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.     

Plasma membrane cholesterol depletion disrupts prechordal plate and affects early forebrain patterning

Cholesterol-rich membrane microdomains (CRMMs) are specialized structures that have recently gained much attention in cell biology because of their involvement in cell signaling and trafficking. However, few investigations, particularly those addressing embryonic development, have succeeded in manipulating and observing CRMMs in living cells. In this study, we performed a detailed characterization of the CRMMs lipid composition during early frog development. Our data showed that disruption of CRMMs through methyl-β-cyclodextrin (MβCD) cholesterol depletion at the blastula stage did not affect Spemann's organizer gene expression and inductive properties, but impaired correct head development in frog and chick embryos by affecting the prechordal plate gene expression and cellular morphology. The MβCD anterior defect phenotype was recapitulated in head anlagen (HA) explant cultures. Culture of animal cap expressing Dkk1 combined with MβCD-HA generated a head containing eyes and cement gland. Together, these data show that during Xenopus blastula and gastrula stages, CRMMs have a very dynamic lipid composition and provide evidence that the secreted Wnt antagonist Dkk1 can partially rescue anterior structures in cholesterol-depleted head anlagen.     

Zebrafish Dkk1, induced by the pre-MBT Wnt signaling, is secreted from the prechordal plate and patterns the anterior neural plate

mRNA injection into the ventral blastomeres of Xenopus embryos of mRNA encoding Wnt pathway genes induces a secondary axis with complete head structures. To identify target genes of the pre-MBT dorsalization pathway that might be responsible for head formation in zebrafish, we have cloned zebrafish dickkopf1 (dkk1), which is expressed in tissues implicated in head patterning. We found that dkk1 blocks the post-MBT Wnt signaling and dkk1 is a target of the pre-MBT Wnt signaling. Dkk1 overexpression in the prechordal plate suggests that Dkk1, secreted from the prechordal plate, expands the forebrain at the expense of the midbrain in the anterior neural plate. Furthermore, dkk1 acts in parallel to the homeobox gene bozozok and bozozok is required for the maintenance of dkk1 expression. The nodal gene squint is also required for the maintenance of dkk1 expression. Among the mutually dependent target genes of the pre-MBT Wnt signaling, dkk1 plays an important role in patterning the anterior head of zebrafish.     

Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification.

Cranial neural crest (CNC) cells migrate extensively, typically in a pattern of cell streams. In Xenopus, these cells express the adhesion molecule Xcadherin-11 (Xcad-11) as they begin to emigrate from the neural fold. In order to study the function of this molecule, we have overexpressed wild-type Xcad-11 as well as Xcad-11 mutants with cytoplasmic (deltacXcad-11) or extracellular (deltaeXcad-11) deletions. Green fluorescent protein (GFP) was used to mark injected cells. We then transplanted parts of the fluorescent CNC at the premigratory stage into non-injected host embryos. This altered not only migration, but also the expression of neural crest markers. Migration of transplanted cranial neural crest cells was blocked when full-length Xcad-11 or its mutant lacking the beta-catenin-binding site (deltacXcad-11) was overexpressed. In addition, the expression of neural crest markers (AP-2, Snail and twist) diminished within the first four hours after grafting, and disappeared completely after 18 hours. Instead, these grafts expressed neural markers (2G9, nrp-I and N-Tubulin). Beta-catenin co-expression, heterotopic transplantation of CNC cells into the pharyngeal pouch area or both in combination failed to prevent neural differentiation of the grafts. By contrast, deltaeXcad-11 overexpression resulted in premature emigration of cells from the transplants. The AP-2 and Snail patterns remained unaffected in these migrating grafts, while twist expression was strongly reduced. Co-expression of deltaeXcad-11 and beta-catenin was able to rescue the loss of twist expression, indicating that Wnt/beta-catenin signalling is required to maintain twist expression during migration. These results show that migration is a prerequisite for neural crest differentiation. Endogenous Xcad-11 delays CNC migration. Xcad-11 expression must, however, be balanced, as overexpression prevents migration and leads to neural marker expression. Although Wnt/beta-catenin signalling is required to sustain twist expression during migration, it is not sufficient to block neural differentiation in non-migrating grafts.     

Xenopus Enhancer of Zeste (XEZ); an anteriorly restricted polycomb gene with a role in neural patterning

We have identified the Xenopus homologue of Drosophila Enhancer of Zeste using a differential display strategy designed to identify genes involved in early anterior neural differentiation. XEZ codes for a protein of 748 amino acids that is very highly conserved in evolution and is 96% identical to both human and mouse EZ(H)2. In common with most other Xenopus Pc-G genes and unlike mammalian Pc-G genes, XEZ is anteriorly restricted. Zygotic expression of XEZ commences during gastrulation, much earlier than other anteriorly localized Pc-G genes; expression is restricted to the anterior neural plate and is confined later to the forebrain, eyes and branchial arches. XEZ is induced in animal caps overexpressing noggin; up-regulation of XEZ therefore represents a response to inhibition of BMP signalling in ectodermal cells. We show that the midbrain/hindbrain junction marker En-2,and hindbrain marker Krox-20, are target genes of XEZ and that XEZ functions to repress these anteroposterior marker genes. Conversely, XEZ does not repress the forebrain marker Otx-2. XEZ overexpression results in a greatly thickened floor of the forebrain. These results implicate an important role for XEZ in the patterning of the nervous system.     

The neural plate specifies somite size in the Xenopus laevis gastrula.

The organizer has traditionally been considered the major source of somite-inducing signals. We show here that signaling from the neural plate specifies somite tissue and regulates somite size in the Xenopus gastrula. Ectopic undifferentiated neural tissue induces massive somite expansion at the expense of intermediate and lateral plate mesoderm. Although the early expanded somite expresses muscle-specific markers, only a portion terminally differentiates, suggesting that myotome development requires additional signals. Explant assays demonstrate that neural tissue induces somite-specific marker expression even in the absence of the organizer. Finally, we demonstrate that neural tissue is required for proper somite development because elimination of neural precursors results in pronounced somite reduction. Thus, an important reciprocal interaction exists between somite and neural tissue that is mutually reinforcing and critical for normal embryonic patterning.     

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.     

A role for xGCNF in midbrain-hindbrain patterning in Xenopus laevis.

Cells in the presumptive neural ectoderm of Xenopus are committed to neural fate through a process called neural induction, which may involve proteins that antagonize BMP signaling pathways. To identify genes that are induced by the BMP antagonists and that may be involved in subsequent neural patterning, we used a suppression PCR-based subtraction screen. Here we investigate the prospective activities and functions of one of the genes, a nuclear orphan receptor previously described as xGCNF. In animal cap assays, xGCNF synergizes with ectopic chordin to induce the midbrain-hindbrain marker engrailed-2 (En-2). In Keller explants, which rely on endogenous factors for neural induction, similar increases in En-2 are observed. Expression in embryos of a dominant interfering form of xGCNF reduces the expression of endogenous En-2 and Krox-20. These gain-of-function and prospective loss-of-function experiments, taken with the observation that xGCNF is expressed in the early neural plate and is elevated in the prospective midbrain-hindbrain region, which subsequently expresses En-2, suggest that xGCNF may play a role in regulating En-2 and thus midbrain-hindbrain identity.     

The role of Xmsx-2 in the anterior-posterior patterning of the mesoderm in Xenopus laevis

Many molecules are involved in defining mesodermal patterning of the Xenopus embryo. In this paper, evidence is provided that a member of the msx family of genes, the Xmsx-2 gene, is involved in anterior-posterior patterning of the mesoderm. A comparison of its sequence to another previously cloned msx-2 Xenopus homolog, Xhox-7.1' [45] showed that they are closely related. The Xmsx-2 gene is first expressed at midgastrulation predominantly in the dorsal part of the embryo. It showed a complex pattern of spatial expression, consistent with a role in patterning of the anterior-posterior axis. This inference is confirmed by gain-of-function experiments in which overexpressed msx-2 mRNA in developing Xenopus embryos resulted in embryos lacking anterior structures. Analysis of markers in mutant embryos showed that genes involved in ventral-posterior patterning such as Xhox-3, Xwnt-8, and Xvent-1 were upregulated, confirming the posteriorized nature of the embryos. We believe that the Xmsx-2 gene is involved in refining the patterning of the anterior-posterior part of the dorsal mesoderm after the initial signals determining the dorsal or ventral nature of the mesoderm have been specified.     

A restrictive role for Hedgehog signalling during otic specification in Xenopus

Vertebrate inner ear development is initiated by the specification of the otic placode, an ectodermal structure induced by signals from neighboring tissue. Although several signaling molecules have been identified as candidate otic inducers, many details of the process of inner ear induction remain elusive. Here, we report that otic induction is responsive to the level of Hedgehog (Hh) signaling activity in Xenopus, making use of both gain- and loss-of-function approaches. Ectopic activation of Hedgehog signaling resulted in the development of ectopic vesicular structures expressing the otic marker genes XPax-2, Xdll-3, and Xwnt-3A, thus revealing otic identity. Induction of ectopic otic vesicles was also achieved by misexpression of two different inhibitors of Hh signaling: the putative Hh antagonist mHIP and XPtc1deltaLoop2, a dominant-negative form of the Hh receptor Patched. In addition, misexpression of XPtc1deltaLoop2 as well as treatment of Xenopus embryos with the specific Hh signaling antagonist cyclopamine resulted in the formation of enlarged otic vesicles. In summary, our observations suggest that a defined level of Hh signaling provides a restrictive environment for otic fate in Xenopus embryos.