Fig. 5

Fig. 5 Deletion of the three enhancer elements T3, C and I results in selective loss of Brachyury protein expression in the notochord at E9.5 and posterior defects at E12.5.

A Overview of wildtype mouse Brachyury/T/TBXTB locus adapted from the UCSC genome browser and deletion alleles generated with CRISPR-Cas9 genome editing. Exact coordinates and sequences of target sites, deletions, and genotyping primer sequences can be found in Supplementary Data 5. B–G Brachyury/T antibody staining (red) of E9.5 embryos. White dashed square in panels represents location of right bottom inserts with 2x magnification. Brachyury/T protein expression in the notochord is dose-dependent on the three enhancer elements. Asterisks in (D–G) mark absent notochord in rostral portion of the embryo. Scale bar in (B): 1 mm, applies to panels (B–G). H–M Overall morphology of E12.5 embryos with different genotypes. Blue lines indicate the location of immunofluorescence and H&E sections. Spina bifida and tail defects are dose-dependent. Arrowheads mark rudimentary tails. White lines mark spina bifida. Scale bar in H: 1 mm, applies to (H–M). N–S Dorsal view of embryos (sectioned at blue line in H–M). White lines mark areas of spina bifida. Arrowheads mark rudimentary tails compared to tails in wildtype control and double knock-out allele. Scale bar in (N): 2.5 mm, applies to panels (N–S). T–Y Immunofluorescence of mouse transverse sections. Anti-Sox2 labels the neural plate, anti-Tbxt the notochord, and DAPI marks nuclei. Sox2 expression is comparable amongst all genotypes, even in the genotypes with spina bifida, while there is loss of Brachyury/T staining in the notochord with increased loss of the enhancers. Arrowheads point to notochord. Asterisks mark absent notochord. Scale bar in (T): 0.2 mm, applies to panels (T–Y). Z–E' H&E staining of transverse sections confirm the dose-dependent loss of the notochord and spina bifida. Arrowheads point to notochord. Asterisks mark absent notochord. Scale bar in (Z): 0.2 mm, applies to (Z–E').