Transcription factors and signaling pathways that regulate stem cells and specialized hormone-producing cells in the pituitary gland have been the subject of intense study and have yielded a mechanistic understanding of pituitary organogenesis and disease. However, the regulation of stem cell proliferation and differentiation, the heterogeneity among specialized hormone-producing cells, and the role of nonendocrine cells in the gland remain important, unanswered questions. Recent advances in single-cell RNA sequencing (scRNAseq) technologies provide new avenues to address these questions. We performed scRNAseq on ∼13,663 cells pooled from six whole pituitary glands of 7-week-old C57BL/6 male mice. We identified pituitary endocrine and stem cells in silico, as well as other support cell types such as endothelia, connective tissue, and red and white blood cells. Differential gene expression analyses identify known and novel markers of pituitary endocrine and stem cell populations. We demonstrate the value of scRNAseq by in vivo validation of a novel gonadotrope-enriched marker, Foxp2. We present novel scRNAseq data of in vivo pituitary tissue, including data from agnostic clustering algorithms that suggest the presence of a somatotrope subpopulation enriched in sterol/cholesterol synthesis genes. Additionally, we show that incomplete transcriptome annotation can cause false negatives on some scRNAseq platforms that only generate 3′ transcript end sequences, and we use in vivo data to recover reads of the pituitary transcription factor Prop1. Ultimately, scRNAseq technologies represent a significant opportunity to address long-standing questions regarding the development and function of the different populations of the pituitary gland throughout life.

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Defects in pituitary gland organogenesis are sometimes associated with congenital anomalies that affect head development. Lesions in transcription factors and signaling pathways explain some of these developmental syndromes. Basic research studies, including the characterization of genetically engineered mice, provide a mechanistic framework for understanding how mutations create the clinical characteristics observed in patients. Defects in BMP, WNT, Notch, and FGF signaling pathways affect induction and growth of the pituitary primordium and other organ systems partly by altering the balance between signaling pathways. The PITX and LHX transcription factor families influence pituitary and head development and are clinically relevant. A few later-acting transcription factors have pituitary-specific effects, including PROP1, POU1F1 (PIT1), and TPIT (TBX19), while others, such as NeuroD1 and NR5A1 (SF1), are syndromic, influencing development of other endocrine organs. We conducted a survey of genes transcribed in developing mouse pituitary to find candidates for cases of pituitary hormone deficiency of unknown etiology. We identified numerous transcription factors that are members of gene families with roles in syndromic or nonsyndromic pituitary hormone deficiency. This collection is a rich source for future basic and clinical studies.

FOXL2 is a forkhead transcription factor expressed in the eye, ovary, and pituitary gland. Loss of function mutations in humans and mice confirm a functional role for FOXL2 in the eye and ovary, but its role in the pituitary is not yet defined. We report that FOXL2 colocalizes with the glycoprotein hormone alpha-subunit (alphaGSU) in quiescent cells of the mouse pituitary from embryonic d 11.5 through adulthood. FOXL2 is expressed in essentially all gonadotropes and thyrotropes and a small fraction of prolactin-containing cells during pregnancy, but not somatotropes or corticotropes. The coincident expression patterns of FOXL2 and alphaGSU suggested that the alphaGSU gene (Cga) is a downstream target of FOXL2. We demonstrate that FOXL2 regulates mouse Cga transcription in gonadotrope-derived (alphaT3-1, LbetaT2), thyrotrope-derived (alphaTSH) and heterologous (CV-1) cells in a context-dependent manner. In addition, a FOXL2-VP16 fusion protein is sufficient to stimulate ectopic Cga expression in transgenic animals. Normal FOXL2 expression requires the transcription factors Lhx3 and Lhx4 but not of Prop1. Thus, FOXL2 expression is affected by mutations in early pituitary developmental regulatory genes, and its expression precedes that of genes necessary for gonadotrope-specific development such as Egr1 and Sf1 (Nr5a1). These data place FOXL2 in the hierarchy of pituitary developmental control and suggest a role in regulation of Cga gene expression.

Impairments in pituitary FSH synthesis or action cause infertility. However, causes of FSH dysregulation are poorly described, in part because of our incomplete understanding of mechanisms controlling FSH synthesis. Previously, we discovered a critical role for forkhead protein L2 (FOXL2) in activin-stimulated FSH β-subunit (Fshb) transcription in immortalized cells in vitro. Here, we tested the hypothesis that FOXL2 is required for FSH synthesis in vivo. Using a Cre/lox approach, we selectively ablated Foxl2 in murine anterior pituitary gonadotrope cells. Conditional knockout (cKO) mice developed overtly normally but were subfertile in adulthood. Testis size and spermatogenesis were significantly impaired in cKO males. cKO females exhibited reduced ovarian weight and ovulated fewer oocytes in natural estrous cycles compared with controls. In contrast, ovaries of juvenile cKO females showed normal responses to exogenous gonadotropin stimulation. Both male and female cKO mice were FSH deficient, secondary to diminished pituitary Fshb mRNA production. Basal and activin-stimulated Fshb expression was similarly impaired in Foxl2 depleted primary pituitary cultures. Collectively, these data definitively establish FOXL2 as the first identified gonadotrope-restricted transcription factor required for selective FSH synthesis in vivo.