Avian photoreceptor homologies and the origin of double cones

Birds possess the most complex photoreceptor system among vertebrates, with one rod and six cone types, including four single cones (violet, blue, green, and red) and two constituent cells of the double cone (DC-P and DC-A). The evolutionary relationships of avian photoreceptors to those of other vertebrate taxa have not been systematically explored. Here, we perform single-cell RNA-seq on retinas of newly hatched chickens to trace cell-type homologies across species. Analysis of differentially expressed transcription factors (TFs) suggests that avian rods and single cone types largely correspond to cognate cell types in fish and placental mammals, whereas the double cone has a distinct origin. We propose that DC-P arose from an ancestral red cone - as revealed by expression of the red cone cell fate determinants thyroid hormone receptor beta (THRB) and SAMD7, whereas DC-A may have arisen from an ancestral blue cone - as suggested by expression of the blue cone TFs FOXQ2 and SKOR1. These expression signatures are shared by DC-P and DC-A of the green anole lizard (Anolis carolinensis), suggesting conservation throughout Sauropsida. Consistent with our hypothesis, CRISPR-mediated knockout of THRB causes loss of red cones and DC-P, but not DC-A, and the appearance of supernumerary rods and green cones, suggestive of direct transfating. Furthermore, cis-regulatory analysis suggests that separate enhancers control red cone opsin expression in DC-P and DC-A, consistent with distinct evolutionary origins. Taken together, our studies trace the evolutionary relationships of avian photoreceptors and suggest an origin of DC-P and DC-A from ancestral red and blue cones, respectively.