Rump white inversion in the mouse disrupts dipeptidyl aminopeptidase-like protein 6 and causes dysregulation of Kit expression

The mouse rump white (Rw) mutation causes a pigmentation defect in heterozygotes and embryonic lethality in homozygotes. At embryonic day (E) 7.5, Rw/Rw embryos are retarded in growth, fail to complete neurulation and die around E 9.5. The Rw mutation is associated with a chromosomal inversion spanning 30 cM of the proximal portion of mouse chromosome 5. The Rw embryonic lethality is complemented by the W19H deletion, which spans the distal boundary of the Rw inversion, suggesting that the Rw lethality is not caused by the disruption of a gene at the distal end of the inversion. Here, we report the molecular characterization of sequences disrupted by both inversion breakpoints. These studies indicate that the distal breakpoint of the inversion is associated with ectopic Kit expression and therefore may be responsible for the dominant pigmentation defect in Rw/+ mice; whereas the recessive lethality of Rw is probably due to the disruption of the gene encoding dipeptidyl aminopeptidase-like protein 6, Dpp6 [Wada, K., Yokotani, N., Hunter, C., Doi, K., Wenthold, R. J. & Shimasaki, S. (1992) Proc. Natl. Acad. Sci. USA 89, 197-201] located at the proximal inversion breakpoint.     

Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase

The ability to generate specific genetic modifications in mice provides a powerful approach to assess gene function. When genetic modifications have been generated in the germ line, however, the resulting phenotype often only reflects the first time a gene has an influence on - or is necessary for - a particular biological process. Therefore, systems allowing conditional genetic modification have been developed (for a review, see [1]); for example, inducible forms of the Cre recombinase from P1 phage have been generated that can catalyse intramolecular recombination between target recognition sequences (loxP sites) in response to ligand [2] [3] [4] [5]. Here, we assessed whether a tamoxifen-inducible form of Cre recombinase (Cre-ERTM) could be used to modify gene activity in the mouse embryo in utero. Using the enhancer of the Wnt1 gene to restrict the expression of Cre-ERTM to the embryonic neural tube, we found that a single injection of tamoxifen into pregnant mice induced Cre-mediated recombination within the embryonic central nervous system, thereby activating expression of a reporter gene. Induction was ligand dependent, rapid and efficient. The results demonstrate that tamoxifen-inducible recombination can be used to effectively modify gene function in the mouse embryo.     

Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos

Embryonic lethality of thrombomodulin-deficient mice has indicated an essential role for this regulator of blood coagulation in murine development. Here, the embryonic expression pattern of thrombomodulin was defined by surveying beta-galactosidase activity in a mouse strain in which the reporter gene was placed under the regulatory control of the endogenous thrombomodulin promoter via homologous recombination in embryonic stem cells. The murine trophoblast was identified as a previously unrecognized anatomical site where TM expression is conserved between humans and mice and may exert a critical function during postimplantation development. Targeted reporter gene expression in mesodermal precursors of the endothelial cell lineage defined thrombomodulin as an early marker of vascular differentiation. Analysis of the thrombomodulin promoter in differentiating ES cells and in transgenic mice provided evidence for a disparate and cell type-specific gene regulatory control mechanism in the parietal yolk sac. The thrombomodulin promoter as defined in this study will allow the targeting of gene expression to the parietal yolk sac of transgenic mice and the initiation of investigations into the role of parietal endoderm in placental function.     

A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans

A novel screen to isolate conditional cell-division mutants in Caenorhabditis elegans has been developed. The screen is based on the phenotypes associated with existing cell-division mutations: some disrupt postembryonic divisions and affect formation of the gonad and ventral nerve cord-resulting in sterile, uncoordinated animals-while others affect embryonic divisions and result in lethality. We obtained 19 conditional mutants that displayed these phenotypes when shifted to the restrictive temperature at the appropriate developmental stage. Eighteen of these mutations have been mapped; 17 proved to be single alleles of newly identified genes, while 1 proved to be an allele of a previously identified gene. Genetic tests on the embryonic lethal phenotypes indicated that for 13 genes, embryogenesis required maternal expression, while for 6, zygotic expression could suffice. In all cases, maternal expression of wild-type activity was found to be largely sufficient for embryogenesis. Cytological analysis revealed that 10 mutants possessed embryonic cell-division defects, including failure to properly segregate DNA, failure to assemble a mitotic spindle, late cytokinesis defects, prolonged cell cycles, and improperly oriented mitotic spindles. We conclude that this approach can be used to identify mutations that affect various aspects of the cell-division cycle.     

Multiple roles of the eyes absent gene in Drosophila

The eyes absent (eya) gene plays an essential role in the events that lead to formation of the Drosophila eye; without expression of eya in retinal progenitor cells, they undergo programmed cell death just prior to the morphogenetic furrow, leading to an eyeless or reduced eye phenotype. The eya gene has recently been found to be highly conserved to humans, defining a new gene family. Insights into the gene's function in the fly, therefore, are likely to be relevant to the role of its homologs in vertebrates. Detailed studies at the subcellular level indicate that the Eya protein is localized to the nucleoplasm, suggesting a role in control of nuclear events. The eya gene shows expression and roles in tissues other than the eye, including subsets of cells of the adult visual system, brain, and ovary, as well as an elaborate expression pattern in the embryo. Various mutations in the eya gene cause loss of ocelli, female sterility, or lethality. Analysis of the embryonic lethal phenotype indicates that mutant alleles show defects in head morphogenesis. These data indicate that eya has critical roles in morphogenesis of a number of tissues in the animal, in addition to its role in early eye formation. Despite multiple roles at multiple stages of development of the fly, both the type I and type II forms of the protein, when expressed ectopically during larval development, can direct eye formation.     

Goosecoid-like (Gscl), a candidate gene for velocardiofacial syndrome, is not essential for normal mouse development

Velocardiofacial syndrome (VCFS) and DiGeorge syndrome (DGS) are characterized by a wide spectrum of abnormalities, including conotruncal heart defects, velopharyngeal insufficiency, craniofacial anomalies and learning disabilities. In addition, numerous other clinical features have been described, including frequent psychiatric illness. Hemizygosity for a 1.5-3 Mb region of chromosome 22q11 has been detected in >80% of VCFS/DGS patients. It is thought that a developmental field defect is responsible for many of the abnormalities seen in these patients and that the defect occurs due to reduced levels of a gene product active in early embryonic development. Goosecoid-like ( GSCL ) is a homeobox gene which is present in the VCFS/DGS commonly deleted region. The mouse homolog, Gscl, is expressed in mouse embryos as early as E8.5. Gscl is related to Goosecoid ( Gsc ), a gene required for proper craniofacial development in mice. GSCL has been considered an excellent candidate for contributing to the developmental defects in VCFS/DGS patients. To investigate the role of Goosecoid-like in VCFS/DGS etiology, we disrupted the Gscl gene in mouse embryonic stem cells and produced mice that transmit the disrupted allele. Mice that are homozygous for the disrupted allele appear to be normal and they do not exhibit any of the anatomical abnormalities seen in VCFS/DGS patients. RNA in situ hybridization to mouse embryo sections revealed that Gscl is expressed at E8.5 in the rostral region of the foregut and at E11.5 and E12.5 in the developing brain, in the pons region and in the choroid plexus of the fourth ventricle. Although the gene inactivation experiments indicate that haploinsufficiency for GSCL is unlikely to be the sole cause of the developmental field defect thought to be responsible for many of the abnormalities in VCFS/DGS patients, its localized expression during development could suggest that hemizygosity for GSCL, in combination with hemizygosity for other genes in 22q11, contributes to some of the developmental defects as well as the behavioral anomalies seen in these patients. The mice generated in this study should help in evaluating these possibilities.     

Antihemophilic factor inhibitors. Management with prothrombin complex concentrates

Prothrombin complex concentrates have been evaluated in the treatment of acute bleeding episodes occurring in 11 hemophilic and two nonhemophilic patients with factor VIII inhibitors. In several of the hemophilic patients, a continued fall in inhibitor levels has been observed coincident with continued use of the concentrate Proplex, and one of these patients is now responsive to factor VIII therapy. The therapeutic effectiveness of prothrombin complex products appears to be related to the presence of small amounts of activated factors or to the continued formation of factor Xa in vivo as a result of the increased levels of prothrombin and factors VII and X. Until high-potency activated products are available, prothrombin concentrates offer an effective means of treating patients with factor VIII inhibitor.     

Using genetically modified mice to study apolipoprotein B

The B apolipoproteins, apo-B48 and apo-B100, are key proteins in mammalian lipoprotein metabolism and are components of all classes of lipoproteins considered to be atherogenic. Our laboratory has generated an array of genetically modified mice for studying apo-B biology. Using gene targeting in mouse embryonic stem cells, we have generated apo-B-deficient mice. Heterozygotes had low plasma levels of apo-B and cholesterol; homozygotes died early in embryonic development, most likely because the absence of lipoprotein secretion by the yolk sac interfered with the delivery of lipid nutrients to the developing embryo. We have also generated human apo-B transgenic mice with an 80-kb genomic DNA fragment spanning the entire human apo-B gene; those mice had markedly increased plasma levels of low density lipoprotein cholesterol and exhibited increased susceptibility to atherosclerosis. The human apo-B transgenic mice have also yielded insights regarding the regulation of apo-B expression in different tissues. Although the 80-kb transgene contained nearly 20 kb of 5' and 3' flanking sequences and was expressed at high levels in the liver, no transgene expression was detectable in the intestine. Subsequent transgenic mouse studies have demonstrated that the expression of the apo-B gene in the intestine is controlled by DNA sequences that are very distant from the structural gene. Transgenic mice have also proved useful for studying apo-B structure/function relationships. By expressing mutant forms of human apo B in transgenic mice, we have examined the structural features of the apo-B molecule that are required for lipoprotein (a) formation. We have demonstrated that the carboxyl terminal cystine residue of apo-B100, cysteine-4326, is required for apo-B100's disulfide linkage with apo(a) to form lipoprotein (a). Finally, we have used gene targeting techniques to generate mice that synthesize exclusively apo-B48 (apo B48-only mice) and mice that synthesize exclusively apo-B100 (apo-B100 only mice): These mice have helped to clarify the unique metabolic roles of the two apo-B proteins.     

Testis-determining H-Y antigen in XO males of the mole-vole (Ellobius lutescens)

The XO sex chromosome constitution has been found in both sexes of the mole-vole (Ellobius lutescens) belonging to the rodent family Microtinae. This enigmatic species has apparently been enduring a 50% zygotic lethality. The current serological study revealed the presence in XO males and the absence from XO females of H-Y (histocompatibility Y) antigen. In all the mammalian species studied thus far, the expression of H-Y antigen strictly coincided with the presence of testicular tissue and not necessarily with the presence of the Y chromosome. The testis-organizing function of the H-Y gene appears to have been confirmed. In the mole-vole, X linkage of the testis-organizing H-Y gene is favored over its autosomal inheritance. Only X linkage of the H-Y gene creates a compelling evolutionary need to change the female sex chromosome constitution from XX to XO, and to abandon the dosage compensation by an X inactivation mechanism, so that the nonproductive XH-YX zygote can be eliminated as an embryonic lethal. With regard to the electrophoretic mobilities of three X-linked marker enzymes, however, a genetic difference between the male-specific XH-Y and the female-specific X was not detected. This might reflect a relatively recent speciation.     

Adrenal tumors and pregnancy

The lats gene has been identified as a tumour suppressor in Drosophila melanogaster using mosaic screens. Mosaic flies carrying somatic cells that are mutant for lats develop large tumours in many organs. The human LATS1 homologue rescues embryonic lethality and inhibits tumour growth in lats mutant flies, demonstrating the functional conservation of this gene. Biochemical and genetic analyses have revealed that LATS1 functions as a negative regulator of CDC2 (ref. 3). These data suggest that mammalian LATS1 may have a role in tumorigenesis. To elucidate the function of mammalian LATS1, we have generated Lats1-/- mice. Lats1-/- animals exhibit a lack of mammary gland development, infertility and growth retardation. Accompanying these defects are hyperplastic changes in the pituitary and decreased serum hormone levels. The reproductive hormone defects of Lats1-/- mice are reminiscent of isolated LH-hypogonadotropic hypogonadism and corpus luteum insufficiency in humans. Furthermore, Lats1-/- mice develop soft-tissue sarcomas and ovarian stromal cell tumours and are highly sensitive to carcinogenic treatments. Our data demonstrate a role for Lats1 in mammalian tumorigenesis and specific endocrine dysfunction.     

Hepatic vascular tumors, angiectasis in multiple organs, and impaired spermatogenesis in mice with conditional inactivation of the VHL gene

von Hippel-Lindau (VHL) disease is a multisystem inherited cancer syndrome characterized by the development of highly vascular tumors including hemangioblastomas of the retina and central nervous system, pheochromocytomas, and clear cell renal carcinoma, which result from somatic inactivation of the wild-type VHL allele in cells harboring a germ-line VHL mutation. Homozygous inactivation of the VHL gene in mice resulted in embryonic lethality. To produce a mouse model that closely mimics human VHL disease and avoids embryonic lethality, we used Cre/lox site-specific recombination technology. We generated mice carrying conditional VHL alleles and a cre transgene under the control of the human beta-actin promoter, which directs cre expression in a mosaic pattern in multiple organs. VHL(f/d)/Cre mice developed multiple, hepatic hemangiomas that led to premature death, as well as angiectasis and angiogenesis in multiple organs. Interestingly, testes of male VHL(f/d)/Cre mice were unusually small with severely reduced sperm count resulting in infertility. Loss of pVHL function in this VHL conditional knockout mouse model results in an extensive abnormal vascular phenotype in multiple mouse organs, which will provide a useful animal model for testing potential antiangiogenic therapies for VHL disease treatment. Importantly, the phenotypic defects in sperm development observed in these mice support a novel role for VHL in spermatogenesis. This VHL conditional knockout mouse model will provide an in vivo system for studying the functional requirement of the VHL gene in reproductive biology.     

Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice

Receptor tyrosine kinases Flt-1 and Flk-1/KDR, and their ligand, the vascular endothelial growth factor (VEGF), were shown to be essential for angiogenesis in the mouse embryo by gene targeting. Flk-1/KDR null mutant mice exhibited impaired endothelial and hematopoietic cell development. On the other hand, Flt-1 null mutation resulted in early embryonic death at embryonic day 8.5, showing disorganization of blood vessels, such as overgrowth of endothelial cells. Flt-1 differs from Flk-1 in that it displays a higher affinity for VEGF but lower kinase activity, suggesting the importance of its extracellular domain. To examine the biological role of Flt-1 in embryonic development and vascular formation, we deleted the kinase domain without affecting the ligand binding region. Flt-1 tyrosine kinase-deficient homozygous mice (flt-1(TK-/-)) developed normal vessels and survived. However, VEGF-induced macrophage migration was strongly suppressed in flt-1(TK-/-) mice. These results indicate that Flt-1 without tyrosine kinase domain is sufficient to allow embryonic development with normal angiogenesis, and that a receptor tyrosine kinase plays a main biological role as a ligand-binding molecule.     

Gene-dose effect on carnitine transport activity in embryonic fibroblasts of JVS mice as a model of human carnitine transporter deficiency

Recently, the marked decline in renal carnitine reabsorption has been thought to account fotr the systemic carnitine deficiency in juvenile visceral steatosis (JVS) mice. We have conducted a kinetic analysis using embryonic fibroblasts derived from normal, heterozygous, and homozygous jvs mice and found that the high-affinity carnitine transporter (Km = 5.5 microM), which shows Na+ and temperature dependency and stereospecificity, is defective in homozygous jvs mice. Moreover, a gene dose-dependent decrease of carnitine transport activity, which was due to a decrease in the number of the transporter molecules, was found in heterozygous jvs mice. Similar phenomena have been observed in human primary carnitine deficiency. Therefore, JVS mice may be useful for understanding this extremely rare human hereditary disorder.     

Aberrant 11beta-hydroxysteroid dehydrogenase-1 activity in the cpk mouse: implications for regulation by the Ke 6 gene

Glucocorticoids have been used to create experimental polycystic kidney disease in rodents and to induce cysts in embryonic kidneys cultures. In addition, the plasma corticosterone levels are higher in a heritable murine model of polycystic kidney disease, cpk mice, in the first postnatal week. Previously, we had shown that the 11beta-hydroxysteroid dehydrogenase-1 (11betaHSD-1) gene is down-regulated in the cpk mice in a coordinated pattern with the Ke 6 gene. In this study, we measured the level of 11betaHSD-1 activity in kidney and liver tissues of cpk homozygote mice and found a reduction in its activity only in the kidney, not in the liver. The activity of the 11betaHSD-1 enzyme appears to be tightly correlated to the level of Ke 6 protein in these tissues. We discuss the possibility that the activity of the 11betaHSD-1 enzyme may be regulated by the Ke 6 enzyme. Ke 6 gene expression has been located to the outer stripe region of rodent kidneys, which is the same region of expression as that for the 11betaHSD-1 gene. These results suggest that down-regulation of the Ke 6 gene may lead to elevated corticosterone levels, mediated through an inhibition of 11betaHSD-1 activity.     

Intravascular coagulation with use of human prothrombin complex concentrates

Prothrombin complex concentrates are used in the treatment of the congenital bleeding disorders associated with Factors II, VII, IX, and X deficiencies. They have also been extensively used to treat acquired coagulation abnormalities secondary to vitamin K deficiency, warfarin ingestion, and various types of liver disease. The reported complications of prothrombin complex concentrates administration include hepatitis, anaphylaxis, and thrombosis. This paper documents the development of disseminated intravascular coagulation in association with the administration of prothrombin complex concentrates to patients with liver disease.