The role of notch signaling in the regulation of proliferation and cell differentiation during embryonic and postnatal pituitary development

The pituitary develops in two distinct waves in mice, one occurring during embryogenesis and a second taking place during early postnatal development. During both periods, progenitor cells proliferate and subsequently differentiate into hormone-producing cells. Maintaining the proper number of progenitors and producing the appropriate number and type of endocrine cells is essential to pituitary development and function. Although signals necessary for initial pituitary commitment and those required for pituitary hormone synthesis have been well characterized, pathways that regulate progenitor proliferation and cell fate remain unclear. Previous studies have implicated the Notch signaling pathway in these processes, both in the pituitary and in other developing tissues. In order to more specifically define the role of Notch signaling during different stages of pituitary development, we utilized pituitary-specific gain- and loss-of-function mouse models, in combination with chemical Notch inhibition. Notch signaling is necessary for progenitor maintenance in many systems. Additionally, loss of the canonical Notch target Hes1 in combination with loss of the pituitary specific Notch target, Prop1, results in premature differentiation of corticotropes. Based on these observations, we hypothesized that Notch signaling is sufficient to inhibit the differentiation of corticotropes and maintain these cells in a progenitor state. To test this hypothesis, we generated mice with persistent expression of activated Notch in POMC-expressing cells. We show that constitutive Notch expression prevents the differentiation of corticotropes and melanotropes, which also contain POMC. These cells instead maintain progenitor markers. In addition, mRNA levels of transcription factors necessary for Pomc expression are downregulated in this model. Cells that aberrantly maintain progenitor markers are lost during early postnatal development, resulting in dysfunction of the hypothalamic-pituitary-adrenal axis. These findings confirm our hypothesis that Notch signaling is sufficient to prevent corticotrope differentiation. Previous data have suggested that Notch signaling may be able to transcriptionally activate Prop1, which is necessary for Pit1 transcription, which is necessary for GH, TSH and Prl synthesis. These findings, in combination with the observation that Notch signaling is sufficient to inhibit differentiation of POMC-expressing cells, led us to hypothesize that Notch signaling may regulate the balance between POMC lineage cells and Prop1/Pit1 lineage cells. Additionally, we hypothesized that alterations in Notch signaling may lead to changes in proliferation and progenitor maintenance, both well characterized roles of Notch. To address these hypotheses, we examined a mouse model with loss of Notch2 prior to pituitary induction. In this model, we observe a decrease in Pit1-positive cell number and an increase in corticotrope cell number, suggesting Notch regulates the choice between these pituitary lineages. Additionally, we observe defects in proliferation and progenitor maintenance, but not until after birth, indicating Notch2 is dispensable for these functions during embryonic development. Because of this observation, we examined the role of Notch signaling during postnatal development using a chemical inhibitor of Notch, which was specifically administered during postnatal. We observe an increase in Pomc mRNA, a decrease in Pit1 mRNA and a decrease in the number of proliferating cells after Notch inhibition. These findings demonstrate that Notch signaling is an important regulator of cell fate and proliferation in the postnatal pituitary. In order to identify targets that may be necessary for these functions, microarray analysis was performed comparing Notch2 ablated mice to controls. Among the target mRNAs that were altered, many have been implicated in cell proliferation and a subset are involved in corticotrope cell signaling. These targets will be areas of future focus in the Raetzman laboratory. Taken together, these studies have defined a role for Notch signaling in the control of cellular proliferation, progenitor maintenance and specification during embryonic and postnatal pituitary development