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doi: 10.1242/10.1242/dev.00474

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Tbx5 and Tbx4 trigger limb initiation through activation of the Wnt/Fgf signaling cascade

Jun K. Takeuchi^*, Kazuko Koshiba-Takeuchi^*, Takayuki Suzuki, Mika Kamimura, Keiko Ogura and Toshihiko Ogura

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan
^* Present address: Cardiovascular Research, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada

View larger version (63K): [in a new window]   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.

View larger version (83K): [in a new window]   Fig. 2. Expression of the dominant-negative Tbx5 (EnTbx5) and Tbx4 (EnTbx4) induces repression of Wnt and Fgf genes, and Pea3. (A) When EnTbx5 was misexpressed in the prospective wing field by in ovo electroporation, Wnt2b expression was repressed 24 hours after electroporation (arrowheads), whereas this gene was normally expressed in the left side. (A') Robust fluorescent signals derived from the co-electroporated EGFP gene were evident where Wnt2b was repressed. (B) By contrast, Wnt8c expression was normal 14 hours after electroporation of EnTbx5 in the prospective leg field. (B') Robust GFP fluorescent signals were observed in the leg field. (C) Expression of Fgf8 was repressed in the hypoplastic right wing buds (red arrowheads). Normal expression of Fgf8 was evident in the left wing buds. (D) Robust Env expression indicated successful EnTbx5 misexpression in the prospective wing field 19 hours after electroporation (arrowheads). In such embryos, expression of Fgf10 (E) and Pea3 (F) was repressed, whereas the normal expression was evident on the opposite side of the embryo. (G) When EnTbx4 electroporation was performed in the prospective leg field, Wnt8c expression was repressed (arrowheads) where robust GFP signals were observed (G'). (H) When EnTbx4 was misexpressed in the prospective wing field, Wnt2b expression was unaffected, although this area showed strong GFP signals (H'). (I) Expression of Fgf10 was repressed in the hypoplastic leg buds (red arrowheads). Normal expression of this gene was evident in the left leg buds. (J) Thirty hours after electroporation, robust env expression was evident in the leg field (red arrowheads). In its serial sections, expression of Fgf8 (K) and Pea3 (L) was repressed (red arrowheads). (M-O) Top row shows wings and bottom row shows legs. Arrowheads indicate repression. (M) When a dominant-negative form of Lef1 (dnLef1) was misexpressed, expression of Fgf10 was weakly repressed in both dnLef1-misexpressed wing and leg fields 24 hours after electroporartion. At this stage, expression of Tbx5 and Tbx4 was normal. (N) Repression of Fgf10 became evident 30 hours after electroporation. At this stage, expression of Tbx5 and Tbx4 was also weakly repressed. (O) Repression of Tbx5 and Tbx4 became evident 36 hours after electroporation.

View larger version (104K): [in a new window]   Fig. 3. Additional limb formation induced by Wnt2b, Tbx5 and Tbx4 in the flank. (A) When Wnt2b-expressing cells were implanted in the flank, a mosaic limb was formed (red arrowheads). (A') Careful examination of this additional limb revealed its mosaic structures with wing-like morphology on the anterior side (W') and leg-like structure on the posterior side (L'). (B) At earlier stages, the Wnt2b-induced additional limb (green arrow) expressed Tbx5 (red arrow) and Tbx4 (blue arrow) in its anterior and posterior sides, respectively. A white circle indicates the position of cell implantation. (C to C'') In the Wnt2b-induced limb buds, leg-specific Hoxb9 (C) and Hoxc9 (C') were expressed in the posterior region, whereas wing-specific Hoxd9 (C'') in the anterior. Arrows correspond to the positions indicated in B. (D) Schematic representation of the Wnt- or Fgf-induced additional limb and its mosaic structures. (E) When CAGGS-Tbx5-EGFP plasmid was electroporated in the flank, wing-like limb was formed (red arrowhead), covered extensively by feather buds. (F) About 16 hours after electroporation, Wnt2b expression was induced (arrowhead) in the electroporated area visualized by the GFP (G). Expression of Fgf10 (H) was observed in the induced limb bud (red arrowhead). L, normal leg bud. (I) About 60 hours after electroporation, an additional limb (red arrowhead) was formed at the caudal side of the normal wing (W). This additional limb bud expressed Fgf8 in the AER (red arrowhead). (J) In the Tbx5-induced limb, expression of leg-specific Hoxb9 was observed weakly only in the posterior margin, excluded form the anterior part of limb. (K) Expression of another leg-specific Hoxc9 was very faint. (L) By contrast, wing-specific Hoxd9 was expressed. Red brackets indicate additional limbs. (M) When the CAGGS-Tbx4-EGFP plasmid was electroporated in the flank, a leg-like limb was formed (red arrowheads). Black arrowheads indicate a boundary between feather and scale domains. When expression of several marker genes was examined, Wnt8c (N, red arrowheads) was induced. Electroporation was monitored by the GFP as shown in O. (P) Likewise, expression of Fgf10 was induced in the additional limb bud (red arrowhead). (Q) In the Tbx4-induced limb bud, expression of Fgf8 (red arrowhead) was observed in the AER. In the additional limb buds, leg-specific Hoxb9 (R) and Hoxc9 (S) were expressed. (T) By contrast, expression of wing-specific Hoxd9 was suppressed, although faint expression was observed at the anterior margin. Red brackets indicate additional limbs.

View larger version (20K): [in a new window]   Fig. 4. Schematic representation of the signal cascade. In early stages of limb development (stages 10 to 12), Tbx5 and Tbx4 activate the Wnt2B/Fgf and Wnt8C/Fgf signals, respectively. Once activated, the Wnt/Fgf cascades feedback on to Tbx5 and Tbx4 genes to establish a tight positive regulatory loop. At later developmental stages, the Wnt and Fgf signaling cascades interact to make a positive feedback loop and maintain outgrowth of limb buds. During later stages, Tbx5 and Tbx4 exert distinct actions to make different limb structures: wing and leg, respectively.