Mutation of the Sry-related Sox10 gene in Dominant megacolon, a mouse model for human Hirschsprung disease

Abstract

The spontaneous mouse mutant Dominant megacolon (Dom) is a valuable model for the study of human congenital megacolon (Hirschsprung disease). Here we report that the defect in the Dom mouse is caused by mutation of the gene encoding the Sry-related transcription factor Sox10. This assignment is based on (i) colocalization of the Sox10 gene with the Dom mutation on chromosome 15; (ii) altered Sox10 expression in the gut and in neural-crest derived structures of cranial ganglia of Dom mice; (iii) presence of a frameshift in the Sox10 coding region, and (iv) functional inactivation of the resulting truncated protein. These results identify the transcriptional regulator Sox10 as an essential factor in mouse neural crest development and as a further candidate gene for human Hirschsprung disease, especially in cases where it is associated with features of Waardenburg syndrome.

Figure 1

(a) Genetic map of the Dom region in the central-terminal part of chromosome 15. Genetic distances are given based on the EUCIB backcross. (b) Physical map of the the Dom region as determined from positive YACs. LE, left end; RE, right end. YAC chimerism is represented by an arrow.

Figure 2

(a) Predicted amino acid sequences of wild-type (C57BL/6J) and mutant Sox10 (Dom). Divergent amino acids are set off by bold type; the DNA-binding HMG-domain is boxed. (b) Analysis of Sox10 expression by Northern blotting. Ten micrograms of total RNA from brain and intestine of newborn wild-type (+/+), heterozygous (Dom/+), and homozygous (Dom/Dom) mice were transferred to filters and sequentially hybridized with probes for Sox10 (Upper) and β-actin (Lower). Molecular sizes are indicated on the left.

Figure 3

(a) Morphological comparison of cranial ganglia in homozygous mutant (Dom/Dom) and heterozygous (Dom/+) embryos at E10.5 by immunohistochemistry using anti-NF160 specific antibodies. Note that at this developmental stage, NF160 is expressed mainly in the peripheral nervous system. Cranial ganglia (trigeminal, v; facial/acoustic, vii/viii; glossopharyngeal, ix; and vagus, x) are marked. (b) Analysis of Sox10 expression in homozygous mutant (Dom/Dom), heterozygous (Dom/+), and wild-type (+/+) embryos at E10.5 by whole-mount in situ hybridization. Lateral and dorsal views are shown for each embryo. Localization of cranial ganglia (v, vii/viii, ix, x) and dorsal root ganglia (drg) are indicated. Hybridization in the otic vesicle (ot) was nonspecific. (c) Higher magnification of homozygous mutant (Dom/Dom) and wild-type (+/+) E10.5 mouse embryos to show Sox10 expression in cranial ganglia (left two panels) and to compare Sox10 expression (Sox10) with NF160 immunoreactivity (NF) in motor nerves (right four panels). (d) Sox10 expression in the intestine at E14.5 studied by in situ hybridization on sections of homozygous mutant (Dom/Dom), heterozygous (Dom/+), and wild-type (+/+) embryos.

Figure 4

(a) Detection of Sox10 proteins (wild type, Sox10; the glutamate¹¹-to-valine substitution mutant, E¹¹→ V; and the Dom frameshift mutant, fs) in nuclear extracts of transiently transfected COS cells by Western blotting using a rabbit antiserum against Sox10. −, mock-transfected. (b) DNA binding of Sox10 proteins as determined by electrophoretic mobility-shift assay using a radioactively labeled consensus binding site for Sox proteins as probe and COS nuclear extracts from a as protein source. Sox10-specific complexes are marked by asterisks. (c) Promoter induction after transient transfection of the luciferase reporter plasmid 3xFXO luc into U138 glioblastoma cells in combination with empty CMV expression plasmid (−), pCMV/Tst-1 (Tst-1), and CMV expression plasmids for Sox10 proteins (Sox10, E¹¹→ V, and fs). (d) Promoter induction after transient transfection of the luciferase reporter plasmid 3xFXP luc into U138 cells in combination with empty CMV expression plasmid (−), pCMV/Pax3 (Pax3), and CMV expression plasmids for Sox10 proteins. Results in c and d show mean (+SE) promoter induction (n = 4).