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).

但是，第一周的胚胎发育可以说是人类发展的最关键时期，但是，与胚胎发生的基本方面有关（谱系隔离和X染色体灭活（XCI））仍然缺乏。当前的知识几乎是使用小鼠模型确定的，但是考虑到出现的差异，从小鼠外推时需要谨慎，从而突出了对人类特定研究的需求。我们最近证明，在胚胎日（e）5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）多能层层细胞（EPI），与小鼠相反。我确定的另一个打击差异是人类胚胎中XCI的一种新型机制。 X-CHR的活性逐渐降低到E7中男性观察到的水平。不确定的是驱动胚泡发育的谱系分离和机制的原因。我们的初步数据证明了在胚胎日（E）5中单个谱系中Wnt和TGF-β途径的信号成分的存在，并且在E5期间，与这些途径相关的大多数基因都会打开/关闭；表明它们在谱系规范中起着重要作用。 Wnt和TGF-ß信号在人类植入前发育中的作用尚不清楚。此外，Porcn（Wnt途径的调节剂）是X连锁基因。鉴于PORCN在Wnt信号传导上的调节功能，我们现在还希望检查X-CHR剂量补偿和Wnt信号传导之间是否存在联系；这可以解释人类的植入前胚胎方法。为了更好地了解人类胚胎中涉及的谱系隔离和潜在XCI的潜在机制，我现在旨在探索使用小分子激动剂/拮抗剂探索Wnt和TGF-ß信号传导的功能作用。除了获得形成的胚胎和谱系的空间概述外，免疫组织化学还将测量成分（易位，磷酸化，细胞位置）和下游靶的变化。单细胞RNA测序将在跨多个发育时间点上的谱系特定分辨率下调节这些途径的影响，并确定与XCI与Smfish一起的关系。这些开创性的研究将对细胞脂肪规格，XCI和胚泡形成的基因调节机制提供首先详细的机械见解，从而为早期人类胚胎发生提供了基本知识。总体而言，了解早期人类发展的规定对于增强生殖生物学和发展领域至关重要。此外，该提案的结果可能导致改善用于干细胞生物学（幼稚和启动条件）和体外受精（IVF）的培养条件或推导方案。