The Molecular Mechanism Of An ENU-induced Hirschsprung Mouse Model

ENU is such a mutagen. This synthetic compound was described as the "most potent mutagen in mice". Following an ENU-mutagenesis screen, a large number of mouse mutants with a variety of phenotypes were recovered. We here describe an ENU-treated B6male mouse, showed a cluster of white spots on the abdomen. Homozygous mutants, generated by intercross of the heterozygous mutants, showed extensive white spotting of the coat and aganglionic megacolon. Our work is divided into three parts based on the mutant mice.1. Generation of Hirschsprung mice using ENU mutagenesisA mouse with a cluster of white spots on the abdomen was discovered by screening G1mice. The mutant mouse was mated with wild-type B6mice:9/25of the progenies showed a similar phenotype. Homozygous mutants, generated by intercross of the heterozygous mutants, showed extensive white spotting of the coat. The shapes of these pigmented patches are irregular and differ at random from mouse to mouse. These mice appeared otherwise healthy in the first2weeks, but after2weeks of age they showed increasing abdominal distention. Eventually, all homozygotes died prematurely.2. Mapping and identification of the mutant gene that cause Hirschsprung diseaseIn order to locate the position of the mutant gene on the chromosome, we outcrossed heterozygotes on the B6genetic background to D2to obtain F1mice and then crossbreed the F1mice to obtain an F2. We tested genomic DNA from24F2samples exhibiting extensive white spotting of the coat and aganglionic megacolon with microsatellite markers across the whole genome. We observed no exchange of the markers D14mit165and D14mit265with the aganglionic megacolon phenotype and found no significant linkages with other chromosomal loci. The region between D14mit165and D14mit265contains the Ednrb gene and the phenotypes of other Ednrb mutants resemble that of the our mutant. Therefore, the Ednrb gene was considered a good candidate for the aganglionic megacolon.On examination of the Ednrb gene, we found a single nucleotide change, a T/C substitution, in exon4at nucleotide position857. This substitution converted Lys-286residue in the fifth transmembrane helix of the G protein-coupled receptor with a proline (L286P).Sequence alignment across multiple species revealed that this aspartic acid residue is highly conserved among vertebrates.3. The effect of Ednrb (L286P) missense mutation on Ca2+transient levels in transfected cellsTo examine whether the L286P mutation has a causal role in HSCR predisposition, we assessed Ednrb receptor ligand-induced intracellular Ca2+responses in a cell line transiently transfected with the mutant receptor. The in vitro mutagenized L286P EDNRB cDNA as well as the wild-type plasmid vector were introduced into CHO-K1cells. Cells were loaded with the fluorescence Ca2+indicator Fluo-4/AM, and ligand-induced intracellular Ca2+increments were monitored.CHO-K1cells transfected with the wild-type EDNRB cDNA responded to the ET-3in a dose-dependent manner. The mutant L286P receptor exhibited a partial impairment of ligand-induced Ca2+transient levels in transfected cells.