Xenopus Meis3 protein lies at a nexus downstream to Zic1 and Pax3 proteins, regulating multiple cell-fates during early nervous system development

In Xenopus embryos, XMeis3 protein activity is required for normal hindbrain formation. Our results show that XMeis3 protein knock down also causes a loss of primary neuron and neural crest cell lineages, without altering expression of Zic, Sox or Pax3 genes. Knock down or inhibition of the Pax3, Zic1 or Zic5 protein activities extinguishes embryonic expression of the XMeis3 gene, as well as triggering the loss of hindbrain, neural crest and primary neuron cell fates. Ectopic XMeis3 expression can rescue the Zic knock down phenotype. HoxD1 is an XMeis3 direct-target gene, and ectopic HoxD1 expression rescues cell fate losses in either XMeis3 or Zic protein knock down embryos. FGF3 and FGF8 are direct target genes of XMeis3 protein and their expression is lost in XMeis3 morphant embryos. In the genetic cascade controlling embryonic neural cell specification, XMeis3 lies below general-neuralizing, but upstream of FGF and regional-specific genes. Thus, XMeis3 protein is positioned at a key regulatory point, simultaneously regulating multiple neural cell fates during early vertebrate nervous system development.     

Sox3 expression is maintained by FGF signaling and restricted to the neural plate by Vent proteins in the Xenopus embryo

The formation of the nervous system is initiated when ectodermal cells adopt the neural fate. Studies in Xenopus demonstrate that inhibition of BMP results in the formation of neural tissue. However, the molecular mechanism driving the expression of early neural genes in response to this inhibition is unknown. Moreover, controversy remains regarding the sufficiency of BMP inhibition for neural induction. To address these questions, we performed a detailed analysis of the regulation of the soxB1 gene, sox3, one of the earliest genes expressed in the neuroectoderm. Using ectodermal explant assays, we analyzed the role of BMP, Wnt and FGF signaling in the regulation of sox3 and the closely related soxB1 gene, sox2. Our results demonstrate that both sox3 and sox2 are induced in response to BMP antagonism, but by distinct mechanisms and that the activation of both genes is independent of FGF signaling. However, both require FGF for the maintenance of their expression. Finally, sox3 genomic elements were identified and characterized and an element required for BMP-mediated repression via Vent proteins was identified through the use of transgenesis and computational analysis. Interestingly, none of the elements required for sox3 expression were identified in the sox2 locus. Together our data indicate that two closely related genes have unique mechanisms of gene regulation at the onset of neural development.     

Sequential actions of Pax3 and Pax7 drive xanthophore development in zebrafish neural crest

The Pax3/7 gene family has a fundamental and conserved role during neural crest formation. In people, PAX3 mutation causes Waardenburg syndrome, and murine Pax3 is essential for pigment formation. However, it is unclear exactly how Pax3 functions within the neural crest. Here we show that pax3 is expressed before other pax3/7 members, including duplicated pax3b, pax7 and pax7b genes, early in zebrafish neural crest development. Knockdown of Pax3 protein by antisense morpholino oligonucleotides results in defective fate specification of xanthophores, with complete ablation in the trunk. Other pigment lineages are specified and differentiate. As a consequence of xanthophore loss, expression of pax7, a marker of the xanthophore lineage, is reduced in neural crest. Morpholino knockdown of Pax7 protein shows that Pax7 itself is dispensable for xanthophore fate specification, although yellow pigmentation is reduced. Loss of xanthophores after reduction of Pax3 correlates with a delay in melanoblast differentiation followed by significant increase in melanophores, suggestive of a Pax3-driven fate switch within a chromatophore precursor or stem cell. Analysis of other neural crest derivatives reveals that, in the absence of Pax3, the enteric nervous system is ablated from its inception. Therefore, Pax3 in zebrafish is required for specification of two specific lineages of neural crest, xanthophores and enteric neurons.     

Neural crest determination by co-activation of Pax3 and Zic1 genes in Xenopus ectoderm

A number of regulatory genes have been implicated in neural crest development. However, the molecular mechanism of how neural crest determination is initiated in the exact ectodermal location still remains elusive. Here, we show that the cooperative function of Pax3 and Zic1 determines the neural crest fate in the amphibian ectoderm. Pax3 and Zic1 are expressed in an overlapping manner in the presumptive neural crest area of the Xenopus gastrula, even prior to the onset of the expression of the early bona fide neural crest marker genes Foxd3 and Slug. Misexpression of both Pax3 and Zic1 together efficiently induces ectopic neural crest differentiation in the ventral ectoderm, whereas overexpression of either one of them only expands the expression of neural crest markers within the dorsolateral ectoderm. The induction of neural crest differentiation by Pax3 and Zic1 requires Wnt signaling. Loss-of-function studies in vivo and in the animal cap show that co-presence of Pax3 and Zic1 is essential for the initiation of neural crest differentiation. Thus, co-activation of Pax3 and Zic1, in concert with Wnt, plays a decisive role for early neural crest determination in the correct place of the Xenopus ectoderm.