INVOLVEMENT OF T-BOX GENES TBX2-TBX5 IN VERTEBRATE LIMB SPECIFICATIONAND DEVELOPMENT

We have recently shown in mice that four members of the T-box family o f transcription factors (Tbx2-Tbx5) are expressed in developing limb b uds, and that expression of two of these genes, Tbx4 and Tbx5, is prim arily restricted to the developing hindlimbs and forelimbs, respective ly. In this report, we investigate the role of these genes in limb spe cification and development, using the chick as a model system. We indu ced the formation of ectopic limbs in the flank of chick embryos to ex amine the relationship between the identity of the limb-specific T-box genes being expressed and the identity of limb structures that subseq uently develop. We found that, whereas bud regions expressing Tbx4 dev eloped characteristic leg structures, regions expressing Tbx5 develope d characteristic wing features. In addition, heterotopic grafts of lim b mesenchyme (wing bud into leg bud, and vice versa), which are known to retain the identity of the donor tissue after transplantation, reta ined autonomous expression of the appropriate, limb-specific T-box gen e, with no evidence of regulation by the host bud. Thus there is a dir ect relationship between the identity of the structures that develop i n normal, ectopic and recombinant limbs, and the identity of the T-box gene(s) being expressed. To investigate the regulation of T-box gene expression during limb development, we employed several other embryolo gical manipulations. By surgically removing the apical ectodermal ridg e (AER) from either wing or leg buds, we found that, in contrast to al l other genes implicated in the patterning of developing appendages, m aintenance of T-box gene expression is not dependent on the continued provision of signals from the AER or the zone of polarizing activity ( ZPA), By generating an ectopic ZPA, by grafting a sonic hedgehog (SHH) -expressing cell pellet under the anterior AER, we found that Tbx2 exp ression can lie downstream of SHH. Finally, by grafting a SHH-expressi ng cell pellet to the anterior margin of a bud from which the AER had been removed, we found that Tbx2 may be a direct, short-range target o f SHH, Our findings suggest that these genes are intimately involved i n limb development and the specification of limb identity, and a new m odel for the evolution of vertebrate appendages is proposed.