Evolution of Hoxa-11 in lineages phylogenetically positioned along the fin-limb transition

HOXA11 is a transcription factor implicated in paired appendage development. To identify signatures of evolutionary change in the structural, and putative functional, domains of HOXA11, we studied its evolution in tetrapod and nontetrapod lineages that represent approximately 1.5 billion years of evolutionary time. Here, Hoxa-11 gene proper sequences were determined for frog (Xenopus tropicalis), coelacanth (Latimeria chalumnae), common zebrafish (Danio rerio; Hoxa-11a and Hoxa-11b paralogs), and giant zebrafish (D. aequipinnatus; Hoxa-11b) and aligned against previously published Hoxa-11 sequences of human, mouse, chick, and newt. Based on aligned Hoxa-11 amino acid sequences, the protein was demarcated into three segments: Domains I (N-terminal) and III (homeobox + C-terminal), which varied slightly in rates and patterns of evolution, and a variable, overall hydrophilic region (HyD), which partially overlaps with Domain I. As judged by character reconstructions of HOXA11 Domains I and III, no significant changes in rates of coding sequence evolution occurred in tetrapods (frog and chick), relative to coelacanth (a lobe-finned fish), i.e., across the fin-limb transition. Accelerated rates of Hoxa-11 coding sequence evolution were observed for the mammalian and newt lineages. This was shown to be a gene-specific phenomenon. The duplicated Hoxa-11a and Hoxa-11b genes of zebrafish exhibited accelerated rates of evolution and accumulated substitutions at sites that are conserved among coelacanth and all tetrapods examined. Amino acid sequence comparisons of the HyD of HOXA11 suggested that a putative repressor subdomain, containing stretches of consecutive alanine residues, emerged within the tetrapods. A high degree of nucleotide conservation in the 5' half of the Hoxa-11 intron was observed for tetrapod and nontetrapod lineages. Using electrophoretic mobility shift assays, a 35-bp intron sequence, which is 100% conserved in all Hoxa-11 loci except for the zebrafish Hoxa-11a paralog, was found to bind protein(s) in HeLa and chick whole-cell extracts.