Yamanaka Factors in the Budding Tunicate Botryllus schlosseri Show a Shared Spatio-Temporal Expression Pattern in Chordates

Abstract

In vertebrates, the four transcription factors Sox2, c-Myc, Pou5f1 and Klf4 are involved in the differentiation of several tissues during vertebrate embryogenesis; moreover, they are normally co-expressed in embryonic stem cells and play roles in pluripotency, self-renewal, and maintenance of the undifferentiated state in adult cells. The in vitro forced co-expression of these factors, named Yamanaka factors (YFs), induces pluripotency in human or mouse fibroblasts. Botryllus schlosseri is a colonial tunicate undergoing continuous stem cell-mediated asexual development, providing a valuable model system for the study of pluripotency in the closest living relatives of vertebrates. In this study, we identified B. schlosseri orthologs of human Sox2 and c-Myc genes, as well as the closest homologs of the vertebrate-specific Pou5f1 gene, through an in-depth evolutionary analysis of the YF gene families in tunicates and other deuterostomes. Then, we studied the expression of these genes during the asexual cycle of B. schlosseri using in situ hybridization in order to investigate their possible involvement in tissue differentiation and in pluripotency maintenance. Our results show a shared spatio-temporal expression pattern consistent with the reported functions of these genes in invertebrate and vertebrate embryogenesis. Moreover, Myc, SoxB1 and Pou3 were expressed in candidate stem cells residing in their niches, while Pou2 was found expressed exclusively in the immature previtellogenic oocytes, both in gonads and circulating in the colonial vascular system. Our data suggest that Myc, SoxB1 and Pou3 may be individually involved in the differentiation of the same territories seen in other chordates, and that, together, they may play a role in stemness even in this colonial ascidian.

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

“…Evidence for this comes from the absence of the core pluripotency factors pou5 , and ventx / nanog in invertebrate genomes, and the finding that cells of pre-blastula tunicate embryos—the sister group to vertebrates—are already restricted to a single lineage 63,64 . Although pou5 and ventx / nanog are not encoded in invertebrate chordate genomes 9,65,66 , invertebrate chordates do express homologs of the neural stem cell factors soxB1 and myc in the blastula 17 . Thus the precursors of the vertebrate pluripotency GRN may have arisen in cells fated to be neural progenitor cells, perhaps explaining why the neural lineage has been found to lie closest in gene regulatory state space to pluripotent blastula cells and why several neural crest regulatory factors have been found to have deep roots in bilaterian neurogenesis 4,67,68 .…”

Manipulating Myc for reparative regeneration

“…Ascidians or sea squirts are marine invertebrate sessile tunicates that belong to phylum Chordata. Ascidians present with a single Myc gene that contains a bHLH-LZ ( Vanni et al, 2022 ). In ascidian species Botryllus schlosseri , Ciona savignyi , and Polyandrocarpa misakiensis , Myc is expressed in early development and disappears in adult tissues ( Kobayashi et al, 2022 ; Vanni et al, 2022 ).…”

“…Ascidians present with a single Myc gene that contains a bHLH-LZ ( Vanni et al, 2022 ). In ascidian species Botryllus schlosseri , Ciona savignyi , and Polyandrocarpa misakiensis , Myc is expressed in early development and disappears in adult tissues ( Kobayashi et al, 2022 ; Vanni et al, 2022 ). Knockdown of Myc in embryonic/larval stages via morpholinos (modified antisense oligonucleotides), RNAi, or a dominant negative version of Myc suppresses mesenchymal and endodermal cell cycle and impairs organogenesis ( Fujiwara et al, 2011 ; Kobayashi et al, 2022 ).…”

Retinoic Acid and POU Genes in Developing Amphioxus: A Focus on Neural Development

“…Class V POU genes are only found in vertebrates and are thought to have originated from an ancient Pou3-like gene [1]. Interestingly, Pou3 is expressed in circulating stem cells of tunicates, the sister group of vertebrates, suggesting that the newly originated vertebrate Oct3/4 might have replaced Pou3 in an ancestral pluripotency program that was in place before the split between tunicates and vertebrates [76]. Although little is known about stem cells in amphioxus, the most prominent stem cell niche so far identified during development is in the tail bud and comprises at least the primordial germ cells [77].…”