WNT/β-catenin signaling mediates human neural crest induction via a pre-neural border intermediate

Abstract

Neural crest (NC) cells arise early in vertebrate development, migrate extensively and contribute to a diverse array of ectodermal and mesenchymal derivatives. Previous models of NC formation suggested derivation from neuralized ectoderm, via meso-ectodermal, or neural-non-neural ectoderm interactions. Recent studies using bird and amphibian embryos suggest an earlier origin of NC, independent of neural and mesodermal tissues. Here, we set out to generate a model in which to decipher signaling and tissue interactions involved in human NC induction. Our novel human embryonic stem cell (ESC)-based model yields high proportions of multipotent NC cells (expressing SOX10, PAX7 and TFAP2A) in 5 days. We demonstrate a crucial role for WNT/β-catenin signaling in launching NC development, while blocking placodal and surface ectoderm fates. We provide evidence of the delicate temporal effects of BMP and FGF signaling, and find that NC development is separable from neural and/ or mesodermal contributions. We further substantiate the notion of a neural-independent origin of NC through PAX6 expression and knockdown studies. Finally, we identify a novel pre-neural border state characterized by early WNT/β-catenin signaling targets that displays distinct responses to BMP and FGF signaling from the traditional neural border genes. In summary, our work provides a fast and efficient protocol for human NC differentiation under signaling constraints similar to those identified in vivo in model organisms, and strengthens a framework for neural crest ontogeny that is separable from neural and mesodermal fates.

Antagonism between the transcription factors NANOG and OTX2 specifies rostral or caudal cell fate during neural patterning transition

“…We confirmed that NANOG was sustained by GSK3β inhibition at early stage of differentiation via WNT signaling ( Figure 4E). Wnt signaling is also important to regulate neural patterning through forming morphogen gradients with other signal factors (7,(51)(52)(53). Our data then extend the role of NANOG in patterning the regional specified neural precursors at early stage of differentiation.…”

Regulatory Logic Underlying Diversification of the Neural Crest

“…Three major signaling pathways, BMP, WNT, and FGF, work combinatorially to drive expression of neural plate border specifier genes [14,16–18]. Crosstalk between signaling pathways result in the formation of the neural plate border; for example, Schille and colleagues in frog, found that WNT/PCP receptor, Ror2, upregulates Gdf6 at the neural plate border [19], thus, linking Wnt/PCP signaling to BMP signaling.…”

Signaling pathways and tissue interactions in neural plate border formation

“…8,10 Just recently, analysis of NC induction in hESCs demonstrated that the differentiation of hESCs into NC cells as indicated by expression of NPB genes including pax3 and msx1 takes place independent of the presence of neural precursors or neuroectoderm. 11 The role of the dorsolateral marginal zone…”

“…19,30,40,41 Likewise, Wnt/b-Catenin signaling is essential for NPB specification in epiblast explants from chick blastulae, where Wnt activity precedes and upregulates bmp4, 17 as well as for expression of NPB genes in hESCs. 11 Steventon and colleagues have analyzed Wnt/ b-Catenin activity in Xenopus embryos using the TOPFlash reporter gene assay. They observed low Wnt/b-Catenin activity at early gastrula stage 10 in the prospective NC, which rises slightly already till late gastrula stage 12 and continuously increases till mid-neurula stage 17.…”

“…This study further demonstrated that NC induction in hESCs requires Wnt/b-Catenin, BMP and FGF signaling, which indicates that the basic mechanisms of early NPB and NC induction are also conserved in humans. 11 It remains to be shown whether the recently discovered role of b-Catenin independent Wnt signaling is likewise conserved. A role of Ror2 in NC development is indicated by the phenotypes of Ror2 knockout mice and humans affected with recessive Robinow syndrome (RRS).…”