Table of contents

Targets of DPP in the dorsal ectoderm

Signals from the BMP family member Decapentaplegic (Dpp) play a role in establishing a variety of positional cell identities in dorsal and lateral areas of the early Drosophila embryo, including the extra-embryonic amnioserosa as well as different ectodermal and mesodermal cell types. Although a reasonably clear picture is available of how Dpp signaling activity is modulated spatially and temporally during these processes, a better understanding of how these signals are executed requires the identification and characterization of a collection of downstream genes that uniquely respond to these signals. Three novel genes, Dorsocross1, Dorsocross2 and Dorsocross3, referred to collectively as Dorsocross, are described that are expressed downstream of Dpp in the presumptive and definitive amnioserosa, dorsal ectoderm and dorsal mesoderm. These genes are good candidates for being direct targets of the Dpp signaling cascade. Dorsocross expression in the dorsal ectoderm and mesoderm is metameric and requires a combination of Dpp and Wingless signals. In addition, a transverse stripe of expression in dorsoanterior areas of early embryos is independent of Dpp. The Dorsocross genes encode closely related proteins of the T-box domain family of transcription factors. All three genes are arranged in a gene cluster, are expressed in identical patterns in embryos, and appear to be genetically redundant. By generating mutants with a loss of all three Dorsocross genes, it has been demonstrated that Dorsocross gene activity is crucial for the completion of differentiation, cell proliferation arrest, and survival of amnioserosa cells. In addition, the Dorsocross genes are required for normal patterning of the dorsolateral ectoderm and, in particular, the repression of wingless and the ladybird homeobox genes within this area of the germ band. These findings extend knowledge of the regulatory pathways during amnioserosa development and the patterning of the dorsolateral embryonic germ band in response to Dpp signals (Reim, 2003).

Since peak levels of Dpp activity are known to be required for cell fate determination at the dorsal midline, the correlation between Dpp activity and dorsal longitudinal Doc expression during blastoderm stages was examined. Double-staining for Doc mRNA and phosphorylated Mad (PMad) indicates a close correlation between cells containing high levels of PMad and Doc products within the dorsal-longitudinal stripe. In addition, faint Doc signals that are modulated in a pair-rule pattern extend into areas that receive lower Dpp inputs and lack detectable PMad. Both PMad and Doc expression in the dorsal-longitudinal stripe, but not the dorsal-transverse head stripe of Doc expression, are absent in dpp-null mutant embryos. Conversely, in blastoderm embryos with ubiquitous Dpp expression (UAS-dpp activated by maternally provided nanos-GAL4), a significant widening is observed of the dorsal-longitudinal stripes of PMad and Doc expression, during which the correlation between high PMad and Doc mRNA levels is still maintained (Reim, 2003).

The expansion of PMad upon uniform ectopic expression of dpp includes the prospective mesoderm, although not ventrolateral areas of the blastoderm embryo. However, high PMad in the prospective mesoderm does not trigger ectopic Doc expression, suggesting either the presence of a ventral repressor or the requirement for a co-activator in dorsal areas. A candidate for a co-activator is the homeobox gene zerknüllt (zen). Double in situ hybridization shows that the appearance of dorsal Doc mRNAs coincides with the time when zen mRNA levels increase in the areas of the presumptive amnioserosa as a result of high Dpp inputs. When the refinement of zen expression is completed, there is an exact correspondence in the widths of the Doc and zen expression domains -- although Doc expression extends more posteriorly, the activity of zen is necessary for normal levels of Doc expression in the dorsal-longitudinal stripe, because in zen mutant embryos there are only low residual levels of Doc products present in this domain. These observations suggest that Doc expression along the dorsal midline of blastoderm embryos requires the combined activities of dpp and zen (Reim, 2003).

The known distribution of dpp mRNA during its second phase of expression in the dorsolateral ectoderm of stage 9-11 embryos suggests that Doc expression in the dorsolateral ectoderm and mesoderm during these stages is also dependent on Dpp activity. As expected from the known fate map shifts in dpp mutants, these domains of Doc expression are missing in dpp-null mutant embryos. Notably, the exact coincidence between the ventral borders of the domains of dorsolateral Doc expression and high nuclear PMad suggests that Doc expression is directly controlled by Dpp-activated Smad proteins in the ectoderm and mesoderm during this stage. Additional evidence for this hypothesis comes from experiments with ectopic expression of dpp in the ventral ectoderm of the Krüppel domain (by virtue of a modified Kr-GAL4 driver), that results in the concomitant expansion of PMad and the Doc expression stripes towards the ventral midline (Reim, 2003).

In addition to the inputs from dpp, metameric Doc expression in dorsolateral areas of the germ band must depend on the activity of segmental regulators. A direct comparison with the expression of engrailed (en) shows that the clusters of Doc expression straddle the compartmental borders. Although Doc expression overlaps with en in the P compartments, about two-thirds of the Doc expressing cells of each cluster are located in posterior areas of the A compartments. In agreement with this allocation, it has been found that the metameric Doc domains are exactly centered on the stripes of Wingless (Wg) expression. The observed correlation of the segmental registers of Wg and Doc makes wg a good candidate for an upstream regulator of Doc. Dorsolateral Doc expression in the ectoderm and mesoderm is shown to be completely absent if wg is inactive. By contrast, deletion of sloppy paired (slp), a known target of wg in the mesoderm and a wg feedback regulator in the ectoderm, results in a reduction, but not a complete loss of metameric DOC expression. Hence, slp probably affects Doc indirectly through its effect on ectodermal wg expression. Altogether, the data suggest that metameric Doc expression in the ectoderm and mesoderm is triggered by the intersecting activities of Wg and Dpp (Reim, 2003).

Targets of DPP during neurulation

An important question in neurobiology is how different cell fates are established along the dorsoventral (DV) axis of the central nervous system (CNS). The origins of DV patterning within the Drosophila CNS have been investigated. The earliest sign of neural DV patterning is the expression of three homeobox genes in the neuroectoderm -- ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) -- which are expressed in ventral, intermediate, and dorsal columns of neuroectoderm, respectively. Previous studies have shown that the Dorsal, Decapentaplegic (Dpp), and EGF receptor (Egfr) signaling pathways regulate embryonic DV patterning, as well as aspects of CNS patterning. This study describes the earliest expression of each DV column gene (vnd, ind, and msh), the regulatory relationships between all three DV column genes, and the role of the Dorsal, Dpp, and Egfr signaling pathways in defining vnd, ind, and msh expression domains. The vnd domain is established by Dorsal and maintained by Egfr, but unlike a previous report vnd is found not to be regulated by Dpp signaling. ind expression requires both Dorsal and Egfr signaling for activation and positioning of its dorsal border, and abnormally high Dpp can repress ind expression. The msh domain is defined by repression: it occurs only where Dpp, Vnd, and Ind activity is low. It is concluded that the initial diversification of cell fates along the DV axis of the CNS is coordinately established by Dorsal, Dpp, and Egfr signaling pathways. Understanding the mechanisms involved in patterning vnd, ind, and msh expression is important, because DV columnar homeobox gene expression in the neuroectoderm is an early, essential, and evolutionarily conserved step in generating neuronal diversity along the DV axis of the CNS (Von Ohlen, 2000).

Early stage 5 embryos express vnd in a narrow domain similar to its final width; ind and msh are not detected. By the end of stage 5, both vnd and ind are expressed with a one to two cell wide gap; again, this expression is seen in domains similar to their final widths. The gap fills in during development resulting in the precise juxtaposition of the vnd and ind domains. Expression of msh in the trunk is not detected until stage 7. Thus, the timing of gene expression progresses from ventral to dorsal: vnd is detected first, ind appears soon after, and msh is observed last (Von Ohlen, 2000).

Although Dpp is not required for any aspect of ind expression in wild type embryos, ectopic Dpp signaling in the neuroectoderm can repress ind expression. This shows that Dpp signaling must be kept low in the intermediate column to allow ind transcription and raises the possibility that the loss of ind expression seen in dorsal embryos is an indirect effect, due to the de-repression of Dpp activity within the neuroectoderm. dorsal;dpp double mutants fail to express ind, however, proving that loss of ind expression in dorsal mutants is not due to de-repression of Dpp within the neuroectoderm. It is proposed that Dorsal must both activate ind expression and repress Dpp signaling to allow ind expression (Von Ohlen, 2000).

msh is expressed in a DV domain that has low Vnd, Ind, and Dpp activity. Overexpression of any of these genes will repress msh expression, and dorsal;dpp embryos that lack all vnd, ind, and dpp expression show ectopic msh expression around the DV axis. Thus, the borders of the msh domain are defined by repression: Vnd and Ind ventrally, and Dpp dorsally. What activates msh expression? msh expression could be activated by 'basal' transcription factors present uniformly in the early embryo. Alternatively, msh expression may be induced by a low level of ubiquitous TGFbeta activity, similar to the observed activation of zebrafish msh homologs. The screw gene encodes a TGFbeta-like protein expressed at low levels throughout the embryo, and although it has no striking CNS phenotype, it would be interesting to see if screw;dpp embryos lose dorsal msh expression, or whether screw;dorsal;dpp embryos lose global msh expression (Von Ohlen, 2000).

Determining the position of ventro-lateral neuroectoderm versus dorsal non neural ectoderm is controlled by maternal (dorsal) and zygotic genes (dpp, sog, brk, sna, twi). SoxNeuro (SoxN) expression is specifically affected in these mutants. dl mutants lack early SoxN expression. Embryos mutants for dpp show a dorsal expansion of SoxN expression, as also observed when misexpressing sog by means of the Gal4 system. Inversely, misexpressing dpp early in embryogenesis leads to severe reduction of SoxN expressing-cells, as observed in sog mutants and sog, brk double mutants. Finally, twi mutants are characterized by a ventral expansion of SoxN expression into the presumptive mesoderm. These experiments are consistent with a role for the D/V patterning genes in the control of SoxN expression, with SoxN being negatively regulated dorsally and ventrally by dpp and mesoderm genes, and positively by sog and brk in the neuroectodermal region. A similar situation has been reported in Xenopus, with SoxD, an essential mediator of neural induction, being negatively regulated by BMP4 and positively by chordin (the vertebrate homologs of dpp and sog, respectively) (Cremazy, 2000).

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)

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).

Targets of DPP during segmentation

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

Different dorsal ectoderm genes were examined in a variety of mutant and transgenic embryos using digoxigenin-labeled RNA probes and in situ hybridization. The normal expression patterns suggest the occurrence of at least three thresholds of gene activity in response to the Dpp/Scw activity gradient. The Race and hindsight/pebbled (hnt) genes are expressed in narrow strips in the dorsalmost regions of the embryo, although the anteroposterior limits of the two patterns are distinct. It is conceivable that early-acting segmentation genes are responsible for repressing Race in posterior regions and hnt in anterior regions. Somewhat broader expression patterns are observed for tup and ush. These patterns encompass the presumptive amnioserosa and dorsal regions of the dorsal epidermis. Broad staining patterns are observed for two genes encoding GATA transcription factors, dGATAc and pnr. pnr is expressed throughout the dorsal ectoderm in the presumptive thorax and abdomen. dGATAc exhibits a nearly reciprocal pattern in anterior and posterior regions; staining is mainly excluded from regions expressing pnr, although a weak patch of staining is detected in a portion of the presumptive amnioserosa. Most of the subsequent analyses on gradient thresholds have focussed on the regulation of hnt, tup and pnr (Ashe, 2000).

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

The dpp stripe results in an expansion in both the hnt and ush expression patterns. The broadening of these patterns is particularly evident in anterior regions in the vicinity of the eve stripe. Increases in dpp+ gene dose do not expand the pnr expression pattern. For example, four copies of dpp+ result in augmented levels of pnr expression, but the dorsoventral limits of expression are essentially normal. The stripe2-dpp transgene has no obvious effect on the early sog and brk expression patterns (Ashe, 2000).

Previous studies have shown that the pnr expression pattern expands into neurogenic regions in brk- mutant embryos. No such expansion was observed for other Dpp/Scw target genes, including ush. To test the role of the Brk repressor in establishing the different responses of Dpp target genes, the brk-coding sequence was attached to the eve stripe 2 enhancer. Transgenic embryos carrying the stripe2-brk transgene exhibit both the normal pattern (lateral stripes) in the neurogenic ectoderm as well as an ectopic stripe of expression. pnr is normally expressed in a series of 5 stripes in the dorsal ectoderm. The anteriormost stripe is lost in transgenic embryos carrying the stripe2-brk fusion gene. This result suggests that Brk is sufficient to repress pnr in an ectopic location in the embryo (Ashe, 2000).

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

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

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

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

Previous studies have identified mutations in the Drosophila homolog of the mammalian CBP histone acetyltransferase gene, nejire. nej is maternally expressed so that the detection of early patterning defects depends on the analysis of embryos derived from females containing nej germline clones. The complete loss of nej+ activity results in a failure to make mature eggs. However, it is possible to obtain embryos from a strong hypomorphic allele, nej1. These embryos exhibit dorsoventral patterning defects. Recent studies have shown that CBP interacts with Smad proteins including the Drosophila protein Mad, a transcription factor downstream of Dpp signaling. In nej mutant embryos, there is a loss of the amnioserosa and other derivatives of the dorsal ectoderm. The expression of target genes requiring peak levels of Dpp signaling is essentially abolished. For example, hnt expression is lost in the presumptive amnioserosa, but persists at the posterior pole where it might be separately regulated by the torso signaling pathway (Ashe, 2000).

There is a similar loss of the dorsal rho pattern in mutant embryos. In contrast, the lateral, neurogenic stripes are unaffected, indicating that the nej mutant does not cause defects in the patterning of the neurogenic ectoderm. Moreover, the fact that the rho stripes are excluded from ventral regions, as seen in wild-type embryos, suggests that the patterning of the mesoderm is also normal. Thus, the nej mutation does not appear to cause a general loss of transcriptional activation, but instead results in specific patterning defects in the dorsal ectoderm. Target genes that are activated by lower levels of Dpp signaling such as ush and pnr are also affected by the nej mutation. In the case of ush, there is a loss of staining in central regions of the dorsal ectoderm. Moreover, the residual staining pattern is narrower than the wild-type pattern. This is reminiscent of the ush pattern seen in dpp/+ heterozygotes. However, the nej mutation also causes a narrowing of the pnr pattern, whereas expression is normal in dpp/+ embryos (Ashe, 2000).

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

Targets of DPP during limb allocation

aristaless is involved in the allocation of cells to the most distal elements of appendages. aristaless is expressed in the thorax at the intersection of Wingless stripes (running dorsal to ventral), with stripes of DPP (running anterior to posterior). Ectopic expression of aristaless is induced by ectopic wingless in regions expressing dpp. One or two cells expressing aristaless then invaginate with the formation of imaginal disc primordia, and are allocated to the distal-most element of appendages (Campbell, 1993).

wingless, dpp and hh are required for allocation of cells to the thoracic imaginal primordia in the germ band extended embryo (corresponding to phase three of dpp expression). Narrow horizontal stripes of DPP intercept vertical stripes of WG secreting cells to form a ladder-like pattern in the ectoderm. It is at points where WG and DPP stripes intersect that wing and leg imaginal discs are specified and distal-less is induced (Cohen, 1993).

A third signaling molecule, Hedgehog, is also required for distal-less induction. HH is secreted from the posterior compartments of imaginal discs. These three secreted signaling molecules, HH, WG and DPP, specify the distal-axis of imaginal discs, and each is required for distal-less induction. (Cohen, 1993 and Diaz-Benjumea, 1994).

Two thoracic limbs of Drosophila, the leg and the wing, originate from a common cluster of cells that include the source of two secreted signaling molecules, Decapentaplegic and Wingless. Wingless, but not Decapentaplegic, is responsible for the initial distal identity specification of the limb primordia. Proximal limb precursors expressing escargot encircle the Distal-less expressing distal primordium. Dll expressing cells show a dynamic cell migration in the early stage of limb formation, migrating basally during stage 12. Cells that have just started to express Dll also express thickveins. This suggests a requirement for regulated Dpp signaling at the level of receptor expression. Limb formation is restricted to the lateral position of the embryo through exertion of negative control by Decapentaplegic and the EGF receptor, both of which determine the global dorsoventral pattern. dpp specifies proximal cell identities. In the absence of dpp Escargot and Snail are lost. A late function of Decapentaplegic locally determines additional cell identities in a dosage dependent manner. Loss of Decapentaplegic activity results in a deletion of the proximal structures of the limb, in contrast to the deletion of distal structures when decapentaplegic mutations affect the imaginal disc. The limb pattern elements appear to be added in a distal to proximal direction in the embryo, which is just the opposite of what is happening in the growing imaginal disc. It is proposed that Wingless and Decapentaplegic act sequentially to initiate the proximodistal axis. This model is contrary to that of Cohen (1993) who argues that Dpp and Wingless are both required to induce the limb. Since Dll expression persists and expands dorsally in the absence of Dpp, it is clear that Dpp plays no role in inducing initial Dll expression but that the dorsoventral limit of Dll expression is defined by repression as a result of Dpp expression. Similarly, EGF-R is required to repress Dll expression in the ventral ectoderm (Goto, 1997).

Primary neurogenesis in the central nervous system of insects and vertebrates occurs in three dorsoventral domains on either side of the neuroectoderm. Among the three dorsoventral domains of the Drosophila neuroectoderm, the medial and lateral columns express the zinc-finger gene escargot (esg), whereas the intermediate column does not. esg expression was examined as a probe to investigate the mechanism of neuroectoderm patterning. The effect of dorsoventral patterning genes on esg expression was studied. decapentaplegic, snail and twist repress esg expression outside the neuroectoderm. The expression of esg in the intermediate column is normally repressed, but is de-repressed when Egfr activity is either elevated or reduced. A neurogenic enhancer of esg was identified, and shown to be separable into a distal region that promotes ubiquitous expression in the neuroectoderm and a proximal region that represses the intermediate expression. It is concluded that decapentaplegic, snail, twist and an activator all act through the distal region to initiate transcription of esg in the neuroectoderm. It is proposed that the combination of opposing gradients of Egfr and its ligand creates a peak of Egfr activity in the intermediate column, where Egfr represses esg transcription through the proximal repressor region. These two kinds of regulation establish the early esg expression that prefigures the neuroectoderm patterning (Yagi, 1997).

Targets of DPP during limb outgrowth

Limb development requires the formation of a proximal-distal axis perpendicular to the main anterior-posterior and dorsal-ventral body axes. The secreted signaling proteins Decapentaplegic and Wingless act in a concentration-dependent manner to organize the proximal-distal axis. Discrete domains of proximal-distal gene expression are defined by different thresholds of Decapentaplegic and Wingless activities. distal-less is expressed in a central domain that corresponds to the presumptive tarsal segments and the distal tibia. The dachshund gene is required for development of the femur and tibia. Dac is expressed in a ring corresponding to the presumptive femur, tibia and first tarsal segment, but is absent from the more distal tarsal segments of the leg disc. Although there is little or no overlap between Dll and Dac domains at early stages, by mid third instar the combination of Dac and Dll expression defines three regions along the P-D axis. Dll and Dac are expressed in circular domains centered on the point at which the ventral Wg domain and the dorsal Dpp domain meet. Dll expression in the center of the disc depends on the combined activities of wg and dpp. Wg and Dpp act directly to induce Dll, as analysis of constitutively active Thick-veins clones has shown (Tkv is the receptor for Dpp); analysis of shaggy/zeste white 3 clones (Sgg is required for transduction of the Wingless signal) reveals that both Wg and Dpp transduction pathways are activated cell autonomously. Continuous signalling is not required to maintain Dll or Dac expression. The spatial domains of Dac and Dll expression are defined by different threshold levels of both Wg and Dpp activities. Both Dpp and Wg act to directly repress Dac in the center of the disc. Dac repression is actively maintained by Wg and Dpp signaling long after Dac and Dll have been induced and are stably expressed in the absence of further signaling. Subsequent modulation of the relative sizes of these domains by growth of the leg is required to form the mature pattern (Lecuit, 1997).

Overexpression of dpp reveals an involvement in wing and leg patterning. High levels of dpp lead to reduction of the scutellum, loss of one or more scutellar bristles, and duplication of posterior wing structures. In leg discs, intermediate increases in dpp cause the loss of ventral leg structures with the concomitant fusion of left and right dorsal forelegs. Supernumerary leg bifurcations are evident, and they arise exclusively from the anterior-ventral region of the leg. The defects in legs and wings appear to arise through dose-dependent effects of dpp on wingless expression. A high level of dpp in the wing disc causes a reduction of wg expression in the presumptive scutellar region. Intermediate dpp levels in leg discs induces the expansion of wg expression into the ventral outgrowth, while high dpp expression eliminates ventral wg expression. It is thought that a critical role for dpp in leg and wing discs is to reduce or eliminate the expression of wg. Consistent with this role of dpp is the observation that ectopic wg expression is detected in imaginal discs when dpp signaling is impaired by lowering the activity of thick veins, one of the three known dpp receptors. Reciprocal interactions between wg and dpp appear to be important in the subdivision of the leg disc between dorsal and ventral compartments. Inhibition of wg expression by dpp in the dorsal anterior portion of the leg disc would help limit the intersection of dpp and wg expression to one site, providing a single distal organizer for the disc (Morimura, 1996).

wingless is transcribed in a ventral sector of the leg disc throughout development while dpp is transcribed in a stripe that spans the disc, although its expression is more intense dorsally than ventrally. DPP and Wingless specify respectively dorsal and ventral cell fates in the leg disc. It is suggested that expression of the ventral DPP half-stripe is nonfunctional and that inhibition of ventral DPP function could be mediated by WG. It is proposed that DPP specificies cell position relative to the dorsal midline in the leg disc, a role analogous to the role of DPP in the early embryo where it specifies fates within the dorsal 40% of the ectoderm relative to the dorsal midline (Held, 1994).

In the leg disc, HH is secreted by posterior cells and acts at short range to induce dorsal anterior cells to secrete DPP and ventral anterior cells to secrete WG. Complementary patterns of dpp and wg expression are maintained by mutual repression. DPP signaling blocks wg transcription and WG signaling attenuates dpp transcription. This repression is essential for normal axial patterning because it ensures that the dorsalizing and ventralizing activities of DPP and WG are restricted to opposite sides of the leg primordium and meet only at the center of the primordium to distalize the appendage. A similar dorsoventral bias in the choice of dpp or wg expression is revealed by eliminating the activity of protein kinase A, an experimental intervention that mimics the reception of the HH signal. Constitutive activation of the WG signal transduction pathway by loss of Zeste white (Shaggy) kinase mimics the reception of WG signal, and is sufficient to bias dorsal cells to express wg rather than dpp. Interfering with DPP signaling with dorsally situated thick veins mutant clones results in the dorsal expression of wg and expression of aristaless, which usually marks the central domain, in the periphery. Interfering with WG signaling with ventrally situated dishevelled mutant clones (dsh is required for transduction of the WG signal), results in the ventral expression of dpp (Jiang, 1996).

Homothorax is shown to limit Dpp and Wg expression. Expression of the Dpp and Wg targets omb and H15 is restricted to those cells that do not express Hth. To determine if hth inhibits target gene activation by Dpp and Wg, hth was either removed from its endogenous domain or either a GFP-Hth fusion protein or the murine hth homolog MEIS-1B was misexpressed in the distal portion of the leg disc. Removing hth function results in the expansion of wg and dpp target gene expression. Dorsally situated hth- clones result in the expansion of omb expression, as marked by the omb-lacZ reporter gene. Does hth repress Distal-less and dachshund? Similar to removing exd function, when hth loss-of-function clones were examined, dac was found to be only partially derepressed, and derepression was found to be more likely to occur in clones that arise near endogenous dac expression. hth- clones have no effect on Dll expression, regardless of where they are situated. However, when clones of GFP-Hth- or MYC-MEIS-expressing cells are generated, both Dll and dac can be repressed. These results suggest that the expression of Dll and dac requires two conditions: (1) the absence of Hth and (2) sufficient activity in the Dpp and Wg pathways. High levels of Wg and Dpp signaling are shown to repress the nuclear localization of Exd by repressing hth transcription. The direct action of both the Wg- and Dpp-signaling pathways is required to specify cell fates along the P/D axis. High levels of Wg and Dpp signaling are required to activate Dll, a determinant of distal cell fates, and to repress expression of dac, a determinant of intermediate fates along the P/D axis. At intermediate levels of Wg and Dpp signaling, dac, but not Dll, is activated. The distal edge of hth expression coincides with the proximal edge of dac expression, suggesting that the threshold of Dpp and Wg signaling required to activate dac is similar to that required to repress hth. To test this idea, either Wg or Dpp signaling was elevated in the hth expression domain by generating clones of cells that express either a membrane-tethered form of Wg or an activated Dpp receptor, Thickveins QD (TKV QD). When Wg-expressing clones were generated dorsally, where endogenous Wg levels are low but where Dpp is present at high concentrations, there was a loss of Hth protein and a shift of Exd protein to the cytoplasm. This suggests that sufficient levels of both Wg and Dpp signaling are required to repress Hth (Abu-Shaar, 1998).

High levels of Wg and Dpp signaling are shown to affect Hth and Exd, at least in part, by repressing hth transcription. The ability of Wg and Dpp to repress hth appears to be indirectly mediated by Dll and dac. Like Dll, Dac appears to have the capacity to repress hth. The expression patterns of Hth and Dll were examined in leg discs that were entirely devoid of dac function. In these discs, the hth domain appears expanded distally and the Dll domain appears to be expanded proximally, consistent with the idea that dac normally represses both hth and Dll (Abu-Shaar, 1998).

The domains of gene expression for Hth, Dac and Dll, as well the regulatory interactions between them, suggest that the leg is functionally divided into two major domains. The first is a proximal domain, which expresses hth, has nuclear Exd and does not express at least some of the potential target genes of the Wg- or Dpp-signaling pathways. The second is a distal domain, which does not express hth, has Exd localized to the cytoplasm, and expresses the targets of Wg, Dpp and Wg+Dpp signaling. These data suggest that the proximal domain is what has been referred to as the coxopodite, or an extension of the body wall, and is distinct from the distal domain, the telopodite. hth expression and nuclear Exd in the coxopodite would restrict the ability of the Wg and Dpp signals to activate their target genes. This idea is consistent with the observation that these two domains differ with respect to their requirement for Hh signaling: unlike the telopodite, which exhibits severe truncations upon the reduction of hh function, the coxopodite is less severely affected. These two domains also appear to have different cell surface properties; cells from one domain prefer not to mix with cells from the other domain. For example, Dll mutant clones almost always relocalize to the hth-expressing domain and hth mutant clones frequently sort into distal regions of the leg disc. This phenomenon is not observed in the wing disc, where hth and Dll are restricted to outside and within the wing pouch, respectively: hth or Dll mutant clones are positioned randomly in this tissue. The mutant phenotypes displayed by the loss of coxopodite gene function are qualitatively different from those displayed by the loss of telopodite gene function. Removal of coxopodite genes such as exd results in either ‘nonsense’ or proximal to distal cell fate transformations, whereas removal of telopodite gene functions such as Dll and dac results in deletions of the appendage. In summary, the data support the idea that the proximal and distal regions of the leg have independent origins and differ from each other primarily due to the expression of hth, which limits or alters the ability of proximal cells to respond to Wg and Dpp signaling (Abu-Shaar, 1998 and references).

dac and Dll are shown to mediate Wg and Dpp mediated repression of hth. The demonstration that Wg and Dpp signaling repressed hth transcription and Exd’s nuclear localization was surprising, because these two signaling molecules induce Exd’s nuclear localization in the endoderm of the embryonic midgut. An investigation was carried out into the possibility that the repression of hth by Wg and Dpp is indirect and perhaps mediated by dac and Dll, which are not expressed in the midgut. TKV QD-expressing clones were generated and Hth, Dll and Dac were examined. Loss of function clones of Dll and dac were generated. When Dll- clones were generated before ~72 hours of development, hth was found to be derepressed and Exd was nuclear. However, clones generated after ~72 hours have no effect on hth or Exd, suggesting that there is an alternative mechanism for maintaining hth repression. Like Dll, Dac appears to have the capacity to repress hth. The ability of Dac to repress hth expression was confirmed by generating dac- clones. These dac- clones suggest that there might be other regulators of hth in addition to dac and Dll. Completely removing dac function results in viable animals that have deletions along the P/D axes of their legs. The expression patterns of Hth and Dll were examined in leg discs that were entirely devoid of dac function. In these discs, the hth domain appears expanded distally and the Dll domain appears to be expanded proximally, consistent with the idea that dac normally represses both hth and Dll. It is an apparent paradox that Wg and Dpp repress hth in the leg disc while these same signals activate hth expression and nuclear Exd in the midgut endoderm. This may be explained because in the leg, Wg and Dpp repress hth indirectly, by activating the hth repressors Dll and dac. In the absence of Dll or dac, hth is derepressed in the leg disc, even in cells that receive high levels of the Wg and Dpp signals. In contrast, in the embryonic endoderm, dac and Dll are not activated by Wg and Dpp, nor are any other known hth repressors, allowing hth to be activated in these cells (Abu-Shaar, 1998).

In the developing wing blade, somatic clones lacking the DPP receptors Punt or Thick veins, or lacking Schnurri, a transcription factor targeted by DPP signaling, fail to grow when induced early in larval development. tkv and shn are also required for vein cell differentiation. Drosophila wing veins are dorsoventrally asymmetrical, in that some protrude on the dorsal wing serface while others proturde on the ventral surface. tkv and shn mutant clones cause loss of veins when they are present on the dominant (protruding) side of the vein. Although dpp is expressed in only a subset of cells in the anterior compartment of the developing wing disc, all cells of the early prospective wing blade require tkv, punt and shn. This implies that DPP must diffuse away from its source to fulfill its function. Secreted DPP molecules would have to travel at least 4 cell diameters in order to signal to all future wing blade cells in the posterior compartment (Burke, 1996).

araucan and caupolican, two members of the iroquois gene complex, are highly related proteins belonging to a new family of homeoproteins. ARA and CAUP regulate the pattern elements (sensory organs and veins) in wing imaginal discs by spatially restricting domains of expression of the proneural genes achaete and scute and the provein gene rhomboid. ara-caup expression is restricted to two symmetrical patches located one at each side of the dorsoventral compartment border. ara-caup expression in these patches is necessary for the specification of the prospective vein L3 and associated sensory organs. Here, ara-caup expression is mediated by the Hedgehog signal through its induction of high levels of Cubitus interruptus in anterior cells near the the AP compartment border. The high levels of CI activate decapentaplegic expression, and together, CI and DPP positively control ara-caup. patched overexpression is equivalent to a reduced hh function in that accumulation of CI and DPP at the AP border are strongly depressed. The wing pouch of patched mutants have much reduced or absent ara-caup L3 patches. dpp by itself is insufficient to account for ara-caup expression (Gómez-Skarmeta, 1996).

Dpp signaling promotes the cuboidal-to-columnar shape transition of Drosophila wing disc epithelia by regulating Rho1

Morphogenesis is largely driven by changes in the shape of individual cells. However, how cell shape is regulated in developing animals is not well understood. This study shows that the onset of TGFbeta/Dpp signaling activity correlates with the transition from cuboidal to columnar cell shape in developing Drosophila melanogaster wing disc epithelia. Dpp signaling is necessary for maintaining this elongated columnar cell shape and overactivation of the Dpp signaling pathway results in precocious cell elongation. Moreover, evidence is provided that Dpp signaling controls the subcellular distribution of the activities of the small GTPase Rho1 and the regulatory light chain of non-muscle myosin II (MRLC). Alteration of Rho1 or MRLC activity has a profound effect on apical-basal cell length. Finally, it was demonstrated that a decrease in Rho1 or MRLC activity rescues the shortening of cells with compromised Dpp signaling. These results identify a cell-autonomous role for Dpp signaling in promoting and maintaining the elongated columnar shape of wing disc cells and suggest that Dpp signaling acts by regulating Rho1 and MRLC (Widmann, 2009).

Cell extrusion was observed when Dpp signaling was locally reduced in tkva12 bsk- clones, but not when it was reduced throughout the dorsal compartment by expression of Dad. This indicates that cell extrusion is a consequence of the sharp boundary of Dpp signaling at the clone border. One of the first morphological consequences of the loss of Dpp signaling in tkva12 bsk- clones was the apical constriction of mutant cells and surrounding control cells. Apical constriction correlated with increased staining intensities of F-actin and P-MRLC, a marker for active non-muscle myosin II, at the apicolateral side of tkva12 bsk- and neighboring wild-type cells. The formation of a similar actin-myosin ring has been previously demonstrated during the extrusion of apoptotic cells, and it has been proposed that contraction of this ring squeezes cells out of the epithelium. It is currently unclear whether these increased staining intensities reflect an increase in the total amount of F-actin and P-MRLC in tkva12 bsk- mutant clones, or whether they are instead merely a consequence of the apical constriction of cells. Nevertheless, these findings are consistent with the view that contraction of an actin-myosin ring might contribute to the extrusion of tkva12 bsk- cells. Apical cell constriction was paralleled with cell shortening along the apical-basal axis. Based on the observation that reduction in Dpp signaling throughout the wing disc pouch resulted in apical-basal cell shortening, but not in apical cell constriction, it is speculated that cell shortening, and thus the formation of an inappropriate cell shape, might be an initial event leading to the extrusion of tkva12 bsk- cells. If so, cell extrusion might not represent a specific response to eliminate slow-growing or apoptotic cells, but rather represents a general response to inappropriate cell function or morphology. In the wild type, cell extrusion might be instrumental in maximizing tissue fitness by removing cells with inappropriate function or morphology (Widmann, 2009).

The basal membrane of tkva12 bsk- cells and neighboring control cells, identified by PSβ-integrin labeling, became apposed. Since this led to a reduction in the lateral contact between mutant and neighboring control cells, this apposition might help to dislodge tkva12 bsk- cells from the remaining epithelium, and thereby, might aid the extrusion process. It is also noted that extruded tkva12 bsk- cells displayed features reminiscent of epithelial-to-mesenchymal transition (EMT). In particular, a strong decrease in E-cadherin, a hallmark of EMT and actin-rich processes were observed in extruded tkva12 bsk- cells. Interestingly, a role for Dpp/BMPs in preventing EMT has also been identified in vertebrates. Mouse BMP7, which is related to Dpp, for example, is required for counteracting EMT associated with renal fibrosis. Decreased E-cadherin levels have also recently been reported following the extrusion of cells deficient for C-terminal Src kinase from Drosophila epithelia, indicating that this might be a more common consequence of cell extrusion (Widmann, 2009).

Reduced apical-basal cell length was observed when Dpp signaling was severely reduced, either in clones or throughout the wing disc pouch; however, apical cell constriction, fold formation and cell extrusion were only detected by clonal reduction of Dpp signaling. Instead, cells were apically widened and did not extrude when Dpp signaling was reduced throughout the dorsal compartment. These experiments therefore allowed the effects of sharp boundaries of Dpp signaling at clone borders to be separated from cell-autonomous functions of Dpp signaling. They demonstrate that the cell-autonomous function of Dpp signaling is not to prevent apical cell constriction, folding and cell extrusion, but rather to maintain proper columnar cell shape. Moreover, three further observations suggest that Dpp signaling has an instructive role that drives cell elongation. (1) In the wild type, an increase in Dpp signal transduction activity correlated with apical-basal cell elongation in second instar larval discs. (2) In wing discs of late third instar larvae, Dpp signal transduction activity correlated with apical-basal cell length along the anteroposterior axis. (3) Activation of Dpp signaling, by expressing the constitutively active Dpp receptor TkvQ-D, resulted in precocious cell elongation and apical cell narrowing during early larval development. These findings indicate that Dpp signaling is an important trigger for the cuboidal-to-columnar transition in cell shape that occurs during mid-larval development (Widmann, 2009).

How does Dpp signaling promote the apical-basal elongation of wing disc cells? Compartmentalization of Rho1 activity has been recognized as being important for shaping cells and tissues. In the wild-type wing disc, Rho1 protein is enriched and the activity of the Rho1 sensor is increased at the apicolateral side, and more moderately at the basal side, of elongated cells. By contrast, Rho1 activity is more uniform in cuboidal cells, and overexpression of RhoGEF2, which leads to uniform distribution of this protein and presumably also uniform Rho1 activity, resulted in a cuboidal cell shape. Rho1, when present at the apicolateral side of cells, might therefore have a function in stabilizing or promoting cell elongation. Since the apicolateral increase in Rho1 sensor activity correlated with an increase of P-MRLC at a similar location, this function of Rho1 might be mediated by myosin II. The observation that a decrease in the bulk of Rho1 activity, either through expression of Rho1N19 or rho1dsRNA, resulted in cell elongation rather than in cell shortening, further suggests that the compartmentalization of Rho1 activity is important for shaping wing disc cells. Future studies will need to examine the morphogenetic consequences of locally modulating the activity of Rho1 (Widmann, 2009).

The results provide strong evidence for a functional link between Dpp signaling and Rho1-myosin II. Shortening of cells with compromised Dpp signaling could be rescued by a decrease in Rho1 or MRLC activity. In particular, the expression of MbsN300, an activated form of a subunit of myosin light chain phosphatase, which in wild-type wing discs did not significantly alter cell length, did rescue the shortening of Dpp-compromised cells. This indicates a specific interaction between Dpp signaling and Mbs-myosin II. The data further suggest that Dpp signaling controls apical-basal cell length by compartmentalizing Rho1 protein abundance and/or activity. (1) In late third instar wing discs, apicolateral enrichment of Rho1 protein and Rho1 sensor activity directly correlated with the local level of Dpp signal transduction activity. (2) Rho1 protein abundance and Rho1 sensor activity were decreased at the apicolateral side of cells when Dpp signal transduction was compromised by expression of Dad. (3) Rho1 protein and Rho1 sensor activity were increased at the apicolateral side and also at the basal side of cells when Dpp signal transduction was activated during early development by expression of TkvQ-D (Widmann, 2009).

Local activation of Rho1 and myosin II can lead to contraction of actin-myosin filaments, which can increase the cortical tension that is important for the shaping of cells during various developmental processes. By compartmentalizing Rho1 activity, Dpp signaling might promote both apical-basal cell elongation and apical cell narrowing. An increase in tension at the apicolateral cell cortex might promote apical cell narrowing. At the same time, a relative decrease in cortical tension laterally, compared with that on the apicolateral side, might allow cells to elongate through intrinsic cytoskeletal forces and/or extrinsic forces imposed by the growth of the epithelium. In this model, Dpp signaling directs the cuboidal-to-columnar shape transition of wing disc cells by increasing the Rho1 and myosin II activities at the apicolateral side of cells. The local increase of Rho1 and myosin II activities might then shift the balance of tension between the apicolateral cell cortex and the lateral cell cortex towards an increased tension at the apicolateral cell cortex (Widmann, 2009).

The results identify a Dpp-Brk-Rho1-myosin II pathway controlling cell shape in the wing disc epithelium. The elimination of Brk function in mad- mutant cells allowed these cells to maintain a normal columnar cell shape, indicating that Dpp controls epithelial morphogenesis through repression of Brk. Since Brk acts as a transcriptional repressor, the link between Brk and Rho1 is most probably established through an unknown Brk-repressible gene. The identification of genes transcriptionally repressed by Brk will thus be important for determination of how Dpp signaling controls Rho1 and thereby, epithelial cell shape. The finding that Dpp signaling has a cell-autonomous morphogenetic function indicates that Dpp signaling provides a connection between cell-fate specification, cell growth and the control of morphogenesis. It, thereby, might help to facilitate the coordination of these processes during wing disc development (Widmann, 2009).

Given the evolutionary conserved functions of Rho and myosin II, it is anticipated that the mechanisms regulating columnar cell shape, which are describe in this study for the wing disc, will also operate in a wide range of other epithelia. Moreover, the role of TGFβ/Dpp signaling in patterned morphogenesis appears to be conserved in vertebrates, raising the possibility that Rho and myosin II are common mediators of TGFβ/Dpp signaling (Widmann, 2009).

Table of contents

decapentaplegic: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effect of mutation | References

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