Recessive cardiac mutant gene c in axolotls (salamanders) provides an excellent model for studying the molecular biology of heart induction. When homozygous, the gene results in a reduction of tropomyosin, an absence of myofibrils, and a failure of the cardiac muscle to initiate contractions. The gene appears to exert its effect via abnormal inductive processes from the anterior endoderm since mutant hearts can be """"""""rescued"""""""" by organ-culturing in the presence of normal endoderm, a known potent heart muscle inductor tissue in vertebrates. Furthermore, it has been determined that the addition of an RNA fraction obtained from normal anterior endoderm or from medium """"""""conditioned"""""""" by the normal endoderm can correct mutant hearts in vitro; these rescued mutant hearts have normal amounts of tropomyosin incorporated into myofibrils that contract normally. The present investigation is designed to elucidate the sequence of cellular and molecular events and mechanism(s) directing normal myofibrillogenesis and to identify, characterize and determine the role of inductive factors which regulate myocyte differentiation.
The specific aims are as follows: (1) we will purify and characterize the RNA produced by normal embryonic anterior endoderm that turns the quiescent mutant hearts into vigorously- contracting """"""""normal"""""""" organs. It is our hypothesis that the normal anterior endoderm in axolotl embryos produces a diffusible RNA which promotes (induces) differentiation of the heart; (2) The gene coding for the active heart inducing RNA will be cloned and sequenced. This will help us test our hypothesis that this single gene mutation alters the inductive capability of the anterior endoderm in mutant axolotls by affecting the production of a diffusible RNA; (3) Tropomyosin, whose expression is apparently modulated by the cardiac lethal mutation, will be analyzed in normal, mutant and rescued-mutant hearts by Northern blot studies, in situ hybridization, and in vitro translation experiments. This research will provide significant new information on the mechanism(s) of inductive interactions responsible for normal myocyte differentiation. The genetic abnormalities of the mutant axolotl system can be used as an important tool in these studies since there is a clearly-defined bioassay end point for the various experiments, namely, normally contracting mutant hearts. Thus, the proposed studies should provide significant insights into the regulation of heart muscle induction and normal myofibrillogenesis at the gene level. The health relevance of understanding the being able to turn a """"""""nonmuscle"""""""" cell into contracting muscle could be tremendous; if this could be applied in humans, people who have damaged tissue in their heart muscle due to myocardial infarcts might be able to have the tissue redifferentiate into functional muscle again. In a broader biological sense, this vertebrate """"""""birth defect"""""""" is potentially capable of providing answers to major unsolved problems in modern biology and medicine related to the control of gene expression during embryonic development.

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National Heart, Lung, and Blood Institute (NHLBI)
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Cardiovascular and Pulmonary Research A Study Section (CVA)
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Upstate Medical University
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