Genetic Characterization of the Cell Types of in Developing Feathers, and the Evolution of Feather Complexity

Feathers are the most complex and diverse epidermal appendages found in vertebrates. Their unique hierarchical organization and development is based on a diversity of cell types and morphologies. Despite being well characterized morphologically and extensive molecular developmental research focusing on candidate genes, little is known about the gene regulatory identities of these presumptive feather cell types. Here, we use single cell and single nuclear RNA sequencing with in situ hybridization to identify and characterize cells types in embryonic chicken feathers. We show that the distinct cell morphologies correspond to feather cell types with distinct gene expression profiles. We also describe a previously unidentified cell type, the basal barb ridge epithelium, which appears to play a role in signaling necessary for barb ridge differentiation and pulp cap production. We also analyze RNA velocity trajectories of developing feather cells, and find distinct developmental trajectories for epidermal cells that constitute the mature feather and those that function only in feather development. Finally, we produce an evolutionary tree of feather cell types based on transcription factor expression in order to test prior developmental hypotheses about feather evolution. Our tree is consistent with the developmental model of feather evolution, and sheds light on the influence of ancestral epidermal stratification on feather cell evolution. This transcriptomic approach to study feather cell types helps lay the ground work for understanding the developmental evolutionary complexity and diversity of feathers. Overall design: We dissected feathers from H&H stage 38, 39, and 40 white leghorn chicken embryos. For the sample labeled, Feathers in Skin, we dissected off a tract of epidermis with feathers embedded in it from the dorsal midline of the embryo. The “Plucked Feather” samples were plucked from the dorsal midline as well. Cells were isolated from these tissues and analyzed using scRNAseq for stage 38 embryos, and snRNAseq for stage 39 and 40 embryos.