Abstract
Hirschsprung disease (HSCR) is characterized by absence of enteric neurons from the distal colon and severe intestinal dysmotility. To understand the pathophysiology and genetics of HSCR we developed a unique zebrafish model that allows combined genetic, developmental and in vivo physiological studies. We show that ret mutant zebrafish exhibit cellular, physiological and genetic features of HSCR, including absence of intestinal neurons, reduced peristalsis, and varying phenotype expressivity in the heterozygous state. We perform live imaging experiments using a UAS-GAL4 binary genetic system to drive fluorescent protein expression in ENS progenitors. We demonstrate that ENS progenitors migrate at reduced speed in ret heterozygous embryos, without changes in proliferation or survival, establishing this as a principal pathogenic mechanism for distal aganglionosis. We show, using live imaging of actual intestinal movements, that intestinal motility is severely compromised in ret mutants, and partially impaired in ret heterozygous larvae, and establish a clear correlation between neuron position and organised intestinal motility. We exploited the partially penetrant ret heterozygous phenotype as a sensitised background to test the influence of a candidate modifier gene. We generated mapk10 loss-of-function mutants, which show reduced numbers of enteric neurons. Significantly, we show that introduction of mapk10 mutations into ret heterozygotes enhanced the ENS deficit, supporting MAPK10 as a HSCR susceptibility locus. Our studies demonstrate that ret heterozygous zebrafish is a sensitized model, with many significant advantages over existing murine models, to explore the pathophysiology and complex genetics of HSCR.
Highlights
The genetic basis of diseases exhibiting simple Mendelian inheritance can be readily uncovered, especially using techniques available in the post-genomic era [1]
Hirschsprung Disease (HSCR) is a common congenital intestinal motility disorder diagnosed at birth by absence of enteric neurons in the distal gut, leading to intestinal obstruction that requires life-saving surgery
We establish that ret mutant zebrafish display key features of HSCR, including absence of intestinal neurons, reduced gut motility and varying phenotype expressivity
Summary
The genetic basis of diseases exhibiting simple Mendelian inheritance can be readily uncovered, especially using techniques available in the post-genomic era [1]. The genetic basis of human diseases exhibiting complex, multifactorial and/or polygenic inheritance is daunting to unravel. Conditions such as autism and schizophrenia, show spectra of phenotypes, making clear association between genotype and phenotype difficult [4]. Genetic studies of such diseases require advanced methodologies, including genome-wide association studies (GWAS), and this analysis throws up many possible candidate loci. Perhaps expectedly, modeling these diseases has proven challenging
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