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. 2017 Nov 15;19(1):118–134. doi: 10.15252/embr.201744201

Figure EV1. Y to A mutations in T and Tbx6 .

Figure EV1

  1. Whole‐mount in situ hybridization for Shh expression reveals the loss of notochordal structures in T Y88A mutant embryos at E9.5–10.0. White arrows point to the remnants of notochord at the rostral end of the embryo. Asterisks depict Shh staining in the foregut. The panels on the right display the kinked neural tube (outlined with a black dashed line) and truncated posterior mesoderm in the T Y88A mutant embryos at E9.5. The T Y88A mutant embryo is the same as that in Fig 1F. Scale bar: 200 μm. Ant, anterior; Pos, posterior.
  2. Schematic illustrating the homologous recombination strategy to insert the Y137A point mutation in the endogenous Tbx6 locus. The hygromycin selection was removed by expression of FlpE recombinase. The orange line depicts the probe used for Southern blotting to confirm integration of the mutation and removal of the hygromycin resistance cassette. BglI restriction enzyme sites used for Southern blotting are depicted.
  3. Southern blot of mESC genomic DNA to detect homologous recombination of the Y137A mutation and removal of the hygromycin cassette using the probe depicted in (B). The 3.2‐kb band corresponds to the Tbx6 Y137A mutant allele containing the hygromycin selection cassette, while the 7.6‐kb band corresponds to the Tbx6 Y137A allele with the selection cassette removed. The 7.0‐kb band corresponds to the Tbx6 Δ null allele.