Supporting InformationZhao et al. 10.1073/pnas.1116035109SI Materials and MethodsIn Situ Hybridization. cDNA clones of genes expressed in zebra-fish photoreceptor cells were purchased from Open Biosystems.1-phenyl-2-thiourea (PTU)-treated zebrafish larvae were fixedat 3 dpf or 5 dpf. RNA probe synthesis, in situ hybridization,washes, and signal detection were carried out by using standardprotocols as described (1).

Genetic Screen. To enhance our ability to identify subtle photore-ceptor defects, we used a transgene that drives EGFP expression inrod photoreceptors from a Xenopus rod opsin promoter (2).

TUNEL Assay. Apoptotic cell death was detected on transverse cry-osections through the central retinaof zebrafish larvaebyusingan InSitu Cell Death Detection kit (Roche) according to the manu-facturer’s instructions.

1. Avanesov A, Malicki J (2010) Analysis of the retina in the zebrafish model. MethodsCell Biol 100:153–204.

2. Fadool JM (2003) Development of a rod photoreceptor mosaic revealed in transgeniczebrafish. Dev Biol 258:277–290.

Fig. S1. Kupffer’s vesicle (KV) cilia and opsin transport in kif3bjj203 mutant larvae. (A) Graph showing average length of KV cilia at the 10-somite stage inembryos collected from crosses between WT animals or kif3bjj203 mutant heterozygotes as indicated. Each dot represents the average KV cilia length in oneembryo. The average length in WT is set as 100%. Red horizontal and vertical bars show average and SD, respectively. “n” indicates the number of embryosused in this analysis. (B) Representative image showing KV cilia visualized via staining with anti-acetylated tubulin antibody in an embryo originating froma cross between two kif3bjj203 heterozygotes. (C) Diagram of opsin transport evaluation method as published previously (1). Upper: Schematic representationof the chimeric GFP-opsin gene construct driven by a heat-shock promoter. 44C, 44 C-terminal amino acids that are known to be sufficient to mediate opsintransport into the ciliary (outer segment) compartment. Lower: Schematic drawing of GFP–opsin localization at three hypothetical time points (t1, t2, t3) afterheat shock. In WT photoreceptors, opsin (green dots) gradually translocates from the cell body to the outer segment. (D) Graph depicting measurements ofGFP–opsin signal intensity in photoreceptor cell bodies of WT and jj203 mutant larvae at three time points after heat shock as indicated. For each data point,four to nine retinae and 19 to 47 photoreceptors were analyzed (P ≤ 0.0001). (E) Representative confocal images of transverse cryosections through centralretinae of WT and jj203 mutant larvae at three time points as indicated. GFP–opsin fluorescence is detected (green). Sections are counterstained with phal-loidin (red). Brackets indicate the photoreceptor cell layer, asterisks indicate outer segments, arrowheads indicate the outer limiting membrane, and arrowsindicate the outer plexiform layer. Signal intensity in the cell body was measured between the outer limiting membrane and the outer plexiform layer. In allimages, apical direction is up.

1. Zhao C, Malicki J (2011) Nephrocystins and MKS proteins interact with IFT particle and facilitate transport of selected ciliary cargos. EMBO J 30:2532–2544.

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Fig. S2. Rods die whereas cones survive in jj203 mutants. (A–C) Whole-mount in situ hybridization on WT and jj203 mutant embryos with probes to genesexpressed in rods or cones at 3 dpf (A) or 5 dpf (B and C) as indicated. (D) Transverse cryosections through the central retina of WT (Left) and kif3bjj203 mutant(Right) larvae stained with anti-rod opsin, anti-Nr2e3, or anti-green cone opsin antibodies at 7 dpf (all in red) as indicated. The green channel (Left) shows GFPtransgene expression driven by the Xenopus rod opsin promoter (2).

Fig. S3. Cell death analysis. Left: Confocal images of transverse cryosections through eyes of WT, jj203, and ovl larvae at four stages as indicated. Red spotscorrespond to apoptotic cells. In A–D′, rod photoreceptor cells are visualized (in green) via GFP transgene driven by the Xenopus rod opsin promoter. Right:Number of dying cells in the photoreceptor cell layer is quantified per section. “n” indicates the number of embryos investigated.

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Fig. S4. kif17mutant phenotype. (A) Top two rows show confocal images of whole embryos stained with anti-acetylated tubulin antibody (green) to visualizecilia in the pronephric duct and the spinal canal (sc) at 30 hpf. Bottom two rows show confocal images of transverse cryosections through the retina at 5 dpfstained with anti-rod opsin or anti-green opsin antibodies (green). (B) Confocal images of kif17 mutant larvae at 3 dpf treated with control morpholino (CMO)or anti-splice kif17 morpholino as indicated. Larvae were stained with anti-acetylated tubulin antibody (green) to visualize cilia in the spinal canal, nasal pit,and ear cristae. When shown, red channel contains phalloidin staining.

Fig. S5. kif17 rescues ciliogenesis defects in the spinal canal of kif3bjj203 mutant larvae. Confocal images of whole embryos stained with anti-acetylatedtubulin antibody (green) to visualize cilia in the nasal pit (Left) and the spinal canal (Right) at 3 dpf. Mutant and WT larvae express control GFP or kif17 mRNAas indicated. kif17 mRNA rescues the formation of spinal canal but not nasal cilia. C and D, and E and F, show the same embryos. Larvae in the right column arecounterstained with phalloidin (red).

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Fig. S6. Phylogenetic relationships between motor subunits of zebrafish and human kinesin-2 genes. The zebrafish kif3c clusters with human and mouse (notshown) genes. kif3c and kif3c-like genes are syntenic to human KIF3C. The sequences were aligned by using the ClustalW2 neighbor-joining method, and thephylogenetic tree was drawn by using NJplot software. Hs, Homo sapiens; Dr, Danio rerio.

Fig. S7. Morpholino knockdown and rescue data for kif3c-like and kif3c genes. (A) Upper: Schematic drawing of the exon/intron structure for the kif3c-likeand kif3c genes. Targeted exons are in color. Morpholino target sites are indicated as red horizontal bars. Lower: RT-PCR amplification results. Based onsequence analysis, knockdown of the kif3c-like gene at 24 hpf results in a smaller amplification product as a result of the loss of exon 4. In kif3c knockdownanimals, we do not detect WT transcript at 24 hpf. In kif3c-like and kif3c knockdown larvae, some WT transcript persists at 3 dpf. Arrowheads indicate WTamplification product. (B) Confocal images of lateral cristae in whole animals stained with anti-acetylated tubulin antibody to visualize kinocilia (green) andcounterstained with phalloidin (red). WT and kif3bjj203 mutant embryos were treated with morpholinos and mRNA as indicated. The number of individualstested that differentiate cilia is indicated in the lower left corner of each panel.

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Fig. S8. Rescue of kif3bjj203 mutant phenotype by kif3c overexpression. (A) Confocal images of transverse cryosections through the retina at 4 dpf stained withanti-rod opsin antibody (in green) and counterstained with phalloidin (red). Brackets indicate the photoreceptor cell layer; arrowheads indicate the outerlimiting membrane. (B) The frequency of photoreceptor cilia in kif3b−/− mutant homozygotes at 4 dpf, following rescue with kif3c or control GFP mRNA asindicated. Each dot represents the number of cilia per an arbitrary segment of the photoreceptor cell layer in a single retina. “n” is the number of retinaeinvestigated.

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Fig. S9. Summary of functional relationships between kinesin-2 family motors in ciliogenesis. (A) In rod photoreceptors, kif3b is necessary for cilia differ-entiation and cell survival. In cone photoreceptors, on the contrary, kif3b is necessary for cilia (outer segment) formation at 3 dpf. Somewhat later, at 5 dpf,kif3b and kif3c function largely redundantly in cone cilia formation. However, kif3c does not entirely compensate for the loss of kif3b function, and, vice versa,kif3b does not seem to entirely compensate for the loss of kif3c. (B) In the ear, two populations of mechanosensory hair cells display very different re-quirements for kinesin function: whereas kif3b is required for ciliogenesis in auditory maculae, kif3b and kif3c function redundantly in the cilia of cristae;either is sufficient to drive ciliogenesis in these cells. (C) In olfactory placodes, both kif3b and kif17 are necessary for normal ciliogenesis. The contribution ofkif3b is much more significant compared with kif17. Kif3a, which most likely heterodimerizes with Kif3b and Kif3c gene products, is not depicted in this figure.Its function has not been studied in this work.

Movie S1. Cilia motility in phenotypically WT zebrafish at 7 dpf.

Movie S1

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Movie S2. Cilia motility in kif17sa0119 mutant homozygotes at 7 dpf.

Movie S2

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