Pinopsin Regulates Melatonin Production and Daily Locomotor Activity: Functional Insights From Gene‐Edited Xenopus Tadpoles

Circadian rhythm alignment depends on environmental light detection via opsins. Pinopsin, originally identified in the pineal organ of birds and later in amphibian pineal complex and eyes, may play a role in this process, though its function has not been genetically tested. Evolutionary analysis suggests pinopsin was independently lost in several vertebrate lineages, including mammals (Synapsida), some reptiles (e.g. snakes and crocodiles), and teleost fish, but retained in birds, turtles, lizards, and non-teleost Actinopterygii. We conducted a detailed genomic search of the pinopsin gene across 95 amphibian species and assessed its function in Xenopus laevis tadpoles using CRISPR/Cas9-mediated knockout. Our survey indicates that pinopsin is highly conserved in salamanders and most anurans, but absent in many caecilians (Gymnophiona), which have a fossorial lifestyle with limited light exposure. To investigate its biological role, we generated X. laevis F0 pinopsin knockout tadpoles and evaluated two light-sensitive responses: (1) day/night melatonin fluctuations inferred from skin pigmentation changes, and (2) locomotor activity over a 24-h photoperiod. We show these responses depend only on pineal light sensitivity and are independent of eye sensitivity at developmental stage 46/47. Our findings reveal: (1) Pinopsin is co-expressed with Aanat, a key enzyme in melatonin synthesis; (2) knockout tadpoles show paler skin during the light phase, suggesting pinopsin suppresses melatonin production in daylight; and (3) reduced daytime locomotor activity in F0 mutants, consistent with melatonin-induced lethargy. Overall, pinopsin emerges as a critical opsin for light-regulated circadian-associated behavior in Xenopus, with likely conserved roles across amphibians (anurans and salamanders in general) and other non-mammalian vertebrates, including birds, turtles, and lizards.