BMP-dependent serosa and amnion specification in the scuttle fly Megaselia abdita

Bone morphogenetic protein (BMP) signaling is an essential factor in dorsoventral patterning of animal embryos but how BMP signaling evolved with fundamental changes in dorsoventral tissue differentiation is unclear. Flies experienced an evolutionary reduction of extra-embryonic tissue types from two (amniotic and serosal tissue) to one (amnionserosal tissue). BMP-dependent amnioserosa specification has been studied in Drosophila melanogaster. However, the mechanisms of serosal and amniotic tissue specification in less diverged flies remain unknown. To better understand potential evolutionary links between BMP signaling and extra-embryonic tissue specification, we examined the activity profile and function of BMP signaling in serosa and amnion patterning of the scuttle fly Megaselia abdita (Phoridae) and compared the BMP activity profiles between M. abdita and D. melanogaster. In blastoderm embryos of both species, BMP activity peaked at the dorsal midline. However, at the beginning of gastrulation, peak BMP activity in M. abdita shifted towards prospective amnion tissue. This transition correlated with the first signs of amnion differentiation laterally adjacent to the serosa anlage. Marker-assisted analysis of six BMP signaling components (dpp, gbb, scw, tkv, sax, sog) by RNA interference revealed that both serosa and amnion specification of M. abdita are dependent on BMP activity. Conversely, BMP gain-of-function experiments caused sharpened expression boundaries of extra-embryonic target genes indicative of positive feedback. We propose that changes in the BMP activity profile at the beginning of gastrulation might have contributed to the reduction of extra-embryonic tissue types during the radiation of cyclorrhaphan flies.     

Distinct functions of the Tribolium zerknüllt genes in serosa specification and dorsal closure

BACKGROUND: In the long-germ insect Drosophila, a single extraembryonic membrane, the amnioserosa, covers the embryo at the dorsal side. In ancestral short-germ insects, an inner membrane, the amnion, covers the embryo ventrally, and an outer membrane, the serosa, completely surrounds the embryo. An early differentiation step partitions the uniform blastoderm into the anterior-dorsal serosa and the posterior-ventral germ rudiment giving rise to amnion and embryo proper. In Drosophila, amnioserosa formation depends on the dorsoventral patterning gene zerknüllt (zen), a derived Hox3 gene. RESULTS: The short-germ beetle Tribolium castaneum possesses two zen homologs, Tc-zen1 and Tc-zen2. Tc-zen1 acts early and specifies the serosa. The loss of the serosa after Tc-zen1 RNAi is compensated by an expansion of the entire germ rudiment toward the anterior. Instead of the serosa, the amnion covers the embryo at the dorsal side, and later size regulation normalizes the early fate shifts, revealing a high degree of plasticity of short-germ development. Tc-zen2 acts later and initiates the amnion and serosa fusion required for dorsal closure. After Tc-zen2 RNAi, the amnion and serosa stay apart, and the embryo closes ventrally, assuming a completely everted (inside-out) topology. CONCLUSIONS: In Tribolium, the duplication of the zen genes was accompanied by subfunctionalization. One of the paralogues, Tc-zen1, acts as an early anterior-posterior patterning gene by specifying the serosa. In absence of the serosa, Tribolium embryogenesis acquires features of long-germ development with a single extraembryonic membrane. We discuss implications for the evolution of insect development including the origin of the zen-derived anterior determinant bicoid.     

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.     

Grasshopper hunchback expression reveals conserved and novel aspects of axis formation and segmentation

While the expression patterns of segment polarity genes such as engrailed have been shown to be similar in Drosophila melanogaster and Schistocerca americana (grasshopper), the expression patterns of pair-rule genes such as even-skipped are not conserved between these species. This might suggest that the factors upstream of pair-rule gene expression are not conserved across insect species. We find that, despite this, many aspects of the expression of the Drosophila gap gene hunchback are shared with its orthologs in the grasshoppers S. americana and L. migratoria. We have analyzed both mRNA and protein expression during development, and find that the grasshopper hunchback orthologs appear to have a conserved role in early axial patterning of the germ anlagen and in the specification of gnathal and thoracic primordia. In addition, distinct stepped expression levels of hunchback in the gnathal/thoracic domains suggest that grasshopper hunchback may act in a concentration-dependent fashion (as in Drosophila), although morphogenetic activity is not set up by diffusion to form a smooth gradient. Axial patterning functions appear to be performed entirely by zygotic hunchback, a fundamental difference from Drosophila in which maternal and zygotic hunchback play redundant roles. In grasshoppers, maternal hunchback activity is provided uniformly to the embryo as protein and, we suggest, serves a distinct role in distinguishing embryonic from extra-embryonic cells along the anteroposterior axis from the outset of development - a distinction made in Drosophila along the dorsoventral axis later in development. Later hunchback expression in the abdominal segments is conserved, as are patterns in the nervous system, and in both Drosophila and grasshopper, hunchback is expressed in a subset of extra-embryonic cells. Thus, while the expected domains of hunchback expression are conserved in Schistocerca, we have found surprising and fundamental differences in axial patterning, and have identified a previously unreported domain of expression in Drosophila that suggests conservation of a function in extra-embryonic patterning.     

Regulation of early expression of Dlx3, a Xenopus anti-neural factor, by beta-catenin signaling

The ectoderm of the pre-gastrula Xenopus embryo has previously been shown to be at least partially patterned along the dorsal-ventral axis. The early expression of the anti-neural homeodomain gene Dlx3 is localized to the ventral ectoderm by a mechanism that occurs prior to gastrulation and is independent of the Spemann organizer. The repression of Dlx3 is mediated by signaling though beta-catenin, but is probably not dependent on the induction of the Xnr3 or chordin genes by beta-catenin. We propose a model in which this early regulation of Dlx3 accounts for the pro-neural bias of dorsal ectoderm.     

BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb.

We demonstrate that signaling via the bone morphogenetic protein receptor IA (BMPR-IA) is required to establish two of the three cardinal axes of the limb: the proximal-distal axis and the dorsal-ventral axis. We generated a conditional knockout of the gene encoding BMPR-IA (Bmpr) that disrupted BMP signaling in the limb ectoderm. In the most severely affected embryos, this conditional mutation resulted in gross malformations of the limbs with complete agenesis of the hindlimbs. The proximal-distal axis is specified by the apical ectodermal ridge (AER), which forms from limb ectoderm at the distal tip of the embryonic limb bud. Analyses of the expression of molecular markers, such as Fgf8, demonstrate that formation of the AER was disrupted in the Bmpr mutants. Along the dorsal/ventral axis, loss of engrailed 1 (En1) expression in the non-ridge ectoderm of the mutants resulted in a dorsal transformation of the ventral limb structures. The expression pattern of Bmp4 and Bmp7 suggest that these growth factors play an instructive role in specifying dorsoventral pattern in the limb. This study demonstrates that BMPR-IA signaling plays a crucial role in AER formation and in the establishment of the dorsal/ventral patterning during limb development.     

Axial patterning interactions in the sea urchin embryo: suppression of nodal by Wnt1 signaling

Wnt and Nodal signaling pathways are required for initial patterning of cell fates along anterior-posterior (AP) and dorsal-ventral (DV) axes, respectively, of sea urchin embryos during cleavage and early blastula stages. These mechanisms are connected because expression of nodal depends on early Wnt/β-catenin signaling. Here, we show that an important subsequent function of Wnt signaling is to control the shape of the nodal expression domain and maintain correct specification of different cell types along the axes of the embryo. In the absence of Wnt1, the posterior-ventral region of the embryo is severely altered during early gastrulation. Strikingly, at this time, nodal and its downstream target genes gsc and bra are expressed ectopically, extending posteriorly to the blastopore. They override the initial specification of posterior-ventral ectoderm and endoderm fates, eliminating the ventral contribution to the gut and displacing the ciliary band dorsally towards, and occasionally beyond, the blastopore. Consequently, in Wnt1 morphants, the blastopore is located at the border of the re-specified posterior-ventral oral ectoderm and by larval stages it is in the same plane near the stomodeum on the ventral side. In normal embryos, a Nodal-dependent process downregulates wnt1 expression in dorsal posterior cells during early gastrulation, focusing Wnt1 signaling to the posterior-ventral region where it suppresses nodal expression. These subsequent interactions between Wnt and Nodal signaling are thus mutually antagonistic, each limiting the range of the other's activity, in order to maintain and stabilize the body plan initially established by those same signaling pathways in the early embryo.     

FGF10 can induce Fgf8 expression concomitantly with En1 and R-fng expression in chick limb ectoderm, independent of its dorsoventral specification

The limb bud has a thickened epithelium at the dorsal-ventral boundary, the apical ectodermal ridge (AER), which sustains limb outgrowth and patterning. A secreted molecule fibroblast growth factor (FGF)10 is involved in inducing Fgf8 expression in the prospective AER and mutual interaction between mesenchymal FGF10 and FGF8 in the AER is essential for limb outgrowth. A secreted factor Wnt7a and a homeobox protein Lmx1 are involved in the dorsal patterning of the limb, whereas a homeobox protein Engrailed 1 (En1) is involved in the dorsal-ventral patterning as well as AER formation. Radical fringe (R-fng), a vertebrate homolog of Drosophila fringe was also found to elaborate AER formation in chicks. However, little is known about the molecular interactions between these factors during AER formation. The present study clarified the relationship between FGF10, Wnt7a, Lmx1, R-fng and En1 during limb development using a foil-barrier insertion experiment. It was found that a foil-barrier inserted into the chick prospective wing mesenchyme lateral to the mesonephric duct blocks AER induction. This experiment was expanded by implanting Fgf10-expressing cells lateral to the barrier and examined whether FGF10 could rescue the expression of the limb-patterning genes reported in AER formation. It was found that FGF10 is sufficient to induce Fgf8 expression in the ectoderm of the foil-inserted limb bud, concomitantly with R-fng and En1 expression. However, FGF10 could not rescue the expression of the dorsal marker genes, Wnt7a or Lmx1. Thus, it is suggested that epithelial factors of En1 and R-fng can induce Fgf8 expression in the limb ectoderm in cooperation with a mesenchymal factor FGF10. Some factor(s) other than FGF10, possibly from the paraxial structures medial to the limb mesoderm, is responsible for the initial dorsal-ventral specification of the limb bud.