Variations in regulatory regions of developmental control genes have been implicated in the divergence of axial morphologies. To find potentially significant changes in cis-regulatory regions, we compared nucleotide sequences and activities of mammalian Hoxc8 early enhancers. The nucleotide sequence of the early enhancer region is extremely conserved among mammalian clades, with five previously described cis-acting elements, A-E, being invariant. However, a 4-bp deletion within element C of the Hoxc8 early enhancer sequence is observed in baleen whales. When assayed in transgenic mouse embryos, a baleen whale enhancer (unlike other mammalian enhancers) directs expression of the reporter gene to more posterior regions of the neural tube but fails to direct expression to posterior mesoderm. We suggest that regulation of Hoxc8 in baleen whales differs from other mammalian species and may be associated with variation in axial morphology.

We have characterized cis-acting elements that direct the early phase of Hoxc-8 expression using reporter gene analysis in transgenic mice. By deletion we show that a 135 bp DNA fragment, located approximately 3 kb upstream of the coding region of Hoxc-8, is capable of directing posterior neural tube expression. This early neural tube (ENT) enhancer consists of four separate elements, designated A, B, C and D, whose nucleotide sequences are similar to binding sites of known transcription factors. Nucleotide substitutions suggest that element A is an essential component of the ENT enhancer. However element A by itself is incapable of directing neural tube expression. This element requires interactions at any two of the other three elements, B, C or D. Thus, the components of the ENT enhancer direct neural tube expression in an interdependent manner. We propose that Hoxc-8 is activated in the neural tube by combinatorial interactions among several proteins acting within a small region. Our transgenic analyses provide a means to identify transcription factors that regulate Hoxc-8 expression during embryogenesis.