CAREER: Specify germ cell lineages from pluripotent state

在小鼠或人类等多细胞生物体中，一个受精卵可以发育成构成身体的具有不同生物功能的所有细胞类型。然而，这些过程是复杂的，尚未得到充分理解。例如，原始生殖细胞（PGCs）是最终发育成卵子和精子以确保生命延续的胚胎前体。PGC形成的破坏会导致不孕。PGC在发育过程中如何与其他细胞类型分化并建立生殖细胞能力仍然是难以捉摸的。基于最近开发的平台，该平台能够实现强大的PGC生成和先进的全基因组基因编辑技术，该项目旨在揭示管理生殖细胞承诺的新型调控因子和基本机制（或“规则”）。研究结果将极大地提高对人类不孕症原因的理解，这影响了全球约15%的夫妇。该项目的结果将进一步造福农业社区，为改善牲畜生殖健康的新战略提供重要信息。研究小组将通过将科学研究与教育和公共宣传活动相结合，最大限度地扩大该项目的广泛影响。他们将把科学进步纳入教育计划，以培养下一代STEM科学家，并与外展合作伙伴密切合作，吸引K-12学生。他们将通过鼓励代表性不足的群体和妇女参与科学来促进多样性，平等和包容。到目前为止，只有少数监管机构（例如，WNT和BMP）在哺乳动物胚胎发育过程中PGC的特化中起着关键作用。控制PGC个体发生的更广泛的基因调控网络仍然定义不清-可行性在很大程度上受到发育期间新生PGC数量少以及大规模鉴定和功能评估候选调控因子的低效方法的限制。多能干细胞（Pluripotent stem cells，PSC）具有在体外无限增殖并分化为包括生殖细胞在内的体内所有细胞谱系的巨大潜力。在试点研究中，研究小组使用了一个强大的小鼠PSC体外分化平台，用生殖系报告基因产生了大量的PGC，随后从全基因组基于CRISPR的激活筛选中鉴定了589种推定的生殖细胞调节因子。基于这些有希望的数据，该项目旨在揭示PSC建立生殖能力的新分子决定因素和功能机制。目标1将在全基因组范围内发现PGC特异性的新型调节剂，具有定制的小向导RNA（sgRNA）文库和逐步验证策略。然后根据候选调节剂的sgRNA富集频率、PGC中的表达水平和本体论，对候选调节剂进行优先级排序以进行个体验证。目的2将揭示一个新发现的候选人通过WNT依赖性和独立途径调节PGC特异性的分子机制。总之，该项目将揭示一个更广泛的管理PGC个体发育的监管网络，这是理解在发育过程中控制谱系规范的分子决定因素的一个重大进步。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

In multi-cell organisms like mice or humans, one fertilized egg can develop into all cell types with diverse biological functions that make up the body. However, these processes are complex and yet to be fully understood. For example, primordial germ cells (PGCs) are the embryonic precursors that ultimately develop into eggs and sperm to ensure the continuation of life. Disruption of PGC formation will cause infertility. It remains elusive how PGCs diverge from other cell types during development and establish germ cell competency. Based on a recently developed platform that enables robust PGC generation and advanced genome-wide gene-editing technologies, this project aims to unveil novel regulators and fundamental mechanisms (or “rules”) that govern germ cell commitment. Research findings will critically improve understanding of the causes of human infertility, which affects about 15% of couples worldwide. Results from this project will further benefit the agricultural community by critically informing novel strategies to improve the reproductive health of livestock animals. The research team will maximize the broader impact of this project by combining scientific research with educational and public outreach activities. They will incorporate scientific advances into educational programs to foster next-generation STEM scientists, and work closely with outreach partners to engage K-12 students. They will promote diversity, equality, and inclusion by encouraging underrepresented groups and women to get involved in sciences. To date, only a few regulators (e.g., WNT and BMPs) have been identified to play crucial roles in PGC specification during mammalian embryonic development. The broader gene-regulatory network that controls PGC ontogeny remains poorly defined – feasibility largely limited by the low numbers of nascent PGCs during development and inefficient approaches to identify and functionally evaluate candidate regulators at large scales. Pluripotent stem cells (PSCs) have the great potential to proliferate unlimitedly in vitro and to differentiate into all lineages of cells in the body, including germ cells. In pilot studies, the research team used a robust in vitro differentiation platform of mouse PSCs with germline reporters to generate a large quantity of PGCs, and subsequently identified 589 putative germ cell regulators from a genome-wide CRISPR-based activation screening. Building on these promising data, this project aims to reveal the novel molecular determinants and functional mechanisms by which PSCs establish germline competency. Objective 1 will discover novel regulators of PGC specification at a genome-wide scale, with a custom-made small guide RNA (sgRNA) library and a stepwise validation strategy. Candidate regulators will then be prioritized for individual validation according to their sgRNA enrichment frequency, expression levels in PGCs, and ontology. Objective 2 will unveil the molecular mechanisms by which a newly discovered candidate from the pilot study regulates PGC specification via both WNT-dependent and independent pathways. In summary, this project will unveil a broader regulatory network that governs PGC ontogeny, a major advance in understanding the molecular determinants that control lineage specification during development.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.