Project description: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.
Project description:Anolis carolinensis embryos were collected 0-1 days post egg laying, and total RNA was extracted for RNA-Seq analysis (Illumina Hi-Seq2000). Transcriptome sequence from these stages in the green anole, equivalent to mouse 9.5-10.5 dpc embryos, will help to improve gene annotations in A. carolinensis and provide expression level information for key organogenesis and patterning processes. Anolis carolinensis embryos were collected 0-1 days post egg laying for RNA-Seq analysis. The two embryos collected were at 28 somite-pair (28S) and 38 somite-pair (38S), equivalent to mouse 9.5 dpc and 10.5 dpc embryos, respectively. Total RNA was extracted using the total RNA component of the mirVana (Ambion) kit, RNA-Seq library prep was carried out using the NuGEN Ovation RNA-Seq kit, and sequencing was carried out on an Illumina HiSeq 2000, following the manufacturer's protocol. The untrimmed data was then aligned to the Anolis carolinensis reference genome (Anocar2.0) using tophat. Published: Eckalbar WL, Lasku E, Infante CR, Elsey RM, Markov GJ, Allen AN, Corneveaux JJ, Losos JB, DeNardo DF, Huentelman MJ, Wilson-Rawls J, Rawls A, Kusumi K. Somitogenesis in the anole lizard and alligator reveals evolutionary convergence and divergence in the amniote segmentation clock. Dev Biol. DOI: 10.1016/j.ydbio.2011.11.021