MON-715 How Heterogeneous Are Pituitary Thyrotropes?

Many transcription factors have been identified that are important for specification and differentiation of the hormone-producing cells of the pituitary gland. For example, POU1F1 is essential for differentiation of thyrotropes, somatotropes, and lactotropes. GATA2 is important for differentiation of both thyrotropes and gonadotropes, and NR5A1 drives gonadotrope differentiation. It is not known whether additional transcription factors are involved in thyrotrope specification. A few POU1F1-independent thyrotropes arise during development at the rostral tip of the pituitary gland, but the majority of thyrotropes are POU1F1-dependent and arise in the caudo-medial area of the anterior pituitary gland. Pou1f1-deficient mice lack this major, critical type of thyrotropes and are severely hypothyroid. Several pieces of evidence suggest that the caudo-medial population of thyrotropes is heterogeneous. First, NR5A1-positive cells exist that express both TSH and FSH during pituitary development. Second, Pou1f1 mRNA is detected in only about half of pituitary cells immuno-positive for TSH. Third, the transcription factor ISL1 is only detected in a fraction of developing thyrotropes. The Helix-Loop-Helix transcription factor ASCL1 is critical for zebrafish pituitary development, but there are conflicting data about its role in pituitary thyrotrope specification in mice. To quantify the role of ASCL1 in thyrotrope development we carried out immunostaining for TSH in pituitaries of Ascl1 deficient mice in late gestation. We did not detect any reduction in TSH immuno-reactive cells at this time, suggesting there are species-specific differences in the requirement of ASCL1 in the developing pituitary. To systematically assess and quantify the heterogeneity of thyrotropes for transcription factor expression, we performed co-immunostaining for TSH and various transcription factors in pituitaries of newborn mice. We found that approximately 75% of TSH-positive cells in the caudo-medial region of the pituitary express POU1F1, and the majority express GATA2. We detected a small portion of cells co-expressing NR5A1 and TSH at this time. To confirm our immunostaining results, we analyzed data from single cell RNA sequencing of postnatal thyrotropes. Three robust clusters of thyrotropes were identified. Approximately 80% expressed Gata2, 85% expressed Pou1f1, and roughly 15% expressed Nr5a1. Taken together, the protein and RNA analysis support that thyrotrope heterogeneity exists during development. The functional significance of this heterogeneity and its variation during development will be the subject of future investigation.