Molecular-Genetic Analysis of the silent heart Mutation in Zebrafish

Abstract 356 Cardiac Development and Gene Regulation Platform, Sunday, 5/2

We have taken a genetic approach using zebrafish to study the molecular basis of cardiac contractility. Recessive lethal mutations affecting cardiac contractility have previously been identified in screens of zebrafish embryos. Our studies have focused on silent heart, a mutation which blocks cardiac contractility and leaves skeletal and smooth muscle function intact. Cellular and molecular analyses of silent heart embryos show that excitation is uncoupled from contraction, sarcomere assembly is disrupted, and the thin filament protein cardiac troponin T (cTnT) is absent in mutant embryo hearts. Although much is known about the biochemical structure and function of cTnT, gaps in knowledge exist regarding its transcriptional regulation and its role during sarcomere assembly. The silent heart mutation provides a unique entry point for investigating these processes. To determine whether the silent heart gene encodes zebrafish cTnT or a regulator of its expression, we have taken a candidate gene approach. Zebrafish cTnT was isolated from an adult zebrafish heart expression library by screening with a monoclonal antibody to cTnT. Using the cloned cTnT as a riboprobe for whole-mount in situ hybridization, the gene expression pattern was evaluated in three alleles of the silent heart mutation. In contrast to the robust cardiac expression pattern seen in wild-type embryos, cTnT expression was dramatically reduced in two alleles of the silent heart mutation, sihb109 and sihtc300b, and absent in a third allele, sihb475. The sihb475 allele results from a large lesion which deletes the distal arm of zebrafish linkage group 23 where the silent heart gene maps. Furthermore, we determined by PCR that the cTnT gene is also deleted in this allele, thus suggesting genetic linkage between the cTnT and silent heart gene loci. We therefore proceeded to sequence the entire coding region of the cTnT gene in both the sihb109 and sihtc300b alleles, but found no base pair changes explaining the phenotype. It currently remains possible that the mutation resides in either a cis-acting element or in a trans-acting factor required for cTnT expression. To definitively evaluate genetic linkage between cTnT and the silent heart mutation, we have raised a mapping cross in the sihb109 background and have identified a restriction polymorphism in the 3' UTR of the cTnT gene. Genetic linkage testing is currently underway and a report of our results will be presented. Isolating the silent heart gene and identifying the causative genetic defect promises to advance our understanding of cTnT regulation and function during development and disease.

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(Spon by: James Bristow)

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Sehnert, A., Fishman, M. & Stainier, D. Molecular-Genetic Analysis of the silent heart Mutation in Zebrafish. Pediatr Res 45, 62 (1999) doi:10.1203/00006450-199904020-00373

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