this action is inhibited by the increased presence of Ski/Sno proteins. Therefore, the molecular mode of action is similar to the human Ski/Sno proteins. The function of Ski and Sno is highly context dependent, as they can fulfill an anti-oncogenic or pro-oncogenic role depending on the cancer type or status of the cancer. This was also observed with fuss overexpressing clones. Only when induced 48h after egg laying, was additional tissue found in late third instar larvae and only in eye imaginal discs, because there, Dpp counteracts the proliferative effects of Wg signaling. When fuss is overexpressed in the wing disc or after induction of the morphogenetic furrow differentiation is inhibited, this results in a wing with truncated veins or in a smooth eye surface. This is also underlined by RNAseq data from eye and wing imaginal discs, where fuss was overexpressed with the GMR-Gal4 and Nub-Gal4 driver line, respectively. In the eye dataset, wg expression in eye imaginal discs is not significantly different from control eye discs, whereas wg expression in fuss overexpression wing discs is significantly reduced in contrast to control wing discs (Rass, 2022).
Thus, this study showed that the Skor protein Fuss in Drosophila melanogaster still retained the function of the Ski/Sno proteins by inhibiting differentiation but inducing hyperproliferation. But the hallmarks of real tumorigenesis are lacking, because at some point during pupal development, proliferation stops, and these cells become protruding head tissue as it could be observed in complex eyes of surviving flies. Furthermore, there was no evidence of an epithelial-mesenchymal transition because fuss overexpressing clones maintained their epithelial fate. It will be of high interest if future studies can find similar results in overexpression studies for the vertebrate Skor proteins or detect increased expression of these proteins in specific cancer types (Rass, 2022).
A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, this study shows that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, it was found that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate (Colonnetta, 2022).
Drosophila melanogaster is one of the classic and most thoroughly studied examples of organisms that use a 'preformation' mechanism for PGC specification. One of the first indications that PGCs in flies are specified by preformation came from experiments nearly 100 years ago in which the germline determinants were inactivated by UV irradiating the posterior of the embryo. The existence of localized and UV-sensitive determinants was subsequently supported by transplantation experiments. It was shown that the sterility of UV irradiated embryos could be rescued by injection of cytoplasm from unirradiated embryos into the posterior region of the UV treated embryos. Importantly, it was found that rescuing activity is localized within the donor; it is present at the posterior, but not the anterior pole. In complementary experiments, cytoplasm from the posterior pole was found to induce PGCs when injected into the anterior. Moreover, these ectopic PGCs were able to generate a functional germline if reinjected into the posterior pole of similarly staged recipients. Subsequent experiments showed that both PGC formation and abdominal patterning requires genes that are active in the mother during oogenesis and that the key maternal factor for the formation of the pole plasm is encoded by osk. Ectopic localization of osk mRNAs engineered by linking it to bcd 3' UTR is sufficient for the assembly of pole plasm at the anterior of the oocyte. In pre-cellular blastoderm embryos, the ectopic pole plasm induces the formation of PGCs at the anterior end. Moreover, these PGCs are able to populate the adult germline if transplanted into the posterior of recipient embryos (Colonnetta, 2022).
While these and other studies clearly demonstrate that maternal determinants localized at the posterior pole of the fly embryo orchestrate PGC specification, they do not establish that the mechanism is exclusively preformation. There is a potentially significant caveat with the experiments showing that PGCs induced at the anterior (either by injection of pole plasm or by the osk-bcd 3' UTR) are capable of generating a functional adult germline: the ectopic PGCs were transplanted into the posterior of recipient embryos and thus are subject to much the same milieu as PGCs formed by the normal mechanisms. This leaves open the possibility that epigenesis might play a role in PGC specification. To address this issue, it was asked whether the BMP signaling pathway is involved in the specification of fly PGCs when they are formed during the pre-cellular blastoderm stages of embryogenesis. This pathway was selected for four reasons. First, this pathway is known to function in PGC specification in animals that rely on epigenesis. Second, though dpp expression in the zygote is restricted to the dorsal side of the embryo by the Dorsal morphogen, both in situ hybridization and antibody staining experiments indicate that dpp expression encompasses the entire posterior pole. Third, experiments have shown that the transcriptional activator, pMad, induced in receiving cells by the BMP signaling pathway is present in the nuclei of pre-cellular blastoderm PGCs. Moreover, pMad accumulation depends not only on the Dpp ligand, but also maternal and zygotic sources of the Tkv receptor. Lastly, in previous studies it was found that BMP signaling is required to maintain PGC identity and their differentiation during mid-embryogenesis in the period leading up to the coalescence of the embryonic gonad (Colonnetta, 2022).
These results show that maternal determinants are insufficient on their own for proper specification of PGCs and that this process is not exclusively cell autonomous as has long been thought. Instead, a hybrid of preformation and epigenesis is deployed to generate a full complement of functional PGCs. When the BMP pathway is disrupted in pre-cellular blastoderm embryos, newly formed PGCs exhibit a variety of defects indicative of a failure in PGC determination. Moreover, the same phenotypes are observed in PGCs in embryos whose mothers are mutant in one or more of the three maternal factors, gcl, pgc and nos, that are known to be required for proper PGC specification. It is also worth noting that the functioning of the BMP pathway was not completely disrupted in these experiments. Thus, the possibility remains open that more dramatic or even some additional phenotypic effects might be observed under conditions where BMP signaling was entirely absent in pre-cellular blastoderm embryos. (For instance, dpp activity may affect PGC cellularization. It will also be of interest to assess if pole plasm anchoring is completely normal in pre-blastoderm embryos if BMP pathway is compromised) (Colonnetta, 2022).
Included in the defects that were observed are the partial loss of Vasa protein, a failure to downregulate Ser2 CTD phosphorylation, changes in the profile of histone modifications and the phosphorylation of the terminal (and EGFR) signaling pathway protein ERK. In WT PGCs, phosphorylation of Ser2 in the CTD domain of the large PolII subunit by pTfb is blocked by the Pgc protein. This block is overridden in embryos homozygous for the partial loss of function dpphr92 allele and in embryos produced by tkv- germline clone mothers. Moreover, two of the known targets for Pgc repression, tll and slam, are expressed in dpphr92 PGCs. However, the misfunctioning of Pgc is not the only defect in establishing transcriptional quiescence. Activation of the Sxl establishment promoter, Sxl-Pe, was observed. Previous studies have shown Sxl-Pe transcription is inappropriately turned on in PGCs in the progeny of gcl and nos mothers, but not in the progeny of pgc mothers. Thus, compromising BMP signaling would seem to broadly impact transcriptional quiescence, leading to the misexpression of genes normally repressed in PGCs by the activity of several different factors. In the case of tll, for example, Pgc is not the only pole plasm component expected to play a role in its repression. tll transcription is activated in somatic nuclei at the anterior and posterior ends of the embryo by the terminal pathway. However, the terminal pathway is normally shut down in WT PGCs by Gcl protein, which mediates the degradation of the Torso receptor. gcl function must also be disrupted either directly or indirectly when the BMP signaling pathway is compromised as it was found that dpERK accumulates in PGC nuclei just as in gcl mutants (Colonnetta, 2022).
These are not the only connections between the BMP pathway, Gcl, and the terminal signaling pathway. In gcl embryos, components of the pole plasm (including Vasa and pgc) are not properly captured by PGCs when they cellularize. Instead, they spread into the posterior region of the embryo and are found associated with somatic nuclei. A similar disruption in the proper distribution of pole plasm during PGC cellularization was found when the terminal pathway was upregulated by blocking degradation of Torso, or by a constitutive allele of the downstream MEK kinase. Moreover, as shown in this study, it was found that Vasa is lost from PGCs when the terminal pathway is optogenetically activated at the posterior of the embryo. In fact, the Vasa loss phenotype induced by optogenetic activation of SOS is quite similar to that seen when the BMP pathway was disrupted by RNAi knockdown of dpp and by dpp and tkv mutants. Taken together, these findings argue that one of the functions of the BMP pathway in PGC specification is to block the terminal signaling pathway. In this context, it is interesting to note that inhibition of EGFR-dependent signaling may be important for PGC specification in mammals. When ESC cells are cultured under conditions that promoted the formation of mesoderm lineages, a PGC-like identity could be induced by adding an inhibitor of the upstream kinase MEK to the culture media (Colonnetta, 2022).
In flies there is a complicated relationship between the BMP and EGFR signaling pathways. In the case of the Torso-dependent terminal pathway, dpERK phosphorylation of Capicua in the anterior and posterior soma counteracts the repression of dpp by Dorsal. In other developmental contexts, the relationship between BMP signaling and the cognate EGFR pathway is complex. In the wing disc, for example, BMP and EGFR signaling is reported to establish a positive feedback loop, reinforcing each other by promoting the synthesis of their respective ligands. In other contexts, however, the interactions between the two pathways is different. Genome wide studies of dorsoventral patterning during embryogenesis indicate that BMP signaling both negatively and positively regulates the expression of components of the EGFR signaling pathway during embryogenesis. EGFR, in turn, was proposed to temper rather than augment the BMP pathway by a dpERK-dependent phosphorylation of dSmad that results in its degradation. In studies on the patterning of the eye field and head epidermis in Drosophila, an antagonistic relationship has been proposed between BMP and EGFR signaling. High levels of Dpp were found to block EGFR signaling by inhibiting dpERK accumulation, while EGFR gain of function mutants suppress Dpp signaling. Though the mechanism for inhibiting dpERK accumulation was not uncovered, the same mechanism could be deployed to block the activation of the terminal pathway during PGC specification in pre-cellular blastoderm embryos. Alternatively, the mechanism of inhibition may be specific to the process of PGC specification. For example, it has been predicted that Torso degradation by Gcl should in itself be sufficient to eliminate both canonical and non-canonical activities of the terminal signaling pathway in PGCs. Thus, it is possible that the BMP pathway might inhibit terminal signaling by potentiating Gcl activity either directly or indirectly (Colonnetta, 2022).
That the BMP signaling pathway is required for the proper functioning of maternally deposited pole plasm components during PGC specification is also suggested by the dominant genetic interactions between osk and dpp. In these experiments, females heterozygous for an osk mutation were mated to males carrying the weak viable dpp allele, dpphr92. Reducing the dose of osk in the mother by itself appears to result in a minor perturbation of pole plasm sequestration in her progeny; however, this defect is substantially enhanced when the progeny are also heterozygous for dpphr92. In addition to failing to completely capture the pole plasm, dpphr92/+;osk/+ PGCs exhibit other abnormalities, including a novel loss of cell:cell adhesion and invasive migration. This synergistic interaction would argue that the BMP pathway collaborates with osk in the process of PGC specification, and in doing so serves to integrate preformation with epigenesis (Colonnetta, 2022).
The fact that one of the classical models of preformation deploys a signaling pathway that is known to play a critical role in PGC specification in species that rely on epigenesis would seem to bolster the argument that epigenesis is the ancestral mode for generating this special cell identity. This view would be supported by the evolutionary history of osk and nos, genes that function cell autonomously in PGC specification in flies. The former is restricted to a subset of insects that utilize preformation in PGC specification and is thought to arise from the fusion of bacterial and eukaryotic sequences. nos, by contrast, is conserved from worms to human and spans species that are classically identified as using either preformation or epigenesis for PGC specification (Colonnetta, 2022).
Maintaining long, energetically demanding axons throughout the life of an animal is a major challenge for the nervous system. Specialized glia ensheathe axons and support their function and integrity throughout life, but glial support mechanisms remain poorly defined. This study identified a collection of secreted and transmembrane molecules required in glia for long-term axon survival in vivo. The majority of components of the TGFβ superfamily are required in glia for sensory neuron maintenance but not glial ensheathment of axons. In the absence of glial TGFβ signaling, neurons undergo age-dependent degeneration that can be rescued either by genetic blockade of Wallerian degeneration or caspase-dependent death. Blockade of glial TGFβ signaling results in increased ATP in glia that can be mimicked by enhancing glial mitochondrial biogenesis or suppressing glial monocarboxylate transporter function. It is proposed that glial TGFβ signaling supports axon survival and suppresses neurodegeneration through promoting glial metabolic support of neurons (Lassetter, 2023).
A missense mutation in Nucleoporin107 (Nup107; D447N) underlies XX-ovarian-dysgenesis, a rare disorder characterized by underdeveloped and dysfunctional ovaries. Modeling of the human mutation in Drosophila or specific knockdown of Nup107 in the gonadal soma resulted in ovarian-dysgenesis-like phenotypes. Transcriptomic analysis identified the somatic sex-determination gene doublesex (dsx) as a target of Nup107. Establishing Dsx as a primary relevant target of Nup107, either loss or gain of Dsx in the gonadal soma is sufficient to mimic or rescue the phenotypes induced by Nup107 loss. Importantly, the aberrant phenotypes induced by compromising either Nup107 or dsx are reminiscent of bone morphogenetic protein (BMP signaling hyperactivation). Remarkably, in this context, the metalloprotease AdamTS-A, a transcriptional target of both Dsx and Nup107, is necessary for the calibration of BMP signaling. As modulation of BMP signaling is a conserved critical determinant of soma-germline interaction, the sex- and tissue-specific deployment of Dsx-F by Nup107 seems crucial for the maintenance of the homeostatic balance between the germ cells and somatic gonadal cells (Shore, 2022).
This study has shown that Nup107 activity in the somatic component of the gonad is necessary for the proper development and function of the ovaries. In which somatic cell type is the activity of Nup107 necessary? The fact that KD of Nup107 using the tj-Gal4 driver resulted in larval and adult aberrant phenotypes indistinguishable from those induced by the Nup107 loss of function mutation, indicates that the ovarian function of Nup107 is primarily required in the Tj-expressing cells. Notably, the tj-Gal4 driver is not expressed in terminal filament (TF) cells either at larval or adult stages. TF cells together with the cap cells (CCs) constitute the ovarian stem cell niche. During larval ovarian development, Tj is expressed in the ICs, CCs, and follicle stem cell (FSC) progenitors. CCs, which are derived from the intermingled cells (ICs), are formed at the base of fully formed TFs at the transition from the third larval instar to prepupal stage. Therefore, the aberrant PGCs and ICs observed in the larval gonads are not due to impairment of Nup107 or Dsx activities in CCs. Likewise, FSC progenitors are also not the candidate cells for the site of action of Nup107 activity as they reside posterior to ICs with a minimal physical contact with only a few posteriorly located PGCs. Together these data imply that Nup107 acts specifically in ICs enabling them to effectively interact with the PGCs. Consistent with this notion, loss of Nup107 affected the behavior of ICs such that these cells showed varying degrees of failure to mingle with the PGCs. A severe failure of ICs and PGCs to interact in the larval gonad is expected to cause an ovarian-dysgenesis-like phenotype as it is essential for the germarium development and ovariole formation. Likewise, a milder failure of intermingling in the larval gonad would allow for the formation of adult ovaries. However, in the adult ovary Nup107 activity is further required in ECs for the formation of their cellular extensions and regulation of differentiation of the GSCs. Thus, it will be important to determine in future studies the functional relationship between Nup107 and the signaling pathways which previously were shown to regulate the formation of these cellular extensions (Shore, 2022).
These studies have revealed that Nup107, a ubiquitously expressed nuclear envelope protein, is a crucial player during female gonad formation. How does an essential housekeeping protein critical for nuclear transport, perform such a sex- and tissue-specific function(s)? Two possible scenarios, not necessarily mutually exclusive, are envisioned to explain how the specific mutation in Nup107 results in ovary-specific aberrant phenotypes. In the first scenario, Nup107 would specifically mediate nucleocytoplasmic translocation of factor(s) or downstream effector(s) required for ovarian development. Indeed, recent studies have demonstrated that Nup107 is involved in translocation of specific factors. For instance, in the event of DNA damage, Nup107 directly interacts with the apoptotic protease activating factor 1 (Apaf-1 also known as Drosophila ARK) and mediates its transport into the nucleus to elicit cell-cycle arrest. Furthermore, it has been shown in tissue culture that specific Nucleoporins, including Nup107, are required for nuclear translocation of SMAD1, an important downstream effector of the Dpp/BMP pathway (Shore, 2022).
Alternatively, accumulating evidence has documented that in addition to their primary function in regulating the exchange of molecules between the nucleus and cytoplasm, NPC components may contribute to genome organization and tissue-specific regulation of gene expression in a nuclear transport-independent manner. Consequently, such moonlighting activities may not be confined to the nuclear envelope which is the primary native location of these proteins. For instance, mammalian Nup107-160 complex (a subcomplex of the NPC of which Nup107 is a key component) has recently been shown to shuttle in and out of GLFG nuclear bodies containing Nup98, a nucleoporin that regulates multiple aspects of gene regulation. Consistently, Nup107 was shown to regulate levels of specific RNAs through gene imprinting. Furthermore, using an RNAi-based assay, Nup107 was identified as a positive regulator of OCT4 and NANOG expression in human ESCs. In this regard, it is noteworthy that the recently published chromatin-binding profile of Nup107 suggested that Nup107 specifically targets active genes. Altogether these data support the possibility that Nup107 affects transcription of specific target genes in a tissue- and sex-specific manner either directly or indirectly (Shore, 2022).
In Drosophila melanogaster, Sex-lethal (Sxl) is the master determinant of somatic sexual identity, regulating a splicing dependent regulatory cascade resulting in the presence of alternatively spliced sex-specific isoforms of Dsx protein, Dsx-F and Dsx-M, in females and males, respectively. Subsequent dimorphic sexual development including sex-specific gonad morphogenesis is under the control of these Dsx isoforms. Consistently, Dsx proteins deploy components of the housekeeping machinery to achieve sex-specific development of the gonads. Thus, such 'maintenance' factors are unlikely to be involved in any regulatory capacity. The data challenge this notion and demonstrate the presence of sexually dimorphic circuitry downstream of a 'housekeeping' nuclear envelope protein, Nup107, which regulates the expression the female form of dsx (Shore, 2022).
The similar sex-specific and ovary-restricted phenotype associated with compromised Nup107 activity in both humans and flies implies common underlying molecular mechanisms. This study has identified Dsx as the primary target acting downstream of Nup107 in Drosophila ovarian development. The mammalian homologues of Dsx, the Dmrt family of transcription factors, also function during sex-specific gonad development. However, in mammals the main function of Dmrt genes in the gonad is to promote male-specific differentiation. While detailed functional analysis is not available, it is plausible that in mammals, another key female-specific transcription factor, like Foxl2 (female-specific forkhead box L2) may act downstream of Nup107 to substitute for DsxF in flies (Shore, 2022).
A previous study showed that the key components of the stem cell niche, that is the hub in males and the TFs in the case of females, are still formed in the absence of dsx, but this happens in a stochastic manner in both XX and XY dsx null mutant individuals. These results indicate that in the context of the developing stem cell niche, Dsx may not act in an instructive manner, but is instead required to ensure that the proper program (male or female) is selected, which does not require Dsx activity for the execution of subsequent sex-specific development. Nevertheless, their findings clearly demonstrated that the resulting adult ovaries and testes are improperly formed, consist of aberrant structures, arguing that Dsx activity, is critical for proper gonad development outside of the stem cell niche. Observations made in this study are consistent with this suggestion. Moreover, the experimental strategies and results differ in two important ways. First, the current experiments have relied on reduction of only the female form of dsx that is dsx-F which allowed for sex determination and thus no 'male' structures or cellular identity transformations were observed. Second, in the current experiments tj-Gal4 driver was used, which is not expressed in the stem cell niche (TF cells) but in other somatic gonadal cells. This experimental design enabled uncovering a novel developmental function of Dsx-F in ICs and their adult descendants, ECs. Supporting this notion it has ben shown that RNAi KD of dsx also resulted in small ovaries (Shore, 2022).
This study found that the secreted metalloprotease AdamTS-A is an important downstream component in the Nup107-Dsx axis, as KD of AdamTS-A results in phenotypes similar to those elicited by loss of either Nup107 or dsx. In the adult ovary, these aberrant phenotypes include loss of EC membrane protrusions and expanded BMP signaling. This raised the question of how AdamTS-A regulates the range of BMP signaling. The ECM, which is produced and secreted by cells, has the structure of a complex fibrillar meshwork and provides structural support and tissue integrity, playing an active role in regulating cell behavior. ECM proteoglycans sequester and modulate chemical signals, including growth factors and guidance molecules. Furthermore, type IV collagens, major components of the ECM, were shown to restrict Dpp signaling in the ovary. This is particularly intriguing, since in C. elegans Gon-1, the homolog of AdamTS-A, was shown to genetically interact with a type IV collagen (EMB-9) in the regulation of gonadogenesis. This raised the possibility that AdamTS-A, secreted by ECs, restricts Dpp movement in the germarium through cleavage of ECM components. However, by knocking down coracle in ECs, this study has shown that disruption of their cellular protrusions, which encapsulate the germ cells, leads to expansion of BMP signaling. This implies that the activity provided by these cellular extensions is necessary and sufficient for restricting the BMP signal (Shore, 2022).
Thus, Nup107, Dsx, and AdamTS-A all function in ECs and are necessary for the formation and maintenance of the cellular protrusions which are required for restricting the BMP signal emanating from the GSC's niche. Further, it appears that Adam-TS-A activity in the ECM is required for the formation and/or maintenance of these cellular protrusions. The results indicate that in this context AdamTS-A regulates BMP signal distribution indirectly via regulation of the cellular protrusion maintenance. It is also possible that AdamTS-A utilizes these cellular extensions in order to reach the ECM away from the ECs in the GSC region, where it acts to restrict Dpp trafficking (Shore, 2022).
Overall, these results support a model where Nup107 regulates the expression of dsx, either directly or indirectly, while Dsx directly regulates the transcription of multiple target genes including AdamTS-A. These observations have also uncovered that DsxF controls somatic niche function by calibrating the range and/or strength of Dpp/BMP signaling, possibly via modulation of the level and/or activity of the ECM components. Thus, it will be critical to elucidate how activities of nonsex-specific components such as Nup107 are coordinated with sex-specific regulation to achieve the precise specification and patterning underlying gonad development. This is of particular significance since modulation of BMP signaling circuitry is inextricably linked with the establishment and maintenance of stem cell fate. Importantly, as in the case of Nup107, BMP signaling is also required in a nonsex-specific manner in a variety of developmental contexts. These observations therefore open new avenues toward the critical examination of how a productive molecular dialog is established between nonsex-specific housekeeping machinery and versatile intersecting developmental pathways, in order to ultimately achieve proper sex-specific gonadogenesis crucial for fertility, and transmission of genetic information (Shore, 2022).
Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. To identify upstream regulators or downstream effectors of CDK8, a dominant modifier genetic screen was performed in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. 26 genomic loci were identified whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led to identification of genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFβ, or Transforming Growth Factor-β) signaling pathway. It was observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFβ signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, this analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila (Li, 2020).
To study the function and regulation of CDK8 in vivo, a genetic system was developed that yields robust readouts for the CDK8-specific activities in developing Drosophila wings. These genetic tools provide a unique opportunity to perform a dominant modifier genetic screen, allowing identification multiple components of the Dpp/TGFβ signaling pathway that can genetically interact with the CDK8-CycC complex in vivo. Subsequent genetic and cellular analyses reveal that CDK8, CycC, and six additional subunits of the Mediator complex, as well as CDK9 and Yki are required for the Mad-dependent transcription in the wing discs. In addition, CDK8 can directly interact with the linker region of Mad. These results have extended the previous biochemical and molecular analyses on how different kinases and transcription cofactors modulate the Mad/Smad-activated gene expression in the nucleus. Further mapping of specific genes uncovered by other deficiency lines may also open up the new directions to advance understanding of the conserved function and regulation of CDK8 during development (Li, 2020).
The Mediator complex functions as a molecular bridge between gene-specific transcription factors and the RNA Pol II general transcription apparatus, and diverse transactivators have been shown to interact directly with distinct Mediator subunits. However, it is unclear whether all Mediator subunits are required by different transactivators to regulate gene expression, or whether Mediator complexes composed of fewer and different combinations of Mediator subunits exist in differentiated tissues or developmental stages. Gene-specific combinations of the Mediator subunits may be required in different transcription processes, as not all Mediator subunits are simultaneously required for all transactivation process. For instance, ELK1 target gene transcription requires Med23, but lacking Med23 does not functionally affect some other ETS transcription factors, such as Ets1 and Ets2 . Similarly, Med15 is required for the expression of Dpp target genes, but does not appear to affect the expression of EGFR (epidermal growth factor receptor) and Wg targets in Drosophila (Li, 2020).
It has been previously reported that the Med15 subunit is required for the Smad2/3-Smad4 dependent transcription, as its removal from the Mediator complex abolishes the expression of Smad-target genes and disrupts Smad2/3-regulated dorsal-ventral axis formation in Xenopus embryos. Further biochemical analyses showed that increased Med15 enhances, while its depletion decreases, the transcription of Smad2/3 target genes, and that the Med15 subunit can directly bind to the MH2 domain of Smad2 or Smad3. In Drosophila, loss or reduction of Med15 reduced the expression of Dpp targets, resulting in smaller wings and disrupted vein patterning (mainly L2). It was also observed that depletion of Med15 or CDK8 reduces the expression of a Mad-target gene. These observations support the idea that CDK8 and Med15 play a conserved and positive role in regulating Mad/Smad-activated gene expression (Li, 2020).
Aside from Med15 and CDK8, it remains unclear whether other Mediator subunits are also involved in Mad/Smad-dependent transcription. This study identified six additional Mediator subunits that are required for the Mad-dependent transcription, including CycC, Med12, Med13, Med23, Med24, and Med31. Interestingly, aside from Med23 and Med24 being specific to metazoans, counterparts of the other six subunits are not essential for cell viability in the budding yeast. The similar effects of the four CKM (CDK8 kinase module) subunits on Mad-activity suggest that they may function together to stimulate Mad-dependent transcription. It is noted that depletion of seven Mediator subunits, Med7, Med8, Med14, Med16, Med17, Med21, and Med22, severely disrupts the morphology of the wing discs, making it difficult to assay their effects on the transcriptional activity of Mad in vivo. Consistently, all corresponding subunits, except Med16, are critical for cell viability in the budding yeast. In contrast, reducing expression of the 15 remaining subunits of the Drosophila Mediator complex did not significantly alter the expression of a Mad-dependent reporter. Med1 and Med25 are loosely associated to the small Mediator complex in human cell lines. A caveat for these negative results is that depleting these subunits using the existing RNAi lines may not be sufficient to affect sal-lacZ expression, even though the majority of these transgenic RNAi lines can generate severe phenotypes in the eye, wing, or both. Further analyses are necessary to validate these negative data in the future. Taken together, the results indicate that not all Mediator subunits are required for the expression of the Mad-target genes that were tested in the developing wing discs (Li, 2020).
Interestingly, Yki/YAP, which can function as a transcriptional co-factor for Mad/Smad, was also reported to associate with several subunits of the Mediator complex to drive transcription. Specifically, Med12, Med14, Med23, and Med24 were identified from a YAP IP-mass spectrometry sample in HuCCT1 cells. Med23 was also reported to regulate Yki-dependent transcription of Diap1 in wing discs. In the current study, Yki, Med12, Med23, and Med24 were also required for Mad-dependent transcription of sal-lacZ. Although the exact molecular mechanisms of how Yki interacts with certain Mediator subunits remain unclear, it is plausible that Yki may further strengthen the binding between Mad and Med15 through interactions with other subunits such as Med12, Med23, and Med24 (Li, 2020).
Based on biochemical analyses of the Smad1 phosphomutants and cell biological analyses using cultured human epidermal keratinocytes (HaCaT cells), several kinases including CDK8, CDK9, and ERK2 were shown to phosphorylate serine residues (Ser, or S) within the linker region of pSmad1 at S186, S195, S206, and S214, or the equivalent sites in pSmad2/3/5. These modifications were proposed to regulate positively Smad1-dependent transcriptional activity. Of these sites, S206 and S214 are both conserved from Drosophila to humans. In addition, studies using Xenopus embryos and cultured L cells suggest that MAPKs may phosphorylate the linker region of Smad1 (including S214) and lead to its degradation. Nevertheless, analyses with Drosophila embryos and wing discs indicate that S212 (equivalent to human pSmad1 S214) is phosphorylated by CDK8, while S204 (unique in Drosophila) and S208 (equivalent to human pSmad1 S210) are phosphorylated by Sgg/GSK3. These studies suggest the following model in explaining how Smads activate the expression of their target genes and how this process is turned off: after Smads are phosphorylated at their C-termini and translocated into the nucleus, CDK8 and CDK9 (potentially also MAPKs) act as the priming kinases to further phosphorylate pSmads in the linker region at S206 and S214. This may facilitate the interaction between pSmads and transcriptional cofactors such as YAP, stimulating the expression of Smads target genes. Overexpression of Yki in Drosophila wing disc increases the expression of the vgQE-lacZ reporter, which validates the role of Yki/YAP in activating Mad/Smad1-dependent gene expression in vivo. Subsequently, pSmads are further phosphorylated by GSK3 within the linker region at T202 and S210, which may facilitate Smad1/5 binding to E3 ubiquitin ligases such as Smurf1 and Nedd4L, causing the degradation of Smads through the ubiquitin-proteasome pathway (Li, 2020).
Although this model is still rather speculative, it serves as a conceptual framework to explain how transactivation of Smads is coupled to its degradation, similar to other transcriptional activators. It is challenging to determine whether these kinases act redundantly or sequentially for different phosphorylation sites, the exact orders of these phosphorylation events, as well as their biological consequences in vivo. Moreover, it remains unexplored whether these regulatory mechanisms are conserved during evolution. The importance of these issues is highlighted by the critical role of TGFβ signaling in regulating the normal development of metazoans and the dysregulation of this pathway in a variety of human diseases such as cancers (Li, 2020).
The precise spatiotemporal activation of the Dpp signaling pathway in the wings discs is critical for proper formation of the stereotypical vein patterns in Drosophila. This model system provides an ideal opportunity to dissect the dynamic regulation of the Mad-activated gene expression in the nucleus. Indeed, depleting CDK8 in wing discs reduces expression of the Mad-dependent sal-lacZ reporter, suggesting that CDK8 positively regulates Mad-dependent transcription. This is consistent with the effects of CDK8 on Smad1/5-dependent transcription in mammals. Depleting CDK8 does not affect the phosphorylation of Mad at its C-terminus as revealed by pMad immunostaining, nor does it affect the physical interaction between CDK8 and the linker region of Mad, supporting the idea that CDK8 may only affect subsequent phosphorylation of Mad, presumably within the linker region (Li, 2020).
Besides CDK8-CycC, depleting CDK9-CycT also decreases the expression of the sal-lacZ reporter, supporting the notion that CDK8-CycC and CDK9-CycT may play non-redundant roles in further phosphorylating pMad in the nucleus. However, no effects of depletion of CDK7 or MAPKs on sal-lacZ expression were observed, suggesting that their role in regulating the transcriptional activity of Smads may not be conserved in Drosophila. Alternatively, the two MAPK/ERK homologs, Rolled and ERK2, may act redundantly in regulating Mad-dependent transcription. Lastly, depleting Sgg/GSK3 in the dorsal compartment of the wing disc increases the size of this compartment, yet the expression level of the sal-lacZ reporter is similar to the ventral compartment. These observations are consistent with previous reports that phosphorylations of Mad/Smad in the linker regions by CDK8-CycC and Sgg/GSK3 regulate the level and range of Mad-dependent gene expression (Li, 2020).
Together with the previous reports, the data support that CDK8-CycC and CDK9-CycT may phosphorylate pMad at the linker region, which may facilitate the binding between Yki and Mad. It is speculated that this interaction may synergize the recruitment of the Mediator complex, presumably at least through the interaction between its Med15 subunit and the MH2 domain of Mad (see Model of Mad/Smad-dependent transcription activation through the CKM and the Mediator complex.). Alternatively, Yki may also facilitate the recruitment of the whole Mediator complex through its interactions with Med12, Med23, and Med24. The synergistic interactions among Mad, Yki, the Mediator complex, and RNA Pol II may be required for the optimal transcriptional activation of the Mad-target genes (Li, 2020).
One of the challenges is to illustrate the dynamic interactions between these factors and diverse protein complexes that couple the transactivation effects of Mad/Smads on gene transcription with their subsequent degradation at the molecular level. Smad3 phosphorylation strongly correlates with Med15 levels in breast and lung cancer tissues; together, they potentiate metastasis of breast cancer cells. Thus, it will be important to test whether additional Mediator subunits that were identified in Drosophila play similar roles in mammalian cells. It will also be interesting to determine whether a partial Mediator complex, composed of a subset of the Mediator subunits, exists and regulates Mad/Smad-dependent gene expression. Furthermore, detailed biochemical analyses may yield mechanistic insights into how CDK8 and Med15 act in concert in stimulating the Mad/Smad-dependent gene expression (Li, 2020).
Wing pouch-specific alteration of CDK8 activity results in two major phenotypes: disrupted vein patterns and altered size of wing blades. While the effects on wing size and cell numbers can be explained by the role of CDK8 in regulating cell proliferation through E2F1, the effects of CDK8 on vein patterning are more complex. The stereotypical wing vein patterns in adult flies are gradually defined by elaborated spatiotemporal interplays among different signaling pathways, including Dpp, EGFR, Hedgehog (Hh), Notch (N), and Wingless (Wg), in the developing wing discs. During the larval and pupal stages, these signaling pathways and their downstream transcriptional targets coordinately control the cell proliferation and differentiation of cell in different parts of the wing disc to form individual veins (Li, 2020).
It is noteworthy that varying CDK8 activities has different effects on different veins: gain of CDK8 causes the loss of the L3 and L4 veins, but the vein patterns of L2 and L5 appear thicker and more diffusive; while the ectopic veins caused by reduction of CDK8 are mainly intertwined with the L2 and L5 veins. This analyses on the genetic interactions between CDK8 and the components of the Dpp signaling pathway led to the discovery of the role of the Mediator complex in Mad-stimulated transcription of sal. However, there is a gap in understanding ohow reduced expression of sal in wing discs is linked to the vein defects in adult wings. It is known that salm and salr (spalt-related), two members of the spalt gene family that encode zinc-finger transcriptional repressors, function downstream of the Dpp signaling pathway during development of the central part of the wing. Depletion of either salm or salr alone resulted in ectopic vein formation around L2 in adult wings, yet depletion or loss of both salm or salr caused loss of vein phenotype. In addition, elimination of L2 in ventral-anterior and ectopic L5 in dorsal-posterior were observed in salm/salr clones at different region of the wing. These observations suggest that the dosage of salm and salr in wing discs does not have a linear relationship with the wing vein patterning at the adult stage (Li, 2020).
Interestingly, it is known that the CKM complex regulates the transcriptional activities of the key transcription factors of these pathways, including N-ICD downstream of N signaling and Mad/Smad proteins. In addition, Med12 (Kohtalo, or Kto in Drosophila) and Med13 (Skuld, or Skd in Drosophila) subunits of the CKM interact with Pangolin (the lymphoid-enhancing factor (LEF)/T cell factor (TCF) homolog in Drosophila), the key transcription factor downstream of Wg signaling, through the transcriptional cofactors such as Pygopus, Legless, and Armadillo. In mammalian cells, Med12 is also known to regulate the activities of Gli proteins, the key transcription factors downstream of Hh signaling. Furthermore, the Mediator subunit Med23 interacts with ETS (E-twenty six transcription factor) proteins, a family of key transcription factors downstream of the EGFR signaling pathway. However, whether CDK8-CycC also regulates TCF-, ETS- or Gli-dependent transcription is still not understood. Nevertheless, these studies in other biological contexts suggest that the effects of CDK8 on wing vein patterning are not likely solely through the Dpp signaling pathway. Therefore, it is speculated that the potential interactions between CDK8 and the aforementioned signaling pathways may contribute to these differential effects on distinct veins. Further analyses of these cross-talks, as well as further mapping of other Df lines that modify the CDK8-specific vein phenotypes, may yield the insights into the molecular and dynamic mechanisms underlying these vein phenotypes (Li, 2020).
To understand how dysregulated CDK8-CycC contributes to a variety of human cancers, it is essential to elucidate the function and regulation of CDK8 in vivo. Given that the CDK8-CycC pair and other subunits of the Mediator complex are conserved in almost all eukaryotes, Drosophila serves as an ideal model system to identify both the upstream regulators and the downstream effectors of CDK8 activity in vivo. The dominant modifier genetic screen is based on the wing vein phenotypes caused by specific alteration of CDK8 activity in the developing wing disc, which serves as a unique in vivo readout for the CDK8-specific activities in metazoans. This screen led to the identification of 26 genomic regions that include loci whose haplo-insufficiency could consistently modify CDK8-CycC depletion or CDK8-overexpression phenotypes. Identification of Dad and genes encoding additional components of the Dpp signaling pathway provides a proof of principle for this approach. Since each of the chromosomal deficiencies uncovers multiple genes, further mapping of the relevant genome regions is expected to identify the specific genetic loci encoding factors that may function either upstream or downstream of CDK8 in vivo. It is hoped that further analyses of the underlying molecular mechanisms in both Drosophila and mammalian systems will advance understanding of how dysregulation of CDK8 contributes to human diseases, thereby aiding the development of therapeutic approaches (Li, 2020).
Morphogen gradients specify cell fates during development, with a classic example being the bone morphogenetic protein (BMP) gradient's conserved role in embryonic dorsal-ventral axis patterning. This study elucidates how the gradient of Dpp is interpreted in the Drosophila embryo by combining live imaging with computational modeling to infer transcriptional burst parameters at single-cell resolution. By comparing burst kinetics in cells receiving different levels of BMP signaling, this study shows that BMP signaling controls burst frequency by regulating the promoter activation rate. Evidence is provided that the promoter activation rate is influenced by both enhancer and promoter sequences, whereas Pol II loading rate is primarily modulated by the enhancer. Consistent with BMP-dependent regulation of burst frequency, the numbers of BMP target gene transcripts per cell are graded across their expression domains. It is suggested that graded mRNA output is a general feature of morphogen gradient interpretation and discuss how this can impact on cell-fate decisions (Hoppe, 2020).
A gradient of bone morphogenetic protein (BMP) signaling patterns ectodermal cell fates along the dorsal-ventral axis of vertebrate and invertebrate embryos. In Drosophila, visualization of Decapentaplegic (Dpp), the major BMP signaling molecule, reveals a shallow graded distribution in early embryos that subsequently refines to a peak of Dpp at the dorsal midline. BMP-receptor activation leads to phosphorylation of the Mothers against dpp (Mad) transcription factor, which associates with Medea (Med) to activate or repress target gene transcription. A stripe of phosphorylated Mad (pMad) and Med centered at the dorsal midline has been visualized in the early Drosophila embryo, similar to that observed for Dpp, although lower nuclear pMad levels are also detectable in a few adjacent dorsal-lateral cells. The BMP/pMad gradient activates different thresholds of gene activity, including the peak target gene hindsight (hnt) and the intermediate target u-shaped (ush) (Hoppe, 2020).
New insights into transcriptional activation have been obtained by studying this process in single cells using quantitative and live imaging approaches, including single-molecule fluorescence in situ hybridization (smFISH) and the MS2/MS2 coat protein (MCP) system (Pichon, 2018). The latter allowed the first direct visualization of pulses or bursts of transcriptional activity. Enhancers have been shown to regulate the frequency of transcriptional bursts, with strong enhancers generating more bursts than weaker enhancers. In addition, the detection of simultaneous bursts of transcription of two linked reporters by a single enhancer argues against the classic enhancer-promoter looping model (Fukaya, 2016; Hoppe, 2020).
Recently, Notch target genes in Drosophila and C. elegans have been shown to undergo transcriptional bursting, with Notch controlling burst size through effects on duration (Falo-Sanjuan, 2019; Lee, 2019). However, it is unclear whether this is a general mechanism by which signals control transcriptional bursting. Therefore, to provide insight into BMP gradient interpretation at single-cell resolution, live imaging and quantitative analysis was used to determine the kinetics of endogenous BMP target gene transcription in the Drosophila embryo. These data reveal that BMP signaling modulates the promoter activation rate and therefore burst frequency. The different burst frequencies of BMP target genes, depending on cellular position, result in a gradient of mRNA numbers per cell. Overall these data reveal how a signaling gradient is decoded with different transcriptional kinetics to impart positional information on cells (Hoppe, 2020).
This study has analyzed the transcriptional burst kinetics of the endogenous hnt and ush genes at single-cell resolution. Cells were shown to interpret different BMP signaling levels by modulating burst frequency via kon . For ush, kon is unchanged when the hnt promoter is introduced, suggesting that features of the enhancer, most likely transcription factor-binding sites, dictate kon. Transcription factor binding is coupled to initiation of a burst (Donovan, 2019), and burst frequency depends on the time it takes a transcription factor to find its binding site (Larson, 2011), providing an explanation for why high BMP/pMad levels increase kon. Consistent with this, other studies have found transcription factor concentration and enhancer strength to regulate kon. For ush, this study provides evidence that kon is at a maximum in cells receiving peak signaling. A burst frequency ceiling has been described for tumor necrosis factor (TNF) targets, although the kon ceiling for ush appears to be gene specific, as it is below that observed for hnt and other Drosophila genes studied (Lammers, 2020; Zoller, 2018). Perhaps kon for ush is limited by slow recruitment of pMad or fewer productive activation events between its enhancer and promoter. Current ideas for enhancer-promoter communication include a dynamic kissing model that invokes transient interactions or models based on the proximity of regulatory elements in space, potentially in phase separated condensates. In these models, the Mediator coactivator may act as a dynamic molecular bridge between the enhancer and promoter, as activator-recruited Mediator at the enhancer can also make contact with the promoter (Hoppe, 2020).
When the hnt and ush promoters are swapped, mean Pol II loading rate is unchanged in both cases suggesting that it is predominantly dictated by the enhancer, although increased variability is detected in loading rate for ush>hnt. This suggests that transcription factors bound to the enhancer control Pol II loading rate, which is consistent with a previous study that found loading rate to be influenced by the strength of the transcription factor's activation domain. Loading rate is unchanged by altered BMP levels, with a constant loading rate also described for bursting of gap genes (Zoller, 2018), suggesting it is not a major regulated step of transcription in the Drosophila embryo. Despite the similar bursting behavior between ush and hnt>ush, introducing the ush promoter into hnt reduces kon. This suggests that the hnt promoter has some feature that is important in the hnt genomic context, for example a TATA box or highly paused Pol II, as the ush promoter lacks both. TATA increases burst frequency in the presence of interferon signaling; however, other studies have linked TATA promoters to burst size, and the hnt promoter does not increase frequency when introduced into ush. Another possibility is that the ability of paused Pol II at the hnt promoter to prevent encroachment of nucleosomes increases kon. In support of this, introduction of nucleosome disfavoring sequences around a promoter and linked transcription factor-binding site was found to increase burst frequency. It has also been shown that forcing interactions between a β-globin enhancer and its promoter increases burst frequency (Bartman, 2019). As enhancer-promoter compatibility has been proposed, perhaps the hnt enhancer is more compatible with its cognate promoter. In terms of compatibility, Mediator and the p300 acetyltransferase, both of which are recruited by Smads, most strongly activate TATA-containing promoters (Haberle, 2019). While further work is required to understand how the core promoter influences bursting, these results suggest a role for both the enhancer and promoter in influencing kon and burst frequency, thereby allowing greater flexibility in controlling the transcriptional response (Hoppe, 2020).
hnt transcriptional bursts are shorter and of higher frequency and amplitude than the ush bursts. The resulting hnt promoter occupancy is around half that for ush, providing a molecular explanation for the observed threshold responses of these genes to the BMP gradient. Unlike for ush, low BMP signaling levels are insufficient to maintain the hnt promoter in an active state, resulting in a narrow expression pattern. Burst duration, although not responsive to Dpp levels, is around 4 times longer for ush than for hnt. Transcription factor dwell time, which is limited by binding site affinity and nucleosomes, controls burst duration. As the Smad proteins have low affinity for DNA and weak specificity, they cooperate with other DNA-binding proteins. The ush and hnt enhancers have yet to be characterized, but the pioneer factor Zelda and homeodomain protein Zerknüllt may be pMad cofactors for intermediate (ush) and peak (hnt) Dpp targets, respectively. It is possible that at the hnt enhancer the Zerknüllt-pMad complex has a shorter residency time, or the pMad-binding sites are weaker affinity, resulting in shorter duration bursts (Hoppe, 2020).
The lack of a contribution of burst duration (1/koff ) to decoding BMP signaling is in stark contrast to the findings that Notch alters the duration, but not frequency, of transcription bursts in Drosophila and C. elegans (Falo-Sanjuan, 2019; Lee, 2019). Increasing gene expression through high kon rates can decrease noise, whereas lengthening burst duration is associated with more noise (Wong, 2018). Regulation of burst frequency may also allow genes to respond more sensitively to activator concentration (Li, 2018). Therefore, perhaps regulation of BMP target genes via kon has the advantage of allowing more sensitive regulation with less noise. It remains to be determined whether other signals will be interpreted through changes in kon and burst frequency or duration (Hoppe, 2020).
The different burst kinetics of BMP target gene transcription across the expression domain explain why cells at the edge are frequently captured with a single active allele in the single molecule florescent in situ hybridization (smFISH) data. sog and brk exhibit transcriptional bursting (Esposito, 2016), and the current data suggest that sog and brk bursting is regulated across their expression domains. Allele by allele repression has been observed in the Drosophila embryo, potentially because repressors are better able to act in the refractory period following a burst (Esposito, 2016). Such allele by allele repression can also explain why nuclei with one active allele are observed at the ventral borders of the sog and brk expression domains, where dorsal activator levels are high. Single-allele transcription has also been reported for zygotic hunchback (hb) transcription, which is activated by the Bicoid gradient, particularly at the borders of the expression domain. It is suggested that infrequent transcriptional bursting, with a concomitant reduction in mRNA number, is a general feature of gradient interpretation for cells receiving low signal (Hoppe, 2020).
The ush mRNA distribution reflects the spatial BMP gradient as the central 6 rows that receive peak BMP signaling have the highest mRNA number/cell, with subsequent declining mRNA numbers mirroring a reduction in Dpp levels. Additionally, modeling suggests that the concentration of BMP-receptor complexes at the dorsal midline doubles between 20 min and 30 min into nc14. This corresponds to the onset times of ush and hnt, respectively, suggesting that hnt transcription requires more activated receptors. Furthermore, BMP-receptor levels peak at ~45 min into nc14, which broadly coincides with the observed maximum fluorescence output detected for ush and hnt (Hoppe, 2020).
It is suggested that altered transcriptional burst kinetics and graded mRNA numbers in response to morphogen gradients can impact on cell-fate decisions. It is proposed that cells on the edge of an expression domain synthesize sufficient mRNAs to adopt a particular cell fate, whereas cells in the center have a surplus of transcripts. This model can explain the lack of robustness when shadow enhancers are deleted. Perturbation of the system, such as removal of a shadow enhancer, would lead to a further reduction in mRNA number per cell so that those on the edge would only just exceed the threshold level. Another challenge, such as high temperature or reduced activator level, would further decrease the transcriptional output such that there are insufficient mRNAs to specify the correct cell fate. It will be interesting in the future to test how the different numbers of mRNAs per cell from key BMP target genes impact on the robustness of dorsal ectoderm cell-fate decisions (Hoppe, 2020).
The relationship between chromatin organization and gene regulation remains unclear. While disruption of chromatin domains and domain boundaries can lead to misexpression of developmental genes, acute depletion of regulators of genome organization has a relatively small effect on gene expression. It is therefore uncertain whether gene expression and chromatin state drive chromatin organization or whether changes in chromatin organization facilitate cell-type-specific activation of gene expression. In this study, using the dorsoventral patterning of the Drosophila melanogaster embryo as a model system, evidence is provided for the independence of chromatin organization and dorsoventral gene expression. Tissue-specific enhancers are defined and linked to expression patterns using single-cell RNA-seq. Surprisingly, despite tissue-specific chromatin states and gene expression, chromatin organization is largely maintained across tissues. The results indicate that tissue-specific chromatin conformation is not necessary for tissue-specific gene expression but rather acts as a scaffold facilitating gene expression when enhancers become active (Ing-Simmons, 2021).
Previous studies produced conflicting results regarding the relationship between gene expression, chromatin state and 3D chromatin organization. This study set out to understand this relationship in the context of embryonic development in Drosophila. Using the well-studied dorsoventral patterning system, it was shown that, despite significant differences in chromatin state and gene expression between tissues along the dorsoventral axis of the embryo, chromatin conformation is largely maintained across tissues. This suggests that cell-type-specific gene regulation does not require cell-type-specific chromatin organization in this context. Nevertheless, developmentally regulated genes and enhancers are organized into chromatin domains. It is suggested that this organization plays a permissive role to facilitate the precise regulation of developmental genes (Ing-Simmons, 2021).
Use was made of maternal effect mutations in the Toll signaling pathway, which lead to embryos that lack the usual patterning of the dorsoventral axis and have long been used as a system to study the specification of mesoderm (Toll10B), neuroectoderm (Tollrm9/rm10) and dorsal ectoderm (gd7) cell fates as well as the regulation of tissue-specific gene expression. However, these embryos are still under the influence of anterior-posterior patterning signals and do not show completely uniform cell identities. This study sought to investigate heterogeneity of cell identity at the single-cell level by using single-cell gene expression profiling. This revealed that certain cell types are indeed maintained in all three Toll pathway mutants, including pole cells and other terminal region cell identities, hemocytes and trachea precursor cells. However, heterogeneity of gene expression is reduced in the mutants, as shown by the loss of cells assigned to mesoderm clusters in gd7 and Toll10B embryos and the depletion of ectoderm subsets in each of the mutants. These datasets showcase the advantages of measuring cellular heterogeneity at the single-cell level and provide a useful resource for further characterization of these embryos and investigation of the regulation of dorsoventral patterning (Ing-Simmons, 2021).
Although the gd7, Toll10B and Toll10B embryos still have heterogeneous gene expression profiles, nevertheless, there are clear differences in chromatin state and overall gene expression between these embryos. This study expanded on previous studies by identifying putative enhancers specific to neuroectoderm in addition to dorsal ectoderm and mesoderm. This allowed the identification of tissue-specific putative enhancer-gene pairs, which correspond well with known dorsoventral patterning enhancers and genes that are differentially expressed (DE across the dorsoventral axis. These regulatory elements and their target genes are located inside chromatin domains, distinct from the enrichment of housekeeping genes at domain boundaries. This is in line with previous results that suggest that 3D chromatin domains act as regulatory domains (Ing-Simmons, 2021).
This domain organization is maintained across tissues, even in cases in which there are significant changes in the local chromatin state and gene expression. This is consistent with earlier results from Hi-C experiments carried out in anterior and posterior embryo halves, which also showed no differences, and with previous studies in Drosophila cell lines and other systems, which suggested that domains are widely conserved across different tissues and even different species. To explain this maintenance of organization across cell lines, it was proposed that active chromatin, especially at broadly expressed genes, is responsible for partitioning the genome into domains. It has been proposed that compartmentalization of active and inactive chromatin, at the level of individual genes, underlies the formation of insulated chromatin domains. This model predicts that, when a developmentally regulated gene is active, its domain would merge with or have increased interactions with neighboring domains containing active genes, such as broadly expressed housekeeping genes. The results do not support this model, as this study found no evidence that differences in domain structure are driven by changes in chromatin state or by active expression of developmentally regulated genes. By contrast, this supports the idea that, similar to mammalian domain architecture, additional factors, such as insulator proteins, modulate domain organization in Drosophila. Therefore, based on current data, it is not believed that active transcription is the key determinant of 3D chromatin organization in this system (Ing-Simmons, 2021).
While overall and locus-specific chromatin organization are maintained across tissues, Hi-C and Micro-C analyses identify a small number of examples of regions that do have changes in organization. However, at these loci, there is no clear relationship between changes in organization and changes in chromatin state or expression, and the vast majority of developmentally regulated loci in this system do not have changes. It will be important for future studies to further investigate these loci to understand what drives these rare changes (Ing-Simmons, 2021).
This study also investigated chromatin organization at the level of enhancer-promoter interactions. Previous studies produced conflicting results about whether these interactions are correlated with tissue-specific activation of gene expression. No evidence was found for widespread enrichment of interactions between enhancers and their target promoters, including in tissues where they are active. This is in contrast with previous studies using 3C approaches that have found evidence of enriched enhancer-promoter interactions, which may precede or correlate with transcriptional activation. Notably, Ghavi-Helm (2019) found that a subset of Drosophila long-range enhancer-promoter pairs do form stable interactions that are enriched above local background19. While these loops are visible in the dataset presented in this study, the results suggest that such loops are not likely to be the primary mechanism of promoter regulation during Drosophila development, perhaps because most enhancers are close to their target promoters. Many stable loops in the Drosophila genome are instead associated with polycomb-mediated repression (Ing-Simmons, 2021).
Hi-C provides information about the average conformation across a population of hundreds of thousands of nuclei, which contain dynamic ensembles of different 3D conformations. While the scRNA-seq results indicate that the mutant embryos contain a range of different cell types, it is believed that the results indicate that the 3D chromatin structures in these cell types are drawn from the same population of possible conformations. This is supported by results from a recent study analyzing the structure of the Doc and sna loci in Drosophila embryos using Hi-M, a high-resolution single-cell imaging approach. Strikingly, this orthogonal technique also reveals chromatin organization that is consistent across different tissues in the embryo, despite differential expression of these genes. Imaging-based approaches directly measure spatial proximity between genomic loci, whereas Hi-C and Micro-C rely on cross-linking to detect chromatin interactions. Therefore, care must be taken when comparing these approaches. Nevertheless, both approaches indicate that genome organization is maintained across different tissues in this system (Ing-Simmons, 2021).
The results are consistent with several recent studies in mammals as well as in Drosophila, which provide evidence that stable enhancer-promoter contacts are not always required for gene activation. This is in line with models in which transient or indirect contacts with a regulatory element are sufficient to activate transcription, such as through the formation of nuclear microenvironments or phase-separated condensates (Ing-Simmons, 2021).
Together, the results indicate that differential chromatin organization is not a necessary feature of cell-type-specific gene expression. It is proposed that chromatin organization into domains instead provides a scaffold or framework for the regulation of developmental genes during and after the activation of zygotic gene expression. This may help render developmental enhancers 'poised' for timely regulation of target genes upon receipt of appropriate cellular signals. Other mechanisms of priming have been described, including paused polymerase (Pol) II at promoters and pioneer factors bound to poised enhancers. Feedback effects, such as downstream modification of chromatin state and additional mechanisms, including looping between polycomb-bound elements and segregation of active and inactive chromatin, may then act as layers on top of the initially established domain structure (Ing-Simmons, 2021).
Developmental patterning is thought to be regulated by conserved signalling pathways. Initial patterns are often broad before refining to only those cells that commit to a particular fate. However, the mechanisms by which pattern refinement takes place remain to be addressed. Using the posterior crossvein (PCV) of the Drosophila pupal wing as a model, into which bone morphogenetic protein (BMP) ligand is extracellularly transported to instruct vein patterning, this study investigate how pattern refinement is regulated. It was found that BMP signalling induces apical enrichment of Myosin II in developing crossvein cells to regulate apical constriction. Live imaging of cellular behaviour indicates that changes in cell shape are dynamic and transient, only being maintained in those cells that retain vein fate competence after refinement. Disrupting cell shape changes throughout the PCV inhibits pattern refinement. In contrast, disrupting cell shape in only a subset of vein cells can result in a loss of BMP signalling. It is proposed that mechano-chemical feedback leads to competition for the developmental signal which plays a critical role in pattern refinement (Toddie-Moore, 2021).
Limiting BMP signalling range in the stem cell niche of the ovary protects against germ cell tumors and promotes germ cell homeostasis. The canonical repressor of BMP signalling in both the Drosophila embryo and wing disc is the Brinker (Brk) transcription factor, yet the expression and potential role of brk in the germarium has not previously been described. This study found that brk expression requires a promoter-proximal element (PPE), to both support long-distance enhancer action as well as to drive expression in the germarium. Furthermore, PPE subdomains have different activities; in particular, the proximal portion acts as a damper to precisely regulate brk levels. Using PPE mutants as well as tissue specific RNAi and overexpression, this study shows that altering brk expression within either the soma or germline affects germ cell homeostasis. Remarkably, it was found that Decapentaplegic (Dpp), the main BMP ligand and Brk's canonical antagonist, is upregulated by Brk in the escort cells of the germarium demonstrating that Brk can positively regulate this pathway (Dunipace, 2022).
A missense mutation in Nucleoporin107 (Nup107; D447N) underlies XX-ovarian-dysgenesis, a rare disorder characterized by underdeveloped and dysfunctional ovaries. Modeling of the human mutation in Drosophila or specific knockdown of Nup107 in the gonadal soma resulted in ovarian-dysgenesis-like phenotypes. Transcriptomic analysis identified the somatic sex-determination gene doublesex (dsx) as a target of Nup107. Establishing Dsx as a primary relevant target of Nup107, either loss or gain of Dsx in the gonadal soma is sufficient to mimic or rescue the phenotypes induced by Nup107 loss. Importantly, the aberrant phenotypes induced by compromising either Nup107 or dsx are reminiscent of bone morphogenetic protein (BMP signaling hyperactivation). Remarkably, in this context, the metalloprotease AdamTS-A, a transcriptional target of both Dsx and Nup107, is necessary for the calibration of BMP signaling. As modulation of BMP signaling is a conserved critical determinant of soma-germline interaction, the sex- and tissue-specific deployment of Dsx-F by Nup107 seems crucial for the maintenance of the homeostatic balance between the germ cells and somatic gonadal cells (Shore, 2022).
Autophagy is a conserved, lysosome-dependent catabolic process of eukaryotic cells which is involved in cellular differentiation. Its specific role in the differentiation of spermatogonial cells in the Drosophila testis was studied. In the apical part of the Drosophila testis, there is a niche of germline stem cells (GSCs), which are connected to hub cells. Hub cells emit a ligand for bone morhphogenetic protein (BMP)-mediated signalling that represses Bam (bag of marbles) expression in GSCs to maintain them in an undifferentiated state. GSCs divide asymmetrically, and one of the daughter cells differentiates into a gonialblast, which eventually generates a cluster of spermatogonia (SG) by mitoses. Bam is active in SG, and defects in Bam function arrest these cells at mitosis. This study shows that BMP signalling represses autophagy in GSCs, but upregulates the process in SG. Inhibiting autophagy in SG results in an overproliferating phenotype similar to that caused by bam mutations. Furthermore, Bam deficiency leads to a failure in downstream mechanisms of the autophagic breakdown. These results suggest that the BMP-Bam signalling axis regulates developmental autophagy in the Drosophila testis, and that acidic breakdown of cellular materials is required for spermatogonial differentiation (Varga, 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).
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).
Maintaining synaptic structure and function over time is vital for overall nervous system function and survival. The processes that underly synaptic development are well understood. However, the mechanisms responsible for sustaining synapses throughout the lifespan of an organism are poorly understood. This study demonstrates that a previously uncharacterized gene, CG31475, regulates synaptic maintenance in adult Drosophila NMJs. CG31475 was named mayday, due to the progressive loss of flight ability and synapse architecture with age. Mayday is functionally homologous to the human protein Cab45 (SDF4 - stromal cell derived factor 4), which sorts secretory cargo from the Trans Golgi Network (TGN). Mayday was found to be required to maintain trans-synaptic BMP signaling at adult NMJs in order to sustain proper synaptic structure and function. Finally, mutations in mayday were shown to result in the loss of both presynaptic motor neurons as well as postsynaptic muscles, highlighting the importance of maintaining synaptic integrity for cell viability (Sidisky, 2021).
Among the most prominent neuromuscular synapses in adult Drosophila are those of the indirect flight muscles. One set of IFMs, the Dorsal Longitudinal Muscles (DLMs), are composed of six large muscle fibers innervated by five motor neurons on each side of the thorax. Once the DLM NMJs are established, these stable structures are present throughout the lifespan of the organism. These NMJs are part of the Giant Fiber (GF) pathway that propels flight behavior. Thus, the activity of DLMs can be monitored by assaying flight behavior as a readout of synaptic integrity. Additionally, the DLM NMJs form a tripartite synapse composed of a presynaptic motor neuron, postsynaptic muscle cell, and associated glial cell, that provide the ability to understand synaptic function at the cellular and molecular level. This model also allows for expression of transgenes in non-essential tissue, particularly the DLM motor neurons that are easily accessible. Together, it is possible assess the morphological and functional properties of adult DLM NMJs to elucidate the mechanisms responsible for sustaining synapses in aging adults, then apply this to understand how synapses deteriorate in neurodegenerative diseases (Sidisky, 2021).
Although the processes involved in maintaining synaptic structure and function may not be understood, there are a few key pathways that are crucial for regulating synaptic growth, organization and stability during synaptic development. Specifically, in Drosophila one key signaling cascade that involves coordination between the presynaptic motor neurons and postsynaptic muscle cells is the bone morphogenic protein (BMP) signaling cascade. The morphogen glass bottom boat (Gbb), the Drosophila ortholog to mammalian BMP7, is secreted in a retrograde manner from the postsynaptic muscle cell to the presynaptic motor neuron. Currently, it is not understood how this pathway could function past development. This suggests that this signaling cascade could play a role in maintaining synaptic integrity (Sidisky, 2021).
Gaining a better understanding of synaptic dysfunction should help to identify strategies involved in maintaining synaptic integrity with age. This study identified Mayday, a resident Golgi protein that is required to maintain trans-synaptic signaling across adult NMJs. Mutations in mayday impair retrograde BMP signaling, resulting in degradation of synaptic structure and function. Finally, this study demonstrates that this sustained trans-synaptic signaling is required to maintain the viability of both presynaptic motor neurons and postsynaptic muscles (Sidisky, 2021).
The current study describes mayday (myd), a previously uncharacterized gene that plays a role in maintaining synaptic integrity with age by promoting trans-synaptic signaling. We found that myd3PM71 mutants have structural and functional deficits in adult DLM NMJs. Through tissue-specific RNAi and rescue experiments, it was determined that Myd is necessary in both postsynaptic muscle tissue and presynaptic motor neurons to maintain synaptic integrity. Myd localizes to the TGN and shares functional homology with human Cab45. Myd sustains retrograde BMP signaling in adult DLM NMJs through genetic interactions with gbb, tkv, wit, and mad mutants and staining of Gbb and pMad markers. Finally, myd sustains the viability of presynaptic motor neurons and postsynaptic muscles (Sidisky, 2021).
From developmental studies, it was learned that Gbb is a morphogen secreted in a retrograde manner trans-synapticaly from postsynaptic muscle tissue to the presynaptic motor neuron in larval NMJs to promote synaptic growth. However, relatively little is known regarding the roles of this pathway in fully developed organisms. Recent evidence demonstrates that sustained BMP signaling is required to maintain FMRFamide expression in a subset of neurons in the Drosophila brain. The current results here further demonstrate that retrograde BMP signaling that regulates NMJ development is required in adult NMJs to sustain synaptic integrity with age. It is also possible that several other signaling pathways crucial for organism development may be required throughout the life of the organism (Sidisky, 2021).
Knockdown and rescue experiments using myd demonstrate that it maintains synaptic integrity through roles in both pre- and postsynaptic tissue. While most studies involving BMP in synaptic growth report a retrograde signaling mechanism, recent evidence suggests that this pathway could also signal in an anterograde fashion. While genetic studies provide support for retrograde BMP signaling, it cannot be ruled out that anterograde BMP signaling also plays an important role in maintaining synaptic integrity. In particular, the levels of pMAD that were observed were present within both presynaptic motor neuron terminals as well as postsynaptic muscles. Further studies aimed at characterizing BMP signal activation within muscle cells should help with our understanding of the mechanisms responsible for synaptic maintenance (Sidisky, 2021).
While trans-synaptic BMP signaling plays a clear role in maintaining synapses, myd mutations likely impair other pathways associated with cargo trafficking. In addition to secretory cargo, Cab45 also has a role in trafficking lysosomal proteases. Given the functional homology shared between Cab45 and Myd, it is possible that the trafficking of these lysosomal hydrolases needed for autophagy could be disrupted. Defects in autophagy have been strongly linked with neurodegenerative diseases. Therefore, it is possible that myd mutants have disruptions in autophagy that lead to the loss of synaptic integrity. It will be interesting to investigate how Myd impacts these other processes that are associated with neuronal dysfunction (Sidisky, 2021).
This assessment of synaptic dysfunction in the current study includes flight performance as a readout of functional integrity, as well as morphological measurements of branch length, branch number, and bouton number using a presynaptic membrane marker. In future studies, it will be helpful to further evaluate synaptic integrity in mayday mutants. Additional functional assays may include electrophysiological measurements of activity across these NMJs, and more structural data could be obtained through the use of a wide array of synaptic markers, as well as ultrastructural analysis using Transmission Electron Microscopy. Together, these types of studies should allow for an even greater understanding of synaptic dysfunction and the mechanisms required to maintain these critical structures (Sidisky, 2021).
Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remain poorly understood. This study shows that σ2-adaptin, one of the subunits of the AP2-complex, genetically interacts with Mad, Medea and Dad (components of BMP signaling) to control neuromuscular junction (NMJ) growth in Drosophila Ultrastructural analysis of σ2-adaptin mutants show an accumulation of large vesicles and membranous structures akin to endosomes at the synapse. Mutations in σ2-adaptin lead to an accumulation of Tkv receptors at the presynaptic membrane. Interestingly, the level of small GTPase Rab11 was significantly reduced in the σ2-adaptin mutant synapses. However, expression of Rab11 does not restore the synaptic defects of σ2-adaptin mutations. A model is proposed in which AP2 regulates Tkv internalization and endosomal recycling to control synaptic growth (Choudhury, 2023).
Secreted modular calcium-binding proteins (SMOCs) are conserved matricellular proteins found in organisms from Caenorhabditis elegans to humans. SMOC homologs characteristically contain 1 or 2 extracellular calcium (EC)-binding domain(s) and 1 or 2 thyroglobulin type-1 (TY) domain(s). SMOC proteins in Drosophila and Xenopus have been found to interact with cell surface heparan sulfate proteoglycans (HSPGs) to exert both positive and negative influences on the conserved bone morphogenetic protein (BMP) signaling pathway. This study used a combination of biochemical, structural modeling, and molecular genetic approaches to dissect the functions of the sole SMOC protein in C. elegans. CeSMOC-1 binds to the heparin sulfate proteoglycan GPC3 homolog LON-2/glypican, as well as the mature domain of the BMP2/4 homolog DBL-1. Moreover, CeSMOC-1 can simultaneously bind LON-2/glypican and DBL-1/BMP. The interaction between CeSMOC-1 and LON-2/glypican is mediated specifically by the EC domain of CeSMOC-1, while the full interaction between CeSMOC-1 and DBL-1/BMP requires full-length CeSMOC-1. Both in vitro biochemical and in vivo functional evidence id provided demonstrating that CeSMOC-1 functions both negatively in a LON-2/glypican-dependent manner and positively in a DBL-1/BMP-dependent manner to regulate BMP signaling. It was further shown that in silico, Drosophila and vertebrate SMOC proteins can also bind to mature BMP dimers. This work provides a mechanistic basis for how the evolutionarily conserved SMOC proteins regulate BMP signaling (DeGroot, 2023).
Combinatorial signaling is key to instruct context-dependent cell behaviors. During embryonic development, adult homeostasis, and disease, bone morphogenetic proteins (BMPs) act as dimers to instruct specific cellular responses. BMP ligands can form both homodimers or heterodimers; however, obtaining direct evidence of the endogenous localization and function of each form has proven challenging. This study made use of precise genome editing and direct protein manipulation via protein binders to dissect the existence and functional relevance of BMP homodimers and heterodimers in the Drosophila wing imaginal disc. This approach identified in situ the existence of Dpp (BMP2/4)/Gbb (BMP5/6/7/8) heterodimers. Gbb was found to be secreted in a Dpp-dependent manner in the wing imaginal disc. Dpp and Gbb form a gradient of heterodimers, whereas neither Dpp nor Gbb homodimers are evident under endogenous physiological conditions. This study found that the formation of heterodimers is critical for obtaining optimal signaling and long-range BMP distribution (Bauer, 2023).
BMP, Chordin, Twsg1, and BMP-1 have been shown to enable ligand shuttling in Drosophila and Xenopus dorsal-ventral patterning. On the other hand, two recent studies in zebrafish embryos showed that BMP ligands were not shuttled despite the presence of all four circuit components, provoking the question of whether shuttling can occur in mammals and what minimal set of components is sufficient to generate it. By systematically reconstituting the shuttling circuit one component at a time, this study was able to identify four distinct behaviors enabled by different combinations of components: BMP alone can form simple monotonically decreasing gradients; Chordin can delay and extend those gradients; Twsg1 with Chordin can suppress gradients; and BMP-1 with the other components can generate shuttling. In this last case, time-lapse movies revealed that gradients form at a distance from the source rather than propagating outward from it (Zhu, 2023].
A simple mathematical model shows how the four-component circuit is sufficient to enable ligand shuttling, notably predicts all of the four behaviors observed in these experiments, and highlights how the qualitative properties of the shuttling gradient depend on BMP-1 expression level. To do so, the model relies only on known interactions and previously measured parameters. Looking ahead, it will be important to identify more complex and developmentally relevant conditions in which the model fails, as additional components are included (Zhu, 2023].
Shuttling appears to be conserved during evolution. Biochemical and genetics studies have shown that interactions among BMP, Chordin, Twsg1, and BMP-1 are largely conserved across metazoans. These observations raise the provocative question of whether hybrid systems with components from distantly related organisms can still function. The reconstituted system allows one to directly examine this question. By substituting Drosophila Sog for mouse Chordin, it was found that some features of the resulting circuit are preserved, including gradient inhibition and lengthening. This substitution degraded the shuttling behavior, eliminating gradient displacement. This could be due to quantitative differences in BMP-Sog interactions, such as weaker binding. Another difference between Drosophila Sog and mammalian Chordin is that Tolloid cleavage of Sog is dependent on BMP binding, while BMP-1 can cleave vertebrate Chordin both in its free form and within a BMP-Chordin complex. While BMP-dependent cleavage is not necessary for shuttling, this mechanism has been suggested to increase the robustness of shuttling. It would be interesting to introduce Drosophila Tolloid into the reconstituted system and compare the robustness of shuttling enabled by Drosophila Sog and Tolloid, and mammalian Chordin and BMP-1. Analysis of other hybrid interspecies circuits across a broader range of expression levels could help to understand how evolutionary changes in molecular interactions cause changes in patterning (Zhu, 2023].
These results show that shuttling can occur with mammalian components but do not imply that all BMP-Chordin dependent behaviors involve shuttling. The BMP-Chordin system appears capable of multiple qualitatively distinct patterning behaviors, depending on factors such as the expression level of circuit components or the expression configurations. For example, the configuration demonstrated here, where BMP and Chordin are expressed at the same region, resembles certain developmental contexts such as mouse vertebral field, and ectopic expression systems in embryos. However, it differs from the configuration in zebrafish early embryos, where BMP and Chordin are expressed from opposing poles, and a source-sink, rather than a shuttling, mechanism has been demonstrated. It is possible that the more linear gradient generated by the source-sink mechanism is more desired in this developmental context. Future studies with the reconstituted system should allow investigation of other developmentally relevant configurations (Zhu, 2023).
While this study focused on shuttling, BMP pathway components generate a much broader range of behaviors, including scaling with embryo size and spatially oscillatory patterning of complex tissues, such as digits. By extending the platform described in this study to incorporate additional components [e.g., other ligands, modulators, and receptors], feedback loops in which signaling regulates pathway components, and other geometric configurations, one could, in principle, reconstitute other gradient behaviors and quantitatively explore these phenomena in a simplified setting. One could also analyze the role of co-occurring combinations of multiple BMP ligands such as BMP2, BMP4, and BMP7 in space and time. In addition, one could investigate the proposed ability of shuttling to enhance the robustness of gradient formation and, within a larger expander-repressor system, to enable gradient scaling. By exploring a wide range of circuits, these experiments could also provide a foundation for the development of synthetic circuits to program multicellular pattern formation within the nascent field of synthetic developmental biology (Zhu, 2023).
Leucine-rich repeats and immunoglobulin-like domains (LRIG) are transmembrane proteins shown to promote bone morphogenetic protein (BMP) signaling in Caenorhabditis elegans, Drosophila melanogaster, and mammals. BMPs comprise a subfamily of the transforming growth factor beta (TGFβ) superfamily, or TGFβ family, of ligands. In mammals, LRIG1 and LRIG3 promote BMP4 signaling. BMP6 signaling, but not BMP9 signaling, is also regulated by LRIG proteins, although the specific contributions of LRIG1 (see Drosophila lambik), LRIG2, and LRIG3 have not been investigated, nor is it known whether other mammalian TGFβ family members are regulated by LRIG proteins. To address these questions, advantage was taken of Lrig-null mouse embryonic fibroblasts (MEFs) with doxycycline-inducible LRIG1, LRIG2, and LRIG3 alleles, which were stimulated with ligands representing all the major TGFβ family subgroups. By analyzing the signal mediators pSmad1/5 and pSmad3, as well as the induction of Id1 expression, it was shown that LRIG1 promoted BMP2, BMP4, and BMP6 signaling and suppressed GDF7 signaling; LRIG2 promoted BMP2 and BMP4 signaling; and LRIG3 promoted BMP2, BMP4, BMP6, and GDF7 signaling. BMP9 and BMP10 signaling was not regulated by individual LRIG proteins, however, it was enhanced in Lrig-null cells. LRIG proteins did not regulate TGFβ1-induced pSmad1/5 signaling, or GDF11- or TGFβ1-induced pSmad3 signaling. Taken together, these results show that some, but not all, TGFβ family ligands are regulated by LRIG proteins and that the three LRIG proteins display differential regulatory effects. LRIG proteins thereby provide regulatory means for the cell to further diversify the signaling outcomes generated by a limited number of TGFβ family ligands and receptors (Abdullah, 2023).
Numerous stages of organismal development rely on the cellular interpretation of gradients of secreted morphogens including members of the Bone Morphogenetic Protein (BMP) family through transmembrane receptors. Early gradients of BMPs drive dorsal/ventral patterning throughout the animal kingdom in both vertebrates and invertebrates. Growing evidence in Drosophila, zebrafish, murine and other systems suggests that BMP ligand heterodimers are the primary BMP signaling ligand, even in systems in which mixtures of BMP homodimers and heterodimers are present. Signaling by heterodimers occurs through a hetero-tetrameric receptor complex comprising of two distinct type one BMP receptors and two type II receptors. This study developed a kinetic model for BMP tetramer formation based on current measurements for binding rates and affinities. This study finds that contrary to a common hypothesis, heterodimer-heterotetramer formation is not kinetically favored over the formation of homodimer-tetramer complexes under physiological conditions of receptor and ligand concentrations and therefore other mechanisms, potentially including differential kinase activities of the formed heterotetramer complexes, must be the cause of heterodimer-heterotetramer signaling primacy. Further, although BMP complex assembly favors homodimer and homomeric complex formation over a wide range of parameters, ignoring these signals and instead relying on the heterodimer improves the range of morphogen interpretation in a broad set of conditions, suggesting a performance advantage for heterodimer signaling in patterning multiple cell types in a gradient (Karim, 2021).
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For maternal genes that establish DV polarity see the dorsal group
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