The Journal of Neuroscience, September 9, 2020 • 40(37):7065–7079 • 7065 Development/Plasticity/Repair Zebrafish Crb1, Localizing Uniquely to the Cell Membranes around Cone Photoreceptor Axonemes, Alleviates Light Damage to Photoreceptors and Modulates Cones’ Light Responsiveness Chuanyu Guo,1 Ciana Deveau,2 Cen Zhang,1 Ralph Nelson,2 and Xiangyun Wei1,3,4 1Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, 15213 Pennsylvania, 2National Institute of Neurological Disorders and Stroke, National Institutes of Health, Rockville, 20852 Maryland, 3Department of Molecular Genetics and Microbiology, University of Pittsburgh School of Medicine, Pittsburgh, 15213 Pennsylvania, and 4Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, 15213 Pennsylvania The crumbs (crb) apical polarity genes are essential for the development and functions of epithelia. Adult zebrafish retinal neuroepithelium expresses three crb genes (crb1, crb2a, and crb2b); however, it is unknown whether and how Crb1 differs from other Crb proteins in expression, localization, and functions. Here, we show that, unlike zebrafish Crb2a and Crb2b as well as mammalian Crb1 and Crb2, zebrafish Crb1 does not localize to the subapical regions of photoreceptors and Müller glial cells; rather, it localizes to a small region of cone outer segments: the cell membranes surrounding the axonemes. Moreover, zebrafish Crb1 is not required for retinal morphogenesis and photoreceptor patterning. Interestingly, Crb1 pro- motes rod survival under strong white light irradiation in a previously unreported non–cell-autonomous fashion; in addition, Crb1 delays UV and blue cones’ chromatin condensation caused by UV light irradiation. Finally, Crb1 plays a role in cones’ responsiveness to light through an arrestin-translocation-independent mechanism. The localization of Crb1 and its functions do not differ between male and female fish. We conclude that zebrafish Crb1 has diverged from other vertebrate Crb pro- teins, representing a neofunctionalization in Crb biology during evolution. Key words: apicobasal polarity; axoneme; Crumbs; light damage; photoreceptor; zebrafish Significance Statement Apicobasal polarity of epithelia is an important property that underlies the morphogenesis and functions of epithelial tissues. Epithelial apicobasal polarity is controlled by many polarity genes, including the crb genes. In vertebrates, multiple crb genes have been identified, but the differences in their expression patterns and functions are not fully understood. Here, we report a novel subcellular localization of zebrafish Crb1 in retinal cone photoreceptors and evidence for its new functions in photore- ceptor maintenance and light responsiveness. This study expands our understanding of the biology of the crb genes in epithe- lia, including retinal neuroepithelium. Introduction Received Mar. 1, 2020; revised June 26, 2020; accepted July 22, 2020. Crumbs (Crb) apical polarity proteins are Type I single-pass Author contributions: C.G., R.N., and X.W. designed research; C.G., C.D., C.Z., and R.N. performed research; C.G., C.D., C.Z., R.N., and X.W. analyzed data; C.G. and X.W. wrote the first draft of the paper; C.G., C.D., R.N., transmembrane proteins. They are essential for a variety of and X.W. edited the paper; C.G. and X.W. wrote the paper. epithelial tissues in both invertebrates and vertebrates. Their The authors declare no competing financial interests. extracellular domains mediate cell-cell interactions, and their This work was supported by EY016099 and EY025638 to X.W. The study utilized the core facility supported cytoplasmic C-terminal domains mediate intracellular signaling by National Institutes of Health Grant P30EY008098 to the Department of Ophthalmology of the University of Pittsburgh, which was funded in part by a grant from the Eye and Ear Foundation of Pittsburgh and an (Pocha and Knust, 2013). The prototype Crb was first discovered unrestricted grant from the Research to Prevent Blindness. This work was supported in part by National in Drosophila melanogaster (Tepass et al., 1990), in which Crb Institute of Neurological Disorders and Stroke, National Institutes of Health Intramural Research Program localizes to the subapical regions (SARs) of cell membranes, api- to R.N. cal to the septate junctions in epithelial cells (Tepass et al., 1990). Correspondence should be addressed to Ralph Nelson at [email protected] or Xiangyun Wei at [email protected]. Subsequently, three classes of Crb orthologs have been identified https://doi.org/10.1523/JNEUROSCI.0497-20.2020 in vertebrates: Crb1, Crb2, and Crb3, with Crb1 and Crb2 carry- Copyright © 2020 the authors ing large extracellular domains like the fly Crb, and Crb3 missing 7066 • J. Neurosci., September 9, 2020 • 40(37):7065–7079 Guo et al. · Crumbs at the Outer Segments of Cone Photoreceptors most part of the extracellular domain (den Hollander et al., 1999; cell type-specific expression pattern, protein localization, and Makarova et al., 2003; Lemmers et al., 2004; van den Hurk et al., functions in the retina remain unknown. 2005; Bulgakova and Knust, 2009). For example, the zebrafish Thus, we here characterize zebrafish Crb1’s retinal expression genome contains five crb genes (crb1, crb2a, crb2b, crb3a,and pattern, subcellular localization, and functions. We show that crb3b)(Omori and Malicki, 2006). In vertebrate epithelial cells, zebrafish Crb1 is not only different from zebrafish Crb2a and Crb proteins normally localize to SARs, immediately apical to Crb2b, but also different from human and mouse Crb1 and the tight junctions (Mehalow et al., 2003; Roh et al., 2003; van de Crb2, suggesting that it may represent a neofunctionalization of Pavert et al., 2004; van den Hurk et al., 2005; Omori and Malicki, Crb proteins during evolution. 2006; Zou et al., 2012; Pellissier et al., 2014). The multiplicity of vertebrate crb genes and their structural variations suggest diver- Materials and Methods sified Crb functions. Zebrafish strains and animal care. Tubingen WT, Tg(À5.5opn1sws1: Indeed, since the identification of Crb in the fruit fly, Crb EGFP)kj transgenic (Takechi et al., 2003), Tg (LCRRH2-RH2-1:GFP)pt112 proteins have been found to play essential roles in many biologi- transgenic (Fang et al., 2013), Tg(À3.7rho:EGFP)kj2 transgenic cal properties and processes, such as apicobasal polarity and in- (Hamaoka et al., 2002), and crb1sa12558 mutant (simplified as crb1 mu- tegrity of epithelia (Letizia et al., 2013), cell-cell adhesions and tant hereafter; Zebrafish International Resource Center, catalog cellular patterning (Grawe et al., 1996; Tepass, 1996; Lemmers et #ZL9088.07; Busch-Nentwich et al., 2012) zebrafish were maintained on al., 2004; Zou et al., 2012), cell proliferation and survival a 14 h light/10 h dark cycle at 28.5°C. Zebrafish embryos were raised in (Pellikka and Tepass, 2017), Hippo signaling (Chen et al., 2010; an incubator at 28.5°C. Animal care and handling were in accordance Pan, 2010; Parsons et al., 2010), protein trafficking (Pocha et al., with the guidelines of the University of Pittsburgh. 2011a), photoreceptor morphogenesis (Izaddoost et al., 2002; Phylogenetic analysis of Crb amino acid sequences. The phylogenetic Pellikka et al., 2002; Nam and Choi, 2003), etc. Given the multi- relationships of the extracellular domain, intracellular domain, and full- plicity of vertebrate crb genes, it is possible that this broad spec- length sequences of Crb proteins of 14 species were analyzed with the MEGA X program (http://www.megasoftware.net/). We use the neigh- trum of Crb functions is deferentially allocated to various crb bor-joining method to generate the unrooted phylogenetic trees. In addi- genes. tion, Crb amino acid sequences were compared pairwise with This possibility of differential allocation of functions among VectorNTI and Genedoc (Nicholas et al., 1997). The animal species crb genes is supported by genetic evidence in humans. For exam- and the GenBank accession numbers of these Crb proteins are as ple, ;10% of Leber’s congenital amaurosis (LCA) and retinitis follows: fruit fly (Drosophila busckii; Crb, ALC47276.1); nematode pigmentosa (RP) are because of the malfunction of human CRB1 (Caenorhabditis elegans; Crb1, CCD66913.1; Crb2a, BAA92157.1; caused by .300 pathogenic mutations (den Hollander et al., Crb2b, BAA92158.1; Crb3, NP_001041224.1); human (Homo sapiens; 1999; Abouzeid et al., 2006; Vallespin et al., 2007; Bujakowska et Crb1, NP_957705.1; Crb2, NP_775960.4; Crb3, AAQ89047.1); rhesus al., 2012; Corton et al., 2013; Tiab et al., 2013). However, LCA monkey (Macaca mulatta; Crb1, XP_014976303.1; Crb2, XP_001083811.3; and RP have not been found to be associated with human CRB2 Crb3, NP_001253010.1); chimpanzee (Pan troglodytes;Crb1,XP_ and CRB3 (Slavotinek, 2016), although some missense mutations 009438564.1; Crb2, XP_528426.4; Crb3, XP_016790312.2); house mouse in CRB2 were linked to reduced visual acuity, mild optic atrophy, (Mus musculus; Crb1, AAL65131.1; Crb2, NP_001157038.1; Crb3, AAH24462.1); Norway rat (Rattus norvegicus; Crb1, NP_001100652.1; and irregular retinal pigmentation (van den Hurk et al., 2005; Crb2, NP_001129233.1; Crb3, NP_001020832.1); white-footed mouse Lamont et al., 2016). The preferential association of CRB1 with (Peromyscus leucopus; Crb1, XP_028745692.1; Crb2, XP_028743732.1; RP and LCA may suggest that, in humans, CRB1 carries more Crb3, XP_028718368.1); Chinese alligator (Alligator sinensis;Crb1,