Localized and transient transcription of Hox genes suggests a link between patterning and the segmentation clock.

During development, Hox gene transcription is activated in presomitic mesoderm with a time sequence that follows the order of the genes along the chromosome. Here, we show that Hoxd1 and other Hox genes display dynamic stripes of expression within presomitic mesoderm. The underlying transcriptional bursts may reflect the mechanism that coordinates Hox gene activation with somitogenesis. This mechanism appears to depend upon Notch signaling, as mice deficient for RBPJk, the effector of the Notch pathway, showed severely reduced Hoxd gene expression in presomitic mesoderm. These results suggest a molecular link between Hox gene activation and the segmentation clock. Such a linkage would efficiently keep in phase the production of novel segments with their morphological specification.

Targeted inversion of a polar silencer within the HoxD complex re-allocates domains of enhancer sharing.

Mammalian Hox genes are clustered at four genomic loci. During development, neighbouring genes are coordinately regulated by global enhancer sequences, which control multiple genes at once, as exemplified by the expression of series of contiguous Hoxd genes in either limbs or gut. The link between vertebrate Hox gene transcription and their clustered distribution is poorly understood. Experimental and comparative approaches have revealed that various mechanisms, such as gene clustering or global enhancer sequences, might have constrained this genomic organization and stabilized it throughout evolution. To understand what restricts the effect of a particular enhancer to a precise set of genes, we generated a loxP/Cre-mediated targeted inversion within the HoxD cluster. Mice carrying the inversion showed a reciprocal re-assignment of the limb versus gut regulatory specificities, suggesting the presence of a silencer element with a unidirectional property. This polar silencer appears to limit the number of genes that respond to one type of regulation and thus indicates how separate regulatory domains may be implemented within intricate gene clusters.

Mechanisms of Hox gene colinearity: transposition of the anterior Hoxb1 gene into the posterior HoxD complex.

Transposition of Hoxd genes to a more posterior (5') location within the HoxD complex suggested that colinearity in the expression of these genes was due, in part, to the existence of a silencing mechanism originating at the 5' end of the cluster and extending towards the 3' direction. To assess the strength and specificity of this repression, as well as to challenge available models on colinearity, we inserted a Hoxb1/lacZ transgene within the posterior HoxD complex, thereby reconstructing a cluster with a copy of the most anterior gene inserted at the most posterior position. Analysis of Hoxb1 expression after ectopic relocation revealed that Hoxb1-specific activity in the fourth rhombomere was totally abolished. Treatment with retinoic acid, or subsequent relocations toward more 3' positions in the HoxD complex, did not release this silencing in hindbrain cells. In contrast, however, early and anterior transgene expression in the mesoderm was unexpectedly not suppressed. Furthermore, the transgene induced a transient ectopic activation of the neighboring Hoxd13 gene, without affecting other genes of the complex. Such a local and transient break in colinearity was also observed after transposition of the Hoxd9/lacZ reporter gene, indicating that it may be a general property of these transgenes when transposed at an ectopic location. These results are discussed in the context of existing models, which account for colinear activation of vertebrate Hox genes.