Rigney, S. | bioRxiv Science

bioRxiv2025

Kruppel-like Factors Play Essential Roles in Regulating Pluripotency and the Formation of Neural Crest Stem Cells

The evolutionary transition from simple chordate body plans to complex vertebrate body plans was driven by the acquisition of the neural crest, a stem cell population that retains broad, multi-germ layer developmental potential long after most embryonic cells have become lineage restricted. We have previously shown that neural crest cells share significant gene regulatory architecture with pluripotent blastula stem cells. Here we examine the roles that Kruppel-like Family (Klf) transcription factors play in these stem cell populations. Although Klf4 has established roles in regulating pluripotency in mammalian stem cells cultures, we find that in Xenopus it is klf2 that is highly expressed in pluripotent blastula stem cells. klf2 expression is down-regulated as cells transition to a neural crest state while a related klf factor, klf17, is significantly up regulated in response to neural crest induction. We used gain and loss of function studies to compare the activities of these closely related factors and found that they have both shared and distinct activities. Inhibition of either klf2 or klf17 activity led to significantly expanded expression of pluripotency, neural plate border and neural crest factors in neurula stage embryos, leading us to hypothesize that klf factors regulate the exit from pluripotency and proper establishment of the boundary of the neural crest domain. To gain further insights into the role of klf factors in the evolution of the neural crest, we examined their expression in the jawless vertebrate, Petromyzon marinus (sea lamprey). We find that lamprey have a klf2/4 and a klf17 gene, but that only klf17 is expressed in blastula and neural crest stem cells. Moreover, ectopic expression of lamprey klf17 in Xenopus embryos phenocopies Xenopus klf17 activity. These data suggest that klf17, rather than klf4, may have been the ancestral klf factor that functioned in these GRNs in stem vertebrates.

developmental biology↗

bioRxiv2023

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.

evolutionary biology↗