A new cell culture resource for investigations of reptilian gene function

Abstract

The recent establishment of CRISPR/Cas9 gene editing in A. sagrei lizards makes it a powerful model system for studies of reptilian gene function. To enhance the versatility of this model, we developed an immortalized lizard fibroblast cell line (ASEC-1) for the exploration of reptilian gene function in cellular processes. We demonstrate the use of this in vitro system by scrutinizing the role of primary cilia in lizard Hedgehog (Hh) signaling. Through CRISPR/Cas9 mutagenesis we disrupted the ift88 gene, which is required for ciliogenesis in diverse organisms. We find that the loss of itf88 from lizard cells results in an absence of primary cilia, a partial derepression of gli1 transcription, and an inability of the cells to respond to the Smoothened agonist, SAG. Through a cross-species analysis of SAG-induced transcriptional responses in cultured limb bud cells, we further determined that [~]46% of genes induced as a response to Hh pathway activation in A. sagrei, are also SAG-responsive in M. musculus limb bud cells. Our results highlight conserved and diverged aspects of Hh signaling in anoles and establish a new resource for investigations of reptilian gene function.