Expression of the chicken homeobox-containing gene GHox-8 during embryonic chick limb development

Abstract

Homeobox-containing genes are thought to be involved in the regulation of pattern formation and specification of positional information during vertebrate limb development. Because of its accessibility to microsurgical manipulation, the developing chick limb bud provides a powerful system for investigating the role of homeobox-containing genes in pattering events. We report the isolation from a chick limb bud cDNA library of a chicken homeobox-containing cDNA, which on the basis of its nucleotide and deduced amino acid sequences has been identified as the chicken cognate of mouse Hox-8. The gene encoding this chicken ( Gallus) homeobox-containing cDNA has been designated GHox-8, and is a member of a family of vertebrate homeobox-containing genes that are highly similar in sequence to the Drosophila msh gene. GHox-8 encodes an mRNA transcript of about 3 kb that is expressed at several early stages of chick limb development. In situ and dot-blot hybridization analyses have revealed that GHox-8 is expressed in limb bud mesoderm in a temporal and spatial fashion consistent with its involvement in specifying anterior positional identity. At early stages (stages 20–21) of chick limb development when positional values along the anterior-posterior (A-P) axis are being specified, GHox-8 is expressed in high amounts in the anterior mesoderm of the wing bud. Little expression of the gene is detectable in the middle region of the wing bud mesoderm or in the posterior mesoderm that contains the zone of polarizing activity, which is thought to be the source of a diffusible morphogen, possibly retinoic acid, that specifies the A-P positional values of the skeletal elements of the limb according to its local concentration. Similarly, at later stages of development (stages 23–25), high expression of GHox-8 is localized to the proximal anterior periphery of the wing bud, with no detectable expression in the proximal dorsal and ventral (myogenic) regions, or in the chondrogenic central core. In the proximal posterior periphery of the wing bud at these later stages of development, expression of GHox-8 is limited to a small region in the mid-proximal periphery corresponding to the posterior necrotic zone in which programmed cell death is occuring. The possible involvement of GHox-8 in programmed cell death during limb development is also suggested by the fact that it is expressed in the necrotic interdigital mesenchyme in 6–7 day (stage 31–32) wing buds. Finally, we have found that GHox-8 is expressed by the apical ectodermal ridge (AER) that extends along the distal periphery of the wing bud. This suggests that GHox-8 may be involved in the function of the AER, which is required for the outgrowth and formation of the various skeletal elements of the limb in their appropriate sequence along the proximodistal axis.