BIOLOGICAL OVERVIEW

Coming face to face with a highly magnified fruit fly's head can easily be confused with an extraterrestrial confrontation. Huge compound eyes protrude on either side of this alien head [Images]. Between them, a pair of swollen bulbous antenna lift and arch, covered with a multitude of short spines, as well as antler-like aristae and a sensory pouch, the sacculus. These non-human organs sit in a broad area on the frons, or fly's forehead. Lower down the "face," one finds the clypeus, analogous to a nose only because of anthropocentric attempts to cognitively absorb the strangeness of the fly. The clypeus neither breaths nor smells, nor serves as a perch for glasses. It is part of a segment that includes the labrum, a short tube used for sucking food that emerges to hang down from the bottom of the clypeus. On either side of the labrum are two small bulbs, the maxilla or maxillary palps. These organs are greatly reduced in the fruit fly, when compared to those of other insects. Below the maxilla lies an incredible structure, the labella or labial palps. These large, furrowed bulbs lie at the end of the proboscis, the fly's main feeding organ. The palps serve to gather liquid food, which moves up a series of collecting channels into the straw-like labrum, connecting to the muscular esophagus (Ferris, 1950).

Deformed (Dfd) is responsible for the normal development of the maxillary segment, consisting of those tiny bulbs in front of the labium. Deformed has other developmental responsibilities, as shown by three lethal effects associated with Deformed mutation that show up during metamorphosis: 1) failure to separate the subesophageal ganglion (SEG) from the thoracic ganglion; 2) structural and functional abnormalities of the proboscis and maxillary palps, and 3) failure of the adult salivary glands to extend into the thorax. The SEG is considered a ventral portion of the brain and also as part of the central nervous system (Restifo, 1994).

Broad-Complex is another transcription factor acting late in development. Deformed and Broad-Complex interact in the formation of these adult structures during the pupal stage. Deformed defects are in fact identical to those of Broad-Complex, a locus regulating metamorphosis. The homeotic gene labial is likewise important in the transition from pupal to adult structures (Restifo, 1994). This underscores the significance of homeotic genes in the final stages of pupal development, the transition from pupa to adult. Current interests in the analysis of pupal development and the intricacies of the final stages of wing, leg and head development have been largely side-tracked in this era of ground-breaking molecular genetics, but resurgent interest is only a matter of time.

The reduced size of the maxillary segment in adults, due no doubt to the Drosophila specialization of the proboscis for food gathering, is not as apparent in the larval and pupal stages. Important mouth parts are missing at these stages (Regulski, 1987), indicating that evolutionary change is much more conservative in the pre-adult morphology. The role of homeotics in neural differentiation has not yet been adequately explored. Dfd will very likely have a role in subesophageal ganglion differentiation and in the ventral nervous system, topics that have not yet received sufficient attention (Lou, 1995).

The cis-regulatory code of Hox function in Drosophila

Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, binding regions were identified for the Hox TF Deformed (Dfd) in the Drosophila genome. This analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. It was also found that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, these results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo (Sorge, 2012).

In order to quantitatively identify genomic regions bound by the Hox TF Dfd in Drosophila, two complementing approaches were employed: ChIP-seq, which has been successfully applied previously to identify stage- and tissue-specific enhancer activities, and computational detection of clusters of TF binding sequences, which allows the identification of cis-regulatory modules irrespective of temporal and spatial context. To generate genome-wide maps of Dfd binding in vivo, ChIP was performed using stage 10-12 Drosophila embryos and a Dfd-specific antibod. Stage-independent in silico Dfd-specific Hox response elements (HREs) were identified by searching for clusters of conserved Dfd binding motifs, as defined by a position weight matrix (PWM), in the non-coding regions of the genomes of 12 distinct Drosophila species. By applying both approaches, 4526 genomic regions containing clusters of Dfd binding sites and 1079 Dfd ChIP-seq enrichment peaks were identified, including two out of the three well-characterized Dfd-HREs, namely rpr-4S3 and Dfd-EAE. To study the regulatory capacity of novel in silico and ChIP-seq detected HREs, cell culture-based enhancer assays were performed for 11 randomly selected HREs, and it was found that reporter expression driven by the identified genomic regions was in all cases dependent on Dfd binding. In vivo activity was tested of 21 arbitrarily selected enhancers in transgenic reporter lines, revealing that 7 out of 11 ChIP-identified and 5 out of 10 in silico-predicted Dfd-HREs recapitulate the spatio-temporal expression of adjacent genes). Most importantly, it was possible to demonstrate Dfd-dependent regulation of both transgenic reporter expression and endogenous gene expression, suggesting that they are bona fide direct Dfd target genes. Thus, the identified Dfd-HREs represent a data set of biologically relevant regulatory regions and an excellent resource to unravel sequence features within Hox responsive enhancers that might be essential for the highly selective Hox target gene regulation (Sorge, 2012).

Transcriptional regulation in many cases relies on the assembly of regulatory protein complexes mediated by closely spaced TF binding sites within a cis-regulatory module and previous studies have shown that Hox proteins employ this mechanism to control target gene activity in small subsets of cells. The novel HREs were systematically scanned for TF binding motifs appearing in close proximity to Dfd binding sites. Using a statistical test for pair-wise distance distributions, w11 overrepresented DNA motifs for known TFs were found adjoining to Dfd binding sites with 5 of the motifs occurring in both the ChIP-seq and in silico-identified Dfd-HREs. When the expression patterns of six of these transcriptional regulators known to bind to the 11 motifs that were identified were examined, colocalization with Dfd was found in different sub-populations of cells in all cases. Colocalization was already known for two TFs, whose binding sites were coupled to Dfd motifs, including Extradenticle (Exd) , which is known to cooperatively bind with Hox proteins to DNA and thereby increase Hox DNA-binding selectivity. It was next asked whether the short-distance arrangements in Dfd-HREs are of biological relevance and translated into the regulation of similar classes of target genes. To this end, the overrepresentation was statistically tested of expression and biological terms of genes associated with HREs harbouring specific combinations of Dfd and close-by motifs. This analysis revealed that only those Dfd-HREs with short distance intervals between the Dfd and adjacent motifs were coupled to similar gene classes, while random distance intervals did not show any correlation. Strikingly, genes associated with specific short-distance HREs had similar expression and functional annotations as the TFs interacting with the Hox adjoining motifs, suggesting that time and place of Hox action is dictated by spatio-temporally restricted co-regulators. Support for this hypothesis stems from the observation that one of the close-distance partners, Optix, regulates similar processes as Dfd, since Dfd and Optix mutants displayed comparable morphological defects in the head region, such as the absence of mouth hooks, a maxillary segment-derived structure known to be specified by Dfd. In addition, one of the genes associated with a Dfd-Optix HRE, the known Dfd target gene reaper (rpr), is expressed in the ventral epidermis primordium as predicted by its HRE architecture, and regulated by Dfd and Optix in ventral-maxillary cells, which also express these factors. A cell-culture assay using the well-established Dfd responsive module responsible for rpr expression in a few anterior-maxillary cells, the rpr-4S3 Dfd-HRE, with wild-type or mutated Dfd binding sites or reduction of Dfd levels by RNAi confirmed the requirement for simultaneous activity of Dfd and Optix on the rpr-4S3 Dfd-HRE for strong reporter gene induction. Optix binding to the rpr-4S3 Dfd-HRE was additionally confirmed by electrophoretic mobility shift assay (EMSA) experiments. Furthermore, transgenic reporter expression induced by the rpr-4S3 Dfd-HRE was lost in Optix mutant embryos or when the Optix binding sites were mutated. These results demonstrate that Optix, one of the newly identified factors, is a Dfd co-regulator required for proper regulation of the important Hox target gene rpr (Sorge, 2012).

Whether The precise spacing between Hox and adjacent binding sites plays a role for enhancer activity was explored. The rpr-4S3 Dfd HRE, which induces gene expression in a few anterior-maxillary cells, has previously been shown to be under the control of Dfd and Glial cells missing (Gcm), a Dfd co-regulator also identified in this study. Dfd and Gcm as well as Optix binding sites within the rpr-4S3 HRE are directly adjacent to each other, thus a 5- and 10-bp spacer was introduced to interfere with potential interactions of the proteins on the enhancer. In all cases, reporter gene expression was strongly reduced or completely abolished, showing that the close-distance arrangements between Dfd and Gcm as well as Dfd and Optix are required for the in vivo activity of the rpr-4S3 enhancer (Sorge, 2012).

While the results regarding the close-distance arrangement of Dfd and Gcm binding sites suggested the formation of a Dfd-Gcm protein complex, like in the case of Dfd and Exd, only independent binding of the two proteins to the rpr-4S3 enhancer was observed in EMSA experiments , supporting the idea of Hox proteins collaborating with other TFs on target HREs in the absence of physical contact. It has been shown before that Hox proteins together with other TFs that bind in the immediate vicinity recruit non-DNA binding cofactors to HREs. To test if such factors could interact with Dfd and the newly identified short distance binding TFs, the modENCODE data set was scanned and it was found that dCBP/Nej, a member of the CBP/p300 family of transcriptional co-activators bearing acetyltransferase activity, binds to the rpr-4S3 enhancer in vivo. As nej has been previously reported to genetically interact with Dfd, its function was examined in Dfd/Gcm-mediated transcriptional activation. Both factors, Dfd and Gcm, are required for transcriptional activation, since expression of Gcm in Drosophila D.Mel-2 cells, which have basal levels of Dfd activity, resulted in strong induction of reporter gene expression, while abolishing Dfd binding to the rpr-4S3 HRE by mutating all Dfd binding sites or by reducing Dfd protein levels in D.Mel-2 cells using RNAi, strongly reduced reporter gene expression in the presence of Gcm. Strikingly, Dfd- and Gcm-mediated reporter gene expression was strongly reduced in nej dsRNA-treated cells, whereas inhibition of protein deacetylation by Trichostatin A (TSA0) restored reporter gene expression. Consistently, rpr expression was abolished in nej mutant embryos. These results demonstrate that dCBP/Nej-mediated protein acetylation/histone modification is important for the combined activity of Dfd and Gcm on the rpr-4S3 HRE. While it was not possible to demonstrate that nej physically interacts with Dfd protein using various assays, EMSA experiments show that nej interacts with Gcm. Furthermore, acetylation of transiently transfected Gcm was detected in cultured Drosophila cells. Acetylation of Gcm is dependent on Nej, as it was reduced upon RNAi-mediated downregulation of nej. These results are consistent with published work demonstrating that in human cells CBP interacts with Gcma, resulting in its acetylation and stimulation of its transcriptional activity. Since about 10% of all Dfd and nej in vivo genomic binding events during embryonic stages 10-12 overlap, the functional interaction of Dfd and nej observed at the rpr locus does not seem an exception. This finding suggests that the interaction of co-activators (and co-repressors) with Hox proteins and close distance binding TFs on enhancer modules could be a commonly used mechanism to achieve highly specific spatio-temporal control of target gene activity. In this scenario, Hox proteins would control downstream genes by direct transcriptional and/or epigenetic regulation depending on HRE composition and thus cofactor identity and recruitment (Sorge, 2012)

Despite very similar DNA binding behaviour in vitro, Hox proteins regulate distinct morphological features along the anterior-posterior body axis in animal systems. To elucidate the mechanistic basis for the differences in their regulatory properties, Dfd-HREs identified in this study were compared to genomic regions bound by the Hox TF Ultrabithorax (Ubx) at identical developmental stages, as identified by the modENCODE consortium. Searching for overrepresented DNA motifs in both enriched ChIP regions, it was found that Dfd and Ubx bind to identical DNA sequences in vivo, reminiscent to in vitro systems. However, individual binding motifs seem to play only a minor role for Hox binding site selection in vivo, since this analysis revealed that Dfd and Ubx exclusively interact with non-overlapping genomic regions in embryonic stages 9-12. Consequently, Dfd- and Ubx-HREs were found to be associated with distinct classes of genes, revealing that genes with roles in the epidermis are primarily under the control of Dfd at the analysed embryonic stages while genes with mesoderm-related functions are predominantly regulated by Ubx. Consistently, it was found that the expression of tartan (trn), one of the genes associated with a Dfd-HRE, is regulated exclusively by Dfd, but not by Ubx, in epidermal cells, while parcas (pcs), one of the genes linked to a Ubx-HRE is under the selective control of Ubx in mesodermal cells. Furthermore, only Ubx-HREs were found to substantially overlap with cis-regulatory elements stage specifically bound by the mesoderm-specifying TFs Myocyte enhancer factor 2 (Mef2), Twist and Tinman. In contrast, the common ability of both Dfd and Ubx to regulate genes involved in nervous system development was underlined by comparable representations of binding motifs for the neuronal-specifying TFs Asense, Deadpan and Snail in Dfd- and Ubx-HREs (Sorge, 2012).

Strikingly, the basic design principles of Dfd- and Ubx-HREs were found to be similar: like in Dfd-HREs, six binding motifs for known TFs were located adjacent to Ubx binding sites and colocalization studies showed that they are expressed in subsets of Ubx-positive cells. Again, Ubx binding sites and motifs for potential co-regulators occurred most frequently in specific short intervals and only those Ubx-HREs with the preferred distance were associated with specific gene classes. This analysis also revealed that four of the six short-distance motifs were specific for Ubx-HREs, which is consistent with the data showing that Hox proteins interact with different and spatially restricted co-regulators to control target gene expression in selected cells. Importantly, in the cases of the close-distance motifs detected in both HREs, namely the binding sites for the TFs Ladybird early (Lbe) and Cut (Ct), the associated target genes were also expressed in non-overlapping tissues. This raised the question of how different Hox proteins can act on distinct target genes, even when their target HREs exhibit similar binding site compositions including short-distance arrangements. Since Lbe is active in both mesodermal and epidermal cells, one Dfd-Lbe and one Ubx-Lbe HRE was exemplarily analysed, and binding of Lbe protein was confirmed to both HREs by EMSAs. As predicted by the presence of Lbe binding sequences. Complex formation between the Hox protein and Lbe was observed in the case of Ubx and Lbe while Dfd and Lbe interact independently with the Dfd-Lbe HRE, indicating that the two Hox proteins employ different mechanisms for binding to the selected HREs. Lbe interaction with the Dfd-Lbe and Ubx-Lbe HREs is essential for in vivo activity, since in both cases ectopic reporter gene expression was observed when Lbe binding sites were mutated. Even more important, reporter gene expression was specifically changed only in segments in which either Dfd or Ubx is active, meaning in the case of the Dfd-Lbe HRE in maxillary cells and in the case of the Ubx-Lbe HRE in abdominal segments A1-A7. Taken together, these results demonstrate that the combined activity of Lbe and the Hox proteins Dfd or Ubx on selected HREs is critical for the precise spatiotemporal and segment-specific control of HRE activity. It was next asked whether additional (DNA- and non-DNA-binding) factors contribute to the predicted cell type-specific expression of the Dfd-Lbe and Ubx-Lbe HREs. Using the Drosophila Interactions Database (DroID; Murali, 2011) and published genome-wide DNA binding studies a search was carried out for unique Dfd-lbe and Ubx-lbe interactors. It was discovered that almost 20% of all Ubx-Lbe HREs but none of the Dfd-Lbe HREs were found to interact with the mesoderm-specifying factor Mef2 in vivo, while H3K9me3 histone marks, which are mediated by one of the unique Dfd-lbe interactors, Enhancer of zeste E(z), are enriched only within Dfd-Lbe HREs. Interestingly, E(z) modifies chromatin also by trimethylating H3K27 residues, a histone mark highly enriched at the genomic region spanning the ChIP-detected Dfd-Lbe HRE. Consistent with the repressive function of this histone modification, loss of Lbe binding to the Dfd-Lbe HRE results in ectopic reporter gene expression, suggesting that Lbe (and Dfd) recruits E(z) to the Dfd-Lbe HRE for cell type-specific target gene repression (Sorge, 2012).

Taken together, these results demonstrate that Hox proteins interact with different regulatory proteins on HREs, which allows them to differentially regulate their target genes despite their similar DNA binding properties. The fact that these interactions occur only in a few cells for a short period of time is very likely one of the major reasons why the identification of factors conferring regulatory precision and specificity to Hox function has met with little success so far (Sorge, 2012).

This study, has identified crucial features of HREs, which are essential for cell type-specific regulation of Hox target genes in vivo. In addition to motif composition the exact spatial arrangement of TF binding elements is critical to translate Dfd function into transcriptional regulation in vivo. These architectural features of Dfd-HREs alone accurately predict target gene function and expression patterns. Furthermore, it was found that epigenetic regulators bind to HREs on a genome-wide scale, suggesting that they generally collaborate with Hox proteins to achieve stable target gene regulation. This is in line with recent findings showing that chromatin modifications at enhancers strongly correlate with functional enhancer activity and tissue specificity. By comparing HREs regulated by Dfd and Ubx, two different Hox proteins with different embryonic regulatory specificities, this study shows that while similar design principles apply, specificity is encoded by distinct sets of co-occurring DNA motifs. Due to the highly dynamic regulatory output of Hox TFs in space and time, cell type-specific approaches are required in future to elucidate all relevant aspects of Hox-chromatin and Hox-cofactor interactions (Sorge, 2012).

Hox function is required for the development and maintenance of the Drosophila feeding motor unit

Feeding is an evolutionarily conserved and integral behavior that depends on the rhythmic activity of feeding muscles stimulated by specific motoneurons. However, critical molecular determinants underlying the development of the neuromuscular feeding unit are largely unknown. This study identified the Hox transcription factor Deformed (Dfd) as essential for feeding unit formation, from initial specification to the establishment of active synapses, by controlling stage-specific sets of target genes. Importantly, Dfd was found to control the expression of functional components of synapses, such as Ankyrin2-XL, a protein known to be critical for synaptic stability and connectivity. Furthermore, Dfd was uncovered as a potential regulator of synaptic specificity, as it represses expression of the synaptic cell adhesion molecule Connectin (Con). These results demonstrate that Dfd is critical for the establishment and maintenance of the neuromuscular unit required for feeding behavior, which might be shared by other group 4 Hox genes (Friedrich, 2016).

Stereotypical motor behaviors are the primary means by which animals interact with their environment, forming the final output of most CNS activity. One such behavior is feeding, a crucial and highly conserved activity in all animals. The motor output consists of coordinated contractions of distinct head muscles in a rhythmic pattern required for chewing, sucking, and swallowing of food. Food uptake in adult flies has recently been shown to be controlled by a single pair of interneurons emanating from the subesophageal ganglion (SEG), an insect brain region primarily associated with taste and feeding. Although a substantial number of neurons are linked to different aspects of feeding behavior in flies, molecular factors critical for the establishment and development of feeding motor patterns have not been identified so far (Friedrich, 2016).

The fruit fly Drosophila melanogaster is an excellent model to study the developmental aspect of feeding behavior for several reasons. First, Drosophila takes up food extensively during its larval stage, when the organism almost exclusively feeds to increase its body weight and size. Additionally, the anatomical framework and motor patterns critical for larval food uptake are well described. Feeding requires the rhythmic extension and retraction of the head skeleton, the cephalopharyngeal skeleton (CPS), coupled with coordinated elevation and depression of the mouth hooks (MHs), mandible-derived structures required for chopping up solid food, and subsequent food ingestion. The repetitive larval feeding movements are controlled by head muscles innervated by CNS nerves emerging from the SEG. CPS protraction and tilting are mediated by protractor muscles receiving input from the prothoracic nerve, while MH motor patterns are controlled by the mouth hook elevator (MHE) and depressor (MHD), which are innervated by the maxillary nerve. Food ingestion is achieved by the cibarial dilator muscle (CDM), which is connected to the CNS via the antennal nerve. The cellular framework of Drosophila larval feeding is established during embryogenesis. Thus, molecular and genetic approaches can be used to identify and analyze factors controlling specification and communication of cell types critical for larval motor patterns. In contrast, neuromuscular units required for motor activities in adult flies develop from stem cell systems during larval and pupal stages in a process called metamorphosis. Due to the limited accessibility of this transitional phase, embryonic stages are better suited to study the development of neuromuscular units required for regional movements (Friedrich, 2016).

The Hox family of transcription factors (TFs) have emerged as key regulators of motor behaviors. One such behavior is locomotion, which Drosophila larvae perform by region-specific contractions of abdominal segments allowing them to crawl on substrate. Segment-specific changes of peristaltic movements in animals carrying mutations in the Hox genes Ultrabithorax (Ubx) and abdominal-A (abd-A) led to the assumption that Hox genes orchestrate the development of regional motor activities. Recent studies have now revealed that Hox proteins perform their task in a very refined manner and seem to have a direct transcriptional input on successive steps of motoneuronal development. As one such example, Hox5 function was shown to be required in motoneurons that control the contraction of breathing muscles in vertebrates: Hox5 deletion in mice leads to progressive death of phrenic motor column (PMC) neurons as well as to the inability of surviving PMC neurons to innervate the diaphragm muscle. However, despite the fact that blocking motoneuron apoptosis did rescue the decline in PMC neuron number, branching and innervation defects were still unchanged under these conditions. These findings imply that Hox5 proteins directly regulate early and late processes in the course of PMC neuron differentiation, a hypothesis still awaiting confirmation (Friedrich, 2016).

Hox genes are segmentally expressed along the anterior-posterior body axis of animals, suggesting that members of this gene family expressed in the head region should control food uptake. Intriguingly, the Drosophila group 4 Hox gene Deformed (Dfd), which is known to specify the SEG, has already been associated with feeding behavior before: animals carrying a hypomorphic Dfd allele starve to death as adult flies due to the inability to move their proboscis, an action crucial for food ingestion. This work reveals that Dfd, which is expressed in many cell types, including a large number of SEG neurons, is functional in motoneurons and muscles that drive the movements critical for hatching and feeding. Most interestingly, it was shown that Dfd exerts its function via direct and stage-specific regulation of target genes. Importantly, Ank2-XL, a microtubule organizing protein required for synaptic stability, was shown to be under direct Dfd control throughout different stages in the animal's life. Furthermore, synchronous expression of Dfd targets with critical function in synaptic target specificity, in particular, the cell adhesion molecule (CAM) Connectin (Con), was found in feeding neurons and muscles. This suggests that Dfd positively and/or negatively regulates different CAMs providing a specificity code required for the establishment of regional motor units (Friedrich, 2016).

Consistent with a temporal requirement of Dfd for motoneuronal development, an over-representation of neuronal genes was found among those genes associated with ChIP-seq identified Dfd binding regions, which were classified as Dfd target genes. Importantly, grouping of these genes based on similar GO annotations showed that they operate at different time points in neuronal development: during neurogenesis and neuronal specification (32/182), when axon outgrowth and guidance decisions occur (86/182), and during synapse-related processes (85/182). To test the temporal control of these genes by Dfd, their expression was analyzed when Dfd function was abolished at two different developmental stages. For early interference, ^Dfd16 loss-of-function embryos was used, while late interference was achieved using animals that carry the temperature-sensitive ^Dfd3 allele and were shifted to the restrictive temperature only during larval stages. Early neurogenesis target genes, including prospero, a gene involved in asymmetric neuroblast division, was found to be mis-localized in ^Dfd16 null mutant embryos. Consequently the expression of genes required for subsequent processes in motoneuronal development, like the axon guidance genes capricious (caps), roundabout 2 (robo2), roundabout 3 (robo3), and Neural Lazarillo (NLaz), were also affected. Thus, ^Dfd16 null mutants are unable to form the neuromuscular unit required for MH movements due to the inability to activate the proper developmental program. In contrast, the MH-associated motor unit of ^Dfd3 animals shifted to the restrictive temperature during larval stages was intact, with respect to outgrowth of maxillary nerve projecting motoneurons and MHE innervation. Accordingly, the expression of early Dfd neuronal targets was unchanged (data not shown). However, compared to the control group the expression of Dfd target genes critical for synapse-related processes was substantially altered in these late-shifted ^Dfd3 third-instar larvae. This includes Ankyrin2 extra large (Ank2-XL), which is encoded in the ank2 locus. Ank2-XL, which is part of a membrane-associated microtubule-organizing complex, is known to be required for the establishment of appropriate synaptic dimensions and release properties (Stephan, 2015). This study found that not only was Ank2 mRNA levels reduced in SEG neurons in late-shifted ^Dfd3 third-instar larvae, but also observed decreased Ank2-XL protein expression in synaptic boutons, axons and their terminals on the MHE. Similar to a recent report (Stephan, 2015), Futsch/MAP1B, a microtubule-associated protein known to form a membrane-associated complex with Ank2-XL, was also found to be reduced in synaptic boutons of late-shifted ^Dfd3 third-instar larvae. Concomitantly, the morphology of synaptic boutons on this muscle was also changed in late-shifted ^Dfd3 third-instar larvae: they were not of uniform size but appeared often dramatically increased compared to boutons of control animals. This is in line with the described phenotype of ank2-XL mutant animals (Stephan, 2015), which was suggested to reflect the failed separation of neighboring boutons. The effects observed are due to Dfd's action in (moto)neurons, as tissue-specific knockdown of Dfd activity in neuronal cells only using the elav-GAL4 driver in combination with two independent UAS-DfdRNAi lines resulted in severe bouton phenotypes and Ank2-XL expression changes, while the muscle architecture was completely normal. Similar results on Ank2-XL expression and synapse morphology were obtained in Dfd13/Df(3R)Scr third-instar larvae that survived to this stage. The effect of Dfd on synapses on the MHE is specific, since neuromuscular junctions on control muscles, like the CDM, were completely normal with respect to their morphology and Ank2-XL expression in late-shifted ^Dfd3 third-instar animals. These results show that Dfd activity is continuously required during the formation of the feeding motor unit, from its specification to the establishment of synaptic connections, and that Dfd executes this function by directly regulating the transcription of phase-specific components. Intriguingly, these findings demonstrate that the Hox TF Dfd is one of the upstream regulators coordinating Ankyrin-dependent microtubule organization and synapse stability and provides evidence that Dfd function is required even after the initial establishment of the motor unit to control synapse-related processes via its synaptic targets, like Ank2-XL. Finally, the results indicate that synaptic stability and plasticity is not only determined by the half-life of synaptic proteins, but is dependent on a robust transcriptional program that provides a continuous supply of essential synaptic components that maintain the system (Friedrich, 2016).

This analysis has shown that Dfd is expressed in SEG motoneurons. In addition, Dfd was found to be present in embryonic muscles, which later form the feeding/hatching motor unit. Defects were observed in the structure and number of the MH-associated muscles in the embryo when Dfd function was abolished. As was the case in the CNS, Dfd seems to execute its muscle-specific function in an immediate manner, since a substantial fraction (7.4%) of the genome-wide identified Dfd target genes is associated with mesoderm-related functions. The innervation of the Dfd-expressing MHE by Dfd-positive motoneurons raised the intriguing possibility that the activity of the Hox protein Dfd provides a code on the functionally connected neurons and muscles crucial for the recognition and matching of the synaptic partners and, thus, the execution of rhythmic motor patterns. Consistent with this hypothesis, 27 of the ChiP-seq identified Dfd target genes encode factors with described functions in muscles and the nervous system, and importantly nine of these genes play an important role in synaptic target recognition, like tartan (trn), Connectin (Con), or capricious (caps). Therefore, the expression of the homophilic cell adhesion molecule Con was analyzed, and it was found to be exclusively expressed in motoneurons and muscles devoid of Dfd protein in wild-type embryos, suggesting that Dfd might function as a suppressor of Con expression. In order to provide vigorous proof for this hypothesis, cells were specifically labeled that were devoid of Dfd function in Dfd mutants, and their ability to now express Con was analyzed. Use was made of the fact that ^Dfd16 mutants that do not produce any functional protein (protein-null mutants) still express Dfd mRNA. Consistent with this hypothesis, de-repression of Con mRNA expression was found in many Dfd mutant neuronal cells that were labeled by the presence of Dfd mRNA. Due to the inability of Dfd mutant embryos to involute their heads (which reorganizes the order of the head muscles) and due to the high variance of the muscle phenotype, Con expression in this tissue was not shown in the Dfd loss-of-function situation. Taken together, these results demonstrate that Dfd is one of the critical upstream regulators, which coordinates the interdependent events of neuromuscular development and connectivity by positively or negatively regulating the expression of synaptic target selection molecules on the interacting motoneurons and muscles. Furthermore, it shows that the expression of synaptic cues is tightly regulated even in neurons located in close or direct proximity, allowing these cells to express different sets of synaptic recognition molecules thereby ensuring that they make the proper connections with their synaptic partners (Friedrich, 2016).

Hox genes have been shown to control several motor activities along the anterior-posterior axis of animals; however, critical determinants regulating feeding movements have not been previously identified. This study has shown that the Drosophila group 4 Hox gene Dfd controls multiple aspects in both the establishment and maintenance of the neural network controlling feeding behavior (Friedrich, 2016).

A crucial finding from this study is that Hox TFs are required throughout the formation of regional motor units and mediate their effect not only through the induction of downstream TFs. In fact, it was shown that Hox factors control distinct effector target genes, which realize stage-specific processes in a very immediate manner. This is true for Ankyrin2-XL, which, along with the MAP1B homolog Futsch, forms a membrane-associated microtubule-organizing complex that determines axonal diameter, supports axonal transport, and controls synaptic dimensions and stability (Stephan, 2015). Interestingly, it was found that Dfd is required for the maintenance of Ank2-XL expression, not only when the motor system is established but also when it is fully operational. In the light of recent findings showing that mis-regulation of Ankyrin 1 (ANK1) has an important role in the neurodegenerative Alzheimer disease, these results raise the intriguing possibility that Hox genes have a neuro-protective function (Friedrich, 2016).

An important question arising from this study is whether the establishment of feeding-related motor patterns is one of the basic functions of group 4 Hox genes and thus conserved in the animal kingdom. Promisingly, it is known that tongue muscles critical for rhythmic feeding movements in mammals are innervated by the hypoglossal nerve. This nerve has its origin in rhombomere 8, which expresses several group 4 Hox genes, including Hoxb4. Preliminary analysis using a previously identified fish Hoxb4 promoter as a reporter in the teleost fish medaka (Oryzias latipes) shows that GFP is expressed in distinct neuronal subpopulations of the post-otic hindbrain and the spinal cord in stable Hoxb4-GFP medaka embryos. Intriguingly, a subset of neurons co-expressing GFP and Hoxb4 project their axons ventrally toward Hoxb4-positive cells within the pharyngeal region. Both the branchial muscles and the pharyngeal jaw specialized for feeding in teleost fish develop from this area. When medaka embryos have developed into hatchlings, axons emerging from GFP-labeled neurons innervate branchial muscles and the sternohyodeus, muscle groups required for mouth opening and food swallowing. Thus, the regulatory and transcriptional network dictating the formation of the respective feeding units in flies and fish could be conserved despite the fact that muscles and bones responsible for the execution of feeding movements are of different origin. In future, more functional studies are needed to validate the potentially conserved role of homology group 4 Hox genes in regulating rhythmic feeding movements throughout the animal kingdom (Friedrich, 2016).

GENE STRUCTURE

labial is the most proximal member of the homeobox genes of the Antennapedia Complex. It is followed in the distal direction by proboscopedia, zerknüllt, bicoid, Deformed, Sex combs reduced, fushi tarazu and Antennapedia (Kaufman, 1990). Genomic length - 11 kb

cDNA clone length - 1758 bp

Bases in 5' UTR - 491

Exons - five

Bases in 3' UTR - 491

PROTEIN STRUCTURE

Amino Acids - 586

Structural Domains

The DFD protein has two N-terminal glycine rich regions with a histidine rich region between them. There is a central acidic domain, a homeodomain, and a C-terminal polyglutamine and polyasparagine domain (Regulksi, 1987).

See four paralogous Hox clusters of mammals for homologies of Deformed with mammalian Hox proteins.

date revised: 25 September 2023

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