Fig. 5. RNA whole-mount in situ hybridisation analysis of limb buds injected with dominant-negative forms of Tbx5. (A) Dorsal view of the control, uninjected limb bud showing Fgf8, expressed in the AER, and Shh, expressed in the ZPA at stage 23. (B) Dorsal view of Tbx5-injected limb. Fgf8 is not expressed in the anterior of the injected limb and Shh expression is not detected in the ZPA. In this example, the injection was carried out at stage 8 to achieve high-level infection. In A, the image of the contralateral uninjected control limb has been reversed to provide a clearer comparison with the respective experimental, injected limb in B. (C) Distal view of an uninjected control limb showing Fgf8 expression in the AER and Shh expression in the ZPA. (D) Distal view of the limb injected with Tbx5 virus at stage 10 and harvested at stage 23. Injection at the later stage produces lower-level infection and a weaker phenotype. Cells expressing Fgf8 are present in the anterior of the limb in a broader region of flattened ectoderm and have not formed a ridge of cells (arrow). (E) A transverse section through the anterior of the limb shown in C. The Fgf8-expressing cells (arrow) form a ridge of cells at the distal tip of the limb. (F) Transverse section through the anterior of the limb shown in D. The Fgf8-expressing cells (arrow) are present in the flattened ectoderm and do not form a ridge. The effect of Tbx5 misexpression is observed primarily in the anterior mesenchyme.

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