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Congenital hypopituitarism is a genetically heterogeneous condition that is part of a spectrum disorder that can include holoprosencephaly. Heterozygous mutations in SIX3 cause variable holoprosencephaly in humans and mice. We identified two children with neonatal hypopituitarism and thin pituitary stalk who were doubly heterozygous for rare, likely deleterious variants in the transcription factors SIX3 and POU1F1. We used genetically engineered mice to understand the disease pathophysiology. Pou1f1 loss-of-function heterozygotes are unaffected; Six3 heterozygotes have pituitary gland dysmorphology and incompletely ossified palate; and the Six3+/−; Pou1f1+/dw double heterozygote mice have a pronounced phenotype, including pituitary growth through the palate. The interaction of Pou1f1 and Six3 in mice supports the possibility of digenic pituitary disease in children. Disruption of Six3 expression in the oral ectoderm completely ablated anterior pituitary development, and deletion of Six3 in the neural ectoderm blocked the development of the pituitary stalk and both anterior and posterior pituitary lobes. Six3 is required in both oral and neural ectodermal tissues for the activation of signaling pathways and transcription factors necessary for pituitary cell fate. These studies clarify the mechanism of SIX3 action in pituitary development and provide support for a digenic basis for hypopituitarism.

The pituitary gland regulates growth, metabolism, reproduction, the stress response, uterine contractions, lactation, and water retention. It secretes hormones in response to hypothalamic input, end organ feedback, and diurnal cues. The mechanisms by which pituitary stem cells are recruited to proliferate, maintain quiescence or differentiate into specific cell types, especially thyrotropes, are not well understood. We utilized single-cell RNA sequencing in juvenile P7 mouse pituitary cells to identify novel factors in pituitary cell populations, with a focus on thyrotropes and rare subtypes. We first observed cells co-expressing markers of both thyrotropes and gonadotropes, such as Pou1f1 and Nr5a1. This was validated in vivo by both immunohistochemistry and lineage tracing of thyrotropes derived from Nr5a1-Cre; mTmG mice and demonstrates that Nr5a1-progenitors give rise to a proportion of thyrotropes during development. Our dataset also identifies novel factors expressed in pars distalis and pars tuberalis thyrotropes, including the Shox2b isoform in all thyrotropes and Sox14 specifically in Pou1f1-negative pars tuberalis thyrotropes. We have therefore used single-cell transcriptomics to determine a novel developmental trajectory for thyrotropes and potential novel regulators of thyrotrope populations.

Epithelial to mesenchymal transition (EMT) is important for many developing organs, and for wound healing, fibrosis, and cancer. Pituitary stem cells undergo an EMT-like process as they migrate and initiate differentiation, but little is known about the input of signaling pathways or the genetic hierarchy of the transcriptional cascade. Prop1 mutant stem cells fail to undergo changes in cellular morphology, migration, and transition to the Pou1f1 lineage. We used Prop1 mutant mice to identify the changes in gene expression that are affiliated with EMT-like processes. BMP and TGF-β family gene expression was reduced in Prop1 mutants and Elf5, a transcription factor that characteristically suppresses EMT, had elevated expression. Genes involved in cell-cell contact such as cadherins and claudins were elevated in Prop1 mutants. To establish the genetic hierarchy of control, we manipulated gene expression in pituitary stem cell colonies. We determined that the EMT inducer, Zeb2, is necessary for robust BMP signaling and repression of Elf5. We demonstrated that inhibition of BMP signaling affects expression of target genes in the Id family, but it does not affect expression of other EMT genes. Zeb2 is necessary for expression of the SHH effector gene Gli2. However, knock down of Gli2 has little effect on the EMT-related genes, suggesting that it acts through a separate pathway. Thus, we have established the genetic hierarchy involved in the transition of pituitary stem cells to differentiation.

Combined pituitary hormone deficiency (CPHD) is not a rare disorder, with a frequency of approximately 1 case per 4,000 live births. However, in most cases, a genetic diagnosis is not available. Furthermore, the diagnosis is challenging because no clear correlation exists between the pituitary hormones affected and the gene(s) responsible for the disorder. Next-generation sequencing (NGS) has recently been widely used to identify novel genes that cause (or putatively cause) CPHD. This review outlines causative genes for CPHD that have been newly reported in recent years. Moreover, novel variants of known CPHD-related genes (POU1F1 and GH1 genes) that contribute to CPHD through unique mechanisms are also discussed in this review. From a clinical perspective, variants in some of the recently identified causative genes result in extra-pituitary phenotypes. Clinical research on the related symptoms and basic research on pituitary formation may help in inferring the causative gene(s) of CPHD. Future NGS analysis of a large number of CPHD cases may reveal new genes related to pituitary development. Clarifying the causative genes of CPHD may help to understand the process of pituitary development. We hope that future innovations will lead to the identification of genes responsible for CPHD and pituitary development.

Objective: to describe the marked variability in clinical and biochemical patterns that are associated with a p.R209H GH1 missense variant in a large Argentinean pedigree, which makes the diagnosis of GHD elusive. Design: We describe a non-consanguineous pedigree composed by several individuals with short stature, including 2 pediatric patients with typical diagnosis of isolated growth hormone deficiency (IGHD) and 4 other siblings with severe short stature, low serum IGF-1 and IGFBP-3, but normal stimulated GH levels, suggesting growth hormone insensitivity (GHI) in the latter group. Results: Patients with classical IGHD phenotype carried a heterozygous variant in GH1: c.626G>A (p.R209H). Data from the extended pedigree suggested GH1 as the initial candidate gene, which showed the same pathogenic heterozygous GH1 variant in the four siblings with short stature and a biochemical pattern of GHI. Conclusions: We suggest considering GH1 sequencing in children with short stature associated to low IGF-1 and IGFBP-3 serum levels, even in the context of normal response to growth hormone provocative testing (GHPT). Fil: Sanguineti, Nora María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Braslavsky, Debora Giselle. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Scaglia, Paula Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Keselman, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Ballerini, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Ropelato, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Suco, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Vishnopolska, Sebastián Alexis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Berenstein, Ariel José. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina Fil: Jasper, Hector Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Domene, Horacio Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Rey, Rodolfo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Pérez Millán, Maria I.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Camper, Sally. University Of Michigan. College Of Literature, Sciences And Arts. Molecular, Cellular And Developmental Biology; Estados Unidos Fil: Bergadá, Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina

Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans., Mutations in components of the MAP kinase pathway are associated with a group of syndromes known as RASopathies. Here, the authors identify gain-of-function mutations in BRAF in patients with RASopathies and congenital hypopituitarisms. This article demonstrates a central role for BRAF in the development of the hypothalamo-pituitary axis leading to endocrine deficiencies in patients with RASopathies.

Recent studies have shown a novel mechanism of combined pituitary hormone deficiency associated with mutations in POU1F1, altering the balance of alternative-splicing, which results in over-expression of the beta isoform of POU1F1. These studies underscore the need for biologists, in the context of routine molecular diagnosis of this condition, to investigate alternative splicing in POU1F1 as well as in other genes.

We report four allelic variants (three novel) in three genes previously established as causal for hypopituitarism or related disorders. A novel homozygous variant in the growth hormone gene, GH1 c.171delT (p.Phe 57Leufs*43), was found in a male patient with severe isolated growth hormone deficiency (IGHD) born to consanguineous parents. A hemizygous SOX3 allelic variant (p.Met304Ile) was found in a male patient with IGHD and hypoplastic anterior pituitary. YASARA, a tool to evaluate protein stability, suggests that p.Met304Ile destabilizes the SOX3 protein (ΔΔG = 2.49 kcal/mol). A rare, heterozygous missense variant in the TALE homeobox protein gene, TGIF1 (c.268C&gt, T:p.Arg90Cys) was found in a patient with combined pituitary hormone deficiency (CPHD), diabetes insipidus, and syndromic features of holoprosencephaly (HPE). This variant was previously reported in a patient with severe holoprosencephaly and shown to affect TGIF1 function. A novel heterozygous TGIF1 variant (c.82T&gt, C:p.Ser28Pro) was identified in a patient with CPHD, pituitary aplasia and ectopic posterior lobe. Both TGIF1 variants have an autosomal dominant pattern of inheritance with incomplete penetrance. In conclusion, we have found allelic variants in three genes in hypopituitarism patients. We discuss these variants and associated patient phenotypes in relation to previously reported variants in these genes, expanding our knowledge of the phenotypic spectrum in patient populations.

International audience; Pituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We found four separate heterozygous missense variants in unrelated individuals with hypopituitarism that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant-negative loss of function. Using a high-throughput splicing reporter assay, we tested 1,070 single-nucleotide variants in POU1F1. We identified 96 splice-disruptive variants, including 14 synonymous variants. In separate cohorts, we found two additional synonymous variants nominated by this screen that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in POU1F1.

Background: The heterotrimeric GTP-binding protein eIF2 forms a ternary complex with initiator methionyl-tRNA and recruits it to the 40S ribosomal subunit for start codon selection and thereby initiates protein synthesis. Mutations in EIF2S3, encoding the eIF2γ subunit, are associated with severe intellectual disability and microcephaly, usually as part of MEHMO syndrome. Methods: Exome sequencing of the X chromosome was performed on three related males with normal head circumferences and mild learning difficulties, hypopituitarism (GH and TSH deficiencies), and an unusual form of glucose dysregulation. In situ hybridisation on human embryonic tissue, EIF2S3-knockdown studies in a human pancreatic cell line, and yeast assays on the mutated corresponding eIF2γ protein, were performed in this study. Findings: We report a novel hemizygous EIF2S3 variant, p.Pro432Ser, in the three boys (heterozygous in their mothers). EIF2S3 expression was detectable in the developing pituitary gland and pancreatic islets of Langerhans. Cells lacking EIF2S3 had increased caspase activity/cell death. Impaired protein synthesis and relaxed start codon selection stringency was observed in mutated yeast. Interpretation: Our data suggest that the p.Pro432Ser mutation impairs eIF2γ function leading to a relatively mild novel phenotype compared with previous EIF2S3 mutations. Our studies support a critical role for EIF2S3 in human hypothalamo-pituitary development and function, and glucose regulation, expanding the range of phenotypes associated with EIF2S3 mutations beyond classical MEHMO syndrome. Untreated hypoglycaemia in previous cases may have contributed to their more severe neurological impairment and seizures in association with impaired EIF2S3. Fund: GOSH, MRF, BRC, MRC/Wellcome Trust and NIGMS funded this study. Keywords: Hypopituitarism, Hypoglycaemia, Glucose dysregulation, Translation initiation, Protein synthesis, EIF2S3

Maternal and fetal sources of thyroid hormone are important for the development of many organ systems. Thyroid hormone deficiency causes variable intellectual disability and hearing impairment in mouse and man, but the basis for this variation is not clear. To explore this variation, we studied two thyroid hormone-deficient mouse mutants with mutations in pituitary-specific transcription factors, POU1F1 and PROP1, that render them unable to produce thyroid stimulating hormone. DW/J-Pou1f1dw/dw mice have profound deafness and both neurosensory and conductive hearing impairment, while DF/B-Prop1df/df mice have modest elevations in hearing thresholds consistent with developmental delay, eventually achieving normal hearing ability. The thyroid glands of Pou1f1 mutants are more severely affected than those of Prop1df/df mice, and they produce less thyroglobulin during the neonatal period critical for establishing hearing. We previously crossed DW/J-Pou1f1dw/+ and Cast/Ei mice and mapped a major locus on Chromosome 2 that protects against hypothyroidism-induced hearing impairment in Pou1f1dw/dw mice: modifier of dw hearing (Mdwh). Here we refine the location of Mdwh by genotyping 196 animals with 876 informative SNPs, and we conduct novel mapping with a DW/J-Pou1f1dw/+ and 129/P2 cross that reveals 129/P2 mice also have a protective Mdwh locus. Using DNA sequencing of DW/J and DF/B strains, we determined that the genes important for thyroid gland function within Mdwh vary in amino acid sequence between strains that are susceptible or resistant to hypothyroidism-induced hearing impairment. These results suggest that the variable effects of congenital hypothyroidism on the development of hearing ability are attributable to genetic variation in postnatal thyroid gland folliculogenesis and function.

We report four allelic variants (3 novel) in three genes previously established as causal for hypopituitarism or related disorders. A novel homozygous variant in the growth hormone gene, GH1 c.171delT (p. Phe 57Leufs * 43), was found in a male patient with severe isolated growth hormone deficiency (IGHD) born to consanguineous parents. A SOX3 allelic variant (p.Met304Ile) was found in a male patient with IGHD and hypoplastic anterior pituitary. YASARA, a tool to evaluate protein stability, suggests that p.Met304Ile destabilizes the SOX3 protein (ΔΔG = 2.49 kcal/mol). A rare, heterozygous missense variant in the TALE homeobox protein gene, TGIF1 (c.268C>T:p.Arg90Cys) was found in a patient with combined pituitary hormone deficiency (CPHD), diabetes insipidus, and syndromic features of holoprosencephaly (HPE). A novel heterozygous TGIF1 variant (c.82T>C:p.Ser28Pro) was identified in a patient with CPHD, pituitary aplasia and ectopic posterior lobe. Both TGIF1 variants have an autosomal dominant pattern of inheritance with incomplete penetrance. In conclusion, we have found allelic variants in 3 genes in hypopituitarism patients. We discuss these variants and associated patient phenotypes in relation to previously reported variants in these genes, expanding our knowledge of the phenotypic spectrum in patient populations.

BackgroundThe pituitary gland is a neuroendocrine organ containing diverse cell types specialized in secreting hormones that regulate physiology. Pituitary thyrotropes produce thyroid-stimulating hormone (TSH), a critical factor for growth and maintenance of metabolism. The transcription factors POU1F1 and GATA2 have been implicated in thyrotrope fate, but the transcriptomic and epigenomic landscapes of these neuroendocrine cells have not been characterized. The goal of this work was to discover transcriptional regulatory elements that drive thyrotrope fate.ResultsWe identified the transcription factors and epigenomic changes in chromatin that are associated with differentiation of POU1F1-expressing progenitors into thyrotropes using cell lines that represent an undifferentiatedPou1f1lineage progenitor (GHF-T1) and a committed thyrotrope line that produces TSH (TαT1). We compared RNA-seq, ATAC-seq, histone modification (H3K27Ac, H3K4Me1, and H3K27Me3), and POU1F1 binding in these cell lines. POU1F1 binding sites are commonly associated with bZIP transcription factor consensus binding sites in GHF-T1 cells and Helix-Turn-Helix (HTH) or basic Helix-Loop-Helix (bHLH) factors in TαT1 cells, suggesting that these classes of transcription factors may recruit or cooperate with POU1F1 binding at unique sites. We validated enhancer function of novel elements we mapped nearCga, Pitx1, Gata2,andTshbby transfection in TαT1 cells. Finally, we confirmed that an enhancer element nearTshbcan drive expression in thyrotropes of transgenic mice, and we demonstrate that GATA2 enhancesTshbexpression through this element.ConclusionThese results extend the ENCODE multi-omic profiling approach to the pituitary gland, which should be valuable for understanding pituitary development and disease pathogenesis.Graphical abstract

Targeted oncogenesis is the process of driving tumor formation by engineering transgenic mice that express an oncogene under the control of a cell-type specific promoter. Such tumors can be adapted to cell culture, providing immortalized cell lines. To make it feasible to follow the process of tumorigenesis and increase the opportunity for generating cell lines, we developed a mouse strain that expresses SV40 T antigens in response to Cre-recombinase. Using CRISPR/Cas9 we inserted a cassette with coding sequences for SV40 T antigens and an internal ribosome entry site with green fluorescent protein cassette (IRES-GFP) into the Rosa26 locus, downstream from a stop sequence flanked by loxP sites: Rosa26LSL-SV40-GFP. These mice were mated with previously established Prop1-cre and Tshb-cre transgenic lines. Both the Rosa26LSL-SV40-GFP/+; Prop1-cre and Rosa26LSL-SV40-GFP/+; Tshb-cre mice developed fully penetrant dwarfism and large tumors by 4 weeks. Tumors from both of these mouse lines were adapted to growth in cell culture. We have established a progenitor-like cell line (PIT-P1) that expresses Sox2 and Pitx1, and a thyrotrope-like cell line (PIT-T1) that expresses Pou1f1 and Cga. These studies demonstrate the utility of the novel, Rosa26 LSL-SV40-GFP mouse line for reliable targeted oncogenesis and development of unique cell lines.

Whole Exome Sequencing (WES) has been a useful tool to improve molecular diagnosis in hypopituitarism, leading to the discovery of at least 8 new genes in the last 7 years. However, some genes associated with hypopituitarism show low coverage in this methodology, limiting its use for molecular diagnosis. Our objective is to compare three library prepping kits, NimbleGen (Roche), SureSelect (Agilent) and Nextera (Illumina) examining the best performance related to sequencing quality, exon extension coverage (≥98%) and base depth read (≥20x) of 44 genes associated with hypopituitarism and 32 involved in pituitary development. Three different groups composed of 2 HapMap samples (Group 1), 2 Brazilian patients with hypopituitarism and their respective mothers (Group 2) and 109 random Brazilian samples (Group 3) were sequenced in Illumina platform. Group 1 and 3 were performed using all three library prepping kits, while group 2 was performed with NimbleGen and SureSelect. Although all technologies covered the selected genes with similar efficiency regarding poor (less than 20%) and rich (more than 80%) GC areas, SureSelect has shown to reach the most uniform coverage in the selected region with a lower level of duplicate reads, as well as a higher number of identified pathogenic variants.

Pituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We found four separate heterozygous missense variants in unrelated hypopituitarism patients that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant negative loss of function. Using a high throughput splicing reporter assay, we tested 1,080 single nucleotide variants inPOU1F1. We identified 113 splice disruptive variants, including 23 synonymous variants. We evaluated separate cohorts of hypopituitarism patients and found two different synonymous splice disruptive variants that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in thePOU1F1gene.

The pituitary gland is an organ with a low rate of cell turnover. While differentiated hormone-producing cells can re-enter the cell cycle, most of these cells are not dividing. There is some evidence that the pituitary can undergo limited regeneration after tissue injury. However, the regeneration is generally insufficient to meet physiologic demand, such that hormone replacement therapy is still needed. There is great interest in uncovering the mechanisms whereby specific hormone-producing cells are generated and to understand the similarities and differences for each cell type. Our understanding of anterior pituitary stem cells and committed progenitor cells has increased in the last several years. In this review, we update our previous version and outline the recent advances in pituitary stem cell biology. We lay the foundation with some background on pituitary development, cite some of the foundation studies on pituitary progenitors, and discuss pituitary stem cells in relation to the disease states of hypopituitarism and pituitary adenoma. We discuss areas of important, ongoing investigation, and future challenges.

Objective The transcription factor OTX2is implicated in ocular, craniofacial, and pituitary development. Design We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action. Methods We screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants. Results Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary. Conclusions OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.

Purpose Congenital hypopituitarism (CH) can present in isolation or with other birth defects. Mutations in multiple genes can cause CH, and the use of a genetic screening panel could establish the prevalence of mutations in known and candidate genes for this disorder. It could also increase the proportion of patients that receive a genetic diagnosis. Methods We conducted target panel genetic screening using single-molecule molecular inversion probes sequencing to assess the frequency of mutations in known hypopituitarism genes and new candidates in Argentina. We captured genomic deoxyribonucleic acid from 170 pediatric patients with CH, either alone or with other abnormalities. We performed promoter activation assays to test the functional effects of patient variants in LHX3 and LHX4. Results We found variants classified as pathogenic, likely pathogenic, or with uncertain significance in 15.3% of cases. These variants were identified in known CH causative genes (LHX3, LHX4, GLI2, OTX2, HESX1), in less frequently reported genes (FOXA2, BMP4, FGFR1, PROKR2, PNPLA6) and in new candidate genes (BMP2, HMGA2, HNF1A, NKX2-1). Conclusion In this work, we report the prevalence of mutations in known CH genes in Argentina and provide evidence for new candidate genes. We show that CH is a genetically heterogeneous disease with high phenotypic variation and incomplete penetrance, and our results support the need for further gene discovery for CH. Identifying population-specific pathogenic variants will improve the capacity of genetic data to predict eventual clinical outcomes.

Pituitary thyrotropes are specialized cells that produce thyroid stimulating hormone (TSH), a critical factor for growth and maintenance of metabolism. The transcription factors POU1F1 and GATA2 have been implicated in thyrotrope fate and transcriptional regulation of the beta subunit of TSH,Tshb, but no transcriptomic or epigenomic analyses of these cells has been undertaken. The goal of this work was to discover key transcriptional regulatory elements that drive thyrotrope fate. We identified the transcription factors and epigenomic changes in chromatin that are associated with differentiation of POU1F1-expressing progenitors into thyrotropes, a process modeled by two cell lines: one that represents an early, undifferentiatedPou1f1lineage progenitor (GHF-T1) and one that is a committed thyrotrope that produces TSH (TαT1). We generated and compared RNA-seq, ATAC-seq, histone modification (including H3K27Ac, H3K4Me1, and H3K27Me3), and transcription factor (POU1F1) binding in these two cell lines to identify regulatory elements and candidate transcriptional regulators. We identified POU1F1 binding sites that were unique to each cell line. POU1F1 binding sites are commonly associated with bZIP transcription factor consensus binding sites in GHF-T1 cells and Helix-Turn-Helix (HTH) or basic Helix-Loop-Helix (bHLH) factors in TαT1 cells, suggesting that these classes of transcription factors may recruit or cooperate with POU1F1 binding to unique sites. We validated enhancer function of novel elements we mapped nearCga, Pitx1, Gata2,andTshbby transfection in TαT1 cells. Finally, we confirmed that an enhancer element nearTshbcan drive expression in thyrotropes of transgenic mice, and we demonstrate that GATA2 enhancesTshbexpression through this element. These results extend the ENCODE multi-omic profiling approach to an organ that is critical for growth and metabolism, which should be valuable for understanding pituitary development and disease pathogenesis.

The transcription factor ISL1 is expressed in pituitary gland stem cells and the thyrotrope and gonadotrope lineages. Pituitary-specific Isl1 deletion causes hypopituitarism with increased stem cell apoptosis, reduced differentiation of thyrotropes and gonadotropes, and reduced body size. Conditional Isl1 deletion causes development of multiple Rathke’s cleft-like cysts, with 100% penetrance. Foxa1 and Foxj1 are abnormally expressed in the pituitary gland and associated with a ciliogenic gene-expression program in the cysts. We confirmed expression of FOXA1, FOXJ1, and stem cell markers in human Rathke’s cleft cyst tissue, but not craniopharyngiomas, which suggests these transcription factors are useful, pathological markers for diagnosis of Rathke’s cleft cysts. These studies support a model whereby expression of ISL1 in pituitary progenitors drives differentiation into thyrotropes and gonadotropes and without it, activation of FOXA1 and FOXJ1 permits development of an oral epithelial cell fate with mucinous cysts. This pituitary-specific Isl1 mouse knockout sheds light on the etiology of Rathke’s cleft cysts and the role of ISL1 in normal pituitary development. Fil: Brinkmeier, Michelle L.. University of Michigan; Estados Unidos Fil: Bando, Hironori. University of Michigan; Estados Unidos Fil: Camarano, Adriana Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Fujio, Shingo. Kagoshima University; Japón Fil: Yoshimoto, Koji. Kagoshima University; Japón Fil: Silva Junqueira de Souza, Flávio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Camper, Sally. University of Michigan; Estados Unidos

The Pit-1/GHF-1 (Pit-1) transcription factor is important for the development of anterior pituitary cells that produce GH and PRL. We examined the expression of Pit-1 mRNA in pituitary tissues from rats and mice. Analysis of pituitaries from normal and GHRH transgenic mice showed that Pit-1 transcripts were readily detected in normal, hyperplastic, and neoplastic pituitaries. A cell line (GHRH-CL1) established from a GhRH transgenic mouse pituitary tumor in our laboratory also expressed Pit-1 mRNA. Normal rat pituitaries and those with estrogen-induced PRL cell hyperplasia expressed Pit-1 mRNA. There was a decrease in Pit-1 mRNA in hyperplastic rat pituitaries concomitant with a decrease In GH mRNA amounts and an increase in PRL mRNA amounts after estrogen treatment. Similarly, analysis of GH3 cells in vitro showed that estrogen and bFGF modulated PRL but not Pit-1 mRNA levels. Pit-1 mRNA was localized by combined in situ hybridization and immunohistochemistry to predominantly GH and PRL cells, although some TSH and LH cells in the rat pituitary also expressed Pit-1 mRNA, indicating wide distribution of the mRNA for this transcription factor in various anterior pituitary cell types. Analysis of cell proliferation in normal rat pituitary and GH3 cells revealed that estrogen and bFGF stimulated cell proliferation in normal pituitaries but inhibited proliferation in GH3 cells, whereas Pit-1 transcripts remained unchanged in both groups of cultured cells. These results indicate that Pit-1 mRNA is readily detected in normal, hyperplastic, and neoplastic rodent pituitaries. Changes in Pit-1 mRNA amounts appear to correlate more closely with changes in GH than PRL mRNA levels in cultured pituitary cells.Endocr Pathol 4:146–154, 1993.

Combined pituitary hormone deficiency (CPHD) is an important clinical problem caused by mutations in more than 30 different genes. Six genes in the Sonic Hedgehog (SHH) signalling pathway are reported to cause CPHD. SHH signaling is essential to induce pituitary cell identity in cells of Rathke’s pouch by stimulating expression of the transcription factors Lhx3 and Lhx4. In the absence of SHH signaling, a repressive isoform of the transcription factor GLI2 (Gli-Kruppel family member 2) suppresses gene expression. In the presence of SHH signaling, the activating form of GLI2 gains access to the nucleus and induces expression of downstream target genes. Heterozygous GLI2 loss of function mutations are found in patients with holoprosencephaly (HPE), HPE-like phenotypes associated with pituitary anomalies, and combined pituitary hormone deficiency with or without other extra-pituitary findings. We sought to identify the cause of CPHD in 171 unrelated patients diagnosed with or without extra-pituitary manifestations that were recruited from several Argentinean medical centers. We conducted panel sequencing, and identified GLI2 heterozygous variants that were rare and predicted to be deleterious in two unrelated patients, (p.L761P and p.1404Lfs) and a single, heterozygous, rare, likely deleterious GLI2 variant identified by exome sequencing (p.A203T). p.L761P and p.A203T variants were previously reported as candidates for HPE/CPHD, no functional studies were carried out to determine the effect of the variants on the gene function. We performed functional analysis of these variants using a mammalian cell line (NIH/3T3-CG) previously engineered to be a sensor for SHH signaling. It was stably transfected with a reporter gene that expresses GFP in response to GLI2 activation by a SHH agonist. We modified this cell line to assay GLI2 variants. We created a homozygous knock out of both endogenous Gli2 genes using CRISPR-Cas9 editing, and individual cell clones were selected for loss of GFP expression in response to SHH agonist treatment by FACS. We verified that transfecting the knockout cells with wild type Gli2 restored SHH responsive GFP expression. We assayed the ability of three patient GLI2 variants to rescue GFP expression and SHH agonist responsiveness and found that all three failed to fully rescue to wild type levels. This supports the hypothesis that the GLI2 variants in three CPHD patients are likely pathogenic. Thus, we identified three likely pathogenic GLI2 mutations in CPHD patients from Argentina. The variable phenotype of patients with GLI2 mutations worldwide could be caused by variation in other genes, environmental exposures, maternal effects, and/or epigenetic factors.

The genetic basis for congenital hypopituitarism and related disorders is beginning to emerge, and over causal 30 genes have been identified, including six in the SHH signaling pathway. Mutations in some of these genes can also cause holoprosencephaly (HPE) or septo-optic dysplasia. SIX3 is a homeodomain protein expressed in the developing brain, pituitary gland, and eye. It activates SHH signaling and represses BMP signaling. Heterozygous mutations in SIX3 cause variable HPE in humans and mice. We identified a rare, heterozygous variant in SIX3 in two children with neonatal GH and TSH deficiency and stalk interruption, p.P74R. Using transient transfection in 3T3 cells, we demonstrated that the variant reduced the ability of SIX3 to transactivate the SHH enhancer and promoter of FOXG1, suggesting that the variant could be deleterious. To understand the role of SIX3 in hypothalamic and pituitary development we used Nkx2.1-cre and Prop1-cre to delete Six3 in mice. The Nkx2.1-cre, Six3flox/flox embryos had no evidence of infundibulum evagination or expression of Fgf10 or Tcf7l2 at e11.5. The oral ectoderm invaginated in mutants, but no definitive Rathke’s pouch formed. There was no evidence of Lhx3 expression and only trace amounts of Pitx1, indicating that pituitary induction failed due to the lack of Six3 in the developing hypothalamus. Similarly, disruption of Six3 expression in Rathke’s pouch using Prop1-cre ablated pituitary development. Together, these data reveal essential roles of Six3 in both the neural and oral ectoderm for hypothalamic and pituitary development, respectively. Heterozygous loss of function variants in SIX3 could be a contributor to multiple pituitary hormone deficiencies in children, especially if there are associated craniofacial abnormalities.

Many transcription factors have been identified that are important for specification and differentiation of the hormone-producing cells of the pituitary gland. For example, POU1F1 is essential for differentiation of thyrotropes, somatotropes, and lactotropes. GATA2 is important for differentiation of both thyrotropes and gonadotropes, and NR5A1 drives gonadotrope differentiation. It is not known whether additional transcription factors are involved in thyrotrope specification. A few POU1F1-independent thyrotropes arise during development at the rostral tip of the pituitary gland, but the majority of thyrotropes are POU1F1-dependent and arise in the caudo-medial area of the anterior pituitary gland. Pou1f1-deficient mice lack this major, critical type of thyrotropes and are severely hypothyroid. Several pieces of evidence suggest that the caudo-medial population of thyrotropes is heterogeneous. First, NR5A1-positive cells exist that express both TSH and FSH during pituitary development. Second, Pou1f1 mRNA is detected in only about half of pituitary cells immuno-positive for TSH. Third, the transcription factor ISL1 is only detected in a fraction of developing thyrotropes. The Helix-Loop-Helix transcription factor ASCL1 is critical for zebrafish pituitary development, but there are conflicting data about its role in pituitary thyrotrope specification in mice. To quantify the role of ASCL1 in thyrotrope development we carried out immunostaining for TSH in pituitaries of Ascl1 deficient mice in late gestation. We did not detect any reduction in TSH immuno-reactive cells at this time, suggesting there are species-specific differences in the requirement of ASCL1 in the developing pituitary. To systematically assess and quantify the heterogeneity of thyrotropes for transcription factor expression, we performed co-immunostaining for TSH and various transcription factors in pituitaries of newborn mice. We found that approximately 75% of TSH-positive cells in the caudo-medial region of the pituitary express POU1F1, and the majority express GATA2. We detected a small portion of cells co-expressing NR5A1 and TSH at this time. To confirm our immunostaining results, we analyzed data from single cell RNA sequencing of postnatal thyrotropes. Three robust clusters of thyrotropes were identified. Approximately 80% expressed Gata2, 85% expressed Pou1f1, and roughly 15% expressed Nr5a1. Taken together, the protein and RNA analysis support that thyrotrope heterogeneity exists during development. The functional significance of this heterogeneity and its variation during development will be the subject of future investigation.

The transcription factor OTX2 is implicated in pituitary, ocular and craniofacial development. Mutations have been described in patients with variable congenital hypopituitarism (CH) ranging from isolated growth hormone deficiency (IGHD) to combined pituitary hormone deficiency (CPHD) with/without an ectopic posterior pituitary (EPP).We aimed (i) to establish the contribution of OTX2 mutations in the etiology of CH in a sub-cohort of patients and to study their functional consequences and (ii) establish a detailed human OTX2 expression profile in a hypothalamo-pituitary (HP) context. We screened 127 patients from national (n=103) and international centers (n=24) on the septo-optic dysplasia (SOD) spectrum with variable eye abnormalities. Eye abnormalities included micro/anophthalmia, retinal dystrophy and/or coloboma in 29 of these patients, with the rest having optic nerve hypoplasia (ONH). An EPP was reported on MRI in 35 patients. The cohort previously tested negative for mutations in HESX1, SOX2, SOX3, PROKR2 and GH1. Transactivation assays involved a dual-luciferase reporter in murine hypothalamic GT1-7 neurons transiently transfected with OTX2 constructs. In situ hybridization was performed to analyze human brain OTX2 expression during embryogenesis. Seven heterozygous OTX2 changes were identified: two chromosomal deletions spanning OTX2 in patients with micro/anophthalmia, GHD and an EPP, with one patient having cerebellar hypoplasia. Three missense substitutions resulting in truncated proteins: (i) the previously reported p.S138* and (ii) p.C170*, in two patients with retinal dystrophy, EPP and IGHD respectively, and (iii) the novel p.E79* in a patient with micro/anophthalmia, EPP and CPHD. A novel insertion-deletion resulting in a truncated protein p.S167* in a patient with microphthalmia, GHD, ONH, EPP and an enlarged abnormal pituitary, and a novel deletion resulting in a frameshift p.Val139Aspfs*39 in a patient with microcephaly, microphthalmia, ONH and an EPP were also identified. The human OTX2 variants caused a significant reduction in transactivation compared to wild type. Our gene expression data identified human OTX2 transcripts in the posterior pituitary, retina, ear, thalamus, choroid plexus, and in the hypothalamus during embryogenesis, but not in RP. To conclude, we identified OTX2 variants in 7 unrelated CH patients with eye abnormalities including 3 with retinal dystrophy and one with a cerebellar abnormality. As OTX2 is involved at multiple levels during HP development, these patients should be monitored for evolving endocrinopathies. Human OTX2 is expressed in the posterior pituitary, the retina and the ear at CS19 and 20 (between 6-7 weeks gestation), and in areas of the hindbrain at CS23, but not in RP at any stage analyzed in this study. The endocrine phenotypes in patients with OTX2 mutations are most likely of hypothalamic origin.

Pituitary and hypothalamic cell lines have been developed by targeted oncogenesis. This involved using cell-specific transcriptional regulatory sequences to drive expression of large and small SV40 T-antigens in transgenic mice. Invariably, tumors develop in some of the mice, and the cells in these tumors can sometimes be adapted to grow in culture into stable, immortalized cell lines that maintain some of the features of differentiated cells. Cell lines that represent pre-gonadotropes (αT3-1), gonadotropes (LβT2), precursors to the POU1F1 lineage (GHFT1, Pit1-zero), differentiated cells of the POU1F1 lineage (Pit1-triple, TaT1, and Pit1-PRL), and GnRH neurons (GT1-1) have been made by this approach. Tumors often develop early and cause infertility or death. To increase the opportunity for generating cell lines and to make it feasible to follow the process of tumorigenesis, we developed a mouse strain that expresses SV40 T-antigens in response to cre-recombinase. Using CRISPR/Cas9 we inserted an 8 kb cassette with coding sequences for SV40 T-antigens and IRES-GFP into the Rosa26 locus, downstream from a stop sequence flanked by loxP sites: Rosa26LSL-SV40-GFP. 30% of the progeny born from hybrid zygotes injected with template DNA, CRISPR/Cas9, and sgRNA had correctly targeted the Rosa26 locus. These mice were mated with previously established Prop1-cre and Tshb-cre transgenic lines. The majority of Rosa26LSL-SV40-GFP/+; Prop1-cre and Rosa26LSL-SV40-GFP/+; Tshb-cre mice developed dwarfism and large tumors by 4 wks. The pituitaries of Rosa26LSL-SV40-GFP/+; Tshb-cre mice appear grossly normal at birth, but they are enlarged and showing evidence of increased vascularization by 2 wks. Flow-sorted GFP-positive cells from Rosa26LSL-SV40-GFP/+; Prop1-cre and Rosa26LSL-SV40-GFP/+; Tshb-cre mice express Prop1 and TSH, respectively. Tumors from Rosa26LSL-SV40-GFP/+; Tshb-cre mice were adapted to growth in cell culture. We have established a thyrotrope-like cell line that expresses Cga and Pou1f1. These studies demonstrate the utility of the novel, Rosa26LSL-SV40-GFP mouse line for reliable targeted oncogenesis and development of unique cell lines. The authors have nothing to disclose.

Background: Growth hormone deficiency (GHD) occurs in ~ 1/8000 individuals, and 14% of the patients have mutations in five major candidate genes. However, over 30 genes have been implicated in hypopituitarism. WES (Whole Exome Sequencing) is a promising approach for molecular diagnosis of patients with GHD because it offers the opportunity to screen for all known genes in addition to novel disease gene discovery. Methods: WES was performed for 13 unrelated patients with congenital hypopituitarism born from consanguineous parents. The variants were filtered assuming autosomal recessive inheritance, rare variants in population databases, in silico analysis predicted as deleterious and pituitary and/or hypothalamus gene expression. To determine whether variants in CDH2 that were predicted to be deleterious were functionally significant, L1 fibroblast lines that have no endogenous CDH2 protein were stably transfected with either human wild type or variant CDH2, the transfected cells were labelled with lipophilic dyes, and cell adhesion properties were assessed. Results: Homozygous pathogenic or likely pathogenic allelic variants were found in 2 of the 13 patients. First, a female patient with GH, TSH, ACTH and LH/ FSH deficiencies presenting ectopic posterior pituitary lobe, non-visualized stalk, and hypoplastic anterior pituitary lobe had two homozygous rare variants predicted as deleterious: PLA2G4A p.Asn703Lys and CDH2 c.865G>A (p.Val289Ile). Only CDH2 is known to be expressed in the pituitary, and Pla2g4a null mice have a pleiotropic phenotype without obvious hypopituitarism. The CDH2 variant is rare and classified as deleterious. Sanger sequencing of CDH2 in four family members of the affected proband revealed that the unaffected parents and two unaffected siblings were heterozygous carriers. The effect of the CDH2 variant on cell aggregation was assessed in cell culture. Large cell aggregates formed in cells transfected with wild type CDH2, but cell aggregates were small or absent in cells that were either non-transfected or transfected with the CDH2 variant. Second, a patient with isolated GHD and no MRI abnormalities was identified with a rare, likely deleterious, homozygous GH1 c.171delT (p. Phe 57Leufs*43) variant. He had a sister who died at the age of 5 and had features of GHD. Conclusion: In a cohort of congenital hypopituitarism from consanguineous parents we had 15% molecular diagnosis using WES. We identified a variant in a known gene, GH1 c.171delT and a variant in a novel gene, CDH2 p.Val289I.

Introduction: Hypopituitarism is defined as a deficiency of one or more pituitary hormones. Pathogenic allelic variants in genes implicated in pituitary development were associated in 15% of the patients with congenital hypopituitarism (CH). To improve the molecular diagnosis we performed whole exome sequencing of ten patients born from consanguineous parents with CH. One patient with GH, TSH, ACTH and LH/FSH deficiencies presented an allelic variant c.865G>A, p.V289I in CDH2 gene (exon 7) in homozygous state that was absent in populational databanks. CDH2 produces an N-cadherin protein implicated in cellular adhesion and is responsible for epithelial-mesenchymal transition during pituitary development and differentiation. Aim: To analyze the CDH2 gene in a cohort of unrelated patients with CH. Methods: We selected 143 patients with CH from a single Brazilian center. Genomic DNA, extracted by salting out technique, was submitted to PCR amplification of 15 coding regions, except CG rich exon 1, of the CDH2 gene followed by the Sanger sequencing. Rare allelic variant frequency (MAFA, p.V289I) was found in heterozygous state in a male patient with short stature diagnosed with GH and TSH deficiencies at the age of 11 that evolved with LH/FSH and ACTH deficiencies. Family segregation showed 3 among 11 normal siblings heterozygous carriers. This variant is rare, in heterozygous state, in populational data bank and it was predicted as deleterious or possibly harmful. The allelic variant c.1202C> A (p.A401D), in exon 9, was found in heterozygous state in a female patient with isolated GH deficiency and intellectual disability. The variant was absent in the databases and predicted as deleterious or disease-causing. The variant was absent in the mother and stepsister and the father was not available for testing. The c.1430_1431delCCinsTG allelic variant (p.P477L) was found in heterozygous state in a patient with septo-optic dysplasia, GH, TSH and ACTH deficiencies. It was absent in the databases and was predicted as deleterious or disease causing. The Human Splicing Finder predicted exonic splicing enhancer breakdown leading to the loss of 93 nucleotides. Normal mother is heterozygous carrier suggesting incomplete penetrance. Conclusion: Heterozygous variants in CDH2 were found in 2% of a cohort of Brazilian patients with congenital hypopituitarism and none in homozygous or compound heterozygous state. Further CDH2 analyses in unrelated patients from different ethnic backgrounds are needed to establish the role CDH2 variants in the etiology of congenital hypopituitarism.

We implemented single-cell RNA sequencing (scRNAseq) technology as a discovery tool to identify factors enriched in differentiated thyrotropes. Thyroid-stimulating hormone (TSH) is produced in the pars distalis of the anterior pituitary (AP) and primarily acts on the thyroid gland to regulate metabolism through T3/T4. However, TSH is also produced by cells in the pars tuberalis (PT), which is comprised of a thin layer of cells that extends rostrally from the pars distalis along the pituitary stalk to the median eminence in the hypothalamus. TSH produced by PT thyrotropes acts on hypothalamic tanycytes to regulate seasonal reproduction. PT thyrotropes likely descend from rostral tip thyrotropes that arise at e12.5 of mouse development, which transcribe the TSH beta subunit (Tshb) without detectable expression of the transcription factor POU1F1. POU1F1 is required for Tshb transcription in thyrotropes of the adenohypophysis, and it acts synergistically with GATA2 to drive cell fate. The molecular mechanisms driving Tshb expression independently of Pou1f1 in PT thyrotropes are unclear. Thyrotropes are the least abundant endocrine cell-type in the pituitary gland. We used genetic labeling and fluorescence-activated cell sorting (FACS) to enrich for thyrotropes for single-cell sequencing. We performed scRNAseq on 7-day-old GFP-positive pituitary cells from Tshb-Cre; R26-LSL-eYFP and intact whole pituitaries, recovering more than 15,000 cells altogether. We observe two distinct populations of cells expressing Tshb. The larger thyrotrope population has approximately twenty fold higher levels of Tshb and five fold higher Cga transcripts than the smaller population, and they are also distinguished by expression of Pou1f1, TSH-releasing hormone receptor (Trhr), and deiodinase 2 (Dio2), consistent with expectations for AP thyrotropes. The smaller thyrotrope population does not express Pou1f1, but those cells are characterized by expression of TSH receptor (Tshr) and melatonin receptor 1A (Mtnr1a), consistent with expectations for PT thyrotropes. They express mildly increased levels of Eya3 and Six1, although these genes are expressed in other cell-types including AP thyrotropes, stem cells, and gonadotropes. They have two-fold higher levels of Gata2 transcripts and uniquely express the transcription factor Sox14. SOX14 is a SoxB2 family transcription factor that counteracts the transcriptional activity of SoxB1 family members, such as Sox2. In conclusion, our scRNAseq has identified novel markers of PT thyrotropes and unveils novel insights into the similarities and differences in the development and function of pituitary thyrotrope subpopulations.

Recent genome wide association studies have begun to identify loci that are risk factors for sporadic pituitary adenomas, but the genes associated with these loci are unknown. In general, ~90% of GWAS hits are in noncoding regions, making it difficult to transition from genetic mapping to a biological understanding of risk factors. Recent studies that identify enhancer regions by undertaking large scale functional genomic annotation of non-coding elements like Encyclopedia of DNA Elements (ENCODE) have begun to yield a better understanding of some complex diseases. Dense molecular profiling maps of the transcriptome and epigenome have been generated for more than 250 cell lines and 150 tissues, but pituitary cell lines or tissues were not included. Epigenetic and gene expression data are emerging for somatotropes, gonadotropes and corticotropes, but there is very little available data on thyrotropes. We identified the transcription factors and epigenetic changes in chromatin that are associated with differentiation of POU1F1-expressing progenitors into thyrotropes using cell lines that represent an early, undifferentiated Pou1f1 lineage progenitor (GHF-T1) and a committed thyrotrope (TαT1). TαT1 is an excellent cell line for this purpose because it responds to TRH, retinoids, and secretes TSH in response to diurnal cues. We have also used genetic labeling and fluorescence activated cell sorting to purify thyrotropes from pituitaries of young mice and analyzed gene expression using single cell transcriptomics. We used the Assay for TransposaseAccessible Chromatin with sequencing (ATACseq) and Cleavage Under Target and Release Using Nuclease (CUT&RUN) to identify POU1F1 binding sites and histone marks associated with active enhancers, H3K27Ac and H3K4Me1, or inactive regions, H3K27Me3, in GHF-T1 and TαT1 cells. We integrated DNA accessibility, histone modification patterns, transcription factor binding and RNA expression data to identify regulatory elements and candidate transcriptional regulators. We identified POU1F1 binding sites that were unique to each cell line. For example, POU1F1 binds sites in and around Cga and Tshb only in TαT1 cells and Twist1 and Gli3 only in GHFT1 cells. POU1F1 binding sites are commonly associated with bZIP factor consensus binding sites in GHFT1 cells and Helix-Turn-Helix or basic Helix-Loop-Helix in TαT1 cells, suggesting classes of transcription factors that may recruit POU1F1 to unique sites. We validated enhancer function of novel elements we mapped near Tshb, Gata2, and Pitx1 by transfection in TαT1 cells. Finally, we confirmed that an enhancer element near Tshb can drive expression in thyrotropes of transgenic mice. These data extend the ENCODE analysis to an organ that is critical for growth and metabolism. This information could be valuable for understanding pituitary development and disease pathogenesis.

Advances in genomic technologies are revolutionizing the practice of medicine by delivering molecular diagnoses that can be informative for prognosis and treatment of genetic disorders. Most of the known genetic causes of multiple pituitary hormone deficiency have been investigated as monogenic disorders. It can be challenging to predict clinical features from genetic data, as loss of function mutations in some genes can present with a spectrum of phenotypes ranging from craniofacial abnormalities, intellectual disability, and neurosensory and neuroendocrine defects to pituitary hormone deficiency with no other abnormalities. Although maternal exposures could be contributing factors, the contribution of rare, deleterious variation in other genes is a likely contributor. In humans, loss of function mutations in the transcription factor SIX3 cause variable, autosomal dominant holoprosencephaly with incomplete penetrance, and mouse models recapitulate some of the clinical features. Because Six3 and Pou1f1 gene expression patterns overlap in pituitary development, we hypothesized that doubly heterozygous mice (Six3+/-; Pou1f1+/dw) might have pituitary anomalies not present in singly heterozygous mice. We intercrossed Six3+/- and Pou1f1+/dw mice to produce doubly heterozygous animals. At e11.5, both Six3+/- and Six3+/-; Pou1f1+/dw exhibited abnormal morphology of the developing infundibulum and Rathke’s pouch, although ventral diencephalon expression of Tle4, Fgf10, and Nkx2.1 appeared normal. Both newborn Six3+/- and Six3+/-; Pou1f1+/dw littermates had abnormal pituitary gland morphology that resembled that of Aes-/-. AES is a co-repressor that interacts with SIX3. Specification of vasopressin neurons and anterior lobe hormone cell types appeared normal. Mice of all genotypes were born in expected Mendelian ratios (N=144, p=0.49), and there were no significant differences in body weight at 3 wks. A portion of the Six3+/- and doubly heterozygous mice developed hydrocephalus, exhibited failure to thrive, and died (6-9% of N=82, 85, respectively). At 6 wks, 25% (N=61) of the Six3+/-; Pou1f1+/dw animals exhibited striking pituitary dysmorphology in which the rostral aspect of the pituitary penetrated the palate. This was not observed in single heterozygotes. These results reveal that haploinsufficiency for Six3 affects Rathke’s pouch formation, resulting in pituitary gland dysmorphology in and around the stem cell niche. A significant portion of the Six3+/-; Pou1f1+/dw doubly heterozygous mice have a more pronounced pituitary phenotype than Six3+/-, supporting the possibility of digenic pituitary disease and highlighting phenotypic variability. Genetically engineered mice provide an excellent tool for assessing the possibility of gene-gene interactions that could enhance the severity of hypopituitarism and associated craniofacial development.

Many transcription factors that regulate pituitary gland development and specification of hormone-producing cell types have been discovered. Here we focus on 13 transcription factors that play key roles in these processes, many of which are mutated in patients with congenital pituitary insufficiency disorders. We present the methods by which each of these transcription factors were identified. Each of the genes we discuss has been well studied in genetically engineered mice, facilitating an understanding of the impact of each single gene defect on the expression of downstream targets. Finally, we highlight technical advances that promise to yield future insights into epigenetic regulation of cell fate.

Transcription factors and signaling pathways that regulate stem cells and specialized hormone-producing cells in the pituitary gland have been the subject of intense study and have yielded a mechanistic understanding of pituitary organogenesis and disease. However, the regulation of stem cell proliferation and differentiation, the heterogeneity among specialized hormone-producing cells, and the role of nonendocrine cells in the gland remain important, unanswered questions. Recent advances in single-cell RNA sequencing (scRNAseq) technologies provide new avenues to address these questions. We performed scRNAseq on ∼13,663 cells pooled from six whole pituitary glands of 7-week-old C57BL/6 male mice. We identified pituitary endocrine and stem cells in silico, as well as other support cell types such as endothelia, connective tissue, and red and white blood cells. Differential gene expression analyses identify known and novel markers of pituitary endocrine and stem cell populations. We demonstrate the value of scRNAseq by in vivo validation of a novel gonadotrope-enriched marker, Foxp2. We present novel scRNAseq data of in vivo pituitary tissue, including data from agnostic clustering algorithms that suggest the presence of a somatotrope subpopulation enriched in sterol/cholesterol synthesis genes. Additionally, we show that incomplete transcriptome annotation can cause false negatives on some scRNAseq platforms that only generate 3′ transcript end sequences, and we use in vivo data to recover reads of the pituitary transcription factor Prop1. Ultimately, scRNAseq technologies represent a significant opportunity to address long-standing questions regarding the development and function of the different populations of the pituitary gland throughout life.

A fundamental question for pituitary development and disease is to understand the mechanisms that regulate proliferation, quiescence, and differentiation of stem cells and endocrine lineage-committed precursor cells. Pituitary stem cells, marked by expression of SOX2, are highly proliferative during development and the early postnatal period. This pool becomes quiescent over time, but stem cells retain the ability to re-enter the cell cycle and differentiate into nascent endocrine cells of all lineages in response to physiological demands. The rodent pituitary gland undergoes substantial growth in the postnatal period, and much of this increase in size is due to proliferation of committed progenitors, such as Pou1f1-expressing cells. We performed single-cell RNA transcriptomics analyses of over 8,000 male and female 4-day-old mouse pituitary cells in order to assess stem cell heterogeneity and to identify novel pituitary stem cell biomarkers. We identified a number of factors enriched in pituitary stem cells relative to differentiating cells, including the transcription factors TGIF1 and NR4A3 as well as several members of the nuclear factor I family (NFIA, NFIB, NFIX). We also detected stem cell-specific expression of the cortisol synthesizing enzyme HSD11B1 and folate receptor FOLR1, suggesting novel roles for these pathways in pituitary stem cells. There were few transcriptomic differences between proliferating and non-proliferating stem cells. However, proliferating stem cells and proliferating committed progenitors shared expression of cell-cycle associated genes and novel transcription factors such as BRCA1 and E2F1. Furthermore, single-cell gene network inference and clustering (SCENIC) analyses demonstrated activation of common gene regulatory networks in both proliferating stem and proliferating endocrine populations, including both the E2f1 and Brca1 regulons. RNA velocity, trajectory, and phylogenetic analyses and find that proliferating stem and endocrine cells are likely independent cellular states. Instead, they support the idea that proliferating stem cells become quiescent and transition to committed progenitors that re-enter the cell cycle and then subsequently activate hormone gene expression. In conclusion, our single-cell gene expression analyses of early postnatal pituitary cells have shed light on the developmental trajectory from proliferating stem cell to quiescent differentiated hormone-producing cells.

Congenital hypopituitarism (CH), septo-optic dysplasia (SOD), and holoprosencephaly (HPE) constitute an important group of structural birth defects that cause significant morbidity and life-long consequences for quality of life and care. The genetic causes are highly overlapping. As such, these disorders can be considered as a spectrum of related disorders. Improved insight into genetic causes would be valuable for patients, families, and medical geneticists. Very few systematic genetic screens have been carried out for patients with CH. We implemented genetic screening using single-molecule molecular inversion probes sequencing to identify causative mutations in a set of 67 genes previously reported in CH patients and the spectrum encompassing SOD and HPE. We captured genomic DNA from 170 Argentinean pediatric patients with CH, and 54% of the patients in this cohort have craniofacial, ophthalmologic, and/or central nervous system defects. We found candidate pathogenic, likely pathogenic and variants uncertain significance (VUS) in 23% of the cases. In order to evaluate the functional consequences of VUS in LHX3, LHX4, and GLI2, we performed in-vitro functional assays to study the activity of the mutated proteins. To test LHX3/4 variants we co-transfected HEK293T cells with wild type (WT) or mutated LHX3/4 variant plasmids and luciferase reporter genes driven by the ɑGSU promoter or GH1 promoter and assayed for luciferase activity. For GLI2 functional analysis we used the cell line NIH/3T3-CG, stably transfected to express GFP under the presence of GLI2 activated form. Endogenous Gli2 was knocked out by CRISPR-Cas9 and clones were selected for absence of GFP expression upon activation of the sonic hedgehog pathway. We tested the ability of transfected WT or mutated GLI2 expression plasmids to restore GFP fluorescence. We concluded that variants LHX3:p.Pro187Ser LHX4:p.Arg84His, p.Gln100His and p.Trp204Leu and GLI2:p.1404Lfs impair activation of the reporter gene, while the LHX3:p.Leu220Met and GLI2:p.L761P have WT activity on their respective assays. Identification of disease-causing variants in CH is complicated by phenotypic variation, incomplete penetrance, and VUS. Functional testing of potentially pathogenic variants is critical to arrive at a definitive molecular diagnosis. A full catalogue of variant effects in known causative genes would be invaluable for clinicians in order to simplify the interpretation of novel variants and reduce the diagnostic odyssey that families often experience.

Pituitary hormone deficiency or hypopituitarism is characterized by a malformed or underdeveloped pituitary gland resulting in an impaired pituitary hormone secretion. Several transcription factors have been described in its etiology, but defects in known genes account for only a small proportion of cases. We sought to identify the cause of hypopituitarism in 171 unrelated patients diagnosed with or without extra-pituitary manifestations that were recruited from several Argentinean medical centers. We conducted panel sequencing, and identified among other genes and variants, de novo heterozygous mutations in FOXA2 and PNPLA6. FOXA2 is a transcription factor member of the forkhead class of DNA-binding proteins, involved in the early development of multiple tissues. FOXA2 is highly expressed throughout the developing hypothalamic-pituitary axis, and regulates GLI2, SHH and NKX2-2 expression. Mutations of FOXA2 have been linked to combined pituitary hormone deficiency (CPHD) in some cases with extra-pituitary phenotypes including hyperinsulinism or gastrointestinal malformations. We found two patients with CPHD and rare FOXA2 variants. Case 1 had GHD, anterior pituitary hypoplasia, mammary hypertelorism and digital anomalies and a heterozygous variant FOXA2 p.Arg228Ser, predicted to be pathogenic. Case 2 had GH and TSH deficiency, craniofacial anomalies and neurodevelopmental delay, and a novel, stop codon mutation FOXA2 p.Ser229* and an heterozygous GLI1 variant (p.Asp1048Asn). Both FOXA2 variants are located within the forkhead domain which may affect the DNA binding ability. We suspect they are likely damaging based on the literature, the in-silico prediction, and their absence in GnomAD. PNPLA6 is a conserved lysophospholipase involved in maintaining nervous system integrity. Mutations in PNPLA6 have been identified in a broad spectrum from pure ataxia to rare neuroendocrine conditions including Gordon Holmes and Oliver McFarlane syndromes. Here, we identified two de novo heterozygous variants in PNPLA6 in children with CPHD. Variant p.W1039R was found in a patient with CPHD, intellectual disability and visual problems. A second variant (p.T1115P) was identified in a 10-year-old girl with CPHD, retinitis pigmentosa and neurodevelopmental delay. According to modelling studies of the protein structure, both variants are expected to be critical for the activity of the NTE as they are located in close proximity to the protein’s catalytic pocket. It is likely that these variants may contribute to our patient’s phenotype. However, as most reported PNPLA6 variants in the literature were found in homozygosity or compound heterozygosity, additional studies are necessary to draw more definitive genotype-phenotype correlations. In summary, in this work we were able to expand our knowledge of pituitary target genes for genetic diagnosis for CH.

The hypothalamic-pituitary axes are the coordinating centers for multiple endocrine gland functions and physiological processes. Defects in the hypothalamus or pituitary gland can cause reduced growth and severe short stature, affecting approximately 1 in 4000 children, and a large percentage of cases of pituitary hormone deficiencies do not have an identified genetic cause. SOX21 is a protein that regulates hair, neural, and trophoblast stem cell differentiation. Mice lacking Sox21 have reduced growth, but the etiology of this growth defect has not been described. We studied the expression of Sox21 in hypothalamic-pituitary development and examined multiple endocrine axes in these mice. We find no evidence of reduced intrauterine growth, food intake, or physical activity, but there is evidence for increased energy expenditure in mutants. In addition, despite changes in pituitary hormone expression, hypothalamic-pituitary axes appear to be functional. Therefore, SOX21 variants may be a cause of non-endocrine short stature in humans.

Dietary vitamin A is metabolized into bioactive retinoic acid (RA) in vivo and regulates the development of many embryonic tissues. RA signaling is active in the oral ectoderm-derived tissues of the neuroendocrine system, but its role there has not yet been fully explored. We show here that RA signaling is active during pituitary organogenesis and dependent on the pituitary transcription factor Prop1. Prop1-mutant mice show reduced expression of the aldehyde dehydrogenase gene Aldh1a2, which metabolizes the vitamin A–intermediate retinaldehyde into RA. To elucidate the specific function of RA signaling during neuroendocrine development, we studied a conditional deletion of Aldh1a2 and a dominant-negative mouse model of inhibited RA signaling during pituitary organogenesis. These models partially phenocopy Prop1-mutant mice by exhibiting embryonic pituitary dysmorphology and reduced hormone expression, especially thyrotropin. These findings establish the role of RA in embryonic pituitary stem cell progression to differentiated hormone cells and raise the question of gene-by-environment interactions as contributors to pituitary development and disease.