Functional and mechanistic characterization of Foxd3 during mouse peri-implantation transitions.

植入后，小鼠胚胎内的外胚层细胞经历了一个关键的细胞命运决定，要么被指定为易腐烂的体细胞，要么被指定为不朽的种系。着床前外胚层表现出naïve多能性，可以产生所有胚胎谱系。植入后，外胚层过渡到启动状态，其特征是naïve多能性丧失和体细胞基因的早期表达。少数着床后的外胚层细胞能够恢复体细胞分化，并经历重大的重编程事件，导致种系分化。使用小鼠胚胎干细胞作为体外分化模型，我们最近发现Foxd3是一种新的调节因子，可以促进从naïve到引物多能性的转变，并随后阻止种系身份的获得。从机制上讲，Foxd3作为一种转录抑制因子，使关键naïve多能性和种系基因表达所必需的增强子失活。然而，Foxd3功能在植入期发育中的体内相关性和进化保守性目前尚不清楚。在这里，我们提出了一些实验，应该提供机制和功能的见解，Foxd3在植入后启动外胚层的作用。我们假设Foxd3可以作为主要的外胚层模式调节因子赋予前身份。使用体外和体内模型，我们将测试这种新的Foxd3功能是否具有进化保守性。此外，我们之前已经在体外证明Foxd3的沉默是小鼠原始生殖细胞（PGCs）规范过程中的一个关键调控事件。在目前的提案中，我们将确定这种沉默事件是否对体内种系规范同样重要，以及它是否也与人类相关。总的来说，我们的工作应该提供更深入的理解协调着床周围发育的分子机制，这可能有助于改善PGCs和配子的体外衍生，并可能为不孕症的治疗开辟新的可能性。

Following implantation, epiblast cells within the mouse embryo undergo a critical cell fate decision, becoming specified towards either the perishable soma or the immortal germline. The pre-implantation epiblast displays naïve pluripotency and can give rise to all embryonic lineages. Upon implantation, the epiblast transits to a primed state, characterized by the loss of naïve pluripotency and the incipient expression of somatic genes. A few post-implantation epiblast cells are able to revert somatic differentiation and undergo major reprogramming events leading to germline specification. Using mouse embryonic stem cells as an in vitro differentiation model, we recently uncovered Foxd3 as a novel regulator that promotes the transition from naïve to primed pluripotency and, subsequently, prevents the acquisition of germline identity. Mechanistically, Foxd3 acts as a transcriptional repressor that decommissions enhancers necessary for the expression of key naïve pluripotency and germline genes. However, the in vivo relevance and evolutionary conservation of Foxd3 function during peri-implantation development are currently unknown. Here we propose a number of experiments that should provide mechanistic and functional insights into the role of Foxd3 in the post-implantation primed epiblast. We hypothesize that Foxd3 can act as a major epiblast patterning regulator conferring anterior identity. Using both in vitro and in vivo models, we will test whether this novel Foxd3 function is evolutionary conserved. Furthermore, we have previously shown in vitro that the silencing of Foxd3 is a critical regulatory event during the specification of mouse primordial germ cells (PGCs). In the current proposal, we will determine if this silencing event is equally important for in vivo germline specification and whether it is also relevant in humans. Overall, our work should provide a deeper understanding of the molecular mechanisms orchestrating peri-implantation development, which could help improving the in vitro derivation of PGCs and gametes and potentially opening new possibilities for the treatment of infertility.