| Abstract |
Zebrafish (Danio rerio) is an excellent organism for modeling human diseases and is widely used for studies during the last two decades. It is amenable to forward and reverse genetic screens, and embryos can be obtained in large numbers, are transparent during early developmental stages. This is an advantage that allows in vivo studies through non-invasive imaging. Most of the organs have simple morphology and are amenable to study at larval stages. Finally it shares a high presentence of homology with the human genome. During a forward genetic screen we identified two novel mutant lines. The first is the s457 line which we used as a model for studying intracardiac flow dynamics in respect to the development of the cardiac valves and the second one is the s266 line which is the first zebrafish Charcot-Marie-Tooth Disease (CMT2D) model.
Valvular heart disease is responsible for considerable morbidity and mortality. Cardiac valves develop as the heart contracts, and they function throughout the lifetime of the organism to prevent retrograde blood flow. Their precise morphogenesis is crucial for cardiac function. Accumulating evidence suggests a role for contractility and intracardiac flow dynamics in cardiac valve development. However, these two factors have proved difficult to uncouple, especially since altering myocardial function affects the intracardiac flow pattern.
Here, we describe a novel zebrafish model of valve defects. We identified a novel mutant allele of southpaw (spaw), a nodal related gene involved in the early establishment of left-right asymmetry, which exhibits randomized heart and endoderm positioning. We show that midline, unlooped hearts have defects in cardiac valve maturation, contrary to mutant hearts that are properly looped or exhibit situs inversus. spaw mutants have no detectable defects in myocardial function. Only the relative position of the two chambers is altered in these mutants, affecting the geometry of the heart and therefore the intracardiac flow pattern. Combined those data with another mutant allele of weak atrium (wea), s459 embryos that carry a point mutation in myosin heavy chain 6 (myh6) and affects the myocardium function causing atrioventricular valves failure in homozygous adult fish, we proved that that intracardiac flow dynamics regulate valve morphogenesis, independently of myocardial contractility.
As mentioned above, in this study we also described the first Charcot-Marie-Tooth type 2D zebrafish model. Charcot-Marie-Tooth (CMT) disease is a genetically heterogeneous group of peripheral neuropathies. Mutations in several amino acyl tRNA synthetase (AARS) genes have been implicated in inherited CMT disease. There are 12 reported CMT-causing mutations dispersed throughout the primary sequence of the human Glycyl t-RNA
synthetase (GARS). While there is strong genetic evidence linking GARS mutations to CMT disease, the molecular pathology that underlies the progression of the neuromuscular phenotype is still not fully understood. In particular, it is unclear whether the mutations result in a toxic gain of function, a partial loss of activity related to translation, or a combination of these mechanisms. During a forward genetic screen, we identified a zebrafish allele of gars (garss266). Homozygous mutant embryos carry a C->A transversion, leading to a non-conservative substitution within the gars gene that changes a threonine to a lysine, residing next to the CMT associated human mutation L129P. We show that the neuromuscular phenotype observed in animals homozygous for T130K is due to a loss of dimerization of the mutated protein and the loss of function and dimer deficient G240R mutant protein is unable to rescue the above phenotype. In addition, G526R Gars dimerizes with the remaining wild-type protein in animals heterozygous for the T130K GARS and reduces the function enough, to elicit a neuromuscular phenotype. Our data indicate that dimerization is required for the dominant neurotoxicity of disease-associated GARS mutations and provide a rapid, tractable model for studying newly identified variants for a role in human disease.
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Μελέτη της ανάπτυξης καρδιακών βαλβίδων χρησιμοποιώντας το zebrafish ως οργανισμό μοντέλο : η επίδραση γενετικών και φαρμακολογικών παραγόντων
