FRaeppli: a multispectral imaging toolbox for cell tracing and dense tissue analysis in zebrafish.

Visualizing cell shapes and interactions of differentiating cells is instrumental for understanding organ development and repair. Across species, strategies for stochastic multicolour labelling have greatly facilitated in vivo cell tracking and mapping neuronal connectivity. Yet integrating multi-fluorophore information into the context of developing zebrafish tissues is challenging given their cytoplasmic localization and spectral incompatibility with common fluorescent markers. Inspired by Drosophila Raeppli, we developed FRaeppli (Fish-Raeppli) by expressing bright membrane- or nuclear-targeted fluorescent proteins for efficient cell shape analysis and tracking. High spatiotemporal activation flexibility is provided by the Gal4/UAS system together with Cre/lox and/or PhiC31 integrase. The distinct spectra of the FRaeppli fluorescent proteins allow simultaneous imaging with GFP and infrared subcellular reporters or tissue landmarks. We demonstrate the suitability of FRaeppli for live imaging of complex internal organs, such as the liver, and have tailored hyperspectral protocols for time-efficient acquisition. Combining FRaeppli with polarity markers revealed previously unknown canalicular topologies between differentiating hepatocytes, reminiscent of the mammalian liver, suggesting common developmental mechanisms. The multispectral FRaeppli toolbox thus enables the comprehensive analysis of intricate cellular morphologies, topologies and lineages at single-cell resolution in zebrafish.

Specialization of the photoreceptor transcriptome by Srrm3-dependent microexons is required for outer segment maintenance and vision.

Retinal photoreceptors have a distinct transcriptomic profile compared to other neuronal subtypes, likely reflecting their unique cellular morphology and function in the detection of light stimuli by way of the ciliary outer segment. We discovered a layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. A subset of these microexons is included exclusively in photoreceptor transcripts, particularly in genes involved in cilia biogenesis and vesicle-mediated transport. This microexon program is regulated by Srrm3, a paralog of the neural microexon regulator Srrm4. Despite the fact that both proteins positively regulate retina microexons in vitro, only Srrm3 is highly expressed in mature photoreceptors. Its deletion in zebrafish results in widespread down-regulation of microexon inclusion from early developmental stages, followed by other transcriptomic alterations, severe photoreceptor defects, and blindness. These results shed light on the transcriptomic specialization and functionality of photoreceptors, uncovering unique cell type-specific roles for Srrm3 and microexons with implications for retinal diseases.

Targeting autophagy impairment improves the phenotype of a novel CLN8 zebrafish model.

CLN8 is an endoplasmic reticulum cargo receptor and a regulator of lysosome biogenesis whose loss of function leads to neuronal ceroid lipofuscinosis. CLN8 has been linked to autophagy and lipid metabolism, but much remains to be learned, and there are no therapies acting on the molecular signatures in this disorder. The present study aims to characterize the molecular pathways involved in CLN8 disease and, by pinpointing altered ones, to identify potential therapies. To bridge the gap between cell and mammalian models, we generated a new zebrafish model of CLN8 deficiency, which recapitulates the pathological features of the disease. We observed, for the first time, that CLN8 dysfunction impairs autophagy. Using autophagy modulators, we showed that trehalose and SG2 are able to attenuate the pathological phenotype in mutant larvae, confirming autophagy impairment as a secondary event in disease progression. Overall, our successful modeling of CLN8 defects in zebrafish highlights this novel in vivo model's strong potential as an instrument for exploring the role of CLN8 dysfunction in cellular pathways, with a view to identifying small molecules to treat this rare disease.

Loss of glutamate transporter eaat2a leads to aberrant neuronal excitability, recurrent epileptic seizures, and basal hypoactivity.

Astroglial excitatory amino acid transporter 2 (EAAT2, GLT-1, and SLC1A2) regulates the duration and extent of neuronal excitation by removing glutamate from the synaptic cleft. Hence, an impairment in EAAT2 function could lead to an imbalanced brain network excitability. Here, we investigated the functional alterations of neuronal and astroglial networks associated with the loss of function in the astroglia predominant eaat2a gene in zebrafish. We observed that eaat2a-/- mutant zebrafish larvae display recurrent spontaneous and light-induced seizures in neurons and astroglia, which coincide with an abrupt increase in extracellular glutamate levels. In stark contrast to this hyperexcitability, basal neuronal and astroglial activity was surprisingly reduced in eaat2a-/- mutant animals, which manifested in decreased overall locomotion. Our results reveal an essential and mechanistic contribution of EAAT2a in balancing brain excitability, and its direct link to epileptic seizures.

Elevated photic response is followed by a rapid decay and depressed state in ictogenic networks.

OBJECTIVE: The switch between nonseizure and seizure states involves profound alterations in network excitability and synchrony. In this study, we aimed to identify and compare features of neural excitability and dynamics across multiple zebrafish seizure and epilepsy models. METHODS: Inspired by video-electroencephalographic recordings in patients, we developed a framework to study spontaneous and photically evoked neural and locomotor activity in zebrafish larvae, by combining high-throughput behavioral tracking and whole-brain in vivo two-photon calcium imaging. RESULTS: Our setup allowed us to dissect behavioral and physiological features that are divergent or convergent across multiple models. We observed that spontaneous locomotor and neural activity exhibit great diversity across models. Nonetheless, during photic stimulation, hyperexcitability and rapid response dynamics were well conserved across multiple models, highlighting the reliability of photically evoked activity for high-throughput assays. Intriguingly, in several models, we observed that the initial elevated photic response is often followed by rapid decay of neural activity and a prominent depressed state. Elevated photic response and following depressed state in seizure-prone networks are significantly reduced by the antiseizure medication valproic acid. Finally, rapid decay and depression of neural activity following photic stimulation temporally overlap with slow recruitment of astroglial calcium signals that are enhanced in seizure-prone networks. SIGNIFICANCE: We argue that fast decay of neural activity and depressed states following photic response are likely due to homeostatic mechanisms triggered by excessive neural activity. An improved understanding of the interplay between elevated and depressed excitability states might suggest tailored epilepsy therapies.

Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations.

Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G > T, both reside in exon 13. Skipping exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2armc1 mutants resulted in the production of usherinΔexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in induced pluripotent stem cell (iPSC)-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection and induced a detectable level of exon skipping until at least 6 months post-injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.

Biochemistry and physiology of zebrafish photoreceptors.

All vertebrates share a canonical retina with light-sensitive photoreceptors in the outer retina. These photoreceptors are of two kinds: rods and cones, adapted to low and bright light conditions, respectively. They both show a peculiar morphology, with long outer segments, comprised of ordered stacks of disc-shaped membranes. These discs host numerous proteins, many of which contribute to the visual transduction cascade. This pathway converts the light stimulus into a biological signal, ultimately modulating synaptic transmission. Recently, the zebrafish (Danio rerio) has gained popularity for studying the function of vertebrate photoreceptors. In this review, we introduce this model system and its contribution to our understanding of photoreception with a focus on the cone visual transduction cascade.

Transgenic zebrafish modeling low-molecular-weight proteinuria and lysosomal storage diseases.

Epithelial cells lining the proximal tubule of the kidney reabsorb and metabolize most of the filtered low-molecular-weight proteins through receptor-mediated endocytosis and lysosomal processing. Congenital and acquired dysfunctions of the proximal tubule are consistently reflected by the inappropriate loss of solutes including low-molecular-weight proteins in the urine. The zebrafish pronephros shares individual functional segments with the human nephron, including lrp2a/megalin-dependent endocytic transport processes of the proximal tubule. Although the zebrafish has been used as a model organism for toxicological studies and drug discovery, there is no available assay that allows large-scale assessment of proximal tubule function in larval or adult stages. Here we establish a transgenic Tg(lfabp::½vdbp-mCherry) zebrafish line expressing in the liver the N-terminal region of vitamin D-binding protein coupled to the acid-insensitive, red monomeric fluorescent protein mCherry (½vdbp-mCherry). This low-molecular-weight protein construct is secreted into the bloodstream, filtered through the glomerulus, reabsorbed by receptor-mediated endocytosis and processed in the lysosomes of proximal tubule cells of the fish. Thus, our proof-of-concept studies using zebrafish larvae knockout for lrp2a and clcn7 or exposed to known nephrotoxins (gentamicin and cisplatin) demonstrate that this transgenic line is useful to monitor low-molecular-weight proteinuria and lysosomal processing. This represents a powerful new model organism for drug screening and studies of nephrotoxicity.

Mutations in ARMC9, which Encodes a Basal Body Protein, Cause Joubert Syndrome in Humans and Ciliopathy Phenotypes in Zebrafish.

Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by hypotonia, ataxia, abnormal eye movements, and variable cognitive impairment. It is defined by a distinctive brain malformation known as the "molar tooth sign" on axial MRI. Subsets of affected individuals have malformations such as coloboma, polydactyly, and encephalocele, as well as progressive retinal dystrophy, fibrocystic kidney disease, and liver fibrosis. More than 35 genes have been associated with JS, but in a subset of families the genetic cause remains unknown. All of the gene products localize in and around the primary cilium, making JS a canonical ciliopathy. Ciliopathies are unified by their overlapping clinical features and underlying mechanisms involving ciliary dysfunction. In this work, we identify biallelic rare, predicted-deleterious ARMC9 variants (stop-gain, missense, splice-site, and single-exon deletion) in 11 individuals with JS from 8 families, accounting for approximately 1% of the disorder. The associated phenotypes range from isolated neurological involvement to JS with retinal dystrophy, additional brain abnormalities (e.g., heterotopia, Dandy-Walker malformation), pituitary insufficiency, and/or synpolydactyly. We show that ARMC9 localizes to the basal body of the cilium and is upregulated during ciliogenesis. Typical ciliopathy phenotypes (curved body shape, retinal dystrophy, coloboma, and decreased cilia) in a CRISPR/Cas9-engineered zebrafish mutant model provide additional support for ARMC9 as a ciliopathy-associated gene. Identifying ARMC9 mutations as a cause of JS takes us one step closer to a full genetic understanding of this important disorder and enables future functional work to define the central biological mechanisms underlying JS and other ciliopathies.

Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors.

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming ciliary-directed vesicles, through organization of the vesicle fusion machinery at the periciliary membrane.

Differential expression of PKCα and -ß in the zebrafish retina.

The retina is a complex neural circuit, which processes and transmits visual information from light perceiving photoreceptors to projecting retinal ganglion cells. Much of the computational power of the retina rests on signal integrating interneurons, such as bipolar cells. Commercially available antibodies against bovine and human conventional protein kinase C (PKC) α and -ß are frequently used as markers for retinal ON-bipolar cells in different species, despite the fact that it is not known which bipolar cell subtype(s) they actually label. In zebrafish (Danio rerio) five prkc genes (coding for PKC proteins) have been identified. Their expression has not been systematically determined. While prkcg is not expressed in retinal tissue, the other four prkc (prkcaa, prkcab, prkcba, prkcbb) transcripts were found in different parts of the inner nuclear layer and some as well in the retinal ganglion cell layer. Immunohistochemical analysis in adult zebrafish retina using fluorescent in situ hybridization and PKC antibodies showed an overlapping immunolabeling of ON-bipolar cells that are most likely of the BON s6 and BON s6L or RRod type. However, comparison of transcript expression with immunolabeling, implies that these antibodies are not specific for one single zebrafish conventional PKC, but rather detect a combination of PKC -α and -ß variants.

Usherin defects lead to early-onset retinal dysfunction in zebrafish.

Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2armc1: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2ab1245: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2armc1, but presence of the ectodomain of usherin at the periciliary membrane of ush2ab1245-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2armc1 as well as ush2ab1245 larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies.

MiR-204 is responsible for inherited retinal dystrophy associated with ocular coloboma.

Ocular developmental disorders, including the group classified as microphthalmia, anophthalmia, and coloboma (MAC) and inherited retinal dystrophies, collectively represent leading causes of hereditary blindness. Characterized by extreme genetic and clinical heterogeneity, the separate groups share many common genetic causes, in particular relating to pathways controlling retinal and retinal pigment epithelial maintenance. To understand these shared pathways and delineate the overlap between these groups, we investigated the genetic cause of an autosomal dominantly inherited condition of retinal dystrophy and bilateral coloboma, present in varying degrees in a large, five-generation family. By linkage analysis and exome sequencing, we identified a previously undescribed heterozygous mutation, n.37 C > T, in the seed region of microRNA-204 (miR-204), which segregates with the disease in all affected individuals. We demonstrated that this mutation determines significant alterations of miR-204 targeting capabilities via in vitro assays, including transcriptome analysis. In vivo injection, in medaka fish (Oryzias latipes), of the mutated miR-204 caused a phenotype consistent with that observed in the family, including photoreceptor alterations with reduced numbers of both cones and rods as a result of increased apoptosis, thereby confirming the pathogenic effect of the n.37 C > T mutation. Finally, knockdown assays in medaka fish demonstrated that miR-204 is necessary for normal photoreceptor function. Overall, these data highlight the importance of miR-204 in the regulation of ocular development and maintenance and provide the first evidence, to our knowledge, of its contribution to eye disease, likely through a gain-of-function mechanism.

The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking.

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment.

Comparative transcriptomic analysis identifies evolutionarily conserved gene products in the vertebrate renal distal convoluted tubule.

Understanding the molecular basis of the complex regulatory networks controlling renal ion transports is of major physiological and clinical importance. In this study, we aimed to identify evolutionarily conserved critical players in the function of the renal distal convoluted tubule (DCT) by a comparative transcriptomic approach. We generated a transgenic zebrafish line with expression of the red fluorescent mCherry protein under the control of the zebrafish DCT-specific promoter of the thiazide-sensitive NaCl cotransporter (NCC). The mCherry expression was then used to isolate from the zebrafish mesonephric kidneys the distal late (DL) segments, the equivalent of the mammalian DCT, for subsequent RNA-seq analysis. We next compared this zebrafish DL transcriptome to the previously established mouse DCT transcriptome and identified a subset of gene products significantly enriched in both the teleost DL and the mammalian DCT, including SLCs and nuclear transcription factors. Surprisingly, several of the previously described regulators of NCC (e.g., SPAK, KLHL3, ppp1r1a) in the mouse were not found enriched in the zebrafish DL. Nevertheless, the zebrafish DL expressed enriched levels of related homologues. Functional knockdown of one of these genes, ppp1r1b, reduced the phosphorylation of NCC in the zebrafish pronephros, similar to what was seen previously in knockout mice for its homologue, Ppp1r1a. The present work is the first report on global gene expression profiling in a specific nephron portion of the zebrafish kidney, an increasingly used model system for kidney research. Our study suggests that comparative analysis of gene expression between phylogenetically distant species may be an effective approach to identify novel regulators of renal function.

Mutations in CSPP1 cause primary cilia abnormalities and Joubert syndrome with or without Jeune asphyxiating thoracic dystrophy.

Joubert syndrome (JBTS) is a recessive ciliopathy in which a subset of affected individuals also have the skeletal dysplasia Jeune asphyxiating thoracic dystrophy (JATD). Here, we have identified biallelic truncating CSPP1 (centrosome and spindle pole associated protein 1) mutations in 19 JBTS-affected individuals, four of whom also have features of JATD. CSPP1 mutations explain ∼5% of JBTS in our cohort, and despite truncating mutations in all affected individuals, the range of phenotypic severity is broad. Morpholino knockdown of cspp1 in zebrafish caused phenotypes reported in other zebrafish models of JBTS (curved body shape, pronephric cysts, and cerebellar abnormalities) and reduced ciliary localization of Arl13b, further supporting loss of CSPP1 function as a cause of JBTS. Fibroblasts from affected individuals with CSPP1 mutations showed reduced numbers of primary cilia and/or short primary cilia, as well as reduced axonemal localization of ciliary proteins ARL13B and adenylyl cyclase III. In summary, CSPP1 mutations are a major cause of the Joubert-Jeune phenotype in humans; however, the mechanism by which these mutations lead to both JBTS and JATD remains unknown.

Genetic approaches to retinal research in zebrafish.

The zebrafish (Danio rerio) possesses a vertebrate-type retina that is extraordinarily conserved in evolution. This well-organized and anatomically easily accessible part of the central nervous system has been widely investigated in zebrafish, promoting general understanding of retinal development, morphology, function and associated diseases. Over the recent years, genome and protein engineering as well as imaging techniques have experienced revolutionary advances and innovations, creating new possibilities and methods to study zebrafish development and function. In this review, we focus on some of these emerging technologies and how they may impact retinal research in the future. We place an emphasis on genetic techniques, such as transgenic approaches and the revolutionizing new possibilities in genome editing.

Important role of endocannabinoid signaling in the development of functional vision and locomotion in zebrafish.

The developmental role of the endocannabinoid system still remains to be fully understood. Here, we report the presence of a complete endocannabinoid system during zebrafish development and show that the genes that code for enzymes that catalyze the anabolism and catabolism (mgll and dagla) of the endocannabinoid, 2-AG (2-arachidonoylglycerol), as well as 2-AG main receptor in the brain, cannabinoid receptor type 1, are coexpressed in defined regions of axonal growth. By using morpholino-induced transient knockdown of the zebrafish Daglα homolog and its pharmacologic rescue, we suggest that synthesis of 2-AG is implicated in the control of axon formation in the midbrain-hindbrain region and that animals that lack Daglα display abnormal physiological behaviors in tests that measure stereotyped movement and motion perception. Our results suggest that the well-established role for 2-AG in axonal outgrowth has implications for the control of vision and movement in zebrafish and, thus, is likely common to all vertebrates.-Martella, A., Sepe, R. M., Silvestri, C., Zang, J., Fasano, G., Carnevali, O., De Girolamo, P., Neuhauss, S. C. F., Sordino, P., Di Marzo, V. Important role of endocannabinoid signaling in the development of functional vision and locomotion in zebrafish.

Zebrafish Models for the Mechanosensory Hair Cell Dysfunction in Usher Syndrome 3 Reveal That Clarin-1 Is an Essential Hair Bundle Protein.

Usher syndrome type III (USH3) is characterized by progressive loss of hearing and vision, and varying degrees of vestibular dysfunction. It is caused by mutations that affect the human clarin-1 protein (hCLRN1), a member of the tetraspanin protein family. The missense mutation CLRN1(N48K), which affects a conserved N-glycosylation site in hCLRN1, is a common causative USH3 mutation among Ashkenazi Jews. The affected individuals hear at birth but lose that function over time. Here, we developed an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype. Immunolabeling demonstrated that Clrn1 localized to the hair cell bundles (hair bundles). The clrn1 mutants generated by zinc finger nucleases displayed aberrant hair bundle morphology with diminished function. Two transgenic zebrafish that express either hCLRN1 or hCLRN1(N48K) in hair cells were produced to examine the subcellular localization patterns of wild-type and mutant human proteins. hCLRN1 localized to the hair bundles similarly to zebrafish Clrn1; in contrast, hCLRN1(N48K) largely mislocalized to the cell body with a small amount reaching the hair bundle. We propose that this small amount of hCLRN1(N48K) in the hair bundle provides clarin-1-mediated function during the early stages of life; however, the presence of hCLRN1(N48K) in the hair bundle diminishes over time because of intracellular degradation of the mutant protein, leading to progressive loss of hair bundle integrity and hair cell function. These findings and genetic tools provide an understanding and path forward to identify therapies to mitigate hearing loss linked to the CLRN1 mutation. SIGNIFICANCE STATEMENT: Mutations in the clarin-1 gene affect eye and ear function in humans. Individuals with the CLRN1(N48K) mutation are born able to hear but lose that function over time. Here, we develop an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype. This approach illuminates the role of clarin-1 and the molecular mechanism linked to the CLRN1(N48K) mutation in sensory hair cells of the inner ear. Additionally, the investigation provided an in vivo model to guide future drug discovery to rescue the hCLRN1(N48K) in hair cells.

Megalencephalic leukoencephalopathy with subcortical cysts protein 1 regulates glial surface localization of GLIALCAM from fish to humans.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by myelin vacuolization and caused by mutations in MLC1 or GLIALCAM. Patients with recessive mutations in either MLC1 or GLIALCAM show the same clinical phenotype. It has been shown that GLIALCAM is necessary for the correct targeting of MLC1 to the membrane at cell junctions, but its own localization was independent of MLC1 in vitro. However, recent studies in Mlc1(-/-) mice have shown that GlialCAM is mislocalized in glial cells. In order to investigate whether the relationship between Mlc1 and GlialCAM is species-specific, we first identified MLC-related genes in zebrafish and generated an mlc1(-/-) zebrafish. We have characterized mlc1(-/-) zebrafish both functionally and histologically and compared the phenotype with that of the Mlc1(-/-) mice. In mlc1(-/-) zebrafish, as in Mlc1(-/-) mice, Glialcam is mislocalized. Re-examination of a brain biopsy from an MLC patient indicates that GLIALCAM is also mislocalized in Bergmann glia in the cerebellum. In vitro, impaired localization of GlialCAM was observed in astrocyte cultures from Mlc1(-/-) mouse only in the presence of elevated potassium levels, which mimics neuronal activity. In summary, here we demonstrate an evolutionary conserved role for MLC1 in regulating glial surface levels of GLIALCAM, and this interrelationship explains why patients with mutations in either gene (MLC1 or GLIALCAM) share the same clinical phenotype.