BMP antagonists and FGF signaling contribute to different domains of the neural plate in Xenopus

In ectodermal explants from Xenopus embryos, inhibition of BMP signaling is sufficient for neural induction, leading to the idea that neural fate is the default state in the ectoderm. Many of these experiments assayed the action of BMP antagonists on animal caps, which are relatively naïve explants of prospective ectoderm, and different results have led to debate regarding both the mechanism of neural induction and the appropriateness of animal caps as an assay system. Here we address whether BMP antagonists are only able to induce neural fates in pre-patterned explants, and the extent to which neural induction requires FGF signaling. We suggest that some discrepancies in conclusion depend on the interpretations of sox gene expression, which we show not only marks definitive neural tissue, but also tissue that is not yet committed to neural fates. Part of the early sox2 domain requires FGF signaling, but in the absence of organizer signaling, this domain reverts to epidermal fates. We also reinforce the evidence that ectodermal explants are naïve, and that explants that lack any dorsal prepattern are readily neuralized by BMP antagonists, even when FGF signaling is inhibited.     

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.     

Cell communication with the neural plate is required for induction of neural markers by BMP inhibition: evidence for homeogenetic induction and implications for Xenopus animal cap and chick explant assays

In Xenopus, the animal cap is very sensitive to BMP antagonists, which result in neuralization. In chick, however, only cells at the border of the neural plate can be neuralized by BMP inhibition. Here we compare the two systems. BMP antagonists can induce neural plate border markers in both ventral Xenopus epidermis and non-neural chick epiblast. However, BMP antagonism can only neuralize ectodermal cells when the BMP-inhibited cells form a continuous trail connecting them to the neural plate or its border, suggesting that homeogenetic neuralizing factors can only travel between BMP-inhibited cells. Xenopus animal cap explants contain cells fated to contribute to the neural plate border and even to the anterior neural plate, explaining why they are so easily neuralized by BMP-inhibition. Furthermore, chick explants isolated from embryonic epiblast behave like Xenopus animal caps and express border markers. We propose that the animal cap assay in Xenopus and explant assays in the chick are unsuitable for studying instructive signals in neural induction.     

Frizzled7 mediates canonical Wnt signaling in neural crest induction

The neural crest is a multipotent cell population that migrates from the dorsal edge of the neural tube to various parts of the embryo where it differentiates into a remarkable variety of different cell types. Initial induction of neural crest is mediated by a combination of BMP, Wnt, FGF, Retinoic acid and Notch/Delta signaling. The two-signal model for neural crest induction suggests that BMP signaling induces the competence to become neural crest. The second signal involves Wnt acting through the canonical pathway and leads to expression of neural crest markers such as slug. Wnt signals from the neural plate, non-neural ectoderm and paraxial mesoderm have all been suggested to play a role in neural crest induction. We show that Xenopus frizzled7 (Xfz7) is expressed in the dorsal ectoderm including early neural crest progenitors and is a key mediator of the Wnt inductive signal. We demonstrate that Xfz7 expression is induced in response to a BMP antagonist, noggin, and that Xfz7 can induce neural crest specific genes in noggin-treated ectodermal explants (animal caps). Morpholino-mediated or dominant negative inhibition of Xfz7 inhibits Wnt induced Xslug expression in the animal cap assay and in the whole embryo leading to a loss of neural crest derived pigment cells. Full-length Xfz7 rescues the morpholino-induced phenotype, as does activated beta-catenin, suggesting that Xfz7 is signaling through the canonical pathway. We therefore demonstrate that Xfz7 is regulated by BMP antagonism and is required for neural crest induction by Wnt in the developing vertebrate embryo.     

Neural induction requires continued suppression of both Smad1 and Smad2 signals during gastrulation

Vertebrate neural induction requires inhibition of bone morphogenetic protein (BMP) signaling in the ectoderm. However, whether inhibition of BMP signaling is sufficient to induce neural tissues in vivo remains controversial. Here we have addressed why inhibition of BMP/Smad1 signaling does not induce neural markers efficiently in Xenopus ventral ectoderm, and show that suppression of both Smad1 and Smad2 signals is sufficient to induce neural markers. Manipulations that inhibit both Smad1 and Smad2 pathways, including a truncated type IIB activin receptor, Smad7 and Ski, induce early neural markers and inhibit epidermal genes in ventral ectoderm; and co-expression of BMP inhibitors with a truncated activin/nodal-specific type IB activin receptor leads to efficient neural induction. Conversely, stimulation of Smad2 signaling in the neural plate at gastrula stages results in inhibition of neural markers, disruption of the neural tube and reduction of head structures, with conversion of neural to neural crest and mesodermal fates. The ability of activated Smad2 to block neural induction declines by the end of gastrulation. Our results indicate that prospective neural cells are poised to respond to Smad2 and Smad1 signals to adopt mesodermal and non-neural ectodermal fates even at gastrula stages, after the conventionally assigned end of mesodermal competence, so that continued suppression of both mesoderm- and epidermis-inducing Smad signals leads to efficient neural induction.     

Cdc2-like kinase 2 (Clk2) promotes early neural development in Xenopus embryos

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways.     

Bone morphogenetic protein acts as a ventral mesoderm modifier in early Xenopus embryos

Mesoderm of early vertebrate embryos gradually acquires dorsal–ventral polarity during embryogenesis. This specification of mesoderm is thought to be regulated by several polypeptide growth factors. Bone morphogenetic protein (BMP), a member of the TGF-β family, is one of the regulators suggested to be involved in the formation of ventral mesoderm. In this paper, the nature of the endogenous BMP signal in dorsal–ventral specification was assessed in early Xenopus embryos using a dominant negative mutant of the Xenopus BMP receptor. In ectodermal explant assays, disruption of endogenous BMP signaling by the mutant receptor changed the competence of the explant cells to mesoderm-inducing factors, activin and basic fibroblast growth factor (bFGF), and led to formation of neural tissue without mesoderm induction. This result suggests that endogenous BMP acts as a ventral mesoderm modifier rather than a ventral mesoderm inducer, and that interactions between endogenous BMP and mesoderm-inducing factors may be important in dorsal–ventral patterning of embryonic mesoderm. In addition, the induction of neural tissue by inhibition of the BMP signaling pathway also suggests involvement of BMP in neural induction.     

A role for frizzled 3 in neural crest development

Wnts are a large family of secreted molecules implicated in numerous developmental processes. Frizzled proteins are likely receptors for Wnts and are required for Wnt signaling in invertebrates. A large number of vertebrate frizzled genes have also been identified, but their roles in mediating specific responses to endogenous Wnts have not been well defined. Using a functional assay in Xenopus, we have performed a large screen to identify potential interactions between Wnts and frizzleds. We find that signaling by Xwnt1, but not other Wnts, can be specifically enhanced by frizzled 3 (Xfz3). As both Xfz3 and Xwnt1 are highly localized to dorsal neural tissues that give rise to neural crest, we examined whether Xfz3 mediates Xwnt1 signaling in the formation of neural crest. Xfz3 specifically induces neural crest in ectodermal explants and in embryos, similar to Xwnt1, and at lower levels of expression, synergizes with Xwnt1 in neural crest induction. Furthermore, loss of Xfz3 function, either by depletion with a Xfz3-directed morpholino antisense oligonucleotide or by expression of an inhibitory form of Xfz3 (Nfz3), prevents Xwnt1-dependent neural crest induction in ectodermal explants and blocks neural crest formation in whole embryos. These results show that Xfz3 is required for Xwnt1 signaling in the formation of the neural crest in the developing vertebrate embryo.     

SNW1 is a critical regulator of spatial BMP activity, neural plate border formation, and neural crest specification in vertebrate embryos

Bone morphogenetic protein (BMP) gradients provide positional information to direct cell fate specification, such as patterning of the vertebrate ectoderm into neural, neural crest, and epidermal tissues, with precise borders segregating these domains. However, little is known about how BMP activity is regulated spatially and temporally during vertebrate development to contribute to embryonic patterning, and more specifically to neural crest formation. Through a large-scale in vivo functional screen in Xenopus for neural crest fate, we identified an essential regulator of BMP activity, SNW1. SNW1 is a nuclear protein known to regulate gene expression. Using antisense morpholinos to deplete SNW1 protein in both Xenopus and zebrafish embryos, we demonstrate that dorsally expressed SNW1 is required for neural crest specification, and this is independent of mesoderm formation and gastrulation morphogenetic movements. By exploiting a combination of immunostaining for phosphorylated Smad1 in Xenopus embryos and a BMP-dependent reporter transgenic zebrafish line, we show that SNW1 regulates a specific domain of BMP activity in the dorsal ectoderm at the neural plate border at post-gastrula stages. We use double in situ hybridizations and immunofluorescence to show how this domain of BMP activity is spatially positioned relative to the neural crest domain and that of SNW1 expression. Further in vivo and in vitro assays using cell culture and tissue explants allow us to conclude that SNW1 acts upstream of the BMP receptors. Finally, we show that the requirement of SNW1 for neural crest specification is through its ability to regulate BMP activity, as we demonstrate that targeted overexpression of BMP to the neural plate border is sufficient to restore neural crest formation in Xenopus SNW1 morphants. We conclude that through its ability to regulate a specific domain of BMP activity in the vertebrate embryo, SNW1 is a critical regulator of neural plate border formation and thus neural crest specification.     

Calcium transients and calcium signalling during early neurogenesis in the amphibian embryo Xenopus laevis

Development of the vertebrate embryonic nervous system is characterized by a cascade of signalling events. In Xenopus, the initial step in this cascade results from signals emanating from the dorsal mesoderm that divert the fate of the ectoderm from an epidermal to a neural lineage. These signals include extracellular antagonists of the bone morphogenetic protein (BMP). Experiments performed with isolated ectoderm suggest that epidermis is induced by BMP, whereas neural fates arise by default following BMP inhibition; however, we show that this mechanism is not sufficient for neural determination. Ca2+ imaging of intact Xenopus embryos reveals patterns of Ca2+ transients in the dorsal ectoderm but not in the ventral ectoderm. These increases in intracellular calcium concentration ([Ca2+](i)), which occur via the activation of dihydropyridine (DHP)-sensitive Ca2+ channels, are necessary and sufficient to orientate the ectodermal cells toward a neural fate. On the one hand, the treatments that antagonize the increase in [Ca2+](i), inhibit neuralization, while on the other hand, an artificial increase in [Ca2+](i), whatever its origin, neuralizes the ectoderm. Using these properties, we have constructed a subtractive cDNA library between untreated ectoderm and caffeine-treated ectoderm. The caffeine stimulates an increase in [Ca2+](i) and thus orientates the cells towards the neural pathway. We have identified early Ca2+ target genes expressed in neural territories. One of these genes, an arginine methyl transferase, controls the expression of the early proneural gene, Zic3. Here, we discuss an alternative model where Ca2+ plays a central regulatory role in early neurogenesis. This model integrates the activation of a Ca2+ -dependent signalling pathway due to an influx of Ca2+ through DHP-Ca2+ channels. While Ca2+ is required for neural determination, epidermal determination occurs when Ca2+ -dependent signalling pathways are inactive.     

Xbra3 elicits the production of neural tissue by a non-BMP-dependent mechanism in Xenopus sp

Ectoderm cells in animal caps from Xenopus embryos develop to form either epidermis or neural tissue depending upon their receipt of intercellular signals. To date, several secreted neural inducers have been identified which act through the local inhibition of bone morphogenetic protein (BMP) signaling, preventing differentiation to epidermis and resulting in adoption of neural fate. In this work, we have exploited an interspecies animal cap assay, which enables detection of the effects of signaling molecules produced by cells of one animal cap and influencing development in a second cap cultured in close apposition in a Holtfreter combination. We show that expression of the T-box protein, Xbra3, in one cap causes the production of a factor, which causes adoption of neural fate by cells of the other animal cap. The action of this factor is not inhibited by the over-expression of BMP in cells of the responding animal cap, or by the inhibition of Wnt signaling. These findings suggest the existence of a secreted signaling molecule that is able to induce ectodermal cells to adopt neural fate by a mechanism independent of the inhibition of the BMP or Wnt signaling pathways.     

TAK1 promotes BMP4/Smad1 signaling via inhibition of erk MAPK: a new link in the FGF/BMP regulatory network

FGFs and BMPs act in concert to regulate a wide range of processes in vertebrate development. In most cases, FGFs and BMPs have opposing effects, and specific developmental outcomes arise out of a balance between the two growth factors. We and others have previously demonstrated that signaling pathways activated by FGFs and BMPs interact via inhibitory crosstalk. Here we demonstrate a role for the BMP effector TGF-β Activated Kinase 1 (TAK1) in the maintenance of Smad1 activity in Xenopus embryos, via the inhibition of erk MAPK. Up- or downregulation of TAK1 levels produces an inverse alteration in the amount of activated erk MAPK. The inhibition of erk MAPK by TAK1 is mediated by p38 and a corresponding decrease in phosphorylation of MEK. TAK1 morphant embryos show a decrease in the nuclear accumulation of Smad1. Conversely, reduction of erk MAPK activity via overexpression of MAP Kinase Phosphatase1 (MKP1) leads to an increase in nuclear Smad1. Both TAK1 morphant ectoderm and ectoderm treated with FGF show a decrease in the expression of several Smad1-inducible genes. Neural-specific gene expression is inhibited in isolated ectoderm coexpressing noggin and TAK1, suggesting that TAK1 is sufficient to inhibit neural specification. Introduction of TAK1 morpholino oligonucleotide expands the expression of organizer genes, disrupts formation of the boundary between organizer and non-organizer mesoderm, and increases the spatial range of MAPK activation in response to localized FGF. Our results indicate that inhibitory interactions between FGF and BMP4 effector pathways increase the robustness of BMP signaling via a feed-forward mechanism.     

A transmembrane protein EIG121L is required for epidermal differentiation during early embryonic development

Epidermal differentiation in the ventral ectoderm of Xenopus embryos is regulated by the bone morphogenetic protein (BMP) pathway. However, it remains unclear how the BMP pathway is activated and induces the epidermal fate in the ventral ectoderm. Here, we identify a novel player in the BMP pathway that is required for epidermal differentiation during Xenopus early embryonic development. We show that Xenopus EIG121L (xEIG121L) protein, an evolutionarily conserved transmembrane protein, is expressed in the ventral ectoderm at the gastrula and neurula stages. Almost complete knockdown of xEIG121L protein with antisense morpholino oligonucleotides in early Xenopus embryos results in severe developmental defects, including the inhibition of epidermal differentiation and the induction of neural genes. Remarkably, our analysis shows that BMP/Smad1 signaling is severely suppressed in the xEIG121L knockdown ectoderm. Moreover, immunoprecipitation and immunostaining experiments suggest that xEIG121L protein physically interacts, and co-localizes, with BMP receptors. Thus, our results identify a novel regulator of the BMP pathway that has a positive role in BMP signaling and plays an essential role in epidermal differentiation during early embryonic development.     

Xenopus Meis3 protein forms a hindbrain-inducing center by activating FGF/MAP kinase and PCP pathways

Knockdown studies in Xenopus demonstrated that the XMeis3 gene is required for proper hindbrain formation. An explant assay was developed to distinguish between autonomous and inductive activities of XMeis3 protein. Animal cap explants caudalized by XMeis3 were recombined with explants neuralized by the BMP dominant-negative receptor protein. XMeis3-expressing cells induced convergent extension cell elongations in juxtaposed neuralized explants. Elongated explants expressed hindbrain and primary neuron markers, and anterior neural marker expression was extinguished. Cell elongation was dependent on FGF/MAP-kinase and Wnt-PCP activities. XMeis3 activates FGF/MAP-kinase signaling, which then modulates the PCP pathway. In this manner, XMeis3 protein establishes a hindbrain-inducing center that determines anteroposterior patterning in the brain.     

Xenopus GDF6, a new antagonist of noggin and a partner of BMPs.

In Xenopus, ectodermal cell fates are determined by antagonistic interaction between the BMP subfamily of TGF-(beta) ligands and the organizer-specific secreted factors (e.g. noggin, chordin and follistatin). Inhibition of BMP function by these factors can convert cells from an epidermal to a neural cell fate. In this study, we report that GDF6, a new member of the Xenopus TGF-(beta) family, can function in antagonistic interaction with neural inducers. GDF6 induces epidermis and inhibits neural tissue in dissociated cells, and this activity is blocked by the presence of noggin. We demonstrate that GDF6 binds directly to the neural inducer noggin. Furthermore, we find that GDF6 and BMP2 can form heterodimers and the process seems to require cotranslation of the proteins in the same cells. In normal embryos, GDF6 and BMP2 are coexpressed in several places, including the edge of the neural plate at early neurula stages, suggesting that GDF6 may synergize with BMPs to regulate patterning of the ectoderm. Our data show for the first time that noggin can bind directly to and inhibit another TGF-(beta) family member: GDF6. In addition, BMP and GDF6 heterodimers may play an important role in vivo to regulate cell fate determination and patterning.     

Neural and head induction by insulin-like growth factor signals.

Evidence is presented for a new pathway participating in anterior neural development. It was found that IGF binding protein 5 (IGFBP-5), as well as three IGFs expressed in early embryos, promoted anterior development by increasing the head region at the expense of the trunk in mRNA-injected Xenopus embryos. A secreted dominant-negative type I IGF receptor (DN-IGFR) had the opposite effect. IGF mRNAs led to the induction of ectopic eyes and ectopic head-like structures containing brain tissue. In ectodermal explants, IGF signals induced anterior neural markers in the absence of mesoderm formation and DN-IGFR inhibited neural induction by the BMP antagonist Chordin. Thus, active IGF signals appear to be both required and sufficient for anterior neural induction in Xenopus.