Neural crest induction by the canonical Wnt pathway can be dissociated from anterior-posterior neural patterning in Xenopus

While Wnt signaling is known to be involved in early steps of neural crest development, the mechanism remains unclear. Because Wnt signaling is able to posteriorize anterior neural tissues, neural crest induction by Wnts has been proposed to be an indirect consequence of posteriorization of neural tissues rather than a direct effect of Wnt signaling. To address the relationship between posteriorization and neural crest induction by Wnt signaling, we have used gain of function and loss of function approaches in Xenopus to modulate the level of Wnt signaling at multiple points in the pathway. We find that modulating the level of Wnt signaling allows separation of neural crest induction from the effects of Wnts on anterior-posterior neural patterning. We also find that activation of Wnt signaling induces ectopic neural crest in the anterior region without posteriorizing anterior neural tissues. In addition, Wnt signaling induces neural crest when its posteriorizing activity is blocked by inhibition of FGF signaling in neuralized explants. Finally, depletion of beta-catenin confirms that the canonical Wnt pathway is required for initial neural crest induction. While these observations do not exclude a role for posteriorizing signals in neural crest induction, our data, together with previous observations, strongly suggest that canonical Wnt signaling plays an essential and direct role in 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.     

Sox9 neural crest determinant gene controls patterning and closure of the posterior frontal cranial suture

Cranial suture development involves a complex interaction of genes and tissues derived from neural crest cells (NCC) and paraxial mesoderm. In mice, the posterior frontal (PF) suture closes during the first month of life while other sutures remain patent throughout the life of the animal. Given the unique NCC origin of PF suture complex (analogous to metopic suture in humans), we performed quantitative real-time PCR and immunohistochemistry to study the expression pattern of the NCC determinant gene Sox9 and select markers of extracellular matrix. Our results indicated a unique up-regulated expression of Sox9, a regulator of chondrogenesis, during initiation of PF suture closure, along with the expression of specific cartilage markers (Type II Collagen and Type X Collagen), as well as cartilage tissue formation in the PF suture. This process was followed by expression of bone markers (Type I Collagen and Osteocalcin), suggesting endochondral ossification. Moreover, we studied the effect of haploinsufficiency of the NCC determinant gene Sox9 in the NCC derived PF suture complex. A decrease in dosage of Sox9 by haploinsufficiency in NCC-derived tissues resulted in delayed PF suture closure. These results demonstrate a unique development of the PF suture complex and the role of Sox9 as an important contributor to timely and proper closure of the PF suture through endochondral ossification.     

Expression of Sox1 during Xenopus early embryogenesis

Sox B1 group genes, Sox1, Sox2, and Sox3 (Sox1-3), are involved in neurogenesis in various species. Here, we identified the Xenopus homolog of Sox1, and investigated its expression patterns and neural inducing activity. Sox1 was initially expressed in the anterior neural plate of Xenopus embryos, with expression restricted to the brain and optic vesicle by the tailbud stage. Expression subsequently decreased in the eye region by the tadpole stage. Sox1 expression in animal cap explants was induced by inhibition of BMP signaling in the same manner as Sox2, Sox3, and SoxD. In addition, overexpression of Sox1 induced neural markers in ventral ectoderm and in animal caps. These results implicate Xenopus Sox1 in neurogenesis, especially brain and eye development.     

Zic2 is required for neural crest formation and hindbrain patterning during mouse development

The Zic genes are the vertebrate homologues of the Drosophila pair rule gene odd-paired. It has been proposed that Zic genes play several roles during neural development including mediolateral segmentation of the neural plate, neural crest induction, and inhibition of neurogenesis. Initially during mouse neural development Zic2 is expressed throughout the neural plate while later on expression in the neurectoderm becomes restricted to the lateral region of the neural plate. A hypomorphic allele of Zic2 has demonstrated that in the mouse Zic2 is required for the timing of neurulation. We have isolated a new allele of Zic2 that behaves as a loss of function allele. Analysis of this mutant reveals two further functions for Zic2 during early neural development. Mutation of Zic2 results in a delay of neural crest production and a decrease in the number of neural crest cells that are produced. These defects are independent of mediolateral segmentation of the neurectoderm and of dorsal neurectoderm proliferation, both of which occur normally in the mutant embryos. Additionally Zic2 is required during hindbrain patterning for the normal development of rhombomeres 3 and 5. This work provides the first genetic evidence that the Zic genes are involved in neural crest production and the first demonstration that Zic2 functions during hindbrain patterning.     

Neural crests are actively precluded from the anterior neural fold by a novel inhibitory mechanism dependent on Dickkopf1 secreted by the prechordal mesoderm

It is known the interactions between the neural plate and epidermis generate neural crest (NC), but it is unknown why the NC develops only at the lateral border of the neural plate and not in the anterior fold. Using grafting experiments we show that there is a previously unidentified mechanism that precludes NC from the anterior region. We identify prechordal mesoderm as the tissue that inhibits NC in the anterior territory and show that the Wnt/beta-catenin antagonist Dkk1, secreted by this tissue, is sufficient to mimic this NC inhibition. We show that Dkk1 is required for preventing the formation of NC in the anterior neural folds as loss-of-function experiments using a Dkk1 blocking antibody in Xenopus as well as the analysis of Dkk1-null mouse embryos transform the anterior neural fold into NC. This can be mimicked by Wnt/beta-catenin signaling activation without affecting the anterior posterior patterning of the neural plate, or placodal specification. Finally, we show that the NC cells induced at the anterior neural fold are able to migrate and differentiate as normal NC. These results demonstrate that anterior regions of the embryo lack NC because of a mechanism, conserved from fish to mammals, that suppresses Wnt/beta-catenin signaling via Dkk1.     

A new role for the Endothelin-1/Endothelin-A receptor signaling during early neural crest specification

The neural crest is induced at the border of the neural plate in a multistep process by signals emanated from the epidermis, neural plate and mesoderm. In this work we show for the first time the existence of a neural crest maintenance step which is dependent on signals released from the mesoderm. We identified Endothelin-1 (Edn1) and its receptor (Ednra) as key players of this signal and we show that Edn1/Ednra signaling is required for maintenance of the neural crest by a dual mechanism of cell specification and cell survival. We show that: (i) Ednra is expressed in prospective neural crest; (ii) loss-of-function experiments with antisense morpholino or with specific chemical inhibitor suppress the expression of early neural crest markers; (iii) gain-of-function experiments expand the neural crest territory; (iv) epistatic experiments show that Ednra/Edn1 is downstream of the early neural crest gene Msx1 and upstream of the late genes Sox9 and Sox10; and (v) Edn1/Ednra signaling inhibits apoptosis and controls cell specification of the neural crest. Together, our results provide insight on a new role of Edn1/Ednra cell signaling pathway during early neural crest development.