Fig. 1. Misexpression of dominant-negative Tbx genes induces a limbless phenotype. (A) When dominant-negative Tbx5 (EnTbx5) was misexpressed by retroviral infection at stages 7 to 10, a wingless phenotype (arrowhead) arose at E12. (A') Skeletal examination of A revealed that wing formation was completely blocked. Legs (L) and the left wing (W) were normal. Formation of the right scapula was completely suppressed with hypoplastic ribs underneath (r') (arrowheads). On the uninfected left side, normal scapula (s) and ribs (r) were evident. (B) In another case, severe distal truncation was obtained at E9 (indicated by a red arrowhead), although this embryo showed normal morphology in the legs (L) and left wing (W). (B') Skeletal examination of B showed truncated wing (W') with hypoplastic scapula (s') and clavicula (c'). In both cases, shoulders were hypomorphic. (C) When dominant-negative Tbx4 (EnTbx4) was similarly misexpressed, a legless phenotype was observed at E8 (red arrowheads). (C') Skeletal preparation of C. Right leg was specifically affected (red arrowheads). Right wing (W) and left leg (L) were normal. This embryo showed the hypomorphic ilium (il') and ischium (is') without pubis formation, making the right side of the pelvis deformed and hypoplastic (red arrowheads). In the left leg (L), normal ilium (il), ischium (is), and pubis (p) were formed. (D) When EnTbx4 viral infection was performed at later stages (11 to 13), truncation of leg (L') structures arose (red arrowheads) with normal wing formation (W). (D') Skeletal preparation of (D) showed a severely distorted leg (red arrowheads). Hypomorphic ilium (il'), ischium (is'), and femur (f') were evident with truncated distal structures (red arrowheads). In this side, the pubis was missing, although femur (f), ilium (il), ischium (is) and pubis (p) were normally formed on the uninfected left side. (E) Misexpression of EnTbx4 in the wing did not induce morphological alteration at E8, although whole-mount in situ hybridization using Env gene in the RCAS vector indicate expression of Tbx4 transgene. (F) Likewise, misexpression of EnTbx5 resulted in normal morphology in the leg, despite of the Env expression.