cis-Regulatory Sequences and Functions

IslH, a 7 kb fragment located 6 to 13 kb upstream of the putative transcriptional start site, can direct expression in a pattern similar to that of the ISL protein. Since two or three of the 20 cells per hemisegment that normally express islet cells do not express protein directed by the IslH fragment, additional islet regulatory elements may lie outside of the genomic region of the IslH fragment (Thor, 1997).

Axon pathfinding and target choice are governed by cell type-specific responses to external cues. In the Drosophila embryo, motorneurons with targets in the dorsal muscle field express the homeobox gene even-skipped and this expression is necessary and sufficient to direct motor axons into the dorsal muscle field. Motorneurons projecting to ventral targets express the LIM homeobox gene islet, which is sufficient to direct axons to the ventral muscle field (Thor, 1997). Thus, even-skipped complements the function of islet, and together these two genes constitute a bimodal switch regulating axonal growth and directing motor axons to ventral or dorsal regions of the muscle field (Landgraf, 1999). The LIM homeobox gene islet is sufficient to direct motor axons via the ventral branch of the ISN (ISNb/d) into the ventral muscle field (Thor, 1997). The implications of the findings with respect to Eve are that together eve and islet might constitute a bimodal switch that directs motor axon growth either to ventral (islet) or dorsal (eve) regions of the muscle field. One prediction of such an interpretation would be that the expression patterns of these two genes in motorneurons are mutually exclusive. In the wild type, this is the case. Moreover, while the expression pattern of Eve remains unchanged when Islet is either absent or ectopically expressed, it is found that ectopic Eve expression throughout the CNS suppresses Islet expression in most motorneurons. In the wild type, Islet is expressed medially in the dorsal RP1, 3, and 4 neurons and one ventral VUM motorneuron, and laterally, in approximately four to five motorneurons. In stage 16 elav-GAL4; UAS-eve embryos, the Islet expression pattern is markedly reduced: medially, Islet expression is consistently lost from the VUM and from two of the three RP motorneurons; laterally, Islet expression is lost from a further four to six cells. However, the Islet expression pattern does not expand when Eve function is removed (Landgraf, 1999).

How do transcriptional regulators such as Eve and Islet direct patterns of axonal growth? The phenotype observed in ectopic Eve embryos (fusion of the main nerve trunks and failure of secondary nerve branching) is similar to, though more severe than, phenotypes produced in embryos where general interaxonal adhesion is increased either by overexpression of the homophilic CAM Fas II or by removal of its antagonist Beaten path (Beat). In such embryos, the two main nerve trunks (SN and ISN) form, but secondary nerves fail to branch off. A test was carried out to see if ectopic Eve increases interaxonal adhesion by downregulating the antiadhesive neural CAM antagonist Beat. No significant changes were seen in the overall pattern or relative levels of BEAT mRNA expression in ectopic Eve embryos. The expression patterns of the major neural CAMs Fas II, Fas III, and Connectin were examined in ectopic Eve or eve mutant embryos, but no changes in their expression patterns were detected. To test if Eve might regulate the expression of another (as yet unidentified) neural CAM, it was reasoned that beat might antagonize interaxonal adhesion mediated by such a CAM, just as beat antagonizes adhesion mediated by Fas II and Connectin. When Eve and beat are ectopically co-expressed, the ectopic Eve phenotype of excessive axonal fasciculation is partially rescued. Thus, Eve directs motor axons to the dorsal region of the muscle field by suppressing expression of the ventrally directing islet gene and by promoting adhesion to the ISN (Landgraf, 1999).

There is an interesting correlation between the expression of islet homologs in vertebrate and invertebrate motorneurons. However, while all vertebrate motorneurons express islet-1 and/or islet-2, only a subset of motorneurons express islet in Drosophila (Thor, 1997). Another subset expresses eve, and there may well be further subsets expressing other genes that direct axons to different parts of the muscle field. For instance, the dorsolateral muscles DO3-5 [11, 19, and 20] and DT1 [18] are innervated by at least four intersegmental motorneurons that express neither eve nor islet. Thus, there may be a third gene that defines the dorsolateral sector of the muscle field as the target area of these motorneurons. Interestingly though, the axonal projections of the DO3-5 [11, 19, and 20] and DT1 [18] motorneurons are frequently affected by loss of Eve function. This suggests that these motorneurons rely on the axons of the Eve-expressing cells for pathfinding. In addition, motorneurons whose axons project through the SN express neither eve nor islet, and their growth patterns are likely to be regulated by other genes. Interestingly, the gene vab-7 in the nematode Caenorhabditis elegans (a homolog of the Drosophila eve gene) is also expressed in a set of motorneurons that go to dorsal targets, and it is required for their correct pathfinding (B. Esmaeili and J. Ahringer, personal communication to Landgraf, 1999). Thus, it appears that the function of eve in directing patterns of motorneuron growth is an ancient one (Landgraf, 1999).

An Org-1-Tup transcriptional cascade reveals different types of alary muscles connecting internal organs in Drosophila

The T-box transcription factor Tbx1 and the LIM-homeodomain transcription factor Islet1 are key components in regulatory circuits that generate myogenic and cardiogenic lineage diversity in chordates. This study shows that Optomotor-blind-related-gene-1 (Org-1) and Tup, the Drosophila orthologs of Tbx1 and Islet1, are co-expressed and required for formation of the heart-associated alary muscles (AMs) in the abdomen. The same holds true for lineage-related muscles in the thorax that have not been described previously, which were named thoracic alary-related muscles (TARMs). Lineage analyses identified the progenitor cell for each AM and TARM. Three-dimensional high-resolution analyses indicate that AMs and TARMs connect the exoskeleton to the aorta/heart and to different regions of the midgut, respectively, and surround-specific tracheal branches, pointing to an architectural role in the internal anatomy of the larva. Org-1 controls tup expression in the AM/TARM lineage by direct binding to two regulatory sites within an AM/TARM-specific cis-regulatory module, tupAME. The contributions of Org-1 and Tup to the specification of Drosophila AMs and TARMs provide new insights into the transcriptional control of Drosophila larval muscle diversification and highlight new parallels with gene regulatory networks involved in the specification of cardiopharyngeal mesodermal derivatives in chordates (Boukhatmi, 2014).

The anatomical organization of the Drosophila larval organs is established during embryogenesis. This study show shere that abdominal AMs and their thoracic counterpart, the TARMs, establish physical connections between the exoskeleton and different internal organs, and that TARMs thus represent a novel type of muscle. The control of AM and TARM development by Org-1 and Tup - as orthologs of Tbx1 and Islet1, two key transcription factors of cardiopharyngeal mesoderm development in vertebrates - suggests that these transcription factors were part of an ancestral regulatory kernel that was redeployed several times during evolution to control different embryonic mesoderm derivatives (Boukhatmi, 2014).

Seven pairs of embryonic abdominal AMs connect to the heart and the aorta. Interestingly, pan-mesodermal expression of either Ubx or AbdA, two abdominal Hox proteins, resulted in the formation of three supernumerary pairs of AMs arranged in a segmental pattern in the three thoracic segments. This suggested the existence of thoracic PCs that fail to give rise to muscles in the absence of proper Hox input. Such a scenario accounted for the lack of DA3 muscle in the T1 segment. Alternatively, these thoracic PCs could give rise to previously unrecorded muscles. Both explanations were found to apply. Thoracic Org-1+/Tup+ PCs give rise to a novel type of muscles, which were call TARMs. Only TARMT1 and TARMT2 form during normal development, whereas TARMT3 differentiation is abortive. An extra pair of muscles expressing both org and tup reporter genes in late embryos attaches to the proventriculus. The PCs at the origin of this extra pair of muscles, which were named TARM*, remain to be identified (Boukhatmi, 2014).

In summary, TARMs and AMs are generated by homologous lineages and form thin, elongated muscles of characteristic morphology and attachment sites that integrate segment-specific information (Boukhatmi, 2014).

Descriptions of AMs in adults of different insects led to proposals of their role in either maintaining the position of the heart, regulating the hemolymph flow and/or controlling heart beat. The requirement of transient contacts between some AMs and the distal tip cell of MTs revealed yet another role of AMs, namely for proper bending and positioning of MTs. Through linking the lateral epidermis to the dorsal vessel, AMs press the dorsal, main branch of the trachea towards the body wall. TARMs also establish intimate topological relations with cephalic branches of the trachea in their trajectory from thoracic lateral epidermis/exoskeleton to specific regions of the midgut. Rotation of the gut at the end of embryogenesis results in stretching and bilateral asymmetry of the left and right TARMs. This suggests that TARMs have elastic properties, as already indicated by their transient deformation upon interaction with the MT tip cell (Boukhatmi, 2014).

Although the function of AMs and TARMs during larval development and growth remains to be fully assessed, the morphological and elastic properties of AMs and TARMs suggest that they could be involved in controlling the position of the heart, trachea and gut during larval foraging movements. Whereas Org-1 and Tup are only co-expressed in the AM/TARM lineage, each is expressed in several other mesodermal or non-mesodermal tissues, precluding the use of mutants to selectively study AM and TARM function. Whether AMs and TARMs represent a new type of muscle with both spring-like and contractile properties is one of the questions to be addressed henceforth (Boukhatmi, 2014).

The morphological properties specific to each Drosophila body wall muscle are determined by the expression of specific combinations of iTFs in each FC and derived muscle. Lineage specificity involves positive and negative cross-regulations between different iTFs, as well as iTF autoregulation. The maintenance of Org-1 and Tup expression in body wall muscles depends upon autoregulation (Boukhatmi, 2012; Schaub, 2012). This study shows that tup autoregulation in dorsal muscles is direct, but does not operate in AMs; here, tup is directly regulated by Org-1. Org-1 directly regulates the expression of two other iTFs, namely slouch and ladybird, in other muscle lineages (Schaub, 2012). The AM/TARM Org-1>Tup hierarchy further underlines the intricate, combinatorial nature of transcriptional regulatory networks specifying Drosophila muscle identity. This study has now identified an Org-1-dependent tup enhancer called tupAME, which is only active in AMs/TARMs and ectodermal cells connecting the frontal sac and pharynx. This CRM should provide a means to specifically target expression and modify AMs/TARMs in order to assess their function in larvae (Boukhatmi, 2014).

Nkx2.5, Tbx1 and Islet1 are major actors in the vertebrate genetic program controlling early heart and pharyngeal muscle development from common progenitors lying in the second heart field (SHF) in chordates. NK4/Nkx2.5 was recently shown to antagonize Tbx1 and repress EBF/COE function to promote cardiac versus pharyngeal muscle fate in the ascidian SHF, with Islet1 being expressed in both derivatives. In the Drosophila embryo, the orthologs of Nkx2.5 and Islet1 are required for the formation of all (in the case of Tin) or some (in the case of Tup) of the dorsal mesodermal derivatives (heart, dorsal body wall muscles, visceral muscles, lymph gland). Moreover, Tup represses the COE ortholog Collier (Col; Knot - FlyBase) in dorsal muscles, and Org-1/Tbx1 is expressed in some body wall muscles, the AMs and the visceral mesoderm, although not in myocardial progenitors. Together, these findings suggest that the Nkx2.5/Tin, Tbx1/Org-1, Islet1/Tup and COE/Col transcription factors are part of an ancestral regulatory kernel controlling diversification of heart and muscle lineages from a common progenitor pool. Future studies will be required to establish which ancestral interactions have been redirected to foster the emergence of insect AMs and TARMs (Boukhatmi, 2014).

No striated muscle has been described so far to connect the (exo)skeleton to the gut, either in insects or in vertebrates. In mammals, lung and heart are separated from visceral organs by the diaphragm muscle. However, a muscular diaphragm is a defining characteristic of mammals that is not found in other vertebrates, and the ancestral origin of this recent innovation is currently unknown. Although highly speculative, it will be interesting to investigate whether the vertebrate muscle/septum and the insect AMs/TARMs could represent two specific adaptations of an ancestral demarcation between the circulatory systems, respiratory systems and visceral organs (Boukhatmi, 2014).

Transcriptional Regulation

The dorsal ectoderm of the Drosophila embryo is subdivided into different cell types by an activity gradient of two TGFbeta signaling molecules, Decapentaplegic and Screw. Patterning responses to this gradient depend on a secreted inhibitor, Short gastrulation and a newly identified transcriptional repressor, Brinker, which are expressed in neurogenic regions that abut the dorsal ectoderm. The expression of a number of Dpp target genes has been examined in transgenic embryos that contain ectopic stripes of Dpp, Sog and Brk expression. These studies suggest that the Dpp/Scw activity gradient directly specifies at least three distinct thresholds of gene expression in the dorsal ectoderm of gastrulating embryos. Brk was found to repress two target genes, tailup/islet and pannier, that exhibit different limits of expression within the dorsal ectoderm. These results suggest that the Sog inhibitor and Brk repressor work in concert to establish sharp dorsolateral limits of gene expression. Evidence is provided that the activation of Dpp/Scw target genes depends on the Drosophila homolog of the CBP histone acetyltransferase (Ashe, 2000).

All of the aforementioned genes are virtually silent in the dorsal ectoderm of dpp-/dpp- embryos, while changes in dpp+ gene dose cause altered patterns of expression. For example, increasing the number of dpp+ copies from two to three to four results in a sequential expansion of the hnt expression pattern, whereas expression is lost in dpp/+ heterozygotes. In contrast, ush is expressed in dpp/+ heterozygotes, although there is a marked narrowing in the expression pattern as compared with wild-type embryos. Three copies of dpp+ cause an expansion of the ush pattern. Similarly, the tup expression pattern is narrower in dpp/+ heterozygotes and expanded in embryos with three copies of dpp. Further evidence that hnt and ush are early targets of the Dpp signaling pathway was obtained by analyzing transgenic embryos that contain the dpp-coding sequence attached to the eve stripe 2 enhancer. These embryos exhibit both the endogenous dpp pattern in the dorsal ectoderm as well as an ectopic stripe of expression (Ashe, 2000).

Additional Dpp/Scw target genes were examined for repression by the stripe2-brk transgene. Those showing altered patterns of expression include tup, rho, hnt and Race. The normal tup expression pattern encompasses both the presumptive amnioserosa and dorsal regions of the dorsal epidermis. In transgenic embryos, there is a gap in the pattern in regions where the stripe2-brk fusion gene is expressed. These results suggest that Brk represses tup, even though it appears to respond to a different threshold of Dpp/Scw signaling than pnr. Additional experiments were done to determine whether Brk directly represses tup expression, or works indirectly by inhibiting Dpp signaling (Ashe, 2000).

To examine the relative contributions of the Sog inhibitor and the Brk repressor in establishing different thresholds of Dpp/Scw signaling activity, target genes were analyzed in gastrulation defective (gd) mutants that express either a stripe2-sog or stripe2-brk transgene. Mutant embryos collected from gd-/gd - females lack a Dl nuclear gradient and therefore lack ventral tissues such as the mesoderm and neurogenic ectoderm. All tissues along the dorsoventral axis form derivatives of the dorsal ectoderm, mainly dorsal epidermis. Hereafter, such embryos are referred to as gd-. These mutants lack endogenous sog and brk products, so that the stripe2 transgenes represent the only source of expression. Although the stripe2-sog transgene inhibits Dpp signaling, it does not cause activation of brk. The pnr and tup expression patterns are derepressed in gd- mutants, and exhibit uniform staining in both dorsal and ventral regions. These expanded patterns correlate with the expanded expression of dpp, which is normally repressed in ventral and lateral regions by the Dl gradient. As seen in wild-type embryos, the stripe2-brk transgene represses the anterior portion of the pnr expression pattern. In contrast, the stripe2-sog transgene has virtually no effect on the pattern. These observations suggest that Brk is the key determinant in establishing the lateral limits of the pnr pattern at the boundary between the dorsal ectoderm and neurogenic ectoderm. The failure of stripe2-sog to inhibit pnr expression might be due to redundancy in the action of the Dpp and Scw ligands. Perhaps either Scw alone or just one copy of dpp+ is sufficient to activate pnr. This would be consistent with the observation that the initial pnr expression pattern is essentially normal in dpp-/dpp- and scw-/scw- mutant embryos (Ashe, 2000).

The limits of the tup expression pattern seem to depend on both Sog and Brk. The introduction of the stripe2-brk transgene leads to a clear gap in the tup expression pattern, although there is a narrow stripe of repression in gd- mutants lacking the transgene. The stripe2-sog transgene causes a more extensive gap in the tup pattern. The stripe2-brk transgene was also found to repress Race, hnt and rho in this assay (Ashe, 2000).

In principle, the gap in the tup pattern caused by the stripe2-brk transgene could be indirect, and caused by the repression of dpp. Previous studies have shown that the early dpp expression pattern expands into the ventral ectoderm in brk- mutant embryos. To investigate this possibility, tup expression was monitored in brk- embryos, and in wild-type embryos carrying both the stripe2-brk and stripe2-dpp transgenes. The tup expression pattern exhibits a transient expansion in brk- mutant embryos. However, this expansion is only seen in early embryos, prior to the completion of cellularization. By the onset of gastrulation, the pattern is essentially normal. The stripe2-brk transgene creates an early gap in the normal dpp expression pattern in wild-type embryos. This observation raises the possibility that the repression of tup and rho is indirectly mediated by the inhibition of Dpp signaling. To test this, the tup pattern was examined in embryos carrying both the stripe2-brk and stripe2-dpp transgenes. As expected, the stripe2-dpp transgene alone causes a local expansion of the tup pattern in the vicinity of the stripe 2 pattern. However, the simultaneous expression of both stripe2-dpp and stripe2-brk leads to a clear gap in the tup expression pattern. Thus, it would appear that Brk can repress tup even in regions containing high levels of Dpp signaling. Similar assays suggest that Race, hnt and rho are not directly repressed by Brk (Ashe, 2000).

A summary is presented of Dpp signaling thresholds in the embryo. The Dpp/Scw activity gradient presumably leads to a broad nuclear gradient of Mad and Medea across the dorsal ectoderm of early embryos. It is conceivable that the early lateral stripes of brk expression lead to the formation of an opposing Brk repressor gradient through the limited diffusion of the protein in the precellular embryo. Peak levels of Dpp and Scw activity lead to the activation of Race and hnt at the dorsal midline. The tup and ush patterns represent another threshold of gene activity. The similar patterns might involve different mechanisms of Dpp signaling since tup is repressed by Brk, whereas ush is not. Finally, the broad pnr pattern represents another threshold of gene activity. It is not inhibited by Sog but is repressed by Brk. It is possible that tup and pnr are differentially repressed by a Brk gradient. Low levels of Brk might be sufficient to direct the lateral limits of the tup pattern, whereas high levels may be required to repress pnr (Ashe, 2000).

Targets of Activity

The Drosophila LIM-homeodomain protein Islet acts at the dorsocentral enhancer of ac/sc to antagonize proneural cell specification in the peripheral nervous system

The pattern of the external sensory organs (SO) in Drosophila depends on the activity of the basic helix-loop-helix (bHLH) transcriptional activators Achaete/Scute (Ac/Sc) that are expressed in clusters of cells (proneural clusters) and provide the cells with the potential to develop a neural fate. In the mesothorax, the GATA1 transcription factor Pannier (Pnr), together with its cofactor Chip, activates ac/sc genes directly through binding to the dorsocentral enhancer (DC) of ac/sc. The LIM-homeodomain (LIM-HD) transcription factor Islet (Isl) was identified by genetic screening and its role in the thoracic prepatterning was investigated. isl loss-of-function mutations result in expanded Ac expression in DC and scutellar (SC) proneural clusters and formation of ectopic sensory organs. Overexpression of Isl decreases proneural expression and suppresses bristle development. Moreover, Isl is coexpressed with Pnr in the posterior region of the mesothorax. In the DC proneural cluster, Isl antagonizes Pnr activity both by dimerization with the DNA-binding domain of Pnr and via competitive inhibition of the Chip-bHLH interaction. It is proposed that sensory organ prepatterning relies on the antagonistic activity of individual Chip-binding factors. The differential affinities of these binding-factors and their precise stoichiometry are crucial in specifying prepatterns within the different proneural clusters (Biryukova, 2005).

During Drosophila development, the expression of transcription factors divides the dorsal thorax into three domains -- one median and two lateral domains. The lateral domains are specified by the homeobox-containing proteins of the iroquois-complex (iro), whereas the GATA factor Pnr is required to establish the median domain. Within the mesothorax, Pnr together with U-shaped (Ush) and Chip plays a key role in dorsal closure. This report presents evidence that Isl is an essential regulator of the dorso-median patterning of the thorax. isl clones generated adjacent to the thoracic midline, induce a strong cleft, suggesting that Isl is required for proper dorsal closure during metamorphosis. Ectopic expression of Pnr leads to wing-to-thorax transformations, consistent with its role as medio-dorsal patterning factor. Ectopic Isl expression does not exhibit this phenotype, excluding the LIM-HD factor from a direct function as a prothoracic selector. Pnr is also known to activate wingless (wg) in dorsal thorax. isl loss-of-function has no significant effect on wg expression. However, overexpressed Isl strongly reduces the size of the wg thoracic stripe. This result is consistent with a repressive activity of Isl on Pnr (Biryukova, 2005).

Iro proteins and Pnr are direct activators of the proneural genes in their respective domains. Pnr binds directly to the DC enhancer of ac/sc, providing therefore region-specific control of the proneural prepatterning. Flies with reduced or lack of Pnr function fail to activate ac/sc and to develop DC and SC sensory organs. The proneural activity of Pnr is antagonized by Ush, the vertebrate homologue of the FOG (friend of GATA). Ush is expressed only in the dorsal-most cells of the medial region. As a consequence, the segregation of the sensory organ precursors occurs along two stripes at the border of the medial domain of Pnr expression, where Ush is absent or insufficient to repress Pnr (Biryukova, 2005).

Several lines of evidence indicate that Isl interferes with the proneural activity of Pnr as a repressor. (1) isl loss-of-function mutants show an opposite phenotype with regard to Pnr or Chip loss-of-function mutants: an excess of DC and SC sensory organs. (2) A genetic synergism exists between PnrD and isl alleles. This genetic interaction is less sensitive than that between PnrD and ush, implying an alternative route for Isl to modulate the Pnr proneural activity. (3) Isl is coexpressed with Pnr within the posterior mesothorax. (4) Isl modulates the activity of a DC:ac-lacZ reporter. Loss-of-function isl mutants expand the DC:ac-lacZ expression as in ush or PnrD constitutive mutants, whereas overexpressed Isl reduces the DC:ac-lacZ expression (Biryukova, 2005).

In the DC region, the regulation of Pnr concentration is critical for the proper position and shape of the DC proneural cluster. Isl expression overlaps with the dorsal-most domain of Pnr and DC proneural activity coincides with the posterior border of Isl expression. Therefore, it proposed that both Isl and Ush restrict Pnr activity in the mesothorax. Interestingly, the regulation of the concentration of the mammalian Pnr ortholog, GATA-1, is similarly critical for proper erythroid, megakaryocytic, eosinophilic and mast cell lineages (Biryukova, 2005).

Ush behaves as either an activator or a repressor of Pnr, depending on developmental context. No evidence was found for a direct Isl-Ush interaction by GST pull down assay: Ush, Pnr and Isl could be co-immunoprecipitated from transient transfected S2 cells. Both Ush and Isl may behave as positive cofactors of Pnr for nonneural activities, such as cardiac development, embryonic dorsal closure and metamorphosis. Several reports emphasize the role of the Pnr homolog, GATA-1 and Isl1 in human blood disorders. It seems likely that GATA:Islet interactions represent a conserved mechanism to specify different cell fates in humans and other organisms (Biryukova, 2005).

Isl proteins are known as positive regulators of transcription in vertebrates. In flies, Isl mediates repression of Pnr-driven proneural activity via binding to the DNA-binding domain of Pnr. Interestingly, these interactions are less specific than for the Pnr-Ush interaction, where the amino-terminal zinc finger of Pnr is specifically involved (Biryukova, 2005).

Genetic analyses of mutants reveal that the DC and the SC proneural clusters show differential sensitivities during neurogenesis. Ush mutants display ectopic DC bristles and a few additional SC bristles. This phenotype is similar to PnrD constitutive mutants, in which Pnr-Ush interactions are greatly reduced. In contrast, isl mutants show the opposite phenotype, with a large excess of SC bristles and a few additional DC bristles. The ChipE mutant exhibits antagonistic phenotypes: lack of DC bristles, reflecting Pnr loss-of-function and an excess of SC bristles, reflecting Isl loss-of-function. The differential topography of DC and SC enhancer binding sites presumably underlies differential transcription-complex binding affinities (Biryukova, 2005).

Chip is the ortholog of Ldb factors that are ubiquitous multiadaptor proteins in vertebrates. Each Ldb-dependent developmental event is specified by modification of the transcriptional complex and is dependent on the stoichiometry of the region-specific Ldb partners. During normal development of the thorax, different partners of Chip (i.e., Isl, Ap and Pnr) are expressed in the same region. The ChipE mutant is highly sensitive to the dosage of these factors. In ChipE flies, removing one copy of either Pnr or Isl causes pupal lethality associated with extreme morphogenetic phenotypes. Removing one copy of Ap, however, rescues the Pnr-dependent phenotypes of ChipE flies. Taken together, these results indicate selective competition between the different partners of Chip, suggesting that hierarchical protein interactions depending on differential affinities and the strict stoichiometry of Chip and its partners, are critical to establish proper transcriptional codes within different proneural fields (Biryukova, 2005).

isl mutants were isolated in genetic screens for dominant enhancers of the ChipE phenotype. This study demonstrates that the LIM-HD transcription factor Isl can bind to the LID of Chip. The binding of the LID domain of Chip with LIM domains has been conserved throughout evolution as has Chip binding with bHLHs proteins. LID contains two subdomains: a small N-terminal hydrophobic β patch (VMVV) followed by a large α helix. ChipE mutation has a single substitution that changes an Arg to Trp (R504W) in the middle of the α helix. This residue is highly conserved among species and mediates high-affinity contact with the LIM domains. Interestingly, the R504W substitution in Chip abolishes, or strongly reduces, both interactions with the bHLHs and also interactions with Isl. This result implies that bHLHs and Isl recognize the same site within the LID domain of Chip. The data argue that competition between bHLHs and Isl for the LID domain of Chip may be critical for modulating the activity of transcription complexes during development. In vertebrates, the NLI homolog of Chip mediates direct coupling of the proneural bHLH factors Ngn2, NeuroM and the LIM-HD transcription factors (Isl1 and Lhx3). This interaction leads to transcriptional synergism and the synchronization of motor neuron subtype specification with neurogenesis in the embryonic spinal cord of chicken. This work demonstrates that Isl is able to interfere with proneural activity of Chip-Pnr-bHLH transcription complex and therefore, Isl is thought to be able to antagonize proneural specification (Biryukova, 2005).

Interestingly, the ChipE mutation has little or no effect on interactions with other LIM-containing factors, such as Ap and dLMO, suggesting that different factors have different affinities with the Chip LID domain. Therefore, the ChipE mutation changes the hierarchy of the affinities among the different partners of Chip in the mesothorax (Biryukova, 2005).

A transcription-complex 'cassette' model is proposed for the specification of region-specific patterns of specialized cell types. In this model, the presence of one of a number of alternative binding factors modifies the specificity of a core transcription complex. This model makes the prediction that, while the core components of the transcription complex will be strongly conserved in evolution, the specificity cassette components will vary significantly between species showing divergent morphogenetic patterns. Comparison of these variable components in related species should provide insights into the fundamental mechanisms of encoding the pattern of differentiated cell types within morphogenetic fields (Biryukova, 2005).

Dorsal vessel morphogenesis in Drosophila melanogaster serves as a superb system with which to study the cellular and genetic bases of heart tube formation. A cardioblast-expressed Toll-GFP transgene was used to screen for additional genes involved in heart development and tailup was identified as a locus essential for normal dorsal vessel formation. tailup, related to vertebrate islet1, encodes a LIM homeodomain transcription factor expressed in all cardioblasts and pericardial cells of the heart tube as well as in associated lymph gland hematopoietic organs and alary muscles that attach the dorsal vessel to the epidermis. A transcriptional enhancer regulating expression in these four cell types was identified and used as a tailup-GFP transgene with additional markers to characterize dorsal vessel defects resulting from gene mutations. Two reproducible phenotypes were observed in mutant embryos: hypoplastic heart tubes with misaligned cardioblasts and the absence of most lymph gland and pericardial cells. Conversely, a significant expansion of the lymph glands and abnormal morphology of the heart were observed when tailup was overexpressed in the mesoderm. Tailup was shown to bind to two DNA recognition sequences in the dorsal vessel enhancer of the Hand basic helix-loop-helix transcription factor gene, with one site proven to be essential for the lymph gland, pericardial cell, and Svp/Doc cardioblast expression of Hand. Together, these results establish Tailup as being a critical new transcription factor in dorsal vessel morphogenesis and lymph gland formation and place this regulator directly upstream of Hand in these developmental processes (Tao, 2007).

Thus, Tup is a newly discovered player in the regulatory network controlling dorsal vessel morphogenesis and hematopoietic organ formation. Tup is expressed in all cardioblast and pericardial cells of the heart tube, prohemocytes of the lymph glands, and alary muscles needed to secure the dorsal vessel to the epidermis. Phenotypic studies demonstrate a requirement for tup function in three of these cells types. tup mutant embryos exhibit a hypoplastic dorsal vessel, with a variable number of cardioblasts that fail to organize into a heart tube structure. It appears that correct numbers of cardioblasts are not specified in mutant embryos, since gaps were observed in the bilateral cardioblast rows early in the process of dorsal vessel formation. Missing cardioblasts included cells of both the Tin- and Svp/Doc-positive subclasses. The late cardioblast misalignment phenotype is likely due to the dorsal closure and germ band retraction defects known to occur in tup embryos (Tao, 2007).

While the degree of cardioblast hypoplasia is variable in mutant embryos, the severe reduction in prohemocytes of the lymph glands and pericardial cells surrounding the contractile tube is fully penetrant. The Collier (Col) protein serves as an excellent marker for lymph gland primordia and the posterior signaling centers of lymph glands associated with the mature dorsal vessel. Since Col expression is normal in tup mutants, Tup function is not required for the early specification of lymph gland primordia within the dorsal mesoderm. However, the severe reduction of several mature lymph gland markers such as tup-GFP, Hand-GFP, Srp, and Odd suggests that either prohemocytes are present within lymph glands with Tup activity essential for expression of all four of these indicator genes or the cells are absent due to defects in prohemocyte proliferation and/or programmed cell death. The latter is an attractive possibility since Hand knockout embryos show ectopic apoptosis among lymph gland progenitor cells (Tao, 2007).

A function for the Hand basic helix-loop-helix transcription factor has been reported for cardioblast, pericardial, and lymph gland cells. This is the same set of dorsal vessel and hematopoietic cells that require Tup function. Through analysis of the Hand cardiac and hematopoietic enhancer, Tup was demonstrated to be a direct transcriptional regulator of Hand in these cell types. Specifically, mutation of the single Tup-2 element in the Hand cardiac and hematopoietic regulatory module resulted in a dramatic loss or reduction of Hand enhancer activity in prohemocytes, pericardial cells, and the Svp/Doc cardioblast subtype. These findings invoke two possibilities. (1) tup phenotypes may be due to the lack of Hand expression and function in cardioblasts, pericardial cells, and lymph gland progenitors. However, Tup function is likely to be even more critical for cardiogenic and hematopoietic events; forced expression of tup results in the production of excess prohemocytes, while the ectopic expression of Hand does not. Thus, Tup can be considered to be a seminal upstream regulator of genetic and cellular events controlling lymph gland formation. (2) Tin and GATA factors have been shown to regulate the Hand cardiac and hematopoietic enhancer. Thus, it is possible the Hand cardiac and hematopoietic transcription occurs due to combinatorial control, specifically via Tup and Doc cofunction in Svp/Doc-expressing cardioblasts and Tup and Srp coactivity in lymph gland progenitors. Ample evidence exists for the function of multiple interacting transcription factors in the regulation of heart and blood cell gene expression in Drosophila. To summarize regulatory aspects of its function, the data showing that Tup is a direct transcriptional activator of Hand expression in lymph glands, pericardial cells, and Svp/Doc-positive cardioblasts through the HCH enhancer module are compelling. Likewise, Tup serves as either a direct or indirect regulator of srp expression in lymph gland cells and odd expression in lymph gland and pericardial cells (Tao, 2007).



islet transcripts are first detectable at embryonic stage 10 in precursors of the heart and aorta (the dorsal vessel), the pharynx (gut ectoderm), and the amnioserosa. By stage 16, this expression is restricted to the heart and aorta, plus the alary and pharyngeal muscles, similar to islet-1 expression in the developing vertebrate pharynx, heart and aorta (Korzh, 1993). Expression in the Drosophila CNS commences at the beginning of germ band retraction (stage 12) in subsets of cells in the ventral cord and brain. ISL transcripts are not present in neuroblasts; based on the dorsal location of the expressing cells within the ventral nerve cord, isl expression appears to be confined to neuronal progeny. Slightly later in development, a second wave of newly expressing islet cells occurs in the ventral cord, such that by stage 13, there are 20 islet-expressing cells per hemisegment (Thor, 1997).

islet expression appears to be restricted to postmitotic neurons. isl expressing cells constitute a subset of motor neurons and interneurons. All of the islet motor neurons innervate muscles located ventrally in the body wall. The majority of the islet motor neurons exit the ventral cord in the segmental nerve branches b and d (SNb and d). Based on their medial position within the ventral cord and their pattern of terminal arborization over ventral muscles 6, 7, and 13, three of the islet motor neurons can be identified as RP1, RP3 and RP4. The remaining SN islet motor neurons lie in more lateral positions within the ventral cord, projecting in SNd to innervate ventral muscles 15, 16 and 17 or in SNb along with the RPs to muscles 6, 7, 12, 14 and 30 (Thor, 1997).

Two additional islet-expressing motor neurons exit the ventral cord in the transverse nerve (TN). The TN is prefigured by two exit glia that lie at the dorsal midline of the ventral cord and extend long processes out to the periphery. The two islet TN motor neurons leave the ventral cord at the dorsal midline and project ipsilaterally along the exit glia. One is a motor neuron innervating ventral muscle 25 (TMN25), while the other (TMNp) contacts the ventral process of the lateral bipolar dendrite neuron (LBD) which in turn innervates the alary muscles attached to the heat and aorta. It is probable that the TMNp directly synapses onto the peripheral LBD neuron, analogous to vertebrate sympathetic preganglionic motor neurons that synapse onto postganglionic neurons lying outside the spinal cord (Thor, 1997).

islet interneurons belong to several different classes based on their morphology. Class I and II interneurons project either ipsi- or contralaterally and extend axons within the connectives, forming two discrete fascicles within the longitudinal connectives. A third class is composed of local interneurons that project across the midline and terminate contralaterally within the same segment. apterous is expressed in a small subset of ipsilaterally projecting interneurons that form a single fascicle in the connective, similar to the Class I and II islet interneurons (Lundgren, 1995). AP and ISL are expressed in nonoverlapping sets of neurons. In addition, both apterous and islet expressing neuronal subsets also project axons along different pathways (Thor, 1997).

Intrinsic control of precise dendritic targeting by an ensemble of transcription factors

Proper information processing in neural circuits requires establishment of specific connections between pre- and postsynaptic neurons. Targeting specificity of neurons is instructed by cell-surface receptors on the growth cones of axons and dendrites, which confer responses to external guidance cues. Expression of cell-surface receptors is in turn regulated by neuron-intrinsic transcriptional programs. In the Drosophila olfactory system, each projection neuron (PN) achieves precise dendritic targeting to one of 50 glomeruli in the antennal lobe. PN dendritic targeting is specified by lineage and birth order, and their initial targeting occurs prior to contact with axons of their presynaptic partners, olfactory receptor neurons. A search was performed for transcription factors (TFs) that control PN-intrinsic mechanisms of dendritic targeting. Two POU-domain TFs, acj6 and drifter have been identified as essential players. After testing 13 additional candidates, four TFs were identified, (LIM-homeodomain TFs islet and lim1, the homeodomain TF cut, and the zinc-finger TF squeeze) and the LIM cofactor Chip, that are required for PN dendritic targeting. These results begin to provide insights into the global strategy of how an ensemble of TFs regulates wiring specificity of a large number of neurons constituting a neural circuit (Komiyama, 2007).

For technical simplicity, larval born GH146-Gal4-positive PNs, originating from three neuroblast lineages, anterodorsal (adPNs), lateral (lPNs), and ventral (vPNs), were studied. Out of ~25 classes defined by their glomerular targets, focus was placed on 17 classes whose target glomeruli are reliably recognized across different animals. The MARCM technique allows visualization and genetic manipulation of PNs in neuroblast and single-cell clones in otherwise heterozygous animals, so PN-intrinsic programs can be studied for dendritic targeting. GH146 is expressed only in postmitotic PNs (Komiyama, 2007).

acj6 and drifter have been identified as lineage-specific regulators of PN dendritic targeting. To identify additional transcription factors (TFs) that regulate dendritic targeting of different PN classes, candidates were tested that have been shown to regulate neuronal subtype specification and targeting specificity and have available loss-of-function mutants. The following was tested; (1) the expression of candidate genes in PNs at 18 hr after puparium formation (APF) when PN dendrites are in the process of completing their initial targeting, and/or (2) their requirement in PNs by examining dendritic targeting in homozygous mutant MARCM clones (Komiyama, 2007).

In addition to the eight genes described below, five other TFs were examined that were not pursued because of the lack of expression in GH146-PNs at 18 hr APF (aristaless and pdm-1) or the lack of targeting defects in homozygous mutant PNs (abrupt [abk02807], kruppel [Kr1], and Dichaete [Dichaete87]) (Komiyama, 2007).

LIM-HD factors and PN targeting: LIM-homeodomain (LIM-HD) TFs are involved in multiple events during neuronal development. Most functions of LIM-HD factors require the LIM domain-binding cofactor, which is represented in Drosophila by ubiquitously expressed Chip. Chip antibody revealed ubiquitous expression of Chip in cells around the antennal lobe (AL) including all GH146-PNs at 18 hr APF (Komiyama, 2007).

The requirement of Chip in PN dendritic targeting was tested. Wild-type adPNs, lPNs, and vPNs target stereotyped sets of glomeruli. PNs homozygous for a Chip null allele (Chipe5.5) failed to target most of the correct glomeruli and occupied inappropriate glomeruli. Most adPN and lPN clones (12/13) also mistargeted a fraction of dendrites to the structure ventral to the AL, the suboesophaegeal ganglion (SOG). Thus, Chip is required for targeting specificity of most, if not all, PN classes studied here, and Chip-interacting proteins including LIM-HD factors likely play important roles in PN dendritic targeting (Komiyama, 2007).

Five LIM-HD factors have been characterized in Drosophila: apterous, arrowhead, islet, lim1, and lim3. apterous, arrowhead, or lim3 were not pursued because they are not expressed in GH146-PNs at 18 hr APF (apterous) or they do not have targeting defects in PNs homozygous for null alleles (lim337Bd6 and awh16) (Komiyama, 2007).

Islet antibody detected Islet expression in ~50% adPNs and most lPNs but not in vPNs at 18 hr APF and adult. isl−/− adPNs failed to target many (but not all) of the normal target glomeruli, including VA1lm, VA3, and VM7. In addition, DA1, a lPN target, was often specifically mistargeted. Defects of isl−/− lPNs were very similar to Chip−/− lPN defects. A fraction of dendrites often mistargeted to the SOG. Within the AL, dendrites were diffusely spread, although DA1 and DL3 were always correctly innervated. Targeting of isl−/− vPNs was normal, consistent with their lack of Islet expression (Komiyama, 2007).

Lim1 antibody revealed Lim1 expression in most or all vPNs, but not in adPNs or lPNs in adults. The expression pattern appears similar at 18 hr APF, although vPNs are difficult to identify unambiguously at early stages. lim1−/− adPNs showed no defects, consistent with the lack of Lim1 expression. lim1−/− lPNs rarely showed a cell number decrease, but in clones in which the cell number was normal, lim1−/− lPNs targeted correct glomeruli. In contrast, lim1−/− vPNs showed a specific targeting defect. Wild-type vPNs innervate DA1 and VA1lm densely because of the single vPNs that specifically innervate these glomeruli, in addition to the diffuse innervation all over the AL contributed by the pan-AL vPN. In lim1−/− vPNs, DA1 innervation was greatly reduced and sometimes undetectable. Therefore, lim1 is required for dendritic targeting by a single vPN class, vDA1, despite its general expression in vPNs. lim1 might be redundant with other factors in non-DA1 vPNs. It was note that phenotypes of islet and lim1 combined are only a subset of the Chip phenotype. Additional Chip phenotype may be explained by non-Lim-HD molecules interacting with Chip (Komiyama, 2007).

Dual role for Drosophila lethal of scute in CNS midline precursor formation and dopaminergic neuron and motoneuron cell fate: lethal of scute, tailup and SoxNeuro act together to control midline cell fate

Dopaminergic neurons play important behavioral roles in locomotion, reward and aggression. The Drosophila H-cell is a dopaminergic neuron that resides at the midline of the ventral nerve cord. Both the H-cell and the glutamatergic H-cell sib are the asymmetric progeny of the MP3 midline precursor cell. H-cell sib cell fate is dependent on Notch signaling, whereas H-cell fate is Notch independent. Genetic analysis of genes that could potentially regulate H-cell fate revealed that the lethal of scute [l(1)sc], tailup and SoxNeuro transcription factor genes act together to control H-cell gene expression. The l(1)sc bHLH gene is required for all H-cell-specific gene transcription, whereas tailup acts in parallel to l(1)sc and controls genes involved in dopamine metabolism. SoxNeuro functions downstream of l(1)sc and controls expression of a peptide neurotransmitter receptor gene. The role of l(1)sc may be more widespread, as a l(1)sc mutant shows reductions in gene expression in non-midline dopaminergic neurons. In addition, l(1)sc mutant embryos possess defects in the formation of MP4-6 midline precursor and the median neuroblast stem cell, revealing a proneural role for l(1)sc in midline cells. The Notch-dependent progeny of MP4-6 are the mVUM motoneurons, and these cells also require l(1)sc for mVUM-specific gene expression. Thus, l(1)sc plays an important regulatory role in both neurogenesis and specifying dopaminergic neuron and motoneuron identities (Stagg, 2011).

In insects, dopaminergic neurons are found in both the nerve cord and brain. One of the best-characterized insect dopaminergic neurons is the H-cell (named for its 'H'-like axonal projections), which is present in the CNS midline cells of the nerve cord. The H-cell was first described in grasshopper as one of the two progeny of the Midline Precursor 3 (MP3) cell, and shown in the moth Manduca sexta to be dopaminergic. The H-cell midline interneuron is also present in Drosophila, and similar to other dopaminergic neurons expresses a set of genes encoding dopamine biosynthetic enzymes, including pale (ple; which encodes tyrosine hydroxylase) and dopa decarboxylase (Ddc). The H-cell also expresses a vesicular monoamine transporter (Vmat), dopamine membrane transporter (DAT) and neurotransmitter receptors that receive input for serotonin (5-HT1A), glutamate (Glu-RI) and neuropeptide F (NPFR1). This characteristic pattern of gene expression and its 'H' axonal projection, to a large degree, constitute the unique character of the H-cell (Stagg, 2011).

Recent work has provided insight into the origins of midline neurons and glia (see Formation of midline precursors (MPs) and MP neurons in Drosophila). Around the time of gastrulation, the single-minded midline master regulatory gene activates the midline developmental program, and soon after 3 MP equivalence groups (MP1, MP3, MP4) of five or six cells/each form. Notch signaling selects one cell from the MP1 group to become an MP1 and the others become midline glia (MG). The same occurs for the MP3 group, with one cell becoming an MP3 and the others MG. Development of the MP4 group is more complex, with sequential Notch-dependent formation of MP4 followed by MP5, MP6 and the median neuroblast (MNB). Each MP undergoes a single division that leads to two neurons. For MP3-6, this involves binary cell fate decisions: MP3 gives rise to the dopaminergic H-cell and glutamatergic H-cell sib interneurons, and MP4-6 each gives rise to a GABAergic iVUM interneuron and glutamatergic/octopaminergic mVUM motoneuron pair. The differences in MP3-6 neuron cell fate are due to the asymmetric localization of the Numb protein, which is high in H-cell and mVUMs, but low in H-cell sib and iVUMs, and differential Sanpodo localization. Although Notch signaling directs H-cell sib and iVUMs to their fates, it is blocked in H-cell and mVUMs due to the presence of Numb. Thus, H-cell sib and iVUM cell fate and gene expression are dependent on Notch signaling, and a different regulatory program governs H-cell and mVUM fates (Stagg, 2011).

This paper asks the question: what regulatory proteins govern Notch-independent H-cell and mVUM fate and gene expression? Also addressed is how the two types of midline precursors, MPs and MNB, form. Proneural genes of the bHLH transcription family have been implicated in controlling neural precursor formation and neuron-specific transcription in both vertebrates and invertebrates. The Drosophila bHLH proneural genes, achaete (ac), scute (sc), lethal of scute [l(1)sc] and atonal have been implicated in the formation of either sensory cell or CNS neuroblast precursors. Proneural bHLH genes can also direct the formation of specific neuronal cell types, as exemplified by studies in the vertebrate spinal cord. Neuronal cell type specification is commonly due to the combinatorial action of proneural and homeodomain-containing proteins. This study demonstrates that three transcription factors: the L(1)sc bHLH protein, Tailup (Tup; Islet) Lim-homeodomain protein and the Sox family protein SoxNeuro (SoxN), work together to control overlapping aspects of H-cell gene expression. In addition, l(1)sc regulates mVUM motoneuron gene expression. All three proneural members of the Drosophila achaete-scute complex (AS-C) [ac, l(1)sc and sc] are expressed in MPs in distinct patterns, and l(1)sc is required for the formation of MP4-6 and the MNB. Thus, l(1)sc controls both midline precursor formation and, in combination with SoxN and tup, controls H-cell-specific gene expression and cell fate. Both the l(1)sc and tup genes may also function together more broadly and control non-midline dopaminergic neuron gene expression (Stagg, 2011).

The formation of midline neural precursors (five MPs and the MNB) is a dynamic, yet stereotyped process. The MPs undergo cellular changes in which their nuclei delaminate from an apical position within the ectoderm and move to the basal (internal) surface. There they divide after orienting their spindles. The precursors arise in a distinct order: MP4/MP3>MP5>MP1>MP6>MNB (Wheeler, 2008). The l(1)sc gene is required for the formation of the MP4-6 and MNB precursors and their neuronal progeny. Since MP4 could not be definitively distinguished from MP5 in Df(1)sc-B57, there is some uncertainty whether both cell types are regulated by l(1)sc. However, as most segments only possess two VUMs, and those are VUM6s in over 60% of segments, it is likely that both MP4 and MP5 are commonly affected in Df(1)sc-B57, in addition to MP6. The ac and sc genes are both expressed in MPs and MNB, yet do not appear to play a significant role in MP and MNB formation. Although l(1)sc is the major proneural gene that controls formation of embryonic neuroblasts, relatively little is known about how it functions and the identity of relevant target genes. In one study, it was shown that morphological changes that accompany neuroblast formation were dependent on l(1)sc function. This is likely to be the case for l(1)sc and MP4-6 and MNB development, as MP4-6 and MNB delamination or division was commonly absent in Df(1)sc-B57. One key question is what activates or maintains l(1)sc expression in MP3-6 and MNB? Signaling by hedgehog (hh) is likely to be important, as no midline l(1)sc expression is present in hh mutant embryos (Stagg, 2011).

Although all MPs and MNB express l(1)sc, only MP4-6 and MNB were affected in mutants - formation of MP1 and MP3 were unaffected. These differences are unlikely to be solely due to different levels of L(1)sc protein or to a combination of Ac, L(1)sc and Sc. L(1)sc protein levels are relatively constant among all five MPs and MNB, both L(1)sc and Sc are present in all MPs and MNB, and Ac, L(1)sc and Sc are present in MP1 as well as MP5,6 and MNB, yet no defects in MP1 and MP3 delamination or cell division were observed. Instead, the ability of l(1)sc to direct development of some MPs and not others may reflect the different cell states (and distinct co-factors) of the precursor populations from which each MP arises. Similarly, l(1)sc controls expression of different genes in the H-cell compared with mVUMs, probably based on their different origins (MP3 versus MP4-6). Variability in the genetic control of midline MP formation extends to the non-midline MP2 cells. The MP2s require both ac and sc for MP formation and differentiation, whereas l(1)sc does not play a role. Thus, MP2 and midline MPs (MP4-6) each require AS-C gene activity for proneural and differentiation functions, but use different AS-C family members (Stagg, 2011).

At least two distinct genetic programs control H-cell gene expression: (1) H-cell-specific gene expression is controlled by l(sc), tup and SoxN, and (2) unknown factors control gene expression that is present in both the H-cell and H-cell sib. All H-cell-specific gene expression requires l(1)sc function. tup acts in parallel to control important aspects of H-cell gene expression, including the DAT, ddc and ple genes. SoxN acts downstream of l(1)sc to control NPFR1 expression. H-cell neural function gene expression begins at stage 13, well after l(1)sc expression is absent, indicating that l(1)sc is unlikely to directly regulate these genes. However, Tup is present after stage 13 and could directly regulate DAT, ddc and ple; SoxN is also present and could directly regulate NPFR1. The l(1)sc gene regulates mVUM gene expression in a manner similar to its control of H-cell expression, but does so independently of tup, which is not expressed in mVUMs. It is noted that L(1)sc protein is present at higher levels in H-cell than mVUMs, although the significance of this is unclear. Expression of genes common to both H-cell and H-cell sib cells, including 5-HT1A, Glu-RI and tup, were not affected in l(1)sc or Notch pathway mutants, indicating a second distinct regulatory pathway. This was also observed for genes expressed in common between mVUMs and iVUMs (Stagg, 2011).

The relationship between l(1)sc and tup in controlling H-cell-specific gene expression is complex. Both genes are initially expressed in the H-cell and H-cell sib after MP3 division, but expression of both is soon restricted to the H-cell. Misexpression of l(1)sc resulted in the ectopic expression of tup in the H-cell sib, similar to other H-cell-specific genes. However, in l(1)sc mutants, tup expression was not absent in the H-cell, but instead tup expression remained present in the H-cell and sometimes in two cells: one was the H-cell and the other was (probably) the H-cell sib. In addition, l(1)sc expression was not affected in tup mutants. These results indicated that: (1) l(1)sc and tup act in parallel in the H-cell to regulate dopaminergic pathway gene transcription; and (2) l(1)sc downregulates tup in the H-cell sib, indicating a role for l(1)sc in H-cell sib development. The best marker for the H-cell sib is CG13565, although it is expressed in wild type in only 54% of segments. In Df(1)sc-B57 mutant embryos, CG13565 was expressed in 46% of segments, similar to wild type. However, given its variability of gene expression in Df(1)sc-B57 mutants and the normal variability of CG13565 expression, it remains possible that l(1)sc (and tup) may play roles in H-cell sib development. Additional experiments are necessary to determine how l(1)sc and tup function together to control H-cell-specific gene expression (Stagg, 2011).

Within midline cells, l(1)sc plays important roles in controlling H-cell and mVUM gene expression, while playing relatively insignificant roles in MP1, H-cell sib and iVUM neuronal gene expression. Whether non-midline neuronal gene regulation is regulated by l(1)sc is currently being addressed. Significantly, Df(1)sc-B57 mutant embryos show a strong reduction in DAT and ple expression in the non-midline dorsal lateral dopaminergic neurons. It has been shown that ple expression in these cells is also reduced in tup mutant embryos. Although more detailed cellular and genetic studies are required to bolster these observations, these data raise the possibility that both l(1)sc and tup may regulate gene expression in both midline and non-midline dopaminergic neurons. More generally, l(1)sc control of neuron-specific gene expression is likely to be uncommon. This is based on the observation that in the developing CNS, there is little L(1)sc protein colocalizing with newly divided Elav+ neurons or GMCs (Stagg, 2011).

Because of the key neurobiological and medical importance of dopaminergic neurons, there has been intensive analysis of the regulatory factors that control their development in vertebrates and C. elegans. Are the regulatory programs involved in dopaminergic neuron differentiation conserved between insects, worms, and mammals? The two key regulatory proteins that control Drosophila H-cell dopamine differentiation are l(1)sc and tup. In vertebrates the bHLH genes mouse achaete-scute homolog [Mash1; homolog of l(1)sc] and neurogenin 2 (Ngn2) play roles in midbrain dopaminergic neuron development, although the role of Mash1 is secondary to Ngn2, which has a key function in dopaminergic differentiation. However, Mash1 (as well as Ngn2) can initiate neurogenic programs of other neuronal cell types. This was emphatically demonstrated in recent work in which forced expression of Mash1 and two other transcription factor genes converted murine fibroblast cells to neurons. The mammalian orthologs of Drosophila tup, Isl1 and Isl2, play important roles in motoneuron differentiation, but have not been reported to influence dopaminergic neuron development and gene expression. Recently, C. elegans and vertebrate ETS family transcription factor genes were shown to directly regulate dopamine pathway gene expression. It will be important to identify the transcription factors in Drosophila that directly regulate dopaminergic neural function genes and connect them to the regulatory genes identified in this paper (Stagg, 2011).

Tup/Islet1 integrates time and position to specify muscle identity in Drosophila

The LIM-homeodomain transcription factor Tailup/Islet1 (Tup) is a key component of cardiogenesis in Drosophila and vertebrates. This study reports an additional major role for Drosophila Tup in specifying dorsal muscles. Tup is expressed in the four dorsal muscle progenitors (PCs) and tup-null embryos display a severely disorganized dorsal musculature, including a transformation of the dorsal DA2 into dorsolateral DA3 muscle. This transformation is reciprocal to the DA3 to DA2 transformation observed in collier (col) mutants. The DA2 PC, which gives rise to the DA2 muscle and to an adult muscle precursor, is selected from a cluster of myoblasts transiently expressing both Tinman (Tin) and Col. The activation of tup by Tin in the DA2 PC is required to repress col transcription and establish DA2 identity. The transient, partial overlap between Tin and Col expression provides a window of opportunity to distinguish between DA2 and DA3 muscle identities. The function of Tup in the DA2 PC illustrates how single cell precision can be reached in cell specification when temporal dynamics are combined with positional information. The contributions of Tin, Tup and Col to patterning Drosophila dorsal muscles bring novel parallels with chordate pharyngeal muscle development (Boukhatmi, 2012).

The pattern of rp298lacZ (a general marker of PCs/FCs) expression and the three-dimensional arrangement of founder cells (FCs) distinguished four groups: dorsal, dorsolateral, lateral and ventral. Whether this topology reflects specific genetic programs has remained unclear. Tup and Col are expressed in the four dorsal and three DL PCs, respectively, supporting the notion of DV regionalization of the somatic mesoderm. This notion was evoked by regional Pox meso (Poxm) expression in most ventral and lateral FCs. As other known iTFs are only expressed in subsets of dorsal PCs/FCs, it raised the possibilty that Tup could reside at the top of dorsal 'identity' transcription factor (iTF) cascades. The data show that this is not the case, as Tup, although required for Kr expression in the DO1 PC and for Col repression in the DA2 PC, is not required for expression of Eve, Runt and Vg in the DA1, DO2 and DA2 and DA1 lineages, respectively. Likewise, Col is required for expression of some iTFs but not others in DL PCs. Together, the patterns of Col, Eve, Kr, Poxm, Runt, Tup and Vg expression in wild-type and tup or col mutant conditions underline the intertwined, combinatorial nature of transcriptional regulatory networks specifying muscle identity. The DA2 PC gives rise to the DA2 muscle/DL adult muscle precursor (AMP) mixed lineage. Each abdominal hemisegment features six AMPs at stereotypical positions. Other AMPs originate from mixed lineages, e.g. the ventral VA3/AMP and lateral SBM/AMP lineages. The VA3/AMP and SBM/AMP PCs express Poxm and S59, and Lb, respectively. Tup expression in the DA2/AMP lineage confirms that different AMPs express different iTFs at the time of specification. Whether, as for somatic muscles, the iTF code confers specific properties to each AMP remains an unresolved issue (Boukhatmi, 2012).

How PCs born at similar positions in the somatic mesoderm come to express different combinations of iTFs and acquire distinct identities has remained elusive. For example, what distinguishes the fate of the two Eve-expressing PCs, which are sequentially born from the same dorsal cluster, is unknown. One other example is the expression of S59 and Lb, each in one of two abutting ventrolateral PCs: the LO1/VT1 and SBM PCs. Activation of both Lb and Slo expression in the two PCs is controlled by the same upstream regulator, Org-1 (Schaub, 2012). Subsequent reciprocal secondary cross-repression results in exclusive S59 or Lb expression, but the nature of the presumed positional bias responsible for the oriented resolution of this cross-repression has not been explored. In the case of the adjacent DA2 and DA3 PCs, this study shows that Tup activation by Tin in the DA2 PC is instrumental in distinguishing between DA2 and DA3 identities. The DA2 PC is selected from a small group of cells at the intersection between Tin and Col expression domains. Thus, the relative registers of tin and col expression along the DV axis provide precise positional information. Another key is timing. The overlap between Tin and Col expression is only transient, such that only the earlier-born Col-expressing PC expresses Tin. This provides a unique temporal window for Tup activation and Col repression. The transient overlap is due to the dorsal restriction of Tin expression to cardial cells during stage 11 This dynamic process is controlled by JAK-STAT signalling activity in the mesoderm, which is itself modulated by Tin activity. The key function of Tup in the DA2 PC, which is to distinguish between two muscle identities, illustrates how cell identity can be specified with single-cell precision when temporal dynamics are combined with positional information (Boukhatmi, 2012).

Some iTFs are expressed during all steps of myogenesis, from promuscular stage to muscle attachment. Schematically, two major phases of expression can be distinguished: (1) PC specification when multiple iTFs are expressed in different PCs and extensive cross-regulation occurs, leading to FC-specific iTF patterns; and (2) muscle differentiation when the FC pattern is maintained and propagated into the syncytial fibre via transcriptional activation of the iTF code in newly fused FCMs . Analysis of col regulation in the DA3 lineage showed that these two phases rely on two separate, early (CRM276) and late (4_0.9) CRMs, the activity of the late CRM requiring Col provided under the control of the early CRM. This auto-regulatory loop has been termed a CRM handover mechanism. This study has provided evidence that tup transcriptional regulation in the DA2 muscle follows the same rule. On the one hand, tup activation by Tin is mediated by an early CRM, IsletH; on the other, tup expression is maintained in differentiating muscles via a late CRM, DME, the activity of which depends upon Tup. It is proposed that this handover relay mechanism could be a widespread mode of iTF regulation, as it efficiently links early steps of muscle specification in response to positional information with final muscle identity (Boukhatmi, 2012).

Tup and Tin are key components of the transcriptional regulatory cascade that controls early cardiogenesis, with Tin acting to activate Tup, the expression of which then persists after Tin has ceased to be expressed. This study now showns that a similar cascade operates in the somatic muscle mesoderm. Tup and Tin expression in both the heart and dorsal somatic muscles recalls Nkx2.5 (Tin ortholog) and Islet1 expression in the pharyngeal mesoderm, which contributes to some head muscles and part of the vertebrate heart. Nkx2.5 is required for deployment of the second heart field (SHF) and Islet1 marks SHF progenitors that contribute both to the right ventricle and the arterial pole of the forming heart and a subset of skeletal pharyngeal muscles. Similarly, in the simple chordate Ciona intestinalis, Nk4 (Tin/NKx2.5) marks the cardio-pharyngeal mesoderm at the origin of the heart and atrian siphon muscles (ASMs) that are evocative of vertebrate pharyngeal muscles. Islet-expressing cells also contribute to ASMs, suggesting an evolutionarily conserved link between cardiac and pharyngeal muscle development. Interestingly, the ascidian Col/EBF ortholog Ci-COE, is expressed in ASM precursors and is a crucial determinant of the ASM fate, reminiscent of Xenopus XCoe2 expression and requirement in pharyngeal arches for aspects of jaw muscle development. It is now well established that distinct genetic networks govern skeletal myogenesis in the vertebrate head and trun. The repertoire of TFs differentially deployed in the head mesoderm includes Tbx1, the Drosophila ortholog of which, Org-1, has recently been shown to act as a muscle iTF . Tin/Nkx2.5, Tup/Isl1, Org-1/Tbx1 and Col/EBF may thus be part of a repertoire of transcription factors co-opted and diversified to regulate muscle patterning in Drosophila trunk and head muscle patterning in chordates (Boukhatmi, 2012).

The Iroquois Complex Is Required in the Dorsal Mesoderm to Ensure Normal Heart Development in Drosophila

Drosophila heart development is an invaluable system to study the orchestrated action of numerous factors that govern cardiogenesis. Cardiac progenitors arise within specific dorsal mesodermal regions that are under the influence of temporally coordinated actions of multiple signaling pathways. The Drosophila Iroquois complex (Iro-C) consists of the three homeobox transcription factors araucan (ara), caupolican (caup) and mirror (mirr). The Iro-C has been shown to be involved in tissue patterning leading to the differentiation of specific structures, such as the lateral notum and dorsal head structures and in establishing the dorsal-ventral border of the eye. A function for Iro-C in cardiogenesis has not been investigated yet. Loss of the whole Iroquois complex, as well as loss of either ara/caup or mirr only, affect heart development in Drosophila. The data indicate that the GATA factor Pannier requires the presence of Iro-C to function in cardiogenesis. A detailed expression pattern analysis of the members of the Iro-C revealed the presence of a possibly novel subpopulation of Even-skipped expressing pericardial cells and seven pairs of heart-associated cells that have not been described before. Taken together, this work introduces Iro-C as a new set of transcription factors that are required for normal development of the heart. As the members of the Iro-C may function, at least partly, as competence factors in the dorsal mesoderm, these results are fundamental for future studies aiming to decipher the regulatory interactions between factors that determine different cell fates in the dorsal mesoderm (Mirzoyan, 2013).

Tissue patterning requires the spatial and temporal coordinated action of signals providing instructive or permissive cues that result in the specification of different cell types and their subsequent differentiation into different lineages. This analyses of Iro-C deficient embryos demonstrate that ara/caup and mirr are required in the dorsal mesoderm for normal heart development. The heart phenotypes could be caused by alterations of the fine balance of the interactions between factors of the cardiac signaling network. In early stage Drosophila embryos the mesoderm is patterned along the anterior-posterior (AP) axis with cardiac and somatic mesodermal domains alternating with visceral mesodermal domains. The tin-positive mesoderm is specified as cardiac and somatic mesoderm under the influence of combined Dpp and Wg signaling. Subsequently, the cardiac and somatic mesodermal domains are further subdivided by the action of the Notch pathway and MAPK signaling activated by EGFR and FGFR. The Eve-expressing cell clusters that give rise to pericardial and DA1 somatic muscle cells, as well as the Doc expression pattern, distinguish the cardiac and somatic mesodermal domain from the visceral mesodermal domain. The early expression pattern of Ara/Caup and Mirr at stages 10/11 suggests a role for Iro-C in patterning the dorsal mesoderm along the AP axis. Consistent with their previously described functions in other developmental contexts, members of the Iro-C may integrate signaling inputs and interact with other transcription factors to specify different dorsal mesodermal derivatives. Activation of the Iro-C by the EGFR pathway is required for the specification of the notum. Mirr was shown to interpret EGFR signaling by eliciting a specific cellular response required for patterning the follicular epithelium. During Drosophila eye development, mirr expression can be regulated by Unpaired, a ligand that activates JAK/Stat signaling. In fact, the JAK/Stat signaling pathway has only recently been added to the signaling pathways that function in Drosophila cardiogenesis. In chromatin immunoprecipitation experiments caup was identified as a target of Stat92E, which is the sole transcriptional effector of the JAK/Stat signaling pathway in Drosophila. Interestingly, the increase of Odd-pericardial cells and the additional Tin-expressing cells that were the characteristic phenotypes in ara/caup (iroDFM1) and in mirr (mirre48) mutants are highly similar to the phenotypes in stat92E mutants described by Johnson (Johnson, 2011). Also, as described for stat92E mutants, cell adhesion defects were noticed in a number of embryos as determined by the distant location of some Tin-expressing cells from the forming heart tube. As for establishing a possible link between JAK/Stat and Iro-C in the dorsal mesoderm and specifically in cardiogenesis, it would be necessary to determine for example whether caup and mirr can rescue the heart phenotype of stat92E mutants. Also, it would be interesting to compare the expression of the other crucial heart marker genes, Tup, Doc and Pnr, in stat92E mutants at early stages to determine to what extent the phenotypes of embryos mutant for Iro-C and for JAK/Stat signaling are similar (Mirzoyan, 2013).

Members of the Iro-C were shown to be positively or negatively regulated by signaling pathways that play crucial roles in heart development. Conversely, the Iro-C factors can also regulate the activity of at least one of these pathways. Specifically, Ara/Caup, as well as Mirr were shown to regulate the expression of the glycosyltransferase fringe and as a consequence modulate Notch signaling activity in the eye. In the dorsal mesoderm, the lateral inhibitory function of Notch signaling establishes the proper number of heart and muscle progenitors. Given the fact that Iro-C can regulate Notch activity it may be that the loss of Iro-C leads to an imbalance of progenitor cell specification resulting in an abnormal number of heart cells. Further studies are required to decipher the molecular mechanism by which Iro-C could integrate diverse signaling inputs and thereby function in the specification and differentiation of the different dorsal mesodermal derivatives (Mirzoyan, 2013).

To determine whether Iro-C can be positioned into the early transcriptional network that determines a cardiac lineage, this study investigated the interdependency between crucial cardiac factors and Iro-C during cardiogenesis. Analyses of the expression of Ara/Caup and Mirr in tin346, Df(3L)DocA, pnrVX6 and tupisl-1 embryos demonstrated the dependency of Ara/Caup and Mirr on all four factors. The strongest loss of Ara/Caup and Mirr expression was observed in tin346 and Df(3L)DocA mutants, which clearly places tin and Doc upstream of Ara/Caup and Mirr. In tupisl-1 and in pnrVX6 mutant embryos, Ara/Caup and Mirr were strongly downregulated, however regarding Ara/Caup, some expression remained in segmental patches suggesting a different level of regulation. The currently available data indicates a positive and a negative regulatory effect of pnr on Iro-C. Whereas pnr restricts Iro-C expression in the dorsal ectoderm and in the wing disc, there is also evidence that pnr can positively regulate Iro-C in the wing disc. Whether Pnr activates or represses Iro-C appears to depend on the presence of U-shaped (Ush), a protein that modulates the transcriptional activity of Pnr. In the wing disc it was shown that an Iro-C-lacZ (IroRE2-lacZ) construct was activated in cells that contained Pnr but were devoid of Ush. The data demonstrate that in the dorsal mesoderm, the expression of Ara/Caup and Mirr depends on pnr. Additionally this analyses show that pnr expression is independent of Iro-C. This finding is intriguing with respect to the downregulation of Tup and Doc in Iro-C mutants. Pnr is required for the maintenance of Doc and for the initiation and/or maintenance of Tup. Since Iro-C mutants exhibit a reduction in Doc-positive cells despite the presence of pnr, members of the Iro-C appear to be required independently or in addition to pnr to maintain expression of Doc. This could be investigated by expressing ara, caup and/or mirr in the mesoderm of pnr mutants to determine whether these factors are able to restore Doc expression. Alternatively, it may be that Iro-C is required indirectly meaning that its main function is to provide a molecular context in which Pnr can be active. For example, it is known that Ush can bind to Pnr thereby inactivating Pnr function. It is conceivable that the absence of Iro-C affects the spatial expression of Ush. If, in the absence of Iro-C, the expression domain of Ush shifts into the Pnr expression domain, Ush could bind to Pnr and inactivate it in the region where Pnr is required to maintain the expression of Tup and Doc. Adding to the complexity of the interpretation of the observed phenotypes is the finding that the majority of embryos that are mutant for ara/caup or for mirr were characterized by supernumerary Tin-positive cells in the cardiac region by stage 11/12. This phenotype could still be observed at later stages when the heart tube forms. The additional Tin-positive cells are pericardial cells as determined by the expression of Prc around the Tin-expressing cells. Also, no increase was observed of Dmef2-positive myocardial cells. Hence, the data suggests a different level of regulation of Tin by the Iro-C. Similar to the findings of Johnson (2011), it may be that Iro-C is normally required to restrict Tin expression at an early stage. The regulation of Tin expression can be divided into four phases. The phenotype this study observed occurs when Tin expression becomes restricted to the myo- and pericardial cells in the cardiac region. In summary, the data adds Iro-C to tin, pnr, Doc and tup whose concerted actions establish the cardiac domains in the dorsal mesoderm. Further studies are required to re-evaluate the current understanding of the interactions between factors of the cardiac transcriptional network (Mirzoyan, 2013).

According to the expression pattern of Ara/Caup and Mirr it was possible to distinguish between an early and late role for these factors, the latter being a role in the differentiation of heart cells (Mirzoyan, 2013).

This analyses of the expression of Ara/Caup and Mirr during embryogenesis led to the identification of hitherto unknown heart-associated cells. Seven pairs of Ara/Caup and Mirr expressing cells and seven pairs of Mirr only expressing cells were detected that were located along the dorsal vessel. No co-expression was detected with any of the known pericardial cell markers. Because there are seven pairs of these cells segmentally arranged, it was tempting to speculate that these cells may function, for example, as additional attachment sites for the seven pairs of alary muscles. The alary muscles attach the heart to the dorsal epidermis and their extensions can be visualized by Prc. Due to the lack of markers little is known about the development of the alary muscles. Previous work demonstrated that the alary muscles attach to the dorsal vessel in the vicinity of the Svp pericardial cells and, in addition, more laterally to one of two distinct locations on the body wall. Maybe it is the Mirr-positive cells that identify the more lateral locations. Clearly, a detailed analysis is needed to identify the function of the Ara/Caup- and Mirr- as well as Mirr-expressing cells that are positioned along the heart tube and whose existence has now been revealed. Additionally, on each side at the anterior end of the dorsal vessel four pericardial cells were identified that co-express Ara/Caup and Eve. Their location at the anterior tip of the heart is intriguing. Further analysis is required to unambiguously determine whether these cells are, for example, the wing heart progenitor cells or the newly identified heart anchoring cells. It is also possible that they represent a yet undefined, novel subpopulation of pericardial cells. In any case, this finding suggests that Ara/Caup plays a role in the diversification of pericardial heart cell types. Future experiments aim to determine the developmental fate of these cells (Mirzoyan, 2013).

Taken together, this investigation of a role for Iro-C in heart development introduces ara/caup and mirr as additional components of the transcriptional network that acts in the dorsal mesoderm and as novel factors that function in the diversification of heart cell types (Mirzoyan, 2013).

The results show that the role of the Iro-C and its individual members, respectively, appears to be rather complex and awaits in-depth analyses. Nevertheless, this work raises important questions regarding the current understanding of interactions between the well-characterized transcription factors that will be addressed in future studies (Mirzoyan, 2013).

Effects of Mutation or Deletion

islet mutants are embryonic lethal and have defects in CNS development as well as in the organization of heart, aorta and alary muscles. Neuron survival and axon extention is normal in islet mutants. Thus the islet gene, like apterous is not necessary for survival of the expressing neurons. However, islet mutant interneurons and motor neurons exhibit striking pathfinding defects. The Class I and II interneurons fail to form their distinct fascicles in the longitudinal connective. Similarly, axons of the Class III local interneurons often appear defasciculated and highly disorganized. In islet mutants, SNb motoneurons project to the ventral muscle area but show a range of defects in target selection. The most common phenotype is a failure to innervate the cleft between muscle 12 and 13. This is often coupled with SNb motor axons leaving the muscle field altogether and joining the transverse nerve, and a frequent lack of innervation of the muscle 6/7 and 6/13 clefts (Thor, 1997).

In almost half the mutant flies, the TMN25 and TMNp neurons fail to enter the TN and instead project inappropriately within the CNS, sending axons either across the midline or into adjacent segements. In many segments, they appear to be stalled along their path. However, the exit glia that pioneer the TN and act as a substrate for the TMN neurons are presentnd appear morphologically normal in mutants, suggesting that the pathfinding defects of the TMNS are due to a failure to recognize the exit glia. The peripheral lateral bipolar dendrite neuron (LBD) neurons, which also normally project in the TN, extend abnormal ventral processes into the ventral muscle area, often joining the SNb. Since no islet expression is detected in the LBD of wild-type embryos, this phenotype is nonautonomous and likely the result of either a failure to synapse within the islet-expressing TMNp neuron or a lack of innervation of the ventral muscles by the SNb motor neurons (Thor, 1997). A similar invasion of the ventral muscle region by projects from the TN has been observed in situations of weak SNb innervation (Chang, 1996 and Kopczynski, 1996).

dHb9 (FlyBase designation: Extra-extra [Exex]), the Drosophila homolog of vertebrate Hb9, encodes a factor central to motorneuron (MN) development. Exex regulates neuronal fate by restricting expression of Lim3 and Even-skipped, two homeodomain (HD) proteins required for development of distinct neuronal classes. Exex and Lim3 are activated independently of one another in a virtually identical population of ventrally and laterally projecting MNs. Surprisingly, Exex represses Lim3 cell nonautonomously in a subset of dorsally projecting MNs, revealing a novel role for intercellular signaling in the establishment of neuronal fate in Drosophila. Evidence is provided that Exex and Eve regulate one another's expression through Groucho-dependent crossrepression. This mutually antagonistic relationship bears similarity to the crossrepressive relationships between pairs of HD proteins that pattern the vertebrate neural tube (Broihier, 2002).

The ISNb MN phenotypes of Exex exhibit similarity to those of Lim3 and Islet. Lim3 and Islet are two LIM-HD proteins that are required for the development of ISNb-projecting axons (Thor, 1997; Thor, 1999). As noted, ISNb-MNs express Exex and require Exex function for their differentiation, suggesting that Exex might interact with Lim3 and Islet to regulate neuronal fate. To investigate this, the relative expression patterns and genetic interactions between these genes were examined. To this end, Lim3- and Islet-specific antibodies were generated because prior expression analyses of Lim3 and Islet used gene-specific reporter constructs (Thor, 1997; Thor, 1999) and such reporter constructs often identify only a subset of a gene's expression profile (Broihier, 2002).

It was found that Lim3 is expressed in about 40 neurons per hemisegment -- this is many more neurons than previously identified by reporter gene expression. Of particular interest, Lim3 is expressed in all Exex-positive neurons as well as in several lateral Exex-negative neurons, including the Eve-positive EL interneurons. Therefore, like Exex, Lim3 is expressed in MNs projecting in the primary and secondary branches of both the SN and ISN. Since previous work has demonstrated that Lim3 is expressed in the TN nerve (Thor, 1999), it is concluded that Lim3 is expressed in all motor axon branches. These results suggest that all ventrally and laterally projecting MNs may express Lim3 (Broihier, 2002).

Despite the near identity of the Exex and Lim3 expression patterns, Exex and Lim3 do not activate each other's expression in these cells. Exex expression initiates normally in lim3 mutants and Lim3 expression in Exex-expressing cells also initiates normally in exex mutants. These data demonstrate that Exex and Lim3 are activated independently of one another in coexpressing cells and suggest that they act in parallel to specify neuronal identity. In addition, the striking similarity of the Exex and Lim3 expression patterns suggests coregulation of Lim3 and Exex by a largely overlapping set of transcriptional regulators (Broihier, 2002).

More limited overlap is found in the expression patterns of Exex and Islet. Islet is expressed in roughly 30 neurons per hemisegment, the majority of which are located laterally in the CNS. Exex and Islet are coexpressed in three discrete neuronal populations: the medial ISNb-projecting RP MNs, a pair of mediolateral interneurons corresponding to the serotonergic interneurons of the CNS, and a compact cluster of six lateral neurons. As observed for Exex and Lim3, Exex and Islet do not regulate each other's expression -- Islet expression is normal in exex mutant embryos and Exex expression is normal in isl mutant embryos. These results indicate that exex and isl do not fall into a simple linear hierarchy and suggest they act in parallel to specify neuronal fate (Broihier, 2002).

To investigate whether exex and Islet act in parallel, isl;exex double mutants were constructed and axonal organization was analyzed in these embryos. isl or exex single mutant embryos exhibit no overt defects in the overall architecture of the CNS. In contrast, isl;exex double mutant embryos exhibit clear defects in the organization of the axonal scaffold. For example, the anterior and posterior commissures are thinner than in wild-type and frequently only one commissure forms per segment. In addition, the longitudinal connectives are thinner than in wild-type and often veer toward or away from the midline (Broihier, 2002).

The defects in axonal organization in isl;exex double mutants have suggested these embryos might exhibit pronounced defects in motor axon projections. Whereas the axonal phenotypes of both single mutants are confined to the ISNb nerve branch, double mutant embryos display widespread defects. In isl;exex double mutants, the organization of motor axons into five nerve branches usually occurs, though axonal outgrowth is substantially delayed relative to wild-type. In addition, the penetrance of ISNb phenotypes in isl;exex double mutant embryos is dramatically higher than in exex single mutants. In 96% of hemisegments, the ISNb either bypasses the ventral muscle domain and extends along the ISN, or stalls shortly after it defasciculates from the ISN. Furthermore, defects are observed in the main ISN branch. In 32% of hemisegments, ISN axons defasciculate inappropriately, giving the ISN a 'frayed' appearance. At lower frequency (5%), the ISNs from adjacent hemisegments fuse. The ISN phenotypes are consistent with the presence of Exex-positive axons in the ISN and demonstrate that like ISNb, the ISN is sensitive to exex levels. Since it is unclear whether Isl is expressed in ISN-projecting neurons, the ISN phenotype in isl;exex embryos may result from loss of isl and exex activity either in common or distinct neuronal populations. In conclusion, the widespread axonal phenotypes in isl;exex double mutant embryos indicate that isl and exex act in parallel to regulate neuronal differentiation. Furthermore, the fact that the isl;exex double mutant reveals a role for exex in regulating ISN-projecting axons suggests that exex may genetically interact with other factors to control the outgrowth of additional motor axon branches (Broihier, 2002).

Expression analyses indicate that Exex and Lim3 are expressed widely in ventrally and laterally projecting MNs. In contrast, Eve has been shown to be expressed in dorsally projecting MNs, suggesting that Exex/Lim3 and Eve might label nonoverlapping MN populations. This is, in fact, what is observed since Exex and Eve label mutually exclusive neuronal subsets. Lim3 and Eve also identify nonoverlapping sets of MNs, since they are only coexpressed in the EL interneurons. Together with other expression analyses, these data show that Exex/Lim3 are expressed in the majority of Eve-negative MNs and demonstrate that Exex/Lim3 and Eve identify distinct MN classes (Broihier, 2002).

tailup, a LIM-HD gene, and Iro-C cooperate in Drosophila dorsal mesothorax specification

The LIM-HD gene tailup has been categorised as a prepattern gene that antagonises the formation of sensory bristles on the notum of Drosophila by downregulating the expression of the proneural achaete-scute genes. tup has an earlier function in the development of the imaginal wing disc; namely, the specification of the notum territory. Absence of tup function causes cells of this anlage to upregulate different wing-hinge genes and to lose expression of some notum genes. Consistently, these cells differentiate hinge structures or modified notum cuticle. The LIM-HD co-factors Chip and Sequence-specific single-stranded DNA-binding protein (Ssdp) are also necessary for notum specification. This suggests that Tup acts in this process in a complex with Chip and Ssdp. Overexpression of tup, together with araucan, a `pronotum' gene of the iroquois complex (Iro-C), synergistically reinforces the weak capacity of either gene, when overexpressed singly, to induce ectopic notum-like development. Whereas the Iro-C genes are activated in the notum anlage by EGFR signalling, tup is positively regulated by Dpp signalling. These data support a model in which the EGFR and Dpp signalling pathways, with their respective downstream Iro-C and tup genes, converge and cooperate to commit cells to the notum developmental fate (de Navascues, 2007).

Tup has been categorised as a prepattern factor that controls the expression of the proneural achaete-scute genes in the third instar wing disc. This study shows that tup functions earlier in the development of the dorsal mesothorax. Loss of tup causes a range of phenotypes, which taken together indicate interference with the assignment of cells to form notum. Thus, depending on the time of induction of the clones and their location multiple effects are observed; the formation of notum-like cuticle with altered cell-cell adhesion properties, the generation of ectopic wing-hinge structures including tegulae, sclerites or sensilla typical of the proximal wing, or even the loss of the entire heminotum. Consistent with these adult phenotypes, in third instar wing discs tup mutant cells can upregulate genes typically expressed at high levels in the wing-hinge territory of the disc, such as zfh2, msh, sal and the lacZ insertion line l(2)09261. Concomitantly, notum-expressed genes such as eyg, ush and pnr are generally repressed, although in some cases tup cells may abnormally express notum and hinge genes together. These data indicate that notum tup cells undergo transformation towards either an altered notum fate or a hinge fate. Moreover, the activation of hinge markers in wild-type cells surrounding some tup clones might reflect the presence of ectopic notum/hinge borders, which are known to promote non-autonomous effects (de Navascues, 2007).

Unequivocal notum-to-hinge transformations are consistently observed in clones induced during the first larval instar. In later-induced clones, this phenotype becomes less manifest and the modified notum cuticle phenotype becomes prevalent. Accordingly, the upregulation of hinge marker genes and the converse downregulation of notum genes in the notum territory are most consistently observed in first instar-induced clones. This suggests that the requirement for the 'pronotum' function of tup progressively decreases as development advances. Lesions associated with tup clones can appear anywhere within the notum, although each particular phenotype shows a degree of topographic specificity. Interestingly, the activation of hinge genes and the repression of notum genes are best shown in early-induced clones located in the presumptive medial notum. Probably, these clones, which are normally large, do not yield adult structures, since the expected large regions of mutant cuticle have not been recovered. The clones might give rise to flies lacking part or most of a heminotum. The dynamic expression pattern of tup fits well with the spatial distribution of these phenotypes and the early requirement for tup function for the development of the notum. Indeed, tup is expressed very early in the wing disc, when it has less than 100 cells, and the expression occurs within the region that will form the notum. It is concluded that, similar to other LIM-HD factors such as Ap and the vertebrate Tup homologue Isl1, Tup is required for the proper specification of not only cell types, but also developing territories (de Navascues, 2007).

Tup is known to bind the co-factor Chip. Since, in dorsal compartment specification, Chip functions in a 2Ap-2Chip-2Sspd hexamer, it was asked whether a similar 2Tup-2Chip-2Sspd complex might mediate Tup function in notum specification. The results support this interpretation. The loss of either Chip or Ssdp upregulates hinge genes (zfh2, msh), represses a notum marker (eyg), and induces cuticular defects similar to those associated with tup clones. Moreover, an excess of Chip would be expected to titrate Tup and/or Ssdp in incomplete complexes and mimic the loss-of-function phenotype of notum-to-hinge transformation, as was experimentally observed (de Navascues, 2007).

By contrast, during the later process of sensory organ formation, Tup appears to act by sequestering both Chip and Pnr, thus preventing activation of the proneural genes achaete-scute. This negative function of Tup does not seem relevant for notum specification, where both Tup and Chip work as positive effectors. Moreover, the Tup homeodomain is dispensable for titrating Chip and Pnr, but this is not the case for its 'pronotum' function. Interestingly, a missense mutation within the LIM-interacting interacting domain of Chip (ChipE) severely reduces its ability to interact with Tup and suppresses the negative regulation by Tup of bristle formation. However, homozygous ChipE flies have no defects in notum specification. This suggests that a residual interaction between ChipE and Tup might persist, as additionally suggested by the suppression of the extra bristles present in ChipE individuals by UAS-tup overexpression. A weak interaction between Tup and Chip, which might only permit the formation of low levels of hexameric complex, might still allow proper notum specification. This suggestion agrees with the fact that tupd03613, a strong hypomorphic allele (as substantiated by its embryonic lethality over the null tupex4, allows proper notum formation in homozygosis (de Navascues, 2007).

Similarly to tup, Iro-C also has a 'pronotum' function. However, their roles are not entirely equivalent. Anywhere within the notum territory, loss of Iro-C during first or second instar induces a clear switch to hinge fate. By contrast, loss of tup causes an assortment of different combinations of derepressed hinge genes and repressed notum genes. Moreover, many tup clones induced during the second larval instar, and even some induced in the first, can develop recognisable notum cuticle. Thus, it is proposed that tup reinforces/stabilises the commitment of cells to develop as notum, a commitment imposed mainly by Iro-C. This reinforcement or stabilisation might be most necessary in the proximal part of the disc, where expression of ara/caup ceases after the second instar, but that of tup persists. This might account for the derepression of hinge genes being most manifest in this region. Depending on the location and time of Tup deprival, its loss may be inconsequential or lead to a partial or even a complete loss of notum commitment. Such diversity of consequences led to an exploration of whether tup might act on target genes by affecting chromatin remodelling. However, no genetic interactions have been found with Polycomb (Pc, Scr+Pcl+esc) or trithorax (trx, osa, brm, Trl, lawc) group genes (de Navascues, 2007).

In contrast to the absolute requirement for Iro-C for notum specification, overexpression of UAS-ara can impose a notum fate only on the wing anlage, and only when provided early in the development of the disc. An extra notum with mirror-image disposition versus the extant notum is generated at the expense of the wing, a phenotype identical to that resulting from early deprivation of Wg function. Since UAS-ara overexpression can interfere with wg expression, Wg deprival probably explains the formation of the extra notum. Thus, by itself, overexpression of UASara probably lacks a genuine potential for imposing the notum fate. Similar notum duplications arise upon early and strong overexpression of UAS-tup (MD638, dpp-Gal4 and ptc-Gal4 drivers) and, again, they probably result from inhibition of Wg activity. Consistent with this interpretation, weaker and later expression of either UAS-tup or UAS-ara (C765 driver) has little or no capacity to promote notum fate. However, when coexpressed, these transgenes are effective in imposing the notum fate and this should not be attributed to Wg depletion. Indeed, the transformation consists of an expansion of the notum tissue, rather than a notum duplication. Moreover, as detected by the onset of the ectopic expression of notum markers (eyg, DC-lacZ), the transformation occurs in late third instar discs (J.deN., unpublished) that have a nearly wild-type morphology and a distinguishable wing pouch. This indicates that these markers are activated in territories previously specified as wing, hinge or pleura, and subsequently forced to acquire notum identity. Moreover, overexpression of the Wg pathway antagonists UAS-Axin or UAS-dTCFDN (dTCF or pan with the same driver failed to transform wing towards notum. Finally, the activation of eyg and the formation of notum tissue in the sternopleurite, a derivative of the leg disc, also attest to the capacity of tup plus ara to commit cells to develop as notum (de Navascues, 2007).

It is well established that signalling by the EGFR pathway is essential for notum development. Its inhibition prevents activation of Iro-C and the growth of the notum territory. By contrast, Dpp negatively regulates Iro-C and restricts its domain of expression at both its distal and proximal borders. The data indicate a novel function of Dpp in notum development; namely, the activation or maintenance of tup expression in second and third instar discs. In the notum region of the early disc, Dpp signalling occurs at low levels, but the results suggest that these are sufficient for activating tup. Expression of tup is largely independent on EGFR signalling. Thus, EGFR and Dpp signalling seem to cooperate in specifying notum identity to the cells of the proximal part of the disc by activating their respective 'pronotum' downstream genes, Iro-C and tup (de Navascues, 2007).

The Drosophila homolog of vertebrate Islet1 is a key component in early cardiogenesis

In mouse, the LIM-homeodomain transcription factor Islet1 (Isl1) has been shown to demarcate a separate cardiac cell population that is essential for the formation of the right ventricle and the outflow tract of the heart. Whether Isl1 plays a crucial role in the early regulatory network of transcription factors that establishes a cardiac fate in mesodermal cells has not been fully resolved. This study analyzed the role of the Drosophila homolog of Isl1, tailup (tup), in cardiac specification and formation of the dorsal vessel. The early expression of Tup in the cardiac mesoderm suggests that Tup functions in cardiac specification. Indeed, tup mutants are characterized by a reduction of the essential early cardiac transcription factors Tin, Pnr and Dorsocross1-3 (Doc). Conversely, Tup expression depends on each of these cardiac factors, as well as on the early inductive signals Dpp and Wg. Genetic interactions show that tup cooperates with tin, pnr and Doc in heart cell specification. Germ layer-specific loss-of-function and rescue experiments reveal that Tup also functions in the ectoderm to regulate cardiogenesis and implicate the involvement of different LIM-domain-interacting proteins in the mesoderm and ectoderm. Gain-of-function analyses for tup and pnr suggest that a proper balance of these factors is also required for the specification of Eve-expressing pericardial cells. Since tup is required for proper cardiogenesis in an invertebrate organism, it is appropriate to include tup/Isl1 in the core set of ancestral cardiac transcription factors that govern a cardiac fate (Mann, 2009).

The specification of a subset of mesodermal cells towards a cardiac fate requires well-orchestrated interactions of a plethora of factors. Drosophila is the model system of choice to decipher the complex transcriptional network that initiates and sustains a cardiac lineage. The data place tup as an essential component in the early transcriptional network that specifies cardiac mesoderm (Mann, 2009).

After the initially broad expression domain of Tin has become restricted to the dorsal mesodermal margin, Tup expression is first seen in the cardiac mesoderm in ~10 small clusters, which co-express Eve. Slightly later, Tup is present throughout the Tin-positive cardiac mesoderm and gene expression analyses in tupisl-1, tin346, pnrVX6 and Df(3L)DocA embryos demonstrate that all four factors are required to maintain each other's expression. Additionally, analyses of cardiac gene expression in embryos that are transheterozygotic for tup and tin, pnr or Doc, showed that these factors interact genetically to specify heart cells (Mann, 2009).

Although it might be expected that Tup expression is lost in tin mutants since these embryos are devoid of heart cells, it is interesting that Tup expression in the early cell clusters is still initiated. This finding is somewhat reminiscent of the observation that the initiation of Doc expression is also independent of tin. According to the temporal appearance of Tup in the cardiac mesoderm with respect to Tin and Doc, tup is required for their maintenance rather than their initiation. By contrast, the onset of mesodermal Pnr and Tup expression appears to coincide. It was not resolved whether Tup is induced by Pnr or directly by Dpp. A direct regulation by Dpp was implicated by the reduced expression of Tup after mesodermal overexpression of UAS-brinker, which is known to bind to dpp-response elements of dpp target genes. Conversely, it was shown that dpp expression depends on tup and the present data suggest that this regulation requires pnr (Mann, 2009).

Germ layer-specific inhibition of Tup using a construct that lacks the homeodomain, but contains the two LIM domains, revealed that Tup can regulate cardiogenesis in the mesoderm as well as from the ectoderm. Since the 69B-Gal4 driver has been reported not to be strictly ectodermal, it is possible that mesodermal Tup function was also interfered with. However, the mesodermal expression of 69B-Gal4 seems to be negligible. The effect of ectodermal Tup inhibition on cardiogenesis in the mesoderm can only be explained if the function of a secreted growth factor is impaired. dpp expression was analyzed, and a slight downregulation of its transcripts was observed in embryos expressing UAS-tupδHD in the ectoderm. Since this effect might not be sufficient to account for the strong Tin phenotype, further experiments will be required to determine whether additional growth factors are affected (Mann, 2009).

To better determine the germ layer-specific contribution of Tup in cardiogenesis, attempts were made to rescue the Tin phenotype by co-expressing the full-length tup cDNA. Somewhat unexpectedly, a better rescue was obtained when both constructs were expressed in the ectoderm rather than in the mesoderm. Since the LIM domains present in tupδHD can sequester LIM-domain-binding proteins, a simple explanation for this finding is that Tup interacts with proteins that are present in the mesoderm but not in the ectoderm. It is reasonable to hypothesize that in the mesoderm the LIM domains of tupδHD not only act as a dominant-negative for Tup, but additionally for another, perhaps as yet unidentified, LIM-domain containing protein. Since it has been shown that Pnr can bind Tup through the LIM domains, it is likely that Pnr function was interfered with by overexpressing UAS-tupδHD. The requirement of the LIM domains for proper cardiac specification is shown by the reduction of Tin-expressing cells after mesodermal expression of the UAS-tupδLIM construct. Further experiments are under way to better resolve the molecular function of Tup in the different tissues (Mann, 2009).

Since the mesodermal expression of UAS-tupδHD resulted in a strong reduction of Tin-expressing cells at early stages of cardiac mesoderm formation, it was surprising to observe a rather low reduction of Dmef2-positive myocardial cells at later stages (15/16). To exclude the possibility that the twi-Gal4 driver does not sufficiently express UAS-tupδHD throughout embryogenesis, this experiment was repeated using the combined mesodermal driver twi-Gal4; 24B-Gal4. However, the phenotypes were not enhanced. A time course for Tin expression in these crosses revealed that Tin appears to recover over time. A similar phenomenon can be seen in tupisl-1 mutants, although it might not be as obvious because the mutants also lack ectodermal tup expression. In any case, the data is suggestive of a different temporal requirement for tup with respect to tin expression. It is known that tin expression depends on different transcriptional activation events. Consistent with the onset of Tup expression in the cardiac mesoderm at mid-stage 11, the earlier phases of Tin expression are unlikely to depend on Tup. Hence, the initial Tin expression at stages 8-10 is sufficient to generate a considerable number of Dmef2-positive myocardial cells at later stages (Mann, 2009).

These analyses further implicate that Tup might act as a transcriptional activator or repressor depending on the cellular context and on the factors with which it is co-expressed. This is most strikingly observed with respect to the Odd-expressing pericardial and lymph gland cells. In tup mutants, Odd-positive cells are missing in both organs. A similar phenotype is seen when Tup is overexpressed in the mesoderm using the twi-Gal4 driver. The loss of Odd-expressing cells in lymph glands is reminiscent of the phenotype observed in tup mutants, although it is less severe. This differential occurrence of the phenotype indicates that tup can differentially regulate factors involved in cardiogenesis versus lymph gland development. This is substantiated by the finding that mesodermal overexpression of tup results in an increase in Hand expression in the lymph glands, while Hand expression throughout the dorsal vessel is only mildly affected. Despite the loss of Odd-positive cells after early mesodermal tup overexpression, Tup is required in the pericardial and lymph gland cells at later stages to maintain Odd expression. Moreover, overexpressing tup in the pericardial cell lineage yields additional Odd-expressing pericardial cells and rescues Odd expression in the lymph glands (Mann, 2009).

To obtain more insight into possible functional interactions with other cardiac transcription factors, tup was overexpressed in combination with pnrD4. The latter is a highly active variant of wild-type pnr that contains an amino acid substitution in the N-terminal zinc finger, which abolishes binding of Ush to Pnr. Mesodermal overexpression of pnrD4 results in robust ectopic activation of Tin and embryos co-overexpressing tup and pnrD4 exhibit the same phenotype. Most likely, a possible influence of Tup on Pnr activity, regardless of whether it is positive or negative, is concealed by the strong gain-of-function pnr allele. However, analysis of Eve expression does provide insight into possible regulatory interactions between Tup and Pnr. Mesodermal overexpression of each factor alone yields opposing phenotypes, and when both factors are co-overexpressed PnrD4 can efficiently counteract Tup activity and prevent the overspecification of Eve cells. Vice versa, Tup can, although only moderately, counteract the effect of PnrD4. It has been shown that during patterning of the thorax, Tup can antagonize the proneural activity of Pnr by forming a heterodimer, and that the physical interaction between Pnr and Tup is mediated by the two zinc fingers of Pnr. Hence, the somewhat weak, but possibly antagonistic, function of Tup towards PnrD4 in Eve-positive cell specification could be due to the amino acid substitution encoded in the pnrD4 allele, which might weaken the interaction between the two factors, as compared with wild-type Pnr. Overexpression of a Tup construct that lacks both LIM domains did not result in expanded Eve-positive clusters, which strongly suggests that the effect of Pnr on Tup activity, as seen when both factors are co-expressed, requires the presence of the LIM domains (Mann, 2009).

In summary, these data demonstrate the crucial role of tup in the proper specification of cardiac mesoderm in an invertebrate organism. Therefore, tup/Isl1 should be added to the core set of ancestral cardiac transcription factors. Consequently, this implicates that the evolution of the vertebrate four-chambered heart does not necessarily require the acquisition of a novel network of cardiac transcription factors. At least, it is unlikely that tup/Isl1 is part of a regulatory network separate from that of tin/Nkx2.5, pnr/Gata4 and Doc/Tbx5/6 because it is an essential factor for the formation of the simple linear heart tube in the fly (Mann, 2009).

Regulation of locomotion and motoneuron trajectory selection and targeting by the Drosophila homolog of Olig family transcription factors

During the development of locomotion circuits it is essential that motoneurons with distinct subtype identities select the correct trajectories and target muscles. In vertebrates, the generation of motoneurons and myelinating glia depends on Olig2, one of the five Olig family bHLH transcription factors. This study investigated the so far unknown function of the single Drosophila homolog Oli. Combining behavioral and genetic approaches, this study demonstrates that oli is not required for gliogenesis, but plays pivotal roles in regulating larval and adult locomotion, and axon pathfinding and targeting of embryonic motoneurons. In the embryonic nervous system, Oli is primarily expressed in postmitotic progeny, and in particular, in distinct ventral motoneuron subtypes. oli mediates axonal trajectory selection of these motoneurons within the ventral nerve cord and targeting to specific muscles. Genetic interaction assays suggest that oli acts as part of a conserved transcription factor ensemble including Lim3, Islet and Hb9. Moreover, oli is expressed in postembryonic leg-innervating motoneuron lineages and required in glutamatergic neurons for walking. Finally, over-expression of vertebrate Olig2 partially rescues the walking defects of oli-deficient flies. Thus, these findings reveal a remarkably conserved role of Drosophila Oli and vertebrate family members in regulating motoneuron development, while the steps that require their function differ in detail (Oyallon, 2012).

The generation of coordinated muscle contractions, enabling animals to perform complex movements, depends on the assembly of functional neuronal motor circuits. Motoneurons lie at the heart of these circuits, receiving sensory input directly or indirectly via interneurons within the central nervous system (CNS) and relaying information to muscles in the periphery. During development neural precursors give rise to progeny that eventually adopt unique motoneuron subtype identities. Their axons each follow distinct trajectories into the periphery to innervate specific target muscles. Understanding of the molecular mechanisms that control the differentiation and respective connectivity of distinct neuronal subtypes is still limited (Oyallon, 2012).

The Olig family of basic Helix-Loop-Helix (bHLH) transcription factors in vertebrates includes the Oligodendrocyte lineage proteins Olig1-3, Bhlhb4 and Bhlhb5. All members play pivotal roles in regulating neural development. Olig2 controls the sequential generation of somatic motoneurons and one type of myelinating glia, the oligodendrocytes, from the pMN progenitor domain in the ventral neural tube. Olig2 mediates progenitor domain formation by cross-repressive transcriptional interactions and motoneuron differentiation upstream of the LIM-homeodomain containing transcription factors Lim3 (Lhx3) and Islet1/2 (Isl1/2). Downregulation of Olig2 enables Lim3 and Isl1/2 together with the proneural bHLH transcription factor Neurogenin2 (Neurog2) to activate the expression of Hb9, a homeodomain protein and postmitotic motoneuron determinant. In addition, Olig2 cooperates with the homeodomain protein Nkx2.2 to promote oligodendrocyte formation from uncommitted pMN progenitors. Olig1 mediates gliogenesis redundantly with Olig2, while Olig3 controls interneuron specification within dorsal neural tube progenitor domains. Recent studies uncovered important requirements of Bhlhb4 in retinal bipolar cell maturation, and Bhlhb5 in regulating the specification of retinal amacrine and bipolar cells, area-specific identity acquisition and axon targeting of cortical postmitotic neurons, as well as differentiation and survival of distinct interneuron subtypes in the spinal cord. In Drosophila, genome-wide data base searches identified one single family member, called Olig family (Oli)), and a recent study described Oli expression in the embryonic ventral nerve cord (VNC). However, despite the central roles of vertebrate Olig family members, the function of their Drosophila counterpart has not been investigated (Oyallon, 2012 and references therein).

In Drosophila, neurons are derived from stem cell-like neuroblasts (NBs). These divide asymmetrically to generate secondary precursor cells, the ganglion mother cells (GMCs), which divide once to produce two postmitotic neurons and/or glia. 15 of 30 embryonic NB lineages give rise to 36 motoneurons in addition to interneurons per abdominal hemisegment. Zfh1 regulates general motoneuron fate acquisition at the postmitotic level. The specification of ventrally projecting motoneuron subtypes is mediated by a combinatorial expression of five transcriptional regulators -- the fly orthologs of Isl, Lim3, Hb9 and Nkx6, as well as the POU protein Drifter (Dfr; Ventral veinless -- FlyBase). Many of these determinants are highly conserved, raising the question as to whether Oli functions as part of this genetic network that shapes motoneuron diversity. Although related molecules in vertebrates and invertebrates appear to mediate late aspects of glial function, factors that regulate early steps of gliogenesis and are molecularly and functionally conserved have so far not been identified. Olig2 is essential for oligodendrocyte development in vertebrates, and a recent study also implicated the C. elegans homolog Hlh-17 in regulating gliogenesis). Thus, Oli is also a potential candidate that could control early glial development in Drosophila (Oyallon, 2012).

This study provides insights into the so far unexplored function of the Oli bHLH transcription factor in the Drosophila nervous system. Oli is not required in glia; however, taking advantage of the well-defined embryonic motoneuron lineages and axonal projections, this study demonstrates that oli controls trajectory selection and muscle targeting of ventral motoneuron subtypes. Moreover, Oli is expressed in postembryonic lineages, which include glutamatergic leg-innervating motoneurons. Loss-of-function experiments revealed that oli is required for larval and adult locomotion. Chick Olig2 can partially rescue these defects in adults, highlighting at least one evolutionarily conserved role of Olig transcription factors in flies and vertebrates (Oyallon, 2012).

Oli protein is mainly expressed in postmitotic neurons, as well as in some GMCs during embryonic development. This is consistent with in situ hybridization labeling detecting high levels of oli mRNA in postmitotic progeny, in addition to transient expression in MP2 and 7.1 NBs. Oli is also expressed in postmitotic progeny of postembryonic lineages. By contrast, vertebrate Olig2 is required in progenitors to promote commitment to a general motoneuron identity. Also Olig1 and 3 largely function in progenitors. Interestingly, Bhlhb4 and Bhlhb5 are expressed and required in postmitotic progeny of the retina, brain and spinal cord. Thus, with respect to its primarily postmitotic expression, Drosophila Oli resembles more that of Bhlhb4 and Bhlhb5 than Olig1–3 in vertebrates (Oyallon, 2012).

The dynamic expression of Drosophila Oli is not consistent with that of a member of the temporal series of transcriptional regulators. With the latter, neurons largely maintain the determinant they expressed at the time of their birth. By contrast, Oli is widely expressed in newly born progeny, but subsequently levels decrease, and only some subtypes show high expression during late stages. Vertebrate Olig2 acts as a transcriptional repressor in homomeric and heteromeric complexes, and expression is downregulated in differentiating motoneurons to enable the activation of postmitotic determinants such as Hb9 by Lim3, Isl1/2 and Neurog2 . Strikingly in flies, Oli expression decreases in RP and lateral ISNb motoneurons during embryogenesis and prolonged high expression of Oli elicits muscle innervation defects, supporting the notion that Oli downregulation is critical for its function in some neurons. Oli could thus act in a dual mode to regulate the differentiation of neuronal subtypes. The first one may rely on downregulation and be a feature shared with vertebrate Olig2, the second one may require persistent activity, and possibly be a feature more in common with Bhlhb4 and Bhlhb5 family members (Oyallon, 2012).

The findings indicate that Drosophila Oli, unlike vertebrate Olig2, does not act as a general early somatic motoneuron determinant. It rather contributes to shaping ventral motoneuron subtype development as part of a postmitotic transcriptional regulatory network in concert with Drosophila Lim3, Isl, Hb9 and Dfr (Drifter/Vvl). This notion is supported by findings that (1) Oli is co-expressed in specific combinations with these determinants in differentiated ISNb and TN motoneuron subtypes; (2) similar to other ventral determinants, oli mutant embryos display distinct axonal pathfinding and muscle targeting defects; (3) oli does not act upstream of hb9, isl, lim3 or Dfr; and (4) oli and hb9 genetically interact, as loss of both enhances phenotypes in ISNb axons. Because of the proximity of oli, isl and lim3 genetic loci, it has so far not been possible to further extend these interaction assays. Some defects observed in oli mutants, such as failure to innervate the clefts of muscles 12/13 or aberrant contacts between ISNb and TN motoneurons are qualitatively similar to those observed in isl, lim3, hb9 and dfr, while the phenotype of isl-τ-myc-positive neurons abnormally exiting the VNC via the SN branch appears characteristic for oli. Moreover, the connectivity phenotypes observed in oli gain-of-function experiments were not reminiscent of trajectories of other motoneuron subtypes. This suggests that although Oli is a member of the combinatorial code, unlike for instance Dfr, it does not act as a simple switch between fates. It may rather act in concert or partially redundantly with these other determinants in regulating the stepwise process of axon guidance to ensure robustness of trajectory selection (Oyallon, 2012).

Individual transcription factors within an ensemble may regulate different biological properties to tightly coordinate the differentiation and synaptic connectivity of a given neuron subtype. As Oli does not act upstream of Isl, Lim3, Hb9 and Dfr, it may control the expression of other yet to be identified transcription factors, or - similar to dfr, Nkx6 and eve in Drosophila and Bhlhb5 in mice - axon guidance determinant or - as reported for Neurog2 - cytoskeletal regulators. Examining Fasciclin 3, N-Cadherin, PlexinA, and Frazzled, no obvious altered expression was observed in the absence of oli). Thus, future studies using approaches such as microarrays will be required to identify oli downstream targets that control subtype-specific axonal connectivity (Oyallon, 2012).

While the role of oli in controlling neuronal development linked to locomotion appears conserved in Drosophila and vertebrates, conservation does not extend to glia. Oli is neither expressed in glia during embryonic or postembryonic development, nor is it essential for basic glial formation in the embryonic VNC or required in glia for locomotion. This also applied to other parts of the nervous system, such as the 3rd instar larval visual system endowed with large glial diversity. hlh-17, the C. elegans Oli homolog, is expressed in cephalic sheath glia in the brain, and interestingly in some motoneurons in the larval CNS. However, as analysis of hlh-17 mutants could not pinpoint any requirement in glial generation and differentiation possibly due to redundancy with related factors, the precise role of the worm homolog remains elusive. Although ensheathing glia can be found in both invertebrates and vertebrates, myelinating glia have so far only been identified in vertebrates. This raises the possibility that the glial requirement of vertebrate Olig family members could be secondary, and Olig2 may have been recruited to collaborate with additional transcriptional regulators to promote the formation of myelinating glia. Indeed, Olig2 promotes motoneuron development together with Neurog2, and subsequently collaborates with Nkx2.2 to enable the generation of oligodendrocyte precursors and differentiating offspring from newly formed, uncommitted pMN progenitors. Interestingly in cell-based assays, Oli can physically interact with the Nkx2.2 homolog Ventral nervous system defective (Vnd). Together with the current observation that Oli is not essential for glial development, this suggests that the potential of these determinants to interact is evolutionarily conserved, while the steps depending on them diverged in flies and vertebrates (Oyallon, 2012).

The locomotion defects in oli mutant larvae are likely the consequence of embryonic wiring defects, whereas the adult phenotypes may be due to an additional or even sole postembryonic requirement. Unlike the so far identified widely expressed determinants Chinmo, Broad Complex or Castor in the postembryonic VNC, Oli expression is restricted to distinct lineages. That these include motoneurons is supported by observations that Oli is detected in postembryonic lineages 20-22 and 15, and expression overlaps with that of OK371-Gal4. Moreover, locomotion defects can be partially rescued by over-expressing oli in glutamatergic neurons with this driver. This initial characterization raises many new questions regarding the specific postembryonic role of Oli. Because of the expression in lineage 15, future experiments will need to specifically test, whether oli contributes to consolidating motoneuron subtype identity by regulating dendritic arbor-formation or leg muscle innervation with single cell resolution. The wider expression of Oli and the partial rescue with OK371-Gal4 further suggest a requirement of oli in lineages that are part of locomotion-mediating neural circuits beyond motoneurons. Because of the expression pattern and the severe walking defects of adult oli escapers, these observations open the door for future functional studies to unravel the mechanisms that shape neural circuits underlying adult locomotion (Oyallon, 2012).


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islet/tailup: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 9 December 2013

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