CROSS-REGULATION IN THE MOUSE HOXB COMPLEX - THE EXPRESSION OF HOXB2 IN RHOMBOMERE-4 IS REGULATED BY HOXB1

Correct regulation of the segment-restricted patterns of Hox gene expr ession is essential for proper patterning of the vertebrate hindbrain. We have examined the molecular basis of restricted expression of Hoxb 2 in rhombomere 4 (r4), by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx bi nding motif is located within this enhancer, and in vitro DNA binding experiments showed that the vertebrate labial-related protein Hoxb1 wi ll cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic express ion of Hoxb1, Hoxa1, and Drosophila labial in transgenic mice. In cont rast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicat ing that in vivo this enhancer preferentially responds to labial famil y members. Mutational analysis demonstrated that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retin oids and ectopic Hox expression. Furthermore, three copies of the Hoxb 2 motif are sufficient to mediate r4 expression in transgenic mouse em bryos and a labial pattern in Drosophila embryos. This reporter expres sion in Drosophila embryos is dependent upon endogenous labial and exd , suggesting that the ability of this Hox/Pbx site to interact with la bial-related proteins has been evolutionarily conserved. The endogenou s Hoxb2 gene is no longer upregulated in r4 in Hoxb1 homozygous mutant embryos. On the basis of these experiments we conclude that the r4-re stricted domain of Hoxb2 in the hindbrain is the result of a direct cr oss-regulatory interaction by Hoxb1 involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxb1 in maintaining r4 identity may be mediated by the Hoxb2 gene.