K Locus Effects In Gray Wolves: Experimental Assessment Of Tlr3 Signaling And The Gene Expression Response To Canine Distemper Virus

In North American gray wolves, black coat color is dominantly inherited via a 3 base pair coding deletion in the canine beta defensin 3 (CBD103) gene. This 3 base pair deletion, called the K-B allele, was introduced through hybridization with dogs and subsequently underwent a selective sweep that increased its frequency in wild wolves. Despite apparent positive selection, K-BB wolves have lower fitness than wolves with the K-yB genotype, even though the 2 genotypes show no observable differences in black coat color.Thus, the K-B allele is thought to have pleiotropic effects on as-yet unknown phenotypes. Given the role of skin-expressed CBD103 in innate immunity, we hypothesized that the K-B allele influences the keratinocyte gene expression response to TLR3 pathway stimulation and/or infection by canine distemper virus (CDV). To test this hypothesis, we developed a panel of primary epidermal keratinocyte cell cultures from 24 wild North American gray wolves of both K-W and K-yB genotypes. In addition, we generated an immortalized K-yy line and used CRISPR/Cas9 editing to produce a K-yB line on the same genetic background. We assessed the transcriptome-wide responses of wolf keratinocytes to the TLR3 agonist polyinosinic:polycytidylic acid (polyl:C), and to live CDV. K locus genotype did not predict the transcriptional response to either challenge, suggesting that variation in the gene expression response does not explain pleiotropic effects of the K-B allele on fitness.This study supports the feasibility of using cell culture methods to investigate the phenotypic effects of naturally occurring genetic variation in wild mammals.