Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system

Mouse embryos deficient in Gata3 die by 11 days post coitum (d.p.c.) from pathology of undetermined origin. We recently showed that Gata3-directed lacZ expression of a 625-kb Gata3 YAC transgene in mice mimics endogenous Gata3 expression, except in thymus and the sympathoadrenal system. As this transgene failed to overcome embryonic lethality (unpublished data and ref. 3) in Gata3-/- mice, we hypothesized that a neuroendocrine deficiency in the sympathetic nervous system (SNS) might cause embryonic lethality in these mutants. We find here that null mutation of Gata3 leads to reduced accumulation of Th (encoding tyrosine hydroxylase, Th) and Dbh (dopamine beta-hydroxylase, Dbh) mRNA, whereas several other SNS genes are unaffected. We show that Th and Dbh deficiencies lead to reduced noradrenaline in the SNS, and that noradrenaline deficiency is a proximal cause of death in mutants by feeding catechol intermediates to pregnant dams, thereby partially averting Gata3 mutation-induced lethality. These older, pharmacologically rescued mutants revealed abnormalities that previously could not be detected in untreated mutants. These late embryonic defects include renal hypoplasia and developmental defects in structures derived from cephalic neural crest cells. Thus we have shown that Gata3 has a role in the differentiation of multiple cell lineages during embryogenesis.     

Crh and Oprm1 mediate anxiety-related behavior and social approach in a mouse model of MECP2 duplication syndrome

Genomic duplications spanning Xq28 are associated with a spectrum of phenotypes, including anxiety and autism. The minimal region shared among affected individuals includes MECP2 and IRAK1, although it is unclear which gene when overexpressed causes anxiety and social behavior deficits. We report that doubling MECP2 levels causes heightened anxiety and autism-like features in mice and alters the expression of genes that influence anxiety and social behavior, such as Crh and Oprm1. To test the hypothesis that alterations in these two genes contribute to heightened anxiety and social behavior deficits, we analyzed MECP2 duplication mice (MECP2-TG1) that have reduced Crh and Oprm1 expression. In MECP2-TG1 animals, reducing the levels of Crh or its receptor, Crhr1, suppressed anxiety-like behavior; in contrast, reducing Oprm1 expression improved abnormal social behavior. These data indicate that increased MeCP2 levels affect molecular pathways underlying anxiety and social behavior and provide new insight into potential therapies for MECP2-related disorders.     

beta-cell-specific deletion of the Igf1 receptor leads to hyperinsulinemia and glucose intolerance but does not alter beta-cell mass

Regulation of glucose homeostasis by insulin depends on the maintenance of normal beta-cell mass and function. Insulin-like growth factor 1 (Igf1) has been implicated in islet development and differentiated function, but the factors controlling this process are poorly understood. Pancreatic islets produce Igf1 and Igf2, which bind to specific receptors on beta-cells. Igf1 has been shown to influence beta-cell apoptosis, and both Igf1 and Igf2 increase islet growth; Igf2 does so in a manner additive with fibroblast growth factor 2 (ref. 10). When mice deficient for the Igf1 receptor (Igf1r(+/-)) are bred with mice lacking insulin receptor substrate 2 (Irs2(-/-)), the resulting compound knockout mice show a reduction in mass of beta-cells similar to that observed in pancreas of Igf1r(-/-) mice (ref. 11), suggesting a role for Igf1r in growth of beta-cells. It is possible, however, that the effects in these mice occur secondary to changes in vascular endothelium or in the pancreatic ductal cells, or because of a decrease in the effects of other hormones implicated in islet growth. To directly define the role of Igf1, we have created a mouse with a beta-cell-specific knockout of Igf1r (betaIgf1r(-/-)). These mice show normal growth and development of beta-cells, but have reduced expression of Slc2a2 (also known as Glut2) and Gck (encoding glucokinase) in beta-cells, which results in defective glucose-stimulated insulin secretion and impaired glucose tolerance. Thus, Igf1r is not crucial for islet beta-cell development, but participates in control of differentiated function.     

Syndecan-1 is required for Wnt-1-induced mammary tumorigenesis in mice

Syndecan-1 is a cell-surface, heparan-sulphate proteoglycan (HSPG) predominantly expressed by epithelial cells. It binds specifically to many proteins, including oncoproteins. For example, it induces the assembly of a signalling complex between FGF ligands and their cognate receptors. But so far there has been no direct evidence that this proteoglycan contributes to tumorigenesis. Here we have examined the role of syndecan-1 (encoded by Sdc1) during mammary tumour formation in response to the ectopic expression of the proto-oncogene Wnt1. We crossed syndecan-1-deficient mice with transgenic mice that express Wnt1 in mammary gland (TgN(Wnt-1)1Hev; ref. 2). Ectopic Wnt-1 expression induces generalized mammary hyperplasia, followed by the development of solitary tumours (median time 22 weeks). We show that in Sdc1-/- mice, Wnt-1-induced hyperplasia in virgin mammary gland was reduced by 70%, indicating that the Wnt-1 signalling pathway was inhibited. Of the 39 tumours that developed in a test cohort of mice, only 1 evolved in the Sdc1-/- background. In addition, we show that soluble syndecan-1 ectodomain purified from mouse mammary epithelial cells stimulates the activity of a Wnt-1 homologue in a tissue culture assay. Our results provide both genetic and biochemical evidence that syndecan-1 can modulate Wnt signalling, and is critical for Wnt-1-induced tumorigenesis of the mouse mammary gland.     

Retinopathy and attenuated circadian entrainment in Crx-deficient mice

Crx, an Otx-like homeobox gene, is expressed specifically in the photoreceptors of the retina and the pinealocytes of the pineal gland. Crx has been proposed to have a role in the regulation of photoreceptor-specific genes in the eye and of pineal-specific genes in the pineal gland. Mutations in human CRX are associated with the retinal diseases, cone-rod dystrophy-2 (adCRD2; refs 3, 4, 5), retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), which all lead to loss of vision. We generated mice carrying a targeted disruption of Crx. Crx-/- mice do not elaborate photoreceptor outer segments and lacked rod and cone activity as assayed by electroretinogram (ERG). Expression of several photoreceptor- and pineal-specific genes was reduced in Crx mutants. Circadian entrainment was also affected in Crx-/- mice.     

Cdc25b phosphatase is required for resumption of meiosis during oocyte maturation

In a wide variety of animal species, oocyte maturation is arrested temporarily at prophase of meiosis I (ref. 1). Resumption of meiosis requires activation of cyclin-dependent kinase-1 (CDK1, p34cdc2), one component of maturation-promoting factor (MPF). The dual specificity phosphatases Cdc25a, Cdc25b and Cdc25c are activators of cyclin-dependent kinases; consequently, they are postulated to regulate cell-cycle progression in meiosis and mitosis as well as the DNA-damage response. We generated Cdc25b-deficient (Cdc25b-/-) mice and found that they are viable. As compared with wildtype cells, fibroblasts from Cdc25b-/- mice grew vigorously in culture and arrested normally in response to DNA damage. Female Cdc25b-/- mice were sterile, and Cdc25b-/- oocytes remained arrested at prophase with low MPF activity. Microinjection of wildtype Cdc25b mRNA into Cdc25b-/- oocytes caused activation of MPF and resumption of meiosis. Thus, Cdc25b-/- female mice are sterile because of permanent meiotic arrest resulting from the inability to activate MPF. Cdc25b is therefore essential for meiotic resumption in female mice. Mice lacking Cdc25b provide the first genetic model for studying the mechanisms regulating prophase arrest in vertebrates.     

Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha

Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane. Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses. The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class IA Pi3ks (ref. 2). Mice lacking only the p85 alpha isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants. Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites. We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class IA Pi3k catalytic subunits; nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.     

Fras1 deficiency results in cryptophthalmos,renal agenesis and blebbed phenotype in mice

Loss of tight association between epidermis and dermis underlies several blistering disorders and is frequently caused by impaired function of extracellular matrix (ECM) proteins. Here we describe a new protein in mouse, Fras1, that is specifically detected in a linear fashion underlying the epidermis and the basal surface of other epithelia in embryos. Loss of Fras1 function results in the formation of subepidermal hemorrhagic blisters as well as unilateral or bilateral renal agenesis during mouse embryogenesis. Postnatally, homozygous Fras1 mutants have fusion of the eyelids and digits and unilateral renal agenesis or dysplasia. The defects observed in Fras1-/- mice phenocopy those of the existing bl (blebbed) mouse mutants, which have been considered a model for the human genetic disorder Fraser syndrome. We show that bl/bl homozygous embryos are devoid of Fras1 protein, consistent with the finding that Fras1 is mutated in these mice. In sum, our data suggest that perturbations in the composition of the extracellular space underlying epithelia could account for the onset of the blebbed phenotype in mouse and Fraser syndrome manifestation in human.     

Pharmacologic rescue of lethal seizures in mice deficient in succinate semialdehyde dehydrogenase.

Succinate semialdehyde dehydrogenase (ALDH5A1, encoding SSADH deficiency is a defect of 4-aminobutyric acid (GABA) degradation that manifests in humans as 4-hydroxybutyric (gamma-hydroxybutyric, GHB) aciduria. It is characterized by a non-specific neurological disorder including psychomotor retardation, language delay, seizures, hypotonia and ataxia. The current therapy, vigabatrin (VGB), is not uniformly successful. Here we report the development of Aldh5a1-deficient mice. At postnatal day 16-22 Aldh5a1-/- mice display ataxia and develop generalized seizures leading to rapid death. We observed increased amounts of GHB and total GABA in urine, brain and liver homogenates and detected significant gliosis in the hippocampus of Aldh5a1-/- mice. We found therapeutic intervention with phenobarbital or phenytoin ineffective, whereas intervention with vigabatrin or the GABAB receptor antagonist CGP 35348 (ref. 2) prevented tonic-clonic convulsions and significantly enhanced survival of the mutant mice. Because neurologic deterioration coincided with weaning, we hypothesized the presence of a protective compound in breast milk. Indeed, treatment of mutant mice with the amino acid taurine rescued Aldh5a1-/- mice. These findings provide insight into pathomechanisms and may have therapeutic relevance for the human SSADH deficiency disease and GHB overdose and toxicity.     

The pallid gene encodes a novel, syntaxin 13-interacting protein involved in platelet storage pool deficiency

Pallid (pa) is 1 of 13 platelet storage pool deficiency (SPD) mouse mutants. pa animals suffer from prolonged bleeding time, pigment dilution, kidney lysosomal enzyme elevation, serum alpha1-antitrypsin activity deficiency and abnormal otolith formation. As with other mouse mutants of this class, characterization of pa mice suggests a defect in organelle biosynthesis. Here we describe the physical mapping, positional cloning, and mutational and functional analysis of the gene that is defective in pa mice. It encodes a ubiquitously expressed, highly charged 172-amino-acid protein (termed pallidin) with no homology to known proteins. We detected a nonsense mutation at codon 69 of this gene in the pallid mutant. In a yeast two-hybrid screen, we discovered that pallidin interacts with syntaxin 13, a t-SNARE protein that mediates vesicle-docking and fusion. We confirmed this interaction by co-immunoprecipitation assay. Immunofluorescence studies corroborate that the cellular distribution of pallidin overlaps that of syntaxin 13. Whereas the mocha and pearl SPD mutants have defects in Ap-3, our findings suggest that pa SPD mutants are defective in a more downstream event of vesicle-trafficking: namely, vesicle-docking and fusion.     

Regulatory defects in liver and intestine implicate abnormal hepcidin and Cybrd1 expression in mouse hemochromatosis

Individuals with hereditary hemochromatosis suffer from systemic iron overload due to duodenal hyperabsorption. Most cases arise from a founder mutation in HFE (845G-->A; ref. 2) that results in the amino-acid substitution C282Y and prevents the association of HFE with beta2-microglobulin. Mice homozygous with respect to a null allele of Hfe (Hfe-/-) or homozygous with respect to the orthologous 882G-->A mutation (Hfe(845A/845A)) develop iron overload that recapitulates hereditary hemochromatosis in humans, confirming that hereditary hemochromatosis arises from loss of HFE function. Much work has focused on an exclusive role for the intestine in hereditary hemochromatosis. HFE deficiency in intestinal crypt cells is thought to cause intestinal iron deficiency and greater expression of iron transporters such as SLC11A2 (also called DMT1, DCT1 and NRAMP2) and SLC11A3 (also called IREG1, ferroportin and MTP1; ref. 3). Published data on the expression of these transporters in the duodenum of HFE-deficient mice and humans are contradictory. In this report, we used a custom microarray to assay changes in duodenal and hepatic gene expression in Hfe-deficient mice. We found unexpected alterations in the expression of Slc39a1 (mouse ortholog of SLC11A3) and Cybrd1, which encode key iron transport proteins, and Hamp (hepcidin antimicrobial peptide), a hepatic regulator of iron transport. We propose that inappropriate regulatory cues from the liver underlie greater duodenal iron absorption, possibly involving the ferric reductase Cybrd1.     

Mice lacking the homologue of the human 22q11.2 gene CRKL phenocopy neurocristopathies of DiGeorge syndrome

Heterozygous deletions within human chromosome 22q11 are the genetic basis of DiGeorge/velocardiofacial syndrome (DGS/VCFS), the most common deletion syndrome (1 in 4,000 live births) in humans. CRKL maps within the common deletion region for DGS/VCFS (ref. 2) and encodes an SH2-SH3-SH3 adapter protein closely related to the Crk gene products. Here we report that mice homozygous for a targeted null mutation at the CrkL locus (gene symbol Crkol for mice) exhibit defects in multiple cranial and cardiac neural crest derivatives including the cranial ganglia, aortic arch arteries, cardiac outflow tract, thymus, parathyroid glands and craniofacial structures. We show that the migration and early expansion of neural crest cells is unaffected in Crkol-/- embryos. These results therefore indicate an essential stage- and tissue-specific role for Crkol in the function, differentiation, and/or survival of neural crest cells during development. The similarity between the Crkol-/- phenotype and the clinical manifestations of DGS/VCFS implicate defects in CRKL-mediated signaling pathways as part of the molecular mechanism underlying this syndrome.     

Atp6i-deficient mice exhibit severe osteopetrosis due to loss of osteoclast-mediated extracellular acidification

Solubilization of bone mineral by osteoclasts depends on the formation of an acidic extracellular compartment through the action of a V-proton pump that has not yet been characterized at the molecular level. We previously cloned a gene (Atp6i, for V-proton pump, H+ transporting (vacuolar proton pump) member I) encoding a putative osteoclast-specific proton pump subunit, termed OC-116kD (ref. 4). Here we show that targeted disruption of Atp6i in mice results in severe osteopetrosis. Atp6i-/- osteoclast-like cells (OCLs) lose the function of extracellular acidification, but retain intracellular lysosomal proton pump activity. The pH in Atp6i-/- liver lysosomes and proton transport in microsomes of Atp6i-/- kidney are identical to that in wild-type mice. Atp6i-/- mice exhibit a normal acid-base balance in blood and urine. Our results demonstrate that Atp6i is unique and necessary for osteoclast-mediated extracellular acidification.     

Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the Carney complex

Carney complex (CNC) is a multiple neoplasia syndrome characterized by spotty skin pigmentation, cardiac and other myxomas, endocrine tumours and psammomatous melanotic schwannomas. CNC is inherited as an autosomal dominant trait and the genes responsible have been mapped to 2p16 and 17q22-24 (refs 6, 7). Because of its similarities to the McCune-Albright syndrome and other features, such as paradoxical responses to endocrine signals, genes implicated in cyclic nucleotide-dependent signalling have been considered candidates for causing CNC (ref. 10). In CNC families mapping to 17q, we detected loss of heterozygosity (LOH) in the vicinity of the gene (PRKAR1A) encoding protein kinase A regulatory subunit 1-alpha (RIalpha), including a polymorphic site within its 5' region. We subsequently identified three unrelated kindreds with an identical mutation in the coding region of PRKAR1A. Analysis of additional cases revealed the same mutation in a sporadic case of CNC, and different mutations in three other families, including one with isolated inherited cardiac myxomas. Analysis of PKA activity in CNC tumours demonstrated a decreased basal activity, but an increase in cAMP-stimulated activity compared with non-CNC tumours. We conclude that germline mutations in PRKAR1A, an apparent tumour-suppressor gene, are responsible for the CNC phenotype in a subset of patients with this disease.     

Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2

The actions of corticotropin-releasing hormone (Crh), a mediator of endocrine and behavioural responses to stress, and the related hormone urocortin (Ucn) are coordinated by two receptors, Crhr1 (encoded by Crhr) and Crhr2. These receptors may exhibit distinct functions due to unique tissue distribution and pharmacology. Crhr-null mice have defined central functions for Crhr1 in anxiety and neuroendocrine stress responses. Here we generate Crhr2-/- mice and show that Crhr2 supplies regulatory features to the hypothalamic-pituitary-adrenal axis (HPA) stress response. Although initiation of the stress response appears to be normal, Crhr2-/- mice show early termination of adrenocorticotropic hormone (Acth) release, suggesting that Crhr2 is involved in maintaining HPA drive. Crhr2 also appears to modify the recovery phase of the HPA response, as corticosterone levels remain elevated 90 minutes after stress in Crhr2-/- mice. In addition, stress-coping behaviours associated with dearousal are reduced in Crhr2-/- mice. We also demonstrate that Crhr2 is essential for sustained feeding suppression (hypophagia) induced by Ucn. Feeding is initially suppressed in Crhr2-/- mice following Ucn, but Crhr2-/- mice recover more rapidly and completely than do wild-type mice. In addition to central nervous system effects, we found that, in contrast to wild-type mice, Crhr2-/- mice fail to show the enhanced cardiac performance or reduced blood pressure associated with systemic Ucn, suggesting that Crhr2 mediates these peripheral haemodynamic effects. Moreover, Crhr2-/- mice have elevated basal blood pressure, demonstrating that Crhr2 participates in cardiovascular homeostasis. Our results identify specific responses in the brain and periphery that involve Crhr2.     

Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production

The gene Ucp2 is a member of a family of genes found in animals and plants, encoding a protein homologous to the brown fat uncoupling protein Ucp1 (refs 1-3). As Ucp2 is widely expressed in mammalian tissues, uncouples respiration and resides within a region of genetic linkage to obesity, a role in energy dissipation has been proposed. We demonstrate here, however, that mice lacking Ucp2 following targeted gene disruption are not obese and have a normal response to cold exposure or high-fat diet. Expression of Ucp2 is robust in spleen, lung and isolated macrophages, suggesting a role for Ucp2 in immunity or inflammatory responsiveness. We investigated the response to infection with Toxoplasma gondii in Ucp2-/- mice, and found that they are completely resistant to infection, in contrast with the lethality observed in wild-type littermates. Parasitic cysts and inflammation sites in brain were significantly reduced in Ucp2-/- mice (63% decrease, P<0.04). Macrophages from Ucp2-/- mice generated more reactive oxygen species than wild-type mice (80% increase, P<0.001) in response to T. gondii, and had a fivefold greater toxoplasmacidal activity in vitro compared with wild-type mice (P<0.001 ), which was absent in the presence of a quencher of reactive oxygen species (ROS). Our results indicate a role for Ucp2 in the limitation of ROS and macrophage-mediated immunity.