Loss ofGrhl3expression in gut endoderm causes NTDs. (A, B) Among litters analysed at E15.5, conditional knockout of Grhl3 in the gut endoderm, using Sox17cre causes spina bifida (SB; example shown also has a tightly curled tail) and/or tail flexion defects (TFD) whereas wild-type and heterozygotes are unaffected (ST; straight tail; scale bars represent 1 mm). The proportion of phenotypes differs significantly with genotype (* different from wild-type and Grhl3 heterozygous genotypes, p<0.001; Fisher Exact). (C) At E11.5, both Sox17+/cre;Grhl3f/- and Grhl3-/- embryos have persistent open, extremely large PNPs (rostral limit indicated by red arrowhead), whereas the PNP is closed in wild-type embryos (scale bars = 0.5 mm). (D) At E10.5, the PNP of wildtype and heterozygous embryos shortens as closure progresses to completion. PNP length of Grhl3 null (Grhl3-/-) is enlarged at all somite stages, and and gut-conditional null (Sox17+/cre;Grhl3f/-) embryos is enlarged from 24 somites onwards compared with wild-type (Grhl3f/+) and heterozygous embryos. # indicates significant difference compared with all other genotypes (p<0.001; ANOVA); n = 15 Grhl3f/+, 57 Grhl3+/-, 28 Grhl3-/-, 31 Sox17cre/+; Grhl3f/- (5–28 embryos per group at each somite stage).