THE CSPG2 GENE, DISRUPTED IN THE HDF MUTANT, IS REQUIRED FOR RIGHT CARDIAC CHAMBER AND ENDOCARDIAL CUSHION FORMATION

The heart defect (hdf) mouse is a recessive lethal that arose from a t ransgene insertional mutation on chromosome 13. Embryos homozygous for the transgene die in utero by embryonic day 10.5 postcoitus and exhib it specific defects along the anterior-posterior cardiac axis. The fut ure right ventricle and conus/truncus of the single heart tube fail to form and the endocardial cushions in the atrioventricular and conus/t runcus regions are absent. Because the hdf mouse mutation provided the opportunity to identify a gene required for endocardial cushion forma tion and for specification or maintenance of the anterior most segment s of the heart, we initiated studies to further characterize the pheno type, clone the insertion site, and identify the gene disrupted. Chrom osome mapping studies first identified the gene, Cspg2 (versican), as a candidate hdf gene. In addition, an antibody recognizing a glycosami noglycan epitope on versican was found to be positive by immunohistoch emistry in the extracellular matrix of normal wild-type embryonic hear ts, but absent in homozygous hearts. Expression analysis of the Cspg2 gene showed that the 6/8, 6/9, and 7/3 Cspg2 exon boundaries were pres ent in mRNA of normal wild-type embryonic hearts but absent in the hom ozygous mutant embryos. DNA sequence flanking the transgene was used t o isolate from a normal mouse library overlapping genomic DNA segments that span the transgene insertion site. The contiguous genomic DNA se gment was found to contain exon 7 of the Cspg2 in a position 3' to the transgene insertion site. These four separate lines of evidence suppo rt the hypothesis that Cspg2 is the gene disrupted by the transgene in sertion in the hdf mouse line. The findings of this study and our prev ious studies of the hdf insertional mutant mouse have shown that norma l expression of the Cspg2 gene is required for the successful developm ent of the endocardial cushion swellings and the embryonic heart segme nts that give rise to the right ventricle and conus/truncus in the out let of the looped heart. (C) 1998 Academic Press.