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International audience; 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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12861-015-0080-y) contains supplementary material, which is available to authorized users.

FOXL2 is a forkhead transcription factor expressed in the eye, ovary, and pituitary gland. Loss of function mutations in humans and mice confirm a functional role for FOXL2 in the eye and ovary, but its role in the pituitary is not yet defined. We report that FOXL2 colocalizes with the glycoprotein hormone alpha-subunit (alphaGSU) in quiescent cells of the mouse pituitary from embryonic d 11.5 through adulthood. FOXL2 is expressed in essentially all gonadotropes and thyrotropes and a small fraction of prolactin-containing cells during pregnancy, but not somatotropes or corticotropes. The coincident expression patterns of FOXL2 and alphaGSU suggested that the alphaGSU gene (Cga) is a downstream target of FOXL2. We demonstrate that FOXL2 regulates mouse Cga transcription in gonadotrope-derived (alphaT3-1, LbetaT2), thyrotrope-derived (alphaTSH) and heterologous (CV-1) cells in a context-dependent manner. In addition, a FOXL2-VP16 fusion protein is sufficient to stimulate ectopic Cga expression in transgenic animals. Normal FOXL2 expression requires the transcription factors Lhx3 and Lhx4 but not of Prop1. Thus, FOXL2 expression is affected by mutations in early pituitary developmental regulatory genes, and its expression precedes that of genes necessary for gonadotrope-specific development such as Egr1 and Sf1 (Nr5a1). These data place FOXL2 in the hierarchy of pituitary developmental control and suggest a role in regulation of Cga gene expression.

Mutations in the transcription factors PIT1 (pituitary transcription factor 1) and PROP1 (prophet of Pit1) lead to pituitary hormone deficiency and hypopituitarism in mice and humans. To determine the basis for this, we performed histological analysis of Pit1- and Prop1-deficient dwarf mouse pituitaries throughout fetal and postnatal development. Pit1-deficient mice first exhibit pituitary hypoplasia after birth, primarily caused by reduced cell proliferation, although there is some apoptosis. To determine whether altered development of the vascular system contributes to hypopituitarism, we examined vascularization from embryonic d 14.5 and throughout development. No obvious differences in vascularization are evident in developing Pit1-deficient pituitaries. In contrast, the Prop1-deficient mouse pituitaries are poorly vascularized and dysmorphic, with a striking elevation in apoptosis. At postnatal d 11, apoptosis-independent caspase-3 activation occurs in thyrotropes and somatotropes of normal but not mutant pituitaries. This suggests that Prop1 and/or Pit1 may be necessary for caspase-3 expression. These studies provide further insight as to the mechanisms of Prop1 and Pit1 action in mice.

The shaker2 (sh2) mouse is a murine model for human non-syndromic deafness DFNB3. The mice have abnormal circling behavior suggesting a balanced disorder, and profound deafness. The insertion of a bacterial artificial chromosome (BAC) transgene containing the Myo15a gene into sh2/sh2 zygotes confers hearing capability and abolishes the circling behavior in 1-month-old transgenic animals. In this study, we investigated both the hearing and the morphology of the cochlea in Myo15a mutants carrying this BAC transgene at two, four, or six months of age. The hearing threshold of these mice is normal, with no physiologically significant differences compared to age-matched heterozygous sh2J mice (with or without the BAC transgene). In six-month-old transgenic mice with the BAC, the morphology of hair cells in the apical and upper basal turns of the cochlea is normal. Hair cells of lower basal turn, however, were missing in some mutant animals. This study demonstrates that BAC transgene correction cannot only maintain normal morphology but also confer stable hearing function in Myo15a mutant mice for as long as 6 months. In addition, excess Myo15a expression has no physiologically significant protective or deleterious effects on hearing of normal mice, suggesting that the dosage of Myo15a may not be problematic for gene therapy.

AML1/Runx1 (Runx1) is a mammalian transcription factor that plays critical roles in regulating the differentiation of a number of different cell types. In the present study, we have utilized mice expressing beta-galactosidase (beta-gal) under the control of the Runx1 promoter to characterize the spatiotemporal expression pattern of Runx1 during retinogenesis. Expression of beta-gal was first detected at embryonic day 13.5 in post-mitotic cells located in the inner retina and overlapped with expression of the early amacrine and ganglion cell marker protein Islet1. During subsequent developmental stages, the number of beta-gal-positive cells increased in a central-to-peripheral gradient until late embryogenesis but then decreased in the early post-natal retina. beta-gal-positive cells were located primarily in the ganglion cell layer by late embryonic/early post-natal stages and were identified as a subpopulation of displaced amacrine cells by the continued expression of Islet1, as well as Pax6, and the coexpression of the amacrine cell subtype-specific markers choline acetyltransferase, calretinin and the 65-kDa isoform of glutamic acid decarboxylase. These findings identify Runx1 as a novel marker for a restricted amacrine cell subtype and suggest a role for this gene in regulating the post-mitotic development of these cells.

The PITX family of transcription factors regulate the development of many organs. Pitx1 mutants have a mild pituitary phenotype, but Pitx2 is necessary for the development of Rathke's pouch, expression of essential transcription factors in gonadotropes, and expansion of the Pit1 lineage. We report that lack of Pitx2 causes the pouch to undergo excessive cell death, resulting in severe pituitary hypoplasia. Transgenic overexpression of PITX2 in the pituitary can increase the gonadotrope population, suggesting that the absolute concentration of PITX2 is important for normal pituitary cell lineage expansion. We show that PITX1 and PITX2 proteins are present in similar expression patterns throughout pituitary development and in the mature pituitary. Both transcription factors are preferentially expressed in adult gonadotropes and thyrotropes, suggesting the possibility of overlap in maintenance of adult pituitary functions within these cell types. Double knockouts of Pitx1 and Pitx2 exhibit severe pituitary hypoplasia and fail to express the transcription factor LHX3. This indicates that these PITX genes are upstream of Lhx3 and have compensatory roles during development. Thus, the combined dosage of these PITX family members is vital for pituitary development, and their persistent coexpression in the adult pituitary suggests a continued role in maintenance of pituitary function.

To study the effects of premature and chronic ligand-mediated luteinizing hormone receptor (LHR) activation on reproductive development, we have generated transgenic mice expressing a genetically engineered, constitutively active yoked hormone–receptor complex (YHR), in which a fusion protein of human chorionic gonadotropin (hCG) is covalently linked to the N-terminus of rat LHR. YHR-expressing mice (YHR+) were analyzed at pre- and post-pubertal ages. Relative to wild type (WT) controls, male mice exhibited prepubertal increases in testosterone levels and seminal vesicle weights, and decreases in serum FSH, serum LH, testes weight, and the size of the seminiferous tubules. In adult male YHR+ mice, testosterone and LH levels are not significantly different from WT controls. However, FSH levels and testes weights remain decreased. Female YHR+ mice undergo precocious puberty with early vaginal opening, accelerated uterine development, enhanced follicular development, including the presence of corpora lutea, and an increase in serum progesterone. At 12 weeks of age, the ovary exhibits a relative increase in the amount of interstitial tissue, comprised of cells that are hypertrophic and luteinized, as well as follicles that are degenerating. Additionally, hemorrhagic cysts develop in approximately 25% of the transgenic mice. These degenerative changes are consistent with an aging ovary suggesting that CG-induced LHR activation in female mice leads to precocious sexual development and ovarian lesions. Taken together, these data indicate that the single chain YHR is functional in vivo and demonstrate that YHR+ mice provide a novel system to further understand the reproductive consequences of aberrant LHR activation.

Mutations in the PROP1 transcription factor gene lead to reduced production of thyrotropin, GH, prolactin, and gonadotropins as well as to pituitary hypoplasia in adult humans and mice. Some PROP1-deficient patients initially exhibit pituitary hyperplasia that resolves to hypoplasia. To understand this feature and to explore the mechanism whereby PROP1 regulates anterior pituitary gland growth, we carried out longitudinal studies in normal and Prop1-deficient dwarf mice from early embryogenesis through adulthood, examining the volume of Rathke’s pouch and its derivatives, the position and number of dividing cells, the rate of apoptosis, and cell migration by pulse labeling. The results suggest that anterior pituitary progenitors normally leave the perilumenal region of Rathke’s pouch and migrate to form the anterior lobe as they differentiate. Some of the cells that seed the anterior lobe during organogenesis have proliferative potential, supporting the expansion of the anterior lobe after birth. Prop1-deficient fetal pituitaries are dysmorphic because mutant cells are retained in the perilumenal area and fail to differentiate. After birth, mutant pituitaries exhibit enhanced apoptosis and reduced proliferation, apparently because the mutant anterior lobe is not seeded with progenitors. These studies suggest a mechanism for Prop1 action and an explanation for some of the clinical findings in human patients.

Pitx2, a homeodomain transcription factor, is essential for normal development of the pituitary gland, craniofacial region, eyes, heart, abdominal viscera, and limbs. Complete loss of Pitx2 in mice (Pitx2(-/-)) results in embryonic lethality by approximately e15 due to cardiac defects, whereas embryos with partial loss of function (Pitx2(neo/-) or Pitx2(neo/neo)) survive until later in development (e17-e19). Pitx2 is expressed in discrete populations of postmitotic neurons in the mouse brain, but its role in mammalian central nervous system (CNS) development is not known. We undertook an analysis of Pitx2-deficient embryos to determine whether loss of Pitx2 affects CNS development. The CNS is normal in hypomorphic e16.5 Pitx2(neo/-) and e18.5 Pitx2(neo/neo) embryos, with no evidence of midline or other defects. Midgestation (e10.5) Pitx2(-/-) embryos have normally formed neural tube structures and cerebral vesicles, whereas older (e14.5) Pitx2(-/-) embryos exhibit loss of gene expression and axonal projections in the subthalamic nucleus (a group of cells in the ventrolateral thalamus) and in the developing superior colliculus of dorsal midbrain. Our results suggest a role for Pitx2 in regulating regionally specific terminal neuronal differentiation in the developing ventrolateral thalamus and midbrain.

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.

Mutations in the prophet of PIT1 gene (PROP1) are the most common cause of multiple pituitary hormone deficiency in humans; however, the mechanism of PROP1 action is not well understood. We report that Prop1 is essential for dorsally restricted expression of a Groucho-related gene, transducin-like enhancer of split 3 (Tle3), which encodes a transcriptional corepressor. Deficiency of a related gene, amino terminal enhancer of split (Aes), causes pituitary anomalies and growth insufficiency. TLE3 and AES have been shown to interact with TCF/LEF (transcripiton factors of the T cell-specific and lymphoid enhancer specific group) family members in cell culture systems. In the absence of TCF4 (Tcf7L2), Prop1 levels are elevated, pituitary hyperplasia ensues and palate closure is abnormal. Thus, we demonstrate that Tcf4 and Aes influence pituitary growth and development, and place Tcf4 and Tle3 in the genetic hierarchy with Prop1.

The unconventional myosin genes Myo15, Myo6 and Myo7a are essential for hearing in both humans and mice. Despite the expression of each gene in multiple organs, mutations result in identifiable phenotypes only in auditory or ocular sensory organs. The pirouette (pi) mouse also exhibits deafness and an inner ear pathology resembling that of Myo15 mutant mice and thus may be functionally related to Myo15. In order to investigate possible interactions between Myo15 and Myo6, Myo7a, and the gene affected in pirouette, we crossed Myo15(sh2/sh2) mice to the three other mutant mouse strains. Hearing in doubly heterozygous mice was similar to age-matched singly heterozygous animals, indicating that partial deficiency for both Myo15 and one of these other deafness genes does not reduce hearing. Viable double mutants were obtained from each cross, indicating that potential overlapping functions between these genes in other organs are not essential for viability. All critical cell types of the cochlear sensory epithelium were present in double mutant mice and cochlear stereocilia exhibited a superimposition of single mutant phenotypes. These data suggest that the function of Myo15 is distinct from that of Myo6, Myo7a or pi in development and/or maintenance of stereocilia.

The mouse serves as a valuable model for treatment leading to the prevention and therapy of inner ear disease. Transgenic correction of genetic inner ear disease in mice may help develop treatment for human genetic inner ear disease. In mutations involving hair cells (HCs) or supporting cells (SCs), it is necessary to insert the wild-type transgenes directly into these cells. We used inner ear explants to characterize the transgenic expression using adenovirus-mediated reporter genes (bacterial lacZ). The variable parameters were the age of the explants (P1-P5), the type of vector (first and advanced generation adenovirus) and the genotype of the mouse (wild-type versus shaker-2 mutant). Transduction of cochlear HCs was detected at P1 and in some of the P3 cochleae. Low efficiency transduction of SCs was observed in P1 explants, but the efficiency increased with age and reached high levels at P5. The pattern of transduction was similar regardless of the genotype and the type of vector used. The data demonstrate that differentiating HCs and SCs in mouse explants can be transduced by adenovirus vectors, suggesting that cultures of mouse ears are a valuable model for developing inner ear gene therapy protocols.

Analysis of an allelic series in mice revealed that the Pitx2 homeobox gene is required at multiple stages of pituitary development. It is necessary for initiating expansion of Rathke’s pouch and maintaining expression of the fetal-specific transcription factors Hesx1 and Prop1. At later stages Pitx2 is necessary for specification and expansion of the gonadotropes and Pit1 lineage within the ventral and caudomedial anterior pituitary. Mechanistically, this is due to the dependence of several critical lineage-specific transcription factors, Pit1, Gata2, Egr1 and Sf1, on a threshold level of PITX2. The related Pitx1 gene has a role in hormone gene transcription, and it is important late in ontogeny for the final expansion of the differentiated cell types. Pitx1 and Pitx2 have overlapping functions in the expansion of Rathke’s pouch, revealing the sensitivity of pituitary organogenesis to the dosage of the PITX family. The model developed for PITX gene function in pituitary development provides a better understanding of the etiology of Rieger syndrome and may extend to other PITX-sensitive developmental processes.

With the reports of the DNA sequence of the human and mouse genomes, research attention is now on questions of protein function and interactions, developmental and physiological pathways, and systems biology. The International Mouse Mutagenesis Consortium proposes goals and outlines plans for the next 10 years for annotating the mouse genome and compiling data on mouse mutations. In addition to the challenges of identifying new assays to probe biological functions, more efficient and reliable methods are needed for archiving, managing, analyzing, displaying, and disseminating the complex phenotype data sets resulting from mutagenesis programs. Achieving these goals will require the biomedical research community to improve efficiencies, to reduce costs, and to coordinate international expertise and resources.

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 α subunit of glycoprotein hormones (αGSU), 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. αGSU-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.

Summary: 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 (αGSU-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, αGSU-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. genesis 30:65–69, 2001. © 2001 Wiley-Liss, Inc.

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.

Summary 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.

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.

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, are12 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.

Pitx2 is a homeodomain transcription factor that is mutated in Rieger syndrome, a haploinsufficiency disorder affecting eyes and teeth. Pitx2 also has a postulated role in left-right axis determination. We assessed the requirements for Pitx2 directly by generating hypomorphic and null alleles. Heterozygotes for either allele have eye abnormalities consistent with Rieger syndrome. The ventral body wall fails to close in embryos homozygous for the null allele, leaving the heart and abdominal organs externalized and the body axis contorted. In homozygotes for either allele, the heart tube undergoes normal, rightward looping and the stomach is positioned normally. In contrast, homozygotes for both alleles exhibit right isomerization of the lungs. Thus, Pitx2 is required for left-right asymmetry of the lungs but not other organs. Homozygotes for either allele exhibit septal and valve defects, and null homozygotes have a single atrium proving that a threshold level of Pitx2 is required for normal heart development. Null homozygotes exhibit arrest of pituitary gland development at the committed Rathke pouch stage and eye defects including optic nerve coloboma and absence of ocular muscles. This allelic series establishes that Pitx2 is required for the development of mulitple organs in a dosage-sensitive manner.

Corticotropin-releasing hormone (CRH) is widely recognized as the primary mediator of the neuroendocrine and behavioral responses to stress, including stress-induced anxiety. The biological activity of CRH and other mammalian CRH-like peptides, such as urocortin, may be modulated by CRH-binding protein (CRH-BP). To assess directly the CRH-BP function, we created a mouse model of CRH-BP deficiency by gene targeting. Basal adrenocorticotropic hormone and corticosterone levels are unchanged in the CRH-BP-deficient mice, and the animals demonstrate a normal increase in adrenocorticotropic hormone and corticosterone after restraint stress. In contrast, adult male CRH-BP-deficient mice show significantly reduced body weight when compared with wild-type controls. CRH-BP-deficient mice also exhibit a significant increase in anxiogenic-like behavior as assessed by the elevated plus maze and defensive withdrawal tests. The increased anorectic and anxiogenic-like behavior most likely is caused by increased “free” CRH and/or urocortin levels in the brain of CRH-BP-deficient animals, suggesting an important role for CRH-BP in maintaining appropriate levels of these peptides in the central nervous system.

The mouse is the model organism for the study of hearing loss in mammals. In recent years, the identification of five different mutated genes in the mouse (Pax3, Mitf, Myo7a, Pou4f3, and Myo15) has led directly to the identification of mutations in families with either congenital sensorineural deafness or progressive sensorineural hearing loss. Each of these cases is reviewed here. In addition to providing a powerful gateway to the identification of human hearing loss genes, the study of mouse deafness mutants can lead to the discovery of critical components of the auditory system. Given the availability of several mouse mutants that affect possible homologues of other human deafness genes, it is likely that the mouse will play a key role in identifying other human hearing loss genes in the years to come.

The important roles of homeobox genes in development of the hindbrain and axial body are well established. More recently, it has become clear that certain subfamilies of homeobox genes play particularly important roles in the development of more anterior structures. These have included the paired gene family in the eye (Gehring, 1996; Hanson and Van Heyningen, 1995; Macdonald and Wilson, 1996; Wehr and Gruss, 1996), the orthodenticle and distalless gene families in the foreand midbrains (Acampora et al., 1996; Acampora et al., 1995; Price et al., 1991; Simeone et al., 1994; Williams, 1998), and the Lhx gene family in the pituitary gland (Sheng et al., 1997; Sheng et al., 1996). This review summarizes the newly identified Pitx gene family and its role in development. This family includes three vertebrate paralogues that have been cloned in multiple organisms, and a fly cognate. Mutations in two members of this gene family lead to human disease or birth defects affecting anterior structures. The nomenclature for this gene family has been complicated by the fact that members have been cloned and uniquely named by more than one laboratory (Table 1). The first member of this family, mouse Ptx1 (pituitary homeobox 1) was isolated as a transcription factor involved in pro-opiomelanocortin gene transcription in anterior pituitary corticotropes (Lamonerie et al., 1996). However, since some pentaxin genes in mouse and human had previously been assigned the Ptx gene symbol, the gene symbols for the three mouse paralogues for this new homeobox gene family are Pitx1, Pitx2, and Pitx3 (Mouse Genome Database). In this review, we have adopted the official nomenclature of the MGD and propose that, for clarity, this nomenclature be adopted for other organisms. Three vertebrate paralogues, Pitx1, Pitx2, and Pitx3, have all been cloned from mouse and human (Table 1 and references therein). Some paralogues have also been cloned from chicken (Pitx1 and Pitx2), xenopus and zebrafish (Pitx2), and rat (Pitx3) (Table 1 and references therein). In two reports, mouse Pitx1 was cloned in functional assays: in a two-hybrid screen using Pit-1 as bait (Szeto et al., 1996) and as noted above. Human PITX2 was identified by positional cloning of the Rieger Syndrome gene (Semina et al., 1996). In the other reports, cloning was the result of using degenerate PCR or low stringency hybridization to detect expressed homeobox sequences in a variety of embryonic and adult tissues. The difficulty in cloning Pitx1 from xenopus and zebrafish has suggested that this orthologue may not be as widely distributed in nature as Pitx2 (Kitamura et al., 1997). However, the recent identification of a fly Pitx gene during a chromosome walk demonstrates that this gene family arose prior to the divergence of vertebrates and invertebrates (Vorbruggen et al., 1997). Each vertebrate paralogue has been mapped genetically in mouse and human (Table 1). The Pitx proteins all belong to the bicoid-related subclass of homeodomain proteins because they encode the defining lysine at residue 50 within the homeodomain. This residue, at residue 9 within the recognition helix of the homeodomain, is the major determinant of DNA binding specificity (Gehring et al., 1994; Hanes and Brent, 1989). Several members of this small subfamily are essential for axis and pattern formation (Ang et al., 1996). Pitx2 expresses multiple protein isoforms as a result of alternative splicing (Gage and Camper, 1997; Kitamura et al., 1997) and the use of different promoters (P. Gage and E. Semina, unpublished results) (Fig. 1). The three vertebrate paralogues are all highly conserved at the amino acid level (Fig. 1). For example, in mouse the Pitx2 and Pitx3 homeodomains are identical while Pitx1 differs by only two amino acids. The paralogues are also conserved Cterminal to the homeodomain (55–70%). In contrast, the N-termini of these proteins are essentially unrelated. The vertebrate orthologues are even more highly conserved. For example, there the mouse and chicken Pitx2a proteins are 96% identical with only ten amino acid substitutions between them. The Drosophila Pitx protein shows high conservation to the vertebrate proteins within the homeodomain (90–93%) and a short region near C-terminus that has been termed the OAR sequence (Furukawa et al., 1997) or the C-peptide (Kitamura et al., 1997). This sequence is present in several homeobox genes. In Pitx2, this domain appears to function as an intrinsic inhibitor of DNA binding activity whose function can be modulated by protein-protein interactions (Amendt et al., 1998). The vertebrate Pitx genes each have unique developmental and tissue-specific expression patterns (Fig. 2 and Table 2). However, there are several significant overlaps in expression pattern (Fig. 2). The most significant may be in the eye, where both Pitx2 and Pitx3 are expressed in the mesenchyme and its derivatives (Semina et al., 1998; Semina et al., 1996; Smidt et al., 1997). Demonstration in humans that mutations to Pitx2 result in Rieger’s Syndrome (Semina et al., 1996) and mutations to Pitx3 result in anterior segment mesenchymal dysgenesis and dominant cataracts (Semina et al., 1998) confirmed the importance of these genes in eye development. These autosomal-dominant conditions each affect the development or maintenance of anterior structures of the eye. Interestingly, mouse Pitx3 maps near aphakia, a recessive mutation resulting in small eyes that lack lenses and fail to develop beyond 11 days of gestation (Semina et al., 1997). Rieger’s Syndrome patients frequently show defects in dental and umbilical development in addition to their ocular defects (Feingold et al., 1969; Rieger, 1935), and subsets of patients also present with isolated growth insufficiency (Feingold et al., 1969). Several observations suggest that Pitx genes are also important for the development and function of other organs. The stomodeum is an ectoderm-derived layer of epithelium that derives from the anterior neural ridge and forms the earliest mouth structures (Couly and Le Douarin, 1985). Pitx1 expression defines the stomodeum and continues within stomodial derivatives, including the nasal pit and Rathke’s pouch (Lanctot et al., 1997). Pitx1 is also expressed more caudally in the posterior lateral plate and extraCorrespondence to: P.J. Gage Mammalian Genome 10, 197–200 (1999).

DFNB3 , a locus for nonsyndromic sensorineural recessive deafness, maps to a 3-centimorgan interval on human chromosome 17p11.2, a region that shows conserved synteny with mouse shaker-2 . A human unconventional myosin gene, MYO15 , was identified by combining functional and positional cloning approaches in searching for shaker-2 and DFNB3 . MYO15 has at least 50 exons spanning 36 kilobases. Sequence analyses of these exons in affected individuals from three unrelated DFNB3 families revealed two missense mutations and one nonsense mutation that cosegregated with congenital recessive deafness.

The shaker-2 mouse mutation, the homolog of human DFNB3 , causes deafness and circling behavior. A bacterial artificial chromosome (BAC) transgene from the shaker-2 critical region corrected the vestibular defects, deafness, and inner ear morphology of shaker-2 mice. An unconventional myosin gene, Myo15 , was discovered by DNA sequencing of this BAC. Shaker-2 mice were found to have an amino acid substitution at a highly conserved position within the motor domain of this myosin. Auditory hair cells of shaker-2 mice have very short stereocilia and a long actin-containing protrusion extending from their basal end. This histopathology suggests that Myo15 is necessary for actin organization in the hair cells of the cochlea.

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. In the last 7 years, evidence has rapidly accumulated that defines the characteristics of pituitary stem cells or progenitor cells present in rodent embryos, early postnatal pups, and adult pituitaries. In some cases the findings have already been reproduced in humans. Here, we will discuss different approaches used from diverse groups in order to study the pituitary stem cell population. These discoveries are providing a mechanistic understanding of normal organ development and disease pathophysiology. In the future, we expect that understanding the roles of pituitary stem cells will help find alternative therapies for pituitary hormone deficiencies and tumors. Many questions remain unsolved, and some enormous challenges will need to be overcome in order to translate this exciting basic knowledge into therapeutic value. It will be important to further define the steps in the regulation of multi-potent progenitors and understand the mechanisms for guidance to specific cell fates. Some of these aspects will be discussed in this review.

A major role of the peptide hormone somatostatin is inhibition of growth hormone secretion. The effects of somatostatin are mediated through five distinct G-protein-coupled receptors, each of which is expressed in the pituitary gland and other tissues. Allelic variation in the five somatostatin receptor genes (Smstr) could contribute to growth rate and overall body size. To evaluate this hypothesis we determined the chromosomal location of the Smstr genes. Restriction fragment length polymorphisms and single-strand conformational polymorphisms were used to follow the segregation of each gene in interspecific mouse backcrosses. Smstr1 through Smstr5 were localized to mouse chromosomes 12, 11, 15, 2, and 17, respectively. None of the Smstr genes colocalized with single gene mutations that affect growth. However, growth is a quantitative trait influenced by many genes and by the environment. Strains of mice selected for high and low growth have been exploited to identify chromosomal regions that modestly influence growth (J. Cheverud et a1., 1996, Genetics 142: 1305-1319). Several Smstr genes map within these regions, suggesting that they be considered candidate genes for these quantitative trait loci.

Many aspects of pituitary development have become better understood in the last two decades. The signaling pathways regulating pituitary growth and shape have emerged, and the balancing interactions between the pathways are now appreciated. Markers for multi-potent 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 3-D 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.

Humans with mutations in SOX3 have panhypopituitarism, but the developmental mechanisms underlying this defect are unknown. Conditional disruption of Sox3 in mice now suggests that anterior pituitary development depends on Sox3 expression in the overlying neural ectoderm, which establishes midline structures and regulates production of inductive BMP and FGF signals.

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.

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.

Vitronectin (VN) is an abundant glycoprotein present in plasma and the extracellular matrix of most tissues. Though the precise function of VN in vivo is unknown, it has been implicated as a participant in diverse biological processes, including cell attachment and spreading, complement activation, and regulation of hemostasis. The major site of synthesis appears to be the liver, though VN is also found in the brain at an early stage of mouse organogenesis, suggesting that it may play an important role in mouse development. Genetic deficiency of VN has not been reported in humans or in other higher organisms. To examine the biologic function of VN within the context of the intact animal, we have established a murine model for VN deficiency through targeted disruption of the murine VN gene. Southern blot analysis of DNA obtained from homozygous null mice demonstrates deletion of all VN coding sequences, and immunological analysis confirms the complete absence of VN protein expression in plasma. However, heterozygous mice carrying one normal and one null VN allele and homozygous null mice completely deficient in VN demonstrate normal development, fertility, and survival. Sera obtained from VN-deficient mice are completely deficient in "serum spreading factor" and plasminogen activator inhibitor 1 binding activities. These observations demonstrate that VN is not essential for cell adhesion and migration during normal mouse development and suggest that its role in these processes may partially overlap with other adhesive matrix components.

Ames dwarf mice have a recessive defect that results in affected mice (df/df) with extremely hypocellular anterior pituitaries that generally lack somatotropes, lactotropes, and thyrotropes. We report detection of rare foci of GH+ cells by immunocytochemistry, suggesting that some commitment to a somatotrope cell fate can occur in the pituitaries of df/df mice. A role for GHRF in regulating somatotrope proliferation is well documented. It has been shown that expression of human GHRF (hGHRF) in transgenic mice resulted in increased somatic growth and somatotrope hyperplasia over nontransgenic littermates. To assess whether overexpression of GHRF during ontogeny might elicit a physiological response in df/df mice, we generated df/df mice expressing the hGhrf transgene. Although the somatic growth of transgenic df heterozygotes was dramatically increased over that of nontransgenic littermates, df/df mice were refractory to excess GHRF. No GHRF receptor (Grfr) transcripts were detectable in df/df fetuses or adult mice by in situ hybridization analysis. In contrast, Grfr expression is detected by e16.5 in df/+ mice. The lack of Grfr expression in df/df fetuses may account for their lack of response to GHRF and implies that the df gene product is required before e16.5.

This review highlights the use of transgenic mice and gene targeting in the study of reproduction, pituitary gene expression, and cell lineage. Since 1980 numerous applications of transgenic animal technology have been reported. Altered phenotypes resulting from transgene expression demonstrated that introduced genes can exert profound effects on animal physiology. Transgenic mice have been important for the study of hormonal and developmental control of gene expression because gene expression in whole animals often requires more DNA sequence information than is necessary for expression in cell cultures. This point is illustrated by studies of pituitary glycoprotein hormone alpha- and beta-subunit gene expression (Kendall et al., Mol Endocrinol 1994; in press [1]. Transgenic mice have also been invaluable for producing animal models of cancer and other diseases and testing the efficacy of gene therapy. In addition, cell-cell interactions and cell lineage relationships have been explored by cell-specific expression of toxin genes in transgenic mice. Recent studies suggest that attenuated and inducible toxins hold promise for future transgene ablation experiments. Since 1987, embryonic stem (ES) cell technology has been used to create numerous mouse strains with targeted gene alterations, contributing enormously to our understanding of the functional importance of individual genes. For example, the unexpected development of gonadal tumors in mice with a targeted disruption of the inhibin gene revealed a potential role for inhibin as a tumor suppressor (Matzuk et al., Nature 1992:360: 313-319 [2]. The transgenic and ES cell technologies will undoubtedly continue to expand our understanding and challenge our paradigms in reproductive biology.

ANTIBODIES to DNA and nucleoproteins are found in sera of individuals with systemic autoimmune disease. In the population (and in the autoimmune mouse strain MRL/lpr) there is a great variety of such antinuclear antibodies, but individuals with systemic lupus erythematosus or single MRL mice express a subset only of the antinuclear specificities found in the population. These observations have been interpreted to mean that these antibodies arise by immunization1. The oligoclonal nature of the autoantibody response and the evidence of selection acting on somatically mutated autoantibodies favour this interpretation2,3. Specific activation of autoantibodies in disease implies either that autoantibodies are regulated in non-diseased individuals or that autoantigen availability is variable. The former has been demonstrated in anti-DNA transgenic mice. In normal mice, transgene-encoded antibodies against double-stranded (ds) DNA are not expressed in serum or on B cells4–6. Here we describe modified anti-dsDNA transgenic mice which allow us to study the site and developmental stage at which such B-cell regulation occurs. This model shows that in normal mice B cells expressing anti-DNA specificity are deleted in the bone marrow at a pre-B to immature B transitional stage.

The genetic map location of the recently discovered imprinted gene U2afbpL has been verified and refined in several mouse crosses. RI strain analysis had previously shown that the gene is located on mouse chromosome 11. This assignment has been verified using interspecific backcrosses. Moreover, the location of the gene relative to a fixed order of markers in the proximal region of mouse chromosome 11 has been established. The location of the gene on mouse chromosome 11 corresponds to a homologous linkage group that is conserved on human chromosome 5q. The location of the human homologue has been determined using both somatic cell hybrid genetic analysis and fluorescence in situ hybridization. These analyses have mapped the human locus U2AFBPL to human chromosome 5q23→q31.

Transient transfection studies have proven useful in unraveling the molecular mechanisms underlying gonadotrope-specific expression and hormonal regulation of the gene encoding the alpha-subunit of the glycoprotein hormones. In contrast, similar studies performed with the LH beta gene have been less informative. When assayed by transient transfection into alpha T3-1 cells, activity of a 776-basepair bovine LH beta promoter-chloramphenicol acetyltransferase fusion gene (bLH beta CAT) was no greater than that of a promoterless control. To determine whether limited activity in vitro reflected the absence of critical regulatory elements, we examined activity of bovine LH beta fusion genes after stable integration in transgenic mice. In contrast to transient transfection studies, the LH beta promoter targeted high levels of CAT expression specifically to the pituitary. In addition, a bLH beta TK fusion gene was active only in gonadotropes. The bLH beta CAT transgene was also evaluated for responsiveness to gonadal steroids and GnRH. Testosterone and 17 beta-estradiol were capable of suppressing activity 70-80% in castrated males, despite the absence of high affinity binding sites for androgen or estrogen receptors. This suggests that feedback inhibition of LH beta CAT transgene expression by gonadal steroids may occur through an indirect mechanism, possibly at the level of the hypothalamus. To address whether the bLH beta CAT transgene could be regulated by GnRH, we treated ovariectomized females with antide, a GnRH antagonist. Antide suppressed transgene activity by 60%. Thus, the proximal promoter of the bovine LH beta subunit gene directs appropriate patterns of cell-specific expression and retains responsiveness to gonadal steroids and GnRH. In light of the robust activity of the LH beta promoter in transgenic mice, we suggest that this animal model can be exploited further to dissect the complex mechanisms that underlie gonadotrope-specific expression and hormonal regulation of the LH beta gene.

Hereditary hyperekplexia, an autosomal dominant neurologic disorder characterized by an exaggerated startle reflex and neonatal hypertonia, can be caused by mutations in the gene encoding the alpha 1 subunit of the inhibitory glycine receptor (GLRA1). Spasmodic (spd), a recessive neurologic mouse mutant, resembles hyperekplexia phenotypically, and the two disease loci map to homologous chromosomal regions. Here we describe a Glra1 missense mutation in spd that results in reduced agonist sensitivity in glycine receptors expressed in vitro. We conclude that spd is a murine homologue of hyperekplexia and that mutations in GLRA1/Glra1 can produce syndromes with different inheritance patterns.

CRH mRNA was detected by in situ hybridization histochemistry in numerous regions of the adult mouse brain, including most prominently the paraventricular nucleus (PVN) of the hypothalamus, the inferior olivary nucleus, and Barrington's nucleus. After adrenalectomy, steady state CRH mRNA levels increased 1.7-fold, specifically in the PVN, consistent with reports of negative glucocorticoid regulation of CRH expression in the rat PVN. Ontogenetic analysis of CRH expression in fetal and neonatal mouse brain demonstrated CRH mRNA in PVN, Barrington's nucleus, olivary complex, and amygdaloid primordia on embryonic day 13.5. In contrast, CRH mRNA was not detectable in the cortex until after birth. CRH expression also exhibited differential regulation in ontogeny. CRH mRNA reached adult levels at markedly different times of development in each brain region, and CRH expression was reduced specifically in the PVN just before birth and the stress hyporesponsive period. High levels of CRH mRNA were present transiently in the developing lung and celiac ganglion. The novel findings of CRH expression in fetal lung during the period of glucocorticoid-induced lung maturation and in celiac ganglion during development of the sympathetic nervous system indicate that CRH may have some important developmental functions in addition to its role in activation of the stress response.

The neurologic mutant mouse, oscillator, is characterized by a fine motor tremor and muscle spasms that begin at 2 weeks of age and progressively worsen, resulting in death by 3 weeks of age. We report the localization of the oscillator mutation to the central region of mouse Chr 11, and demonstrate its allelism with spasmodic, a recessive viable neurological mutation which displays excessive startle. Oscillator is caused by a microdeletion in the gene coding for the alpha 1 subunit of the adult glycine receptor (Glra1). Glra1 assembles into a pentameric complex with the beta subunit of the glycine receptor (3 alpha (1)2 beta 5) to form a glycine-gated chloride channel. This receptor is the major adult glycine receptor, and the site of action of the poison strychnine. The oscillator deletion causes a frameshift resulting in loss of the highly conserved third cytoplasmic loop and fourth transmembrane domain of the protein. Membranes isolated from oscillator homozygote spinal cords display a 90% reduction in glycine-displaceable strychnine binding. This lack of ligand binding function confirms that oscillator is a complete loss of function allele. The oscillator mutation provides evidence that although at least four different alpha subunits exist for the glycine receptor, none of the other subunits can compensate for the loss of alpha 1 function. Mutations which impair GLRA1 function in humans have been shown to cause dominant familial startle disease. The identification of the oscillator mutation suggests that severe loss of function alleles in humans would result in prenatal or neonatal lethality.

The transcription factor GATA-2 plays vital roles in quite diverse developmental programs, including hematopoietic stem cell (HSC) survival and proliferation. We previously identified a vascular endothelial (VE) enhancer that regulates GATA-2 activity in pan-endothelial cells. To more thoroughly define the in vivo regulatory properties of this enhancer, we generated a tamoxifen-inducible Cre transgenic mouse line using the Gata2 VE enhancer (Gata2 VECre) and utilized it to temporally direct tissue-specific conditional loss of Gata2. Here, we report that Gata2 VECre–mediated loss of GATA-2 led to anemia, hemorrhage, and eventual death in edematous embryos. We further determined that the etiology of anemia in conditional Gata2 mutant embryos involved HSC loss in the fetal liver, as demonstrated by in vitro colony-forming and immunophenotypic as well as in vivo long-term competitive repopulation experiments. We further documented that the edema and hemorrhage in conditional Gata2 mutant embryos were due to defective lymphatic development. Thus, we unexpectedly discovered that in addition to its contribution to endothelial cell development, the VE enhancer also regulates GATA-2 expression in definitive fetal liver and adult BM HSCs, and that GATA-2 function is required for proper lymphatic vascular development during embryogenesis.

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.

Pituitary adenomas are common in older people, representing approximately 10% of intracranial neoplasias (1). They tend to be slow-growing, and are usually benign, but they can compress the brain and optic chiasm and cause abnormalities in pituitary hormone production. A fraction of pituitary adenomas become invasive and are resistant to treatment with surgery, radiation, and pharmacotherapy. Progress is being made in identification of genes that enhance the risk of pituitary adenoma formation, but the etiology of many adenomas remains unexplained (1, 2). Common adenomas are sporadic and clonal in origin. Craniopharyngiomas differ from most sporadic pituitary adenomas in that they occur in children, tend to be resistant to treatment, and cause significant morbidity. Histopathology analyses suggested that craniopharyngiomas arise from embryonic pituitary tissue and implicated β-catenin as a marker. In PNAS, Gaston-Massuet and colleagues provide proof that craniopharyngiomas arise from activation of β-catenin in pituitary progenitors during embryogenesis (3). Their elegant studies in patient tumor samples and genetically engineered mice lend support to the cancer stem cell hypothesis. They also provide insight into the basic nature of pituitary progenitors, the stem cell niche, and normal regulation of the transition from self-renewal to differentiation. Now the pituitary gland joins a collection of other tissues in which β-catenin signaling can affect this critical decision point (4).