Single-Cell Gene Expression Analysis Reveals Gene Regulatory Networks Driving Proliferation in Pituitary Stem and Endocrine Cells

A fundamental question for pituitary development and disease is to understand the mechanisms that regulate proliferation, quiescence, and differentiation of stem cells and endocrine lineage-committed precursor cells. Pituitary stem cells, marked by expression of SOX2, are highly proliferative during development and the early postnatal period. This pool becomes quiescent over time, but stem cells retain the ability to re-enter the cell cycle and differentiate into nascent endocrine cells of all lineages in response to physiological demands. The rodent pituitary gland undergoes substantial growth in the postnatal period, and much of this increase in size is due to proliferation of committed progenitors, such as Pou1f1-expressing cells. We performed single-cell RNA transcriptomics analyses of over 8,000 male and female 4-day-old mouse pituitary cells in order to assess stem cell heterogeneity and to identify novel pituitary stem cell biomarkers. We identified a number of factors enriched in pituitary stem cells relative to differentiating cells, including the transcription factors TGIF1 and NR4A3 as well as several members of the nuclear factor I family (NFIA, NFIB, NFIX). We also detected stem cell-specific expression of the cortisol synthesizing enzyme HSD11B1 and folate receptor FOLR1, suggesting novel roles for these pathways in pituitary stem cells. There were few transcriptomic differences between proliferating and non-proliferating stem cells. However, proliferating stem cells and proliferating committed progenitors shared expression of cell-cycle associated genes and novel transcription factors such as BRCA1 and E2F1. Furthermore, single-cell gene network inference and clustering (SCENIC) analyses demonstrated activation of common gene regulatory networks in both proliferating stem and proliferating endocrine populations, including both the E2f1 and Brca1 regulons. RNA velocity, trajectory, and phylogenetic analyses and find that proliferating stem and endocrine cells are likely independent cellular states. Instead, they support the idea that proliferating stem cells become quiescent and transition to committed progenitors that re-enter the cell cycle and then subsequently activate hormone gene expression. In conclusion, our single-cell gene expression analyses of early postnatal pituitary cells have shed light on the developmental trajectory from proliferating stem cell to quiescent differentiated hormone-producing cells.