An examination of chiropteran HOXD regulation from an evolutionary perspective

Here we present our efforts to develop a model system that is suitable for laboratory experiments addressing Chiropteran development, genetics and evolution. The bat, Myotis lucifugus was selected as a model organism to study Chiropteran biology. To build a model system, Myotis lucifugus specimens were collected from several locations across the United States. From the collected specimens, embryos and adult tissues were used to produce an assessment of embryonic development, primary cell cultures, and various genetic libraries. These resources were then used to investigate the Chiropteran HoxD complex. The HoxD 13 genomic locus was cloned and sequenced to derive a protein coding sequence, which showed strong conservation with the Human and mouse HOXD 13 proteins. HoxD 13 expression, however, was expanded in the Myotis lucifugus forelimb when compared to mouse. We therefore investigated the regulation of the HoxD complex by cloning and sequencing two Chiropteran global control regions (GCR) belonging to Myotis lucifugus and Rhinolophus ferrumequinum. The two Chiropteran GCRs were used in a series of unbiased triangulation alignments, looking for sequence changes that were shared between the two bat species but not found in Human or mouse GCRs. A number of exclusive Chiropteran changes were found regarding sequence order, large insertions, large deletions and small nucleotide changes. The GCR was finally examined in a mouse transgenic assay, to determine its activity domains. As compared with the previously reported Human GCR activity assay, we found new Chiropteran GCR activity domains in the stylopod and zeugopod that correlate with the expansion of HoxD 13 in the Chiropteran forelimb. Previously reported domains in the fore- and midbrains were absent, which also correlate with a mutation in a highly conserved tetrapod specific GCR sequence. In light of the proposed hypothesis that the highly clustered structure of Hox genes in tetrapods acts as a constraint against evolution in the absence of duplication events, our results suggest that the Hox genes are evolving in a circuitous route in the absence of duplications while respecting ancestral constraints.