PROJECT SUMMARY FOR DIVERSITY SUPPLEMENT IS SAME AS PARENT APPLICATION PROJECT SUMMARY Primordial germ cells seed the somatic gonad early in vertebrate development, and develop together to yield a functional testis replete with spermatogonial stem cells (SSCs). This population of SSCs maintains itself while generating astonishing numbers of differentiated sperm across the lifetime of the animal. We lack an understanding of the cellular and molecular mechanisms in SSCs that maintain an active population of stem cells. Studies of SSCs in vertebrates have been limited to population or single-cell snapshot studies, which fail to capture the contributions of individual SSCs to this process. Here, I propose to apply recently developed CRISPR lineage tracing, CRISPR mutagenesis, and single-cell RNA sequencing methods to uncover the mechanisms that maintain the vertebrate testis. First, we will investigate the dynamics of individual germline stem cells in testis homeostasis. We will use CRISPR lineage tracing in zebrafish to determine the contributions of individual SSCs to self-renewal and differentiation, and serial sampling from individual animals to track long-term dynamics. Second, we will characterize new regulators of SSC homeostasis. Using single-cell atlases of SSC gene expression, we will use our rapid CRISPR methods to mutagenize candidate regulator genes and phenotype using single-cell phenotyping and high-resolution imaging. Third, we will examine the evolutionary mechanisms that have generated testis phenotypic diversity across the vertebrate subphylum. We will generate single-cell, spatially resolved atlases of the juvenile, adult and aged testis from species across a phylogenetic tree of teleosts, and use comparative gene expression analysis to determine cellular and molecular differences in SSCs and niche cells. These projects will define the molecular and cellular mechanisms that maintain stem cell populations within the testis, with implications for human infertility. They will also uncover general principles of stem cell homeostasis in tissues and organs.