DataSheet4_Yamanaka Factors in the Budding Tunicate Botryllus schlosseri Show a Shared Spatio-Temporal Expression Pattern in Chordates.FASTA

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.在脊椎动物中，四种转录因子（transcription factor）Sox2、c-Myc、Pou5f1及Klf4参与脊椎动物胚胎发生过程中多种组织的分化；此外，这类因子通常在胚胎干细胞（embryonic stem cells）中共表达，并在成体细胞的多能性、自我更新以及未分化状态维持中发挥作用。体外强制共表达这些被命名为山中因子（Yamanaka factors, YFs）的因子，可诱导人类或小鼠成纤维细胞（fibroblast）获得多能性。聚柄海鞘（Botryllus schlosseri）是一类群居被囊动物（tunicate），可持续进行干细胞介导的无性发育，是研究脊椎动物现存近亲类群多能性的优质模型系统。本研究通过对被囊动物及其他后口动物（deuterostome）的山中因子基因家族开展深入进化分析，鉴定出了聚柄海鞘的人类Sox2与c-Myc基因直系同源基因，以及脊椎动物特异性Pou5f1基因的最近缘同源基因。随后，本研究利用原位杂交（in situ hybridization）技术检测了这些基因在聚柄海鞘无性发育周期中的表达模式，以探究其在组织分化与多能性维持中的潜在作用。研究结果显示，这些基因呈现出共有的时空表达模式，与已报道的它们在无脊椎动物与脊椎动物胚胎发生中的功能相符。此外，Myc、SoxB1与Pou3在位于其微环境中的候选干细胞中表达，而Pou2则仅在未成熟卵黄发生前卵母细胞中表达，这类卵母细胞既存在于性腺中，也可在群居体的血管系统内循环。本研究数据表明，Myc、SoxB1与Pou3可能分别参与其他脊索动物中相同区域的组织分化，且三者协同可在这类群居海鞘中发挥维持干细胞干性的作用。