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

Limb-type-specific expression of Tbx5/Tbx4 plays a key role in drawing distinction between a forelimb and a hindlimb. Here, we show insights into specification and determination during commitment of limb-type identity, in particular that median tissues regulate Tbx expressions. By using the RT-PCR technique on chick embryos, the onset of specific Tbx5/Tbx4 expression in the wing/leg region was estimated to be stage 13. Specification of the limb-type identity is thought to occur before stage 9, since all explants from stage 9 through 14 expressed the intrinsic Tbx gene autonomously in a simple culture medium. The results of transplantation experiments revealed that axial structures medial to the lateral plate mesoderm at the level of the wing region are capable of transforming leg identity to wing identity, suggesting that a factor(s) from the median tissues is involved in the limb-type determination. Nevertheless, the transplanted wing region was not converted to leg identity. The results of the transplantation experiments also suggested that wing-type identity is determined much earlier than is leg-type identity. Finally, we also found that inhibitory effects of median tissues mediate the specific expression of Tbx5/Tbx4 in the presumptive wing/leg region. We propose a model for limb-type identification in which inhibitory regulation is involved in restricting one Tbx gene expression by masking the other Tbx expression there.