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. 2022 Jul;32(7):1242–1253. doi: 10.1101/gr.276196.121

Figure 5.

Figure 5.

Deletions in Hdac9 that alter Twist1 regulatory elements lead to dysregulation of SHH pathway genes. (A) Whole-mount in situ hybridization of E11.5 mouse embryos showing reduced Twist1 expression pattern in eTw5-7Δ/Δ and CTCF-5Δ/Δ. (B) Whole-mount in situ of E11.5 hindlimb mouse embryos for Twist1 and its target SHH pathway genes, Hand2 and Alx4. Twist1 expression is reduced, especially in the anterior limb bud. Hand2 and Alx4 expression along the anterior/posterior axes is lost in the mutants. (C) Quantitative real-time PCR analyses in E11.5 HL buds show a significantly decreased Twist1 expression in anterior limb buds of CTCF-5Δ/Δ (P = 9 × 10−4) and in the entire limb bud of eTw5-7Δ/Δ (P = 5 × 10−4). No expression of Hdac9 in the limb buds of wild-type, CTCF-5Δ/Δ, or eTw5-7Δ/Δ embryos. Significant decrease of Hand2 expression in posterior limb buds of CTCF-5Δ/Δ (P = 3 × 10−2) and eTw5-7Δ/Δ (P = 1 × 10−5) embryos. Significant increase of Alx4 expression in posterior limb buds of CTCF-5Δ/Δ embryos (P = 8.8 × 10−3). Expression levels were normalized to actin, beta expression (Student's t-test, [*] P-value <0.05, [**] P-value <0.01, [***] P-value <0.001).