Unraveling Fundamental Aspects of Preimplantation Development using Single Cell Genomics

使用单细胞基因组学揭示植入前发育的基本方面

• 批准号: RGPIN-2019-05423
• 负责人: Petropoulos, Sophie
• 金额: $ 2.4万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760598
• 关键词: Unraveling; Fundamental; Aspects; Preimplantation; Development

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-ß 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-ß 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-ß 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 (naïve and primed conditions) and In Vitro Fertilization (IVF).

第一周的胚胎发育可以说是人类发育最关键的时期，然而，目前几乎完全缺乏与该窗口期胚胎发生基本方面（谱系分离和 X 染色体失活 (XCI)）相关的信息。使用小鼠模型确定，但鉴于不断出现的差异，从小鼠推断时需要谨慎，因此强调了对人类特定研究的需要，我们最近证明，在胚胎第 5 天 (E) 期间，建立了第一个谱系： 1) 滋养外胚层 (TE)、2) 原始内胚层 (PE) 和 3) 多能外胚层细胞 (EPI)，这与小鼠相反，我发现的另一个显着差异是 XCI 在人类胚胎活性中的新机制；来自两个 X-chrs 的信号逐渐降低至 E7 在雄性中观察到的水平。驱动谱系分离和囊胚发育的机制尚未确定。我们的初步数据表明信号传导的存在。胚胎日 (E)5 中各个谱系中 WNT 和 TGF-β 通路的组成部分，并且参与这些通路的大多数基因在 E5 期间打开/关闭；表明它们在谱系规范中发挥着重要作用。 (s) WNT 和 TGF-β 信号传导在人类植入前发育中的作用仍不清楚。此外，PORCN（WNT 通路调节因子）是一个 X 连锁基因，考虑到 PORCN 对 WNT 信号传导的调节功能。现在还想检查 X-chr 剂量补偿和 WNT 信号传导之间是否存在联系，这可以解释人类植入前胚胎的 XCI 方法，从而更好地了解谱系分离所涉及的潜在机制。以及人类胚胎中潜在的 XCI，我现在的目标是使用小分子激动剂/拮抗剂来探索 WNT 和 TGF-β 信号传导的功能作用，免疫组织化学将测量成分的变化（易位、除了获得胚胎和形成的谱系的空间概览之外，单细胞 RNA 测序还将测量跨多个发育时间点的谱系特异性分辨率调节这些途径的影响。确定与 XCI 和 smFISH 的关系。这些开创性的研究将为细胞命运规范、XCI 和囊胚形成的基因调控机制提供第一个详细的机制见解，从而为早期人类胚胎发生提供基础知识。人类发育对于加强生殖生物学和发育领域具有重要意义。此外，该提案的结果可能会改善干细胞生物学（初始条件和引发条件）和体外受精（IVF）中的培养条件或衍生方案。 。