Animals heterozygous for Dominant megacolon (Dom/+) exhibit multiple defects in neural crest development including reduced numbers of melanocytes in the skin and an absence of myenteric ganglion in the colon. A human congenital disorder, Hirschsprung disease also exhibits rectocolic aganglionosis and can be associated with hypopigmentation. Thus Dom/+ mice, as well as the piebald and lethal spotting mutants, serve as mouse models for this disease. Dom arose and has been maintained on a C57BL/6JLe X C3HeB/FeJLe-a/a (B6C3F1) hybrid background, however the severity of the phenotype is dramatically influenced by genetic background. Using continuous backcrossing to different inbred strains of mice, we have found that both spotting and survival are increased in Dom/C3 lines in comparison to Dom/B6 lines. Genotype analysis of Dom/B6 N7 and Dom/C3 N11 backcross pedigrees with simple sequence repeat markers defined the region containing Dom to 5.6 cM, bounded by D15Mit68 and D15Mit189. Additional analysis in inter- and intra-subspecific crosses has narrowed the interval containing Dom to less than or equal to 0.001 cM (2 recombinants/1700 meioses). A physical map has been assembled from the region and Dom has been restricted to about 150 kb segment. We are currently using positional and candidate gene approaches to identify the gene that is altered in Dom and are setting up crosses to determine the genes responsible for modifier effects in the different genetic backgrounds. To evaluate the phenotype of Dom/Dom homozygous mice, the most closely linked markers were applied to genotype intercross progeny. Previous reports indicated that Dom/Dom homozygotes die in utero, prior to embryonic day 13.5. Consistent with this finding, no Dom/Dom pups have been identified at weaning (0/160 mice); however Dom/Dom embryos were identified at e16.5, suggesting that the time of embryonic lethality, like spotting and post-natal survival, is variable. Future studies in this area will include in situ hybridization analyses to describe neural crest development in Dom/Dom mutant embryos. Our in vitro neural crest culture system and aggregation chimera analyses will be used to determine if the Dom defect acts intrinsic to the neural crest derived melanocytes. Investigation of the involvement of Dom in Hirschsprung disease will be explored in collaboration with Aravinda Chachravarti (Extramural) through the use of human pedigree analysis.
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