muscle segment homeobox
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).
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).
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).
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).
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).
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).
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