The products of Hox genes function in assigning positional identity along the 
anterior-posterior body axis during animal development. In mouse embryos, Hox 
genes located at the 3' end of HoxA and HoxB complexes are expressed in nested 
patterns in the progenitors of the secondary heart field during early 
cardiogenesis and the combined activities of both of these clusters are required 
for proper looping of the heart. Using Hox bacterial artificial chromosomes 
(BACs), transposon reporters, and transgenic analyses in mice, we present the 
identification of several novel enhancers flanking the HoxB complex which can 
work over a long range to mediate dynamic reporter expression in the endoderm 
and embryonic heart during development. These enhancers respond to exogenously 
added retinoic acid and we have identified two retinoic acid response elements 
(RAREs) within these control modules that play a role in potentiating their 
regulatory activity. Deletion analysis in HoxB BAC reporters reveals that these 
control modules, spread throughout the flanking intergenic region, have 
regulatory activities that overlap with other local enhancers. This suggests 
that they function as shadow enhancers to modulate the expression of genes from 
the HoxB complex during cardiac development. Regulatory analysis of the HoxA 
complex reveals that it also has enhancers in the 3' flanking region which 
contain RAREs and have the potential to modulate expression in endoderm and 
heart tissues. Together, the similarities in their location, enhancer output, 
and dependence on retinoid signaling suggest that a conserved cis-regulatory 
cassette located in the 3' proximal regions adjacent to the HoxA and HoxB 
complexes evolved to modulate Hox gene expression during mammalian cardiac and 
endoderm development. This suggests a common regulatory mechanism, whereby the 
conserved control modules act over a long range on multiple Hox genes to 
generate nested patterns of HoxA and HoxB expression during cardiogenesis.