Table of contents

Targets of DPP in sense organ patterning

Wingless and Decapentaplegic cell signaling pathways act synergistically in their contribution to macrochaete (sense organ) patterning on the notum of Drosophila. The analysis of the origin of sense organ precursor prepatterning has focussed on the specification and positioning of the anterior and posterior dorsocentral macrochaetes (aDC, pDC) two large mechanosensory organs located in precise positions relative to surrounding rows of microchaetes. The aDC and pDC SOPs form sequentially on the proximal edge of a single DC proneural cluster where Achaete and Scute expression depends on a cis-activating enhancer sequence, the DC enhancer. Ac expression in the DC proneural cluster requires the activity of wingless. The DC SOPs form adjacent to the stripe of cells expressing wg in the presumptive notum during the third larval instar. To probe the nature of gene interaction required for macrochaetae formation, the Wingless-signaling pathway was ectopically activated by removing Shaggy activity (the homolog of vertebrate glycogen synthase kinase 3) in mosaics. Proneural activity is asymmetric within the Shaggy-deficient clone of cells and shows a fixed polarity with respect to body axis, independent of the precise location of the clone. This asymmetric response indicates the existence in the epithelium of a second signal, possibly Decapentaplegic. Ectopic expression of Decapentaplegic induces extra macrochaetes only in cells that also receive the Wingless signal. Outside the Wg-activated domain, in the medial scutum and prescutum, clones that ectopically express Dpp make only microchaetes. In the Wg-activated domain, within and lateral to the DC meridian, clones of cells ectopically expressing dpp are associated with many extra macrochaetes, which are formed both within and around the Dpp-expressing clones. It is concluded that in areas of the notum where the WG transduction pathway is inactive, Dpp alone is insufficient for macrochaete formation. Activation of Hedgehog signaling generates a long-range signal (Dpp) that can promote macrochaete formation in the Wingless activity domain. This signal depends on decapentaplegic function. Autonomous activation of the Wingless signal response in cells causes them to attenuate or sequester this signal. Extramacrochaetae (a proneural antagonist) is required to limit the anterior/posterior extent of this cluster. If the level of emc is reduced, extra macrochaetes form primarily anterior but also posterior to the normal DCs along the proximal edge of the wg stripe. Further reduction of emc results in additional extra macrochaetes along the dorsal edge of the stripe. These results suggest a novel patterning mechanism that determines sense organ positioning in Drosophila (Phillips, 1999).

The Bar homeobox genes function as latitudinal prepattern genes in the developing Drosophila notum. In Drosophila notum, the expression of achaete-scute proneural genes and bristle formation have been shown to be regulated by putative prepattern genes expressed longitudinally. The two Bar locus genes may belong to a different class of prepattern genes expressed latitudinally: it is suggested that the developing notum consists of checker-square- like subdomains, each governed by a different combination of prepattern genes. BarH1 and BarH2 are coexpressed in the anterior-most notal region and regulate the formation of microchaetae within the region of BarH1/BarH2 expression through activating achaete-scute. Presutural macrochaetae formation also requires Bar gene activity. Bar gene expression is restricted in dorsal and posterior regions by Decapentaplegic signaling, while the ventral limit of the expression domain of Bar genes is determined by wingless, whose expression is under the control of Decapentaplegic signaling (Sato, 1999).

In developing Drosophila notum, wingless expression is regulated by positive and negative Decapentaplegic signaling so that only notal cells receiving optimal levels of Decapentaplegic signal express wingless. This Decapentaplegic-dependent regulation of notal wingless expression includes multiple mechanisms involving pannier and u-shaped. In the medial notum, Pannier and U-shaped form a complex. The expression of pannier and u-shaped is positively regulated by Decapentaplegic signals emanating from the dorsal-most region. The Pannier/U-shaped complex serves as a repressor and a transcriptional activator, respectively, for wingless and u-shaped expression. In the more lateral region, wingless expression is up-regulated by U-shaped-unbound Pannier. wingless expression is also weakly regulated by its own signaling (Sato, 2000).

To further clarify the notion that notal wg expression occurs only in cells receiving optimal levels of Dpp signal, wg expression was examined in Mothers against dpp (Mad) and thickveins (tkv) mutant clones generated at two different stages. Mad encodes a transactivator acting downstream of Dpp signals, while tkv encodes a type I receptor for Dpp. Early and late clones were generated at late first instar and late second instar, respectively; resultant clones were observed in late third instar. A dorsal shift of wg expression was detected in both early and late clones homozygous for Mad1-2, a hypomorphic mutant allele of Mad. In contrast, in the case of tkva12 (a strong hypomorphic mutant allele of tkv), a dorsal shift of wg expression was observed only in late clones. Little or no wg expression could be detected in any of the early clones. These results suggest that Dpp-signaling activity in cells within early tkva12 clones is much lower than that in Mad1-2 and late tkva12 clones, and hence, medial-notal cells in Mad1-2 and late tkva12 clones but not early tkva12 clones possess residual levels of Dpp-signaling activity, sufficient to induce wg expression. Consistent with this, twin spot analysis in the wing pouch where Dpp signals are autonomously required for cell proliferation has showen that Mad1-2 mutant clones are recovered much more frequently than tkva12 clones. The absence of wg misexpression in late medial tkva12 clones situated along the anterior notal edge is possibly due to Bar-dependent repression of wg expression in the future anterior notum (Sato, 2000).

Notal wg expression is regulated not only by dpp signaling but also by Pnr and Ush. Thus, pnr and ush expression may be under the control of Dpp signaling or conversely, Dpp signaling is regulated by pnr and ush. The second possibility, however, seems to be unlikely, since neither pnr nor ush mutant clones exhibit any appreciable change in brinker (brk)-LacZ expression. brk is a general Dpp target gene whose expression is negatively regulated by Dpp signaling. Loss of Dpp signaling causes cell-autonomous brk misexpression in the wing pouch and notum of wing imaginal discs. To determine the feasibility of the first possibility, pnr and ush expression was examined in tkva12, Mad1-2 or tkvQ253D(tkvQD) clones; tkvQD is a constitutively active form of tkv. pnr and ush are misexpressed in lateral UAS-tkvQD clones generated in late second instar, an observation indicating that pnr and ush expression is under the control of Dpp signaling. Unlike wg expression, pnr and ush expression are abolished not only in early tkva12 clones but also in late tkva12 and early Mad1-2 clones, both expressing wg, suggesting that pnr and ush expression requires higher levels of Dpp-signaling activity than those required for wg expression. Loss of ush expression in tkva12 and Mad1-2 clones might be a secondary effect due to the loss of pnr expression, since the maintenance of ush expression requires both pnr and ush activities. pnr and ush expression may be independently initiated by Dpp signaling, since pnr expression normally occurs in ush mutant clones and no ush misexpression is induced by ubiquitous pnr expression. It is concluded that the graded expression of pnr and ush is determined by Dpp signaling and hence, Pnr and Ush act downstream of Dpp (Sato, 2000).

In the larval notal region, dpp expression is not continuous but is broken by the authentic wg expression domain, thus suggesting that notal development could be regulated by Dpp signals emanating separately from dorsal and ventral sources up to the wg expression domain. As anticipated, the expression of dad (dad-LacZ), a downstream component of Dpp signaling whose expression is positively regulated by Dpp signaling, is detected not only in medial but also in lateral notum. However, double-staining of dad-LacZ and either PNR or USH RNA expression shows that unlike dad-LacZ, pnr and ush are not induced in the postero-lateral notum in spite of the presence of active Dpp signals. In addition, ectopic wg expression induced by tkvQD is restricted to the antero-lateral notum. It may thus follow that an unidentified factor represses the expression of a fraction of Dpp target genes, which include pnr, ush and wg but not dad, in the postero-lateral notum (Sato, 2000).

A summary is presented of wg regulation in the notum. In both future medial and lateral notal regions, dpp is expressed and Dpp signaling is active. However, ventral Dpp signals are neutralized by an unknown mechanism as far as pnr, ush and wg expression is concerned. Notal wg expression, except for that in the scutellum, is regulated through four different pathways, three under the control of Dpp signals emanating from the dorsal-most region. pnr and ush expression is up-regulated by Dpp signaling, but ush expression is much narrower than that of pnr, possibly because of the requirement of higher Dpp-signaling activity for ush expression than that for pnr expression. In the future medial notum, Pnr and Ush form a complex repressing wg expression, while Ush-unbound Pnr activates lateral wg expression. The authentic wg domain and the medial notum abut one another. Unlike wg expression, ush expression in the future medial notum is positively regulated by the Pnr/Ush complex. This regulation appears required for the maintenance of medial ush expression. Dpp signaling is also capable of activating notal wg expression through an unidentified factor X. This route includes neither Pnr nor Ush. In addition, wg expression is weakly up-regulated by its own signaling in the lateral notum (Sato, 2000).

The nuclear proteins Spalt and Spalt-related belong to a conserved family of transcriptional regulators characterized by the presence of double zinc-finger domains. In the wing, they are regulated by the secreted protein Decapentaplegic and participate in the positioning of the wing veins. Regulatory regions in the spalt/spalt-related gene complex have been identifed that direct expression in the wing disc. The regulatory sequences are organized in independent modules, each of them responsible for expression in particular domains of the wing imaginal disc. In the thorax, spalt and spalt-related are expressed in a restricted domain that includes most proneural clusters of the developing sensory organs in the notum, and are regulated by the signaling molecules Wingless, Decapentaplegic and Hedgehog. spalt/spalt-related are found to participate in the development of sensory organs in the thorax, mainly in the positioning of specific proneural clusters. Later, the expression of at least spalt is eliminated from the sensory organ precursor cells and this is a requisite for the differentiation of these cells. It is postulated that spalt and spalt-related belong to a category of transcriptional regulators that subdivide the thorax into expression domains (prepattern) required for the localized activation of proneural genes (de Celis, 1999).

The sal and salr genes are expressed in only part of the thorax in three domains that have been defined with reference to en, wg and ci: the thoracic posterior compartment marked by En, an adjacent stripe anterior to the anteroposterior compartment boundary corresponding to the stripe of maximal accumulation of Ci, and a zone between the stripe of wg expression and the hinge. A fourth domain in the central thorax, from where only microchaetae develop, does not express sal/salr. To explore the regulatory mechanisms that localize sal/salr expression with respect to the anteroposterior compartment boundary and wg, experiments were performed in which genes that function in developmental signaling were expressed ectopically using the Gal4 system. A series of experiments led to the conclusion that, in the thorax, dual hh signaling is required to induce sal/salr expression: signaling through dpp and signaling that is dpp independent. Thus, expression of hh in clones within the central thorax (presumably accompanied by induction of dpp) leads to ectopic expression of sal/salr; interestingly, this ectopic expression is observed both in hh-expressing cells and in adjacent cells. In contrast, ectopic expression of dpp does not result in activation of sal transcription in the thorax or in the hinge, but it does so in the wing blade. The transcription factor Cubitus interruptus (Ci), a key mediator of Hedgehog signaling, was also experimentally mis-expressed in clones of cells. Ci is only able to activate sal ectopically in the wing blade, a place where ectopic expression of Ci results in novel expression of dpp, but not in the central thorax or wing hinge. In any other tissue studied to date, Hh signaling depends on Ci; since hh positively regulates sal in the thorax, the failure of ectopic Ci to activate sal expression there may be ascribed to the presence of countermanding repressors. However, even though dpp is not sufficient to induce sal/salr in the thorax, it is required. Thus, mitotic clones of Pka (corresponding to constitutive activation of hh signaling show cell autonomous expression of sal; in contrast, Pka;dpp double mutant clones do not express sal, indicating that, close to the Dpp source, hh and dpp signaling must cooperate to activate sal expression in the thorax. In agreement with this, the expression of sal can be reduced in tkv mutant cells, which have reduced levels of a Dpp receptor. The requirement of dpp function for induction of sal differs in different parts of the thorax. In the central thorax, where sal is normally not expressed, tkv clones have no effect. In region 2, where dpp is normally expressed, tkv clones result in reduced expression of sal. In other regions of the thorax, expression of sal is unaffected by the reduction of tkv (de Celis, 1999).

Targets of DPP during oogeneis

Each of the somatic cell types of the gonad arises from mesodermal cells that constitute the embryonic gonad. The functions of the homeotic genes abdominal A and Abdominal B are both required for the development of gonadal precursors. Each plays a distinct role. abd A activity alone specifies anterior somatic gonadal precursor (SGP) fates, whereas abd A and Abd B act together to specify a posterior subpopulation of gonadal precursors. Once specified, gonadal precursors born within posterior parasegments move to the site of gonad formation. clift has been identified as a regulator of Drosophila gonadogenesis. When cloned, clift turned out to be identical to eyes absent. Mutations in clift abolish gonad formation and produce female sterility. Just as with abdominal A, clift is expressed within SGP as these cells first form, demonstrating that 9-12 cells are selected as SGP within each of three posterior parasegments at early stages in gonadogenesis. Using clift as a marker, it has been shown that the anteroposterior and dorsoventral position of the somatic gonadal precursor cells within a parasegment are established by the secreted growth factor Wingless, acting from the ectoderm, coupled with a gene regulatory hierarchy involving abd A within the mesoderm. While loss of wg abolishes gonadal precursors, ectopic expression expands the population such that most cells within lateral mesoderm adopt gonadal precursor fates. Initial dorsoventral positioning of somatic gonadal precursors relies on a regulatory cascade that establishes dorsal fates within the mesoderm. tinman appears to mediate the role of ectodermally expressed decapentaplegic; in tinman mutants few or no SGP cells are detected. clift expression is subsequently refined through negative regulation by bagpipe, a gene that specifies nearby visceral mesoderm. Thus, these studies identify essential regulators of gonadal precursor specification and differentiation and reveal novel aspects of the general mechanism whereby somatic gonadal cell fate is allocated within the mesoderm (Boyle, 1997).

During Drosophila oogenesis, Gurken, a protein associated with the oocyte nucleus, activates the Drosophila EGF receptor in the follicular epithelium. Gurken first specifies posterior follicle cells, which in turn signal back to the oocyte to induce the migration of the oocyte nucleus from a posterior to an anterior-dorsal position. From this location Gurken signals again to specify dorsal follicle cells, which give rise to dorsal chorion structures, including the dorsal appendages. If Gurken signaling is delayed and starts after stage 6 of oogenesis, the nucleus remains at the posterior pole of the oocyte. Eggs develop with a posterior ring of dorsal appendage material that is produced by main-body follicle cells expressing the gene Broad-Complex. They encircle terminal follicle cells expressing variable amounts of the TGFbeta homolog, decapentaplegic. By ectopically expressing decapentaplegic and using clonal analysis with Mothers against dpp, it has been shown that Decapentaplegic signaling is required for Broad-Complex expression. Thus, the specification and positioning of dorsal appendages along the anterior-posterior axis depends on the intersection of both Gurken and Decapentaplegic signaling. This intersection also induces rhomboid expression and thereby initiates the positive feedback loop of EGF receptor activation, which positions the dorsal appendages along the dorsal-ventral egg axis (Peri, 2000).

The Drosophila BMP homolog DPP can function as a morphogen, inducing multiple cell fates across a developmental field. However, it is unknown how graded levels of extracellular DPP are interpreted to organize a sharp boundary between different fates. Opposing DPP and EGF signals are shown to set the boundary for an ovarian follicle cell (FC) fate. First, DPP regulates gene expression in the follicle cells that will create the operculum of the eggshell. Global increase in DPP levels, using heat-shock-GAL4 to drive UAS-dpp expression throughout all FCs gives rise to eggs that show expanded opercula and reduced dorsal appendages. In other respects, the eggshells are normal. At the extreme anterior, normal micropyles were formed. The mutant opercula generally have a normal organization of large cell imprints surrounded by a raised structure, the collar. Significantly, expansion of the operculum always occurs over the dorsal side of the egg, indicating that dorsal-ventral patterning is unperturbed. DPP induces expression of the enhancer trap reporter A359 and represses expression of bunched, which encodes a protein similar to the mammalian transcription factor TSC-22. Second, DPP signaling indirectly regulates A359 expression in these cells by downregulating expression of bunched. Reduced bunched function restores A359 expression in cells that lack the Smad protein Mad; ectopic expression of Bunched suppresses A359 expression in this region. Importantly, reduction of bunched function leads to an expansion of the operculum and loss of the collar at its boundary. Third, EGF signaling upregulates expression of bunched. The bunched expression pattern requires the EGF receptor ligand Gurken. Activated EGF receptor is sufficient to induce ectopic bunched expression. Thus, the balance of DPP and EGF signals sets the boundary of bunched expression. It is proposed that the juxtaposition of cells with high and low Bunched activity organizes a sharp boundary for the operculum fate (Dobens, 2000).

Gurken signaling through the Egfr is necessary for normal bunched-lacZ expression in the dorsal anterior FC. Ectopic expression of activated Egfr is sufficient to induce ectopic bunched-lacZ in the centripetal migrating FCs. Conversely, Dpp signaling is both necessary and sufficient to repress bunched-lacZ in columnar FCs. Thus the dorsal anterior boundary of bunched-lacZ expression is set by a balance of positive EGF and negative Dpp signals. Dpp also sets the anterior boundary for Broad-Complex expression; however, the regulation of this gene by EGF signaling is more complex. In summary, a model is proposed where the boundary for the operculum is set by the boundary of Bunched activity, which is positioned by opposing activity of Dpp and EGF signals in the dorsal FCs. Dorsal anterior FC are exposed to high levels of EGF ligands Grk, Spitz and Vein, and thus have elevated bunched expression. High anterior Dpp signaling represses bunched expression. The close apposition of these signals in the dorsal anterior FCs creates a sharp boundary of bunched expression. BUN-1 functions to repress A359 and define the boundary to centripetal migrating FC fates, including the operculum. These data indicate that the ventral operculum boundary is also set by bunched; however, another signal appears to promote ventral bunched expression at late stages. The normal operculum border is defined by the eggshell collar. This structure is lost as bunched activity is lowered, suggesting that the boundary of bunched expression may serve to further organize cell fates at the operculum boundary (Dobens, 2000).

Although the data suggest that EGF signals antagonize operculum patterning, EGF signaling is essential for operculum formation. (1) grk and Egfr mutant eggs have no opercula. (2) Overexpression of activated Egfr can result in operculum expansion, although interpretation of the specific phenotype is not straightforward. Thus, it is expected that Dpp does not prevent all Egfr-induced events in the operculum-forming FCs. It is likely that EGF signaling is active in cells that lack Bunched activity, and that Dpp inactivation of Bunched modifies the response of these cells to EGF signals. In cultured mammalian cells, RTK signaling can directly antagonize BMP signaling by preventing nuclear accumulation of Smad protein, offering a possible molecular mechanism for these interactions (Dobens, 2000).

The data presented here indicate that the A359 locus may be a direct target for negative regulation by Bunched. The sensitive A359 repression assay also shows that the TSC domain is critical to Bunched function: altering these amino acids makes BUN-1 inactive. The role of bunched in other tissues is poorly understood. Embryos homozygous for bunched mutations die with morphological defects in the peripheral neurons and subtle defects in cuticle pattern. bunched maternal effect phenotypes are pleiotropic, ranging from very early defects to segmental defects in the embryonic cuticle. During eye development, bunched promotes photoreceptor differentiation, and shows genetic interactions with dpp, wingless, hedgehog and components of the Egfr signaling pathway. It remains to be determined whether bunched has a similar role throughout development, for example as an RTK target gene or a repressor of Dpp target genes. Bunched antagonizes Dpp function in the follicle cells. This finding is surprising, for mutations in the dpp and bunched genes synergize to severely arrest eye development. A mechanistic interpretation of this genetic interaction awaits better understanding of Dpp functions during eye development. It has been noted that the bunched eye phenotype is rescued by the BUN-2 transcript, whereas BUN-1 is the antagonist of Dpp in the FC. The BUN-2 transcript is expressed in the operculum-forming FC, raising the possibilities that this isoform has a distinct role, or that it is subject to post-transcriptional regulation. Further studies of the functions of the two isoforms will be needed to resolve these differences (Dobens, 2000 and references therein).

It is proposed here that the boundary to a Dpp-induced fate in the follicle cells is set by transcriptional regulation of a downstream transcriptional repressor, Bunched. Recently, a similar role has been proposed for the gene brinker in setting threshold gene expression responses to Dpp in the Drosophila wing. Thus, Dpp induction of gene expression through negative regulation of a negative regulator may be a common theme in development. Regulation of the expression of these key downstream repressors provides a powerful mechanism to modulate responses to Dpp signaling (Dobens, 2000 and references therein).

The Drosophila fos/kayak gene is a key regulator of epithelial cell morphogenesis during dorsal closure of the embryo and fusion of the adult thorax. It is also required for two morphogenetic movements of the follicular epithelium during oogenesis: (1) it is necessary for the proper posteriorward migration of main body follicle cells during stage 9; (2) it controls, from stage 11 onwards, the morphogenetic reorganization of the follicle cells that are committed to secrete the respiratory appendages. Egfr pathway activation and a critical level of Dpp signaling are required to pattern a high level of transcription of kayak at the anterior and dorsal edges of the two groups of cells that will give rise to the respiratory appendages. In addition, evidence is provided that, within the dorsal-anterior territory, the level of paracrine Dpp signaling controls the commitment of follicle cells towards either an operculum or an appendage secretion fate. kayak is required in follicle cells for the dumping of the nurse cell cytoplasm into the oocyte and the subsequent apoptosis of nurse cells. This suggests that in somatic follicle cells, kayak controls the expression of one or several factors that are necessary for these processes in underlying germinal nurse cells (Dequier, 2001).

The data show that determination and localization of the kayak columnar expression pattern requires both Egfr pathway activation and a precise level of paracrine Dpp signaling. The alteration of kayak expression in mutants affecting different components of the Egfr pathway shows clearly that Grk-dependent Egfr activation and secondary Spitz- dependent Egfr amplification and refinement are necessary to determine the kayak columnar expression pattern. Nonetheless, colchicine feeding experiments demonstrate that Grk-dependent Egfr activation is not sufficient to induce kayak transcription in CFC, as is the case for the Egfr target gene kekkon. However, alteration of kayak expression resulting from either a reduction of the Dpp level or its overincrease throughout the columnar epithelium, provides direct evidence that this signaling process is also required for proper patterning of kayak expression (Dequier, 2001).

In C532-GAL4/UAS-dpp females grown at 18°C, a slight increase in the level of Dpp accumulation in CFC induces multiple patches of cells showing a pattern of BR-C Z1 and Kayak accumulation reminiscent of that of respiratory appendage secreting units in wild-type egg chambers. Strikingly, these patches are located at the lateral and posterior peripheries of the dorsal-anterior follicle cell territory, which is consistent with the hypothesis that the central-most CMFC are the localized source of a Dpp gradient. In addition, these results indicate that ectopically provided Dpp in FLP-out clones represses BR-C Z1 and Kayak accumulation in both dpp-expressing cells and those located within a radius of one to two cells, thus providing a direct evidence that Dpp acts in a paracrine manner to repress expression of the BR-C Z1 and kayak genes. The observation that the Dpp-dependent repression of BR-C Z1 is restricted to DAFC suggests that it is mediated by a component of the Dpp-signaling pathway, i.e., either a Dpp receptor or a Smad cofactor expressed differentially in DAFC. It has been shown that among the known Dpp receptors, Saxophone and Punt are ubiquitously expressed in CFC whereas Thick-vein is expressed in a row of anterior follicle cells. In a preliminary investigation of the pattern of expression of the Drosophila Smad genes in follicle cells, it has been observed that medea is expressed from stage 11 onwards in two patches of CFC that may correspond to RASFC. However, whereas the medea gene is required for kayak transcription in the main body follicle cells during stage 9, it appears to be fully dispensable for the kayak columnar expression pattern. Work is currently in progress to investigate the pathway involved in the restriction of the Dpp-dependent repression of BR-C Z1 to DAFC (Dequier, 2001).

Other targets of DPP

dpp may indirectly regulate twisted gastrulation, a secreted protein made in dorsal midline cells. Structural analysis of the tsg gene reveals features of a secreted protein suggesting an extracellular site of action. Dorsal midline cell fate is specified by the combination of both a TSG and a DPP signal to which the dorsal midline cells are uniquely competent to respond (Mason, 1994).

Mutations in EGF-receptor result in the expansion of muscle segment homeobox ectodermal 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).

An effect on the early stripe of Goosecoid expression is observed in sloppy-paired, orthodenticle, tailless and decapentaplegic mutants. Both slp and otd affect Gsc in a similar way: the early stripe of Gsc appears normally but at the end of the cellularization stage, there is no reinforcement of its expression and it is prematurely lost. dpp is necessary to bring aboud Gsc repression in the dorsal-most region of the embryo, while tll is required to promote Gsc expression in the lateral region, or to prevent its repression by the dorsoventral patterning system (Goriely, 1996).

DPP target gene zerknullt (zen) activates the amnioserosa-specific expression of a downstream target gene, Race (Related to angiotensin converting enzyme), the earliest known marker gene for the amnioserosa. Two TGF-beta growth factors, dpp and screw, function synergistically to subdivide the dorsal ectoderm into two embryonic tissues, the amnioserosa and dorsal epidermis. Previous studies have shown that peak dpp activity is required for the localized expression of zen. ZEN in turn directly activates the amnioserosa-specific expression of a downstream target gene, Race. A 533 bp enhancer from the Race promoter region is shown to mediate selective expression in the amnioserosa, as well as the anterior and posterior midgut rudiments. This enhancer contains three ZEN protein binding sites, and mutations in these sites virtually abolish the expression of an otherwise normal Race-lacZ fusion gene in the amnioserosa, but not in the gut. Genetic epistasis experiments suggest that ZEN is not the sole activator of Race, although a hyperactivated form of ZEN (a zen-VP16 fusion protein) can partially complement reduced levels of dpp activity. These results suggest that dpp regulates multiple transcription factors, which function synergistically to specify the amnioserosa. It is unknown whether the dpp pathway leads to the post-translational modification of ZEN or whether an unknown transcription factor serves as a DPP substrate and then participates in the activation of zen (Rusch, 1997).

In the early blastoderm embryo, dpp mediates the subdivision of the dorsal ectoderm into two embryonic tissues: the amnioserosa and the dorsal epidermis. High Dpp levels in the dorsal-most cells specify amnioserosa while lower Dpp levels in dorsolateral regions specify epidermis. Expression of the gene Race (related to angiotensin converting enzyme; the earliest known marker for the amnioserosa) in the dorsal blastoderm embryo depends on dpp signaling. Thus it was asked whether the activity of the Race enhancer depends on CBP function. This enhancer mediates lacZ staining in the presumptive amnioserosa and in the anterior midgut primodium: the former, but not the latter, staining requires dpp. In nejire mutants embryos, there is no detectable lacZ staining in the presumptive amnioserosa, although staining remains, and is even slightly enhanced, in the head and in the anterior midgut primordium. This demonstrates that the Race enhancer depends on an activating function of CBP exclusively in a subset of the blastoderm cells, namely in the dorsal-most cells of the embryonic trunk. It suggests that CBP is required for the response of this enhancer to dpp (Waltzer, 1999).

Pannier, a GATA family transcription factor expressed in the dorsal portion of the embryo just after cellularization, lies downstream of decapentaplegic and zerknüllt. In embryos null for dpp, no pannier is expressed (Winick, 1993).

The ventral nervous system defective/NK-2 gene is not expressed in the mesodermal anlage due to repression by Snail, in mesectodermal cells due to repression by Single-minded, or in the lateral neuroectodermal and/or dorsal epidermal anlagen due to repression mediated indirectly by DPP. Twist either activates vnd gene in the posterior portion of the embryo or is a coactivator with dorsal (Mellerick, 1995).

labial (lab) is induced to high levels of localised expression in the endoderm of Drosophila embryos by the indirect action of dpp. Dissection of lab 5' flanking sequences reveals two types of response elements. One of these mediates lab dependent activity, providing evidence that lab induction in the endoderm is autoregulatory. The other element, to a large extent independent of lab function, responds to dpp (Tremml, 1992).

A stepwise morphogenetic program of cell division and cell fate determination generates the precise neuronal architecture of the visual centers of the Drosophila brain. The assembly of the target structure for ingrowing retinal axons involves cell-cell interactions mediated by the secreted product of the wingless gene. wg, expressed in two symmetrical domains of the developing brain, is required to induce and maintain the expression of the secreted decapentaplegic gene product in adjacent domains. Both wg and dpp function are required for target field neurons to adopt their proper fates and to send axons into the developing target structure (Kaphingst, 1994).

The foregut of the larval fly is subdivided into the pharynx, the esophagus and the proventriculus. The proventriculus is located at the caudal end of the esophagus and serves as a valve in regulating passages of food to the midgut. It is composed of an ectodermal epithelial layer surrounded by visceral mesoderm. The proventriculus is a multi-folded muscular organ of the foregut formed from a simple epithelial tube, whose function is grinding and masticating food. Coordinated cell movements are critical for tissue and organ morphogenesis in animal development. hedgehog and wingless, made in ectodermal cells, and the gene myospheroid, which encodes a beta subunit of the integrins, are required for epithelial morphogenesis during proventriculus development. In contrast, this morphogenetic process is indirectly suppressed by the decapentaplegic gene. Thus integrins may serve as potential effectors for hh, wg and dpp signaling. (Pankratz, 1995).

During Drosophila embryogenesis the two halves of the lateral epidermis migrate dorsally over a surface of flattened cells, the amnioserosa, and meet at the dorsal midline in order to form the continuous sheet of the larval epidermis. During this process of epithelial migration, known as dorsal closure, signaling from a Jun-amino-terminal-kinase cascade causes the production of the secreted Tgf-beta-like ligand, Decapentaplegic. Binding of Decapentaplegic to the putative Tgf-beta-like receptors Thickveins and Punt activates a Tgf-beta-like pathway that is also required for dorsal closure. Mutations in genes involved in either the Jun-amino-terminal-kinase cascade or the Tgf-beta-like signaling pathway can disrupt dorsal closure. Although these pathways are linked they are not equivalent in function. Signaling by the Jun-amino-terminal-kinase cascade may be initiated by the small Ras-like GTPase Drac1 and acts to assemble the cytoskeleton and specify the identity of the first row of cells of the epidermis prior to the onset of dorsal closure. Signaling in the Tgf-beta-like pathway is mediated by Dcdc42, and acts during the closure process to control the mechanics of the migration process, most likely via its putative effector kinase DPAK (Ricos, 1999).

The Drosophila wing is divided into two compartments along its anteroposterior (A/P) axis. The compartment boundary between these regions serves as the source of an organizing activity that patterns both anterior and posterior compartments. This activity is mediated, at least in part, by the long-range action of Dpp, which is expressed by cells along the A/P compartment boundary. Dpp is thought to act as a morphogen to inform target cells of their position along the A/P axis, but as yet, little is known about how cells interpret the distribution of Dpp protein. An enhancer trap screen was conducted to identify genes whose transcription is controlled by Dpp. Two enhancer trap lines in the same locus (89E/F), P1883 and 1(3)1E4, were identified whose expression patterns are similar to those of Dpp during embryonic and imaginal development. The gene whose expression is reflected in these enhancer traps has been named Daughters against dpp (Dad). In these enhancer trap lines, beta-galactosidase is expressed in a wide stripe that straddles the A/P compartment boundary of the imaginal discs, in contrast to Dpp, whose expression is confined to the anterior side. This pattern of expression suggests that Dad expression is positively regulated by the secreted Dpp molecule. To test whether Dad responds to Dpp signaling, its expression has been examined in P1883 wing discs in which a UAS-dpp transgene was transcribed in a ring around a wing pouch under the control of a Gal4 driver. Ectopic Dpp expression results in abnormally large discs and in ectopic expression of Dad in a broad ring around a wing pouch. Identical results were obtained when another transgene was used -- UAS-tkv Q253D -- which encodes a constitutively active form of the major type-I Dpp receptor, Thick veins. In addition, expression of Dad is not detected in cells that lack a functional Tkv Dpp receptor. These results indicate that Dpp signaling is necessary and sufficient for Dad expression in the developing wing (Tsuneizumi, 1997).

In the Drosophila ventral nerve cord, a small number of neurons express the LIM-homeodomain gene apterous (ap). These ap neurons can be subdivided based upon axon pathfinding and their expression of neuropeptidergic markers. ap, the zinc finger gene squeeze, the bHLH gene dimmed, and the BMP pathway are all required for proper specification of these cells. Here, using several ap neuron terminal differentiation markers, how each of these factors contributes to ap neuron diversity has been resolved. These factors interact genetically and biochemically in subtype-specific combinatorial codes to determine certain defining aspects of ap neuron subtype identity. However, it was also found that ap, dimmed, and squeeze additionally act independently of one another to specify certain other defining aspects of ap neuron subtype identity. Therefore, within single neurons, single regulators acting in numerous molecular contexts differentially specify multiple subtype-specific traits (Allan, 2005).

The TGFβ/BMP signal transduction pathway plays critical roles during a number of developmental events, and mutants affecting the Drosophila BMP pathway show dramatic defects in embryonic development. In contrast, in the Tv neuron, BMP signaling plays a much more subtle role, and although it is critical for dFMRFa expression, no effects were found upon the expression of sqz, ap, or dimm or on the general peptidergic marker PHM in wit mutants. Although these studies cannot rule out other roles for the BMP pathway in Tv neurons, it is tempting to speculate that target-derived BMP signaling in neurons may have quite a limited set of nuclear readouts in each specific neuronal subclass (Allan, 2005).

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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