BET activity plays an essential role in control of stem cell attributes in Xenopus.

Neural crest cells are a stem cell population unique to vertebrate embryos that retains broad multi-germ layer developmental potential through neurulation. Much remains to be learned about the genetic and epigenetic mechanisms that control the potency of neural crest cells. Here, we examine the role that epigenetic readers of the BET (bromodomain and extra terminal) family play in controlling the potential of pluripotent blastula and neural crest cells. We find that inhibiting BET activity leads to loss of pluripotency at blastula stages and a loss of neural crest at neurula stages. We compare the effects of HDAC (an eraser of acetylation marks) and BET (a reader of acetylation) inhibition and find that they lead to similar cellular outcomes through distinct effects on the transcriptome. Interestingly, loss of BET activity in cells undergoing lineage restriction is coupled to increased expression of genes linked to pluripotency and prolongs the competence of initially pluripotent cells to transit to a neural progenitor state. Together these findings advance our understanding of the epigenetic control of pluripotency and the formation of the vertebrate neural crest.

Small molecule-mediated reprogramming of Xenopus blastula stem cells to a neural crest state.

Neural crest cells are a stem cell population unique to vertebrates that give rise to a diverse array of derivatives, including much of the peripheral nervous system, pigment cells, cartilage, mesenchyme, and bone. Acquisition of these cells drove the evolution of vertebrates and defects in their development underlies a broad set of neurocristopathies. Moreover, studies of neural crest can inform differentiation protocols for pluripotent stem cells and regenerative medicine applications. Xenopus embryos are an important system for studies of the neural crest and have provided numerous insights into the signals and transcription factors that control the formation and later lineage diversification of these stem cells. Pluripotent animal pole explants are a particularly powerful tool in this system as they can be cultured in simple salt solution and instructed to give rise to any cell type including the neural crest. Here we report a protocol for small molecule-mediated induction of the neural crest state from blastula stem cells and validate it using transcriptome analysis and grafting experiments. This is an powerful new tool for generating this important cell type that will facilitate future studies of neural crest development and mutations and variants linked to neurocristopathies.

Shared features of blastula and neural crest stem cells evolved at the base of vertebrates.

The neural crest is vertebrate-specific stem cell population that helped drive the origin and evolution of the vertebrate clade. A distinguishing feature of these stem cells is their multi-germ layer potential, which has drawn developmental and evolutionary parallels to another stem cell population-pluripotent embryonic stem cells (animal pole cells or ES cells) of the vertebrate blastula. Here, we investigate the evolutionary origins of neural crest potential by comparing neural crest and pluripotency gene regulatory networks (GRNs) in both jawed ( Xenopus ) and jawless (lamprey) vertebrates. Through comparative gene expression analysis and transcriptomics, we reveal an ancient evolutionary origin of shared regulatory factors between neural crest and pluripotency GRNs that dates back to the last common ancestor of extant vertebrates. Focusing on the key pluripotency factor pou5 (formerly oct4), we show that the lamprey genome encodes a pou5 ortholog that is expressed in animal pole cells, as in jawed vertebrates, but is absent from the neural crest. However, gain-of-function experiments show that both lamprey and Xenopus pou5 enhance neural crest formation, suggesting that pou5 was lost from the neural crest of jawless vertebrates. Finally, we show that pou5 is required for neural crest specification in jawed vertebrates and that it acquired novel neural crest-enhancing activity after evolving from an ancestral pou3 -like clade that lacks this functionality. We propose that a pluripotency-neural crest GRN was assembled in stem vertebrates and that the multi-germ layer potential of the neural crest evolved by deploying this regulatory program.