CAREER: Specify germ cell lineages from pluripotent state

在像老鼠或人类这样的多细胞生物中，一个受精卵可以发育成构成身体的所有具有不同生物功能的细胞类型。然而，这些过程是复杂的，尚未完全理解。例如，原始生殖细胞（PGCs）是胚胎的前体，最终发育成卵子和精子，以确保生命的延续。PGC形成的破坏会导致不孕。PGCs在发育过程中如何从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。基于最近开发的一个平台，该平台能够生成强大的PGC和先进的全基因组基因编辑技术，该项目旨在揭示控制生殖细胞承诺的新的调节因子和基本机制（或“规则”）。研究结果将极大地提高对人类不孕症原因的了解，全世界约有15%的夫妇受到不孕症的影响。该项目的结果将通过为改善牲畜生殖健康的新策略提供重要信息，进一步造福农业社区。研究小组将把科学研究与教育和公众宣传活动结合起来，最大限度地扩大这个项目的影响。他们将把科学进步纳入教育项目，培养下一代STEM科学家，并与外展伙伴密切合作，吸引K-12学生。它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。迄今为止，只有少数调节因子（如WNT和bmp）被确定在哺乳动物胚胎发育过程中对PGC规范起关键作用。控制PGC个体发生的更广泛的基因调控网络仍然不明确，其可行性在很大程度上受到发育过程中新生PGC数量少以及大规模识别和功能评估候选调控因子的低效方法的限制。多能干细胞（PSCs）在体外具有无限增殖和分化为体内所有细胞谱系的巨大潜力，包括生殖细胞。在初步研究中，研究小组使用强大的小鼠PSCs体外分化平台与生殖系报告细胞产生大量的PGCs，随后通过基于全基因组crispr的激活筛选确定了589个假定的生殖细胞调节因子。基于这些有希望的数据，本项目旨在揭示PSCs建立种系能力的新分子决定因素和功能机制。目标1将利用定制的小向导RNA （sgRNA）文库和逐步验证策略，在全基因组范围内发现新的PGC规范调节因子。候选调节因子将根据其sgRNA富集频率、PGCs中的表达水平和本体优先进行个体验证。目的2将揭示中试研究中新发现的候选物质通过wnt依赖和独立途径调节PGC规范的分子机制。总之，该项目将揭示一个更广泛的控制PGC个体发生的调控网络，这是理解在发育过程中控制谱系规范的分子决定因素的重大进展。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.