Pituitary Tumors and Immortalized Cell Lines Generated by Cre-Inducible Expression of SV40 T Antigen.

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, Rosa26LSL-SV40-GFP mouse line for reliable targeted oncogenesis and development of unique cell lines.

Genetic variation in thyroid folliculogenesis influences susceptibility to hypothyroidism-induced hearing impairment.

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.

Next generation sequencing panel based on single molecule molecular inversion probes for detecting genetic variants in children with hypopituitarism.

BACKGROUND: Congenital Hypopituitarism is caused by genetic and environmental factors. Over 30 genes have been implicated in isolated and/or combined pituitary hormone deficiency. The etiology remains unknown for up to 80% of the patients, but most cases have been analyzed by limited candidate gene screening. Mutations in the PROP1 gene are the most common known cause, and the frequency of mutations in this gene varies greatly by ethnicity. We designed a custom array to assess the frequency of mutations in known hypopituitarism genes and new candidates, using single molecule molecular inversion probes sequencing (smMIPS). METHODS: We used this panel for the first systematic screening for causes of hypopituitarism in children. Molecular inversion probes were designed to capture 693 coding exons of 30 known genes and 37 candidate genes. We captured genomic DNA from 51 pediatric patients with CPHD (n = 43) or isolated GH deficiency (IGHD) (n = 8) and their parents and conducted next generation sequencing. RESULTS: We obtained deep coverage over targeted regions and demonstrated accurate variant detection by comparison to whole-genome sequencing in a control individual. We found a dominant mutation GH1, p.R209H, in a three-generation pedigree with IGHD. CONCLUSIONS: smMIPS is an efficient and inexpensive method to detect mutations in patients with hypopituitarism, drastically limiting the need for screening individual genes by Sanger sequencing.

Genetics of Combined Pituitary Hormone Deficiency: Roadmap into the Genome Era.

The genetic basis for combined pituitary hormone deficiency (CPHD) is complex, involving 30 genes in a variety of syndromic and nonsyndromic presentations. Molecular diagnosis of this disorder is valuable for predicting disease progression, avoiding unnecessary surgery, and family planning. We expect that the application of high throughput sequencing will uncover additional contributing genes and eventually become a valuable tool for molecular diagnosis. For example, in the last 3 years, six new genes have been implicated in CPHD using whole-exome sequencing. In this review, we present a historical perspective on gene discovery for CPHD and predict approaches that may facilitate future gene identification projects conducted by clinicians and basic scientists. Guidelines for systematic reporting of genetic variants and assigning causality are emerging. We apply these guidelines retrospectively to reports of the genetic basis of CPHD and summarize modes of inheritance and penetrance for each of the known genes. In recent years, there have been great improvements in databases of genetic information for diverse populations. Some issues remain that make molecular diagnosis challenging in some cases. These include the inherent genetic complexity of this disorder, technical challenges like uneven coverage, differing results from variant calling and interpretation pipelines, the number of tolerated genetic alterations, and imperfect methods for predicting pathogenicity. We discuss approaches for future research in the genetics of CPHD.

Cocaine-and Amphetamine Regulated Transcript (CART) Peptide Is Expressed in Precursor Cells and Somatotropes of the Mouse Pituitary Gland.

Cocaine-and Amphetamine Regulated Transcript (CART) peptide is expressed in the brain, endocrine and neuroendocrine systems and secreted into the serum. It is thought to play a role in regulation of hypothalamic pituitary functions. Here we report a spatial and temporal analysis of Cart expression in the pituitaries of adult and developing normal and mutant mice with hypopituitarism. We found that Prop1 is not necessary for initiation of Cart expression in the fetal pituitary at e14.5, but it is required indirectly for maintenance of Cart expression in the postnatal anterior pituitary gland. Pou1f1 deficiency has no effect on Cart expression before or after birth. There is no 1:1 correspondence between CART and any particular cell type. In neonates, CART is detected primarily in non-proliferating, POU1F1-positive cells. CART is also found in some cells that express TSH and GH suggesting a correspondence with committed progenitors of the POU1F1 lineage. In summary, we have characterized the normal temporal and cell specific expression of CART in mouse development and demonstrate that postnatal CART expression in the pituitary gland requires PROP1.

PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells.

Mutations in PROP1 are the most common cause of hypopituitarism in humans; therefore, unraveling its mechanism of action is highly relevant from a therapeutic perspective. Our current understanding of the role of PROP1 in the pituitary gland is limited to the repression and activation of the pituitary transcription factor genes Hesx1 and Pou1f1, respectively. To elucidate the comprehensive PROP1-dependent gene regulatory network, we conducted genome-wide analysis of PROP1 DNA binding and effects on gene expression in mutant mice, mouse isolated stem cells and engineered mouse cell lines. We determined that PROP1 is essential for stimulating stem cells to undergo an epithelial to mesenchymal transition-like process necessary for cell migration and differentiation. Genomic profiling reveals that PROP1 binds to genes expressed in epithelial cells like Claudin 23, and to EMT inducer genes like Zeb2, Notch2 and Gli2. Zeb2 activation appears to be a key step in the EMT process. Our findings identify PROP1 as a central transcriptional component of pituitary stem cell differentiation.

ß-catenin is required in the neural crest and mesencephalon for pituitary gland organogenesis.

BACKGROUND: The pituitary gland is a highly vascularized tissue that requires coordinated interactions between the neural ectoderm, oral ectoderm, and head mesenchyme during development for proper physiological function. The interactions between the neural ectoderm and oral ectoderm, especially the role of the pituitary organizer in shaping the pituitary precursor, Rathke's pouch, are well described. However, less is known about the role of head mesenchyme in pituitary organogenesis. The head mesenchyme is derived from definitive mesoderm and neural crest, but the relative contributions of these tissues to the mesenchyme adjacent to the pituitary are not known. RESULTS: We carried out lineage tracing experiments using two neural crest-specific mouse cre lines, Wnt1-cre and P0-cre, and determined that the head mesenchyme rostral to the pituitary gland is neural crest derived. To assess the role of the neural crest in pituitary development we ablated it, using Wnt1-cre to delete Ctnnb1 (ß-catenin), which is required for neural crest development. The Wnt1-cre is active in the neural ectoderm, principally in the mesencephalon, but also in the posterior diencephalon. Loss of ß-catenin in this domain causes a rostral shift in the ventral diencephalon, including the pituitary organizer, resulting in pituitary dysmorphology. The neural crest deficient embryos have abnormally dilated pituitary vasculature due to a loss of neural crest derived pericytes. CONCLUSIONS: ß-catenin in the Wnt1 expression domain, including the neural crest, plays a critical role in regulation of pituitary gland growth, development, and vascularization.

Deletion of OTX2 in neural ectoderm delays anterior pituitary development.

OTX2 is a homeodomain transcription factor that is necessary for normal head development in mouse and man. Heterozygosity for loss-of-function alleles causes an incompletely penetrant, haploinsufficiency disorder. Affected individuals exhibit a spectrum of features that range from developmental defects in eye and/or pituitary development to acephaly. To investigate the mechanism underlying the pituitary defects, we used different cre lines to inactivate Otx2 in early head development and in the prospective anterior and posterior lobes. Mice homozygous for Otx2 deficiency in early head development and pituitary oral ectoderm exhibit craniofacial defects and pituitary gland dysmorphology, but normal pituitary cell specification. The morphological defects mimic those observed in humans and mice with OTX2 heterozygous mutations. Mice homozygous for Otx2 deficiency in the pituitary neural ectoderm exhibited altered patterning of gene expression and ablation of FGF signaling. The posterior pituitary lobe and stalk, which normally arise from neural ectoderm, were extremely hypoplastic. Otx2 expression was intact in Rathke's pouch, the precursor to the anterior lobe, but the anterior lobe was hypoplastic. The lack of FGF signaling from the neural ectoderm was sufficient to impair anterior lobe growth, but not the differentiation of hormone-producing cells. This study demonstrates that Otx2 expression in the neural ectoderm is important intrinsically for the development of the posterior lobe and pituitary stalk, and it has significant extrinsic effects on anterior pituitary growth. Otx2 expression early in head development is important for establishing normal craniofacial features including development of the brain, eyes and pituitary gland.

Pituitary gland development and disease: from stem cell to hormone production.

Many aspects of pituitary development have become better understood in the past two decades. The signaling pathways regulating pituitary growth and shape have emerged, and the balancing interactions between the pathways are now appreciated. Markers for multipotent progenitor cells are being identified, and signature transcription factors have been discovered for most hormone-producing cell types. We now realize that pulsatile hormone secretion involves a 3D integration of cellular networks. About a dozen genes are known to cause pituitary hypoplasia when mutated due to their essential roles in pituitary development. Similarly, a few genes are known that predispose to familial endocrine neoplasia, and several genes mutated in sporadic pituitary adenomas are documented. In the next decade, we anticipate gleaning a deeper appreciation of these processes at the molecular level, insight into the development of the hypophyseal portal blood system, and evolution of better therapeutics for congenital and acquired hormone deficiencies and for common craniopharyngiomas and pituitary adenomas.

Aged PROP1 deficient dwarf mice maintain ACTH production.

Humans with PROP1 mutations have multiple pituitary hormone deficiencies (MPHD) that typically advance from growth insufficiency diagnosed in infancy to include more severe growth hormone (GH) deficiency and progressive reduction in other anterior pituitary hormones, eventually including adrenocorticotropic hormone (ACTH) deficiency and hypocortisolism. Congenital deficiencies of GH, prolactin, and thyroid stimulating hormone have been reported in the Prop1(null) (Prop1(-/-)) and the Ames dwarf (Prop1(df/df)) mouse models, but corticotroph and pituitary adrenal axis function have not been thoroughly investigated. Here we report that the C57BL6 background sensitizes mutants to a wasting phenotype that causes approximately one third to die precipitously between weaning and adulthood, while remaining homozygotes live with no signs of illness. The wasting phenotype is associated with severe hypoglycemia. Circulating ACTH and corticosterone levels are elevated in juvenile and aged Prop1 mutants, indicating activation of the pituitary-adrenal axis. Despite this, young adult Prop1 deficient mice are capable of responding to restraint stress with further elevation of ACTH and corticosterone. Low blood glucose, an expected side effect of GH deficiency, is likely responsible for the elevated corticosterone level. These studies suggest that the mouse model differs from the human patients who display progressive hormone loss and hypocortisolism.

Candidate genes for panhypopituitarism identified by gene expression profiling.

Mutations in the transcription factors PROP1 and PIT1 (POU1F1) lead to pituitary hormone deficiency and hypopituitarism in mice and humans. The dysmorphology of developing Prop1 mutant pituitaries readily distinguishes them from those of Pit1 mutants and normal mice. This and other features suggest that Prop1 controls the expression of genes besides Pit1 that are important for pituitary cell migration, survival, and differentiation. To identify genes involved in these processes we used microarray analysis of gene expression to compare pituitary RNA from newborn Prop1 and Pit1 mutants and wild-type littermates. Significant differences in gene expression were noted between each mutant and their normal littermates, as well as between Prop1 and Pit1 mutants. Otx2, a gene critical for normal eye and pituitary development in humans and mice, exhibited elevated expression specifically in Prop1 mutant pituitaries. We report the spatial and temporal regulation of Otx2 in normal mice and Prop1 mutants, and the results suggest Otx2 could influence pituitary development by affecting signaling from the ventral diencephalon and regulation of gene expression in Rathke's pouch. The discovery that Otx2 expression is affected by Prop1 deficiency provides support for our hypothesis that identifying molecular differences in mutants will contribute to understanding the molecular mechanisms that control pituitary organogenesis and lead to human pituitary disease.

A modifier gene alleviates hypothyroidism-induced hearing impairment in Pou1f1dw dwarf mice.

Thyroid hormone has pleiotropic effects on cochlear development, and genomic variation influences the severity of associated hearing deficits. DW/J-Pou1f1dw/dw mutant mice lack pituitary thyrotropin, which causes severe thyroid hormone deficiency and profound hearing impairment. To assess the genetic complexity of protective effects on hypothyroidism-induced hearing impairment, an F1 intercross was generated between DW/J-Pou1f1dw/+ carriers and an inbred strain with excellent hearing derived from Mus castaneus, CAST/EiJ. Approximately 24% of the (DW/J×CAST/EiJ) Pou1f1dw/dw F2 progeny had normal hearing. A genome scan revealed a locus on chromosome 2, named modifier of dw hearing, or Mdwh, that rescues hearing despite persistent hypothyroidism. This chromosomal region contains the modifier of tubby hearing 1 (Moth1) locus that encodes a protective allele of the microtubule-associated protein MTAP1A. DW/J-Pou1f1dw/+ carriers were crossed with the AKR strain, which also carries a protective allele of Mtap1a, and we found that AKR is not protective for hearing in the (DW/J×AKR) Pou1f1dw/dw F2 progeny. Thus, protective alleles of Mtap1a are not sufficient to rescue DW/J-Pou1f1dw/dw hearing. We expect that identification of protective modifiers will enhance our understanding of the mechanisms of hypothyroidism-induced hearing impairment.

Birthdating studies reshape models for pituitary gland cell specification.

The intermediate and anterior lobes of the pituitary gland are derived from an invagination of oral ectoderm that forms Rathke's pouch. During gestation proliferating cells are enriched around the pouch lumen, and they appear to delaminate as they exit the cell cycle and differentiate. During late mouse gestation and the postnatal period, anterior lobe progenitors re-enter the cell cycle and expand the populations of specialized, hormone-producing cells. At birth, all cell types are present, and their localization appears stratified based on cell type. We conducted a birth dating study of Rathke's pouch derivatives to determine whether the location of specialized cells at birth is correlated with the timing of cell cycle exit. We find that all of the anterior lobe cell types initiate differentiation concurrently with a peak between e11.5 and e13.5. Differentiation of intermediate lobe melanotropes is delayed relative to anterior lobe cell types. We discovered that specialized cell types are not grouped together based on birth date and are dispersed throughout the anterior lobe. Thus, the apparent stratification of specialized cells at birth is not correlated with cell cycle exit. Thus, the currently popular model of cell specification, dependent upon timing of extrinsic, directional gradients of signaling molecules, needs revision. We propose that signals intrinsic to Rathke's pouch are necessary for cell specification between e11.5 and e13.5 and that cell-cell communication likely plays an important role in regulating this process.

Genetics, gene expression and bioinformatics of the pituitary gland.

Genetic cases of congenital pituitary hormone deficiency are common and many are caused by transcription factor defects. Mouse models with orthologous mutations are invaluable for uncovering the molecular mechanisms that lead to problems in organ development and typical patient characteristics. We are using mutant mice defective in the transcription factors PROP1 and POU1F1 for gene expression profiling to identify target genes for these critical transcription factors and candidates for cases of pituitary hormone deficiency of unknown aetiology. These studies reveal critical roles for Wnt signalling pathways, including the TCF/LEF transcription factors and interacting proteins of the groucho family, bone morphogenetic protein antagonists and targets of notch signalling. Current studies are investigating the roles of novel homeobox genes and pathways that regulate the transition from proliferation to differentiation, cell adhesion and cell migration. Pituitary adenomas are a common human health problem, yet most cases are sporadic, necessitating alternative approaches to traditional Mendelian genetic studies. Mouse models of adenoma formation offer the opportunity for gene expression profiling during progressive stages of hyperplasia, adenoma and tumorigenesis. This approach holds promise for the identification of relevant pathways and candidate genes as risk factors for adenoma formation, understanding mechanisms of progression, and identifying drug targets and clinically relevant biomarkers.

Pitx2 deletion in pituitary gonadotropes is compatible with gonadal development, puberty, and fertility.

This report introduces a gonadotrope-specific cre transgenic mouse capable of ablating floxed genes in mature pituitary gonadotropes. Initial analysis of this transgenic line, Tg(Lhb-cre)1Sac, reveals that expression is limited to the pituitary cells that produce luteinizing hormone beta, beginning appropriately at e17.5. Cre activity is detectable by a reporter gene in nearly every LHbeta-producing cell, but the remaining hormone-producing cell types and other organs exhibit little to no activity. We used the Tg(Lhb-cre)1Sac strain to assess the role Pitx2 in gonadotrope function. The gonadotrope-specific Pitx2 knockout mice exhibit normal expression of LHbeta, sexual maturation, and fertility, suggesting that Pitx2 is not required for gonadotrope maintenance or for regulated production of gonadotropins.