In their seminal 1983 paper, Gans and Northcutt proposed that evolution of the vertebrate 'new head' was made possible by the advent of the neural crest and cranial placodes. The neural crest is a stem cell population that arises adjacent to the forming CNS and contributes to important cell types, including components of the peripheral nervous system and craniofacial skeleton and elements of the cardiovascular system. In the past few years, the new head hypothesis has been challenged by the discovery in invertebrate chordates of cells with some, but not all, characteristics of vertebrate neural crest cells. Here, we discuss recent findings regarding how neural crest cells may have evolved during the course of deuterostome evolution. The results suggest that there was progressive addition of cell types to the repertoire of neural crest derivatives throughout vertebrate evolution. Novel genomic tools have enabled higher resolution insight into neural crest evolution, from both a cellular and a gene regulatory perspective. Together, these data provide clues regarding the ancestral neural crest state and how the neural crest continues to evolve to contribute to the success of vertebrates as efficient predators.

Premigratory neural crest cells arise within the dorsal neural tube and subsequently undergo an epithelial-to-mesenchymal transition (EMT) to leave the neuroepithelium and initiate migration. Draxin is a Wnt modulator that has been shown to control the timing of cranial neural crest EMT. Here we show that this process is accompanied by three stages of remodeling of the basement membrane protein laminin, from regression to expansion and channel formation. Loss of Draxin results in blocking laminin remodeling at the regression stage, whereas ectopic maintenance of Draxin blocks remodeling at the expansion stage. The latter effect is rescued by addition of Snail2, previously shown to be downstream of Draxin. Our results demonstrate an essential function for the Wnt modulator Draxin in regulating basement membrane remodeling during cranial neural crest EMT.

During vertebrate development, the central (CNS) and peripheral nervous systems (PNS) arise from the neural plate. Cells at the margin of the neural plate give rise to neural crest cells, which migrate extensively throughout the embryo, contributing to the majority of neurons and all of the glia of the PNS. The rest of the neural plate invaginates to form the neural tube, which expands to form the brain and spinal cord. The emergence of molecular cloning techniques and identification of fluorophores like Green Fluorescent Protein (GFP), together with transgenic and electroporation technologies, have made it possible to easily visualize the cellular and molecular events in play during nervous system formation. These lineage-tracing techniques have precisely demonstrated the migratory pathways followed by neural crest cells and increased knowledge about their differentiation into PNS derivatives. Similarly, in the spinal cord, lineage-tracing techniques have led to a greater understanding of the regional organization of multiple classes of neural progenitor and post-mitotic neurons along the different axes of the spinal cord and how these distinct classes of neurons assemble into the specific neural circuits required to realize their various functions. Here, we review how both classical and modern lineage and marker analyses have expanded our knowledge of early peripheral nervous system and spinal cord development.

Neural crest cells undergo a spatiotemporally regulated epithelial-to-mesenchymal transition (EMT) that proceeds head to tailward to exit from the neural tube. In this study, we show that the secreted molecule Draxin is expressed in a transient rostrocaudal wave that mirrors this emigration pattern, initiating after neural crest specification and being down-regulated just before delamination. Functional experiments reveal that Draxin regulates the timing of cranial neural crest EMT by transiently inhibiting canonical Wnt signaling. Ectopic maintenance of Draxin in the cranial neural tube blocks full EMT; while cells delaminate, they fail to become mesenchymal and migratory. Loss of Draxin results in premature delamination but also in failure to mesenchymalize. These results suggest that a pulse of intermediate Wnt signaling triggers EMT and is necessary for its completion. Taken together, these data show that transient secreted Draxin mediates proper levels of canonical Wnt signaling required to regulate the precise timing of initiation and completion of cranial neural crest EMT.

Elk3/Net/Sap2 (here referred to as Elk3) is an Ets ternary complex transcriptional repressor known for its involvement in angiogenesis during embryonic development. Although Elk3 is expressed in various tissues, additional roles for the protein outside of vasculature development have yet to be reported. Here, we characterize the early spatiotemporal expression pattern of Elk3 in the avian embryo using whole mount in situ hybridization and quantitative RT-PCR and examine the effects of its loss of function on neural crest development. At early stages, Elk3 is expressed in the head folds, head mesenchyme, intersomitic vessels, and migratory cranial neural crest (NC) cells. Loss of the Elk3 protein results in the retention of Pax7+ precursors in the dorsal neural tube that fail to upregulate neural crest specifier genes, FoxD3, Sox10 and Snail2, resulting in embryos with severe migration defects. The results putatively place Elk3 downstream of neural plate border genes, but upstream of neural crest specifier genes in the neural crest gene regulatory network (NC-GRN), suggesting that it is critical for the progression from progenitor to definitive neural crest cell.

Characterizing endogenous protein expression, interaction and function, this study identifies in vivo interactions and competitive balance between N-cadherin and E-cadherin in developing avian (Gallus gallus) neural and neural crest cells. Numerous cadherin proteins, including neural cadherin (Ncad) and epithelial cadherin (Ecad), are expressed in the developing neural plate as well as in neural crest cells as they delaminate from the newly closed neural tube. To clarify independent or coordinate function during development, we examined their expression in the cranial region. The results revealed surprising overlap and distinct localization of Ecad and Ncad in the neural tube. Using a proximity ligation assay and co-immunoprecipitation, we found that Ncad and Ecad formed heterotypic complexes in the developing neural tube, and that modulation of Ncad levels led to reciprocal gain or reduction of Ecad protein, which then alters ectodermal cell fate. Here, we demonstrate that the balance of Ecad and Ncad is dependent upon the availability of β-catenin proteins, and that alteration of either classical cadherin modifies the proportions of the neural crest and neuroectodermal cells that are specified.

The neural crest, an embryonic stem cell population, initially resides within the dorsal neural tube but subsequently undergoes an epithelial-to-mesenchymal transition (EMT) to commence migration. Although neural crest and cancer EMTs are morphologically similar, little is known regarding conservation of their underlying molecular mechanisms. We report that Sip1, which is involved in cancer EMT, plays a critical role in promoting the neural crest cell transition to a mesenchymal state. Sip1 transcripts are expressed in premigratory/migrating crest cells. After Sip1 loss, the neural crest specifier gene FoxD3 was abnormally retained in the dorsal neuroepithelium, whereas Sox10, which is normally required for emigration, was diminished. Subsequently, clumps of adherent neural crest cells remained adjacent to the neural tube and aberrantly expressed E-cadherin while lacking N-cadherin. These findings demonstrate two distinct phases of neural crest EMT, detachment and mesenchymalization, with the latter involving a novel requirement for Sip1 in regulation of cadherin expression during completion of neural crest EMT.

The neural crest is an embryonic population of multipotent stem cells that form numerous defining features of vertebrates. Due to lack of reliable techniques to perform transcriptional profiling in intact tissues, it remains controversial whether the neural crest is a heterogeneous or homogeneous population. By coupling multiplex single molecule fluorescence in situ hybridization with machine learning algorithm based cell segmentation, we examine expression of 35 genes at single cell  resolution in vivo. Unbiased hierarchical clustering reveals five spatially distinct subpopulations within the chick dorsal neural tube. Here we identify a neural crest stem cell niche that centers around the dorsal midline with high expression of neural crest genes, pluripotency factors, and lineage markers. Interestingly, neural and neural crest stem cells express distinct pluripotency signatures. This Spatial Genomic Analysis toolkit provides a straightforward approach to study quantitative multiplex gene expression in numerous biological systems, while offering insights into gene regulatory networks via synexpression analysis.

Directional migration of neural crest (NC) cells is essential for patterning the vertebrate embryo, including the craniofacial skeleton. Extensive filopodial protrusions in NC cells are thought to sense chemo-attractive/repulsive signals that provide directionality. To test this hypothesis, we generated null mutations in zebrafish fascin1a (fscn1a), which encodes an actin-bundling protein required for filopodia formation. Homozygous fscn1a zygotic null mutants have normal NC filopodia due to unexpected stability of maternal Fscn1a protein throughout NC development and into juvenile stages. In contrast, maternal/zygotic fscn1a null mutant embryos (fscn1a MZ) have severe loss of NC filopodia. However, only a subset of NC streams display migration defects, associated with selective loss of craniofacial elements and peripheral neurons. We also show that fscn1a-dependent NC migration functions through cxcr4a/cxcl12b chemokine signaling to ensure the fidelity of directional cell migration. These data show that fscn1a-dependent filopodia are required in a subset of NC cells to promote cell migration and NC derivative formation, and that perdurance of long-lived maternal proteins can mask essential zygotic gene functions during NC development.

During gastrulation, neural crest cells are specified at the neural plate border, as characterized by Pax7 expression. Using single-cell RNA sequencing coupled with high-resolution in situ hybridization to identify novel transcriptional regulators, we show that chromatin remodeler Hmga1 is highly expressed prior to specification and maintained in migrating chick neural crest cells. Temporally controlled CRISPR-Cas9-mediated knockouts uncovered two distinct functions of Hmga1 in neural crest development. At the neural plate border, Hmga1 regulates Pax7-dependent neural crest lineage specification. At premigratory stages, a second role manifests where Hmga1 loss reduces cranial crest emigration from the dorsal neural tube independent of Pax7. Interestingly, this is rescued by stabilized ß-catenin, thus implicating Hmga1 as a canonical Wnt activator. Together, our results show that Hmga1 functions in a bimodal manner during neural crest development to regulate specification at the neural plate border, and subsequent emigration from the neural tube via canonical Wnt signaling.