Heterozygous variants in SIX3 and POU1F1 cause pituitary hormone deficiency in mouse and man.

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.

High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency.

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.

Otx2b mutant zebrafish have pituitary, eye and mandible defects that model mammalian disease.

Combined pituitary hormone deficiency (CPHD) is a genetically heterogeneous disorder caused by mutations in over 30 genes. The loss-of-function mutations in many of these genes, including orthodenticle homeobox 2 (OTX2), can present with a broad range of clinical symptoms, which provides a challenge for predicting phenotype from genotype. Another challenge in human genetics is functional evaluation of rare genetic variants that are predicted to be deleterious. Zebrafish are an excellent vertebrate model for evaluating gene function and disease pathogenesis, especially because large numbers of progeny can be obtained, overcoming the challenge of individual variation. To clarify the utility of zebrafish for the analysis of CPHD-related genes, we analyzed the effect of OTX2 loss of function in zebrafish. The otx2b gene is expressed in the developing hypothalamus, and otx2bhu3625/hu3625 fish exhibit multiple defects in the development of head structures and are not viable past 10 days post fertilization (dpf). Otx2bhu3625/hu3625 fish have a small hypothalamus and low expression of pituitary growth hormone and prolactin (prl). The gills of otx2bhu3625/hu3625 fish have weak sodium influx, consistent with the role of prolactin in osmoregulation. The otx2bhu3625/hu3625 eyes are microphthalmic with colobomas, which may underlie the inability of the mutant fish to find food. The small pituitary and eyes are associated with reduced cell proliferation and increased apoptosis evident at 3 and 5 dpf, respectively. These observations establish the zebrafish as a useful tool for the analysis of CPHD genes with variable and complex phenotypes.

Variants in myelin regulatory factor (MYRF) cause autosomal dominant and syndromic nanophthalmos in humans and retinal degeneration in mice.

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor (MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98, another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development.

Multi-omic profiling of pituitary thyrotropic cells and progenitors.

BACKGROUND: The 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. RESULTS: We 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 undifferentiated Pou1f1 lineage 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 near Cga, Pitx1, Gata2, and Tshb by transfection in TαT1 cells. Finally, we confirmed that an enhancer element near Tshb can drive expression in thyrotropes of transgenic mice, and we demonstrate that GATA2 enhances Tshb expression through this element. CONCLUSION: These results extend the ENCODE multi-omic profiling approach to the pituitary gland, which should be valuable for understanding pituitary development and disease pathogenesis.

Genetic redundancy of GATA factors in the extraembryonic trophoblast lineage ensures the progression of preimplantation and postimplantation mammalian development.

GATA transcription factors are implicated in establishing cell fate during mammalian development. In early mammalian embryos, GATA3 is selectively expressed in the extraembryonic trophoblast lineage and regulates gene expression to promote trophoblast fate. However, trophoblast-specific GATA3 function is dispensable for early mammalian development. Here, using dual conditional knockout mice, we show that genetic redundancy of Gata3 with paralog Gata2 in trophoblast progenitors ensures the successful progression of both pre- and postimplantation mammalian development. Stage-specific gene deletion in trophoblasts reveals that loss of both GATA genes, but not either alone, leads to embryonic lethality prior to the onset of their expression within the embryo proper. Using ChIP-seq and RNA-seq analyses, we define the global targets of GATA2/GATA3 and show that they directly regulate a large number of common genes to orchestrate stem versus differentiated trophoblast fate. In trophoblast progenitors, GATA factors directly regulate BMP4, Nodal and Wnt signaling components that promote embryonic-extraembryonic signaling cross-talk, which is essential for the development of the embryo proper. Our study provides genetic evidence that impairment of trophoblast-specific GATA2/GATA3 function could lead to early pregnancy failure.

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.

Advances in Skeletal Dysplasia Genetics.

Skeletal dysplasias result from disruptions in normal skeletal growth and development and are a major contributor to severe short stature. They occur in approximately 1/5,000 births, and some are lethal. Since the most recent publication of the Nosology and Classification of Genetic Skeletal Disorders, genetic causes of 56 skeletal disorders have been uncovered. This remarkable rate of discovery is largely due to the expanded use of high-throughput genomic technologies. In this review, we discuss these recent discoveries and our understanding of the molecular mechanisms behind these skeletal dysplasia phenotypes. We also cover potential therapies, unusual genetic mechanisms, and novel skeletal syndromes both with and without known genetic causes. The acceleration of skeletal dysplasia genetics is truly spectacular, and these advances hold great promise for diagnostics, risk prediction, and therapeutic design.

LINE-1 Mediated Insertion into Poc1a (Protein of Centriole 1 A) Causes Growth Insufficiency and Male Infertility in Mice.

Skeletal dysplasias are a common, genetically heterogeneous cause of short stature that can result from disruptions in many cellular processes. We report the identification of the lesion responsible for skeletal dysplasia and male infertility in the spontaneous, recessive mouse mutant chagun. We determined that Poc1a, encoding protein of the centriole 1a, is disrupted by the insertion of a processed Cenpw cDNA, which is flanked by target site duplications, suggestive of a LINE-1 retrotransposon-mediated event. Mutant fibroblasts have impaired cilia formation and multipolar spindles. Male infertility is caused by defective spermatogenesis early in meiosis and progressive germ cell loss. Spermatogonial stem cell transplantation studies revealed that Poc1a is essential for normal function of both Sertoli cells and germ cells. The proliferative zone of the growth plate is small and disorganized because chondrocytes fail to re-align after cell division and undergo increased apoptosis. Poc1a and several other genes associated with centrosome function can affect the skeleton and lead to skeletal dysplasias and primordial dwarfisms. This mouse mutant reveals how centrosome dysfunction contributes to defects in skeletal growth and male infertility.

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.

Gonadotrope-specific deletion of Dicer results in severely suppressed gonadotropins and fertility defects.

Pituitary gonadotropins follicle-stimulating hormone and luteinizing hormone are heterodimeric glycoproteins expressed in gonadotropes. They act on gonads and promote their development and functions including steroidogenesis and gametogenesis. Although transcriptional regulation of gonadotropin subunits has been well studied, the post-transcriptional regulation of gonadotropin subunits is not well understood. To test if microRNAs regulate the hormone-specific gonadotropin ß subunits in vivo, we deleted Dicer in gonadotropes by a Cre-lox genetic approach. We found that many of the DICER-dependent microRNAs, predicted in silico to bind gonadotropin ß subunit mRNAs, were suppressed in purified gonadotropes of mutant mice. Loss of DICER-dependent microRNAs in gonadotropes resulted in profound suppression of gonadotropin-ß subunit proteins and, consequently, the heterodimeric hormone secretion. In addition to suppression of basal levels, interestingly, the post-gonadectomy-induced rise in pituitary gonadotropin synthesis and secretion were both abolished in mutants, indicating a defective gonadal negative feedback control. Furthermore, mutants lacking Dicer in gonadotropes displayed severely reduced fertility and were rescued with exogenous hormones confirming that the fertility defects were secondary to suppressed gonadotropins. Our studies reveal that DICER-dependent microRNAs are essential for gonadotropin homeostasis and fertility in mice. Our studies also implicate microRNAs in gonadal feedback control of gonadotropin synthesis and secretion. Thus, DICER-dependent microRNAs confer a new layer of transcriptional and post-transcriptional regulation in gonadotropes to orchestrate the hypothalamus-pituitary-gonadal axis physiology.

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.

HESX1 mutations in patients with congenital hypopituitarism: variable phenotypes with the same genotype.

INTRODUCTION: Mutations in the transcription factor HESX1 can cause isolated growth hormone deficiency (IGHD) or combined pituitary hormone deficiency (CPHD) with or without septo-optic dysplasia (SOD). So far there is no clear genotype-phenotype correlation. PATIENTS AND RESULTS: We report four different recessive loss-of-function mutations in three unrelated families with CPHD and no midline defects or SOD. A homozygous p.R160C mutation was found by Sanger sequencing in two siblings from a consanguineous family. These patients presented with ACTH, TSH and GH deficiencies, severe anterior pituitary hypoplasia (APH) or pituitary aplasia (PA) and normal posterior pituitary. The p.R160C mutation was previously reported in a case with SOD, CPHD and ectopic posterior pituitary (EPP). Using exome sequencing, a homozygous p.I26T mutation was found in a Brazilian patient born to consanguineous parents. This patient had evolving CPHD, normal ACTH, APH and normal posterior pituitary (NPP). A previously reported patient homozygous for p.I26T had evolving CPHD and EPP. Finally, we identified compound heterozygous mutations in HESX1, p.[R159W];[R160H], in a patient with PA and CPHD. We showed that both of these mutations abrogate the ability of HESX1 to repress PROP1-mediated transcriptional activation. A patient homozygous for p.R160H was previously reported in a patient with CPHD, EPP, APH. CONCLUSION: These three examples demonstrate that HESX1 mutations cause variable clinical features in patients, which suggests an influence of modifier genes or environmental factors on the phenotype.

The pattern of congenital heart defects arising from reduced Tbx5 expression is altered in a Down syndrome mouse model.

BACKGROUND: Nearly half of all individuals with Down Syndrome (DS) have some type of congenital heart defect (CHD), suggesting that DS sensitizes to CHD but does not cause it. We used a common mouse model of DS, the Ts65Dn mouse, to study the contribution of Tbx5, a known modifier of CHD, to heart defects on a trisomic backgroun. Mice that were heterozygous for a Tbx5 null allele were crossed with Ts65Dn mice. Thoraxes of progeny were fixed in 10% formalin, embedded in paraffin, and sectioned for analysis of CHD. Gene expression in embryonic hearts was examined by quantitative PCR and in situ hybridization. A TBX5 DNA binding site was verified by luciferase assays. METHODS: Mice that were heterozygous for a Tbx5 null allele were crossed with Ts65Dn mice. Thoraxes of progeny were fixed in 10% formalin, embedded in paraffin, and sectioned for analysis of CHD. Gene expression in embryonic hearts was examined by quantitative PCR and in situ hybridization. A TBX5 DNA binding site was verified by luciferase assays. RESULTS: We crossed mice that were heterozygous for a Tbx5 null allele with Ts65Dn mice. Mice that were trisomic and carried the Tbx5 mutation (Ts65Dn;Tbx5 (+/-) ) had a significantly increased incidence of overriding aorta compared to their euploid littermates. Ts65Dn;Tbx5 (+/-) mice also showed reduced expression of Pitx2, a molecular marker for the left atrium. Transcript levels of the trisomic Adamts1 gene were decreased in Tbx5 (+/-) mice compared to their euploid littermates. Evidence of a valid binding site for TBX5 upstream of the trisomic Adamts1 locus was also shown. CONCLUSION: Haploinsufficiency of Tbx5 and trisomy affects alignment of the aorta and this effect may stem from deviations from normal left-right patterning in the heart. We have unveiled a previously unknown interaction between the Tbx5 gene and trisomy, suggesting a connection between Tbx5 and trisomic genes important during heart development.

A novel loss-of-function mutation in Npr2 clarifies primary role in female reproduction and reveals a potential therapy for acromesomelic dysplasia, Maroteaux type.

We discovered a new spontaneous mutant allele of Npr2 named peewee (pwe) that exhibits severe disproportionate dwarfism and female infertility. The pwe phenotype is caused by a four base-pair deletion in exon 3 that generates a premature stop codon at codon 313 (L313X). The Npr2(pwe/pwe) mouse is a model for the human skeletal dysplasia acromesomelic dysplasia, Maroteaux type (AMDM). We conducted a thorough analysis of the female reproductive tract and report that the primary cause of Npr2(pwe/pwe) female infertility is premature oocyte meiotic resumption, while the pituitary and uterus appear to be normal. Npr2 is expressed in chondrocytes and osteoblasts. We determined that the loss of Npr2 causes a reduction in the hypertrophic and proliferative zones of the growth plate, but mineralization of skeletal elements is normal. Mutant tibiae have increased levels of the activated form of ERK1/2, consistent with the idea that natriuretic peptide receptor type 2 (NPR2) signaling inhibits the activation of the MEK/ERK mitogen activated protein kinase pathway. Treatment of fetal tibiae explants with mitogen activated protein kinase 1 and 2 inhibitors U0126 and PD325901 rescues the Npr2(pwe/pwe) growth defect, providing a promising foundation for skeletal dysplasia therapeutics.

The Histone Methyltransferase Gene Absent, Small, or Homeotic Discs-1 Like Is Required for Normal Hox Gene Expression and Fertility in Mice.

Chromatin remodeling influences gene expression in developing and adult organisms. Active and repressive marks of histone methylation dictate the embryonic expression boundaries of developmentally regulated genes, including the Hox gene cluster. Drosophila ash1 (absent, small or homeotic discs 1) gene encodes a histone methyltransferase essential for regulation of Hox gene expression that interacts genetically with other members of the trithorax group (TrxG). While mammalian members of the mixed lineage leukemia (Mll) family of TrxG genes have roles in regulation of Hox gene expression, little is known about the expression and function of the mammalian ortholog of the Drosophila ash1 gene, Ash1-like (Ash1l). Here we report the expression of mouse Ash1l gene in specific structures within various organs and provide evidence that reduced Ash1l expression has tissue-specific effects on mammalian development and adult homeostasis. Mutants exhibit partially penetrant postnatal lethality and failure to thrive. Surviving mutants have growth insufficiency, skeletal transformations, and infertility associated with developmental defects in both male and female reproductive organs. Specifically, expression of Hoxa11 and Hoxd10 are altered in the epididymis of Ash1l mutant males and Hoxa10 is reduced in the uterus of Ash1l mutant females. In summary, we show that the histone methyltransferase Ash1l is important for the development and function of several tissues and for proper expression of homeotic genes in mammals.

Pituitary stem cells: past, present and future perspectives.

Pituitary cells that express the transcription factor SOX2 are stem cells because they can self-renew and differentiate into multiple pituitary hormone-producing cell types as organoids. Wounding and physiological challenges can activate pituitary stem cells, but cell numbers are not fully restored, and the ability to mobilize stem cells decreases with increasing age. The basis of these limitations is still unknown. The regulation of stem cell quiescence and activation involves many different signalling pathways, including those mediated by WNT, Hippo and several cytokines; more research is needed to understand the interactions between these pathways. Pituitary organoids can be formed from human or mouse embryonic stem cells, or from human induced pluripotent stem cells. Human pituitary organoid transplantation is sufficient to induce corticosterone release in hypophysectomized mice, raising the possibility of therapeutic applications. Today, pituitary organoids have the potential to assess the role of individual genes and genetic variants on hormone production ex vivo, providing an important tool for the advancement of exciting frontiers in pituitary stem cell biology and pituitary organogenesis. In this article, we provide an overview of notable discoveries in pituitary stem cell function and highlight important areas for future research.

Knockout mice with pituitary malformations help identify human cases of hypopituitarism.

BACKGROUND: Congenital hypopituitarism (CH) and its associated syndromes, septo-optic dysplasia (SOD) and holoprosencephaly (HPE), are midline defects that cause significant morbidity for affected people. Variants in 67 genes are associated with CH, but a vast majority of CH cases lack a genetic diagnosis. Whole exome and whole genome sequencing of CH patients identifies sequence variants in genes known to cause CH, and in new candidate genes, but many of these are variants of uncertain significance (VUS). METHODS: The International Mouse Phenotyping Consortium (IMPC) is an effort to establish gene function by knocking-out all genes in the mouse genome and generating corresponding phenotype data. We used mouse embryonic imaging data generated by the Deciphering Mechanisms of Developmental Disorders (DMDD) project to screen 209 embryonic lethal and sub-viable knockout mouse lines for pituitary malformations. RESULTS: Of the 209 knockout mouse lines, we identified 51 that have embryonic pituitary malformations. These genes not only represent new candidates for CH, but also reveal new molecular pathways not previously associated with pituitary organogenesis. We used this list of candidate genes to mine whole exome sequencing data of a cohort of patients with CH, and we identified variants in two unrelated cases for two genes, MORC2 and SETD5, with CH and other syndromic features. CONCLUSIONS: The screening and analysis of IMPC phenotyping data provide proof-of-principle that recessive lethal mouse mutants generated by the knockout mouse project are an excellent source of candidate genes for congenital hypopituitarism in children.

Efficient, specific, developmentally appropriate cre-mediated recombination in anterior pituitary gonadotropes and thyrotropes.

Tissue-specific expression of cre recombinase is a well-established genetic tool to analyze gene function, and it is limited only by the efficiency and specificity of available cre mouse strains. Here, we report the generation of a transgenic line containing a cre cassette with codon usage optimized for mammalian cells (iCre) under the control of a mouse glycoprotein hormone α-subunit (αGSU) regulatory sequences in a bacterial artificial chromosome genomic clone. Initial analysis of this transgenic line, Tg(αGSU-iCre), with cre reporter strains reveals onset of cre activity in the differentiating cells of the developing anterior pituitary gland at embryonic day 12.5, with a pattern characteristic of endogenous αGSU. In adult mice, αGSU-iCre was active in the anterior lobe of the pituitary gland and in the cells that produce αGSU (gonadotropes and thyrotropes) with high penetrance. Little or no activity was observed in other tissues, including skeletal and cardiac muscle, brain, kidney, lungs, testis, ovary, and liver. This αGSU-iCre line is suitable for efficient, specific, and developmentally regulated deletion of floxed alleles in anterior pituitary gonadotropes and thyrotropes.

Pituitary Stem Cell Regulation by Zeb2 and BMP Signaling.

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-ß 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.