Science of Life Laboratories and Department of Evolutionary Biology, Uppsala University, SE-752 36 Uppsala, Sweden
Department of Pharmaceutical Biosciences, Uppsala University, SE-752 36 Uppsala, Sweden.
J Exp Biol. 2019 Mar 8;222(Pt 5):jeb194431. doi: 10.1242/jeb.194431.
Functional validation of candidate genes involved in adaptation and speciation remains challenging. Here, we 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 histological context of growing feather follicles. Expression of Tyrosinase Related Protein 1 (), Solute Carrier Family 45 member 2 () and Hematopoietic Prostaglandin D Synthase () was melanocyte-limited and significantly reduced in follicles from hooded crow, explaining the substantially lower eumelanin content in grey versus black feathers. The central upstream Melanocyte Inducing Transcription Factor () 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.
功能验证候选基因参与适应和物种形成仍然具有挑战性。在这里，我们举例说明了一种方法的实用性，该方法定量测量个体 mRNA 转录本，揭示了在乌鸦早期物种形成过程中羽毛色素合成分化的分子基础。我们使用套锁探针检测结合滚环扩增，在生长羽毛滤泡的组织学背景下定量细胞特异性基因表达。酪氨酸酶相关蛋白 1 ()、溶质载体家族 45 成员 2 () 和造血前列腺素 D 合酶 () 的表达仅限于黑色素细胞，并且在 Hooded Crow 的滤泡中显著减少，这解释了灰色与黑色羽毛之间黑色素含量显著降低的原因。中央上游黑素细胞诱导转录因子 () 仅显示黑色素细胞特异性的差异表达——这是批量 RNA-seq 无法捕捉到的特征。总的来说，这项研究深入了解了色素合成这一进化年轻转变的分子基础，并展示了组织学上明确、统计学上合理的单细胞基因表达定量在自然种群中进行功能遗传推断的强大功能。
