Harmonin (Ush1c) is required in zebrafish Müller glial cells for photoreceptor synaptic development and function

Phillips, Jennifer B.; Blanco-Sánchez, Bernardo; Lentz, Jennifer J; Tallafuß, Alexandra; Khanobdee, Kornnika; Sampath, Smita; Jacobs, Zachary G.; Han, Philip F.; Mishra, Monalisa; Titus, Tom A.; Williams, David S.; Keats, Bronya J.B.; Washbourne, Philip; Westerfield, Monte

doi:10.1242/dmm.006429

Cited by 67 publications

(87 citation statements)

References 81 publications

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“…Of the eight tested, three genes (cdh23, dfna5b, and ush1c) displayed a loss of startle response directly linked to the genotype of the fish (Table 3). All three of these genes have published phenotypes from either mutants or morpholino inhibition (Busch-Nentwich et al 2004;Sollner et al 2004;Phillips et al 2011). Our observed phenotypes for cdh23 and ush1c matched the published mutant phenotypes exactly; however, our observed phenotype for dfna5b was significantly less severe than the morpholino inhibition phenotype reported in Busch-Nentwich et al (2004).…”

Section: Wwwgenomeorgsupporting

confidence: 77%

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

et al. 2015

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The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis.Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloningfree single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F 1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/ Cas9 is sixfold more efficient than other techniques. We show that the majority of published "rules" for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5 ′ end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.

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Section: Wwwgenomeorgsupporting

confidence: 77%

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

et al. 2015

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“…The harmonin (ush1c) mutant presented with defective photoreceptor function attributed to a primary defect in Müller glial cells and subsequent ribbon synapse stability and function. 84 Interestingly, PDZ domaincontaining 7 (PDZD7) encodes a ciliary protein with homology to the USH1C and USH2D proteins. Although morpholino-induced knockdown of ush2a or pdzd7a alone resulted in moderate levels of photoreceptor cell death in the retina, combined morpholino-induced partial ush2a; pdzd7a, or pdzd7a;gpr98 knockdown exacerbated photoreceptor death, consistent with the possibility of human digenic inheritance of USH-associated mutations and retinal disease modifiers in patients with USH2A.…”

Section: Retinitis Pigmentosamentioning

confidence: 99%

The zebrafish eye—a paradigm for investigating human ocular genetics

Richardson¹,

Tracey‐White²,

Webster

et al. 2016

Eye

147

162

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Although human epidemiological and genetic studies are essential to elucidate the aetiology of normal and aberrant ocular development, animal models have provided us with an understanding of the pathogenesis of multiple developmental ocular malformations. Zebrafish eye development displays in depth molecular complexity and stringent spatiotemporal regulation that incorporates developmental contributions of the surface ectoderm, neuroectoderm and head mesenchyme, similar to that seen in humans. For this reason, and due to its genetic tractability, external fertilisation, and early optical clarity, the zebrafish has become an invaluable vertebrate system to investigate human ocular development and disease. Recently, zebrafish have been at the leading edge of preclinical therapy development, with their amenability to genetic manipulation facilitating the generation of robust ocular disease models required for large-scale genetic and drug screening programmes. This review presents an overview of human and zebrafish ocular development, genetic methodologies employed for zebrafish mutagenesis, relevant models of ocular disease, and finally therapeutic approaches, which may have translational leads in the future.

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“…Finally, the zebrafish genome has been sequenced, and many human genes are conserved in zebrafish. Particularly, the zebrafish has been used as a unique vertebrate model for studying hearing and balance disorders (Nicolson et al 1998;Ernest et al 2000;Whitfield 2002;Söllner et al 2004;Kappler et al 2004;Nicolson 2005;Shen et al 2008;Gleason et al 2009;Phillips et al 2011;Yariz et al 2012), with its externally exposed hair cells of the lateral line being used for study in hair cell death and regeneration, ototoxicity, and drug screens (Chiu et al 2008;López-Schier et al 2004;Owens et al 2009;Coffin et al 2010;Buck et al 2012).…”

Section: Introductionmentioning

confidence: 99%

Early Development of Hearing in Zebrafish

DeSmidt

2013

JARO

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The zebrafish (Danio rerio) has become a valuable vertebrate model for human hearing and balance disorders because it combines powerful genetics, excellent embryology, and exceptional in vivo visualization in one organism. In this study, we investigated auditory function of zebrafish at early developmental stages using the microphonic potential method. This is the first study to report ontogeny of response of hair cells in any fish during the first week post fertilization. The right ear of each zebrafish embedded in agarose was linearly stimulated with a glass probe that was driven by a calibrated piezoelectric actuator. Using beveled micropipettes filled with standard fish saline, extracellular microphonic potentials were recorded from hair cells in the inner ear of zebrafish embryos or larvae in response to 20, 50, 100, and 200-Hz stimulation. Saccular hair cells expressing green fluorescent protein of the transgenic zebrafish from 2 to 7 days post fertilization (dpf) were visualized and quantified using confocal microscopy. The otic vesicles' areas, otoliths' areas, and saccular hair cell count and density increased linearly with age and standard body length. Microphonic responses increased monotonically with stimulus intensity, stimulus frequency, and age of zebrafish. Microphonic threshold at 200 Hz gradually decreased with zebrafish age. The increases in microphonic response and sensitivity correlate with the increases in number and density of hair cells in the saccule. These results enhance our knowledge of early development of auditory function in zebrafish and provide the control data that can be used to evaluate hearing of young zebrafish morphants or mutants.

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Harmonin (Ush1c) is required in zebrafish Müller glial cells for photoreceptor synaptic development and function

Cited by 67 publications

References 81 publications

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

The zebrafish eye—a paradigm for investigating human ocular genetics

Early Development of Hearing in Zebrafish

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