There is considerable interest in understanding how cis-regulatory modifications drive morphological changes across species. Because developmental regulatory genes, including Hox genes, are remarkably conserved, their noncoding regulatory regions are likely sources for variations. Modifications of Hox cis-regulatory elements have potential to alter Hox gene expression and, hence, axial morphologies. In vertebrates, differences in the axial levels of Hox gene expression correlate with differences in the number and relative position of thoracic vertebrae. Variation in cis-regulatory elements of Hox genes can be identified by comparative sequence and reporter gene analyses in transgenic mouse embryos. Using these approaches, we show a remarkable divergence of the Hoxc8 early enhancers between mammals and fishes representing diverse axial morphologies. Extensive restructuring of the Hoxc8 early enhancer including nucleotide substitutions, inversion, and divergence result in distinct patterns of reporter gene expression along the embryonic axis. Our results provide an evolutionary perspective on how the enhancer elements are engineered and support the hypothesis that remodeling of Hox regulatory elements in different species has played a significant role in generating morphological diversity.

Activated Raf is a potent inhibitor of skeletal muscle gene transcription and myocyte formation through stimulation of downstream MAPK. However, the molecular targets of elevated MAPK with regard to myogenic repression remain elusive. We examined the effects of activated Raf on myogenin gene expression in avian myoblasts. Overexpression of activated Raf in embryonic chick myoblasts prevented myogenin gene transcription and myocyte differentiation. Treatment with PD98059, an inhibitor of MAPK kinase (MEK), restored myogenin expression but did not reinstate the myogenic program. Using a panel of myogenin promoter deletion mutants, we were unable to identify a region within the proximal 829-bp promoter that confers responsiveness to MEK. Interestingly, our experiments identified MEF2A as a target of Raf-mediated inhibition in mouse myoblasts but not in avian myogenic cells. Embryonic myoblasts overexpressing activated Raf were unable to drive transcription from a minimal myogenin promoter reporter, containing a single E-box and MEF2 site, to levels comparable with controls. Unlike mouse myoblasts, forced expression of MEF2A did not synergistically enhance transcription from the myogenin promoter in chick myoblasts, indicating that additional molecular determinants of the block to myogenesis exist. Results of these experiments further exemplify specie differences in the mode of Raf-mediated inhibition of muscle differentiation.

Hoxc8 early enhancer controls the initiation and establishment phase of Hoxc8 expression in the mouse. Comparative studies indicate the presence of Hoxc8 early enhancer sequences in different vertebrate clades including mammals, birds and fish. Previous studies have shown differences between teleost and mammalian Hoxc8 early enhancers with respect to sequence and organization of protein binding elements. This raises the question of when the Hoxc8 early enhancer arose and how it has become modified in different vertebrate lineages. Here, we describe Hoxc8 early enhancer from the Indonesian coelacanth, Latimeria menadoensis. Coelacanths are the only extant lobefinned fish whose genome is tractable to genome analysis. The Latimeria Hoxc8 early enhancer sequence more closely resembles that of the mouse than that of Fugu or zebrafish. When assayed for enhancer activity by reporter gene analysis in transgenic mouse embryos, Latimeria Hoxc8 early enhancer directs expression to the posterior neural tube and mesoderm similar to that of the mouse enhancer. These observations support a close relationship between coelacanths and tetrapods and place the origin of a common Hoxc8 early enhancer sequence within the sarcopterygian lineage. The divergence of teleost (actinopterygii) Hoxc8 early enhancer may reflect a case of relaxed selection or other forms of instability induced by genome duplication events.