A novel C-to-T nucleotide substitution (G-to-A on forward strand) was identified at -21,350 downstream of the start codon at position 135,594,774 (GRCm38) in the promoter/enhancer region of mutant genomic DNA, compared with the partially congenic wild-type strain which carries a region of SWR encompassing the ct locus. The C to T nucleotide substitution is not a known polymorphism according to dbSNP. The wild-type sequence, corresponding to nucleotide C, was also present in C57/BL6 and four other strains. A bacterial artificial chromosome (BAC), containing the complete Grhl3 gene together with 120 kb of upstream sequence that encompasses the site of the putative ct mutation, completely rescued the curly tail phenotype when expressed in mutant mice. Grhl3 expression was examined in caudal regions isolated from ct/ct embryos and equivalent somite staged ct/ct embryos that were matched for genetic background. At the 28-29 and 30-31 somite stages, which correspond to the final stages of spinal neurulation, Grhl3 mRNA was reduced in abundance in ct/ct embryos compared with wild-type by 56 and 41%, respectively, whereas no difference was apparent at the 25-27 somite stage. These findings support the idea that insufficient expression of Grhl3, in the hindgut endoderm located ventrally to the PNP, could be causally related to spina bifida in curly tail embryos. (J:129979)
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