ANALYSIS OF HOX GENE-EXPRESSION IN THE CHICK LIMB BUD

The vertebrate Hox genes have been shown to be important for patternin g the primary and secondary axes of the developing vertebrate embryo. The function of these genes along the primary axis of the embryo has b een generally interpreted in the context of positional specification a nd homeotic transformation of axial structures. The way in which these genes are expressed and function during the development of the second ary axes, particularly the limb, is less clear. In order to provide a reference for understanding the role of the Hox genes in limb patterni ng, we isolated clones of 23 Hox genes expressed during limb developme nt, characterized their expression patterns and analyzed their regulat ion by the signalling centers which pattern the limb. The expression p atterns of the Abd-B-related Hoxa and Herd genes have previously been partially characterized; however, our study reveals that these genes a re expressed in patterns more dynamic and complex than generally appre ciated, only transiently approximating simple, concentric, nested doma ins. Detailed analysis of these patterns suggests that the expression of each of the Nora and Herd genes is regulated in up to three indepen dent phases. Each of these phases appears to be associated with the sp ecification and patterning of one of the proximodistal segments of the limb (upper arm, lower arm and hand). Interestingly, in the last of t hese phases, the expression of the Herd genes violates the general rul e of spatial and temporal colinearity of Hox gene expression with gene order along the chromosome. In contrast to the Abd-B-related Horn and Herd genes, which are expressed in both the fore and hind limbs, diff erent sets of Hoxc genes are expressed in the two limbs. There is a co rrelation between the relative position of these genes along the chrom osome and the axial level of the limb bud in which they are expressed. The more 3' genes are expressed in the fore limb bud while the 5' gen es are expressed in the hind limb bud; intermediate genes are transcri bed in both limbs, However, there is no clear correlation between the relative position of the genes along the chromosome and their expressi on domains within the limb. With the exception of Hoxc-11, which is tr anscribed in a posterior portion of the hind limb, Hoxc gene expressio n is restricted to the anterior/proximal portion of the limb bud. Impo rtantly, comparison of the distributions of Hoxc-6 RNA and protein pro ducts reveals posttranscriptional regulation of this gene, suggesting that caution must be exercised in interpreting the functional signific ance of the RNA distribution of any of the vertebrate Hox genes. To un derstand the genesis of the complex patterns of Hox gene expression in the limb bud, we examined the propagation of Hox gene expression rela tive to cell proliferation. We find that shifts in Hox gene expression cannot be attributed to passive expansion due to cell proliferation. Rather, phase-specific Hox gene expression patterns appear to result f rom a context-dependent response of the limb mesoderm to Sonic hedgeho g. Sonic hedgehog (the patterning signal from the Zone of Polarizing A ctivity) is known to be able to activate Herd gene expression in the l imb. Although we find that Sonic hedgehog is capable of initiating and polarizing Herd gene expression during both of the latter two phases of Hox gene expression, the specific patterns induced are not determin ed by the signal, hut depend upon the temporal context of the mesoderm receiving the signal. Misexpression of Sonic hedgehog also reveals th at Hoxb-9, which is normally excluded from the posterior mesenchyme of the leg, is negatively regulated by Sonic hedgehog and that Hoxc-11, which is expressed in the posterior portion of the leg, is not affecte d by Sonic hedgehog and hence is not required to pattern the skeletal elements of the lower leg.