Divergence of Hoxc8 early enhancer parallels diverged axial morphologies between mammals and fishes

Abstract

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.

Comparison of diverged Hoxc8 early enhancer activities reveals modification of regulatory interactions at conserved cis‐acting elements

“…1B). We have previously described the reporter gene expression directed by mouse and Fugu Hoxc8 early enhancers in day 9.5 transgenic mouse embryos (Anand et al, 2003). They direct reporter gene expression to the posterior neural tube and mesoderm, but to distinct anterior boundaries ( Fig.…”

“…The mouse and Fugu reporter gene constructs carrying 399 bp Hoxc8 early enhancer elements have been previously described (Shashikant and Ruddle, '96;Anand et al, 2003). Alterations in the 399 bp reporter construct were introduced by an overlapping PCR strategy using synthetic oligonucleotide primers containing appropriate changes in the nucleotide sequence (Shashikant et al,'95).…”

“…These elements either singly or in combination contribute to posterior neural tube and mesoderm expression in the developing mouse embryo. Comparative studies have indicated the presence of the Hoxc8 early enhancer sequence among different vertebrate lineages including mammals, birds and fish (Belting et al, '98;Shashikant et al, '98, 2004;Anand et al, 2003). A survey of Hoxc8 early enhancer sequences in about 30 mammalian species belonging to different clades revealed a high degree of sequence similarity with all the previously identified cis-acting elements being invariant.…”

“…In contrast, Hoxc8 early enhancers of teleosts including Fugu, Sphoeroides and zebrafish (belonging to the actinopterygean lineage) diverged significantly from the mouse enhancer in their sequence and activity (Anand et al, 2003;Shashikant et al, 2004). For instance, the Fugu Hoxc8 early enhancer lacks mouse cis-acting elements F-H, yet showed remarkably distinct and robust reporter gene expression, in posterior neural tube and mesoderm when assayed in transgenic mouse embryos (Anand et al, 2003). Expression in the somites was to a more anterior level compared to that directed by the mouse enhancer.…”

“…In general, Hoxc8 early enhancers isolated from the sarcopterygean lineages shared high degree of sequence similarity, similar elemental composition yet subtly different enhancer activities when assayed in mouse embryos. In contrast, Hoxc8 early enhancers of teleosts including Fugu, Sphoeroides and zebrafish (belonging to the actinopterygean lineage) diverged significantly from the mouse enhancer in their sequence and activity (Anand et al, 2003;Shashikant et al, 2004). For instance, the Fugu Hoxc8 early enhancer lacks mouse cis-acting elements F-H, yet showed remarkably distinct and robust reporter gene expression, in posterior neural tube and mesoderm when assayed in transgenic mouse embryos (Anand et al, 2003).…”