The Nucleoside Diphosphate Kinase Gene Nme3 Acts as Quantitative Trait Locus Promoting Non-Mendelian Inheritance

The t-haplotype, a variant form of the t-complex region on mouse chromosome 17, acts as selfish genetic element and is transmitted at high frequencies (>95%) from heterozygous (t/+) males to their offspring. This phenotype is termed transmission ratio distortion (TRD) and is caused by the interaction of the t-complex responder (Tcr) with several quantitative trait loci (QTL), the t-complex distorters (Tcd1 to Tcd4), all located within the t-haplotype region. Current data suggest that the distorters collectively impair motility of all sperm derived from t/+ males; t-sperm is rescued by the responder, whereas +-sperm remains partially dysfunctional. Recently we have identified two distorters as regulators of RHO small G proteins. Here we show that the nucleoside diphosphate kinase gene Nme3 acts as a QTL on TRD. Reduction of the Nme3 dosage by gene targeting of the wild-type allele enhanced the transmission rate of the t-haplotype and phenocopied distorter function. Genetic and biochemical analysis showed that the t-allele of Nme3 harbors a mutation (P89S) that compromises enzymatic activity of the protein and genetically acts as a hypomorph. Transgenic overexpression of the Nme3 t-allele reduced t-haplotype transmission, proving it to be a distorter. We propose that the NME3 protein interacts with RHO signaling cascades to impair sperm motility through hyperactivation of SMOK, the wild-type form of the responder. This deleterious effect of the distorters is counter-balanced by the responder, SMOKTcr, a dominant-negative protein kinase exclusively expressed in t-sperm, thus permitting selfish behaviour and preferential transmission of the t-haplotype. In addition, the previously reported association of NME family members with RHO signaling in somatic cell motility and metastasis, in conjunction with our data involving RHO signaling in sperm motility, suggests a functional conservation between mechanisms for motility control in somatic cells and spermatozoa

Molecular and Cellular Basis of Microvascular Perfusion Deficits Induced by Clostridium perfringens and Clostridium septicum

Reduced tissue perfusion leading to tissue ischemia is a central component of the pathogenesis of myonecrosis caused by Clostridium perfringens. The C. perfringens α-toxin has been shown capable of inducing these changes, but its potential synergy with perfringolysin O (θ-toxin) is less well understood. Similarly, Clostridium septicum is a highly virulent causative agent of spontaneous gas gangrene, but its effect on the microcirculation has not been examined. Therefore, the aim of this study was to use intravital microscopy to examine the effects of C. perfringens and C. septicum on the functional microcirculation, coupled with the use of isogenic toxin mutants to elucidate the role of particular toxins in the resultant microvascular perfusion deficits. This study represents the first time this integrated approach has been used in the analysis of the pathological response to clostridial toxins. Culture supernatants from wild-type C. perfringens induced extensive cell death within 30 min, as assessed by in vivo uptake of propidium iodide. Furthermore, significant reductions in capillary perfusion were observed within 60 min. Depletion of either platelets or neutrophils reduced the alteration in perfusion, consistent with a role for these blood-borne cells in obstructing perfusion. In addition, mutation of either the α-toxin or perfringolysin O structural genes attenuated the reduction in perfusion, a process that was reversed by genetic complementation. C. septicum also induced a marked reduction in perfusion, with the degree of microvascular compromise correlating with the level of the C. septicum α-toxin. Together, these data indicate that as a result of its ability to produce α-toxin and perfringolysin O, C. perfringens rapidly induces irreversible cellular injury and a marked reduction in microvascular perfusion. Since C. septicum induces a similar reduction in microvascular perfusion, it is postulated that this function is central to the pathogenesis of clostridial myonecrosis, irrespective of the causative bacterium

Notch Lineages and Activity in Intestinal Stem Cells Determined by a New Set of Knock-In Mice

The conserved role of Notch signaling in controlling intestinal cell fate specification and homeostasis has been extensively studied. Nevertheless, the precise identity of the cells in which Notch signaling is active and the role of different Notch receptor paralogues in the intestine remain ambiguous, due to the lack of reliable tools to investigate Notch expression and function in vivo. We generated a new series of transgenic mice that allowed us, by lineage analysis, to formally prove that Notch1 and Notch2 are specifically expressed in crypt stem cells. In addition, a novel Notch reporter mouse, Hes1-EmGFPSAT, demonstrated exclusive Notch activity in crypt stem cells and absorptive progenitors. This roster of knock-in and reporter mice represents a valuable resource to functionally explore the Notch pathway in vivo in virtually all tissues

An Inducible and Reversible Mouse Genetic Rescue System

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications

Fatal haemorrhage and incomplete block to embryogenesis in mice locking coagulation factor V

COAGULATION factor V is a critical cofactor for the activation of prothrombin to thrombin, the penultimate step in the generation of a fibrin blood clot(1,2). Genetic deficiency of factor V results in a congenital bleeding disorder (parahaemophilia)(3), whereas inheritance of a mutation rendering factor V resistant to inactivation is an important risk factor for thrombosis(4,5). We report here that approximately half of homozygous embryos deficient in factor V (F upsilon(-/-)), which have been generated by gene targeting, die at embryonic day (E) 9-10, possibly as a result of an abnormality in the yolk-sac vasculature. The remaining F upsilon(-/-) mice progress normally to term, but die from massive haemorrhage within 2 hours or birth. Considered together with the milder phenotypes generally associated with deficiencies of other clotting factors(6,7), our findings demonstrate the primary role of the common coagulation pathway and the absolute requirement for functional factor V for prothrombinase activity. They also provide direct evidence for the existence of other critical haemostatic functions for thrombin in addition to fibrin clot formation, and identify a previously unrecognized role for the coagulation system in early mammalian development.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62813/1/384066a0.pd

Chromosomal deletion complexes in mice by radiation of embryonic stem cells.

Chromosomal deletions (deficiencies\u27) are powerful tools in the genetic analysis of complex genomes. They have been exploited extensively in Drosophila melanogaster, an organism in which deficiencies can be efficiently induced and selected. Spontaneous deletions in humans have facilitated the dissection of phenotypes in contiguous gene syndromes and led to the positional cloning of critical genes. In mice, deletion complexes created by whole animal irradiation experiments have enabled a systematic characterization of functional units along defined chromosomal regions. However, classical mutagenesis in mice is logistically impractical for generating deletion sets on a genome-wide scale. Here, we report a high-throughput method for generating radiation-induced deletion complexes at defined regions in the genome using ES cells. Dozens of deletions of up to several centiMorgans, encompassing a specific locus, can be created in a single experiment and transmitted through the germline. The ability to rapidly create deletion complexes along chromosomes will facilitate systematic functional analyses of the mammalian genome

Inactivation of Notch 1 in immature thymocytes does not perturb CD4 or CD8T cell development.

Notch proteins influence cell-fate decisions in many developing systems. Several gain-of-function studies have suggested a critical role for Notch 1 signaling in CD4-CD8 lineage commitment, maturation and survival in the thymus. However, we show here that tissue-specific inactivation of the gene encoding Notch 1 in immature (CD25+CD44-)T cell precursors does not affect subsequent thymocyte development. Neither steady-state numbers nor the rate of production of CD4+ and CD8+ mature thymocytes is perturbed in the absence of Notch 1. In addition, Notch 1-deficient thymocytes are normally sensitive to spontaneous or glucocorticoid-induced apoptosis. In contrast to earlier reports, these data formally exclude an essential role for Notch 1 in CD4-CD8 lineage commitment, maturation or survival

Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation.

Tie-1 and Tie-2 define a new class of receptor tyrosine kinases that are specifically expressed in developing vascular endothelial cells. To study the functions of Tie-1 and Tie-2 during vascular endothelial cell growth and differentiation in vivo, targeted mutations of the genes in mice were introduced by homologous recombination. Embryos deficient in Tie-1 failed to establish structural integrity of vascular endothelial cells, resulting in oedema and subsequently localized haemorrhage. However, analyses of embryos deficient in Tie-2 showed that it is important in angiogenesis, particularly for vascular network formation in endothelial cells. This result contrasts with previous reports on Tie-2 function in vasculogenesis and/or endothelial cell survival. Our in vivo analyses indicate that the structurally related receptor tyrosine kinases Tie-1 and Tie-2 have important but distinct roles in the formation of blood vessels