Uncovering the Mechanisms that Promote and Maintain Germ Cell Identity

Project Summary/Abstract Germ cells have the potential to generate every cell type in the body. Specified in the embryo, germ cells face the challenge of protecting and maintaining this poised totipotency for the entirety of an organism’s reproductive lifespan. Despite the fundamental importance of this protection for the fertility and health of the next generation,

the molecular programs that promote and protect germ cell identity are poorly understood. A specific program for germ cell transcriptional activation has yet to be described, largely because a ‘master regulator transcription factor’ for germ cell fate has not been identified in any organism. Instead, primordial germ cells (PGCs) rely on

conserved networks of RNA regulators that repress somatic differentiation programs and protect the germline genetic program, a strategy that is common to PGCs of worms, flies, mice, and humans. Yet how these RNA regulators intersect with the gene regulatory network of PGCs is unknown. My proposed research program seeks

to uncover the molecular mechanisms that initiate and maintain germ cell identity by revealing the PGC gene regulatory network. The goal of the research proposed here is to (a) uncover mechanisms that ‘repress’ the somatic program thereby allowing the PGC program to develop and (b) identify the ‘instructive’ cues that actively

control PGC fate. In Aim 1, I focus on discovering the dynamics of germline gene activation during embryogenesis by identifying specific, temporally regulated sets of genes that define the PGC transcriptional program using single-cell RNA sequencing. Using tissue-specific interference, I will distinguish between cell-

intrinsic and non-autonomous signaling mechanisms regulating the PGC transcriptional program. In Aim 2, I will probe the ‘instructive’ cues of germ cell identity by uncovering PGC-specific cis-regulatory sequences using ATAC-seq. I will identify transcriptional regulators (such as transcription factors/chromatin factors) that bind these

cis-regulatory sequences and test their role in the PGC transcriptional program. In Aim 3, I address the ‘repressive’ model by examining the function of a critical regulator of germ cell fate, the conserved RNA-binding protein Nanos. Using the RNA target identification method HyperTRIBE, I will identify Nanos targets and ask

whether these targets promote the germline program and/or repress somatic programs. I will use Drosophila as my model system, expanding my prior training in biochemistry and molecular biology to encompass organismal biology, development, and genetics and gaining expertise in bioinformatics and gene regulation. Together, this

work will uncover the regulatory landscape of primordial germ cells, providing a fundamental understanding of the mechanisms that specify germ cell fate, promote fertility, and ultimately protect the survival of the species.