Figure S1. Pineal Photoreceptors Are Present in Retinal Photoreceptor Degeneration Mutants
Coronal sections of the forebrain at 7 dpf were stained with DAPI (A, C, E, and G) and zpr1, a marker of both retinal and pineal photoreceptors (B, D, F, and H). Pineal photoreceptors (arrow and inset) were consistently present in mutants in which retinal photoreceptors were depleted (D, F, and H). Scale bar is 100 μm for A–J and 25 μm for the insets.
(1.2 MB PDF)
Figure S2. Dorsal RGCs Are Present and Properly Differentiated in darl Mutants
Sagittal sections of WT (A and C) and darls327 retina (B and D) were stained with DAPI (A and B) and zn5 (C and D), a marker for differentiated RGCs. RGCs are present in the dorsal part of the retina and sending out axons into the optic nerve head in the mutant. The mutant eyes are reduced in size compared to WT.
(513 KB PDF)
Figure S3. The Tectum of exa Mutants Has an Abnormal Shape
RGC axon tracing, following whole-eye DiI fills at 7 dpf, reveals a subtle extension of the tectal neuropil (delineated by DAPI counterstaining) at the ventral-posterior margin (arrow). Scale bar is 50 μm
(1.7 MB PDF)
Figure S4. Retinal Axon Outgrowth Is Delayed in shir Mutants
Lateral views of the retinal ganglion cell axons labeled with DiO. Anterior to the left, dorsal to the top.
(A and B) At 7 dpf, the retinofugal projection in shirs362 (B) appears similar to WT (A), although the anterior portion may be less dense (arrow).
(C and D) At 5 dpf, RGC axon outgrowth in shirs362 (D) evidently lags behind WT (C). Scale bar is 100 μm.
(1.6 MB PDF)
Video S1. Optomotor Response
The movie shows a close-up of part of a racetrack tank during OMR testing. A visible light filter has been used to remove the stimulus, and the fish are visualized using infrared light (Sony TRV-9 video camera, night vision mode). The stimulus is represented below. Initially, a converging grating brings the fish into the field of view. After 8 s, the stimulus changes to a rightward-moving grating, and all the fish swim to the right, out of the field of view. At 18 s, the converging movie reappears, and the fish return. Playback in Quicktime runs at twice the actual speed.
(2.3 MB WMV)
Video S2. Optokinetic Response
The WT larva is on the left, and a zats125 mutant is on the right. For the first 60 s no stimulus is shown, and both fish show spontaneous eye movements. After 60 s, a clockwise-rotating striped pattern is projected on the drum around the fish. The WT fish responds by tracking the pattern slowly to the right and making fast reset saccades to the left. The mutant continues to make undirected spontaneous eye movements.
(2.21 MB MOV)
Accession Numbers
The GenBank (http://www.ncbi.nlm.nih.gov/) accession numbers of the Danio rerio genes discussed in this paper are retinal guanylyl cyclase 3 (gc3) (AY050505) and phenylalanine hydroxylase (pah) (BC056537).
Acknowledgments
We thank D. Stainier and S. Baraban, and their labs for collaboration in the screen, in particular L. D'Amico, B. Jungblut, I. Scott, D. Beis, P. Castro, and S. Jin. In addition, S. Brockerhoff, C. B. Chien, S. Horne, and J. Malicki kindly provided mutant carriers for complementation tests. We are grateful to W. Harris, P. Goldsmith, T. Roeser, and M. Taylor for advice and support and to K. Deere, A. Mrejeru, E. Janss, K. Menuz, B. Bogert, H. Haeberle, B. Griffin, M. Dimapasoc, and K. Takahashi for their assistance at various stages of the project. Doctoral and postdoctoral fellowship support came from Naito Foundation (AM), Uehara Memorial Foundation (AM), Howard Hughes Medical Institute (MBO), National Science Foundation (MCS, JNK, LMN), a National Research Service Award from the National Institutes of Health (NIH) (EG), American Heart Association (MCS), University of California, San Francisco Chancellor's Fund (MCS), an Achievement Reward for College Scientist/ARCS (AMW), American Association of University Women Educational Foundation (AMW), National Alliance for Research on Schizophrenia and Depression (PSPM), and an NIH neuroscience postdoctoral training grant (TX). HB was supported by the NIH (EY12406, EY13855, NS42328), by the Sandler Family, by the Sloan Foundation, by the Klingenstein Foundation, and by the David and Lucile Packard Foundation.
Author contributions. HB conceived the project. AM, MBO, and HB designed the experiments. AM, MBO, AMW, MCS, JNK, PSPM, EG, TX, LMN, NJG, WS, KFB, and HB performed the experiments. AM, MBO, JNK, and HB analyzed the data. AM, MBO, MCS, PSPM, KFB, and HB contributed reagents/materials/analysis tools. AM and HB wrote the paper with input from all authors.
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