Identification of Hedgehog signaling responsive genes in A. sagrei and M. musculus via transcriptional profiling

The brown anole lizard, Anolis sagrei, is an emerging squamate model for developmental and functional genetic studies. To develop additional tools and resources for mechanistic studies of signaling pathways and cellular processes in A. sagrei, we established an in vitro system. Using this approach, we studied Hedgehog (Hh) signaling, one of the key developmental signaling pathways, which has evolved across the metazoa. We investigated Hh pathway-induced transcriptional changes in two evolutionarily distinct tetrapods: A. sagrei, and M. musculus, to identify the species-specific and evolutionarily shared responses. We report that ~45 % of genes induced as a response to Hh pathway activation in A. sagrei, are shared with M. musculus. To further increase the versatility of A. sagrei as a squamate model system for gene editing and genomic studies, we established and characterized a new immortalized A. sagrei embryonic fibroblast cell line ASEC-1. We performed whole-genome sequencing analysis to annotate the set of polymorphisms within this cell line. We conclude that transcriptome characterization of the ASEC-1 cell line would permit further investigations dissecting the complex biological and evolutionary aspects of Hh signaling. Overall design: The Hedgehog pathway was activated by treating 48 hr serum-starved cells with optimum concentration of a pharmacological compound, Smoothened agonist (SAG). Total RNA was collected after SAG exposure. SAG-induced transcriptome from primary embryonic fibroblasts was generated by deep sequencing using 5 biological replicates and from the ASEC-1 cell line by deep sequencing using 4 replicates.