Notch signaling in postnatal pituitary expansion: proliferation, progenitors, and cell specification.

Mutations in PROP1 account for up to half of the cases of combined pituitary hormone deficiency that result from known causes. Despite this, few signaling molecules and pathways that influence PROP1 expression have been identified. Notch signaling has been linked to Prop1 expression, but the developmental periods during which Notch signaling influences Prop1 and overall pituitary development remain unclear. To test the requirement for Notch signaling in establishing the normal pituitary hormone milieu, we generated mice with early embryonic conditional loss of Notch2 (conditional knockout) and examined the consequences of chemical Notch inhibition during early postnatal pituitary maturation. We show that loss of Notch2 has little influence on early embryonic pituitary proliferation but is crucial for postnatal progenitor maintenance and proliferation. In addition, we show that Notch signaling is necessary embryonically and postnatally for Prop1 expression and robust Pit1 lineage hormone cell expansion, as well as repression of the corticotrope lineage. Taken together, our studies identify temporal and cell type-specific roles for Notch signaling and highlight the importance of this pathway throughout pituitary development.

Prenatal exposure to low doses of bisphenol A increases pituitary proliferation and gonadotroph number in female mice offspring at birth.

The pituitary gland is composed of hormone-producing cells essential for homeostasis and reproduction. Pituitary cells are sensitive to endocrine feedback in the adult and can have altered hormonal secretion from exposure to the endocrine disruptor bisphenol A (BPA). BPA is a prevalent plasticizer used in food and beverage containers, leading to widespread human exposure. Although prenatal exposure to BPA can impact reproductive function in the adult, the effects of BPA on the developing pituitary are unknown. We hypothesized that prenatal exposure to low doses of BPA impacts gonadotroph cell number or parameters of hormone synthesis. To test this, pregnant mice were administered 0.5 µg/kg/day of BPA, 50 µg/kg/day of BPA, or vehicle beginning on Embryonic Day 10.5. At parturition, pituitaries from female offspring exposed in utero to either dose of BPA had increased proliferation, as assessed by mKi67 mRNA levels and immunohistochemistry. Coincidently, gonadotroph number also increased in treated females. However, we observed a dichotomy between mRNA levels of Lhb and Fshb. Female mice exposed to 0.5 µg/kg/day BPA had increased mRNA levels of gonadotropins and the gonadotropin-receptor hormone (GNRH) receptor (Gnrhr), which mediates GNRH regulation of gonadotropin production and release. In contrast, mice treated with 50 µg/kg/day of BPA had decreased gonadotropin mRNA levels, Gnrhr and Nr5a1, a transcription factor required for gonadotroph differentiation. No other pituitary hormones were altered on the day of birth in response to in utero BPA exposure, and male pituitaries showed no change in the parameters tested. Collectively, these results show that prenatal exposure to BPA affects pituitary gonadotroph development in females.

p21, an important mediator of quiescence during pituitary tumor formation, is dispensable for normal pituitary development during embryogenesis.

A delicate balance between proliferation and differentiation must be maintained in the developing pituitary to ensure the formation of the appropriate number of hormone producing cells. In the adult, proliferation is actively restrained to prevent tumor formation. The cyclin dependent kinase inhibitors (CDKIs) of the CIP/KIP family, p21, p27 and p57, mediate cell cycle inhibition. Although p21 is induced in the pituitary upon loss of Notch signaling or initiation of tumor formation to halt cell cycle progression, its role in normal pituitary organogenesis has not been explored. In wildtype pituitaries, expression of p21 is limited to a subset of cells embryonically as well as during the postnatal proliferative phase. Mice lacking p21 do not have altered cell proliferation during early embryogenesis, but do show a slight delay in separation of proliferating progenitors from the oral ectoderm. By embryonic day 16.5, p21 mutants have an alteration in the spatial distribution of proliferating pituitary progenitors, however there is no overall change in proliferation. At postnatal day 21, there appears to be no change in proliferation, as assessed by cells expressing Ki67 protein. However, p21 mutant pituitaries have significantly less mRNA of Myc and the cyclins Ccnb1, Ccnd1, Ccnd2 and Ccne1 than wildtype pituitaries. Interestingly, unlike the redundant role in cell cycle inhibition uncovered in p27/p57 double mutants, the pituitary of p21/p27 double mutants has a similar proliferation profile to p27 single mutants at the time points examined. Taken together, these studies demonstrate that unlike p27 or p57, p21 does not play a major role in control of progenitor proliferation in the developing pituitary. However, p21 may be required to maintain normal levels of cell cycle components.

N-cadherin loss in POMC-expressing cells leads to pituitary disorganization.

Pituitary tumors are the third most common intracranial tumor in humans and can cause altered hormone secretions leading to hypercortisolism, acromegaly, and infertility. Reduced expression of the cell adhesion molecule N-cadherin has been linked with the formation of pituitary tumors, but its role in normal pituitary gland physiology or tumor initiation is unknown. In the murine pituitary, N-cadherin expression is detected in virtually all cells of the posterior, intermediate, and anterior lobes. N-cadherin may function to initiate important cues such as controlling proliferation, directing cell placement, and promoting formation of cell networks that coordinately release hormones into the bloodstream. To address this, we generated mice lacking N-cadherin in proopiomelanocortin-expressing melanotrope and corticotrope cells of the intermediate and anterior lobes of the pituitary. We observed that intermediate lobe cells can aberrantly displace SOX2-containing progenitor cells in the N-cadherin conditional knockout mice at postnatal d 1. By postnatal d 30, although a reduction in α- and ß-catenin membrane staining occurs, there is little effect on intermediate lobe architecture with N-cadherin loss. Also, despite these changes in adherens junction molecules, no alterations in cell proliferation occur. In contrast, loss of N-cadherin in the corticotropes leads to aberrant cell clustering and a reduction in Pomc mRNA. Taken together, our data reveal important roles of N-cadherin in pituitary cell placement and that loss of N-cadherin alone does not lead to pituitary tumor formation.

Premature differentiation and aberrant movement of pituitary cells lacking both Hes1 and Prop1.

In the pituitary, the transition from proliferating progenitor cell into differentiated hormone producing cell is carefully regulated in a time-dependent and spatially-restricted manner. We report that two targets of Notch signaling, Hes1 and Prop1, are needed to maintain progenitors within Rathke's pouch and for the restriction of differentiated cells to the ventral pituitary. We observed ACTH and alphaGSU producing cells that had prematurely differentiated within Rathke's pouch along with correlated ectopic expression of Mash1 only when both Prop1 and Hes1 were lost. We also discovered that downregulation of N-cadherin expression in cells as they transition from Rathke's pouch to the anterior lobe appears to be essential for their movement. In the Prop1 mutant, cells are trapped in Rathke's pouch and N-cadherin expression remains high. Also, Slug, a marker of epithelial-to-mesenchymal transition, is absent in the dorsal anterior lobe. When Hes1 is lost in the Prop1 mutant, N-cadherin is downregulated and cells are able to exit Rathke's pouch but have lost their migrational cues and form ectopic foci surrounding Rathke's pouch. Our data reveal important overlapping functions of Hes1 and Prop1 in cell differentiation and movement that are critical for pituitary organogenesis.