RESEARCH ARTICLE Disease Models & Mechanisms 4, 786-800 (2011) doi:10.1242/dmm.006429
Harmonin (Ush1c) is required in zebrafish Müller glial cells for photoreceptor synaptic development and function
Jennifer B. Phillips1,*, Bernardo Blanco-Sanchez1,*, Jennifer J. Lentz2,3, Alexandra Tallafuss1, Kornnika Khanobdee4,‡, Srirangan Sampath2, Zachary G. Jacobs1, Philip F. Han1, Monalisa Mishra4, Tom A. Titus1, David S. Williams4,5, Bronya J. Keats2,§, Philip Washbourne1 and Monte Westerfield1,¶
SUMMARY Usher syndrome is the most prevalent cause of hereditary deaf-blindness, characterized by congenital sensorineural hearing impairment and progressive photoreceptor degeneration beginning in childhood or adolescence. Diagnosis and management of this disease are complex, and the molecular changes underlying sensory cell impairment remain poorly understood. Here we characterize two zebrafish models for a severe form of Usher syndrome, Usher syndrome type 1C (USH1C): one model is a mutant with a newly identified ush1c nonsense mutation, and the other is a morpholino knockdown of ush1c. Both have defects in hearing, balance and visual function from the first week of life. Histological analyses reveal specific defects in sensory cell structure that are consistent with these behavioral phenotypes and could implicate Müller glia in the retinal pathology of Usher syndrome. This study shows that visual defects associated with loss of ush1c function in zebrafish can be detected from the onset of vision, and thus could be applicable to early diagnosis for USH1C patients. DMM
INTRODUCTION profound deafness seen in USH1, and hearing aids can compensate Usher syndrome (USH) is characterized by recessively inherited for some of the hearing loss in USH2 and USH3, but there is deaf-blindness. Previous estimates of the worldwide prevalence of currently no treatment to counter the vision loss resulting from USH ranged from 1/17,000 to 1/25,000 (Boughman et al., 1983; retinitis pigmentosa. Early diagnosis is important for genetic Rosenberg et al., 1997). However, a r-ecent study of deaf and hard- counseling and preparation for late-onset blindness. of-hearing children suggested a prevalence of 1/6000 (Kimberling At least 11 different loci are implicated in USH. The nine et al., 2010). USH1 is the most severe of the three clinical subtypes, identified USH genes encode structurally and functionally with profound congenital deafness and onset of vision loss usually heterogeneous proteins (Keats and Savas, 2004; Reiners et al., 2006; before age 10. Vestibular dysfunction is also typical in USH1 cases. Ebermann et al., 2007), but most possess protein interaction USH2, the most common clinical subtype worldwide, is domains that suggest the ability to form molecular complexes with characterized by moderate to severe congenital hearing impairment one another. Biochemical studies have confirmed these binding and the onset of visual defects usually in the second decade of life. propensities (Reiners et al., 2005; Pan et al., 2009). Colocalization No balance problems are associated with USH2. USH3 is rare in of the USH proteins in subcellular domains of auditory hair cells most populations, and distinct from the other clinical subtypes in and retinal cells, along with evidence of mislocalization of USH Disease Models & Mechanisms that both hearing and vision impairments are progressive, and proteins in various USH mutant backgrounds, suggest that at least vestibular dysfunction is variable (for reviews, see Saihan et al., 2009; some USH proteins directly interact with one another (Adato et Yan and Liu, 2010). Cochlear implants are indicated in cases of al., 2005; Reiners et al., 2005; Lefevre et al., 2008; Bahloul et al., 2010; Yang et al., 2010; Caberlotto et al., 2011). Previous studies of USH proteins have concentrated on their 1 Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254, USA potential functions in the region of the connecting cilium of the 2Department of Genetics, LSU Health Sciences Center, New Orleans, LA 70112, USA 3Neuroscience Center, LSU Health Sciences Center, New Orleans, LA 70112, USA photoreceptor (Liu et al., 2007; van Wijk et al., 2006; Maerker et al., 4Department of Pharmacology and Neuroscience, UCSD, La Jolla, CA 92093-0636, 2008; Yang et al., 2010) and the stereocilia of hair cells (Adato et al., USA 2005; Delprat et al., 2005; Seiler et al., 2005; Söllner et al., 2004; 5Jules Stein Eye Institute, UCLA, Los Angeles, CA 90095-7000, USA *These authors contributed equally to this work Kikkawa et al., 2005; Prosser et al., 2008; Lefevre et al., 2008). In the ‡Present address: School of Biology, Institute of Science, Suranaree University of ear, several studies have provided evidence for colocalization of USH Technology, Muang, Nakhon Ratchasima 30000, Thailand proteins in the tip and ankle-linking regions of the stereocilia and at § Present address: Research School of Biology, Australian National University, hair cell ribbon synapses (Mburu et al., 2003; Siemens et al., 2004; Canberra, ACT 2601, Australia ¶Author for correspondence ([email protected]) Reiners et al., 2005; Michalski et al., 2007; Kazmierczak et al., 2007; Lefevre et al., 2008). Most of the USH proteins have been shown to Received 30 July 2010; Accepted 23 May 2011 colocalize in the retina at the connecting cilium and/or at the © 2011. Published by The Company of Biologists Ltd photoreceptor synapse (Liu et al., 1997; Reiners et al., 2006; Liu et This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), which al., 2007; Overlack et al., 2008; Yang et al., 2010). In addition to these permits unrestricted non-commercial use, distribution and reproduction in any medium provided that the original work is properly cited and all further distributions of the work or adaptation are regions of colocalization, many of the USH proteins are found subject to the same Creative Commons License terms. individually at discrete subcellular locations (Hasson et al., 1995;
786 dmm.biologists.org ush1c in zebrafish visual function RESEARCH ARTICLE
Reiners et al., 2006; Overlack et al., 2008; Williams et al., 2009), swimming and balance defects consistent with the auditory and suggesting that at least some have functions independent of the vestibular component of the human disease (Ernest et al., 2000; proposed complex. In hair cells, USH proteins are thought to Söllner et al., 2004; Seiler et al., 2005). Although expression in the stabilize the growth and orientation of stereocilia during development retina has been reported for each of these genes, only the (for reviews, see Brown et al., 2008; Yan and Liu, 2010), whereas USH morpholino knockdown of pcdh15b has been reported to have an proteins in the region of the connecting cilium are proposed to effect on visual function (Seiler et al., 2005). Photoreceptor contribute to the trafficking of molecular cargo from the organization is severely affected in pcdh15b morphants, and photoreceptor inner segment to the outer segment (Maerker et al., electroretinogram (ERG) traces are notably attenuated. Here, we 2008; Liu et al., 2007). USH proteins have also been immunolocalized describe two zebrafish models for USH1C that exhibit a strong at the ribbon synapses of these sensory cells, but their potential USH-like phenotype at early developmental stages. Young fish with functions in this region are unknown. depleted ush1c function have penetrant and specific visual defects The USH1C gene encodes the PDZ-domain-containing protein as well as compromised hair cell development, resulting in hearing harmonin (Verpy et al., 2000), which has been proposed to function and balance impairment. Detailed analyses of these phenotypes as a key scaffolding molecule to which most other USH proteins have confirmed a requirement for harmonin in zebrafish hair cell can bind (Reiners et al., 2005). Patients with mutations in USH1C morphogenesis, and have implicated Müller glial cells in supporting exhibit the classic USH1 pathology, with profound congenital harmonin-mediated ribbon synapse stability and function. USH has hearing impairment, vestibular dysfunction, and clinically traditionally not been considered a developmental disease of vision. appreciable retinal degeneration in childhood or early adolescence. The results of this study, however, show that visual defects can be USH1C mutations resulting in non-syndromic deafness have also detected from the onset of vision in zebrafish with depleted ush1c been reported (Ouyang et al., 2002). The deaf circler mouse function, thus providing a rationale for examination of Müller cell (Johnson et al., 2003), harboring an in-frame deletion in the function in USH1C patients. murine Ush1c gene, has provided a valuable model for the
DMM mechanosensory hair cell defects that cause deafness in USH1C RESULTS patients, but does not exhibit a retinal phenotype (Williams et al., ush1c expression in zebrafish inner ear sensory patches, the lateral 2009). Similarly, the Ush1c knockout mouse exhibits profound line and retina deafness without notable retinal defects (Tian et al., 2010). We used the human harmonin protein sequence in tBLASTn Interestingly, the c.216G>A mouse model of USH1C searches to identify a single ortholog of USH1C in the zebrafish (Ush1c216AA), a knock-in of the human exon 3 USH1C c.216G>A genome (Fig. 1A). We then used a radiation hybrid panel (Hukriede mutation (plus surrounding sequence) cloned from an Acadian et al., 1999) to position this gene on chromosome 25. Previous USH1C patient (Lentz et al., 2007), shows both the well- studies of the mouse and human genes have indicated that the characterized auditory defects of the other USH1C mouse models, mammalian genes express three splice forms, which vary slightly as well as visual defects comparable to the human disease. In in structure and subcellular localization (Reiners et al., 2003; addition to the disorganized stereocilia and hair cell degeneration Reiners et al., 2005; Williams et al., 2009). The most abundant observed in the cochleae of USH1C c.216G>A mice, reduced visual zebrafish transcript found in whole larvae or adult retinas function was noted after 1 month of age and progressive loss of corresponded to isoform A, containing 21 exons that encode a 548 rod photoreceptors was observable after 6.5 months of age (Lentz amino acid protein with three PDZ domains and a coiled-coil et al., 2010). These intriguing differences in retinal pathology domain (Fig. 1A). Zebrafish harmonin isoform A was found to be between the Ush1c knockout and the USH1C c.216G>A knock-in structurally similar to the corresponding human isoform, with 70% Disease Models & Mechanisms could indicate differences between the functions of endogenous amino acid identity overall and highest similarity in the PDZ mouse and human harmonin in the retina. domains and the N-terminal region (Pan et al., 2009). A In vitro assays have demonstrated that many of the known USH hydrophobicity plot of the human and zebrafish proteins revealed proteins bind preferentially to one or more of the three PDZ domains a high level of conservation, even where residue substitutions occur in harmonin, hence its depiction as a central scaffold upon which (Fig. 1A). Using reverse transcriptase (RT)-PCR and RACE primers an USH protein complex can form (Adato et al., 2005). Although the within and between regions encoding the second and third PDZ subcellular localization of harmonin and other USH proteins in hair domains, we identified unique transcripts that might correspond cells is compatible with this hypothesis, there have been conflicting to the B or C isoforms of the gene, although no variant encoding reports of localization of other USH proteins at the photoreceptor a second coiled-coil or PST domain, as would be predicted for an synapse, where harmonin localization is undisputed (Reiners et al., isoform B transcript, was identified. All transcripts other than that 2005; Liu et al., 2007; Yang et al., 2010). Moreover, harmonin is of isoform A were in low abundance such that we were unable to notably absent from the connecting cilium region (Reiners et al., 2005; obtain sequence definitively identifying them as belonging to a Williams et al., 2009), where the presence of most other USH proteins particular isoform class. The probes generated from these partial has been well documented (Liu et al., 2007; Maerker et al., 2008; sequences showed weak, generalized signal in all tissues and time Yang et al., 2010). Thus, the normal cellular functions of harmonin, points analyzed (data not shown). the types of complexes it forms with other USH proteins in vivo, and cRNA probes directed against either the unique sequence of the early, pre-degenerative manifestations of the retinal pathology isoform A or the 5Ј region predicted to be common to all splice in USH1C remain mysterious. forms yielded identical expression patterns in larval tissues. RNA Previously, zebrafish mutants for myo7a (ush1b), cdh23 (ush1d) expression was first detected in the ear at 27 hours post-fertilization and pcdh15 (ush1f) have been described as exhibiting severe (hpf), when the first hair cells are specified (data not shown), and
Disease Models & Mechanisms 787 RESEARCH ARTICLE ush1c in zebrafish visual function
Fig. 1. Zebrafish ush1c is expressed in retina and the inner ear. (A)The predominant splice form of ush1c corresponds to mammalian isoform A, with 21 exons that produce a 1.6 kb mRNA. A hydrophobicity plot of zebrafish (red) and human (blue) isoform A protein sequence shows conserved residue polarity. % amino acid identity with human protein is indicated below the plot. The positions of the ush1cfh293 nonsense mutation (red arrowhead), the MO target (green DMM arrowhead) and the region recognized by the zebrafish harmonin antibody (red bar) are indicated. (B)At 5 dpf, ush1c transcripts are abundant in the inner retina (black arrow), the sensory patches of the ear (white arrows), the intestinal tract (i) and the swim bladder (sb). Fainter expression in the brain (b) and trunk muscles (m) is also observed. (C)High-magnification view of a 5 dpf whole-mount zebrafish ear showing ush1c expression in all sensory patches (ac, lc, pc: anterior, lateral and posterior cristae, respectively; am: anterior macula) and an adjacent head neuromast (n). (D)ush1c transcripts are restricted to cells in the inner nuclear layer in 5 dpf sectioned retina. (E)Central section of an adult zebrafish retina showing continued strong expression in the inner retina; expression is detected in the outer nuclear layer as well. onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer. Scale bars: 100m (B); 20m (C); 50m (D,E).
hair cell expression persisted through all later larval stages examined 2 boundary (MO1; green arrowhead, Fig. 1A) and one that [5 days post-fertilization (dpf) shown in Fig. 1B,C]. We also recognizes the translation initiation site (MO2). We obtained observed strong expression in the neuromasts of the lateral line identical phenotypes with both morpholinos, but found that the (Fig. 1C). This neuromast expression was consistent with previous exon-2-targeting morpholino (MO1) provided more consistent reports of the zebrafish USH gene orthologs myo7a, cdh23 and results; thus, MO1 was used for all morpholino experiments and pcdh15a (Ernest et al., 2000; Söllner et al., 2004; Seiler et al., 2005). all uses of ‘morpholino’, ‘MO’ or ‘morphant’ refer to MO1. Using Retinal expression was present beginning at 48 hpf, when retinal RT-PCR analysis of MO-injected embryos and larvae, we identified lamination is complete and cell specification is underway. By 72 an aberrant splice form in which exon 2 is skipped, producing out- Disease Models & Mechanisms hpf, strong expression was observed in a subset of cells in the inner of-frame splicing of exons 1 and 3 that results in an early nuclear layer, which persisted through all larval stages examined termination codon (supplementary material Fig. S1). This altered (5 dpf, shown in Fig. 1D). In situ hybridization of adult retinal tissue splice form predominates for the first 4 dpf, and persists through using all described probes showed persistent, strong expression in 6 dpf, although recovery of the normal splice form is evident by 5 the inner nuclear layer as well as expression in the outer nuclear dpf (Fig. 2A). layer (Fig. 1E). We also observed strong expression of ush1c in the We obtained a polyclonal rabbit antibody raised against the first gut and swim bladder, and weaker expression in the brain and trunk 100 amino acids of zebrafish harmonin (red bar, Fig. 1A), a region muscles (Fig. 1B), consistent with reported expression in human common to all predicted isoforms, and probed protein extracts on tissues (Verpy et al., 2000; Hirai et al., 2004). a western blot to test antibody specificity and to determine the extent of protein depletion in morphant and mutant larvae (Fig. Mutation or morpholino knockdown of ush1c depletes harmonin 2B). In wild-type controls, we observed a single band in the 3 dpf protein in zebrafish larvae sample, which was slightly smaller than the corresponding bands We obtained a nonsense mutation in ush1c from the Zebrafish in the 4 dpf and 5 dpf samples. In addition to this band, which, at TILLING Consortium by screening for mutations in the first PDZ- approximately 60 kDa, corresponded to the predicted size of domain-encoding region of the gene (PDZ1). We recovered a line, zebrafish harmonin isoform A, we noted progressively increasing ush1cfh293, that harbors a nonsense mutation in the fifth exon, which numbers of smaller bands in the 4 dpf and 5 dpf controls. Consistent encodes the C-terminal portion of the PDZ1 domain (red with the time course of morpholino efficacy revealed by RT-PCR arrowhead, Fig. 1A; supplementary material Fig. S1). (Fig. 2A), strong knockdown of harmonin protein levels was We also designed morpholino oligonucleotides to knock down observed at 3-4 dpf in ush1c morphants. Moreover, although the the function of ush1c: one to block splicing at the exon-2–intron- upper band was restored nearly to wild-type levels in the 5 dpf
788 dmm.biologists.org ush1c in zebrafish visual function RESEARCH ARTICLE DMM Fig. 2. Mutation and morpholino knockdown of ush1c produce behavioral defects. (A)RT-PCR of control and splice-blocking morpholino-injected embryos from 1 to 5 dpf reveals a smaller splice form lacking exon 2 that predominates for the first 4 days of development and persists through the fifth day. (B)Western blots from extracts of control + ush1c MO-injected embryos from 3 to 5 dpf (left) and ush1cfh293 homozygous mutant embryos alongside wild-type siblings (right) confirm morpholino efficacy and antibody specificity. Samples from 3 dpf to 5 dpf control larvae probed with the zebrafish harmonin antibody show a 60 kDa band consistent with the predicted size of isoform A. Additional smaller bands are visible faintly in the 4 dpf control and are more pronounced and abundant in the 5 dpf control. The band corresponding to the isoform A variant is undetectable in 3 dpf and 4 dpf morphant samples. The major band reappears at 5 dpf at a slightly smaller weight, and there is a reduced number of smaller bands in this sample. In extracts from 5 dpf ush1cfh293 homozygous mutants, no bands are observed. Extracts from wild-type siblings from the same clutch show the characteristic banding pattern of the 5 dpf uninjected controls, allowing for the slightly longer running time for this gel. A pan-actin antibody was used as a loading control. (C)Behavioral assays of control, mutant and morpholino-injected larvae show (top) impaired startle response and balance, and (bottom) reduced optokinetic response, tracking an average of 50% or fewer of the dark stripes (average targets tracked for wild type: 11.5±1.27; ush1c mutants: 6.13±2.78, P<0.0001; ush1c MO: 5.13±2.85, P<0.0001; n30 for each group).
sample, the size was slightly reduced, consistent with the control exons of ush1c. We observed reduced probe signal in all ush1c- band from the 3 dpf time point (Fig. 2B). These temporal changes expressing tissues examined (ear and gut tissue shown in in size and abundance of the major band, recapitulated by the supplementary material Fig. S2), suggesting that this mutant Disease Models & Mechanisms recovering morphant protein extracts, might reflect a period of transcript is unstable. post-translational modification. We performed RT-PCR experiments on zebrafish cDNA from Reduced harmonin function produces defects in vestibular, multiple developmental stages and discovered five distinct auditory and visual function expressed sequence tags (ESTs), but no complete sequences of splice At 5 dpf, wild-type fish exhibited rapid evasive swimming behavior variants other than isoform A. Thus, we cannot definitively identify when startled by a tap on the Petri dish. We tested the startle reflex the smaller bands on the western blots, but postulate that they of 5 dpf offspring from natural crosses between ush1c–/+ might be additional isoforms or products of protein degradation heterozygous carriers (n952 larvae) and found that 25% (n236) of isoform A, the largest running band. Similar, smaller bands were of these larvae had abnormal responses (Fig. 2C, top). Within this detected by western analysis of mouse harmonin (Williams et al., group, 83% did not exhibit any evasive swimming behavior, and 2009). We did not observe any bands larger than ~60 kDa at any those that did respond swam in circles. Subsequent genotyping larval time point examined. This, combined with the RT-PCR and revealed that 100% of the larvae exhibiting these defects were RACE results described above, suggests that isoform B splice homozygous ush1cfh293 mutants. variants are not active through 5 dpf. Free-swimming 5 dpf ush1c morphant larvae exhibited vestibular No bands were detected in protein samples from ush1c mutant defects identical to ush1c mutants, with only 30% of the larvae larvae at any time point examined (5 dpf shown alongside wild- responding to tapping on the Petri dish, and all responders swimming type sibling in Fig. 2B). However, the ush1c nonsense mutation in circles (n400). These defects are similar to those reported for in the fifth exon could potentially produce a truncated protein other zebrafish USH gene mutations or knockdowns (Ernest et al., that would be recognized by the antibody. We investigated RNA 2000; Söllner et al., 2004; Seiler et al., 2005) and indicate reduced levels in ush1c mutants using a riboprobe against the first six function of both the auditory and vestibular systems.
Disease Models & Mechanisms 789 RESEARCH ARTICLE ush1c in zebrafish visual function
DMM Fig. 3. Harmonin is required in otic sensory patches for normal bundle development. (A-C)Phalloidin-stained F-actin (green) and anti-zebrafish harmonin antibody labeling (red) in 80-hpf wild-type ears show localization of harmonin relative to the stereocilia in the five otic sensory patches (ac, anterior crista; am, anterior macula; lc, lateral crista; pc, posterior crista; pm, posterior macula) and in the neuromasts (n). The anti-harmonin antibody signal is reduced in sensory patches and in neuromasts in ush1c mutants (B) and ush1c-MO-injected larvae (C). (D-L)High-magnification view of phalloidin-stained hair bundles in the anterior and posterior maculae of 5 dpf larvae. Hair bundle number is reduced and organization of the existing bundles is disrupted in mutant (G-I) and morphant (J-L) anterior (G,J) and posterior (H,K) maculae, compared with the anterior and posterior maculae of wild-type controls (D and E, respectively). Close- up images of hair bundles of the anterior maculae (F,I,L) show bent and splayed bundles in mutants and morphants. For mutant analysis, anterior macular wild- type average54.6±3.5 bundles; n8; ush1c mutant average33±3.5; n7; P<0.0001. Posterior macula wild-type average68.2±2.6 bundles; n9; ush1c mutant average40.5±4.2; n10; P<0.0001. For the morpholino experiment, anterior macula control average41.8±4.8 bundles; MO average21.8±3.8; n5 for each group; P<0.0001. Posterior macula control average71.8±4.6 bundles; MO average48.2±6.9; n5 for each; P<0.0001, 5 dpf. Scale bars: 50m (A-C); 10m (D-L).
To assess visual function in mutant and morphant animals, we injected animals, we found a reduced number of hair bundles, with used the optokinetic response (OKR) assay. At 5 dpf, ush1c mutants 48% fewer bundles in the anterior maculae and 33% fewer hair and MO-treated larvae produced significantly fewer saccades, bundles in the posterior maculae of 5 dpf morphants compared tracking only 40-50% of the targets compared with wild type (Fig. with controls. Stereocilia were also disorganized, although to a 2C, bottom). lesser extent than we observed in the mutant. Bundles appeared Disease Models & Mechanisms bent and disoriented relative to the plane of the cuticular plate, but Loss of harmonin results in morphological defects in the splaying or absence of tapering at the tips was less pronounced mechanosensory hair cells than in ush1c mutants (Fig. 3J-L). To gain insights into the cause of the hearing and balance defects We next investigated the subcellular localization of harmonin in harmonin-deficient larvae, we used immunofluorescence to in the anterior maculae of 5 dpf larvae (Fig. 4). In wild-type hair examine the mechanosensory hair cells of the otic sensory patches. cells, harmonin localized to both stereocilia and kinocilia as well Harmonin localized to sensory patches and neuromasts by 80 hpf as within cell bodies and at synapses (Fig. 4A-G). In ush1c (Fig. 3A). We detected no harmonin signal at 80 hpf in ush1c mutants (Fig. 4H-J), we detected reduced levels of harmonin in mutants (Fig. 3B) or morphants (Fig. 3C), confirming the strong stereocilia and kinocilia. Protein was also detected within cell knockdown or depletion indicated by the western blot results. Using bodies, but the distribution appeared abnormal compared with phalloidin to visualize stereocilia, we examined individual sensory controls. Harmonin levels in hair cells of morphant larvae (Fig. patches. At 5 dpf, wild-type hair bundles had a characteristic shape 4K-M) were markedly diminished at 5 dpf, although some signal and orientation, projecting more or less orthogonally from the plane could be detected apical to the hair bundle in a position consistent of each cuticular plate and appearing tightly tapered at their tips with kinocilia localization. When we injected embryos from (Fig. 3D-F). All otic sensory patches in the mutant larvae displayed ush1c+/– heterozygous parents with the ush1c morpholino, raised reduced numbers of hair bundles, as well as defects in stereocilia the larvae to 5 dpf and analyzed harmonin staining in sensory organization at 5 dpf (Fig. 3G-I). We observed 40% fewer hair patches, residual staining was undetectable in injected bundles than in wild-type siblings in the anterior and posterior genotypically ush1c–/– animals, suggesting that a truncated form maculae, and existing stereocilia were splayed and bent, with a of harmonin is produced in the mutants. This truncated protein notable lack of tapering of the hair bundles. In morpholino- might be too low in abundance or too unstable to be detected in
790 dmm.biologists.org ush1c in zebrafish visual function RESEARCH ARTICLE
Fig. 4. Subcellular localization of harmonin in mechanosensory hair cells. Co-labeling of wild-type 5 dpf anterior macula hair cells (A-D) shows anti-zebrafish harmonin localization (red) in the hair bundles (green), kinocilia (arrowheads), cell bodies and at synaptic boutons (arrow in C and D). Incubation conditions favoring hair bundle visualization (E-P) show strong colocalization of harmonin within the bundle structure as well as in the cuticular plate in 5 dpf wild types (E-G). Kinocilia labeling can be seen above the bundle region (asterisks). Enhanced signal detection in mutant hair cells (H- J) shows localization persisting in the kinocilia and in existing bundle structures. Kinocilia are more visible in these panels owing to the reduced bundle architecture in the mutant. Protein distribution seems globular and disorganized in the cell bodies (arrowheads). Hair cells of ush1c MO larvae (K-M) show strong knockdown of harmonin, with localization persisting only in kinocilia at this stage, and no signal observed within hair bundles. Injecting the morpholino into the ush1c–/– mutants reduces subcellular localization of harmonin to undetectable levels in hair cells (N-P). Scale bars: 10m. DMM
a western blot, but was detectable in fixed tissue using enhanced cell fate specification cannot be the primary causes of bundle loss. signal detection protocols. Together, these results confirm a requirement for harmonin in hair To investigate cell degeneration or reduced cell proliferation as bundle development. Additionally, the milder stereocilia possible causes for the observed reduction in hair bundle number, disorganization phenotype in ush1c morphants observed at 5 dpf, Disease Models & Mechanisms we stained larvae at stages between 1 and 5 dpf with TUNEL to in conjunction with the partial recovery of harmonin localization label dying cells or BrdU to label dividing cells. The results in both at 5 dpf observed in the region of the kinocilia, suggests that cases were indistinguishable between morphants and controls (not harmonin also plays a role in maintaining hair bundle integrity. shown), and were consistent with previously reported normal cell death levels in the ear (Bever and Fekete, 1999). Mutant cell Harmonin localizes to Müller glial cells in the retina proliferation was also unaffected at these stages, as were cell death Retinal architecture appeared unaffected in ush1c mutants and levels through 4 dpf. In 5 dpf mutants, we noted a small but morphants compared with wild type (Fig. 5A,F,K; supplementary significant increase in TUNEL-labeled cells for both the anterior material Fig. S3); however, because visual function defects were macula (0.1±0.32 for wild type vs 1.2±1.03 for mutant; n10 for observed, we also examined harmonin protein localization in each group; P0.008) and posterior macula (0.1±0.32 for wild type retinas. We observed abundant protein levels specifically in the cell vs 1.3±0.95 for mutant; n10 for each; P0.003). Although bodies and processes of Müller glia at all larval stages examined statistically significant, the numbers of dead cells were insufficient (Fig. 5B-E; supplementary material Fig. S4), consistent with the to account for the extent of the reduction in bundles. To confirm restricted inner retinal expression noted by in situ hybridization that hair cells were being specified properly in mutant larvae, we with our ush1c riboprobes at 5 dpf (Fig. 1D). We did not observe used a probe to the hair-cell-specification factor atoh1b and noted specific photoreceptor labeling with this antibody between 5 and no differences in labeling of sensory patches in wild type compared 8 dpf. with mutant (data not shown). Thus, we conclude that the defects We examined harmonin antibody labeling in wild-type, mutant in hair bundle formation occur post-mitotically, consistent with and morphant retinas at 5 dpf. Harmonin protein levels in the previously reported studies in mammals (Lefevre et al., 2008), and retinas of ush1c–/– homozygous mutants were markedly reduced, that increased cell death, reduced cell proliferation, or impaired although Müller cells were present and appeared morphologically
Disease Models & Mechanisms 791 RESEARCH ARTICLE ush1c in zebrafish visual function
Fig. 5. Harmonin localization in the larval retina is restricted to Müller glia. Semi-thin plastic sections stained with toluidine blue show consistent size, lamination and organization of 5 dpf wild-type, mutant and morphant retinas (A,F,K). 16-m larval retina sections (B,G,L) show colocalization of the Müller cell marker glutamine synthetase (GS; green) and anti-zebrafish harmonin (red) in wild type (B). Both antibodies label Müller cell processes throughout the retina. Anti-zebrafish harmonin labeling is reduced in mutant (G) and morphant (L) Müller cells. Müller cell number and morphology is unaffected. Nonspecific signal associated with photoreceptor outer segments is observed in all panels at this magnification. See supplementary material Fig. S4 for comparison. High- magnification images of anti-zebrafish harmonin and GS labeling in Müller cell processes at the OPL (large arrow) and OLM (small arrow) of the 5 dpf retina are shown (wild type, C-E; mutant, H-J; morphant, M-O). Scale bars: 50m (A,B,F,G,K,L); 20m (C-E,H-J,M-O).
normal (Fig. 5G-J). Similarly, after morpholino injection, we possibility of a phylogenetically conserved role for harmonin in
DMM observed a strong reduction of harmonin labeling in Müller cells retinal glia. through 5 dpf (Fig. 5L-O). We noted that the duration of protein Harmonin was recently detected in the postsynaptic processes depletion was prolonged in the eye, compared with our results from of murine bipolar cells and horizontal cells, using immunoelectron morphant hair cells, in which we observed rebounding harmonin microscopy (Williams et al., 2009). With our immunofluorescence localization in some kinocilia by 5 dpf (Fig. 3F). We therefore protocols, we found no evidence for harmonin localization within examined harmonin labeling in 6 dpf morphants, at which time cell bodies of bipolar or horizontal cells at any time points examined we observed increased harmonin labeling in Müller cells of the in zebrafish (Fig. 5; Fig. 6A,C). Owing to the strong harmonin peripheral retina, adjacent to the ciliary marginal zone labeling in Müller glial processes, we cannot determine whether (supplementary material Fig. S4). synaptic processes of horizontal or bipolar cells might also be Owing to the apparent presence of ush1c transcript in adult positive for harmonin in the outer plexiform layer (OPL). photoreceptors (Fig. 1E), we evaluated the subcellular localization Given the retinal degeneration experienced by USH1C patients, of harmonin in 6 month adult retinas (Fig. 6A-F). We continued we examined retinal cell death in zebrafish ush1c mutants and to see strong harmonin localization within Müller cells in the adult morphants at 5 and 8 dpf, using an anti-active caspase-3 antibody. retina, extending past the outer limiting membrane (OLM) and into Death rates by retinal cell layer were as follows (n10 for all groups; the glial processes in the subapical region between photoreceptor see also supplementary material Fig. S4): in the photoreceptor layer, cell bodies (Fig. 6, asterisks). In some adult retinas we also observed wild-type animals at 5 dpf had 0.20±0.41 labeled cells compared Disease Models & Mechanisms what might be a low level of protein localization at the with 0.31±0.60 in morphant retinas (P0.5) and 3.09±1.3 in mutants photoreceptor synapses (Fig. 6A, arrow). Occasionally, signal was (P<0.0001); in the inner nuclear layer, no labeled cells were observed detected in photoreceptor inner or outer segments at a lower level in wild type, compared with 1.86±1.23 in morphants (P<0.0001) and with inconsistent distribution compared with the robust and 1.88±0.73 in mutants (P<0.0001); in the ganglion cell layer, staining observed in Müller glia (Fig. 6D, arrow), suggesting that 0.062±0.25 cells were labeled in controls, compared with 0.25±0.45 this signal was due to nonspecific labeling or autofluorescence, in morphants (P0.16) and 0.09±0.30 in mutants (P0.79). Noting although we were unable to confirm this with our current the small but significant elevation of inner nuclear layer (INL) cell immunofluorescence methods. death in 5 dpf mutants and morphants, we co-labeled sectioned 5 Because Müller cell localization of harmonin has not been dpf retinas with caspase-3 and glutamine synthetase to determine reported previously, we used antibodies against murine (Fig. 6G- what proportion of the dying cells were Müller glia (n10 for each I) and human (Fig. 6J-L) harmonin to label retinas from postnatal group). In ush1c–/– animals, exhibiting an average of 1.88 labeled day 10-11 mouse pups. In addition to the previously described cells per eye, 67% of the cells labeled with caspase-3 were also labeling in photoreceptors, we also observed harmonin positive for glutamine synthetase. Similarly, 61% of caspase-labeled immunoreactivity in Müller glial cell bodies and processes cells were co-labeled with glutamine synthetase in ush1c morphant extending throughout the retina. The anti-human harmonin retinas, in which an average of 1.85 cells in the INL were labeled. antibody (Fig. 6J-L) also cross-reacted in 5 dpf zebrafish retinas, In both cases, the remaining labeled cells could not be positively labeling Müller cells, but not photoreceptors (data not shown). identified as Müller cells, based on either their position within the Given that the observed localization of harmonin in murine Müller nuclear layer or by labeling with the Müller cell marker. Although cells differs from published work (Reiners et al., 2005), further our current experiments do not rule out the possibility that a small investigation is needed, but our results are consistent with the subset (<0.7 cells per eye) of caspase-positive cells in the INL might
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Harmonin is required for photoreceptor synaptic organization and function The specific depletion of harmonin from Müller cells in larval mutant and morphant retinas provided an opportunity to study the role of harmonin in this cell type. Because the morphants presented no significant photoreceptor cell death at 5 dpf despite strong protein knockdown, we used morphants for the following analyses. We began by examining synaptic integrity in morphant larvae at 5 dpf, using antibodies against the pre- and postsynaptic proteins SV2 and GluR2/3, respectively. In control retinas, these markers exhibited an organized, parallel and largely colocalized alignment within the OPL (Fig. 7A). In ush1c morphants, both proteins were present in the OPL, but they were remarkably misaligned and less colocalized, indicating a disruption in synaptic organization (Fig. 7B). To investigate this phenotype further, we examined the ultrastructure of cone pedicles in morphant larvae via transmission electron microscopy. We examined cone pedicles from retinas of two uninjected controls at 6 dpf in a quantitative comparison with cone pedicle ultrastructure from retinas of four stage-matched ush1c MO larvae. We found that cone pedicles in uninjected controls (Fig. 7C,E) contained an average of 1.667±0.299 ribbons
DMM per pedicle (n36 pedicles) and a large number of mature-appearing ribbon synapses, with 55% of the synaptic ribbon profiles anchored by an arciform density (ribbons with arciform density per pedicle1.08±0.238); 85% of these membrane-anchored ribbons were associated with postsynaptic processes from horizontal and bipolar cells in a triad (triads per pedicle0.97±0.128). In morphant retinas (Fig. 7D,F), the average number of ribbons per pedicle was consistent with controls (1.659±0.607; n44 pedicles), but only 38% of the ribbon profiles appeared anchored by arciform densities (ribbons with arciform densities per pedicle0.636±0.647; P0.0002). Synaptic ribbons unanchored by an arciform density Fig. 6. Harmonin localizes in Müller cells through all zebrafish life stages seemed to ‘float’ in the pedicle, and postsynaptic processes, when and is evolutionarily conserved. (A-F) Localization of anti-zebrafish present, frequently appeared distant from these floating ribbons harmonin (red) in Müller cells persists in adult zebrafish retinas. Some low level (Fig. 7D,F). As in the controls, most (83%) anchored ribbons in signal is sometimes observed in photoreceptor synapses (arrow in A) and in photoreceptor outer segments (arrow in D). The strongest signal within the morphants were incorporated into triads (triads per photoreceptor layer is associated with Müller cell processes projecting into the pedicle0.523±0.412), suggesting a specific defect in ribbon Disease Models & Mechanisms subapical region (asterisks). Nonspecific fluorescence of photoreceptor outer association with the membrane. To investigate whether the segments at this stage can be compared with panel H in supplementary observed defects could be attributed to a developmental delay, we material Fig. S4. (G-L)Anti-mouse harmonin (SDI; G,I) and anti-human also examined sections of ribbon synapses in cone pedicles of two harmonin (Santa Cruz; J,L) detect localization in photoreceptors and Müller wild-type and three morphant retinas at 8 dpf. The number of cells [glutamine synthetase (GS) is labeled in green; harmonin antibodies in ribbons observed per pedicle (1.34±0.56; n41) was significantly red] in retinas from P10-P11 mouse pups. The outer nuclear layer (onl), inner reduced in 8 dpf morphants compared with controls (1.70±0.22; nuclear layer (inl) and ganglion cell layer (gcl) are labeled in merged panels. Scale bars: 50m (A-C,G-L); 20m (D-F). n43; P0.0002). As seen in the 5 dpf analysis, significantly fewer ribbons in the 8 dpf morphants (52%; n55 ribbons) compared with controls (82%; n73 ribbons; P<0.0001) appeared to be anchored be second order neurons, it is also possible that these cells were to the membrane. The percentage of anchored ribbons associated apoptotic Müller glia that no longer produced glutamine synthetase. with triads was comparable between morphants and controls (68% At 8 dpf, we noted increased photoreceptor cell death in the and 65%, respectively), again suggesting a primary defect in the ush1c mutant retinas, compared with wild-type animals (wild type: formation of arciform densities. Retinal morphology was otherwise 0 labeled photoreceptors; mutant: 5.33±1.32 labeled unaffected, and significant retinal cell death was not observed in photoreceptors; P<0.0001; see supplementary material Fig. S4). No 8 dpf morphants in any retinal cell layers by anti-active caspase-3 statistically significant differences were observed between mutant labeling. Thus, we conclude that temporary depletion of harmonin and wild-type siblings in either the inner nuclear or ganglion cell from Müller glia during retinal development has a lasting, adverse layers at this stage, nor were there significant differences seen in impact on maturation of cone ribbon synapses. the labeling of morpholino and control animals in any cell layers To determine the effect of the morphological defect on synaptic at 8 dpf. transmission, we recorded ERGs from 5 dpf morphant larvae (n6)
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Fig. 7. Synaptic maturation is impaired in ush1c morphants. (A,B)Anti-SV2 (red) and -GluR2/3 (green) antibodies mark the pre-and postsynaptic regions of the OPL, respectively. Tightly associated localization of these factors is apparent in the control retina (A), and is notably disrupted in the morphant retina (B) at 5 dpf. (C-F)Electron micrographs of cone pedicles in 6 dpf control and morphant larvae. In control retinas (C,E), the presence of multiple triads (boxed areas), consisting of a synaptic ribbon (r), arciform density (arrow) and postsynaptic processes from bipolar (b) and horizontal (h) cells, indicate normal synaptic maturation. An enlargement of a triad is shown in E. In morphant retinas (D,F), fewer triads are present (boxed areas in D; enlarged in panel F), and floating ribbons (r) and distant postsynaptic processes (h) are observed in cone pedicles. Scale bars: 20m (A,B); 1m (C,D); 500 nm (E,F). DMM
and compared them with uninjected larvae (n7). Rod in the b-wave amplitude. Thus, our data suggest that loss of photoreceptors are detectable molecularly by 3 dpf and harmonin in Müller cells results in decreased function of the morphologically by 5 dpf. However, physiological analyses photoreceptor ribbon synapses. demonstrate that rods are not functional until 15 dpf (Morris and Fadool, 2005). ERGs performed at 5 dpf show a cone dominant DISCUSSION response in both light-adapted and dark-adapted conditions (Bilotta In this study, we describe the requirement for harmonin in zebrafish et al., 2001; Seeliger at el., 2002), but dark-adapted retinas at this mechanosensory hair cell and retinal development, providing the stage are less susceptible to bleaching and exhibit a more sensitive first evidence that USH proteins function in photoreceptor synaptic ON response than in light-adapted conditions (Makhankov et al., transmission and ribbon synapse development. Our studies also 2004). Thus, we chose to perform our experiments under scotopic implicate Müller glial cells as contributors to the retinal pathology conditions, in which we observed that the b-wave amplitude, an of USH. indicator of synaptic transmission, was markedly reduced in the The localization of harmonin in hair cells and lateral line Disease Models & Mechanisms MO-injected animals (Fig. 8A) compared with controls. The a- neuromasts indicates a phylogenetically conserved role in wave, which is primarily a read out of photoreceptor membrane mechanosensory cells, and our studies of the zebrafish inner ear depolarization as a result of phototransduction, is often quenched reveal defects in hair bundle organization and development, by conditions conducive to generating b-wave amplitudes and is consistent with the stereocilia elongation and organization defects therefore not observable on the same trace. Thus, we isolated the described in Ush1c mutant mice (Lefevre et al., 2008). Our results a-wave pharmacologically with the synaptic transmission inhibitors indicate that, in zebrafish, the predominant splice variant and L-AP4 and TBOA (Trombley and Westbrook, 1992; Huang and protein isoform present during larval development is harmonin Bordey, 2004; Wong et al., 2005), whereupon we observed a slight isoform A. The severe disruption of hair bundle development seen reduction in isolated a-wave amplitude compared with controls in ush1c mutant and morphant zebrafish provides an intriguing (P0.01), but less of an effect overall than that observed from the contrast to the absence of hair bundle defects reported in some b-wave experiments (P0.0007; Fig. 8B). Plotting a-wave and b-wave colonies of dfcr mice, which harbor a deletion affecting harmonin amplitudes against each other shows that the b-wave to a-wave ratio isoform B (Grillet et al., 2009). is significantly diminished in the morphant group (P<0.0007; Fig. The presence of the USH protein harmonin in Müller glia from 8C). These data, taken together with the disorganization of synaptic an early time point in retinal development and the specific proteins in the OPL (Fig. 7B), indicate that postsynaptic activity is defects in synaptic structure and function in ush1c morphants reduced relative to the level of the phototransduction response to prior to detectable ush1c transcript or protein synthesis in light stimulus (Takada et al., 2008). Although we observed a very photoreceptors suggest a non-cell-autonomous role for harmonin small increase in the number of caspase-positive cells in the INL, in facilitating synaptic maturation in cone pedicles. Müller glial the majority of which were identified as Müller glia, we would not cells have established roles in the maintenance of retinal neurons expect the loss of so few cells to account for such a large reduction via their regulation of glutamate neurotransmitter levels and their
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Fig. 8. Deficits in synaptic function of photoreceptors in ush1c morphants. (A,B)Representative larval zebrafish ERG recordings showing b-wave amplitudes (A) or a-wave amplitudes (B) of uninjected control (black) and MO-injected (gray) larvae at unattenuated light intensity. (A)b-wave amplitudes are significantly reduced in morphant animals. (B)The a-wave of these same subjects was isolated by treatment with L-AP4 and TBOA to block synaptic transmission. a-waves are only slightly attenuated in morphant animals compared with controls. Light stimulus duration is 800 mseconds. (C)Comparison of the relative changes of b- and a-wave amplitudes in morphant and control larvae obtained at 0, –1 and –2 log units of light intensity relative to unattenuated light intensity. The decreased b- wave:a-wave ratio indicates a specific synaptic defect. Steeper slopes of the trend lines (dashed lines) represent reduced synaptic efficacy.
expression of neurotrophic factors (Fields and Stevens-Graham, were surprised by the lack of harmonin localization in larval 2002; Newman, 2004; de Melo Reis et al., 2008). Although a role photoreceptors. It is puzzling that there would be no requirement for Müller glia in retinal synaptogenesis has been suggested by for harmonin function in larval zebrafish photoreceptors, especially several studies (Georges et al., 2006; Diaz et al., 2007), little is in light of the abundant antibody signal detected in the
DMM known about how this putative function might be accomplished. photoreceptors of young mice. An as-yet-undetected duplicate In the brain, glutamate receptor function has been shown to be ush1c gene in zebrafish could account for the apparent protein important for synaptogenesis (Han and Stevens, 2009; Kakegawa localization discrepancies between mammals and zebrafish. et al., 2009), so glutamate uptake by Müller cells might similarly However, our searches of the zebrafish genome reveal only one be important for synaptic development of photoreceptors. locus for ush1c, and we find only one copy of this gene in the Interestingly, a recent zebrafish study (Williams et al., 2010) used Tetraodon, Takifugu and Threespine stickleback genome a Müller-cell-specific line to ablate individual glia and observe assemblies, suggesting that only a single copy of the ush1c gene effects on synaptogenesis and the behavior of neighboring glia. has been retained in the teleost lineage. Their data indicated that localized loss of Müller cells did not Another possible explanation for the absence of harmonin in affect the maturation of cone synapses at 5 dpf. These data are larval photoreceptors is the presence of ohnologous genes for two consistent with our findings, because they argue against the small other PDZ-domain-containing proteins: Cip98 and Pdzd7. and transient levels of Müller cell death being the cause of the Alignments with the human CIP98 (whirlin) protein sequence, reduced synaptic function and structural defects observed, and causative of USH2D, uncovered two orthologs in the zebrafish suggest instead that harmonin in Müller cells has a paracrine effect genome assembly. Both of these transcripts, cip98a and cip98b, are on cone synapse stability, such that the sustained absence of this expressed in photoreceptors from early larval time points (J.B.P., protein results in the observed dysfunction and progressive unpublished); thus, the presence of an additional PDZ protein with Disease Models & Mechanisms photoreceptor cell death. similar binding affinities to harmonin (van Wijk et al., 2006) might The cone synapse phenotype of ush1c morphants is similar to be able to compensate for the absence of ush1c expression in these that of the zebrafish nrc mutant, in which the synaptojanin gene cells. However, given that the N-terminal regions of harmonin and is disrupted (Allwardt et al., 2001; Van Epps et al., 2004). The nrc Cip98 are not functionally interchangeable (Pan et al., 2009), any mutant has a diminished b-wave and defects in photoreceptor functional substitution of harmonin by Cip98 in the photoreceptor synapse formation, with floating ribbons and unassociated must not rely on protein interactions in the N-terminal region. postsynaptic processes in cone pedicles. Synaptojanin has been Although PDZD7 is not currently recognized as a monogenic cause shown to be required for cytoskeletal organization, endocytosis, of human USH, it has been shown to act as a modifier of USH gene Ca2+ signaling and post-synaptic glutamate transport (Sakisaka function and to interact physically with USH proteins. Both et al., 1997; Schuske et al., 2003; Gong and DeCamilli, 2008). zebrafish genes, pdzd7a and pdzd7b, are expressed in Although no link between synaptojanin and USH has been photoreceptors at larval stages (Ebermann et al., 2010). proposed previously, nrc mutants have swimming and balance Our ERG recordings from ush1c morphant larvae reveal a defects in addition to blindness (Van Epps et al., 2004), similar specific reduction in b-wave amplitude in the absence of significant to the USH-like phenotype we observe in ush1c mutants and retinal degeneration, which might provide an explanation for the morphants. More recently, a report describing three new mutant early cellular dysfunctions that precede retinitis pigmentosa in alleles of zebrafish synaptojanin, isolated by screening for USH1C patients. A retrospective ERG analysis of USH1 patients vestibular defects, illustrates synaptic transmission defects in the as young as 17 months revealed abnormal waveforms prior to hair cells (Trapani et al., 2009). clinically detectable retinitis pigmentosa (Flores-Guevara et al., Given the distinct, penetrant defects in visual function and 2009), although other studies of USH1 patients in which photoreceptor synapse formation in harmonin-depleted larvae, we photoreceptor loss was already clinically evident revealed no
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synaptic dysfunction in the remaining viable photoreceptors METHODS (Jacobson et al., 2008; Williams et al., 2009). Notably, the Animal strains and maintenance Ush1c216AA knock-in mouse (Lentz et al., 2010) exhibits an All zebrafish studies were conducted with Oregon AB wild-type attenuated ERG as early as 1 month of age, prior to any histologically larvae or ush1cfh293 mutants in the AB background. Animals were appreciable cell loss. However, in contrast to our results with larval raised in a 10-hour dark and 14-hour light cycle, and maintained zebrafish, both a- and b-waves were attenuated in Ush1c216AA as described (Westerfield, 2007). Owing to the swimming and mice. It could be that, in human manifestations of USH1C, the later balance defects exhibited by ush1c mutants and morphants, all consequence of diminished harmonin levels in photoreceptors is experimental and control larvae for experiments occurring up to so severe that it obscures comparatively subtle early defects that 8 dpf were raised without food so as not to give normal swimmers might result from impaired synaptic activity. a nutritional advantage over their swimming-impaired siblings. Our data also indicate that sustained absence of harmonin in Animals were staged according to the standard series (Kimmel et Müller cells, as seen in ush1c mutants, results in progressive retinal al., 1995), or by hpf or dpf. All animal-use protocols were IACUC degeneration. It is not clear whether this cell death results from approved. synaptic defects or from some other impairment of Müller cell function, but the non-cell-autonomous nature of this degeneration Cloning of zebrafish ush1c cDNA and sequencing is notable. Clinical analyses of the retinal pathology of USH patients tBLASTn queries with the human harmonin protein sequence were might provide insight into the link between Müller cell function used to identify the zebrafish sequence, and primers based on this and retinal integrity. Ophthalmic examinations of USH1B patients sequence were used to amplify the full-length open reading frame (who have mutations in the MYO7A gene) using high-resolution of isoform A and partial sequence from additional isoforms from optical coherence tomography (Jacobson et al., 2009) showed that whole larvae or adult retinal cDNA (see supplementary material Table morphological changes in the OLM (the apical terminus of Müller S1 for primer sequences). PCR products were purified using gel cell processes) marked a transitional zone between regions in which electrophoresis and sequenced on an ABI 3100 using the fluorescent
DMM photoreceptors were diseased or depleted and morphologically dideoxy terminator method. The gene is cataloged in NCBI under normal regions, suggesting that such alterations in OLM integrity accession number NC_007136. All novel transcripts were submitted might precede photoreceptor degeneration. Our understanding of to the NCBI dbEST, with accession numbers HO243997-HO244001 the role that the OLM plays in maintaining photoreceptor integrity and HS587020. Gene and protein names follow the zebrafish is incomplete; however, numerous junction proteins, including nomenclature guidelines (https://wiki.zfin.org/display/general/ members of the Crumbs complex, have been shown to localize to ZFIN+Zebrafish+Nomenclature+Guidelines). the OLM (for reviews, see Gosens et al., 2008; Omri et al., 2010) and, in particular, to the subapical region, perhaps contributing to Genetic mapping of zebrafish ush1c junctions within a semipermeable barrier. Mutations in Crumbs The zebrafish ush1c locus was mapped by PCR using the LN54 orthologs in mammals cause progressive retinal degeneration and collection of radiation hybrids (obtained from Marc Ekker, the retinal pathology reveals diminished contacts between University of Ottawa), which contain the zebrafish AB9 cell DNA photoreceptors and the OLM (Mehalow et al., 2003; van de Pavert, and mouse B78 cell DNA (Hukriede et al., 1999). 2004). USH proteins have not been reported in the OLM, or in Müller cells in general, before this study, although whirlin (USH2D) ush1c mutant discovery has been linked biochemically to the Crumbs pathway (Gosens et Labeled primers (supplementary material Table S1) to PCR amplify al., 2007). a 500 bp region surrounding exon 5 of ush1c were generated and Disease Models & Mechanisms Although the extent to which Müller cells might contribute to used to screen 8448 genomes in a TILLING screen as described the USH retinal pathology remains to be investigated, our (Moens et al., 2008). Sperm from the identified carrier sample were histological analyses of both zebrafish and mouse retinas, and our thawed and used to fertilize wild-type AB eggs. Adult F1 carriers of functional studies in ush1c morphants and mutants, suggest that the mutation were identified by fin-clip genotyping, and identified additional investigation of Müller cell function is required to heterozygotes were crossed to produce homozygous offspring. understand the complex etiology of USH. As gene replacement therapies are developed for USH patients, it will be important to Morpholino injections know whether retinal cell types in addition to photoreceptors Antisense morpholinos (GeneTools, Philomath, OR) were injected should be targeted for optimal rescue of the vision defects. The into one-cell-stage embryos as described (Nasciveus and Ekker, ability to isolate Müller-cell-associated pathology in our zebrafish 2000). Embryos were injected with 1.8-2.0 nM of ush1c MO1 USH1C model will allow further investigation of the roles of targeted to the splice donor site of exon 2 (5Ј-TAGAATTG - Müller cells as well as investigations of USH protein function at AGACTTACTGATGGTAC-3Ј), or 0.6-1.0 nM of ush1c MO2 the OLM. targeted to the translation start site (5Ј-TTTTCAGTCCGT - In summary, we have characterized the hearing and vision defects TGGTCGTCTTGCT-3Ј). in a zebrafish ush1c mutant and morpholino knockdown. Our findings support a phylogenetically conserved developmental role RNA isolation and RT-PCR for harmonin isoform A in hair bundle organization, implicate Total RNA was isolated from euthanized larvae at 24 hpf to 7 dpf Müller cells as contributors to retinal dysfunction and using Trizol (Invitrogen, Carlsbad, CA). Reverse transcription was photoreceptor cell death, and demonstrate that visual defects can performed according to manufacturer instructions using the be detected from the onset of vision in these zebrafish models. Superscript Reverse Transcriptase II kit (Invitrogen).
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Western blot glutamine synthetase (Millipore), 1:500; mouse zpr1 (Zebrafish Embryos were homogenized in 10 mM Tris-HCl, pH 7.4, 150 mM International Resource Center, Eugene, OR), 1:200; mouse anti-SV2 NaCl, 1 mM EDTA, 0.1% Triton X-100, 0.1% SDS and 5 mg/ml (DSHB), 1:2000; rabbit anti-GluR2/3 (Millipore), 1:50; rabbit anti- protease inhibitor cocktail (Pierce, Rockford, IL). Samples were run active caspase-3 (BD Biosciences, San Jose, CA), 1:200; mouse anti- on an 8% acrylamide gel and transferred to nitrocellulose acetylated tubulin (Sigma), 1:500. Goat anti-mouse, chicken anti- membranes with Nupage reagents (Invitrogen). Membranes were goat and goat anti-rabbit Alexa-Fluor-488, -568 and -633 secondary washed in PBS twice, each for 15 minutes, incubated in blocking antibodies (Molecular Probes) were used at 1:300 and the anti- solution (3% NFDM in PBS) for 1 hour at room temperature, then rabbit biotinylated antibody was used at 1:500. Alexa-Fluor- in primary antibodies [rabbit anti-zebrafish harmonin (SDIX, 488–phalloidin (Invitrogen) was diluted to a final concentration of Newark, DE) 1:300; mouse monoclonal pan-actin (Millipore, 2.5 M. Billerica, MD) 1:5000] in blocking solution overnight at 4°C. Membranes were rinsed three times for 5 minutes each in PBS, Electron microscopy incubated in donkey anti-rabbit HRP (Jackson, West Grove, PA) Tissue was prepared as described previously (Schmitt and Dowling, for 1 hour at room temperature, then rinsed three times for 5 1999). Ultrathin sections were obtained from Epon-embedded minutes each in PBS. HRP was detected using the ECL plus tissue, stained with uranyl acetate and lead citrate, and examined detection system (Pierce). To detect actin, membranes were by conventional transmission electron microscopy. rehydrated and incubated in donkey anti-mouse HRP for 1 hour, rinsed three times for 5 minutes each in PBS, and labeled with the Optokinetic response ECL plus detection system (Pierce). To measure optokinetic response, we used a modification of the method of Brokerhoff (Brokerhoff, 2006). Larvae were placed in a In situ hybridization and immunohistochemistry small Petri dish containing 3% methylcellulose and mounted on a In situ hybridization on whole and sectioned tissue and antibody stationary platform surrounded by a rotating drum of 8 cm in
DMM labeling on sectioned tissue was performed as described (Jensen diameter. The drum interior displays ten alternating black and white et al., 2001; Ebermann et al., 2010). For imaging of subcellular stripes, each subtending an angle of 28°. The larvae were oriented protein localization in sectioned retinas, incubation with primary in an upright position, illuminated with fiber optic lights and viewed antibodies and a goat anti-mouse Alexa-Fluor-488 to visualize the through a dissecting microscope positioned over the drum, which glutamine synthetase antibody was followed by incubation with an rotated at 5 rpm clockwise and then counter clockwise, for 30 anti-rabbit biotinylated antibody diluted in block (10% NGS, 2% seconds in each direction. Eye movements were recorded during BSA in PBS-T) overnight at 4°C. Sections were then rinsed four this time period, after which the larvae were washed out of the times for 5 minutes each in PBS and signal was detected with the methylcellulose and returned to embryo medium. Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA) and TSA kit (Perkin-Elmer, Waltham, MA). For whole-mount ERG recordings imaging of stereocilia, 5 dpf larvae were fixed in ‘BT’ fix (4% PFA, ERG recordings (Makhankov et al., 2004) were made to examine 0.15 mM CaCl2, 4% sucrose in PBS) for 48 hours, rinsed four times retinal function of morphants (morpholino-injected larvae) for 5 minutes each in PBS + 0.01% Triton X-100, and then compared with control larvae. 5 dpf larvae were incubated in permeabilized with 2.5% Triton X-100 in PBS at room temperature Ringer’s solution for at least 1 hour prior to the experiment and until otoliths dissolved. Larvae were rinsed four times for 5 minutes dark-adapted for a minimum of 30 minutes. Under dim red each in PBS + 0.01% Triton X-100, blocked for 2 hours at room illumination, larvae anesthetized with tricaine (Sigma Aldrich) and Disease Models & Mechanisms temperature in 10% NGS, 10% BSA in PBS + 0.01% Triton X-100, paralyzed with 300 M pancuronium bromide (Sigma Aldrich) then incubated with Alexa-Fluor-488–phalloidin diluted in block were placed on a piece of sponge so that the pupil of one eye was for 24 hours at 4°C. Following four 5-minute PBS + 0.01% Triton on axis with the stimulus light beam. A recording electrode was X-100 washes, larvae were blocked using the Avidin/Biotin blocking placed on the cornea to measure the change in voltage induced by kit (Molecular Probes, Carlsbad, CA), and incubated in antibody stimulation. The reference electrode was a AgCl-coated silver wire solutions overnight at 4°C. Larvae were rinsed four times for 30 inserted into the sponge. The recording electrode was connected minutes each in PBS-Triton, followed by signal detection with the via a AgCl-coated silver wire to a low noise recording amplifier Vectastain ABC Elite kit (Vector) and TSA (Perkin-Elmer) kit. After (Axopatch 200A with a CV201A head stage), and data were rinsing four times for 5 minutes each in PBS-Triton, larvae were acquired and stored digitally (Digidata 1200 interface and pClamp7 immersed in Vectashield (Vector) and mounted in a lateral and pClamp8 software; recording hardware and software from orientation on chambered slides. Images were captured on a Zeiss Axon Instruments). Signals were filtered at 2 kHz. LSM5 confocal or BioRad Radiance 2100 confocal microscope. The following antibodies and concentrations were used in this study: Stimulation rabbit anti-harmonin (SDIX) antibody generated against aa1-100 Recordings were performed in a Faraday cage housing a Zeiss UEM of zebrafish harmonin, 1:200 for sections, 1:1000 for whole mount; microscope, with a 4ϫ objective lens used for visualizing the eye rabbit anti-harmonin (SDIX) antibody generated against aa306-363 and delivering light stimuli. An ultra bright white LED array (Atlas of mouse harmonin, 1:200; goat anti-harmonin (Santa Cruz series high brightness module NT-42D0-0426 from Lamina Biotechnology, Santa Cruz, CA) against the N-terminus of the Lighting, with an LED driver circuit locally designed and human protein, 1:40; rabbit anti-harmonin (Sigma-Aldrich, St constructed by the technical support group) was used to present Louis, MO) against aa42-118 of human harmonin, 1:40; mouse anti- the stimulus. Stimulus durations were 800 mseconds. The light
Disease Models & Mechanisms 797 RESEARCH ARTICLE ush1c in zebrafish visual function
L-(+)-2-amino-4-phosphonobutyric acid (L-AP4; 30-300 M), DL- TRANSLATIONAL IMPACT threo-b-Benzyloxyaspartic acid (DL-TBOA; 15 M) and Ringer’s solution with 5% DMSO as control. After 15-30 minutes, the a- Clinical issue Usher syndrome (USH) is a recessively inherited disease of combined deafness wave traces were recorded. and blindness. Hearing impairment is usually apparent from birth, whereas vision loss is slow and progressive, beginning in the first or second decade of Data analysis life. Cochlear implants and hearing aids can compensate for some of the The amplitudes of the b-waves were measured from the bottom of hearing deficits, but there is currently no treatment to counter the vision loss, the a-wave to the peak of the b-wave; the a-wave was measured which is a form of retinitis pigmentosa. Early diagnosis is important for genetic from the baseline to the peak of the a-wave. We used larvae that counseling and preparation for late onset blindness. had a consistent response to a test flash delivered (±10%) at the There is a great deal of clinical variability among patients with USH, and beginning and the end of the session. Unhealthy or damaged wild- nine genes that can cause this disease when mutated have been identified to date. These genes encode a wide variety of proteins that have complex and type larvae that showed a decreased ERG or changed waveform at dynamic localization patterns within the cells of the inner ear and the retina the end of the experiment compared with the beginning were not that are affected in USH. Discovering more about how these mutations cause included in the analysis. For statistics, we used a linear mixed model deafness and blindness at the cellular and molecular level will enhance our approach and a Student’s t-test. understanding of the etiology of this disease, and contribute to the ACKNOWLEDGEMENTS development of effective treatments. The authors thank David Raible, Kelly Owens and John Constable for help with Results experimental design and reagents, the Cecilia Moens laboratory for their work on the TILLING screen, and Jeremy Wegner for technical assistance. Support was In this paper, the authors describe a zebrafish model for Usher type 1C provided by NIH EY07042, DC004186, DC010447, RR020833 and HD22486, and by (USH1C), a severe form of USH in humans that is characterized by profound the Foundation Fighting Blindness. J.B.P. was supported by an American Heart congenital deafness, balance problems and a relatively early onset of Association Postdoctoral Fellowship. D.S.W. is a Jules and Doris Stein RPB Professor. progressive vision loss. The authors disrupt the normal function of the ush1c gene by using morpholino oligonucleotides to interfere with mRNA splicing. In COMPETING INTERESTS
DMM The authors declare that they do not have any competing or financial interests. addition, they identify an ush1c zebrafish mutant by screening DNA from individual mutagenized fish to find a lesion in the ush1c gene. Both AUTHOR CONTRIBUTIONS morpholino-injected animals and ush1c mutants have distinct defects in J.B.P. designed and performed experiments, analyzed data and wrote the hearing, balance and vision that are apparent in the first days of life. The manuscript with input from the other authors. B.B.-S. designed and performed experiments and analyzed data. J.J.L. designed and performed experiments and authors examine the sensory cells involved in these behaviors – contributed experimental reagents. A.T., K.K. and M.M. performed experiments and mechanosensory hair cells and retinal cells – and discover defects in their analyzed data. S.S., Z.G.J. and P.F.H. performed experiments. D.S.W., P.W., B.J.K. and development and function. In the ear, the structural portion of the M.W. conceived and designed the experiments. mechanosensory hair cells that are important for intercepting sound waves does not form properly. Surprisingly, the authors discover that, in the retina, SUPPLEMENTARY MATERIAL Supplementary material for this article is available at the ush1c gene functions in Müller glia rather than in photoreceptors, the cell http://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.006429/-/DC1 type that has been reported to express Ush1c in mammals. Subsequently, they show that this gene is also expressed in mammalian Müller glia. Importantly, REFERENCES although zebrafish ush1c is not active in photoreceptors, loss of ush1c function Adato, A., Michel, V., Kikkawa, Y., Reiners, J., Alagramam, K. N., Weil, D., in Müller cells leads to compromised photoreceptor function and cell death. Yonekawa, H., Wolfrum, U., El-Amraoui, A. and Petit, C. (2005). Interactions in the network of Usher syndrome type 1 proteins. Hum. Mol. Genet. 14, 347-356. Implications and future directions Allwardt, B. A., Lall, A. B., Brockerhoff, S. E. and Dowling, J. E. (2001). Synapse nrc J. Further insights into the role of ush1c in the development and function of formation is arrested in retinal photoreceptors of the zebrafish mutant. Neurosci. 21, 2330-2342. sensory cells are vital to providing early diagnosis and intervention to forestall
Disease Models & Mechanisms Bahloul, A., Michel, V., Hardelin, J.-P., Nouaille, S., Hoos, S., Houdusse, A., England, the devastating effects of this disease. This work contributes to the P. and Petit, C. (2010). Cadherin-23, myosin VIIa and harmonin, encoded by Usher understanding of USH1C pathology by providing information about the syndrome type 1 genes, form a ternary complex and interact with membrane specific molecules involved in the formation of stereocilia in mechanosensory phospholipids. Hum. Mol. Genet. 19, 3557-3565. hair cells, and by identifying a new retinal cell type through which ush1c Bever, M. M. and Fekete, D. M. (1999). Ventromedial focus of cell death is absent functions to facilitate and maintain synaptic connections between during development of Xenopus and zebrafish inner ears. J. Neurocytol. 28, 81-93. photoreceptor cells and interneurons of the retina, namely Müller glia. USH Bilotta, J., Saszik, S. and Sutherland, S. E. (2001). Rod contributions to the electroretinogram of the dark-adapted developing zebrafish. Dev. Dyn. 222, 564-570. has traditionally not been considered a developmental disorder of vision. The Boughman, J. A., Vernon, M. and Shaver, K. A. (1983). Usher syndrome: definition results of this study, however, show that visual defects in the zebrafish model and estimate of prevalence from two high-risk populations. J. Chronic Dis. 36, 595- of USH1C can be detected from the onset of vision, and thus might have 603. clinical relevance for the early diagnosis of USH1C in humans. Brockerhoff, S. E. (2006). Measuring the optokinetic response of zebrafish larvae. Nat. Protoc. 1, 2448-2451. 2 Brown, S. D., Hardisty-Hughes, R. E. and Mburu, P. (2008). Quiet as a mouse: intensity of the unattenuated beam was 1500 W/cm . Appropriate dissecting the molecular and genetic basis of hearing. Nat. Rev. Genet. 9, 277-290. neutral density filters were inserted into the light beam to achieve Caberlotto, E., Michel, V., Foucher, I., Bahloul, A., Goodyear, R. J., Pepermans, E., accurate light stimulus intensities over a 5 log10 unit range. Three Michalski, N., Perfettini, I., Alegria-Prévot, O., Chardenoux, S. et al. (2011). Usher to five responses per intensity level were averaged and filtered for type 1G protein sans is a critical omponent of the tip-link complex, a structure controlling actin polymerization in stereocilia. Proc. Natl. Acad. Sci. USA 108, 5825- 60 Hz noise. 5830. de Melo Reis, R. A., Ventura, A. L., Schitine, C. S., de Mello, M. C. and de Mello, F. G. Isolation of the a-wave component (2008). Muller glia as an active compartment modulating nervous activity in the vertebrate retina: neurotransmitters and trophic factors. Neurochem. Res. 33, 1466- Directly after recording the b-wave (in Ringer’s solution), 1474. pharmacological compounds were applied externally to the eye Delprat, B., Michel, V., Goodyear, R., Yamasaki, Y., Michalski, N., El-Amraoui, A., using an Eppendorf pipette. The compounds used in this study were Perfettini, I., Legrain, P., Richardson, G., Hardelin, J. P. et al. (2005). Myosin XVa
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