PROJECT SUMMARY/ABSTRACT The germline provides an eternal cellular link between generations and in metazoan a male's genetic contribution to the next generation is delivered via sperm. Thus, the genesis of sperm (i.e. spermatogenesis) is critical for the continuity and diversity of a species. This project is designed to advance our understanding of the developmental origins of the sperm producing (i.e. spermatogenic) lineage. In particular, it seeks to uncover a cell autonomous trigger driving formation of the foundational spermatogonial stem cell (SSC) pool which is required for continuity of spermatogenesis and male fertility in adulthood. In mammals, the SSC pool is known to arise in early postnatal life from a subset of the prospermatogonial precursor population but the mechanism underpinning the process is grossly undefined. Indeed, no single molecule as has been identified, to date, to be obligatory for this process. Understanding how the foundational SSC pool is built from prospermatogonial precursors is scientifically and clinically important. Progress in this area of study has been limited because the tools for investigating the process in fine detail have been lacking. Over the past several years, we discovered genes that are expressed by SSCs (e.g. Id4) and generated multi-transgenic reporter mouse models to label germ cell subsets through fetal and neonatal development. Using these tools, we have been able to describe the kinetics underlying the building of the foundational SSC pool, determine that the process is triggered at a defined age point in neonatal development (P0-3 in mice), and profile transcriptome dynamics as the single cell level. Outcomes of these studies uncovered a unique expression profile for the transcription factor Msx1; temporal induction in a subset of the prospermatogonial population at P0 that based on trajectory predictions from scRNA-seq analyses are fated to become SSCs. The current project is designed to define the potency of temporal Msx1 expression on SSC fate specification and explore the mechanism of action for Msx1 in prospermatogonia. Results are expected to provide a wealth of information from which we can define how SSC fate is triggered in a subset of prospermatogonia during neonatal development. This information may be clinically and scientifically useful for understanding the etiology of male infertility, and through transient ectopic expression approaches to reprogram somatic cells to adopt an SSC fate or rejuvenate stem cell capacity in aged or imperfect SSCs.