Unraveling Fundamental Aspects of Preimplantation Development using Single Cell Genomics

第一周的胚胎发育可以说是人类发育最关键的时期，然而，关于这一窗口期胚胎发生的基本方面(谱系分离和X染色体失活(XCI))的信息仍然缺乏。目前的知识几乎完全是通过老鼠模型来确定的，但考虑到新出现的差异，从老鼠推断时需要谨慎，从而突出了对人类进行特定研究的必要性。我们最近证实，在胚胎第5天，第一个谱系被建立：1)滋养外胚层(TE)，2)原始内胚层(PE)，3)多能上皮细胞(EPI)，这与小鼠不同。我发现的另一个显著差异是人类胚胎中XCI的一种新机制；两种X-ChR的活性逐渐降低到E7在男性中观察到的水平。是什么驱动了谱系分离和囊胚发育的机制(S)尚未确定。我们的初步数据表明，在胚胎第5天的个体谱系中存在WNT和TGF-通路的信号成分，并且这些通路中的大多数基因在胚胎5天期间被打开/关闭；这表明它们在谱系指定中起着重要的作用。WNT和转化生长因子信号在人类植入前发育中的作用(S)尚不清楚。此外，PORCN(WNT途径的调节因子)是一个X连锁基因。鉴于PORCN对WNT信号的调节功能，我们现在还想检查X-CHR剂量补偿和WNT信号之间是否存在联系；这可能解释人类植入前胚胎对XCI的处理。因此，为了更好地了解人类胚胎中涉及谱系分离和潜在的XCI的潜在机制(S)，我现在的目标是探索WNT和转化生长因子信号在小分子激动剂/拮抗剂中的功能作用(S)。免疫组织化学将在蛋白质水平上测量成分(转位、磷酸化、细胞位置)和下游靶标的变化，此外还将获得胚胎和血统形成的空间概况。单细胞RNA测序将以跨多个发育时间点的谱系特定分辨率来衡量调节这些通路的影响，并与smFISH一起确定与XCI的关系。这些开创性的研究将首次提供对细胞命运指定、XCI和囊胚形成背后的基因调控机制的详细机制见解，为人类早期胚胎发育提供基础知识。总体而言，了解控制早期人类发育的因素对于加强生殖生物学和发育领域非常重要。此外，这一建议的结果可能导致改进培养条件或衍生方案，应用于干细胞生物学(初始条件和启动条件)和体外受精(IVF)。

Embryonic development during the first week is arguably the most critical period of human development, however, information pertaining to fundamental aspects of embryogenesis (lineage segregation and X-chromosome inactivation (XCI)) during this window are still lacking. Current knowledge has almost exclusively been determined using mouse models, yet caution is warranted when extrapolating from the mouse, given the emerging discrepancies, thus highlighting the need for human specific studies. We have recently demonstrated that during embryonic day (E) 5, the first lineages are established: 1) trophectoderm (TE), 2) primitive endoderm (PE), and 3) pluripotent epiblast cells (EPI), which is in contrast to the mouse. Another striking difference I identified is a novel mechanism of XCI in the human embryo; activity from both X-chrs is gradually reduced to levels observed in the male by E7. What drives lineage segregation and mechanism(s) of blastocyst development is undetermined. Our preliminary data demonstrate the presence of signalling components of both WNT and TGF- pathways in the individual lineages at embryonic day (E)5 and that the majority of genes involved in these pathways are turned on/off during E5; suggesting that they play an important role in lineage specification. The role(s) of WNT and TGF- signalling in human preimplantation development remains unclear. Further, PORCN (regulator of WNT pathway) is an X-linked gene. Given the regulatory function of PORCN on WNT signalling, we would also now like to examine whether there is a link between X-chr dosage compensation and WNT signalling; which may explain the human preimplantation embryo approach to XCI. Thus, to gain a better understanding of the underlying mechanism(s) involved in lineage segregation and potentially XCI in the human embryo, I now aim to explore the functional role(s) of WNT and TGF- signalling using small molecule agonists/antagonist. Immunohistochemistry will measure changes in components (translocation, phosphorylation, cellular location) and downstream targets at the protein level in addition to obtaining a spatial overview of the embryo and lineages formed. Single-cell RNA sequencing will measure the impact of modulating these pathways, at lineage specific resolution across multiple developmental timepoints and also determine relationship with XCI together with smFISH. These pioneering studies will provide first detailed mechanistic insights into the gene regulatory mechanisms underlying cell fate specification, XCI and blastocyst formation, providing fundamental knowledge toward early human embryogenesis. Overall, understanding what regulates early human development is of great importance for enhancing the fields of Reproductive Biology and Development. Further, results from this proposal may lead to improved culture conditions or derivation protocols in application to stem cell biology (nave and primed conditions) and In Vitro Fertilization (IVF).