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.     

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.     

Rohon-Beard sensory neurons are induced by BMP4 expressing non-neural ectoderm in Xenopus laevis

Rohon-Beard mechanosensory neurons (RBs), neural crest cells, and neurogenic placodes arise at the border of the neural- and non-neural ectoderm during anamniote vertebrate development. Neural crest cells require BMP expressing non-neural ectoderm for their induction. To determine if epidermal ectoderm-derived BMP signaling is also involved in the induction of RB sensory neurons, the medial region of the neural plate from donor Xenopus laevis embryos was transplanted into the non-neural ventral ectoderm of host embryos at the same developmental stage. The neural plate border and RBs were induced at the transplant sites, as shown by expression of Xblimp1, and XHox11L2 and XN-tubulin, respectively. Transplantation studies between pigmented donors and albino hosts showed that neurons are induced both in donor neural and host epidermal tissue. Because an intermediate level of BMP4 signaling is required to induce neural plate border fates, we directly tested BMP4′s ability to induce RBs; beads soaked in either 1 or 10 ng/ml were able to induce RBs in cultured neural plate tissue. Conversely, RBs fail to form when neural plate tissue from embryos with decreased BMP activity, either from injection of noggin or a dominant negative BMP receptor, was transplanted into the non-neural ectoderm of un-manipulated hosts. We conclude that contact between neural and non-neural ectoderm is capable of inducing RBs, that BMP4 can induce RB markers, and that BMP activity is required for induction of ectopic RB sensory neurons.     

FGF/MAPK signaling is required in the gastrula epiblast for avian neural crest induction

Neural crest induction involves the combinatorial inputs of the FGF, BMP and Wnt signaling pathways. Recently, a two-step model has emerged where BMP attenuation and Wnt activation induces the neural crest during gastrulation, whereas activation of both pathways maintains the population during neurulation. FGF is proposed to act indirectly during the inductive phase by activating Wnt ligand expression in the mesoderm. Here, we use the chick model to investigate the role of FGF signaling in the amniote neural crest for the first time and uncover a novel requirement for FGF/MAPK signaling. Contrary to current models, we demonstrate that FGF is required within the prospective neural crest epiblast during gastrulation and is unlikely to operate through mesodermal tissues. Additionally, we show that FGF/MAPK activity in the prospective neural plate prevents the ectopic expression of lateral ectoderm markers, independently of its role in neural specification. We then investigate the temporal participation of BMP/Smad signaling and suggest a later involvement in neural plate border development, likely due to widespread FGF/MAPK activity in the gastrula epiblast. Our results identify an early requirement for FGF/MAPK signaling in amniote neural crest induction and suggest an intriguing role for FGF-mediated Smad inhibition in ectodermal development.     

Role of BMP signaling and the homeoprotein Iroquois in the specification of the cranial placodal field

Different types of placodes originate at the anterior border of the neural plate but it is still an unresolved question whether individual placodes arise as distinct ectodermal specializations in situ or whether all or a subset of the placodes originate from a common preplacodal field. We have analyzed the expression and function of the homeoprotein Iro1 in Xenopus and zebrafish embryos, and we have compared its expression with several preplacodal and placodal markers. Our results indicate that the iro1 genes are expressed in the preplacodal region, being one of the earliest markers for this area. We show that an interaction between the neural plate and the epidermis is able to induce the expression of several preplacodal markers, including Xiro1, by a similar mechanism to that previously shown for neural crest induction. In addition, we analyzed the role of BMP in the specification of the preplacodal field by studying the expression of the preplacodal markers Six1, Xiro1, and several specific placodal markers. We experimentally modified the level of BMP activity by three different methods. First, we implanted beads soaked with noggin in early neurula stage Xenopus embryos; second, we injected the mRNA that encodes a dominant negative of the BMP receptor into Xenopus and zebrafish embryos; and third, we grafted cells expressing chordin into zebrafish embryos. The results obtained using all three methods show that a reduction in the level of BMP activity leads to an expansion of the preplacodal and placodal region similar to what has been described for neural crest regions. By using conditional constructs of Xiro1, we performed gain and loss of function experiments. We show that Xiro1 play an important role in the specification of both the preplacodal field as well as individual placodes. We have also used inducible dominant negative and activator constructs of Notch signaling components to analyze the role of these factors on placodal development. Our results indicate that the a precise level of BMP activity is required to induce the neural plate border, including placodes and neural crest cells, that in this border the iro1 gene is activated, and that this activation is required for the specification of the placodes.     

Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest induction.

The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. Here, the roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, was transformed into neural crest by posteriorizing signals. This transformation was mimicked by bFGF, Wnt-8, or retinoic acid treatment and was also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The transformation of the anterior neural plate border into neural crest cells was also achieved in whole embryos, by retinoic acid treatment or by use of a constitutively active form of the retinoic acid receptor. By analyzing the expression of mesodermal markers and various graft experiments, the expression of the mutant retinoic acid receptor was shown to directly affect the ectoderm. We thereby propose a two-step model for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal specification induce neural folds with an anterior character along the entire neural plate border. Subsequently, the most posterior region of this anterior neural plate border is transformed into the neural crest by the posteriorizing activity of FGFs, Wnts, and retinoic acid signals. We discuss a unifying model where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest 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.     

A balance of FGF, BMP and WNT signalling positions the future placode territory in the head

The sensory nervous system in the vertebrate head arises from two different cell populations: neural crest and placodal cells. By contrast, in the trunk it originates from neural crest only. How do placode precursors become restricted exclusively to the head and how do multipotent ectodermal cells make the decision to become placodes or neural crest? At neural plate stages, future placode cells are confined to a narrow band in the head ectoderm, the pre-placodal region (PPR). Here, we identify the head mesoderm as the source of PPR inducing signals, reinforced by factors from the neural plate. We show that several independent signals are needed: attenuation of BMP and WNT is required for PPR formation. Together with activation of the FGF pathway, BMP and WNT antagonists can induce the PPR in na?ve ectoderm. We also show that WNT signalling plays a crucial role in restricting placode formation to the head. Finally, we demonstrate that the decision of multipotent cells to become placode or neural crest precursors is mediated by WNT proteins: activation of the WNT pathway promotes the generation of neural crest at the expense of placodes. This mechanism explains how the placode territory becomes confined to the head, and how neural crest and placode fates diversify.     

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.     

Delta/Notch signaling promotes formation of zebrafish neural crest by repressing Neurogenin 1 function

In zebrafish, cells at the lateral edge of the neural plate become Rohon-Beard primary sensory neurons or neural crest. Delta/Notch signaling is required for neural crest formation. ngn1 is expressed in primary neurons; inhibiting Ngn1 activity prevents Rohon-Beard cell formation but not formation of other primary neurons. Reducing Ngn1 activity in embryos lacking Delta/Notch signaling restores neural crest formation, indicating Delta/Notch signaling inhibits neurogenesis without actively promoting neural crest. Ngn1 activity is also required for later development of dorsal root ganglion sensory neurons; however, Rohon-Beard neurons and dorsal root ganglion neurons are not necessarily derived from the same precursor cell. We propose that temporally distinct episodes of Ngn1 activity in the same precursor population specify these two different types of sensory neurons.     

The role of BMP signaling in outgrowth and patterning of the Xenopus tail bud.

Tail bud formation in Xenopus depends on interaction between a dorsal domain (dorsal roof) expressing lunatic fringe and Notch, and a ventral domain (posterior wall) expressing the Notch ligand Delta. Ectopic expression of an activated form of Notch, Notch ICD, by means of an animal cap graft into the posterior neural plate, results in the formation of an ectopic tail-like structure containing a neural tube and fin. However, somites are never formed in these tails. Here, we show that BMP signaling is activated in the posterior wall of the tail bud and is involved in the formation of tail somites from this region. Grafts into the posterior neural plate, in which BMP signaling is activated, will form tail-like outgrowths. Unlike the Notch ICD tails, the BMP tails contain well-organized somites as well as neural tube and fin, with the graft contributing to both somites and neural tube. Through a variety of epistasis-type experiments, we show that the most likely model involves a requirement for BMP signaling upstream of Notch activation, resulting in formation of the secondary neural tube, as well as a Notch-independent pathway leading to the formation of tail somites from the posterior wall.     

Different downstream pathways for Notch signaling are required for gliogenic and chondrogenic specification of mouse mesencephalic neural crest cells

We examined the roles of Notch signaling and fibroblast growth factors (FGFs) in the gliogenesis of mouse mesencephalic neural crest cells. The present study demonstrated that Notch activation or FGF treatment promotes the differentiation of glia expressing glial fibrillary acidic protein. Notch activation or FGF2 exposure during the first 48 h in culture was critical for glial differentiation. The promotion of gliogenesis by FGF2 was significantly suppressed by the inhibition of Notch signaling using Notch-1 siRNA. These data suggest that FGFs activate Notch signaling and that this activation promotes the gliogenic specification of mouse mesencephalic neural crest cells. Notch activation and FGF treatment have been shown to participate in the chondrogenic specification of these cells [Nakanishi, K., Chan, Y.S., Ito, K., 2007. Notch signaling is required for the chondrogenic specification of mouse mesencephalic neural crest cells. Mech. Dev. 124, 190–203]. Therefore, we analyzed whether or not there were differences between gliogenic and chondrogenic specifications in the downstream pathway of the Notch receptor. Whereas the activation of only the Deltex-mediated pathway was sufficient to promote glial specification, the activation of both RBP-J- and Deltex-dependent pathways was required for chondrogenic specification. These results suggest that the different downstream pathways of the Notch receptor participate in the gliogenic and chondrogenic specification of mouse mesencephalic neural crest cells.     

Differential distribution of competence for panplacodal and neural crest induction to non-neural and neural ectoderm

It is still controversial whether cranial placodes and neural crest cells arise from a common precursor at the neural plate border or whether placodes arise from non-neural ectoderm and neural crest from neural ectoderm. Using tissue grafting in embryos of Xenopus laevis, we show here that the competence for induction of neural plate, neural plate border and neural crest markers is confined to neural ectoderm, whereas competence for induction of panplacodal markers is confined to non-neural ectoderm. This differential distribution of competence is established during gastrulation paralleling the dorsal restriction of neural competence. We further show that Dlx3 and GATA2 are required cell-autonomously for panplacodal and epidermal marker expression in the non-neural ectoderm, while ectopic expression of Dlx3 or GATA2 in the neural plate suppresses neural plate, border and crest markers. Overexpression of Dlx3 (but not GATA2) in the neural plate is sufficient to induce different non-neural markers in a signaling-dependent manner, with epidermal markers being induced in the presence, and panplacodal markers in the absence, of BMP signaling. Taken together, these findings demonstrate a non-neural versus neural origin of placodes and neural crest, respectively, strongly implicate Dlx3 in the regulation of non-neural competence, and show that GATA2 contributes to non-neural competence but is not sufficient to promote it ectopically.     

Dynamic alterations in gene expression after Wnt-mediated induction of avian neural crest

The Wnt signaling pathway is important in the formation of neural crest cells in many vertebrates, but the downstream targets of neural crest induction by Wnt are largely unknown. Here, we examined quantitative changes in gene expression regulated by Wnt-mediated neural crest induction using quantitative PCR (QPCR). Induction was recapitulated in vitro by adding soluble Wnt to intermediate neural plate tissue cultured in collagen, and induced versus control tissue were assayed using gene-specific primers at times corresponding to premigratory (18 and 24 h) or early (36 h) stages of crest migration. The results show that Wnt signaling up-regulates in a distinct temporal pattern the expression of several genes normally expressed in the dorsal neural tube (slug, Pax3, Msx1, FoxD3, cadherin 6B) at "premigratory" stages. While slug is maintained in early migrating crest cells, Pax3, FoxD3, Msx1 and cadherin 6B all are down-regulated by the start of migration. These results differ from the temporal profile of these genes in response to the addition of recombinant BMP4, where gene expression seems to be maintained. Interestingly, expression of rhoB is unchanged or even decreased in response to Wnt-mediated induction at all times examined, though it is up-regulated by BMP signals. The temporal QPCR profiles in our culture paradigm approximate in vivo expression patterns of these genes before neural crest migration, and are consistent with Wnt being an initial neural crest inducer with additional signals like BMP and other factors maintaining expression of these genes in vivo. Our results are the first to quantitatively describe changes in gene expression in response to a Wnt or BMP signal during transformation of a neural tube cell into a migratory neural crest cell.