Keeping the family together

In vertebrates, homeobox (Hox) gene clustering is necessary for collinear gene activation during the development of the major body axis and for the coordinated expression of these genes in different tissues. François Spitz and colleagues now show that the recruitment of global enhancer sequences positioned on either sides of the cluster contributes to the tight regulation of this gene family.

To dissect the complex Hox genomic organization, the authors designed a simple strategy to separate the mouse Hoxd cluster into two independent pieces. They developed a simplified version of the CRE–loxP recombination system, known as the sequential targeted recombination-induced genomic (STRING) strategy. Using STRING, large chromosomal rearrangements can be efficiently produced without the need for technically demanding embryonic stem cell manipulation. Briefly, the approach is based on the generation of mice that contain loxP sites in cis in selected regions that flank a chromosomal locus of interest, which allows the separation of the two regions through insertion of an inverted allele.

Because Hoxd11 and Hoxd10 have similar expression patterns and respond to the same set of enhancers, the authors selected a breakpoint between these two genes and split the vertebrate Hoxd cluster into two parts (the Hoxd^split10–11 allele). To allow detection of Hoxd10 and Hoxd11 transcripts encoded by the in verted allele exclusively, Hoxd^{split10–11/+} mice were crossed to mice carrying engineered deficiencies of the Hoxd cluster. Both Hoxd11 and Hoxd10 are expressed in the distal and proximal limb buds, in the genital bud and in the intestinal hernia in wild-type fetuses. Intriguingly, a strict and precise partition of these expression domains became apparent after cluster separation. Hoxd11 was expressed in the genital and distal limb buds, whereas Hoxd10 showed a complementary pattern and was expressed in the intestinal hernia and proximal limb bud. These results indicate that Hoxd genes are controlled by a set of enhancer sequences that globally regulate all genes in a cluster; the distal limb and genital enhancers are centromeric to Hoxd, whereas the hernia and proximal limb enhancers are located on the telomeric side.

The authors postulate a clear distinction between these two regulatory mechanisms, and suggest that in the course of tetrapod evolution, the appearance of digits was not achieved through the use of pre-existing regulatory mechanisms but rather through the emergence of novel regulation of posterior Hoxd genes. In addition, their results indicate that the clustered organization of Hox genes provided the basis for recruiting other regulatory mechanisms, such as enhancer sequences. Establishing such large regulatory landscapes might have contributed to the clustered organization, leading to the tight organization of this gene family in vertebrates.