The conserved family of homeobox (Hox) genes is well known for specifying patterning along the metazoan anterior–posterior axis. Equally ancient is the peculiar correspondence between the order of the Hox genes on the chromosome and their expression domains along the body axis, known as collinearity. With the emergence of novel morphological structures, however, some Hox genes have been recruited for functions other than axial specification — limb development in vertebrates, for example. For these genes, the collinearity rule is not always respected, with the genes being controlled instead by shared enhancers. By molecularly dissecting the mouse and human Hoxd clusters, François Spitz and colleagues have shown that collinear expression is dictated by control elements within or close to the Hoxd cluster, whereas non-collinear expression is under the control of more remote elements.

The proteins encoded by the nine genes in the mouse and human Hoxd cluster are highly conserved. In the first of two approaches to map the regulatory potential of this cluster, the authors monitored the expression and functional properties of the human HOXD cluster when introduced into mice on a 120-kb PAC. The human HOXD transcripts recapitulated the expression of their mouse counterparts, and functionally rescued the trunk phenotypes of inactive mouse Hoxd11–13 genes. Significantly, however, no phenotypic rescue of the developing limbs occurred, nor was human HOXD expression present in the limb domain, indicating that the elements required for limb expression (and in some other organs) lie outside the 120-kb genomic fragment. A similar effect was observed when a larger 215-kb construct containing the mouse Hoxd cluster was used.

The remote location of the enhancers required for Hox gene expression in the limb was confirmed by generating nested deletions of the mouse Hoxd cluster and following the expression of a Hoxd11–lacZ reporter gene that replaced the deleted sequences. Although the progressive removal of the cluster led to the loss of lacZ expression in the trunk as expected, its expression in the limb was unaffected, even when the entire Hoxd cluster was deleted.

This study shows that Hox expression domains can be divided into those that reflect ancient functions, which are controlled by sequences within the cluster itself, and others that have been recruited for new functions, which depend on enhancers outside the cluster (at least 100 kb upstream from Hoxd, the authors propose). The recruitment of Hox genes to new capacities required the capture or formation of new control elements; their remote location can probably be explained by the difficulty of incorporating new regulatory sequences into the tightly organized Hox cluster without disturbing the finely honed assembly of this ancient module.