Myotubularin is a ubiquitously expressed phosphatase that acts on phosphatidylinositol 3-monophosphate PI(3)P, a lipid implicated in intracellular vesicle trafficking and autophagy. It is encoded by ...the MTM1 gene, which is mutated in X-linked myotubular myopathy (XLMTM), a muscular disorder characterized by generalized hypotonia and muscle weakness at birth leading to early death of most affected males. The disease was proposed to result from an arrest in myogenesis, as the skeletal muscle from patients contains hypotrophic fibers with centrally located nuclei that resemble fetal myotubes. To understand the physiopathological mechanism of XLMTM, we have generated mice lacking myotubularin by homologous recombination. These mice are viable, but their lifespan is severely reduced. They develop a generalized and progressive myopathy starting at around 4 weeks of age, with amyotrophy and accumulation of central nuclei in skeletal muscle fibers leading to death at 6–14 weeks. Contrary to expectations, we show that muscle differentiation in knockout mice occurs normally. We provide evidence that fibers with centralized myonuclei originate mainly from a structural maintenance defect affecting myotubularin-deficient muscle rather than a regenerative process. In addition, we demonstrate, through a conditional gene-targeting approach, that skeletal muscle is the primary target of murine XLMTM pathology. These mutant mice represent animal models for the human disease and will be a valuable tool for understanding the physiological role of myotubularin.
Identification of factors regulating myocardial structure and function is important to understand the pathogenesis of heart disease. Because little is known about the molecular mechanism of cardiac ...functions triggered by serotonin, the link between downstream signaling circuitry of its receptors and the heart physiology is of widespread interest. None of the serotonin receptor (5-HT(1A), 5-HT(1B), or 5-HT(2C)) disruptions in mice have resulted in cardiovascular defects. In this study, we examined 5-HT(2B) receptor-mutant mice to assess the putative role of serotonin in heart structure and function.
We have generated G(q)-coupled 5-HT(2B) receptor-null mice by homologous recombination. Surviving 5-HT(2B) receptor-mutant mice exhibit cardiomyopathy with a loss of ventricular mass due to a reduction in number and size of cardiomyocytes. This phenotype is intrinsic to cardiac myocytes. 5-HT(2B) receptor-mutant ventricles exhibit dilation and abnormal organization of contractile elements, including Z-stripe enlargement and N-cadherin downregulation. Echocardiography and ECG both confirm the presence of left ventricular dilatation and decreased systolic function in the adult 5-HT(2B) receptor-mutant mice.
Mutation of 5-HT(2B) receptor leads to a cardiomyopathy without hypertrophy and a disruption of intercalated disks. 5-HT(2B) receptor is required for cytoskeleton assembly to membrane structures by its regulation of N-cadherin expression. These results constitute, for the first time, strong genetic evidence that serotonin, via the 5-HT(2B) receptor, regulates cardiac structure and function.
Preimplantation genetic diagnosis (PGD) allows the detection of genetic defects before implantation, thus circumventing the possible need for abortion. France's current legislation allows the ...practice of PGD under certain circumstances which include the prerequisite agreement of the French health authority. Unfortunately, to enact the pending 'bioethical law', voted in July 1994, a decree still needs to be published. So, for the moment, although we know that PGD should be authorized, its practice is currently impossible in France. In order to prepare for licensing, we are setting up the relevant technologies, by performing biopsy on mouse embryos and fluorescent in-situ hybridization (FISH) experiments on human lymphoblast cells. We briefly describe the French legal situation with regard to PGD and the work we have performed in this context to obtain the licensing to offer PGD to patients. After a period of preparation, 95.9% of biopsies were successful and up to 95.4% of the biopsied blastomeres were properly spread onto slides. Biopsied and control mouse embryos were reimplanted into pseudopregnant females and similar birth rates were obtained (34.4 and 30.9% respectively). In these experiments we noticed a birth delay of 12-24 h for the biopsied embryos compared with the controls. Furthermore, scanning electron microscopy of the biopsied embryos allowed assessment of the hole made by the Tyrode's acid. By intercrossing adults derived from biopsied embryos for two successive generations, it was shown that the biopsy did not generate defects affecting their reproductive ability. FISH experiments were performed using specific probes for chromosomes X, Y and 1 on nuclei spread by a conventional protocol or a Tween/HCl blastomere spreading protocol; in the latter case, slides with 1-5 cells were prepared. A similar percentage of correct X,Y,1,1 signal was obtained from all three types of spreading, varying from 85.5 to 89.9%.
Background —Identification of factors regulating myocardial structure and function is important to understand the pathogenesis of heart disease. Because little is known about the molecular mechanism ...of cardiac functions triggered by serotonin, the link between downstream signaling circuitry of its receptors and the heart physiology is of widespread interest. None of the serotonin receptor (5-HT 1A , 5-HT 1B , or 5-HT 2C ) disruptions in mice have resulted in cardiovascular defects. In this study, we examined 5-HT 2B receptor–mutant mice to assess the putative role of serotonin in heart structure and function. Methods and Results —We have generated G q -coupled 5-HT 2B receptor–null mice by homologous recombination. Surviving 5-HT 2B receptor–mutant mice exhibit cardiomyopathy with a loss of ventricular mass due to a reduction in number and size of cardiomyocytes. This phenotype is intrinsic to cardiac myocytes. 5-HT 2B receptor–mutant ventricles exhibit dilation and abnormal organization of contractile elements, including Z-stripe enlargement and N-cadherin downregulation. Echocardiography and ECG both confirm the presence of left ventricular dilatation and decreased systolic function in the adult 5-HT 2B receptor–mutant mice. Conclusions —Mutation of 5-HT 2B receptor leads to a cardiomyopathy without hypertrophy and a disruption of intercalated disks. 5-HT 2B receptor is required for cytoskeleton assembly to membrane structures by its regulation of N-cadherin expression. These results constitute, for the first time, strong genetic evidence that serotonin, via the 5-HT 2B receptor, regulates cardiac structure and function.
During embryogenesis, serotonin has been reported to be involved in craniofacial and cardiovascular morphogenesis. The detailed molecular mechanisms underlying these functions, however remain ...unknown. From mouse and human species, we have recently reported the cloning of 5-HT2B receptors which share signal transduction pathways with other 5-HT2 receptor subtypes (5-HT2A and 5-HT2C). In addition to phospholipase C stimulation, it appears that these three subtypes of receptor transduce a common serotonin-induced mitogenic activity, which could be important for cell differentiation and proliferation. We have first investigated the expression of 5-HT2 receptor mRNAs in the mouse embryo. Interestingly, a peak of 5-HT2B receptor mRNA expression was detected 8-9 days postcoitum, whereas there was only low level 5-HT2A and no 5-HT2C receptor mRNA expression at this stage. Expression of this receptor was confirmed by binding assays using a 5-HT2-specific ligand which revealed a peak of binding to membrane preparations from 9 days postcoitum embryos. In addition, whole mount in situ hybridisation and immunohistochemistry on similar stage embryos detected 5-HT2B expression in neural crest cells, heart myocardium and somites. The requirement for functional 5-HT2B receptors between 8 and 9 days postcoitum is supported by culture of embryos exposed to 5-HT2-specific ligands; 5-HT2B high-affinity antagonist such as ritanserin, induced morphological defects in the cephalic region, heart and neural tube. These antagonistic treatments interfere with cranial neural crest cell migration, induce their apoptosis, and are responsible for abnormal sarcomeric organisation of the subepicardial layer and for the absence of the trabecular cell layer in the ventricular myocardium. This report indicates for the first time that 5-HT2B receptors are actively mediating the action of serotonin on embryonic morphogenesis, probably by preventing the differentiation of cranial neural crest cells and myocardial precursor cells.
Fifty-eight Culicoides species were recorded in Israel, the Golan Heights, and Sinai. Culicoides arabiensis and C. near iranica were recorded for the first time. Culicoides kurensis was identified ...instead of the misidentified C. badooshensis. Of the Culicoides species that appeared as distinct species in previous publications, it is now recognized that C. subravus is a synonym of C. ravus, C. cubitalis is a synonym of C. kibunensis, and C. saevanicus is a synonym of C. griseidorsum. The potential medical/veterinary importance of the vector species is reviewed.
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