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.

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.

Altered anxiety and weight gain in corticotropin-releasing hormone-binding protein-deficient mice.

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.

Implementing transgenic and embryonic stem cell technology to study gene expression, cell-cell interactions and gene function.

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.

Expression of corticotropin-releasing hormone transgenes in neurons of adult and developing mice.

The DNA sequences important for cell-specific expression and developmental regulation of corticotropin-releasing hormone (CRH) were analyzed in transgenic mice. A construct containing 0.5 kb of CRH 5' flanking DNA linked to the chloramphenicol acetyltransferase reporter gene was expressed in many brain regions and in several ectopic peripheral sites, suggesting that this portion of the CRH gene contains basal promoter activity but lacks DNA elements necessary for appropriate tissue specificity. Cell specificity of transgene expression was examined with a CRH-beta-galactosidase reporter construct containing the same 0.5-kb CRH promoter fragment, but also including the CRH structural gene and 2 kb of CRH 3' flanking DNA. Transgene expression was observed in inappropriate regions of the brain, but no expression was detected in peripheral tissues, suggesting that these additional CRH sequences suppress inappropriately high levels of peripheral expression. Cell-specific expression improved significantly with the inclusion of 8.7 kb of CRH 5' flanking DNA. Individual transgenic lines exhibited expression in a number of the major CRH neuronal groups including the paraventricular nucleus, medial geniculate nucleus, inferior olivary nucleus, and Barrington's nucleus. Transgene expression was properly activated in Barrington's nucleus during development. This study demonstrates that the regulatory control of cell-specific and developmentally appropriate CRH expression is complex, utilizing multiple DNA sequence elements located upstream and downstream of the CRH transcription start site.

Differential expression of corticotropin-releasing hormone in developing mouse embryos and adult brain.

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.

Linkage analysis of peripheral neurofibromatosis to DNA markers on chromosome 8.

Linkage relationships of the gene for peripheral neurofibromatosis (NF) were assessed in a large American Caucasian pedigree using two DNA markers located on chromosome 8. Linkage to the thyroglobulin locus, located at 8q24, was excluded (lod less than or equal to -2.0) to 21 cM. Data obtained for the tissue plasminogen activator locus, located at 8p12, excluded linkage to 4 cM. These results exclude between 20 to 30% of chromosome 8 as a possible map location for the NF gene in this family. Comparison of the two DNA markers excluded their linkage to 0.5 cM.