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Fig. 1. Fictive locomotion in prometamorphic X. laevis larvae is sensitive to light. (A, i) Cartoon of stage 56 larva showing the approximate location of the central nervous system. (A, ii) Schematic depicting preparation with glass suction electrodes on ventral motor roots. (B, i) Extracellular record from three ventral motor roots showing spontaneous episodes of fictive locomotion. (B, ii) Expanded time base to show coordination of spontaneous rhythm and various parameters, including burst duration (BD), cycle period (CP), and episode duration (ED). Spontaneous activity is sensitive to ambient light levels. In the light, episodes of activity occur regularly every few minutes, whereas in the dark (gray box) the preparation falls silent. (B, iii) Graph of time spent active in light and dark, expressed as a percentage of total recording period, for 23 larval preparations (light-gray lines). The population mean is shown in black. (B, iv) Other parameters of fictive motor activity are unaffected; BD (n = 18), CP (n = 16), and ED (n = 23) are expressed as mean percentage in light relative to dark. (B, v) Graph of mean latency to motor activity from nine different preparations where at least three transitions between dark and light were recorded. In each example, the latency to activity was measured following 10 min in the dark. (Upper) Example response from a stage 54 larva following 10 min in the dark (gray box). Error bars represent ±SEM; ***P < 0.01.
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Fig. 2. Photosensitivity is tuned to short wavelengths. (A, i) Schematic depicting brainstem and caudal diencephalon. Approximate area illuminated is shown by the black dotted line. (A, ii) Ventral root trace from a stage 55 larva showing 200 s before and 200 s after a sequence of transitions from dark (gray box) to light. In each case, the preparation was illuminated following 10 min in the dark; the wavelength and intensity of light are shown. (A, iii) Graph displaying average data for time spent active 200 s before, during, and after 200-s illumination for each wavelength (UV, red, green, and blue; n = 7). (B, i) Sequence of responses to different intensities of UV light following 10 min in the dark. (B, ii) Graph showing average data for time spent active during responses to UV light at maximum (Max; 39 lx), medium high (21 lx), medium low (10 lx), and minimum (Min; 5 lx) intensity (n = 4). (B, iii) Graph showing average data for latency to first activity after illumination with UV light of different intensities. Error bars represent ±SEM; ***P < 0.01; *P < 0.05.
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Fig. 3. Photosensitive tissue resides within the caudal diencephalon. (A, i) Schematic of the dissection performed: The forebrain is removed apart from a small portion of diencephalon caudal to the dorsal opening of the third ventricle. Both dorsal and sagittal aspects are depicted. (Scale bar, 200 µm.) (A, ii) Ventral root recording from a stage 54 larva showing three consecutive responses to illumination with UV light (400 nm; 39 lx). (A, iii) Graph of average data comparing time spent active 200 s before, during, and after illumination (n = 7). (B, i) Schematic illustrating the dissection made flush with the optic tectum to completely remove the diencephalon. (B, ii and iii) Equivalent data to A displayed for preparations lacking the diencephalon (n = 4). (C, i) Schematic illustrating the approximate location of focal illumination of three areas of isolated nervous system. (C, ii) Sequence of responses to illumination of these different areas with UV light following 10 min in the dark (gray box). (C, iii and iv) Graphs showing average data for time spent active (C, iii) and mean episode number (C, iv) for illumination of each area; comparison of 200 s before, during, and after illumination are plotted (n = 4). (D, i) Schematic illustrating isolated nervous system before (Upper) and after (Lower) removal of the ventral diencephalon containing the hypothalamus and pituitary. (D, ii) Ventral root recording from a stage 56 larva before (Upper) and after (Lower) the dissection was performed. (D, iii) Graph illustrating data from three different preparations. Swim % is shown both before (black line) and after (gray lines) removal of the ventral diencephalon. Error bars represent ±SEM; *P < 0.05.
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Fig. 4. UV-sensitive proteins are located within the tadpole caudal diencephalon. (A) Schematic of a Xenopus tadpole brain showing the approximate position of sections taken for imaging. (B) OPN5-positive neurons within the caudal diencephalon of a stage 55 tadpole. (B, ii) A cluster of neurons is located in the ventral half of the diencephalon in proximity to the hypothalamic ventricle (hv); expanded view of the boxed area (B, iii) and a second more ventral image from a different preparation (B, iv). Examples of neuron cell bodies at arrows in B, iii and B, iv. (Scale bars, 100 μm.)
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Fig. S1. Controls for secondary antibodies. Negative controls lacking primary antibody. (i) Texas red secondary. (ii) FITC secondary. (Scale bar, 100 μm.)
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Fig. S2. CRY2 is found ubiquitously but is localized within nonneuronal tissue. Immunohistochemistry showing widespread CRY2 expression, which is most intense in blood vessels (arrow). (Scale bar, 100 μm.)
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Fig. S3. OPN5 and CRY1 are expressed throughout the nervous system. (A) Schematic of a Xenopus tadpole brain showing the approximate position of sections taken for imaging. (B–E) Immunohistochemistry showing OPN5 and CRY1 expression within the ventral half of the preparation at three distinct anatomical levels moving caudally from the region of light sensitivity. d, dorsal; v, ventral. (C, ii) CRY1 is strongly expressed within the hypothalamus (hyp), the region surgically removed in Fig. 3D, without loss of light sensitivity. [Scale bars, 200 μm (A) and 100 μm (B–E).]
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Fig. S4. OPN5 is not found within dopaminergic neurons within the light-sensitive region of the diencephalon. The OPN5 neurons in the caudal diencephalon are in close proximity to the rostral extent of the dopaminergic neurons of the posterior tuberculum. OPN5-positive neurons (i, Left) and TH-positive neurons (i, Right) are found in close proximity to each other within the caudal diencephalon. The TH-positive neurons are the rostral-most members of the dopaminergic neurons of the posterior tuberculum (pt) shown more caudally in the same animal (ii; magnification in iii). (Scale bars, 100 μm.)
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Fig. S5. Western blot for OPN5 detection in X. laevis protein samples. Stage 51 (lane 1) and adult (lane 2) Xenopus protein samples were run on 4–12% polyacrylamide gels and transferred to nitrocellulose membrane for Western blotting to detect OPN5 using the antibody described above at 1:5,000. Positive bands at ∼40 kDa were obtained for both samples. This finding revealed that the antibody specifically detected a protein of the correct size for Xenopus OPN5.
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Fig. S6. Positive immunoreactivity for OPN5 in the Xenopus nervous system. OPN5-positive immunoreactivity was observed in specific regions of the nervous system, including the cells in the spinal cord (ii). A control section stained with preabsorbed OPN5 is also shown (i). (Scale bar, 150 μm.)
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Fig. S7. Western blots for the detection of CRY1 and CRY2 in Xenopus samples. Stage 51 (lane 1) and adult (lane 2) Xenopus protein samples were run on 4–12% polyacrylamide gels and transferred to nitrocellulose membrane for Western blotting to detect CRY1 or CRY2 using a commercially sourced antibody (Aviva Systems Biology; 1:5,000 for CRY1 and 1:1,000 for CRY2). Positive bands at ∼70 and 65 kDa were obtained for both samples. This result revealed that the antibody specifically detected proteins of the correct size for Xenopus cryptochromes.
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