Additional file 1 of Insights into the evolutionary origin of the pineal color discrimination mechanism from the river lamprey

Additional file1: Figure S1-S6 and Table S1. Figure S1. Phylogenetic position of lamprey parietopsin. The tree was inferred by the neighbor-joining method using the Opn3 homologs as an outgroup. The lamprey opsin isolated in the current study is classified into parietopsin (PT) group. Abbreviations indicating subgroups of opsins are as follows: PT, parietopsin; PPL, parapinopsin-like; PP, parapinopsin; VA/VAL, VA- and VAL-opsin; P, pinopsin; LWS, long wavelength-sensitive opsin; SWS1, short wavelength-sensitive type1 opsin; SWS2, short wavelength-sensitive type2 opsin; MWS, middle wavelength-sensitive opsin; RH, rhodopsin. The position of lamprey parietopsin is shown with bold character. Bootstrap probabilities (≥80%) are indicated at each branch node. The scale bar indicates 0.1 substitutions per site. Figure S2. The distribution patterns of parapinopsin and parietopsin in the lamprey organ (Related to Figure 1). (A, B) Images by in situ hybridization analyses, showing expression patterns of parapinopsin (A), and parietopsin (B). (C) The double stained image with the antibodies against parapinopsin (magenta) and parietopsin (green), indicating biased expression of parietopsin in the peripheral and its surroundings region rather than in the dorsal central region of the pineal organ. (D)The schematic presentation showing the expression patterns of parapinopsin (magenta) and parietopsin (green) in the lamprey pineal organ. The dotted line indicates the location of the pineal slice used in Fig. 2G. Scale bars = 100 μm. Figure S3. Details of extracellular recording analyses (Related to Figure 2B and C). The relative response profiles of the visible light-sensitive component in pineal chromatic responses. The six color traces (open circles) were obtained with six-repeated set of light stimuli (460-640 nm). The black closed circles represent the mean values obtained from the six traces and are the same values as shown in Fig. 2C. Figure S4. Parietopsin-expressing cells contain β-arrestin. (A-C) Fluorescence double staining images by immunohistochemical analyses showing expression patterns of parietopsin (A, green), β-arrestin (B, magenta). Merged image (C) shows parietopsin-expressing cells contain β-arrestin. Arrowheads show the outer segments of parietopsin-expressing cells. Scale bar = 20 μm. Figure S5 Co-expression of two opsins and two G proteins in the iguana parietal eye photoreceptor cells. (A-C) Fluorescence double staining images by immunohistochemical analyses showing the co-expression of molecular components in photoreceptor cells of iguana parietal eyes. The images show (A) the co-expression of Go-type (Go, green) and Gt/gust-type (Gt/gust, magenta), (B) the co-expression of Gt/gust-type G protein (green) and parapinopsin (PP, magenta), and (C) the co-expression of parietopsin (PT, green) and Go-type G protein (magenta) in the iguana parietal eye photoreceptor cells. OS indicates the layer of outer segments of the photoreceptor cells. Scale bar = 20 μm. Figure S6. Changes in cAMP levels of parapinopsin-expressing cultured cells depending on color of light. Changes in cAMP levels in lamprey parapinopsin-expressing HEK293S cells using GloSensor. The adenylyl cyclase activator forskolin (3.5 μM) was added to the culture medium (35-mm dish, ~1.2 × 106 HEK cells) to elevate the intracellular cAMP level (black arrow). The arrowheads show ~400-nm (magenta, ~2.5 × 1015 photon/cm2/s), ~455-nm (blue, ~4.9 × 1016 photon/cm2/s), and ~505-nm (green, ~2.6 × 1016 photon/cm2/s) monochromatic light stimuli (duration, 5s). The profile of cAMP changes clearly shows color-dependent-manner responses evoked with photoreceptions involving lamprey parapinopsin dark state and photoproduct. Table S1. Primer list for 3’ and 5’ RACE (PDF 733 kb)