In situ quantification of individual mRNA transcripts in melanocytes discloses gene regulation of relevance to speciation

Functional validation of candidate genes for adaptation and speciation remains challenging. We here exemplify the utility of a method quantifying individual mRNA transcripts in revealing the molecular basis of divergence in feather pigment synthesis during early-stage speciation in crows. Using a padlock probe assay combined with rolling circle amplification we quantified cell-type specific gene expression in the native, histological context of growing feather follicles. Expression of Tyrosinase related protein 1 (TYRP1), Solute carrier family 45 member 2 (SLC45A2) and Hematopoietic prostaglandin D synthase (HPGDS) was melanocyte-limited and significantly reduced in follicles from hooded crow explaining the substantially lower melanin content in grey vs. black feathers. The central upstream transcription factor Microphthalmia-associated transcription factor (MITF) only showed differential expression specific to melanocytes - a feature not captured by bulk RNA-seq. Overall, this study provides insight into the molecular basis of an evolutionary young transition in pigment synthesis, and demonstrates the power of histologically explicit, statistically substantiated single-cell gene expression quantification for functional genetic inference in natural populations.