Human Pituitary Research Articles

8574 Single cell multiomics suggests tumor cell energy metabolism heterogeneity in non-functioning gonadotroph pituitary adenomas

Abstract Disclosure: F.M. Ruf-Zamojski: None. Z. Zhang: None. W. Cheng: None. G.R. Smith: None. M. Zamojski: None. N. Mendelev: None. Y. Ge: None. D. Marrero-Rodríguez: None. K. Taniguchi-Ponciano: None. C.L. Andoniadou: None. E. Zaslavsky: None. X. Chen: None. O.G. Troyanskaya: None. M. Mercado: None. S.C. Sealfon: None. Pituitary adenomas cause hormonal dysregulation and severe morbidity. The absence of clear biomarkers for prognosis/treatment and the high risk of recurrence make management challenging. Non-functioning gonadotroph pituitary adenomas (NFPAs) represent proliferation of gonadotroph lineage cells without an increase in secretion of gonadotropins. Detection is often delayed until mass effects cause visual defects. Higher resolution molecular study may improve classification, diagnosis, treatment, and provide insight into adenoma biology. We analyzed four NFPAs using unbiased genome-wide same-cell single nucleus 10X Genomics multiome assay (transcriptome and chromatin accessibility) to define individual cell identity/states in comparison with normal gonadotrope cell references from twelve age/sex-matched human post-mortem pituitaries. In total, we generated high quality multiomics single-cell data for 33,832 adenoma cells and 5,451 normal gonadotropes. We identified tumor, immune, vascular, and proliferative cells confirming cellular heterogeneity. Multidimensional scaling analysis using Manhattan distances between cell types showed that NFPA gonadotropes were closer in gene expression to healthy gonadotropes among all pituitary cell types. In addition, we observed differences in the macrophage and endothelial cell populations between tumors and healthy pituitaries. Next, we studied gene regulatory circuitry (regulated gene, modulating transcription factor and its chromatin interaction site) using the new Control of Regulation Extracted from Multiomics Assays (CREMA, [1]) multiome analysis method which showed high variation between normal and tumor gonadotropes. Within each adenoma, the NFPA tumor cells encompassed two main subgroups of cells, one group (GT+ marker tumor cells) expressing established gonadotrope markers and another group not (GT- marker tumor cells). GT+ and GT- tumor cells formed a cluster distinct from healthy gonadotropes. GT+ and GT- tumor cells differed by their level of expression of mitochondrial genes, suggesting decreased aerobic capacity associated with the GT- tumor subgroup. Interestingly, differential analyses between the healthy and the NFPA gonadotropes from same-cell multiome data highlighted dissimilar gene expression and chromatin accessibility patterns.Our newly collected single cell data characterize the difference between NFPA tumor cells and normal gonadotrope cells. Our results suggest a Warburg-effect like difference in aerobic respiratory capacity among tumor cells within the same pituitary adenoma. Overall, this study brings new insights into the molecular characteristics of NFPAs, including tumor cell heterogeneity, and represents a new resource for the field. [1] PMID: 38084892. Presentation: 6/1/2024

Isolated hypoprolactinemia: The rarest of the rare?

Isolated hypoprolactinemia (IHP) can be defined as the presence of consistently low serum levels of prolactin in the absence of other anterior pituitary hormone abnormalities. It is an extremely rare condition, and consequently incompletely understood and unrecognized. A recent study has reported the first cases of IHP caused by mutation in the PRL gene. There are also cases where the pathogenesis of IHP is likely secondary to an autoimmune response against the PRL-secreting cells. But most published cases are acquired and idiopathic. The best characterized function of PRL is to facilitate milk production in the puerperium. Analysis of the GTEX data repository, however, shows that PRL is the most abundantly expressed gene in the human pituitary, independently of gender and age, suggesting the presence of additional roles for PRL. Newer studies have indeed revealed a much larger spectrum of PRL functions and will likely uncover novel clinical phenotypes associated with severe PRL deficiency.

Glucagon does not directly stimulate pituitary secretion of ACTH, GH or copeptin

Glucagon is best known for its contribution to glucose regulation through activation of the glucagon receptor (GCGR), primarily located in the liver. However, glucagon’s impact on other organs may also contribute to its potent effects in health and disease. Given that glucagon-based medicine is entering the arena of anti-obesity drugs, elucidating extrahepatic actions of glucagon are of increased importance. It has been reported that glucagon may stimulate secretion of arginine-vasopressin (AVP)/copeptin, growth hormone (GH) and adrenocorticotrophic hormone (ACTH) from the pituitary gland. Nevertheless, the mechanisms and whether GCGR is present in human pituitary are unknown. In this study we found that intravenous administration of 0.2 mg glucagon to 14 healthy subjects was not associated with increases in plasma concentrations of copeptin, GH, ACTH or cortisol over a 120-min period. GCGR immunoreactivity was present in the anterior pituitary but not in cells containing GH or ACTH. Collectively, glucagon may not directly stimulate secretion of GH, ACTH or AVP/copeptin in humans but may instead be involved in yet unidentified pituitary functions.

Single-cell transcriptomic analysis reveals tumor cell heterogeneity and immune microenvironment features of pituitary neuroendocrine tumors.

Pituitary neuroendocrine tumors (PitNETs) are one of the most common types of intracranial tumors. Currently, the cellular characteristics of normal pituitary and various other types of PitNETs are still not completely understood. We performed single-cell RNA sequencing (scRNA-seq) on 4 normal samples and 24 PitNET samples for comprehensive bioinformatics analysis. Findings regarding the function of PBK in the aggressive tumor cells were validated by siRNA knockdown, overexpression, and transwell experiments. We first constructed a reference cell atlas of the human pituitary. Subsequent scRNA-seq analysis of PitNET samples, representing major tumor subtypes, shed light on the intrinsic cellular heterogeneities of the tumor cells and tumor microenvironment (TME). We found that the expression of hormone-encoding genes defined the major variations of the PIT1-lineage tumor cell transcriptomic heterogeneities. A sub-population of TPIT-lineage tumor cells highly expressing GZMK suggested a novel subtype of corticotroph tumors. In immune cells, we found two clusters of tumor-associated macrophages, which were both highly enriched in PitNETs but with distinct functional characteristics. In PitNETs, the stress response pathway was significantly activated in T cells. While a majority of these tumors are benign, our study unveils a common existence of aggressive tumor cells in the studied samples, which highly express a set of malignant signature genes. The following functional experiments confirmed the oncogenic role of selected up-regulated genes. The over-expression of PBK could promote both tumor cell proliferation and migration, and it was also significantly associated with poor prognosis in PitNET patients. Our data and analysis manifested the basic cell types in the normal pituitary and inherent heterogeneity of PitNETs, identified several features of the tumor immune microenvironments, and found a novel epithelial cell sub-population with aggressive signatures across all the studied cases.

PMON29 A Comparative Analysis of Musashi-Dependent Control of Mouse and Human Pituitary mRNA Translation

Abstract Specialized cell lineages within the anterior pituitary produce specific hormones which, in turn, regulate diverse biological processes ranging from growth, metabolism, and reproduction to stress. Interestingly, these specialized cell lineages exhibit a high degree of cellular plasticity, readily altering their differentiation state to produce different hormones in response to organismal demand. Underlying molecular mechanisms controlling cellular plasticity in the pituitary are not fully defined but appear to involve post-transcriptional regulation exerted by the Musashi family of sequence-specific RNA binding proteins. Musashi is a bifunctional regulator of target mRNAs and can exert either repression or activation of translation in a context-dependent manner. Our recent work has implicated a role for Musashi-dependent repression of key pituitary mRNAs in the mouse, including a lineage-specific transcription factor (Pou1f1), the gonadotropin releasing hormone receptor (Gnrhr), and two anterior pituitary hormones (Prl and Tshb). In this study, we look to determine the potential relevance of Musashi-dependent mRNA translational control to the human pituitary. Reflecting our findings in the adult mouse, a re-analysis of published human fetal pituitary single cell RNA sequencing data revealed Musashi1 (MSI1) and Musashi2 (MSI2) expression in both stem/progenitor cell populations as well as in all hormone-producing cell lineages of the anterior pituitary. We further report that the human homologs of identified Musashi-target mRNAs in the mouse pituitary also possess Musashi binding elements (MBEs) in their 3' untranslated regions (UTRs). In the case of the human Pou1f1 3' UTR, we were able to verify Musashi-dependent mRNA translational repression in reporter assays as seen with the murine Pou1f1 3' UTR. Interestingly, a germline Pou1f1 mutation identified in human patients, disrupts a consensus MBE in the human Pou1f1 3' UTR and abolishes Musashi-dependent repression. We developed a bioinformatic pipeline to characterize pathogenic human mutations catalogued in the Geno2MP database for disruption or creation of consensus MBEs in pituitary-specific mRNA 3' UTRs. Our findings suggest an evolutionarily conserved role for Musashi in the post-transcriptional regulation of pituitary mRNAs in both mice and humans and identify several human mutations which may perturb Musashi regulation of human pituitary function. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

RF13 | PMON143 BRF1-Mediated Paracrine Signalling by a Subset of SOX2-Expressing Stem Cells is Required for Normal Development of the Stem Cell Compartment and Terminal Differentiation of Pituitary Committed Progenitors

Abstract Introduction Hormone-producing pituitary cell lineages are derived from a population of embryonic precursors expressing SOX2. These cells maintain multipotency into early postnatal life, acting as the resident population of pituitary stem cells (PSCs) and contributing extensively to all the endocrine cell lineages. In addition to this direct contribution to pituitary turnover, paracrine signalling from PSCs has been shown to be important for cell proliferation of neighbouring progenitors (PMC7803373). It is not known if SOX2+ PSCs are involved in the regulation of additional cell attributes during normal physiology and if there is functional heterogeneity among the SOX2+ PSC population. Experimental Methods We have carried out single-cell RNA-Sequencing of SOX2+ PSCs from Sox2Egfp/+ mouse pituitaries at three postnatal stages from P3 to P56 and used computational approaches to analyse their molecular signatures. A novel conditional mouse model expressing a constitutively active mutant form of the RNA binding factor BRF1 (R26stop-mBRF1) has been used to attenuate the expression of several cytokines and chemokines in SOX2+ cells embryonically and postnatally (PMC4589897). Results We show that the SOX2+ PSC population consists of three subgroups (SC1, SC2 and SC3). We reveal that SC1-SC2 express abundant cytokines and secreted factors, suggesting paracrine function. In contrast, SC3 is characterised by robust expression of Lef1, is identified as a committing PSC cluster, and its presence diminishes with age. Key markers of PSC clusters SC1-SC2 include the RNA binding factor BRF1. We show that BRF1 is highly expressed in PSCs and validate its expression by immunohistochemistry in both mouse and human pituitaries. Secondly, we show that the dysregulation of BRF1 in embryonic SOX2+ cells using the Hesx1-Cre driver (PMC3461924) results in pituitary hypoplasia and severe hypopituitarism due to a failure of the PIT1 and SF1 cell-lineage committed progenitors to terminally differentiate into hormone-producing cells. Additionally, there is a significant reduction of the stem cell compartment, manifested by lower numbers of SOX2/SOX9+ stem cells. This phenotype is recapitulated when using a Sox2-CreERT2 driver (PMID24094324). The differentiation failure can be rescued in vitro through co-culture of mutant cells with wild-type stem cells, as well as in vivo, in mutant pituitaries where activation of constitutively active BRF1 is restricted to few SOX2+ PSCs in a mosaic manner. Finally, we identify key ligands underlying this differentiation phenotype and demonstrate a partial restoration of terminal differentiation in the mutant, when cultured in the presence of these ligands. Conclusion We provide evidence indicating the presence of functionally distinct groups of SOX2+ pituitary stem cells and reveal a critical role for a PSC subset in the development of the stem cell compartment and in driving terminal differentiation of committed progenitors. Presentation: Sunday, June 12, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Pathology of Crooke Cells in the Human Pituitaries: A Timely Review.

Crooke cell change was first found in the regressed and suppressed corticotroph (adrenocorticotropic hormone-producing) cells, and now is known to occur in pituitary tumors. The tumor cells of this type can be recognized by morphology with immunohistochemistry, and are well known to predict aggressive behavior such as invasion and rare metastases. This is one of the representative neuroendocrine tumors in the pituitary which is now considered to have malignant potential as proposed in the pancreas and gastrointestinal tracts. It is important to emphasize the pituitary tumor pathology such as Crooke cell change for prognostication and appropriate therapies. This review article describes the evolution from the Crooke cells to Crooke cell tumors which is timely along with the Fifth WHO classification 2022 published online.

Characteristic distribution and molecular properties of normal cellular prion protein in human endocrine and exocrine tissues

Prion disease is an infectious and fatal neurodegenerative disease. Human prion disease autopsy studies have revealed abnormal prion protein (PrPSc) deposits in the central nervous system and systemic organs. In deer, chronic wasting disease has also become a global problem, with PrPSc in saliva and feces. Therefore, understanding normal cellular prion proteins (PrPc) characteristics in human systemic organs is important since they could be a PrPSc source. This study used western blotting and immunohistochemistry to investigate endocrine and exocrine tissues, such as the human pituitary, adrenal, submandibular glands and the pancreas. All tissues had 30–40 kDa PrP signals, which is a slightly higher molecular weight than normal brain tissue. Most cytoplasmic PrP-positive adenohypophyseal cells were immunopositive for nuclear pituitary-specific positive transcription factor 1. The adrenal medulla and islet cells of the pancreas were PrP-positive and colocalized with chromogranin A. The duct epithelium in the submandibular gland and pancreas were immunopositive for PrP. This study reports the characteristic molecular properties and detailed tissue localization of PrPc in endocrine and exocrine tissues, which is important for infection control and diagnosis.

Aberrant Nuclear Translocation of E2F1 and Its Association in Cushing's Disease.

Nonsurgical medical treatments are often performed for Cushing's disease due to high recurrence rates. However, current medical treatment that targets corticotroph adenomas are limited. To develop a treatment that specifically targets corticotrophs in Cushing's disease, it is necessary to identify corticotroph lineage-specific proteins, which are involved in the Cushing's tumor phenotype. We have previously reported that the expression of E2F transcription factor 1 (E2F1), one of the cell cycle regulatory proteins, was increased in corticotrophs in Cushing's disease model mice and was involved in the regulation of POMC gene expression. Phosphorylation of Ser337 of E2F1 (pS337-E2F1) facilitates its binding to the POMC promoter, which was suggested to contribute to elevated POMC expression in corticotrophs. Here, we report that E2F1 expression is specific to the corticotroph lineage in normal human pituitaries and that the E2F1 protein is localized in the cytosol in normal corticotrophs. We show that pS337-E2F1 is localized in the nucleus specifically in Cushing's tumors, while it is localized in the perinuclear cytoplasm in the normal pituitary. This observation demonstrates that pS337 is a marker for Cushing's tumors and suggests that phosphorylation of E2F1 may be a target for developing a novel pharmacological treatment for tumorigenesis and hormone dysregulation of Cushing's disease.

Morphometric analysis of somatotropic and folliculostellate cells of human anterior pituitary during ageing

Introduction/Objective. In this study, we have pointed out the immunohistomorphometric characteristics of somatotropic (GH) and folliculostellate (FS) cells of the male pituitary gland during ageing. Methods. On histological sections of the pituitary gland of 14 male cadavers of different ages, the GH and FS cells were immunohistochemically labeled with corresponding antibodies, monoclonal anti-GH antibody, and polyclonal anti-S100 antibody, respectively. Immunopositive GH- and FS-cells were further morphometrically analyzed using ImageJ software. Results. The obtained results of morphometric analysis showed that the surface area of GH cells increased significantly with age. In these cells, the nuclear?cytoplasmic ratio gradually decreased and became significantly higher after the age of 70 years. The volume density of GH cells has not changed during ageing, while in FS cells this parameter significantly increased in the cases older than 70 years. The nuclear?cytoplasmic ratio of GH cells is negatively correlated with the volume density of FS cells. Conclusion. Based on the obtained results, we concluded that hypertrophy of GH and FS cells occurs in men with ageing and that correlation between the morphometric parameters of these two cell types indicates their mutual interaction.

Synthesis of potent antagonists of receptors for growth hormone-releasing hormone with antitumor and anti-inflammatory activity

The syntheses and biological evaluation of GHRH antagonists of AVR series with high anticancer and anti-inflammatory activities are described. Compared to our previously reported GHRH antagonist 602 of MIAMI series, AVR analogs contain additional modifications at positions 0, 6, 8, 10, 11, 12, 20, 21, 29 and 30, which induce greater antitumor activities. Five of nineteen tested AVR analogs presented binding affinities to the membrane GHRH receptors on human pituitary, 2-4-fold better than MIA-602. The antineoplastic properties of these analogs were evaluated in vitro using proliferation assays and in vivo in nude mice xenografted with various human cancer cell lines including lung (NSCLC-ADC HCC827 and NSCLC H460), gastric (NCI-N87), pancreatic (PANC-1 and CFPAC-1), colorectal (HT-29), breast (MX-1), glioblastoma (U87), ovarian (SK-OV-3 and OVCAR-3) and prostatic (PC3) cancers. In vitro AVR analogs showed inhibition of cell viability equal to or greater than MIA-602. After subcutaneous administration at 5 μg/day doses, some AVR antagonists demonstrated better inhibition of tumor growth in nude mice bearing various human cancers, with analog AVR-353 inducing stronger suppression than MIA-602 in lung, gastric, pancreatic and colorectal cancers and AVR-352 in ovarian cancers and glioblastoma. Both antagonists induced greater inhibition of GH release than MIA-602 in vitro in cultured rat pituitary cells and in vivo in rats. AVR-352 also demonstrated stronger anti-inflammatory effects in lung granulomas from mice with lung inflammation. Our studies demonstrate the merit of further investigation of AVR GHRH antagonists and support their potential use for clinical therapy of human cancers and other diseases.

Requirement of FAT and DCHS protocadherins during hypothalamic-pituitary development

Pituitary developmental defects lead to partial or complete hormone deficiency and significant health problems. The majority of cases are sporadic and of unknown cause. We screened 28 patients with pituitary stalk interruption syndrome (PSIS) for mutations in the FAT/DCHS family of protocadherins that have high functional redundancy. We identified seven variants, four of which putatively damaging, in FAT2 and DCHS2 in six patients with pituitary developmental defects recruited through a cohort of patients with mostly ectopic posterior pituitary gland and/or pituitary stalk interruption. All patients had growth hormone deficiency and two presented with multiple hormone deficiencies and small glands. FAT2 and DCHS2 were strongly expressed in the mesenchyme surrounding the normal developing human pituitary. We analyzed Dchs2-/- mouse mutants and identified anterior pituitary hypoplasia and partially penetrant infundibular defects. Overlapping infundibular abnormalities and distinct anterior pituitary morphogenesis defects were observed in Fat4-/- and Dchs1-/- mouse mutants but all animal models displayed normal commitment to the anterior pituitary cell type. Together our data implicate FAT/DCHS protocadherins in normal hypothalamic-pituitary development and identify FAT2 and DCHS2 as candidates underlying pituitary gland developmental defects such as ectopic pituitary gland and/or pituitary stalk interruption.

Pituitary Somatotroph Adenoma-derived Exosomes: Characterization of Nonhormonal Actions.

ContextThe identification and biological actions of pituitary-derived exosomes remain elusive.ObjectiveThis work aimed to validate production of exosomes derived from human and rat pituitary and elucidate their actions.MethodsIsolated extracellular vesicles (EVs) were analyzed by Nanoparticle Tracking Analysis (NTA) and expressed exosomal markers detected by Western blot, using nonpituitary fibroblast FR and myoblast H9C2 cells as controls. Exosome inhibitor GW4869 was employed to detect attenuated EV release. Exosomal RNA contents were characterized by RNA sequencing. In vitro and in vivo hepatocyte signaling alterations responding to GH1-derived exosomes (GH1-exo) were delineated by mRNA sequencing. GH1-exo actions on protein synthesis, cAMP (3′,5′-cyclic adenosine 5′-monophosphate) response, cell motility, and metastases were assessed.ResultsNTA, exosomal marker detection, and GW4869 attenuated EV release, confirming the exosomal identity of pituitary EVs. Hydrocortisone increased exosome secretion in GH1 and GH3 cells, suggesting a stress-associated response. Exosomal RNA contents showed profiles distinct for pituitary cells, and rat primary hepatocytes exposed to GH1-exo exhibited transcriptomic alterations distinct from those elicited by growth hormone or prolactin. Intravenous GH1-exo injection into rats attenuated hepatic Eif2ak2 and Atf4 mRNA expression, both involved in cAMP responses and amino acid biosynthesis. GH1-exo suppressed protein synthesis and forskolin-induced cAMP levels in hepatocytes. GH1-exo–treated HCT116 cells showed dysregulated p53 and mitogen-activated protein kinase (MAPK) pathways and attenuated motility of malignant HCT116 cells, and decreased tumor metastases in nude mice harboring splenic HCT116 implants.ConclusionOur findings elucidate biological actions of somatotroph-derived exosomes and implicate exosomes as nonhormonal pituitary-derived messengers.

Imaging Pituitary Vasopressin 1B Receptor in Humans with the PET Radiotracer 11C-TASP699.

Arginine vasopressin is a hormone that is synthesized mainly in the hypothalamus and stored in the posterior pituitary. Receptors for vasopressin are categorized into at least 3 subtypes (V1A, V1B, and V2). Among these subtypes, the V1B receptor (V1BR), highly expressed in the pituitary, is a primary regulator of hypothalamic-pituitary-adrenal axis activity and thus a potential target for treatment of neuropsychiatric disorders such as depression and anxiety. N-tert-butyl-2-[2-(6-methoxypyridine-2-yl)-6-[3-(morpholin-4-yl)propoxy]-4-oxopyrido[2,3-d]pyrimidin-3(4H)-yl]acetamide (TASP699) is a novel PET radiotracer with high affinity and selectivity for V1BR. The purpose of this study was to characterize the pharmacokinetic and binding profiles of 11C-TASP699 in humans and determine its utility in an occupancy study of a novel V1BR antagonist, TS-121. Methods: Six healthy subjects were scanned twice with 11C-TASP699 to determine the most appropriate kinetic model for analysis of imaging data and test-retest reproducibility of outcome measures. Nine healthy subjects were scanned before and after administration of TS-121 (active component: THY1773) to assess V1BR occupancy. Metabolite-corrected arterial input functions were obtained. Pituitary time-activity curves were analyzed with 1- and 2-tissue-compartment (1TC and 2TC, respectively) models and multilinear analysis 1 (MA1) to calculate distribution volume (VT). Relative test-retest variability (TRV) and absolute TRV were calculated. Since no brain region could be used as a reference region, percentage change in VT after TS-121 administration was computed to assess its receptor occupancy and correlate with plasma concentrations of the drug. Results:11C-TASP699 showed high uptake in the pituitary and no uptake in any brain region. The 2TC model provided better fits than the 1TC model. Because the MA1 VT estimates were similar to the 2TC VT estimates, MA1 was the model of choice. The TRV of VT was good (TRV, -2% ± 14%; absolute TRV, 11%). THY1773 reduced VT in a dose-dependent fashion, with a half-maximal inhibitory concentration of 177 ± 52 ng/mL in plasma concentration. There were no adverse events resulting in discontinuation from the study. Conclusion:11C-TASP699 was shown to display appropriate kinetics in humans, with substantial specific binding and good reproducibility of VT Therefore, this tracer is suitable for measurement of V1BR in the human pituitary and the V1BR occupancy of TS-121, a novel V1BR antagonist.

Checkpoint Inhibitor Pneumonitis Induced by Programmed Death-1 Antibody: A Case Report and Literature Review

Immune Checkpoint Inhibitors (ICIs) are effective treatment therapies for majority advanced tumours. However, the immune-related Adverse Events (irAEs) triggered by ICIs may affect human organs, including skin, lungs, pituitary, thyroid, blood and digestive system, leading to immunotoxicity in these organs. Checkpoint Inhibitor Pneumonitis (CIP) is one of the irAEs that could cause mortality, thereby needs special attention from the clinical physicians. In the present manuscript, the CIP occurred in a unilateral lung in one patient case with advanced oesophageal cancer treated with anti-PD-1 was analysed. Furthermore, a literature review was conducted to investigate its pathogenesis, clinical features, associated risk factors, prevention and the correlation with ICI efficacy, providing valuable information for clinical physicians.

Single Nucleus Transcriptome and Chromatin Accessibility Landscapes of Human Pituitaries

The pituitary gland regulates key physiological functions, including growth, sexual maturation, reproduction, and lactation. Here, we present a paired single-nuclei (sn) transcriptome and chromatin accessibility characterization of six post-mortem human pituitaries. These samples were from juvenile, adult, and elderly male and female subjects. Well-correlated snRNAseq and snATACseq datasets facilitated robust identification of the major pituitary cell types in each sample. Using latent variable pathway analysis, we uncovered previously unreported coordinated gene expression modules and chromatin accessibility programs for each major cell type as well as an age-specific program across all the endocrine cell types. These largely appear to be congruent between human and mouse datasets. Given the importance of murine models in the study of human pituitary disorders and pituitary physiology, we next sought to compare expression profiles of pituitary cell types in mouse vs. human. Murine and human cell types were well correlated, exemplified by coordinated gene expression programs, especially for undifferentiated stem cells (SCs). In both species, we identified clusters corresponding to naive and committing SCs. All human SC clusters expressed the established SC markers SOX2 and SOX9, as well as genes involved in SC regulatory pathways (WWTR1, YAP1 andPITX2). Additional markers previously reported in murine pituitary SCs were also found in human SC, including WIF1, LGR5, FOS, CDH1, EGFR, LGR4, and WLS. Remarkably, in human, the main naive SC cluster was roughly divided into a high-JUN and a low-JUN expressing subgroup, whereas Jun expression was less pronounced in the murine SC cluster. In both species, committing SC clusters expressed the endocrine markers for POU1F1, TSHB, or POMC, while SCs committing to an intermediate lobe/melanotrope cell identity were distinguishable based on PAX7 expression. In addition, in the human datasets we identify a population of cells as originating from the pars tuberalis. We offer a range of markers that can be utilized for in vivo validation of these cells. Overall, the characterization of the murine and human pituitary SCs strongly suggests the co-existence of subpopulations with different lineage commitments in addition to a single uncommitted SC population. This sn atlas of the human pituitary is a valuable resource that will be made web-accessible.

Paracrine Signalling From SOX2-Expressing Pituitary Embryonic Cells Is Required for Terminal Differentiation of Hormone-Producing Cells

The pituitary gland is the master regulator of the endocrine system, housing six major hormone producing cell types. This gland is derived from Rathke’s Pouch, an invagination of the oral ectoderm. Hormone-producing pituitary cell lineages are derived from a population of embryonic cells expressing SOX2. ZFP36L1/Butyrate Response Factor 1 (BRF1) is an RNA binding protein that binds and targets mRNAs of various cytokines and chemokines for degradation prior to translation, attenuating secretion of inflammatory factors (Herranz et al. 2015). Here, we show that BRF1 is a novel marker expressed in SOX2+ cells in human and mouse pituitaries, suggesting that these cells may have a secretory profile. To investigate this possibility, we have combined molecular and genetic studies in vivo. We have used a novel mouse model, R26lsl-mBRF1 that allows the expression of a mutant, constitutively active BRF1 protein upon Cre-mediated recombination, alongside our lab’s models (Hesx1Cre/+ and Sox2CreERT2/+), to express mutant BRF1 in HESX1+ and SOX2+ cells during development and postnatally. This approach results in pituitary hypoplasia and severe hypopituitarism due to a failure of cell-lineage specified cells to differentiate into hormone-producing cells. Hormone production in these mutant cells, however, can be rescued in vitro through co-culture with WT pituitaries and in vivo in chimeric pituitaries, highlighting a cell non-autonomous mechanism underlying the phenotype. Single cell RNA sequencing of WT and Sox2CreERT2/+;R26lsl-mBRF1 murine embryonic pituitaries, as well as use publicly available human pituitary single cell datasets, have allowed us to identify specific cytokines and chemokines secreted by SOX2+ cells, as well as downstream intracellular signalling pathways in differentiating cells (Zhang et al. 2020), which may be responsible for controlling terminal differentiation of hormone-producing cells within the developing pituitary. Together with our recently published data, these results support the notion that SOX2+ pituitary stem cells play a critical paracrine role in controlling progenitor cell proliferation and terminal differentiation (Russell et al. 2021).

Natriuretic Peptide Expression and Function in GH3 Somatolactotropes and Feline Somatotrope Pituitary Tumours.

Patients harbouring mutations in genes encoding C-type natriuretic peptide (CNP; NPPC) or its receptor guanylyl cyclase B (GC-B, NPR2) suffer from severe growth phenotypes; loss-of-function mutations cause achondroplasia, whereas gain-of-function mutations cause skeletal overgrowth. Although most of the effects of CNP/GC-B on growth are mediated directly on bone, evidence suggests the natriuretic peptides may also affect anterior pituitary control of growth. Our previous studies described the expression of NPPC and NPR2 in a range of human pituitary tumours, normal human pituitary, and normal fetal human pituitary. However, the natriuretic peptide system in somatotropes has not been extensively explored. Here, we examine the expression and function of the CNP/GC-B system in rat GH3 somatolactotrope cell line and pituitary tumours from a cohort of feline hypersomatotropism (HST; acromegaly) patients. Using multiplex RT-qPCR, all three natriuretic peptides and their receptors were detected in GH3 cells. The expression of Nppc was significantly enhanced following treatment with either 100 nM TRH or 10 µM forskolin, yet only Npr1 expression was sensitive to forskolin stimulation; the effects of forskolin and TRH on Nppc expression were PKA- and MAPK-dependent, respectively. CNP stimulation of GH3 somatolactotropes significantly inhibited Esr1, Insr and Lepr expression, but dramatically enhanced cFos expression at the same time point. Oestrogen treatment significantly enhanced expression of Nppa, Nppc, Npr1, and Npr2 in GH3 somatolactotropes, but inhibited CNP-stimulated cGMP accumulation. Finally, transcripts for all three natriuretic peptides and receptors were expressed in feline pituitary tumours from patients with HST. NPPC expression was negatively correlated with pituitary tumour volume and SSTR5 expression, but positively correlated with D2R and GHR expression. Collectively, these data provide mechanisms that control expression and function of CNP in somatolactotrope cells, and identify putative transcriptional targets for CNP action in somatotropes.

Chapter 10 - Congenital isolated central hypothyroidism: Novel mutations and their functional implications

Congenital hypothyroidism is the most frequent endocrine disorder in newborns, occurring in 1 per 3000-4000 newborns. In the Netherlands, the neonatal screening program is based primarily on heel prick thyroxine (T4). In contrast to thyroid-stimulating hormone-based programs, this approach allows for the detection of both primary and central congenital hypothyroidism. Over the past decade, the identification of families with isolated congenital central hypothyroidism enabled the identification of novel genetic causes of this condition, in addition to mutations in the TSHβ-subunit gene and thyrotropin-releasing hormone receptor gene reported earlier. In 2012, loss-of-function mutations in the immunoglobulin superfamily, member 1 (IGSF1) gene, were reported as a genetic cause of a syndrome including X-linked congenital central hypothyroidism and adult macroorchidism. IGSF1 encodes a hypothalamic plasma membrane glycoprotein. Mutations in IGSF1 represent the most prevalent genetic cause of isolated central hypothyroidism to date. In 2016, mutations in the transducin β-like 1X (TBL1X) gene were identified in patients with a combination of mild central hypothyroidism and sensorineural hearing loss. TBL1X is an essential subunit of the NCoR/SMRT corepressor complex and expressed in many tissues including the human hypothalamus and pituitary. In 2018, mutations in the insulin receptor substrate 4 (IRS4) gene were reported in cases of familial isolated central hypothyroidism. IRS4 encodes a hypothalamic protein that is part of the insulin and leptin signaling cascade. These recent developments will broaden our understanding of the role of the hypothalamus in hypothalamus-pituitary-thyroid axis regulation and will help to improve diagnosis and treatment of isolated central hypothyroidism.

Requirement of FAT and DCHS protocadherins during hypothalamic-pituitary development.

Pituitary developmental defects lead to partial or complete hormone deficiency and significant health problems. The majority of cases are sporadic and of unknown cause. We screened 28 patients with pituitary stalk interruption syndrome for mutations in the FAT/DCHS family of protocadherins that have high functional redundancy. We identified 7 variants, 4 of which are putatively damaging, in FAT2 and DCHS2 in 6 patients with pituitary developmental defects recruited through a cohort of patients with mostly ectopic posterior pituitary gland and/or pituitary stalk interruption. All patients had growth hormone deficiency, and 2 presented with multiple hormone deficiencies and small glands. FAT2 and DCHS2 were strongly expressed in the mesenchyme surrounding the normal developing human pituitary. We analyzed Dchs2–/– mouse mutants and identified anterior pituitary hypoplasia and partially penetrant infundibular defects. Overlapping infundibular abnormalities and distinct anterior pituitary morphogenesis defects were observed in Fat4–/– and Dchs1–/– mouse mutants, but all animal models displayed normal commitment to anterior pituitary cell types. Together our data implicate FAT/DCHS protocadherins in normal hypothalamic-pituitary development and identify FAT2 and DCHS2 as candidates underlying pituitary gland developmental defects such as ectopic pituitary gland and/or pituitary stalk interruption.