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Specification and determination of limb identity: evidence for inhibitory regulation of Tbx gene expression

Biological Institute, Graduate School of Life Science, Tohoku University, Aobayama Aoba-ku, Sendai 980-8578, Japan

View larger version (34K): [in a new window]   Fig. 1. (A) Amplification profiles for Tbx5/Tbx4 and ß-actin. The intensity of PSL of each band was measured using a molecular imager system and plotted on a semilogarithmic scale against the cycle number. The black square depicted in each graph indicates the cycle number used for quantitative analysis of each gene expression. Two independent sets of experiments using different RNAs gave essentially the same results. (B) Tbx5/Tbx4 expression in the presumptive wing region and the wing bud from embryos of stages 9-23. (C) Tbx5/Tbx4 expression in the presumptive leg region and the leg bud from embryos of stages 9-23. (D,E) The relative amount of Tbx expression after being standardized with ß-actin. The expression of each gene in the presumptive limb region at stage 9 was taken as 1.0.

View larger version (37K): [in a new window]   Fig. 2. (A) Tbx5/Tbx4 expression in cultured explants. Lateral tissues at the level of the presumptive wing and leg regions were prepared from embryos at stages 9 to 14. After incubation for 24 hours, RT-PCR and Southern blot analysis with Tbx5 or Tbx4 probes were performed. The relative amount of each gene was plotted (B,C) after being standardized with ß-actin. The gene expression in stage 9 non-cultured explants was taken as 1.0 for a control. Each experiment was repeated 5 times. Error bars indicate standard deviation.

View larger version (66K): [in a new window]   Fig. 3. (A) Schematic representation of the experimental procedure. As graft tissues for transplantations, the presumptive limb region was taken from wing level (W and blue arrows) or leg level (L and red arrows), and the lateral plate mesoderm with overlying ectoderm was separated from endoderm. Then, the donor graft was inserted in the cavity of the host and placed between the dorsal medial ectoderm and the underlying neural tube and somites at the level of the forelimb or hindlimb region. (B) An example of a wing graft in the leg-level median region. Tbx5 is expressed in the grafted limb bud (white arrowhead) as well as in the host wing bud. (C) Another example of a wing graft in the leg-level median region. Tbx4 is expressed in the host leg bud but is not detected in the grafted limb bud (arrowhead). (D) Expression of Tbx5/Tbx4 in a wing graft at the leg level, analyzed by double in situ hybridization. Tbx5 (turquoise) and Tbx4 (red) are detectable in the host wing and leg bud, respectively. Only blue staining for Tbx5 is obvious in the graft (arrowhead). (E) An example of a leg graft in the wing-level median region. Tbx5 is clearly detectable in the grafted limb bud (arrow). (F) Another example of a leg graft at the wing level. Tbx4 is detectable in the grafted limb bud. Note that Tbx4 expression in the leg graft (arrow) appears weaker than that in the host leg bud. (G) Expression of Tbx5/Tbx4 in a leg graft at the wing level. Chimeric staining with blue (for Tbx5) and red (for Tbx4) is apparent in the graft (arrows). (H,I) Tbx5/Tbx4 expression in serial sections of a leg graft at the wing level. In the area to the right of the arrowheads Tbx4 expression is much weaker than on the left of arrowheads, and the expression of Tbx5 is reversed.

View larger version (70K): [in a new window]   Fig. 4. Cartilage pattern of additional limbs derived from grafted presumptive limb regions. (A) A specimen of a wing graft (arrow) on the leg-level median region has a wing-type skeletal pattern that includes wing digits 2, 3 and 4. (B) A specimen of a leg graft (arrow) at the wing level has a wing-type pattern that includes wing digits 3 and 4. Compare them with leg digits (indicated by I, II, III, IV in A). (C,D) Specimens of a leg graft at the wing level have defective patterns that include leg-like digits. We classified these incomplete types of skeletal patterns (C,D) as unidentified in Table 2.

View larger version (39K): [in a new window]   Fig. 5. (A) Schematic representation of the experimental procedure. Regions 1, 2, 3, 4 and 5 represent neck level- (yellow), wing level- (turquoise), flank level- (blue) and leg level- (pink) median tissues of stage 12 and the presumptive leg region of stage 10 (pink), respectively. (B) Tbx5/Tbx4 expression in cultured explants. Lanes 1, 2, 3 and 4 represent Tbx5/Tbx4 expression of each level-median tissue when regions 1, 2, 3 and 4 were cultured separately. Lanes 1+5, 2+5, 3+5 and 4+5 represent Tbx5/Tbx4 expression of each level-median tissues and the presumptive leg region when these two regions were co-cultured. Forty-eight hours after incubation, RT-PCR and Southern blot analyses were performed. The relative amount of expression of each gene was plotted (C,D) after being standardized with ß-actin. The amount of each Tbx gene expression in 2+5 explants was taken as 1.0. Each experiment was repeated 5 times. Error bars indicate s.d..

View larger version (30K): [in a new window]   Fig. 6. (A) Schematic representation of the experimental procedure. Sample 1 represents a non-cultured ‘unseparated’ explant. Sample 2 is a 24-hour-cultured ‘separated’ explant. Sample 3 is a 24-hour-cultured ‘unseparated’ explant. (B) Tbx5/Tbx4 expressions in non-cultured and cultured explants. Lanes 1, 2 and 3 correspond to Tbx5/Tbx4 expressions in samples 1, 2 and 3, respectively. After incubation, RT-PCR and Southern blot analyses were performed. The relative amount of expression of each gene was plotted (C,D) after being standardized with ß-actin. The amount of each Tbx gene expression in 1 was taken as 1.0. Each experiment was repeated 5 times. Error bars indicate s.d.

View larger version (25K): [in a new window]   Fig. 7. A schematic model of wing/leg development. Turquoise and pink rectangles in the diagrams of the embryos represent the presumptive wing and leg region, respectively. The size of a letter indicates the degree of gene expression. In the presumptive wing region, wing-type identity is specified and determined before stage 9. This determination is maintained until stage 13, and this region begins to exhibit a wing phenotype, as Tbx5 expression, at stage 13. In this process, the abilities to induce Tbx5 and to repress Tbx4 in the presumptive wing level-median tissues contribute to determination and maintenance of wing-type identity. In the presumptive leg region, leg-type identity is specified before stage 9 and determined at stage 12/13, and this region soon begins to differentiate into a leg bud with Tbx4 up-regulation at stage 13. Tbx5-repressing and Tbx4-inducing abilities in the presumptive leg level-median tissues contribute to the determination of leg-type identity.

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