|Abstract:||The pituitary acts as a central mediator of the endocrine system, integrating both hypothalamic and peripheral signals to coordinate physiological functions through the release of hormones from distinct endocrine cell types within the gland. During development, the endocrine cell types are derived from a common pituitary progenitor cell. To maintain the correct number of each cell type, it is essential that the pituitary coordinate progenitor maintenance and proliferation with the differentiation of the hormone producing cells. Disruption of the developmental pathways that are necessary for pituitary cell fate determination can result in pituitary disorders such as hypopituitarism, which is associated with several detrimental health conditions including growth deficiencies, adrenal insufficiency, infertility, and hypothyroidism. To understand the etiology of pituitary disorders we must elucidate the factors and molecular pathways that regulate pituitary cell fate choice and lineage specification. Previous studies from our laboratory have demonstrated that the Notch signaling pathway is an important regulator of pituitary cell fate choice. Specifically, Notch signaling is necessary for pituitary progenitor maintenance and coordinates the balance of endocrine cell differentiation. However, the mechanism by which the Notch signaling pathway regulates cell fate choice in the pituitary remains unclear. To identify novel downstream targets of Notch signaling that mediate its effect on pituitary development, a transcriptomic analysis was performed comparing pituitaries from mice with a conditional loss of the Notch2 receptor to controls. From this analysis, we identified the novel Notch regulated genes, grainyhead-like 2 (Grhl2) and 11-beta hydroxysteroid dehydrogenase type 1 (Hsd11b1) as potential downstream effectors of the Notch signaling pathway that may influence pituitary development.
The Notch signaling pathway has been shown to regulate progenitor cell proliferation and self-renewal capacity in several tissues. Previous studies in the pituitary demonstrate that loss of Notch signaling results in decreased progenitor cell number and proliferation, as well as a misplacement of the progenitor cell niche. Therefore, Notch signaling is necessary for progenitor cell function and presumably helps to maintain the extra-cellular environment required for progenitor cell localization. In this work, we identify Grhl2 as a novel pituitary progenitor factor that is dependent on Notch signaling in the developing pituitary. GRHL2 is present in several epithelial tissues where it has been shown to regulate plasticity by promoting cellular proliferation, inhibiting differentiation and maintaining cellular adhesion. Based on the functions of GRHL2 in other tissues, we hypothesize that Notch regulation of GRHL2 is a potential mechanism by which the pathway influences progenitor cell maintenance. Our studies demonstrate GRHL2 is expressed in pituitary progenitor cells throughout pituitary gland development, with its most pronounced expression during early postnatal development. Utilizing Notch2 cKO mice and chemical Notch inhibitors, we are the first to demonstrate that Grhl2 is dynamically regulated by Notch signaling in the postnatal pituitary. We observe that loss of Notch signaling results in a decrease in GRHL2 expression and also its direct transcriptional targets such as E-cadherin, an important progenitor cell adhesion molecule. Taken together, these data suggest a role for GRHL2 in progenitor cells and regulation of this gene may be a mechanism by which Notch influences progenitor maintenance in the developing pituitary.
Notch signaling also regulates the balance of endocrine cell differentiation in the developing pituitary. Previous studies suggest that Notch signaling promotes the differentiation of the PIT1 lineage comprised of somatotropes, lactotropes and thyrotropes, while suppressing differentiation of the corticotrope lineage. Notch is thought to promote PIT1 lineage development by regulating the expression of Prop1 which is required for Pit1 transcription. In contrast, the mechanism by which Notch suppresses corticotrope differentiation remains poorly understood. Similar to Notch signaling, several studies have demonstrated that the glucocorticoid signaling pathway also suppresses corticotrope differentiation. We therefore sought to identify Notch regulated genes that are associated with the glucocorticoid signaling pathway. In our study, we identify Hsd11b1 as a novel Notch signaling target gene. 11β-HSD1 is widely expressed and in the majority of tissues and functions to convert inactive forms of glucocorticoids into their biologically active forms. We hypothesize that Notch may induce the expression of Hsd11b1 to increase pituitary-specific levels of active glucocorticoids that in turn suppress corticotrope expansion.
In this work we utilize Notch2 cKO mice, in vivo and in vitro chemical Notch inhibition, as well as reporter gene assays in primary pituitary cultures, to demonstrate Hsd11b1 is a novel Notch signaling target in the postnatal pituitary. To help elucidate how Notch regulation of this gene may influence pituitary development we characterized the expression of Hsd11b1 and conducted functional studies in which 11β-HSD1 was chemically and genetically inhibited during early pituitary development. Our study demonstrates Hsd11b1 is expressed in both pituitary progenitor and endocrine cell types. In addition, its expression peaks during early postnatal development, a time in which Notch signaling is crucial for pituitary gland expansion. Our functional studies indicate that loss of 11β-HSD1 function results in an increase in corticotrope lineage gene expression, similar to the phenotype that is observed in Notch2 cKO mice. Our study suggests Notch regulation of Hsd11b1 may be a part of the mechanism by which Notch signaling inhibits corticotrope lineage gene expression. Taken together, these works help to define the transcriptional network of the Notch signaling pathway in the developing pituitary and suggest that Notch signaling can coordinate a vast number of targets to regulate pituitary cell fate decisions.