CAREER: Specify germ cell lineages from pluripotent state

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.