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的最关键时期，然而，关于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小鼠模型确定的，但考虑到新出现的差异，在从小鼠中推断时需要谨慎，因此强调了对人类特定研究的需求。我们最近证明，在胚胎期（E) 5，第一个谱系建立：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。我发现的另一个显著差异是人类胚胎中XCI的新机制；两个X-chrs的活性逐渐降低到E7在男性中观察到的水平。是什么驱动了胚泡发育的谱系分离和机制尚不确定。我们的初步数据表明，在胚胎期(E)5，个体谱系中存在WNT和TGF-ß信号通路的信号成分，并且这些通路中涉及的大多数基因在E5期间被打开/关闭；提示它们在谱系规范中起着重要的作用。WNT和TGF-ß信号在人类着床前发育中的作用尚不清楚。此外，PORCN （WNT通路的调节因子）是一个x连锁基因。鉴于PORCN对WNT信号传导的调节功能，我们现在还想研究X-chr剂量补偿与WNT信号传导之间是否存在联系；这也许可以解释人类植入前胚胎对XCI的影响。因此，为了更好地了解人类胚胎谱系分离和潜在XCI的潜在机制，我现在的目标是利用小分子激动剂/拮抗剂探索WNT和TGF-ß信号传导的功能作用。免疫组织化学将在蛋白质水平上测量组分（易位、磷酸化、细胞位置）和下游靶点的变化，并获得胚胎和形成的谱系的空间概况。单细胞RNA测序将在多个发育时间点以谱系特定分辨率测量调节这些途径的影响，并确定与XCI和smFISH的关系。这些开创性的研究将首次为细胞命运规范、XCI和囊胚形成的基因调控机制提供详细的机制见解，为早期人类胚胎发生提供基础知识。综上所述，了解人类早期发育的调控机制对于加强生殖生物学和发育领域的研究具有重要意义。此外，该提案的结果可能会改善干细胞生物学（naïve和primed条件）和体外受精（IVF）应用的培养条件或衍生方案。