Convergence of dorsal, dpp, and egfr signaling pathways subdivides the drosophila neuroectoderm into three dorsal-ventral columns

An important question in neurobiology is how different cell fates are established along the dorsoventral (DV) axis of the central nervous system (CNS). Here we investigate the origins of DV patterning within the Drosophila CNS. 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. Here we describe 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. We confirm that the vnd domain is established by Dorsal and maintained by Egfr, but unlike a previous report we show that vnd is not regulated by Dpp signaling. We show that ind expression requires both Dorsal and Egfr signaling for activation and positioning of its dorsal border, and that abnormally high Dpp can repress ind expression. Finally, we show that the msh domain is defined by repression: it occurs only where Dpp, Vnd, and Ind activity is low. We conclude 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.     

Axis specification in the spider embryo: dpp is required for radial-to-axial symmetry transformation and sog for ventral patterning

The mechanism by which Decapentaplegic (Dpp) and its antagonist Short gastrulation (Sog) specify the dorsoventral pattern in Drosophila embryos has been proposed to have a common origin with the mechanism that organizes the body axis in the vertebrate embryo. However, Drosophila Sog makes only minor contributions to the development of ventral structures that hypothetically correspond to the vertebrate dorsum where the axial notochord forms. In this study, we isolated a homologue of the Drosophila sog gene in the spider Achaearanea tepidariorum, and characterized its expression and function. Expression of sog mRNA initially appeared in a radially symmetrical pattern and later became confined to the ventral midline area, which runs axially through the germ band. RNA interference-mediated depletion of the spider sog gene led to a nearly complete loss of ventral structures, including the axial ventral midline and the central nervous system. This defect appeared to be the consequence of dorsalization of the ventral region of the germ band. By contrast, the extra-embryonic area formed normally. Furthermore, we showed that embryos depleted for a spider homologue of dpp failed to break the radial symmetry, displaying evenly high levels of sog expression except in the posterior terminal area. These results suggest that dpp is required for radial-to-axial symmetry transformation of the spider embryo and sog is required for ventral patterning. We propose that the mechanism of spider ventral specification largely differs from that of the fly. Interestingly, ventral specification in the spider is similar to the process in vertebrates in which the antagonism of Dpp/BMP signaling plays a central role in dorsal specification.     

Dpp represses eagle expression at short-range, but can repress its expression at a long-range via EGFR signal repression

Nervous system development takes place after positional information has been established along the dorsal-ventral (D/V) axis. The initial subdivision provided by a gradient of nuclear dorsal protein is maintained by the zygotic genes expressed along the D/V axis. In this study, an investigation was conducted to determine the range of Dpp function in repressing the expression of eagle (eg) that is present in intermediate neuroblasts defective (ind) and muscle specific homeobox (msh) gene domain. eg is expressed in neuroblast (NB) 2-4, 3-3 and 6-4 of the msh domain, and NB7-3 of the ind domain at the embryonic stage 11. In decapentaplegic (dpp) loss-of-function mutant embryos, eg was ectopically expressed in the dorsal region, while in dpp gain-of-function mutants produced by sog or sca-GAL4/UAS-dpp, eg was repressed by Dpp. It is worthy of note that Dpp produced from sim;;dpp embryos showed that Dpp could function at long range. However, Dpp produced from en-GAL4/UAS-dpp or wg-GAL4/UAS-dpp primarily acted at short-range. This result demonstrated that this discrepancy seems to be due to the repression of Dpp to EGFR signaling in sim;;dpp embryos. Taken together, these results suggest that Dpp signaling works at short-range, but can function indirectly at long-range by way of repression of EGFR signaling during embryonic neurogenesis.     

Drosophila Tbx6-related gene, Dorsocross, mediates high levels of Dpp and Scw signal required for the development of amnioserosa and wing disc primordium

Regional differentiation along the dorsoventral (DV) axis of the Drosophila embryo primarily depends on a graded BMP signaling activity generated by Decapentaplegic (Dpp) and Screw (Scw). We have identified triplicated Dpp and Scw target genes Dorsocross1, 2 and 3 (Doc1, 2, 3) that have a conserved T-box domain related to the vertebrate Tbx6 subfamily and act redundantly to induce dorsal structures. Doc genes are expressed in the dorsal region in the early blastoderm. After gastrulation, newly expressed Doc appears in a segmental pattern in the ectoderm. This expression correlates spatially with the second phase of Dpp expression in the ectoderm. Doc expression in the early blastoderm is abolished in either dpp or scw mutant embryos, whereas the ectodermal segmented expression depends only on Dpp. Inactivation of Doc genes with RNAi dramatically affected the development of amnioserosa and wing disc primordia, both of which depend on high levels of BMP signaling, although leg disc primordium, which depends on low levels of BMP, remained intact. Doc1 mRNA expressed in Xenopus embryos induced ventral mesoderm, suppressed activin-induced events and induced Xvent genes, which are analogous to the effects of native Tbx6 and its upstream regulator, BMP-4. These results suggest that the Tbx6 subfamily act in the BMP signaling pathway required for embryonic patterning in both animals.     

The Ca-dependent protease Calpain A regulates Cactus/IκB levels during Drosophila development in response to maternal Dpp signals

Regulation of NFκB activity is central to many processes during development and disease. Activation of NFκB family members depends on degradation of inhibitory IκB proteins. In Drosophila, a nuclear gradient of the NFκB/c-rel protein Dorsal subdivides the embryonic dorsal–ventral axis, defining the extent and location of mesodermal and ectodermal territories. Activation of the Toll pathway directs Dorsal nuclear translocation by inducing proteosomal degradation of the IκB homologue Cactus. Another mechanism that impacts on Dorsal activation involves the Toll-independent pathway, which regulates constitutive Cactus degradation. We have shown that the BMP protein Decapentaplegic (Dpp) inhibits Cactus degradation independent of Toll. Here we report on a novel element of this pathway: the calcium-dependent protease Calpain A. CalpainA knockdowns increase Cactus levels, shifting the Dorsal gradient and dorsal–ventral patterning. As shown for mammalian IκB, this effect requires PEST sequences in the Cactus C-terminus, implying a conserved role for calpains. Alteration of CalpainA or dpp results in similar effects on Dorsal target genes. Epistatic analysis confirms CalpainA activity is regulated by Dpp, indicating that Dpp signals increase Cactus levels through Calpain A inhibition, thereby interfering with Dorsal activation. This mechanism may allow coordination of Toll, BMP and Ca²⁺ signals, conferring precision to Dorsal-target expression domains.     

Processing of the Drosophila Sog protein creates a novel BMP inhibitory activity

Structurally unrelated neural inducers in vertebrate and invertebrate embryos have been proposed to function by binding to BMP4 or Dpp, respectively, and preventing these homologous signals from activating their receptor(s). In this study, we investigate the functions of various forms of the Drosophila Sog protein using the discriminating assay of Drosophila wing development. We find that misexpression of Drosophila Sog, or its vertebrate counterpart Chordin, generates a very limited vein-loss phenotype. This sog misexpression phenotype is very similar to that of viable mutants of glass-bottom boat (gbb), which encodes a BMP family member. Consistent with Sog selectively interfering with Gbb signaling, Sog can block the effect of misexpressing Gbb, but not Dpp in the wing. In contrast to the limited BMP inhibitory activity of Sog, we have identified carboxy-truncated forms of Sog, referred to as Supersog, which when misexpressed cause a broad range of dpp(-) mutant phenotypes. In line with its phenotypic effects, Supersog can block the effects of both misexpressing Dpp and Gbb in the wing. Vertebrate Noggin, on the other hand, acts as a general inhibitor of Dpp signaling, which can interfere with the effect of overexpressing Dpp, but not Gbb. We present evidence that Sog processing occurs in vivo and is biologically relevant. Overexpression of intact Sog in embryos and adult wing primordia leads to the developmentally regulated processing of Sog. This in vivo processing of Sog can be duplicated in vitro by treating Sog with a combination of the metalloprotease Tolloid (Tld) plus Twisted Gastrulation (Tsg), another extracellular factor involved in Dpp signaling. In accord with this result, coexpression of intact Sog and Tsg in developing wings generates a phenotype very similar to that of Supersog. Finally, we provide evidence that tsg functions in the embryo to generate a Supersog-like activity, since Supersog can partially rescue tsg(-) mutants. Consistent with this finding, sog(- )and tsg(-) mutants exhibit similar dorsal patterning defects during early gastrulation. These results indicate that differential processing of Sog generates a novel BMP inhibitory activity during development and, more generally, that BMP antagonists play distinct roles in regulating the quality as well as the magnitude of BMP signaling.     

Local inhibition of Drosophila homeobox-containing neural dorsoventral patterning genes by Dpp

An important step in Drosophila neurogenesis is to establish the neural dorsoventral (DV) patterning. Here we describe how dpp loss-of- and gain-of-function mutation affects the homeobox-containing neural DV patterning genes expressed in the ventral neuroectoderm. Ventral nervous system defective (vnd), intermediate neuroblast defective (ind), muscle-specific homeobox (msh), and orthodenticle (otd) genes participate in development of the central nervous system and peripheral nervous system, and encode homeodomain proteins. otd and msh genes were ectopically expressed in dpp loss-of-function mutation, but vnd and ind were not affected. However, when dpp was ectopically expressed in the ventral neuroectoderm by rho-GAL4/UAS-dpp system, it caused the repression of vnd, and msh expressions in ventral and dorsal columns of the neuroectoderm, respectively, but not that of ind. The later expression pattern of otd was also restricted by Dpp. The expression pattern of msh, vnd and otd in dpp loss-of-function and gain-of-function mutation indicates that Dpp activity does not reach to the ventral midline and it works locally to establish the dorsal boundary of the ventral neuroectoderm.     

Localized enhancement and repression of the activity of the TGF-beta family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo.

Seven zygotically active genes are required for normal patterning of the dorsal 40% of the Drosophila embryo. Among these genes, decapentaplegic (dpp) has the strongest mutant phenotype: in the absence of dpp, all cells in the dorsal and dorsolateral regions of the embryo adopt fates characteristic of more ventrally derived cells (Irish and Gelbart (1987) Genes Dev. 1, 868-879). Here we describe the phenotypes caused by alleles of another of this set of genes, tolloid, and show that tolloid is required for dorsal, but not dorsolateral, pattern. Extragenic suppressors of tolloid mutations were isolated that proved to be mutations that elevate dpp activity. We studied the relationship between tolloid and dpp by analyzing the phenotypes of tolloid embryos with elevated numbers of the dpp gene and found that doubling the dpp+ gene dosage completely suppressed weak tolloid mutants and partially suppressed the phenotypes of tolloid null mutants. We conclude that the function of tolloid is to increase dpp activity. We also examined the effect of doubling dpp+ gene dosage on the phenotypes caused by other mutations affecting dorsal development. Like tolloid, the phenotypes of mutant embryos lacking shrew gene function were suppressed by elevated dpp, indicating that shrew also acts upstream of dpp to increase dpp activity. In contrast, increasing the number of copies of the dpp gene enhanced the short gastrulation (sog) mutant phenotype, causing ventrolateral cells to adopt dorsal fates. This indicates that sog gene product normally blocks dpp activity ventrally. We propose that the tolloid, shrew and sog genes are required to generate a gradient of dpp activity, which directly specifies the pattern of the dorsal 40% of the embryo.     

A genetic screen for maternal-effect suppressors of decapentaplegic identifies the eukaryotic translation initiation factor 4A in Drosophila

The Dpp signaling pathway is essential for many developmental processes in Drosophila and its activity is tightly regulated. To identify additional regulators of Dpp signaling, we conducted a genetic screen for maternal-effect suppressors of dpp haplo-insufficiency. We screened approximately 7000 EMS-mutagenized genomes and isolated and mapped seven independent dominant suppressors of dpp, Su(dpp), which were recovered as second-site mutations that resulted in viable flies in trans-heterozygous with dpp(H46), a dpp null allele. Most of the Su(dpp) mutants exhibited increased cell numbers of the amnioserosa, a cell type specified by the Dpp pathway, suggesting that these mutations may augment Dpp signaling activity. Here we report the unexpected identification of one of the Su(dpp) mutations as an allele of the eukaryotic translation initiation factor 4A (eIF4A). We show that Su(dpp)(YE9) maps to eIF4A and that this allele is associated with a substitution, arginine 321 to histidine, at a well-conserved amino acid and behaves genetically as a dominant-negative mutation. This result provides an intriguing link between a component of the translation machinery and Dpp signaling.