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.     

Tsukushi controls ectodermal patterning and neural crest specification in Xenopus by direct regulation of BMP4 and X-delta-1 activity

In Xenopus, ectodermal patterning depends on a mediolateral gradient of BMP signaling, higher in the epidermis and lower in the neuroectoderm. Neural crest cells are specified at the border between the neural plate and the epidermis, at intermediate levels of BMP signaling. We recently described a novel secreted protein, Tsukushi (TSK), which works as a BMP antagonist during chick gastrulation. Here, we report on the Xenopus TSK gene (X-TSK), and show that it is involved in neural crest specification. X-TSK expression accumulates after gastrulation at the anterior-lateral edges of the neural plate, including the presumptive neural crest region. In gain-of-function experiments, X-TSK can strongly enhance neural crest specification by the dorsolateral mesoderm or X-Wnt8 in ectodermal explants, while the electroporation of X-TSK mRNA in the lateral ectoderm of embryos after gastrulation can induce the expression of neural crest markers in vivo. By contrast, depletion of X-TSK in explants or embryos impairs neural crest specification. Similarly to its chick homolog, X-TSK works as a BMP antagonist by direct binding to BMP4. However, X-TSK can also indirectly regulate BMP4 mRNA expression at the neural plate border via modulation of the Delta-Notch signaling pathway. We show that X-TSK directly binds to the extracellular region of X-delta-1, and modulates Delta-dependent Notch activity. We propose that X-TSK plays a key role in neural crest formation by directly regulating BMP and Delta activities at the boundary between the neural and the non-neural ectoderm.     

Evolution of the dorsal-ventral patterning network in the mosquito, Anopheles gambiae

The dorsal-ventral patterning of the Drosophila embryo is controlled by a well-defined gene regulation network. We wish to understand how changes in this network produce evolutionary diversity in insect gastrulation. The present study focuses on the dorsal ectoderm in two highly divergent dipterans, the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae. In D. melanogaster, the dorsal midline of the dorsal ectoderm forms a single extra-embryonic membrane, the amnioserosa. In A. gambiae, an expanded domain forms two distinct extra-embryonic tissues, the amnion and serosa. The analysis of approximately 20 different dorsal-ventral patterning genes suggests that the initial specification of the mesoderm and ventral neurogenic ectoderm is highly conserved in flies and mosquitoes. By contrast, there are numerous differences in the expression profiles of genes active in the dorsal ectoderm. Most notably, the subdivision of the extra-embryonic domain into separate amnion and serosa lineages in A. gambiae correlates with novel patterns of gene expression for several segmentation repressors. Moreover, the expanded amnion and serosa anlage correlates with a broader domain of Dpp signaling as compared with the D. melanogaster embryo. Evidence is presented that this expanded signaling is due to altered expression of the sog gene.     

Transgenic Xenopus embryos reveal that anterior neural development requires continued suppression of BMP signaling after gastrulation.

In vertebrates, BMP signaling before gastrulation suppresses neural development. Later in development, BMP signaling specifies a dorsal and ventral fate in the forebrain and dorsal fate in the spinal cord. It is therefore possible that a change in the competence of the ectoderm to respond to BMP signaling occurs at some point in development. We report that exposure of the anterior neural plate to BMP4 before gastrulation causes suppression of all neural markers tested. To determine the effects of BMP4 after gastrulation, we misexpressed BMP4 using a Pax-6 promoter fragment in transgenic frog embryos and implanted beads soaked in BMP4 in the anterior neural plate. Suppression of most anterior neural markers was observed. We conclude that most neural genes continue to require suppression of BMP signaling into the neurula stages. Additionally, we report that BMP4 and BMP7 are abundantly expressed in the prechordal mesoderm of the neurula stage embryo. This poses the paradox of how the expression of most neural genes is maintained if they can be inhibited by BMP signaling. We show that at least one gene in the anterior neural plate suppresses the response of the ectoderm to BMP signaling. We propose that the suppressive effect of BMP signaling on the expression of neural genes coupled with localized suppressors of BMP signaling result in the fine-tuning of gene expression in the anterior neural plate.     

Conditional BMP inhibition in Xenopus reveals stage-specific roles for BMPs in neural and neural crest induction

Bone morphogenetic protein (BMP) inhibition has been proposed as the primary determinant of neural cell fate in the developing Xenopus ectoderm. The evidence supporting this hypothesis comes from experiments in explanted "animal cap" ectoderm and in intact embryos using BMP antagonists that are unregulated and active well before gastrulation. While informative, these experiments cannot answer questions regarding the timing of signals and the behavior of cells in the more complex environment of the embryo. To examine the effects of BMP antagonism at defined times in intact embryos, we have generated a novel, two-component system for conditional BMP inhibition. We find that while blocking BMP signals induces ectopic neural tissue both in animal caps and in vivo, in intact embryos, it can only do so prior to late blastula stage (stage 9), well before the onset of gastrulation. Later inhibition does not induce neural identity, but does induce ectopic neural crest, suggesting that BMP antagonists play temporally distinct roles in establishing neural and neural crest identity. By combining BMP inhibition with fibroblast growth factor (FGF) activation, the neural inductive response in whole embryos is greatly enhanced and is no longer limited to pre-gastrula ectoderm. Thus, BMP inhibition during gastrulation is insufficient for neural induction in intact embryos, arguing against a BMP gradient as the sole determinant of ectodermal cell fate in the frog.     

The spatio-temporal distribution of single-stranded breaks in nuclear DNA in sections of clawed toad embryos during gastrulation and neurulation

Spatial and temporal pattern and quantities of nicks in nuclear DNA during gastrulation and neurulation was studied using nick-translation in sections of Xenopus laevis embryos. Specific changes in the number of nicks in different mesoderm and ectoderm regions were detected during embryogenesis. Dorso-ventral gradient of nuclear labelling was observed in mesoderm and inner ectoderm layer of early and middle gastrula. The gradient was inverted during transition from gastrula to neurula. At the same time dorso-ventral (in mesoderm) and ventro-dorsal (in outer ectoderm layer) gradients of nuclear labelling were increased. The intensity of nuclear labelling in all parts of embryo as a whole was remarkably higher during neurulation as compared with gastrulation. Dorso-ventral gradient of nuclear labelling was observed in mesoderm and ectoderm during neurulation. A connection between the nicks and differentiation status of the cells during early embryogenesis in amphibians is suggested.     

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.     

BMP/SMAD signaling regulates the cell behaviors that drive the initial dorsal-specific regional morphogenesis of the otocyst

During development of the otocyst, regional morphogenesis establishes a dorsal vestibular chamber and a ventral auditory chamber, which collectively constitute the membranous labyrinth of the inner ear. We identified the earliest morphogenetic event heralding the formation of the vestibular chamber, a rapid thinning and expansion of the dorsolateral wall of the otocyst, and showed that this process is generated by changes in otocyst cell shape from columnar to squamous, as opposed to changes in other cell behaviors, such as localized changes in cell proliferation or cell death. Moreover, we showed that thinning and expansion of the dorsolateral otocyst is regulated by BMP/SMAD signaling, which is both sufficient and necessary for localized thinning and expansion. Finally, we showed that BMP/SMAD signaling causes fragmentation of E-cadherin in the dorsolateral otocyst, occurring concomitantly with cell shape change, suggesting that BMP/SMAD signaling regulates cell-cell adhesion during the initial morphogenesis of the otocyst epithelium. Collectively, our results show that BMP signaling via SMADs regulates the cell behaviors that drive the initial dorsal-specific morphogenesis of the otocyst, providing new information about how regional morphogenesis of a complex organ rudiment, the developing membranous labyrinth, is initiated.     

Neural induction in Xenopus requires early FGF signalling in addition to BMP inhibition

Neural induction constitutes the first step in the generation of the vertebrate nervous system from embryonic ectoderm. Work with Xenopus ectodermal explants has suggested that epidermis is induced by BMP signals, whereas neural fates arise by default following BMP inhibition. In amniotes and ascidians, however, BMP inhibition does not appear to be sufficient for neural fate acquisition, which is initiated by FGF signalling. We decided to re-evaluate in the context of the whole embryo the roles of the BMP and FGF pathways during neural induction in Xenopus. We find that ectopic BMP activity converts the neural plate into epidermis, confirming that this pathway must be inhibited during neural induction in vivo. Conversely, inhibition of BMP, or of its intracellular effector SMAD1 in the non-neural ectoderm leads to epidermis suppression. In no instances, however, is BMP/SMAD1 inhibition sufficient to elicit neural induction in ventral ectoderm. By contrast, we find that neural specification occurs when weak eFGF or low ras signalling are combined with BMP inhibition. Using all available antimorphic FGF receptors (FGFR), as well as the pharmacological FGFR inhibitor SU5402, we demonstrate that pre-gastrula FGF signalling is required in the ectoderm for the emergence of neural fates. Finally, we show that although the FGF pathway contributes to BMP inhibition, as in other model systems, it is also essential for neural induction in vivo and in animal caps in a manner that cannot be accounted for by simple BMP inhibition. Taken together, our results reveal that in contrast to predictions from the default model, BMP inhibition is required but not sufficient for neural induction in vivo. This work contributes to the emergence of a model whereby FGF functions as a conserved initiator of neural specification among chordates.     

The Snail repressor positions Notch signaling in the Drosophila embryo

The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. We have investigated the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, Notch(IC), in transgenic embryos using the eve stripe2 enhancer. The Notch(IC) stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in 'na?ve' embryos that contain low uniform levels of Dorsal. We suggest that these dual activities of Snail, repression of Notch target genes and stimulation of Notch signaling, help define precise lines of sim expression within the neurogenic ectoderm.     

Sequence and expression of FoxB2 (XFD-5) and FoxI1c (XFD-10) in Xenopus embryogenesis

We report on the temporal and spatial expression pattern of two novel genes of the Xenopus fork head/winged helix family, xFoxB2 and xFoxI1c. xFoxB2 is activated at the late blastula stage and first expressed within the dorsolateral ectoderm except for the organiser territory. During gastrulation, xFoxB2 is found in two ectodermal stripes adjacent to the dorsal midline. Expression is completely down-regulated during neurulation. However, two distinct sets of cells expressing xFoxB2 re-appear in the rhombencephalon of swimming tadpoles. xFoxI1c is initially expressed at the early neurula stage in an epidermal ring around the neural field. Subsequent expression is found to be increased, and is exclusively localised to placodal precursor cells. The placodal expression remains until stage 40, when it is restricted to a distinct region in the lateral body wall behind the gills.     

Cell movements during epiboly and gastrulation in zebrafish

Beginning during the late blastula stage in zebrafish, cells located beneath a surface epithelial layer of the blastoderm undergo rearrangements that accompany major changes in shape of the embryo. We describe three distinctive kinds of cell rearrangements. (1) Radial cell intercalations during epiboly mix cells located deeply in the blastoderm among more superficial ones. These rearrangements thoroughly stir the positions of deep cells, as the blastoderm thins and spreads across the yolk cell. (2) Involution at or near the blastoderm margin occurs during gastrulation. This movement folds the blastoderm into two cellular layers, the epiblast and hypoblast, within a ring (the germ ring) around its entire circumference. Involuting cells move anteriorwards in the hypoblast relative to cells that remain in the epiblast; the movement shears the positions of cells that were neighbors before gastrulation. Involuting cells eventually form endoderm and mesoderm, in an anterior-posterior sequence according to the time of involution. The epiblast is equivalent to embryonic ectoderm. (3) Mediolateral cell intercalations in both the epiblast and hypoblast mediate convergence and extension movements towards the dorsal side of the gastrula. By this rearrangement, cells that were initially neighboring one another become dispersed along the anterior-posterior axis of the embryo. Epiboly, involution and convergent extension in zebrafish involve the same kinds of cellular rearrangements as in amphibians, and they occur during comparable stages of embryogenesis.     

Signals derived from the underlying mesoderm are dispensable for zebrafish neural crest induction

Signals from the non-neural ectoderm, the neural ectoderm, and the underlying mesoderm have all been implicated in the induction of neural crest. Bone morphogenetic protein (BMP) signaling in particular has an important role in this process; however, it is unclear whether this activity of BMP is due to its effects on patterning the underlying mesoderm, to its ability to establish a competent neural plate boundary zone, or to the direct specification of neural crest at intermediate levels of activity within a BMP gradient. We show neural crest induction occurs in zebrafish in the absence of involuted mesoderm, indicating that this tissue and signals derived from it are dispensable for the formation of neural crest. Dorsal-involuted mesoderm is a major source of secreted BMP antagonists, and the activity of BMP signaling is thought to depend on the presence of the opposing activity of these antagonists. We find that the three BMP antagonists known to be expressed during gastrulation in zebrafish, noggin1, follistatin, and chordin, are dispensable for neural crest induction. These results suggest that mechanisms for restricting the spatio-temporal pattern of BMP expression may compensate for the loss of secreted BMP antagonist activity in establishing dorso-ventral patterning, neural induction, and the neural crest.     

Sonic hedgehog and the molecular regulation of mouse neural tube closure

Neural tube closure is a fundamental embryonic event whose molecular regulation is poorly understood. As mouse neurulation progresses along the spinal axis, there is a shift from midline neural plate bending to dorsolateral bending. Here, we show that midline bending is not essential for spinal closure since, in its absence, the neural tube can close by a 'default' mechanism involving dorsolateral bending, even at upper spinal levels. Midline and dorsolateral bending are regulated by mutually antagonistic signals from the notochord and surface ectoderm. Notochordal signaling induces midline bending and simultaneously inhibits dorsolateral bending. Sonic hedgehog is both necessary and sufficient to inhibit dorsolateral bending, but is neither necessary nor sufficient to induce midline bending, which seems likely to be regulated by another notochordal factor. Attachment of surface ectoderm cells to the neural plate is required for dorsolateral bending, which ensures neural tube closure in the absence of sonic hedgehog signaling.     

Homeogenetic neural induction in Xenopus

Neural induction is known to involve an interaction of ectoderm with dorsal mesoderm during gastrulation, but several kinds of studies have argued that competent ectoderm can also be neutralized via an interaction with previously neuralized tissue, a process termed homeogenetic neural induction. Although homeogenetic neural induction has been proposed to play an important role in the normal induction of neural tissue, this process has not been subjected to detailed study using tissue recombinants and molecular markers. We have examined the question of homeogenetic neural induction in Xenopus embryos, both in transplant and recombinant experiments, using the expression of two neural antigens to assay the response. When ectoderm that is competent to be neuralized is transplanted to the region adjacent to the neural plate of early neurula embryos, it forms neural tissue, as assayed by staining with antibodies against the neural cell adhesion molecule, N-CAM. Transplants to the ventral region, far from the neural plate, do not express N-CAM, indicating that neuralization is not occurring as a result of the transplantation procedure itself. Because this response might be occurring as a result of interactions of ectoderm with either adjacent neural plate tissue, or with underlying dorsolateral mesoderm, recombinant experiments were performed to determine the source of the neuralizing signal. Ectoderm cultured in combination with neural plate tissue alone expresses neural markers, while ectoderm cultured in combination with dorsolateral mesoderm does not. We conclude that neural tissue can homeogenetically induce competent ectoderm to form neural tissue and argue that this induction occurs via planar signaling within the ectoderm, a mechanism that, in normal development, may be involved in interactions within presumptive neural ectoderm or in specifying structures that lie near the neural plate.