Hypopituitarism is a condition defined as a shortage of one or more kinds of pituitary hormones, which can then lead to various symptoms in the individual, including growth restriction, metabolic deficiencies, and reproductive dysfunction (Bosch i Ara et al., 2021). Genetic causes of hypopituitarism have been extensively investigated and multiple genes have been identified to be associated with this condition. Most of them are critical transcription factors regulating the development of the pituitary including Lhx3, Lhx4, Prop1, Pit1/Pou1f1, and Hesx1 (Fang et al., 2016). However, other than the already known genetic mutations leading to hypopituitarism, the causes of over 80% of the cases remain unclear (Fang et al., 2016). This indicates other mechanisms yet to be discovered could be involved with this condition.

The NOTCH signaling pathway is crucial for pituitary development. Specifically, the NOTCH signaling pathway is necessary for mediating pituitary proliferation and differentiation, as NOTCH target gene Hes1 mutant mice had reduced pituitary proliferation and cell specification during embryogenesis (Raetzman et al., 2007). NOTCH signaling is also important for postnatal pituitary progenitor cell maintenance and expansion. Treatment with NOTCH inhibitor, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), resulted in decreased progenitor cell number and postnatal proliferation, as well as decreased postnatal anterior pituitary volume (Nantie et al., 2014). Among different NOTCH receptors, Notch2 is expressed in the pituitary gland and specifically in pituitary stem/progenitor cells (Raetzman et al., 2004). Our lab's previous study reported that the phenotypes of a Notch2 conditional knockout mouse model (Notch2-cKO) are very similar to Prop1 mutant mice, which is a known model for hypopituitarism (Bartke and Brown-Borg 2004). The shared phenotypes between Notch2-cKO mice and Prop1 mutant mice include anterior pituitary hypoplasia and reduced lactoroph, thyrotroph and somatotroph differentiation, leading to reduced body size compared to littermates (Nantie et al., 2014). Although we see the hypopituitarism-like phenotypes in Notch2-cKO mice, the downstream targets of Notch2 signaling in the pituitary and the mechanisms of how they regulate the functions of the pituitary that lead to the phenotype remain unclear.

To further investigate the downstream targets of NOTCH signaling pathway in the pituitary, our lab performed microarray analysis on Notch2-cKO mice and found mRNA of the gene encoding glycoprotein hormone subunit alpha 2 (Gpha2) was significantly decreased in Notch2-cKO pituitaries compared to wild-type (WT) pituitaries (unpublished data). Gpha2 has been reported to be expressed in the eye and pituitary gland in rodents (Nagasaki et al., 2006; Fletcher et al., 2019). As for its functions, GPHA2 was recently identified as a novel marker for corneal quiescent stem cells in mice (Altshuler et al., 2021), and it was found to support the undifferentiated state of corneal stem cells in human (Collin et al., 2021). GPHA2 also can heterodimerize with glycoprotein hormone subunit beta 5 (GPHB5) to form the hormone thyrostimulin. Thyrostimulin can bind to thyroid stimulating hormone receptor (TSHR) with higher affinity than its canonical ligand thyroid stimulating hormone (TSH) (Nakabayashi et al., 2002; Sun et al., 2010). Thyrostimulin has been shown to signal through TSHR in cell lines and organs, including the ovary, activating multiple downstream signaling pathways including PKA, PKC, PI3K/AKT, MAPK/ERK, and trans-activated EGFR (Nagasaki et al., 2006; Okada et al., 2006; Sun et al., 2010; Huang et al., 2016; Wang et al., 2018). TSHRs are expressed in numerous tissues including the anterior pituitary gland where they are believed to be involved in paracrine signaling via TSH from thyrotrope cells (Prummel et al., 2000; Theodoropoulou et al., 2000; Maso Previde et al., 2021). Despite the presence of GPHA2 and putative target receptor TSHR in the pituitary gland, downstream signaling and actions as a monomer are largely unknown.

In this study, we hypothesize: 1) Gpha2 is one of the downstream target genes of NOTCH signaling in the pituitary; 2) Gpha2 is present in quiescent stem cells, potentially controlling their proliferation; and 3) GPHA2 acts through TSHR in the pituitary in a paracrine manner. We aim to: 1) understand the expression pattern and NOTCH2 regulation of Gpha2 in the developing pituitary in mice; and 2) discover the target receptor and signaling pathway of GPHA2 in the pituitary and its possible role in the pituitary development. We mined single cell RNA sequencing (scRNA-seq) datasets from mouse developing and adult pituitaries, detected the expression of Gpha2 mRNA in Notch2-cKO mouse pituitaries compared to WT pituitaries using both qRT-PCR and in situ hybridization (ISH), and treated pituitaries with GPHA2 peptide and/or TSHR inhibitors to investigate downstream signaling pathways that GPHA2 activates in the pituitary.