ORF45-01 - Single-Cell Multiomic Network Prediction and Experimental Validation Reveal a Role for Pioneer Factors in Driving Neonatal Gonadotrope Differentiation

Pituitary stem cells (PSCs) can differentiate into any of the hormone-producing cell types, but in the neonate they become predominantly gonadotropes. This transient wave of differentiation during mini-puberty establishes the gonadotrope population that regulates adult reproductive function, and thus represents a critical developmental window for fertility. However, the trigger that directs PSC differentiation specifically towards the gonadotrope lineage and the underlying mechanisms remain unclear.
We hypothesized that this process is driven by the activation of distinct transcription factors (TFs), including pioneer factors that remodel the chromatin landscape to induce gonadotrope-specific gene expression. To explore this, we combined paired single-cell transcriptome and chromatin accessibility analysis of PSC-derived cells from neonatal mice, with perturbations in cell lines. Our dataset captures a continuous differentiation trajectory from PSCs through pre-gonadotropes, that express key TFs such as Foxl2 and Nr5a1, to mature gonadotropes. Gene-regulatory network (GRN) analysis, using SCENIC+, revealed stage-specific TF programs, and their associated target genes and candidate regulatory elements.
Focusing on the regulation of Foxl2, which is expressed early during differentiation of the gonadotrope and thyrotrope lineage, our analysis revealed a pioneer-factor-driven mechanism acting at distal enhancers. We predicted the involvement of three genomic loci in regulating Foxl2 expression, through binding of the pioneer TF, Ascl1. Accessibility at each of these loci increases along the differentiation, in concert with Foxl2 promoter accessibility and Foxl2 expression. Ascl1 was found to bind these sites in gonadotrope ChIP-seq experiments, and its overexpression significantly elevated Foxl2 mRNA levels, suggesting its role as a master regulator. Furthermore, CRISPR deletion of one of these elements reduced Foxl2 mRNA and protein levels, and diminished activating histone marks at the Foxl2 promoter, revealing a mechanistic link between enhancer activation and promoter chromatin state. Importantly, these enhancer sequences are evolutionarily conserved and accessible in human gonadotropes, suggesting common regulatory organization.
Neonatal gonadotrope specification thus requires a pioneer factor-dependent regulatory program acting at distal enhancers, as we have shown for Foxl2. Our findings demonstrate the power of combined single-cell network inference and functional validation in revealing events governing differentiation of PSCs along the gonadotrope lineage. Additional GRNs identified in this way, and similar experimental analyses, are providing mechanistic insight into how these networks define gonadotrope lineage commitment and establish the cell population that regulates adult reproductive function.