ISL1 Is Necessary for Maximal Thyrotrope Response to Hypothyroidism.

ISLET1 is a homeodomain transcription factor necessary for development of the pituitary, retina, motor neurons, heart, and pancreas. Isl1-deficient mice (Isl1(-/-)) die early during embryogenesis at embryonic day 10.5 due to heart defects, and at that time, they have an undersized pituitary primordium. ISL1 is expressed in differentiating pituitary cells in early embryogenesis. Here, we report the cell-specific expression of ISL1 and assessment of its role in gonadotropes and thyrotropes. Isl1 expression is elevated in pituitaries of Cga(-/-) mice, a model of hypothyroidism with thyrotrope hypertrophy and hyperplasia. Thyrotrope-specific disruption of Isl1 with Tshb-cre is permissive for normal serum TSH, but T4 levels are decreased, suggesting decreased thyrotrope function. Inducing hypothyroidism in normal mice causes a reduction in T4 levels and dramatically elevated TSH response, but mice with thyrotrope-specific disruption of Isl1 have a blunted TSH response. In contrast, deletion of Isl1 in gonadotropes with an Lhb-cre transgene has no obvious effect on gonadotrope function or fertility. These results show that ISL1 is necessary for maximal thyrotrope response to hypothyroidism, in addition to its role in development of Rathke's pouch.

PITX2 AND PITX1 regulate thyrotroph function and response to hypothyroidism.

Pitx2 is a homeodomain transcription factor required in a dose-dependent manner for the development of multiple organs. Pitx2-null homozygotes (Pitx2(-/-)) have severe pituitary hypoplasia, whereas mice with reduced-function alleles (Pitx2(neo/neo)) exhibit modest hypoplasia and reduction in the developing gonadotroph and Pou1f1 lineages. PITX2 is expressed broadly in Rathke's pouch and the fetal pituitary gland. It predominates in adult thyrotrophs and gonadotrophs, although it is not necessary for gonadotroph function. To test the role of PITX2 in thyrotroph function, we developed thyrotroph-specific cre transgenic mice, Tg(Tshb-cre) with a recombineered Tshb bacterial artificial chromosome that ablates floxed genes in differentiated pituitary thyrotrophs. We used the best Tg(Tshb-Cre) strain to generate thyrotroph-specific Pitx2-deficient offspring, Pitx2(flox/-;)Tg(Tshb-cre). Double immunohistochemistry confirmed Pitx2 deletion. Pitx2(flox/-);Tg(Tshb-cre) mice have a modest weight decrease. The thyroid glands are smaller, although circulating T(4) and TSH levels are in the normal range. The pituitary levels of Pitx1 transcripts are significantly increased, suggesting a compensatory mechanism. Hypothyroidism induced by low-iodine diet and oral propylthiouracil revealed a blunted TSH response in Pitx2(flox/-);Tg(Tshb-cre) mice. Pitx1 transcripts increased significantly in control mice with induced hypothyroidism, but they remained unchanged in Pitx2(flox/-);Tg(Tshb-cre) mice, possibly because Pitx1 levels were already maximally elevated in untreated mutants. These results suggest that PITX2 and PITX1 have overlapping roles in thyrotroph function and response to hypothyroidism. The novel cre transgene that we report will be useful for studying the function of other genes in thyrotrophs.

Molecular mechanisms of pituitary organogenesis: In search of novel regulatory genes.

Defects in pituitary gland organogenesis are sometimes associated with congenital anomalies that affect head development. Lesions in transcription factors and signaling pathways explain some of these developmental syndromes. Basic research studies, including the characterization of genetically engineered mice, provide a mechanistic framework for understanding how mutations create the clinical characteristics observed in patients. Defects in BMP, WNT, Notch, and FGF signaling pathways affect induction and growth of the pituitary primordium and other organ systems partly by altering the balance between signaling pathways. The PITX and LHX transcription factor families influence pituitary and head development and are clinically relevant. A few later-acting transcription factors have pituitary-specific effects, including PROP1, POU1F1 (PIT1), and TPIT (TBX19), while others, such as NeuroD1 and NR5A1 (SF1), are syndromic, influencing development of other endocrine organs. We conducted a survey of genes transcribed in developing mouse pituitary to find candidates for cases of pituitary hormone deficiency of unknown etiology. We identified numerous transcription factors that are members of gene families with roles in syndromic or non-syndromic pituitary hormone deficiency. This collection is a rich source for future basic and clinical studies.

Skeletal dysplasia and male infertility locus on mouse chromosome 9.

In mice and humans, growth insufficiency and male infertility are common disorders that are genetically and phenotypically complex. We describe a spontaneously arising mouse mutant, chagun, that is affected by both dwarfism and male infertility. Dwarfism disproportionately affects long bones and is characterized by a defect in the proliferative zone of chondrocytes in the growth plate. Gonads of mutant males are small, with apparent germ cell loss and no evidence of mature sperm. The locus responsible for chagun is recessive and maps to distal chromosome 9, in a region homologous to human chromosome 3. This location is consistent with chagun defining a novel locus. Identification of the mutant gene will uncover the basis for another type of skeletal dysplasia and male infertility.

Developmental regulation of Notch signaling genes in the embryonic pituitary: Prop1 deficiency affects Notch2 expression.

Normal development of the pituitary gland requires coordination between the maintenance of a progenitor cell pool and the selection of progenitor cells for differentiation. As Notch signaling controls progenitor cell differentiation in many embryonic tissues, we investigated the involvement of this important developmental pathway in the embryonic pituitary. We report that expression of Notch signaling genes is spatially and temporally regulated in pituitary embryogenesis and implicate Notch2 in the differentiation of several cell lineages. Notch2, Notch3, and Dll1 are initially expressed by most cells within the pituitary primordium and become restricted to a subset of the progenitor cell pool as differentiated pituitary cells begin to appear. Mutations in the transcription factor Prop1 interfere with pituitary growth and cell specification, although the mechanism is unknown. Notch2 expression is nearly absent in the developing pituitaries of Prop1 mutant mice, but unaltered in some other panhypopituitary mutants, revealing that Prop1 is directly or indirectly required for normal Notch2 expression. Transgenic overexpression of Prop1 is not sufficient for enhancement of endogenous Notch2 expression, indicating that there are multiple inputs into this pathway. Dll3 is expressed only in the presumptive corticotrope and melanotrope cells. Analysis of Dll3 null mutants indicates that Dll3 is not required for specification of these two cell types, although there may be functional overlap with Dll1. The spatial and temporal expression patterns of Notch signaling genes in the pituitary suggest overlapping roles in pituitary growth and cell specification.

Thyroid hormone-responsive pituitary hyperplasia independent of somatostatin receptor 2.

Mice homozygous for the targeted disruption of the glycoprotein hormone alpha-subunit (alphaGsu) display hypertrophy and hyperplasia of the anterior pituitary thyrotropes. Thyrotrope hyperplasia results in tumors in aged alphaGsu(-/-) mice. These adenomatous pituitaries can grow independently as intrascapular transplants in hypothyroid mice, suggesting that they have progressed beyond simple hyperplasia. We used magnetic resonance imaging to follow the growth and regression of thyrotrope adenomatous hyperplasia in response to thyroid hormone treatment and discovered that the tumors retain thyroid hormone responsiveness. Somatostatin (SMST) and its diverse receptors have been implicated in cell proliferation and tumorigenesis. To test the involvement of SMST receptor 2 (SMSTR2) in pituitary tumor progression and thyroid hormone responsiveness in alphaGsu(-/-) mutants, we generated Smstr2(-/-), alphaGsu(-/-) mice. Smstr2(-/-), alphaGsu(-/-) mice develop hyperplasia of thyrotropes, similar to alphaGsu(-/-) mutants, demonstrating that SMSTR2 is dispensable for the development of pituitary adenomatous hyperplasia. Thyrotrope hyperplasia in Smstr2(-/-), alphaGsu(-/-) mice regresses in response to T4 treatment, suggesting that SMSTR2 is not required in the T4 feedback loop regulating TSH secretion.

Hypomorphic phenotype in mice with pituitary-specific knockout of steroidogenic factor 1.

The bacteriophage Cre recombinase provides a powerful approach for tissue-specific gene inactivation. Using a Cre transgene driven by the common alpha subunit of glycoprotein hormones (alphaGSU-Cre), we have previously inactivated steroidogenic factor 1 (SF-1) in the anterior pituitary, causing hypogonadotropic hypogonadism with sexual infantilism, sterility, and severe gonadal hypoplasia. We now explore the molecular mechanisms underlying a hypomorphic gonadal phenotype in mice carrying two floxed SF-1 alleles (F/F) relative to mice carrying one recombined and one floxed allele (F/R). Because their Cre-mediated disruption of the locus encoding SF-1 was less efficient, alphaGSU-Cre, F/F mice retained some gonadotropin-expressing cells in the anterior pituitary, thereby stimulating some gonadal function. This novel in vivo model for exploring the effects of differing levels of gonadotropins on gonadal development highlights the need for careful genotype-phenotype comparisons in studies using Cre recombinase to produce tissue-specific knockouts.

Mouse models of altered CRH-binding protein expression.

CRH is the key physiological mediator of the endocrine, autonomic, and behavioral responses to stress. The recent characterization of urocortin, a new mammalian CRH-like ligand, adds to the complexity of the CRH system. Both CRH and urocortin mediate their endocrine and/or synaptic effects via two classes of CRH receptors. Similarly, both CRH and urocortin bind to the CRH-binding protein (CRH-BP). This secreted binding protein is smaller than the CRH receptors, but binds CRH and urocortin with an affinity equal to or greater than that of the receptors, and blocks CRH-mediated ACTH release in vitro. Several regions of CRH-BP expression colocalize with sites of CRH synthesis or release, suggesting that this binding protein may have a profound impact on the biological activity of CRH (or urocortin). While in vitro and in vivo studies have characterized the biochemical properties and regulation of the CRH-BP, animal models of altered CRH-BP expression can provide additional information on the in vivo role of this important modulatory protein. This review focuses on three mouse models of CRH-BP overexpression or deficiency. These animal models show numerous physiological changes in the HPA axis and in energy balance, with additional alterations in anxiogenic behavior. These changes are consistent with the hypothesis that CRH-BP plays an important in vivo modulatory role by regulating levels of "free" CRH and other CRH-like peptides in the pituitary and central nervous system.

Persistent Prop1 expression delays gonadotrope differentiation and enhances pituitary tumor susceptibility.

The 'paired'-like homeodomain transcription factor Prop1 is essential for the expansion of the pituitary primordia and for the differentiation and/or function of the hormone-producing cells of the anterior pituitary gland. Prop1 expression is normally extinguished before transcription of most differentiation markers is initiated. We report that constitutive expression of Prop1 interferes with anterior pituitary cell differentiation and increases the susceptibility for pituitary tumors. The terminal differentiation of pituitary gonadotropes is delayed, resulting in transient hypogonadism and a delay in the onset of puberty. Thyrotrope differentiation occurs normally, but thyrotrope function is impaired resulting in mild hypothyroidism. Aged mice exhibit defects consistent with misregulation of pituitary cell proliferation, including adenomatous hyperplasia with the formation of Rathke's cleft cysts and tumors. Thus, silencing Prop1 is important for normal pituitary development and function. These data suggest that gain-of-function mutations in PROP1 could contribute to the most common human pituitary endocrinopathies and tumors.

Steroidogenic factor 1 (SF1) is essential for pituitary gonadotrope function.

Knockout mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF1) exhibit a complex endocrine phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and absence of the ventromedial hypothalamic nucleus (VMH). These multiple defects complicate efforts to delineate primary versus secondary effects of SF1 deficiency in different tissues, such that its direct role in gonadotropes remains uncertain. To define this role, we have expressed Cre recombinase driven by the promoter region of the common alpha subunit of glycoprotein hormones (alpha GSU), thereby inactivating a loxP-modified SF1 locus in the anterior pituitary gland. Although pituitary-specific SF1 knockout mice were fully viable, they were sterile and failed to develop normal secondary sexual characteristics. Their adrenal glands and VMH appeared normal histologically, but their testes and ovaries were severely hypoplastic. alpha GSU-Cre, loxP mice had normal levels of most pituitary hormones, but had markedly decreased expression of LH and FSH. Treatment with exogenous gonadotropins stimulated gonadal steroidogenesis, inducing germ cell maturation in males and follicular and uterine maturation in females--establishing that the gonads can respond to gonadotropins. The pituitary-specific SF1 knockout mice are a novel genetic model of hypogonadotropic hypogonadism that establishes essential role(s) of SF1 in pituitary gonadotropes.

Identification of members of the Wnt signaling pathway in the embryonic pituitary gland.

Prop1 is one of several transcription factors important for the development of the pituitary gland. Downstream targets of PROP1 and other critical pituitary transcription factors remain largely unknown. We have generated a partial expression profile of the developing pituitary gland containing over 350 transcripts, using cDNA subtractive hybridization between Prop1(df/df) and wild-type embryonic pituitary gland primordia. Numerous classes of genes including transcription factors, membrane associated molecules, and cell cycle regulators were identified in this study. Of the transcripts, 34% do not have sequence similarity to known genes, but are similar to ESTs, and 4% represent novel sequences. Pituitary gland expression of a number of clones was verified using in situ hybridization. Several members of the Wnt signaling pathway were identified in the developing pituitary gland. The frizzled2 receptor, Apc, beta-catenin, groucho, and a novel isoform of TCF4 (officially named Tcf7l2) were identified in developing pituitary libraries. Three N-terminal alternatively spliced Tcf7l2 isoforms are reported here, each of which lacks a DNA-binding domain. Functional studies indicate that these isoforms can act as endogenous inhibitors of Wnt signaling in some contexts. This is the first report of Tcf7l2 and Fzd2 expression in the developing pituitary. These molecules may be important in mediating Wnt signaling during pituitary ontogeny. We expect other transcripts from these libraries to be involved in pituitary gland development.

Cre-mediated recombination in the pituitary gland.

Organ-specific expression of a cre recombinase transgene allows for the analysis of gene function in a particular tissue or cell type. Using a 4.6 kb promoter from the mouse glycoprotein hormone alpha-subunit (alphaGSU or Cga) gene, we have generated and characterized a line of transgenic mice that express cre recombinase in the anterior and intermediate lobes of the pituitary gland. Utilizing a cre-responsive reporter transgene, alphaGSU-cre transgene expression was detected in the pituitary primordium and in all five cell types of the adult anterior pituitary. alphaGSU-cre transgene activity was also detected in the cardiac and skeletal muscle. Little or no activity was evident in the gonads, adrenal glands, brain, ventromedial hypothalamus, or kidneys. The alphaGSU-cre transgenic mice characterized here will be a valuable tool for examining gene function in the pituitary gland.

Characterisation and genetic mapping of a new X linked deafness syndrome.

BACKGROUND: Hereditary forms of hearing loss are classified as syndromic, when deafness is associated with other clinical features, or non-syndromic, when deafness occurs without other clinical features. Many types of syndromic deafness have been described, some of which have been mapped to specific chromosomal regions. METHODS: Here we describe a family with progressive sensorineural hearing loss, cognitive impairment, facial dysmorphism, and variable other features, transmitted by apparent X linked recessive inheritance. Haplotype analysis of PCR products spanning the X chromosome and direct sequencing of candidate genes were used to begin characterising the molecular basis of features transmitted in this family. Comparison to known syndromes involving deafness, mental retardation, facial dysmorphism, and other clinical features was performed by review of published reports and personal discussions. RESULTS: Genetic mapping places the candidate locus for this syndrome within a 48 cM region on Xq1-21. Candidate genes including COL4A5, DIAPH, and POU3F4 were excluded by clinical and molecular analyses. CONCLUSIONS: The constellation of clinical findings in this family (deafness, cognitive impairment, facial dysmorphism, variable renal and genitourinary abnormalities, and late onset pancytopenia), along with a shared haplotype on Xq1-21, suggests that this represents a new form of syndromic deafness. We discuss our findings in comparison to several other syndromic and non-syndromic deafness loci that have been mapped to the X chromosome.

The motor and tail regions of myosin XV are critical for normal structure and function of auditory and vestibular hair cells.

Recessive mutations in myosin 15, a class XV unconventional myosin, cause profound congenital deafness in humans and both deafness and vestibular dysfunction in mice homozygous for the shaker 2 and shaker 2(J) alleles. The shaker 2 allele is a previously described missense mutation of a highly conserved residue in the motor domain of myosin XV. The shaker 2(J) lesion, in contrast, is a 14.7 kb deletion that removes the last six exons from the 3"-terminus of the Myo15 transcript. These exons encode a FERM (F, ezrin, radixin and moesin) domain that may interact with integral membrane proteins. Despite the deletion of six exons, Myo15 mRNA transcripts and protein are present in the post-natal day 1 shaker 2(J) inner ear, which suggests that the FERM domain is critical for the development of normal hearing and balance. Myo15 transcripts are first detectable at embryonic day 13.5 in wild-type mice. Myo15 transcripts in the mouse inner ear are restricted to the sensory epithelium of the developing cristae ampularis, macula utriculi and macula sacculi of the vestibular system as well as to the developing organ of Corti. Both the shaker 2 and shaker 2(J) alleles result in abnormally short hair cell stereocilia in the cochlear and vestibular systems. This suggests that Myo15 may be important for both the structure and function of these sensory epithelia.

Thyroid hormone resistance and increased metabolic rate in the RXR-gamma-deficient mouse.

Vitamin A and retinoids affect pituitary-thyroid function through suppression of serum thyroid-stimulating hormone (TSH) levels and TSH-beta subunit gene expression. We have previously shown that retinoid X receptor-selective (RXR-selective) ligands can suppress serum TSH levels in vivo and TSH-beta promoter activity in vitro. The RXR-gamma isotype has limited tissue distribution that includes the thyrotrope cells of the anterior pituitary gland. In this study, we have performed a detailed analysis of the pituitary-thyroid function of mice lacking the gene for the RXR-gamma isotype. These mice had significantly higher serum T4 levels and TSH levels than did wild-type (WT) controls. Treatment of RXR-gamma-deficient and WT mice with T3 suppressed serum TSH and T4 levels in both groups, but RXR-gamma-deficient mice were relatively resistant to exogenous T3. RXR-gamma-deficient mice had significantly higher metabolic rates than did WT controls, suggesting that these animals have a pattern of central resistance to thyroid hormone. RXR-gamma, which is also expressed in skeletal muscle and the hypothalamus, may have a direct effect on muscle metabolism, regulation of food intake, or thyrotropin-releasing hormone levels in the hypothalamus. In conclusion, the RXR-gamma isotype appears to contribute to the regulation of serum TSH and T4 levels and to affect peripheral metabolism through regulation of the hypothalamic-pituitary-thyroid axis or through direct effects on skeletal muscle.

Partial transcriptome of the developing pituitary gland.

The anterior lobe of the pituitary gland is composed of five hormone-producing cell types and develops from Rathke's pouch, an invagination of oral ectoderm. In mice, rapid cell proliferation occurs in the pouch from embryonic day 12.5 (e12.5) to e14.5, preceding the appearance of most hormone transcripts. Cell-type-specific commitment probably occurs prior to e14.5, but cell differentiation can be demonstrated only by detection of hormone transcripts. Although several transcription factors critical for pouch expansion are known, few of their target genes have been identified. To identify putative transcription factor target genes and cell-type-specific markers, we used differential display PCR analysis of RNA prepared from e12.5 and e14.5 Rathke's pouches. We present an expression profile of the developing pituitary gland including 83 transcripts, 40% of which are novel. The tissue distribution, cell specificity, and developmental regulation were determined for a subset of the transcripts.

Hair cells in the inner ear of the pirouette and shaker 2 mutant mice.

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions "cytocauds." The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.

Characterization of the human and mouse unconventional myosin XV genes responsible for hereditary deafness DFNB3 and shaker 2.

Mutations in myosin XV are responsible for congenital profound deafness DFNB3 in humans and deafness and vestibular defects in shaker 2 mice. By combining direct cDNA analyses with a comparison of 95.2 kb of genomic DNA sequence from human chromosome 17p11.2 and 88.4 kb from the homologous region on mouse chromosome 11, we have determined the genomic and mRNA structures of the human (MYO15) and mouse (Myo15) myosin XV genes. Our results indicate that full-length myosin XV transcripts contain 66 exons, are >12 kb in length, and encode 365-kDa proteins that are unique among myosins in possessing very long approximately 1200-aa N-terminal extensions preceding their conserved motor domains. The tail regions of the myosin XV proteins contain two MyTH4 domains, two regions with similarity to the membrane attachment FERM domain, and a putative SH3 domain. Northern and dot blot analyses revealed that myosin XV is expressed in the pituitary gland in both humans and mice. Myosin XV transcripts were also observed by in situ hybridization within areas corresponding to the sensory epithelia of the cochlea and vestibular systems in the developing mouse inner ear. Immunostaining of adult mouse organ of Corti revealed that myosin XV protein is concentrated within the cuticular plate and stereocilia of cochlear sensory hair cells. These results indicate a likely role for myosin XV in the formation or maintenance of the unique actin-rich structures of inner ear sensory hair cells.