muscle segment homeobox


Transcriptional Regulation

Mutations in EGF-receptor result in the expansion of muscle segment homeobox domains ventrally, and their ventral margins become graded rather than forming a sharp border. In decapentaplegic mutants, msh expression expands dorsally and extends all the way to the dorsal midline, showing that dpp normally represses msh in the dorsal 30% of the circumference. In short gastrulation mutants, with four copies of dpp, there is a complete repression of msh. Thus the early msh domains in the lateral neuroectoderm are delimited through dorsal repression by DPP and ventral repression by the active EGF-receptor (D'Alessio, 1996).

Ectodermal and mesodermal msh expression depend on wingless and hedgehog. The intricate pattern of msh expression in segmentally arranged clusters during stages 10 and 11, is altered in segment polarity mutants. Mutation of hh affects the intermediate column of msh expressing clusters. In hh mutant embryos, ectodermal msh expression is absent at these positions and the mesodermal expression in fat body precursors is strongly reduced. In contrast, in mutants for wingless the intermediate clusters of msh are normal, whereas the dorsal clusters, both from ectoderm and the mesoderm are completely absent. As a consequence, later stage embryos lack msh expression both in dorsal muscles and around chordotonal organs (D'Alessio, 1996).

Mesodermal msh expression is not found in either twist or snail mutants. The effect of neurogenic mutants, such as Delta, is to expand the number of msh expressing cells (Lord, 1995). daughterless mutants fail to express msh in neuroblasts, but mesodermal expression is unaffected (Lord, 1995).

intermediate neuroblasts defective mutations were isolated by a mutagenesis screen for altered even-skipped (eve) expression in the CNS (J. Skeath and C.Q. Doe, unpubl.). In addition, three ind alleles were obtained by mobilizing a P element located next to the ind locus. The earliest ind mutant phenotype is observed in stage 7 embryonic neuroectoderm, when msh expression occurs both in its normal locations in the dorsal columns and in the adjacent intermediate columns. Thus ind represses transcription of msh directly or indirectly within intermediate column neuroectoderm. Normally the ind and msh expression domains are adjacent but nonoverlapping, consistent with negative regulation of msh by ind. During the earliest stage of neurogenesis (stage 8 of development), wild-type embryos show expression of the proneural gene achaete in rows 3 and 7 of the neuroectoderm, with expression restricted to the ventral and dorsal columns and excluded from the intermediate column. ind expression in the intermediate column precisely abuts these clusters of achaete-expressing cells without overlapping them. In ind mutant embryos, derepression of achaete expression is observed within the intermediate column of neuroectoderm in rows 3 and 7 . This is consistent with a transformation of intermediate to dorsal neuroectoderm msh marker. It is concluded that ind represses msh and achaete gene expression directly or indirectly, and that ind is necessary for establishing proper intermediate-column identity within the neuroectoderm (Weiss, 1998).

Sequential patterns of vnd, ind, and msh expression respond to distinct thresholds of the Dorsal gradient

A nuclear concentration gradient of the maternal transcription factor Dorsal establishes three tissues across the dorsal-ventral axis of precellular Drosophila embryos: mesoderm, neuroectoderm, and dorsal ectoderm. Subsequent interactions among Dorsal target genes subdivide the mesoderm and dorsal ectoderm. The subdivision of the neuroectoderm by three conserved homeobox genes, ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) has been investigated. These genes divide the ventral nerve cord into three columns along the dorsal-ventral axis. Sequential patterns of vnd, ind, and msh expression are established prior to gastrulation and evidence is presented that these genes respond to distinct thresholds of the Dorsal gradient. Maintenance of these patterns depends on cross-regulatory interactions, whereby genes expressed in ventral regions repress those expressed in more dorsal regions. This 'ventral dominance' includes regulatory genes that are expressed in the mesectoderm and mesoderm. At least some of these regulatory interactions are direct. For example, the misexpression of vnd in transgenic embryos represses ind and msh, and the addition of Vnd binding sites to a heterologous enhancer is sufficient to mediate repression. The N-terminal domain of Vnd contains a putative eh1 repression domain that binds Groucho in vitro. Mutations in this domain diminish Groucho binding and also attenuate repression in vivo. The significance of ventral dominance is discussed with respect to the patterning of the vertebrate neural tube, and ventral dominance is compared with the previously observed phenomenon of posterior prevalence, which governs sequential patterns of Hox gene expression across the anterior-posterior axis of metazoan embryos (Cowden, 2003).

The ability of Vnd to repress msh in addition to ind raises the possibility that transcriptional repressors expressed in ventral regions of the embryo can inhibit repressors active in more dorsal regions. Support for this hypothesis came from using the Krüppel enhancer to misexpress both ind and msh along the anterior-posterior axis. Ectopic Ind failed to repress vnd expression, while ectopic Msh did not repress either vnd or ind expression. To determine if 'ventral dominance' is restriced to the neuroectoderm, the mesodermal repressor snail was misexpressed in transgenic embryos using the even-skipped (eve) stripe 2 enhancer. The stripe2-snail transgene creates an ectopic domain of snail along the anterior-posterior axis. This ectopic expression leads to a gap in the sim expression pattern. The transgene also causes a gap in the vnd pattern, confirming the model that Snail excludes vnd expression in the ventral mesoderm and restricts expression to the neuroectoderm. The stripe2-snail transgene also creates a gap in the ind pattern. These results support the ventral dominance model, whereby repressors located in ventral regions inhibit repressors expressed in more dorsal regions. Consistent with this 'directionality' of repression, ectopic expression of Vnd, Ind, or Msh does not repress snail (Cowden, 2003).

Further support for ventral dominance of the Snail repressor was obtained by analyzing mutant embryos derived from CtBP germline clones. CtBP is a maternally deposited corepressor protein essential for snail-mediated repression. Removal of this corepressor results in ventral derepression of sim and vnd into the presumptive mesoderm due to loss of Snail mediated repression. However, this ventral expansion of vnd does not result in a transformation of mesoderm into medial neuroblasts. Instead, the expanded vnd pattern is lost at slightly later stages, and expression becomes restricted to lateral regions, similar to the endogenous expression pattern. This lateral restriction is consistent with the observation that neuroblasts are formed in lateral regions of CtBP- mutants, and not in ventral regions that normally form the mesoderm. Neuroblast segregation can be visualized using a snail antisense RNA probe, which stains all neuroblasts following gastrulation. Sim may be responsible for the late repression of vnd, because vnd expands into the ventral midline of sim mutant embryos. Repression of vnd by Sim is probably indirect because a Krüppel-sim transgene does not alter vnd expression in the lateral neuroectoderm. Perhaps Sim activates an unknown repressor that ultimately inhibits vnd expression in the midline (Cowden, 2003).

It is conceivable that the cross-regulatory interactions among the Snail, Vnd, Ind, and Msh repressors are indirect. For example, perhaps Vnd activates an unknown repressor, which in turn inhibits the expression of ind and msh in medial neuroblasts. Several experiments were done to determine whether Vnd functions as a transcriptional repressor. The first examined whether Vnd binding sites mediate activation or repression in transgenic embryos (Cowden, 2003).

The IAB5 enhancer drives the expression of a lacZ reporter gene in a series of three adjacent bands in the presumptive abdomen of cellularizing embryos. This staining pattern is maintained through gastrulation and germ band elongation. Vnd binding sites were introduced into this IAB5-lacZ transgene by inserting a 220 bp genomic DNA fragment between the IAB5 enhancer and lacZ reporter. This genomic fragment is located 3' of the ind gene and contains three Vnd binding sites. Insertion of this fragment caused a ventrolateral gap in the IAB5-lacZ staining pattern. This gap coincides with the endogenous vnd expression pattern and is maintained during germ band elongation. At this stage, there is a clear loss of lacZ expression in medial regions of the developing ventral nerve cord. The importance of the Vnd binding sites in mediating this repression was examined by mutagenizing all three sites within the 220 bp DNA fragment. Each site was converted from the 5'-CAAGTG-3' consensus to 5'-CCCGGG-3'. The mutagenized IAB5-lacZ transgene exhibits expanded expression in medial regions of the presumptive nerve cord. This observation suggests that Vnd functions as a sequence-specific transcriptional repressor (Cowden, 2003).

Further evidence that Vnd is a repressor was obtained using an in vivo repression assay in transgenic embryos. The N-terminal region of Vnd contains a putative eh1 Groucho-interaction motif, FxIxxIL. This eh1 motif is present in two known transcriptional repressors, Engrailed and Goosecoid. It is also found in the Ind and Msh proteins. GST pull-down assays suggest that this motif mediates interaction between Vnd and Groucho. A GST-VEH1 fusion protein containing amino acid residues 183 to 226 from Vnd binds S35-labeled Groucho protein produced via in vitro translation. This binding is lost when the GST-Vnd fusion protein is mutagenized to replace the phenylalanine in the FxIxxIL motif with an alanine. Various positive and negative controls were included in these experiments. For example, Groucho does not bind a GST-Ind fusion protein containing the Ind homeodomain. Weak binding is observed with a GST-Eve fusion protein containing the FKPY Groucho-interaction motif (Cowden, 2003 and references therein).

A Gal4-Vnd fusion gene containing the Gal4 DNA binding domain and the N-terminal 543 codons of Vnd was placed under the control of the Krüppel 5' regulatory region. The resulting fusion gene is expressed in central regions of cellularizing embryos. Similar levels of expression were obtained with a mutagenized version of the fusion gene that contains multiple alanine substitutions in the FxIxxIL motif. The regulatory activities of the two Gal4-Vnd fusion proteins were monitored with a lacZ reporter gene that contains a modified version of the rhomboid NEE lateral stripe enhancer. The modified NEE enhancer contains three Gal4 binding sites (UAS) and lacks Snail repressor sites. The reporter gene is expressed in ventral regions, including the mesoderm and portions of the lateral neuroectoderm (Cowden, 2003).

The unmutagenized Gal4-Vnd fusion protein containing an intact FxIxxIL motif attenuates expression of the NEE-lacZ reporter gene. This result suggests that the fusion protein binds UAS sites in the modified NEE enhancer and mediates transcriptional repression, either by direct repression of the core promoter, or quenching Dorsal and other activators within the NEE. In contrast, the mutagenized Gal4-Vnd fusion protein (DeltaVEH1) fails to repress expression from the lacZ reporter gene. This result suggests that the FxIxxIL motif is essential for the repression activity of the normal Gal4-Vnd fusion protein. Altogether, these experiments, along with the analysis of Vnd binding sites, suggest that Vnd functions as a sequence-specific transcriptional repressor that might recruit the Groucho corepressor protein (Cowden, 2003).

Thus the Dorsal gradient directly subdivides the neuroectoderm into separate dorsal-ventral compartments through the differential regulation of three conserved homeobox genes, vnd, ind, and msh. Maintenance of sequential patterns of gene expression depends on cross-regulatory interactions, whereby repressors expressed in ventral regions inhibit repressors active in more dorsal regions. This ventral dominance is evocative of the posterior prevalence phenomenon that governs sequential patterns of Hox gene expression across the anterior-posterior axis of metazoan embryos. At least one of the cross-regulatory interactions is direct and evidence was presented that Vnd functions as a sequence-specific transcriptional repressor (Cowden, 2003).

The Dorsal gradient establishes at least three thresholds of gene expression across the dorsal-ventral axis of early embryos. High concentrations activate target genes such as twist and snail in ventral regions that form the mesoderm. Intermediate concentrations activate the rhomboid gene in ventral regions of the neuroectoderm. Finally, low levels of the gradient activate the sog gene in both ventral and dorsal regions of the neuroectoderm. The same low levels of Dorsal repress target genes important for the differentiation of the dorsal ectoderm, including dpp, zen, and tolloid (Cowden, 2003).

Mutant embryos lacking Dorsal fail to activate early expression of either vnd or ind. Conversely, ectopic Dorsal activity leads to a corresponding dorsal shift in the vnd and ind expression patterns. The lateral stripes of vnd expression encompass ventral regions of the neuroectoderm, similar to the rhomboid (rho) pattern. rho is a direct Dorsal target gene that is expressed in the neuroectoderm and encodes a membrane-associated protease that processes the EGFR ligand spitz. Like rho, vnd appears to be a direct target of the Dorsal gradient: an intronic enhancer containing clustered Dorsal and Twist binding sites directs lateral stripes of expression in transgenic embryos. The ind lateral stripes appear to straddle the region between the vnd/rhomboid ventrolateral stripes and the broad sog lateral stripes, and previous studies suggest that ind may be regulated in a different manner from vnd. The regulation of ind relies on both the Dorsal gradient and the EGF signaling pathway. Removal of either Dorsal or the EGF receptor results in the loss of ind expression from the neuroectoderm. It is unclear whether Dorsal directly activates ind or simply establishes a domain of EGF signaling through the regulation of rhomboid (rho). However, given the early onset of ind expression and the misexpression of ind by ectopic Dorsal, it is likely that Dorsal is essential for its regulation. Consistent with the possibility that early ind expression pattern might reflect a threshold readout of the Dorsal gradient is the finding that the low levels of Dorsal present in Tollrm9/Tollrm10 embryos are sufficient to activate ind, but not msh. Moreover, the ind lateral stripes do not extend beyond the sog expression pattern, which is known to be directly activated by vanishingly low levels of the Dorsal gradient. Finally, a 3' ind enhancer that encompasses the three Vnd binding sites used in this study contains optimal Dorsal and Twist binding sites, suggesting that it is directly regulated by the Dorsal and Twist gradients (Cowden, 2003).

The initial compartmentalization of the neuroectoderm appears to depend on threshold readouts of the Dorsal gradient. This strategy is different from the subdivision of the other two primary embryonic tissues, the mesoderm and dorsal ectoderm. Patterning the mesoderm depends on interactions between twist and dpp. The Snail repressor establishes the limits of mesoderm invagination, while the localized expression of Dpp restricts induction of the lateral mesoderm to dorsal-lateral regions. Similarly, subdivision of the dorsal ectoderm depends on the differential regulation of the Dorsal target genes sog and dpp. Both genes respond to the same low levels of the Dorsal gradient, but sog is activated by Dorsal, while dpp is repressed. Subsequent protein-protein interactions between Sog and Dpp establish a broad Dpp signaling gradient in the dorsal ectoderm (Cowden, 2003).

Transcriptional repression of ind by Vnd was predicted from previous genetic studies but lateral repression of msh was somewhat unexpected. Previous studies have shown that ectopic Vnd represses msh expression in the procephalic neuroectoderm, where the vnd and msh expression patterns overlap. This result was extended in the present study using a Krüppel-vnd transgene. It would appear that Vnd represses both ind and msh to specify medial neuroblasts. A similar result was seen using the eve stripe 2 enhancer to misexpress snail. Previous studies have shown that Snail acts as a transcriptional repressor to create the boundary between mesoderm and neuroectoderm. As expected, ectopic snail repressed vnd expression but surprisingly, ind was also repressed. These results suggest that the Dorsal gradient separates domains along the dorsal-ventral axis by activating a series of localized transcriptional repressors. According to this model, repressors located in ventral regions selectively repress those located more dorsally, while dorsal repressors do not inhibit ventral repressors. For example, ectopic Vnd represses ind but not snail, while ectopic Ind fails to repress vnd or snail. According to this model, ectopic Ind should repress msh expression. However, because none of the transgenic Krüppel-ind lines persisted until germband elongation when msh expression is uniform, it was not possible to determine if ectopic Ind repressed msh. Similarly, while ectopic Msh failed to repress snail, vnd, or ind expression, the lack of early target genes that are regulated by Msh prevents any definitive conclusions regarding its role as a transcriptional repressor. Both Ind and Msh contain putative eh1 domains, suggesting that they may function as Groucho dependent repressors and previous work supports such a role for Ind and Msh in the ventral nerve cord (Cowden, 2003).

'Ventral dominance' might govern the patterning of the ventral nerve cord in older embryos, in addition to the prepatterning of the neuroectoderm in pregastrulating embryos. Sim might exclude vnd, ind, and msh expression in the ventral midline. In embryos lacking maternal CtBP products, Snail fails to act as a repressor, allowing the ventral expansion of sim and vnd into the presumptive mesoderm. However, vnd expression is ultimately lost from ventral regions, while sim expression persists. As a result, ventral regions form an expanded mesectoderm, while neuroblasts arise from lateral regions. These observations suggest that Sim excludes vnd expression from ventral regions in CtBP mutants, either directly by acting through a CNS specific enhancer or indirectly by activating an unknown repressor. This putative repressor probably does not rely on the CtBP corepressor, as it is still capable of repressing vnd in CtBP germ line clones. According to a ventral dominance scenario, the misexpression of this unknown repressor should inhibit the expression of vnd, ind, and msh in the ventral midline. One potential target for the indirect repressor could be the EGF pathway. The ventral midline is a well-characterized source of EGF signaling and both vnd and ind rely upon EGF signaling for maintenance of expression. By eliminating EGF activation, this midline repressor could prevent vnd and ind expression (Cowden, 2003).

It is conceivable that the ventral dominance model governing cross-regulatory interactions among Vnd, Ind, Msh, Snail, and possibly sim, also applies to the patterning of the vertebrate neural tube. The vertebrate homolog of vnd, Nkx2.2, is expressed in ventral regions of the neural tube, while the homologs of ind (Gsh) and msh (Msx) are expressed in intermediate and dorsal regions, respectively. These neural tube expression patterns match the dorsal-to-ventral positions of vnd, ind, and msh in the ventral nerve cord of Drosophila. Furthermore, the vertebrate homolog of Vnd, Nkx2.2, also functions as a Groucho-dependent transcriptional repressor. A clear prediction of this study is that the misexpression of Nkx2.2 throughout the vertebrate neural tube should lead to the repression of both Gsh and Msx. In contrast, the misexpression of Gsh should repress Msx, but not Nkx2.2. Thus, a cascade of homologous localized transcriptional repressors could subdivide both the vertebrate and invertebrate CNS (Cowden, 2003).

Threshold-dependent BMP-mediated repression: a model for a conserved mechanism that patterns the neuroectoderm

Subdivision of the neuroectoderm into three rows of cells along the dorsal-ventral axis by neural identity genes is a highly conserved developmental process. While neural identity genes are expressed in remarkably similar patterns in vertebrates and invertebrates, previous work suggests that these patterns may be regulated by distinct upstream genetic pathways. This study asked whether a potential conserved source of positional information provided by the BMP signaling contributes to patterning the neuroectoderm. This question was addressed in two ways: (1) it was asked whether BMPs can act as bona fide morphogens to pattern the Drosophila neuroectoderm in a dose-dependent fashion, and (2), whether BMPs might act in a similar fashion in patterning the vertebrate neuroectoderm was examined. In this study, it was shown that graded BMP signaling participates in organizing the neural axis in Drosophila by repressing expression of neural identity genes in a threshold-dependent fashion. Evidence is also provided for a similar organizing activity of BMP signaling in chick neural plate explants, which may operate by the same double negative mechanism that acts earlier during neural induction. It is proposed that BMPs played an ancestral role in patterning the metazoan neuroectoderm by threshold-dependent repression of neural identity genes (Mazutani, 2006; full text of article).

The neural identity genes vnd, ind, and msh are expressed in a series of non-overlapping DV domains in the Drosophila embryo. These genes are expressed in a highly dynamic fashion and are activated in a ventral-to-dorsal sequence. The BMP antagonist Sog is expressed throughout the neuroectoderm; prior to the activation of neural identity gene expression and fades dorsally as the Dorsal gradient collapses. By the time msh is expressed in a single contiguous dorsal stripe, sog expression is largely lost from these dorsal-most cells. During this same period, the BMP2/4 homolog Dpp is expressed in adjacent dorsal cells, where it represses the expression of neural genes and acts in a graded fashion to pattern the non-neural ectoderm. It is possible that Dpp also signals to the neuroectoderm, although previous single and double mutant analyses of the dpp pathway have not resolved whether Dpp acts in a graded fashion to help establish the order of the neural domains. In none of these studies, was it possible to sort out the contribution of BMP signaling from that of the Dorsal gradient. To answer whether Dpp acts as a morphogen to pattern the Drosophila neuroectoderm, a system was developed for selectively analyzing its effects in the absence of other DV cues (Mazutani, 2006).

In order to separate the potential patterning effect of BMP signaling in Drosophila from that imposed by the Dorsal gradient, a genetic system was designed that allowed replacement of the normal ventral-to-dorsal gradient of nuclear Dorsal with a uniform neuroectodermal level of Dorsal along the entire DV axis of the embryo. These lateralized embryos were created by first eliminating polarized DV maternal patterning acting upstream of Toll signaling and then adding back uniform adjusted levels of Dorsal across the entire DV axis using activated alleles of the Toll receptor. Uniform maternal Toll signaling was adjusted to specific levels using activated Toll alleles of differing strengths and by altering the dose of maternal Dorsal. In such lateralized embryos, the response was then tested of neural genes to an ectopic BMP gradient formed along the AP axis. This BMP gradient was created by expressing dpp under the control of the even-skipped stripe 2 enhancer of dpp (st2-dpp) construct (Mazutani, 2006).

In lateralized embryos, pan-neuroectodermal markers such as sog are expressed around the entire circumference of the embryo. As expected from the threshold-dependent activity of Dorsal, mesodermal, and dorsal ectodermal markers are absent in these same embryos. The consistent and uniform amounts of Dorsal produced in these lateralized embryos correspond to mid-neuroectodermal levels as revealed by expression of ind along the full DV axis and the absence of vnd expression. The AP limits of ind expression are similar to those in wild-type embryos. Within this domain, msh expression is not detectable, presumably because Ind is acting in a ventral-dominant fashion to repress it. However, in more anterior cells abutting the ind domain, where msh expression normally extends further than ind, msh is expressed in a ring around the embryo. These initial studies indicate that both ind and msh can be expressed in mid-neuroectodermal lateralized embryos, and that Ind efficiently excludes msh from its domain (Mazutani, 2006).

Once conditions were established for reliably producing lateralized embryos, whether it was possible to induce a graded Dpp response by crossing a st2-dpp construct into the lateralized background was tested. The sole source of dpp expression in these embryos is provided by st2-dpp, except at the poles where endogenous dpp expression is independent of Dorsal regulation. The expected pattern of BMP pathway activation in such embryos, assessed by in situ phosphorylation of the signal transducer, phosphorylated form of Mothers against dpp (pMAD), is a broad band centered over the st2-dpp stripe. Expression of the epidermal Dpp target gene u-shaped (ush) was also tested as a second marker for BMP activation. Because lateralized embryos ubiquitously express the BMP inhibitor sog, neither pMAD nor ush expression could be detected near the stripe of dpp expression. However, when sog function was eliminated in st2-dpp lateralized embryos, pMAD was activated in a broad domain extending approximately eight cell diameters beyond the narrower dpp stripe. In addition, ush expression was also activated in this region. These results indicate that Dpp diffusing from a sharp stripe can elicit a graded response over significant distances (Mazutani, 2006).

The effect of graded Dpp activity on the relative patterns of ind and msh expression was examined. Multiplex in situ hybridization methods were used to examine the simultaneous expression of msh, ind, and ush, while scoring for the sog+ versus sog− genotype of the embryos. These experiments revealed a clear dose-dependent repression of ind expression characterized by strong repression near the source of dpp and graded reduction in expression extending approximately 20 cell diameters posteriorly. In contrast, the opposite effect was observed with regard to msh expression, resulting in its activation in cells expressing the lowest levels of ind. In control sog+ lateralized embryos, where BMP signaling is blocked, st2-dpp had no discernable effect on the pattern or intensity of either msh or ind expression. These results can be understood if Dpp signaling preferentially represses expression of ind in sog−; st2-dpp lateralized embryos, thereby relieving ind-mediated repression of msh in cells near the Dpp source. The induction of msh expression near the Dpp stripe followed by a zone of ind expression mimics the wild-type configuration of gene expression and provides the first evidence that BMP signaling can influence the pattern of neuroectodermal gene expression in the absence of other DV cues such as the Dorsal gradient. Similar long-range inhibition of ind and short-range induction of ectopic msh expression can be observed in sog−; eve2-dpp embryos with an intact Dorsal gradient, indicating that ind is also likely to be more sensitive than msh to BMP-mediated repression in wild-type embryos. The fact that the zone of ind repression extends considerably further from the dpp stripe than the region of msh activation indicates that msh is not responsible for ind repression, consistent with existing evidence that msh does not regulate ind. It seems likely, therefore, that BMP signaling acts directly to repress ind expression. These data support the prevailing ventral-dominant model for cross-regulation of neural identity genes, and exclude an alternative model in which Dpp signaling activates msh, which in turn inhibits ind (Mazutani, 2006).

Previous studies of the ventral-most neural identity gene, vnd, reported only a mild expansion of its expression domain in dpp− mutants, or no consistent effect. The sensitive lateralized system was exploited to re-examine the BMP response of vnd in order to resolve these existing ambiguities. st2-dpp was expressed in embryos with uniform levels of Dorsal corresponding to the ventral neuroectoderm, which are sufficient to induce ubiquitous expression of vnd. In such 'ventro-lateralized' embryos, both ind and msh expression are absent, presumably due to repression by vnd. Elimination of sog function in these embryos resulted in activation of BMP signaling as judged by the localized activation of the epidermal marker ush; however, vnd expression remained unaltered. When the function of both sog and the transcriptional repressor of BMP signaling, brinker (brk), was eliminated, stronger and expanded expression of ush and potent repression of vnd was observed in a broad zone centered over st2-dpp. These results indicate that vnd is indeed sensitive to BMP-mediated repression and that Brk can block the repressive as well as activating functions of BMP signaling. In analogy to what was observed in mid-lateralized embryos, it might have been expected that relief of Vnd repression in ventro-lateralized embryos would result in activation of ind in cells lacking vnd expression. However, no expression of either ind or msh was detected in these embryos, even near the edges of the vnd repression domain. These data suggest that the high levels of Dpp signaling generated under these experimental conditions are sufficient to repress vnd, as well as ind and msh. Such strong BMP signaling, which is similar to that acting in the non-neural ectoderm of wild-type embryos, may obscure potential differences in the relative sensitivities of these genes to BMP-mediated repression by repressing expression of all neural genes. Although it remains to be determined what the relative sensitivity of vnd is to BMP repression, the fact that vnd is subject to such repression raises the possibility that Dpp might also regulate vnd expression along its dorsal border in wild-type embryos, despite the low levels of Dpp that diffuse into that region. Since the concentration of Dorsal is limiting with regard to activating vnd in cells along this border, these cells would be expected to be the most susceptible to BMP-mediated repression (Mazutani, 2006).

This analysis of BMP signaling in lateralized embryos showed that Dpp can regulate the expression of ind and msh in a dose-dependent fashion along the AP axis, and can also repress vnd expression. To test whether Dpp plays a similar dosage-sensitive role in the regulation of neural identity genes along the DV axis in the presence of an intact gradient of nuclear Dorsal, an experiment was devised to locally inhibit the response of neural genes to Dpp within the neuroectoderm of embryos with normal DV polarity. Because Brk can suppress BMP-mediated repression of vnd, it was reasoned that mis-expression of brk with the eve-st2 enhancer might also relieve BMP repression of ind and msh. This localized expression of the st2-brk construct has the advantage of providing an internal comparison of gene expression domains within the same embryo. In embryos carrying the st2-brk construct, all three neural domains shifted dorsally at the site of brk over-expression. msh expression was de-repressed in a stripe dorsally as has been observed previously in dpp minus mutants, and the border between msh and ind shifted dorsally by approximately 4-6 cells. The dorsal shift in ind expression was observed prior to initiation of msh expression, consistent with their normal ventral-to-dorsal sequence of activation. In addition, a modest but consistent dorsal shift of 1-2 cells was observed in the ind/vnd border within the zone of st2-brk expression. The domains of msh and ind expression also shift in other situations where BMP signaling is altered in the context of an intact Dorsal gradient, which reinforces the view that BMP signaling plays a role in determining the positions and extents of these expression domains in wild-type embryos (Mazutani, 2006).

The results described above indicate that graded Dpp activity normally plays an important role in establishing the position of the border between the msh and ind domains, and to a lesser degree influences the ind/vnd border, which forms 10-12 cells from the dorsal source of Dpp. The co-ordinate shifts in the borders of neural identity gene expression in st2-brk embryos are consistent with the known ventral-dominant chain of repression among vnd, ind, and msh. This analysis also provides additional support for cis-acting vnd sequences being sensitive to BMP repression and suggests that the dorsal border of vnd expression is normally determined by balancing the opposing influences of Dorsal activation and BMP-mediated repression. It is noted that the dorsal expansion of vnd expression in st2-brk embryos does not necessarily imply that vnd is more sensitive to BMP-mediated repression than ind or msh, but instead that at limiting levels of Dorsal, even low levels of BMP signaling can exert a repressive effect on vnd expression (Mazutani, 2006).

Mutual repression between msh and Iro-C is an essential component of the boundary between body wall and wing in Drosophila

During development, the imaginal wing disc of Drosophila is subdivided into territories separated by developmental boundaries. The best characterized boundaries delimit compartments defined by cell-lineage restrictions. This study analyzes the formation of a boundary that does not rely on such restrictions, namely, that which separates the notum (body wall) and the wing hinge (appendage). It is known that the homeobox genes of the Iroquois complex (Iro-C) define the notum territory and that the distal limit of the Iro-C expression domain demarks the boundary between the notum and the wing hinge. However, it is unclear how this boundary is established and maintained. msh, a homeobox gene of the Msx family, is strongly expressed in the territory of the hinge contiguous to the Iro-C domain. Loss- and gain-of-function analyses show that msh maintains Iro-C repressed in the hinge, while Iro-C prevents high level expression of msh in the notum. Thus, a mutual repression between msh and Iro-C is essential to set the limit between the contiguous domains of expression of these genes and therefore to establish and/or maintain the boundary between body wall and wing. In addition, msh is found to be necessary for proper growth of the hinge territory and the differentiation of hinge structures. msh also participates in the patterning of the notum, where it is expressed at low levels (Villa-Cuesta, 2005).

msh is known to be involved in different processes. Thus, it participates in regional specification of muscle progenitors/founders; together with vnd and ind, it helps subdivide the embryonic neuroectoderm along the dorsoventral axis, and it confers dorsal identity to the dorsal bristles of the anterior margin of the wing. This study reports additional functions of msh, namely, the formation/maintenance of the subdivision between the territories of the wing disc that will give rise to the notum (dorsal mesothoracic trunk) and the dorsal hinge (appendix), the proper growth of the dorsal hinge, and the patterning of this region and of the notum (Villa-Cuesta, 2005).

In the developing wing disc, msh is expressed most strongly in the territory of the dorsal hinge, the region between the notum and the dorsal wing blade territories. Removal of msh in clones results in malformations that range from small defects, such as an outheld wing, to partial or even complete loss of most hinge structures. In the latter cases, the hinge may be posteriorly misplaced and ectopically attached to the scutellum. In addition, in a fraction of flies ectopic notum tissue appears contiguous to the extant hinge. Because at least a large part of the hinge tissue is still present, it is surmised that the absence of recognizable hinge structures is due to the failure of their proper differentiation. This phenotype correlates well with that observed in third instar wing discs displaying msh- clones. Indeed, even large clones that remove msh from most of the dorsal hinge territory allow the specification of this territory, as demonstrated by the relatively unmodified characteristic patterns of expression of genes such as wg, zfh-2, hth and tsh, and the presence of recognizable proneural clusters of sc expression. Moreover, the presence in mutant hinges of relatively well resolved clusters of sc expression indicate that the prepatterning of the hinge can proceed to a large extent in the absence of msh. It is concluded that msh is largely dispensable for specification of the dorsal hinge territory, but it is required for the final stages of its patterning and differentiation (Villa-Cuesta, 2005).

Mosaic analyses aimed at studying the patterns of cell proliferation in the wing disc have disclosed the presence of the anterior, posterior, dorsal and ventral compartments of the wing with borders that imposed absolute restrictions to cell proliferation. A border of this type has been suggested to exist between the notum and dorsal hinge, as well as between the pleura and the ventral hinge, but the complex morphology of these regions and the unavailability of appropriate cuticular markers made the proposal uncertain. In fact, analyses performed in the wing disc, has shown that clones can straddle the notum/dorsal hinge boundary, this being defined by the distal border of the Iro-C domain. Hence, at this boundary, the descendants of a cell adopt their developmental fate not according to lineage, but depending on the side of the boundary they were located. The issue thus arises of how the boundary between the notum and the dorsal hinge territories is established and maintained. Considering that the extent of the notum territory is defined by the expression of the Iro-C, this issue can be largely resolved by explaining how the distal border of the Iro-C domain of expression is defined (Villa-Cuesta, 2005).

So far, several genetic interactions have been identified that together permit to suggest a mechanism that partially answers this question. In the second instar disc, the EGFR pathway activates ap and Iro-C. The distinct but overlapping domains of expression of these genes, the dorsal compartment (ap) and the notum territory (Iro-C), may be defined by differential sensitivity to EGFR signaling or, alternatively, in the case of Iro-C, by Dpp signaling. In these early stages, Dpp signaling is active only in the distal part of the disc, where it represses the Iro-C and sets its distal limit of expression. Hence the antagonistic actions of the EGFR and the Dpp pathways define the position of the distal limit of the Iro-C domain, and therefore the position of the notum/hinge subdivision (Villa-Cuesta, 2005).

At approximately the time Iro-C starts to be expressed in the more proximal part of the disc, i.e., that which will become the notum, expression of msh (by means of ap) is turned on in the adjacent dorsal hinge territory. These essentially complementary patterns of expression are maintained, with some qualifications, in the third instar disc. Loss- and gain-of-function experiments show that msh prevents ara/caup from being expressed in the hinge, and ara/caup restrain msh from being expressed in the notum at the high levels typical of the hinge (although it is expressed at a low level in part of the notum). This mutual repression also occurs late during development (Villa-Cuesta, 2005).

How relevant is this mutual repression for the establishment of the notum/dorsal hinge territorial subdivision? As indicated above, in ~19% of flies with msh clones, the removal of Msh from the hinge induces extra notum tissue. In the remaining cases, this removal does not substantially affect the identity of the hinge territory. Thus, the mutual repression between msh and Iro-C is crucial for the notum/hinge territorial subdivision in only a small but substantial fraction of the discs. This indicates that additional agents, probably expressed in the hinge, participate in effecting the subdivision. By contrast, notum cells that lose Iro-C always change their fate to hinge cells and, consequently, depending on position, they modify the notum/hinge subdivision or create an ectopic notum/hinge boundary. Hence, the relevance of the msh/Iro-C mutual repression to define/maintain that subdivision relies mainly on its defining/maintaining the border of the Iro-C domain, and thereby preventing the expression of hinge genes within the notum territory. Thus, a 'pronotum' gene (Iro-C) and a 'hinge differentiation' gene (msh), despite their different positions within the genetic hierarchies that govern the development of their respective domains, cross-regulate each other and participate in the early definition of their respective territories. The current data do not permit distinguishing between the possibilities that the mutual repression between msh and Iro-C is instrumental in establishing this territorial border, or, alternatively, that it stabilizes a previous border defined by the antagonistic actions of EGFR and Dpp on the Iro-C (Villa-Cuesta, 2005).

The relevance of the mutual repression between Iro-C and msh is also manifested by their respective overexpression. Ectopic Iro-C products in the hinge impair the proper differentiation of hinge structures (R. Diez del Corral, PhD thesis, Universidad Autónoma de Madrid, 1998). High levels of Msh in the notum turn on a hinge-specific marker like zfh-2 and are detrimental for notum development (Villa-Cuesta, 2005).

In the third instar disc, the distal border of the Iro-C domain is no longer straight and displays a pronounced 'bay' where ara/caup are downregulated. This roughly coincides with the area of highest expression of msh in the lateral notum. msh is probably responsible for this downregulation of ara/caup, as the 'bay' disappears in msh clones. Moreover, the abutting domains of msh and Iro-C in the ventral hinge and pleura, respectively, suggest that a similar mutual repression may occur there to establish the subdivision between these neighboring regions. Finally, the removal of msh does not activate Iro-C in the anterior part of the hinge territory, suggesting again that agents other than msh and Dpp help maintain Iro-C expression confined to the notum territory (Villa-Cuesta, 2005).

Iro-C clones located within the medial notum not only undergo an autonomous transformation to dorsal hinge. They also become surrounded by a fold similar to that which separates the notum and hinge territories, and they modify the expression of several markers in the surrounding wild-type tissue in a way consistent with a transformation of this tissue towards lateral notum. These nonautonomous effects suggest that signals emerge from the Iro-C clones, and that these signals alter the fate of the aposed notum tissue. Hence, it was inferred that, in the wild-type disc, signaling would take place across the hinge/notum boundary and this would help pattern at least the lateral notum. This is reminiscent of the DV and AP compartment boundaries, where signaling mediated by the diffusible molecules Wg, and Hh and Dpp, respectively, are key to stimulating the growth and pattern of the wing disc. However, in the hinge/notum boundary, the signaling agents have not been identified. They could be either diffusible molecules or cell-bound molecules that mediate this cell to cell communication (Villa-Cuesta, 2005).

The imaginal disc territories flanking the notum/hinge border are reduced in size when they are mutant for msh. It is not known whether this effect is due to decreased cell proliferation, increased cell death or both, and whether it mostly affects the hinge or the lateral notum. However, it is clear that by removing msh and allowing Iro-C to be expressed in the hinge, the msh clones suppress the confrontation of proper hinge cells with notum cells. It is tempting to speculate that this could affect the net growth of the territory by removing positional values and/or by suppressing or making ineffective the postulated signaling associated with the hinge/notum border. Consistently, a reduced size of the notum plus hinge region (and a simplification of the patterning) is also observed in discs overexpressing UAS-ara in the dorsal compartment, a condition that removes most msh expression from the hinge. The failure of Iro-C clones within the notum territory to grow and survive when they are also depleted of Msh might result from the absence of proper signaling across a boundary where wild-type notum cells confront Iro-C msh cells. Considering that the activity of the EGFR signaling pathway is necessary for notum cell proliferation, it would be of interest to examine whether this pathway is involved in, or is modulated by, the presence of the notum/hinge boundary (Villa-Cuesta, 2005).

In Drosophila, the Iro-C genes and msh respectively participate in the DV subdivision of the eye and of the neuroectoderm. In vertebrates, although no instance of mutual repression between homologs of Iro-C and msh has been described, members of each family participate in establishing borders by repression with other genes in the spinal cord, the brain and between rhombomeres. Clearly, both genes are used frequently to subdivide territories and establish alternative differentiation pathways at each side of the border that separates them (Villa-Cuesta, 2005).

Throughout the third instar, msh is expressed at relatively low levels in the posterior notum territory. Here, removal of msh most often results in impaired growth of the scutellum, absence of the scutellum/scutum suture and alterations of the bristle pattern. Interestingly, the lateral/anterior notum macrochaetae are often missing, even though they arise in a region apparently devoid of msh expression. This suggests that either msh is expressed there at very low but functional levels, or that the suppression of macrochaetae results from non-autonomous effects of the absence of Msh from neighboring territories. It should be noted that non-autonomous macrochaetae suppression is also associated with Iro-C clones that cause notum to hinge transformations. This has suggested that modification of the putative signaling across the notum/hinge boundary interferes with macrochaetae patterning at the notum. It is possible that the msh clones might also interfere, as indicated above, with signaling from this border. If so, the presence of clusters of sc expression at the anterior lateral notum within large msh clones suggest that this interference might occur at a stage later than the emergence of the proneural clusters (Villa-Cuesta, 2005).

The absence of msh function does not modify the expression of Iro-C in the lateral notum or the characteristic patterns of expression of eyg and hth, genes that are high in the hierarchy that control notum development. But it removes the scutum/scutellar suture and promotes development of extra bristles in the dorsocentral and scutellar regions. Again, these are phenotypes suggestive of an interference with the late patterning and differentiation of these structures (Villa-Cuesta, 2005).

Genetic control of dorsoventral patterning and neuroblast specification in the Drosophila central nervous system

The Drosophila embryonic CNS develops from the ventrolateral region of the embryo, the neuroectoderm. Neuroblasts arise from the neuroectoderm and acquire unique fates based on the positions in which they are formed. Previous work has identified six genes that pattern the dorsoventral axis of the neuroectoderm: Drosophila epidermal growth factor receptor (Egfr), ventral nerve cord defective (vnd), intermediate neuroblast defective (ind), muscle segment homeobox (msh), Dichaete and Sox-Neuro (SoxN). The activities of these genes partition the early neuroectoderm into three parallel longitudinal columns (medial, intermediate, lateral) from which three distinct columns of neural stem cells arise. Most of the knowledge of the regulatory relationships among these genes derives from classical loss of function analyses. To gain a more in depth understanding of Egfr-mediated regulation of vnd, ind and msh and investigate potential cross-regulatory interactions among these genes, loss of function was combined with ectopic activation of Egfr activity. Ubiquitous activation of Egfr expands the expression of vnd and ind into the lateral column and reduces that of msh in the lateral column. This work has identified the genetic criteria required for the development of the medial and intermediate column cell fates. ind appears to repress vnd, adding an additional layer of complexity to the genetic regulatory hierarchy that patterns the dorsoventral axis of the CNS. This study also demonstrates that Egfr and the genes of the achaete-scute complex act in parallel to regulate the individual fate of neural stem cells (Zhao, 2007).

Ems and Nkx6 are central regulators in dorsoventral patterning of the Drosophila brain

In central nervous system development, the identity of neuroblasts critically depends on the precise spatial patterning of the neuroectoderm in the dorsoventral (DV) axis. This study has uncovered novel gene regulatory network underlying DV patterning in the Drosophila brain; the cephalic gap gene empty spiracles (ems) and the Nk6 homeobox gene (Nkx6) encode key regulators. The regulatory network implicates novel interactions between these and the evolutionarily conserved homeobox genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind) and muscle segment homeobox (msh). Msh cross-repressively interacts with Nkx6 to sustain the boundary between dorsal and intermediate neuroectoderm in the tritocerebrum (TC) and deutocerebrum (DC), and Vnd positively regulates Nkx6 by suppressing Msh. Remarkably, Ems is required to activate Nkx6, ind and msh in the TC and DC, whereas later Nkx6 and Ind act together to repress ems in the intermediate DC. Furthermore, the initially overlapping expression of Ems and Vnd in the ventral/intermediate TC and DC resolves into complementary expression patterns due to cross-repressive interaction. These results indicate that the anteroposterior patterning gene ems controls the expression of DV genes, and vice versa. In addition, in contrast to regulation in the ventral nerve cord, cross-inhibition between homeodomain factors (between Ems and Vnd, and between Nkx6 and Msh) is essential for the establishment and maintenance of discrete DV gene expression domains in the Drosophila brain. This resembles the mutually repressive relationship between pairs of homeodomain proteins that pattern the vertebrate neural tube in the DV axis (Seibert, 2009).

This study shows that the evolutionarily conserved homeodomain protein Ems is an integral component of the gene regulatory network that governs DV patterning in the posterior brain neuromeres, the TC and DC. This novel function is surprising because ems has hitherto been exclusively connected with patterning functions along the AP axis. It has been proposed that the combined activities of the gap genes ems, buttonhead and orthodenticle (ocelliless - FlyBase) generate head segments and that ems mutants exhibit defects in the formation of the intercalary and antennal segment as well as in the corresponding TC and DC in accordance with the early pattern of ems expression. ems probably also has a homeotic function in specifying aspects of intercalary segment identity. This study provides evidence that another crucial function of Ems is its cross-repressive interaction with Vnd. Previously, it was shown that vnd expression is dynamic and exhibits specific differences in the TC and DC. This study demonstrates that Ems is involved in the regulation of brain-specific differences in vnd expression, and that Vnd acts to repress ems in complementary parts of the TC and DC. These interactions help to refine the pattern into mutually exclusive domains at the onset of neurogenesis, which is important as both genes provide positional information that subsequently specifies the identity of individual brain NBs. Depending on the context, Vnd/Nkx2 can act as a transcriptional activator or repressor, as determined by physical interaction with the co-repressor Groucho, which enhances repression. Interestingly, it was observed that Ems also regulates the expression of two Nkx genes in an opposing manner: it represses vnd/Nkx2 but is necessary to activate Nkx6. The repressor function of Ems most likely also depends on Groucho; Ems has been reported to bind Groucho in vitro (Seibert, 2009).

In ems mutants, defects in proneural gene expression (lethal of scute and achaete) are restricted to NE regions where ems is normally expressed during early neurogenesis, leading to the loss of a subset of NBs in the TC and DC. This contrasts with the phenotype of the late embryonic ems mutant brain, which exhibits a severe reduction, or entire elimination, of the TC and DC, suggesting that the proper development of a larger NE domain and/or fraction of NBs in the TC and DC must be affected. However, in ems mutants the organization of the early procephalic NE appears normal until stages 9/10 and apoptosis is not detected. A possible explanation for the subsequent complete loss of TC and DC is that in ems mutants, vnd becomes derepressed in the ventral/intermediate NE of both neuromeres, and expression of msh, ind and Nkx6 is not activated. It has been shown that ectopic vnd prevents the expression of many NB identity genes. Indeed, the expression of a number of molecular markers has been reported to be absent in the ems mutant brain. It is therefore conceivable that in the TC and DC of ems mutants, as a consequence of lacking ems and ectopic vnd (and the absence of proneural gene activation), some NBs do not form. Additionally, owing to mis-specification of the NE (where neural identity gene expression is absent or altered), the other NBs and their progeny might still form but degenerate at later stages (Seibert, 2009).

It has been largely unclear how expression of Nkx6 is regulated in the brain NE, although Vnd has been suggested to act as a positive regulator. At the blastodermal stage, coexpression of ems and vnd is only observed in the intermediate and ventral NE of the TC and DC, which might account for early Nkx6 expression being limited to the respective NE in the brain and absent from the trunk. The data indicate that Ems and Vnd together facilitate the activation of Nkx6. Ems expression closely prefigures the domain of Nkx6 expression in the TC and DC, and together with the fact that Nkx6 is completely abolished in ems mutants, this suggests that Ems might act as a direct activator to regulate the extension of the Nkx6 domain along the AP axis. Vnd indirectly regulates the enlargement of the Nkx6 domain along the DV axis by repressing the Nkx6-repressor Msh. That DV patterning in the brain NE integrates AP signals is additionally supported by the fact that Ems is also necessary for activation of ind and msh, indicating that ems is a key regulator in DV patterning of the TC and DC. Evidence is also provided for a negative-feedback control in the DV regulatory network, in which Ems is needed to activate its own later-stage repressors, Nkx6 and Ind. Together, these data suggest not only that Ems regulates the expression of all DV genes (activating Nkx6, ind, msh and repressing vnd), but also that DV factors (Nkx6, Ind and Vnd) control expression of ems, indicating that integration of DV and AP patterning signals takes place at different levels in the DV genetic network (Seibert, 2009).

Nkx6 has been identified as specifically involved in DV patterning of the TC and DC. In addition to later suppression of ems (in concert with Ind), a further pivotal function of Nkx6 is to maintain the suppression of msh in the intermediate/ventral TC and DC that was initiated by Vnd. Since in both neuromeres the expression of Nkx6 starts before and persists longer than that of ind, and because msh is ventrally derepressed in Nkx6 but not in ind mutants, this implies that Nkx6 (but not Ind) is the major msh suppressor necessary to prevent intermediate/ventral NE and the descending NBs from adopting dorsal fates. Consequently, Nkx6 indirectly regulates the proper specification of brain NB identity by suppressing msh (and ems). Further experiments are required to show whether Nkx6 is also more directly involved in the fate specification of NBs and progeny cells in the brain, as has been shown in the VNC, where Nkx6 promotes the fate of ventrally projecting, and represses the fate of dorsally projecting, motoneurons (Seibert, 2009).

Additionally, cross-inhibitory interactions were observed between Nkx6 and Msh. It is assumed that this mutually repressive regulation in the TC and DC is necessary to stabilize the boundary between dorsal and intermediate NE, and to ensure the regionalized expression of msh and Nkx6 over time. It is likely that Nkx6 and Msh/Msx interact with the co-repressor Groucho to repress each other at the transcriptional level. Interestingly, aspects of the genetic interactions between Nkx6 and Msh/Msx seem to be evolutionarily conserved, since Msx1, which is expressed in the vertebrate midbrain and functions as a crucial determinant in the specification of dopamine neurons, represses Nkx6.1 in ventral midbrain dopaminergic progenitors of mice (Seibert, 2009).

It had not been shown until now that domains of DV gene expression in the Drosophila brain become established through cross-repressive regulation, and it is possible that such genetic interactions are more common than previously thought (e.g. Ind and Msh act as mutual inhibitors). This suggests that in the fly brain, cross-inhibition between pairs of homeodomain transcription factors is fundamental for establishing and maintaining DV neuroectodermal and corresponding stem cell domains. By contrast, in the NE of the VNC, where DV patterning is much better understood, cross-repressive interactions of homeobox genes are largely omitted. There, DV patterning is proposed to be conducted by a strict ventral-dominant hierarchy according to which ventral genes repress more-dorsal genes. However, one exception to the rule seems to be the cross-inhibitory interaction between Vnd and Ind. Interestingly, in the developing vertebrate neural tube, cross-repressive interactions of homeodomain proteins are common and indeed crucial for the establishment of discrete DV progenitor domains. This bears a marked resemblance to the mutually antagonistic relationship between pairs of homeodomain proteins that dorsoventrally pattern the fly brain (Seibert, 2009).

A predominant feature of the brain-specific DV genetic network described in this study, and a general design feature of gene regulatory networks, is the extensive use of transcriptional repression to regulate target gene expression in spatial and temporal dimensions. All factors involved in the network operate as repressors (except Ems, which may also serve as an activator), via mutual repression (between Ems and Vmd, and between Nkx6 and Msh), a double-negative mechanism (Vnd represses Msh, which represses Nkx6), and a negative-feedback loop (Ems is needed to activate Nkx6 and Ind, which in turn repress Ems). The spatial and temporal complexity of the regulatory interactions that have been deciphered implies similar complexity in the underlying cis-regulatory control of these factors. For example, the domain of msh expression is regulated by the input of at least two transcriptional repressors acting in subsequent time windows (Vnd early and Nkx6 late), and the input of at least three repressors regulates the dynamics of ems expression (Vnd early, Ind and Nkx6 late). The brain-specific DV patterning network probably comprises further genes in addition to those that have been identified, and it is likely that interactions with other putative regulators (e.g. Dorsal, Egfr, Dpp) will complement the present model. Altogether, these data provide the basis for a systematic comparison of the genetic processes underlying DV patterning of the brain between different animal taxa at the level of gene regulatory networks (Seibert, 2009).

The genetic factors considered in this study in the developing fly brain are expressed in similar NE domains from early embryonic stages onwards in the anterior neural plate in vertebrates. Emx2, for example, is expressed in the laterodorsal region, and Nkx2 genes in the ventral region, of the early vertebrate forebrain. At the four-somite stage (~E8), these two domains exhibit a common border, similar to that observed in Drosophila after Ems and Vnd have, through cross-repression, regulated their mutually exclusive expression domains. Moreover, whereas Msx genes are mainly expressed in dorsal regions of the posterior forebrain, midbrain and hindbrain, expression of Nkx6 genes is reported in more lateroventral regions, overlapping ventrally with the expression of Nkx2 genes. However, even though these patterns of gene expression exhibit certain similarities between insects and vertebrates, it remains to be shown whether their genetic interactions are also conserved (Seibert, 2009).

Identification of Ind transcription activation and repression domains required for dorsoventral patterning of the CNS

Specification of cell fates across the dorsoventral axis of the central nervous system in Drosophila involves the subdivision of the neuroectoderm into three domains that give rise to three columns of neural precursor cells called neuroblasts. Ventral nervous system defective (Vnd), intermediate neuroblasts defective (Ind) and muscle segment homeobox (Msh) are expressed in the three columns from ventral to dorsal, respectively. The products of these genes play multiple important roles in formation and specification of the embryonic nervous system. Ind, for example, is known to play roles in two important processes. First, Ind is essential for formation of neuroblasts in conjunction with SoxB class transcription factors. Sox class transcription factors are known to specify neural stem cells in vertebrates. Second, Ind plays an important role in patterning the CNS in conjunction with, vnd and msh, which is also similar to how vertebrates pattern their neural tube. This work focuses two important aspects of Ind function. First, multiple approaches were used to identify and characterize specific domains within the protein that confer repressor or activator ability. Currently, little is known about the presence of activation or repression domains within Ind. This study shows that transcriptional repression by Ind requires multiple conserved domains within the protein, and that Ind has a transcriptional activation domain. Specifically, a novel domain, the Pst domain, was identified that has transcriptional repression ability and appears to act independent of interaction with the co-repressor Groucho. This domain is highly conserved among insect species, but is not found in vertebrate Gsh class homeodomain proteins. Second, it was shown that Ind can and does repress vnd expression, but does so in a stage specific manner. It is concluded from this that the function of Ind in regulating vnd expression is one of refinement and maintenance of the dorsal border (Von Ohlen, 2009).

The function of Ind in development of the embryonic nervous system is multifold. Initially, Ind serves to define the intermediate column of the neuroectoderm, this subsequently leads to formation of the corresponding neuroblasts. This study shows that transcriptional repression activity by Ind involves at least two transcriptional repression domains, suggesting that Ind represses transcription via Groucho-dependent and Groucho-independent mechanisms. There are two highly conserved domains in the N-terminal region of the Ind protein. Both appear to be essential for maximal repression activity of Ind. In addition, a third domain was identified that is capable of conferring transcriptional activation ability on a heterologous DNA-binding domain. Also, data is presented demonstrating that Ind functions to define and maintain this domain via transcriptional repression of other columnar genes vnd and msh. Suggesting that, depending on which enhancer it is bound to and possibly association with co-factors, Ind can act as either a transcriptional repressor on as activator. Finally, an Ind protein lacking the Eh1 domain but retaining the Pst domain fails to physically interact with purified Groucho protein. Furthermore, the Gal4-IndδEh1 protein was still a strong repressor of transcription in cultured cells. These results strongly support the hypothesis that the Pst domain confers repressor activity independent of Groucho interaction. However, the possibility that the Pst domain also plays a role in stabilizing the interaction with Groucho or association with other co-factors in vivo cannot be ruled out (Von Ohlen, 2009).

It is not surprising that Ind has incorporated additional repressor activities that are independent of Groucho activity. Formation of the intermediate column of neuroblasts is also dependent on the activity of the Egfr signaling pathway. Specifically, in egfr mutant embryos the intermediate column of neuroblasts fails to form because Ind is not expressed. The readout for activation of the Egfr pathway is the presence of the activated form of Map kinase (dpErk). DpErk is detected in the ventral and intermediate columns of the neuroectoderm at the early stages of development. Interestingly, the activation of DpErk appears to correlate with down-regulation of Groucho activity. Specifically, Map kinase directly phosphorylates Groucho and this phosphorylation of Groucho results in reduced co-repressor activity (Cinnamon, 2008). Since Groucho activity is down-regulated in the region where Ind is expressed and Ind is a Groucho-dependent transcriptional repressor, additional repression activity may be necessary to overcome the effects of Egfr signaling on Groucho activity (Von Ohlen, 2009).

Formation of the proper complement of neuroblasts in the embryonic nervous system of Drosophila and other insects is essential for the proper development of the organism. Initially the neuroblasts form in three columns that correspond to the domains of vnd, ind and msh expression. Therefore, formation of the stripes of homeobox gene expression is essential for the ultimate formation of the CNS. While there is an apparent ventral dominance mechanism in place to initiate the expression of these genes, there is also a cross repressive relationship that is essential for maintaining the boundaries between the domains of gene expression. Ind represses vnd only at stages 9 and 10 of embryonic development and not earlier. This coincides with differences in the ability of Ind to repress vnd reporter gene expression. Thus, Ind can repress transcription from enhancer elements located upstream that regulate expression in neuroblasts. However, Ind is unable to repress transcription of lacZ message from reporter constructs that include the vnd NEE, which is essential for initiation of vnd expression. In conclusion, the temporal differences in the ability of Ind to repress transcription of vnd reflect a role for Ind maintaining the boundary between Vnd and Ind domains. However, Ind was not required for establishing the dorsal border of vnd expression at the earliest stages of embryogenesis (Von Ohlen, 2009).

Role of en and novel interactions between msh, ind, and vnd in dorsoventral patterning of the Drosophila brain and ventral nerve cord

Subdivision of the neuroectoderm into discrete gene expression domains is essential for the correct specification of neural stem cells (neuroblasts) during central nervous system development. This study extends knowledge on dorsoventral (DV) patterning of the Drosophila embryonic brain and uncovers novel genetic interactions that control expression of the evolutionary conserved homeobox genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh). Cross-repression between Ind and Msh was shown to stabilize the border between intermediate and dorsal tritocerebrum and deutocerebrum, and both transcription factors are competent to inhibit vnd expression. Conversely, Vnd segment-specifically affects ind expression; it represses ind in the tritocerebrum but positively regulates ind in the deutocerebrum by suppressing Msh. These data provide further evidence that in the brain, in contrast to the trunk, the precise boundaries between DV gene expression domains are largely established through mutual inhibition. Moreover, it was found that the segment-polarity gene engrailed (en) regulates the expression of vnd, ind, and msh in a segment-specific manner. En represses msh and ind but maintains vnd expression in the deutocerebrum, is required for down-regulation of Msh in the tritocerebrum to allow activation of ind, and is necessary for maintenance of Ind in truncal segments. These results indicate that input from the anteroposterior patterning system is needed for the spatially restricted expression of DV genes in the brain and ventral nerve cord (Seibert, 2010)

The spatial and temporal order in which the DV genes (vnd, ind, and msh) are activated in neuromeres of the brain differs from their appearance in the trunk neuroectoderm, and those differences seem to be basic for the segment-specific regulation of vnd, ind, and msh expression. In the early trito- and deutocerebrum, Vnd is expressed not only in the ventral but also in the intermediate neuroectoderm, where cross-repression between Vnd and dorsally expressed Msh establishes the border between intermediate and dorsal neuroectoderm. Since Msh was found to be an ind repressor, the repression of msh via Vnd is a prerequisite for ind to become activated in the intermediate tritocerebrum (anterior) and deutocerebrum. In the trunk, ind expression in the intermediate neuroectoderm starts before that of msh in the dorsal neuroectoderm, and msh and vnd domains do not abut; accordingly, repressive interaction between Msh and Vnd is not required (Seibert, 2010)

In the tritocerebrum, Vnd not only acts as repressor of msh but also of ind, in contrast to the deutocerebrum. When the level of Vnd protein in the intermediate tritocerebrum declines with time (down-regulated through the activity of Ems), ind becomes subsequently activated. In the trito- and deutocerebrum, instead of Vnd, increasing levels of Ind, together with the recently uncovered msh-repressor Nkx6, still keep msh expression limited to the dorsal neuroectoderm. Since it was found that Nkx6 expression starts earlier and persists longer than that of ind in both brain neuromeres, and additionally, that msh is expanded into the intermediate neuroectoderm in Nkx6 but not in ind mutants, it is proposes that Nkx6 represses msh more efficiently (Seibert, 2010)

The most striking difference in DV gene regulation leads to the question how vnd and ind can be co-expressed in the anterior deutocerebrum (during stages 6–9), if Vnd is a repressor of ind and, vice versa, Ind is also capable of preventing vnd expression in the neuroectoderm. It has been reported recently that the repressor activity of Ind on vnd seems to be stage-specific, not taking place before stage 9. By contrast, Vnd repression of ind seems independent of the developmental period. In this context, it is interesting that activity of Vnd can be modified by EGFR signalling, which is supposed to affect the selective interaction of Vnd with co-factors necessary to mediate repression or activation of target genes. Availability of co-factors might also account for the specific situation of vnd and ind co-expression in the anterior deutocerebrum that was observed specifically during early stages of development (Seibert, 2010)

Involvement of en, which can act as transcriptional repressor as well as activator, has been implicated in diverse developmental processes in Drosophila such as compartmentalization in the early embryo, modulation of Hox gene expression, or regulation of molecules that directly govern axon growth (e.g. frazzled). This study demonstratea a novel function for En in the early embryo, that is to control the spatially restricted expression of the DV genes in the neuromeres of the posterior brain (trito- and deutocerebrum) and ventral nerve cord. In the posterior compartment of the deutocerebrum, En represses expression of msh and ind, but maintains expression of vnd. Since it was found that Ind (later) becomes a vnd repressor, this indicates that En maintains expression of vnd by repressing ind. In the posterior compartment of the tritocerebrum, En is also required for down-regulation of Msh, but opposite to the deutocerebrum, En is necessary for activation of ind. This study shows that Msh is an ind repressor, its repression by En seems to allow for activation of ind; yet, it cannot be excluded that En in addition directly activates ind expression. Similar to the situation in the tritocerebrum, En seems to negatively regulate expression of msh and to positively regulate expression of ind (as a maintenance factor) in the neuroectoderm of the ventral nerve cord. Together, these data suggest that the AP patterning gene engrailed is crucially involved in fine-tuning the regionalized expression of distinct DV genes in the posterior compartment of neuromeres in the brain and ventral nerve cord. En may act as a positive or negative transcriptional regulator depending on the gene that is regulated and on the segmental context. For DV genes it is known that they control formation and specification of brain neuroblasts. Since all the genetic interactions between En and DV genes take place during the period when neuroblasts develop, it is likely that En, via regulation of DV genes, controls formation and fate specification of neuroblasts in the brain (Seibert, 2010)

It was observed that cross-repressive interaction between pairs of DV gene factors in the brain (i.e. in trito- and deutocerebrum) is essential for the establishment and maintenance of discrete DV gene expression domains. Early, cross-repression between Ems/Vnd pre-patterns the ventral and intermediate neuroectoderm in both neuromeres. Mutual repression between Msh/Nkx6 and Msh/Ind maintains the dorsal/intermediate neuroectodermal border in trito- and deutocerebrum, and between Ind/Vnd the intermediate/ventral border in the tritocerebrum. All these genetic interactions, and the observation that Msh and Vnd act as mutual repressors, are not in compliance with the concept of ventral dominance (as proposed in the neuroectoderm of the ventral nerve cord where the more ventral gene represses the gene expressed more dorsally) but rather support the model that in the brain cross-repression between DV factors is crucial for stabilizing these borders (Seibert, 2010)

However, despite the ability of Msh and Ind to repress vnd, neither factor seems to be sufficient to define the dorsal border of vnd expression in trito- and deutocerebrum, as has been shown for Ind in the ventral nerve cord (from stage 9 onwards). Instead of reinforcing this border through repressive interaction, vnd expression in the brain could also be limited by a (too) low concentration or absence of an activator, like Dorsal (as has been speculated for the trunk), or be regulated by BMP signalling in a dosage-dependent fashion. Neuromere-specific differences are also observed regarding limitation of ind and msh expression domains along the DV axis. Vnd establishes the ventral border of ind expression in the trunk and tritocerebrum, but not in the deutocerebrum or protocerebrum (where the expression domains of ind and vnd do not abut). ind expression was found to be limited dorsally by repression through Msh in the trito- and deutocerebrum, but not in the protocerebrum (where msh is not expressed before stage 11) or trunk, although evidence is available that Msh might act in rendering the dorsal border of ind expression more precisely in the ventral nerve cord. Taking into account that ind expression does not expand into the complete dorsal neuroectoderm of trito- and deutocerebrum in msh mutants, this may also indicate an involvement of the nuclear Dorsal gradient, possibly in concert with graded activity of EGFR (as was shown for the trunk neuroectoderm), or BMP (which can repress ind in the trunk neuroectoderm), in establishing a rough dorsal border of ind expression that is further defined and stabilized via repression by Msh. Whereas Vnd is initially responsible for keeping msh expression confined to the dorsal neuroectoderm in trito- and deutocerebrum, it is only indirectly involved in defining the ventral border of msh expression in the trunk neuroectoderm. Later in development Ind helps to maintain repression of msh in trito- and deutocerebrum (together with Nkx6), which is in contrast to the trunk where Ind directly establishes the ventral limit of msh expression from the beginning (Seibert, 2010)

DV neuroectodermal and corresponding stem cell domains in the Drosophila brain become established and maintained through cross-repressive regulation, and it has been speculated that such genetic interactions are more common in the fly brain. This study has presented further examples supporting this hypothesis. Notably, this is a feature that bears similarity to DV patterning in the neural tube of vertebrates where cross-repressive interactions of homeodomain proteins are common and indeed crucial for the establishment of discrete DV progenitor domains (Seibert, 2010)

All interactions between DV genes in the brain identified so far are based on the interplay of transcriptional repressors. Likewise, this study shows that Vnd does not act as a direct activator to positively regulate ind, but according to a double-negative mechanism, it suppresses the ind-repressor Msh. It has been shown previously, that interactions of the AP patterning gene ems with the DV genes (vnd, ind, msh, and Nkx6) are indispensable for proper development of the trito- and deutocerebrum. This study demonstrates that the segmentation gene en is significantly involved in regionalization of DV gene expression domains, thus representing a further example of an AP patterning gene integrating into the DV gene regulatory network that patterns the brain. This study has shown that En acts differently on the respective DV genes, but no evidence is available that, vice versa, DV genes control en, as has been observed for expression of ems. DV genes, as well as En and Ems, all contain an Eh1 repressor domain and are able to interact with the co-repressor Groucho (Gro), and thus are capable of mediating repression on target genes (including each other). But how could it be possible that all DV genes interact with the same co-factor to stabilize expression domains by conferring repression onto genes expressed in neighboring domains? In the first place, the DV genes display spatio-temporal differences in their respective expression. In addition, conformational changes of the protein seem to be necessary to enable binding of Gro which has been at least shown for Nkx6. It has been observed that Vnd can be phosphorylated by activated MAPK and is present in different isoforms in the developing embryo, which most likely leads to a change in its binding partners. Another critical point could be inactivation of the co-repressor, in case of Gro also through phosphorylation by activated MAPK, or modification of target genes so that binding of the repressor complex is impaired. Still, that the DV genes are able to interact with Groucho, does not exclude that their repressor activity is Gro-independent, since also other repressor domains have been reported for these genes, as well as activator domains, at least for Vnd, Ind, and Nkx6. Whether the DV gene products function as repressors or activators seems to depend on co-factor availability as well as on the respective target gene, since not only the presence of a transcriptional binding site, but also its accessibility is limiting in this context (Seibert, 2010)

The female-specific doublesex isoform regulates pleiotropic transcription factors to pattern genital development in Drosophila.

Regulatory networks driving morphogenesis of animal genitalia must integrate sexual identity and positional information. Although the genetic hierarchy that controls somatic sexual identity in Drosophila is well understood, there are very few cases in which the mechanism by which it controls tissue-specific gene activity is known. In flies, the sex-determination hierarchy terminates in the doublesex (dsx) gene, which produces sex-specific transcription factors via alternative splicing of its transcripts. To identify sex-specifically expressed genes downstream of dsx that drive the sexually dimorphic development of the genitalia, genome-wide transcriptional profiling was performed of dissected genital imaginal discs of each sex at three time points during early morphogenesis. Using a stringent statistical threshold, 23 genes that have sex-differential transcript levels at all three time points were identified, of which 13 encode transcription factors, a significant enrichment. This study focused on three sex-specifically expressed transcription factors encoded by lozenge (lz), Drop (Dr) and AP-2. In female genital discs, Dsx activates lz and represses Dr and AP-2. It was further shown that the regulation of Dr by Dsx mediates the previously identified expression of the fibroblast growth factor Branchless in male genital discs. The phenotypes observed upon loss of lz or Dr function in genital discs explain the presence or absence of particular structures in dsx mutant flies and thereby clarify previously puzzling observations. This time course of expression data also lays the foundation for elucidating the regulatory networks downstream of the sex-specifically deployed transcription factors (Chatterjee, 2011).

A common theme in the evolution of development is that a limited 'toolkit' of regulatory factors is deployed for different purposes during morphogenesis. It is therefore not surprising that the key regulators of genital morphogenesis that this study identified are pleiotropic factors with roles in other developmental processes (Chatterjee, 2011).

Two genes that are expressed sex-differentially in the genital disc, branchless (bnl) and dachshund (dac), provide the best picture of how dsx controls genital morphogenesis. Bnl, which is the fly fibroblast growth factor (FGF), is expressed in two bowl-like sets of cells in the A9 primordium in male discs; there is no expression in female discs because DsxF cell-autonomously represses bnl. Bnl recruits mesodermal cells expressing the FGF receptor Breathless (Btl) to fill the bowls; these Btl-expressing cells develop into the vas deferens and accessory glands (Chatterjee, 2011 and references therein).

Dac, a transcription factor, is expressed in male discs in lateral domains of the A9 primordium and in female discs in a medial domain of the A8 primordium. These lateral and medial domains correspond to regions exposed to high levels of the morphogens Decapentaplegic (Dpp) and Wingless (Wg), respectively. Dsx determines whether these signals activate or repress dac. Male dac mutants have small claspers with fewer bristles and lack the single, long mechanosensory bristle. Female dac mutants have fused spermathecal ducts (Chatterjee, 2011 and references therein).

As with bnl and dac, it remains to be determined whether these downstream genes are direct Dsx targets. Each contains at least one match within an intron to the consensus Dsx binding sequence ACAATGT. Future work will determine whether these matches are indeed contained within Dsx-regulated genital disc enhancers. Moreover, efforts are underway to define Dsx binding locations genome-wide through experiments rather than bioinformatics (B. Baker and D. Luo, personal communication to Chatterjee, 2011); combined with the current expression data, these binding data could speed the discovery of a large number of sex-regulated genital disc enhancers (Chatterjee, 2011).

An important future direction will be to determine how spatial and temporal cues are integrated with dsx to regulate downstream genes. Because lz is expressed in the anterior medial region of the female disc, it is hypothesized that, like dac, it is activated by Wg and repressed by Dpp. Such combinatorial regulation could explain the spatially restricted competence of cells in the male disc to activate lz in response to DsxF. Although Dr, AP-2 and lz are expressed at L3, P6 and P20, many other genes are differentially expressed at only one or two of these time points. How these timing differences are regulated is an important unanswered question, especially for genes such as ac, which shifts from highly female biased at P6 to highly male biased at P20. The finding that Dsx binding sites are most enriched in genes with sex-biased expression at L3 suggests that indirect regulation through a cascade of interactions might contribute to expression timing differences (Chatterjee, 2011).

It has already been shown that DsxF indirectly represses bnl by repressing Dr. To date, Dr has been shown to repress, but not activate, transcription. Therefore, activation of bnl by Dr might itself be indirect, via repression of a repressor. The regulation of bnl by Dr is sufficient to explain the sex-specific expression of bnl. However, upstream of bnl are two sequence clusters that match the consensus binding motif of Dsx. Thus, bnl might be repressed both directly and indirectly by Dsx, in a coherent feed-forward loop (FFL). FFLs attenuate noisy input signals. An FFL emanating from Dsx could provide a mechanism of robustly preventing bnl activation in female discs, despite potential fluctuations in DsxF levels (Chatterjee, 2011).

Understanding how Dr controls the morphogenesis of external structures is also important. The posterior lobe will be of particular interest because it is the most rapidly evolving morphological feature between D. melanogaster and its sibling species. Mutations in Poxn and sal also impair posterior lobe development. Understanding how these two regulators work with Dr to specify and pattern the developing posterior lobe could substantially advance efforts to understand its morphological divergence. Likewise, understanding how lz governs spermathecal development could advance evolutionary studies, as this organ also shows rapid evolution (Chatterjee, 2011).

The extent to which the regulators that were identified play deeply conserved roles in genital development remains to be determined. Although sex-determination mechanisms evolve rapidly, some features are shared by divergent animal lineages. The observation that FGF signaling is crucial to male differentiation in mammals, or that mutations in a human sal homolog cause anogenital defects, could reflect ancient roles in genital development or convergent draws from the toolkit (Chatterjee, 2011).

Whether AP-2, Dr and lz play conserved roles in vertebrate sexual development is similarly uncertain. In mice, an AP-2 homolog is expressed in the urogenital epithelium (albeit in both sexes) and at least one AP-2 homolog shows sexually dimorphic expression (albeit in the brain). The mouse Dr homolog Msx1 is expressed in the genital ridge and Msx2 functions in female reproductive tract development. In chick embryos, Msx1 and Msx2 are expressed male specifically in the Müllerian ducts. The mouse lz homolog Aml1 (Runx1) is expressed in the Müllerian ducts and genital tubercle. As more data accumulate on the genetic mechanisms controlling genital development in other taxa, the question of how deeply these mechanisms are conserved might be resolved (Chatterjee, 2011).

BMPs regulate msx gene expression in the dorsal neuroectoderm of Drosophila and vertebrates by distinct mechanisms

In a broad variety of bilaterian species the trunk central nervous system (CNS) derives from three primary rows of neuroblasts. The fates of these neural progenitor cells are determined in part by three conserved transcription factors: vnd/nkx2.2, ind/gsh and msh/msx in Drosophila melanogaster/vertebrates, which are expressed in corresponding non-overlapping patterns along the dorsal-ventral axis. While this conserved suite of 'neural identity' gene expression strongly suggests a common ancestral origin for the patterning systems, it is unclear whether the original regulatory mechanisms establishing these patterns have been similarly conserved during evolution. In Drosophila, genetic evidence suggests that Bone Morphogenetic Proteins (BMPs) act in a dosage-dependent fashion to repress expression of neural identity genes. BMPs also play a dose-dependent role in patterning the dorsal and lateral regions of the vertebrate CNS, however, the mechanism by which they achieve such patterning has not yet been clearly established. This report examined the mechanisms by which BMPs act on cis-regulatory modules (CRMs) that control localized expression of the Drosophila msh and zebrafish (Danio rerio) msxB in the dorsal central nervous system (CNS). This analysis suggests that BMPs act differently in these organisms to regulate similar patterns of gene expression in the neuroectoderm: repressing msh expression in Drosophila, while activating msxB expression in the zebrafish. These findings suggest that the mechanisms by which the BMP gradient patterns the dorsal neuroectoderm have reversed since the divergence of these two ancient lineages (Esteves, 2014; Open access).

A 700 bp msh CRM (referred to as ME for Msh Element) has been identified that is directly repressed by Ind. The response of the ME to BMP-mediated regulation has not yet been investigated, however. As is the case for the endogenous msh gene, the expression of a ME-lacZ construct expands throughout the dorsal region of the embryo in dpp- mutants. In order to determine whether Dpp regulates msh directly or indirectly, BMP regulation of the ME element was analyzed. Consistent with a direct role of BMP signaling on this CRM, genome wide chromatin immune precipitation (ChIP) data revealed DNA binding sites for the BMP effectors Mad, Medea and Shn within the ME region in blastoderm stage embryos. The involvement of Shn in regulating msh within the neuroectoderm was confirmed by examining homozygous zygotic shn- mutant embryos, which exhibit a partial dorsal expansion of msh expression (Esteves, 2014)

Targets of Activity

Ectopic expression of msh in the mesoderm results in altered expression of the S59 and nau/Dmyd genes leading to a loss of some muscles and defects in the patterning of others, suggesting that the muscle defects are at the level of recruitment and/or patterning of muscle precursor cells (Lord, 1995).

Snail, a zinc-finger transcriptional repressor, is a pan-neural protein, based on its extensive expression in neuroblasts. Previous results have demonstrated that Snail and related proteins, Worniu and Escargot, have redundant and essential functions in the nervous system. The Snail family of proteins control central nervous system development by regulating genes involved in asymmetry and cell division of neuroblasts. Whether the neuroblast expression of snail and worniu is regulated by proneural genes was examined. Such a result would place the snail family in the well established genetic hierarchy that controls early neuroblast differentiation. The scuteB57 deletion mutant uncovers the three pro-neural genes: achaete, scute and lethal of scute. In this mutant, the expression of worniu in neuroblasts is significantly reduced. Only a few neuroblasts within each segment exhibit staining, and the expression level is substantially lower than in the wild type. The expression of worniu is also regulated by vnd and ind, such that in these mutant embryos the whole ventral and intermediate columns of staining are missing. In the mshDelta68 mutant, no abnormal expression of worniu was detected. Previous results have shown that the neuroblast expression of snail is slightly affected in achaete-scute and vnd mutants but is not affected in a daughterless mutant. In ind and msh mutants, Snail protein expression was observed in many neuroblasts but the spatial pattern was rather disorganized. In summary, most of the proneural genes tested have profound effects on the expression of worniu, and have detectable but lesser effects on that of snail. The predominant expression of snail and worniu in neuroblasts and their regulation by proneural genes suggests that the snail family genes may have important functions within neuroblasts (Ashraf, 2001).

muscle segment homeobox: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of mutation | References

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