Deletion of long-range sequences at Sox10 compromises developmental expression in a mouse model of Waardenburg-Shah (WS4) syndrome

The transcription factor SOX10 is mutated in the human neurocristopathy Waardenburg–Shah syndrome(WS4), which is characterized by enteric aganglionosis and pigmentation defects. SOX10 directly regulatesgenes expressed in neural crest lineages, including the enteric ganglia and melanocytes. Although someSOX10 target genes have been reported, the mechanisms by which SOX10 expression is regulated remainelusive. Here, we describe a transgene-insertion mutant mouse line (Hry) that displays partial enteric agan-glionosis, a loss of melanocytes, and decreased Sox10 expression in homozygous embryos. Mutation analy-sis of Sox10 coding sequences was negative, suggesting that non-coding regulatory sequences aredisrupted. To isolate theHry molecular defect, Sox10 genomic sequences werecollected from multiplespecies, comparative sequence analysis was performed and software was designed (ExactPlus) to identifyidentical sequences shared among species. Mutation analysis of conserved sequences revealed a 15.9 kbdeletion located 47.3 kb upstream of Sox10 in Hry mice. ExactPlus revealed three clusters of highly con-served sequences within the deletion, one of which shows strong enhancer potential in cultured melano-cytes. These studies: (i) present a novel hypomorphicSox10 mutation that results in a WS4-likephenotype in mice; (ii) demonstrate that a 15.9 kb deletion underlies the observed phenotype and likelyremoves sequences essential for Sox10 expression; (iii) combine a novel in silico method for comparativesequence analysis within vitro functional assays to identify candidate regulatory sequences deleted inthis strain. These studies will direct further analyses of Sox10 regulation and provide candidate sequencesfor mutation detection in WS4 patients lacking a SOX10-coding mutation.INTRODUCTIONThe neural crest (NC) is a multi-potent, migratory populationof cells that arise from the neural tube boundary duringembryonic development. The NC gives rise to a widevariety of structures, including the craniofacial skeleton,neurons and Schwann cells of the peripheral nervoussystem and melanocytes (1). Delineating the molecular path-ways and transcriptional hierarchies that mediate NC celldevelopment is key toward understanding NC-relatedhuman diseases (neurocristopathies) as well as normalhuman development.Published by Oxford University Press 2005.