The transcription factor cMyb has well known functions in vertebrate hematopoiesis, but little was known about its distribution or function at early developmental stages. Here, we show that cMyb transcripts are present at the neural plate during gastrulation in chick embryos. cMyb expression then resolves to the cranial neural folds and is maintained in early migrating cranial neural crest cells during and after neurulation. Morpholino-mediated knock-down of cMyb reduces expression of Pax7 and Twist at the neural plate border, as well as reducing expression of neural crest specifier gene Slug/Snail2 and completely eliminating expression of Ets1. On the other hand, its loss results in abnormal maintenance of Zic1, but little or no effect on other neural crest specifier genes like FoxD3 or Sox9. These results place cMyb in a critical hierarchical position within the cranial neural crest cell gene regulatory network, likely directly inhibiting Zic1 and upstream of Ets1 and some, but not all, neural crest specifier genes.

The critical stem cell transcription factor FoxD3 is expressed by the premigratory and migrating neural crest, an embryonic stem cell population that forms diverse derivatives. Despite its important role in development and stem cell biology, little is known about what mediates FoxD3 activity in these cells. We have uncovered two FoxD3 enhancers, NC1 and NC2, that drive reporter expression in spatially and temporally distinct manners. Whereas NC1 activity recapitulates initial FoxD3 expression in the cranial neural crest, NC2 activity recapitulates initial FoxD3 expression at vagal/trunk levels while appearing only later in migrating cranial crest. Detailed mutational analysis, in vivo chromatin immunoprecipitation, and morpholino knock-downs reveal that transcription factors Pax7 and Msx1/2 cooperate with the neural crest specifier gene, Ets1, to bind to the cranial NC1 regulatory element. However, at vagal/trunk levels, they function together with the neural plate border gene, Zic1, which directly binds to the NC2 enhancer. These results reveal dynamic and differential regulation of FoxD3 in distinct neural crest subpopulations, suggesting that heterogeneity is encrypted at the regulatory level. Isolation of neural crest enhancers not only allows establishment of direct regulatory connections underlying neural crest formation, but also provides valuable tools for tissue specific manipulation and investigation of neural crest cell identity in amniotes.

Neural crest cells are induced at the neural plate border by the combined action of transcription factors and signaling molecules. Here, we show that Axud1, a downstream effector of Wnt signaling, represents a critical missing link that integrates signaling and transcriptional cues to mediate neural crest formation. Axud1 is a transcription factor expressed in neural crest progenitors in a Wnt1/β-catenin-dependent manner. Axud1 loss leads to downregulation of multiple genes involved in neural crest specification, similar to the effects of Wnt1 knockdown. Importantly, Axud1 is sufficient to rescue neural crest formation after disruption of Wnt signaling. Furthermore, it physically interacts with neural plate border genes Pax7 and Msx1 in vivo to directly activate transcription of stem cell factor FoxD3, initiating the neural crest program. Thus, Axud1 integrates Wnt signaling with transcriptional inputs to endow the neural crest with its unique molecular signature.

The 'neural plate border' of vertebrate embryos contains precursors of neural crest and placode cells, both defining vertebrate characteristics. How these lineages segregate from neural and epidermal fates has been a matter of debate. We address this by performing a fine-scale quantitative temporal analysis of transcription factor expression in the neural plate border of chick embryos. The results reveal significant overlap of transcription factors characteristic of multiple lineages in individual border cells from gastrula through neurula stages. Cell fate analysis using a Sox2 (neural) enhancer reveals that cells that are initially Sox2+ cells can contribute not only to neural tube but also to neural crest and epidermis. Moreover, modulating levels of Sox2 or Pax7 alters the apportionment of neural tube versus neural crest fates. Our results resolve a long-standing question and suggest that many individual border cells maintain ability to contribute to multiple ectodermal lineages until or beyond neural tube closure.