Valerie C. Fleisch

Visual Behavior in Zebrafish

The zebrafish mainly uses the sense of light to hunt for food and avoid predators. This reliance on vision necessitates the rapid development of the visual system. Therefore the early larva already exhibits a number of visually- mediated behaviors that can be used for a genetic analysis of vision. The present article is an overview of the properties of the zebrafish visual system in the context of its use for behavioral based screens.

Mutation of the bone morphogenetic protein GDF3 causes ocular and skeletal anomalies

Ocular mal-development results in heterogeneous and frequently visually disabling phenotypes that include coloboma and microphthalmia. Due to the contribution of bone morphogenetic proteins to such processes, the function of the paralogue Growth Differentiation Factor 3 was investigated. Multiple mis-sense variants were identified in patients with ocular and/or skeletal (Klippel-Feil) anomalies including one individual with heterozygous alterations in GDF3 and GDF6. These variants were characterized, individually and in combination, through integrated biochemical and zebrafish model organism analyses, demonstrating appreciable effects with western blot analyses, luciferase based reporter assays and antisense morpholino inhibition. Notably, inhibition of the zebrafish co-orthologue of GDF3 accurately recapitulates patient phenotypes. By demonstrating the pleiotropic effects of GDF3 mutation, these results extend the contribution of perturbed BMP signaling to human disease and potentially implicate multi-allelic inheritance of BMP variants in developmental disorders.

Subfunctionalization of a retinoid-binding protein provides evidence for two parallel visual cycles in the cone-dominant zebrafish retina.

In vertebrates, the absorption of a photon results in an 11-cis to all-trans isomerization of the retinylidene chromophore of cone and rod visual pigments. To sustain vision, metabolic pathways (visual cycles) have evolved that recycle all-trans-retinal back to 11-cis-retinal. The canonical visual cycle takes place in photoreceptor cells and the adjacent retinal pigment epithelium (RPE). Biochemical analyses provided evidence for the existence of an additional cone-specific visual cycle involving Müller glia cells, but none of its molecular components has yet been identified. Here we took advantage of the zebrafish retina to investigate the role of the cellular retinaldehyde-binding protein CRALBP in this process. We found that the zebrafish genome encodes two cralbp paralogs: cralbp a and cralbp b. These paralogs are differentially expressed in the retina. Cralbp a is exclusively expressed in the RPE, and Cralbp b is localized to Müller cells. We used an antisense morpholino approach to knock down each cralbp paralog. Analysis of 11-cis-retinal levels revealed that visual chromophore regeneration is diminished under both conditions. Visual performance, as assessed by electroretinography, revealed reduced light sensitivity in both Cralbp a- and Cralbp b-deficient larvae, but it was more pronounced in Cralbp b-deficient larvae. Cralbp b-deficient larvae also exhibited significant deficits in their visual behavior. Together, these data demonstrate that Cralbp expression in Müller cells is essential for cone vision, thereby providing evidence that both the canonical and the alternative visual cycle depend on the same type of retinoid-binding protein.

Investigating regeneration and functional integration of CNS neurons: lessons from zebrafish genetics and other fish species.

Zebrafish possess a robust, innate CNS regenerative ability. Combined with their genetic tractability and vertebrate CNS architecture, this ability makes zebrafish an attractive model to gain requisite knowledge for clinical CNS regeneration. In treatment of neurological disorders, one can envisage replacing lost neurons through stem cell therapy or through activation of latent stem cells in the CNS. Here we review the evidence that radial glia are a major source of CNS stem cells in zebrafish and thus activation of radial glia is an attractive therapeutic target. We discuss the regenerative potential and the molecular mechanisms thereof, in the zebrafish spinal cord, retina, optic nerve and higher brain centres. We evaluate various cell ablation paradigms developed to induce regeneration, with particular emphasis on the need for (high throughput) indicators that neuronal regeneration has restored sensory or motor function. We also examine the potential confound that regeneration imposes as the community develops zebrafish models of neurodegeneration. We conclude that zebrafish combine several characters that make them a potent resource for testing hypotheses and discovering therapeutic targets in functional CNS regeneration. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.

The zebrafish mutant lbk/vam6 resembles human multisystemic disorders caused by aberrant trafficking of endosomal vesicles

The trafficking of intracellular vesicles is essential for a number of cellular processes and defects in this process have been implicated in a wide range of human diseases. We identify the zebrafish mutant lbk as a novel model for such disorders. lbk displays hypopigmentation of skin melanocytes and the retinal pigment epithelium (RPE), an absence of iridophore reflections, defects in internal organs (liver, intestine) as well as functional defects in vision and the innate immune system (macrophages). Positional cloning, an allele screen, rescue experiments and morpholino knock-down reveal a mutation in the zebrafish orthologue of the vam6/vps39 gene. Vam6p is part of the HOPS complex, which is essential for vesicle tethering and fusion. Affected cells in the lbk RPE, liver, intestine and macrophages display increased numbers and enlarged intracellular vesicles. Physiological and behavioural analyses reveal severe defects in visual ability in lbk mutants. The present study provides the first phenotypic description of a lack of vam6 gene function in a multicellular organism. lbk shares many of the characteristics of human diseases and suggests a novel disease gene for pathologies associated with defective vesicle transport, including the arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, the Hermansky-Pudlak syndrome, the Chediak-Higashi syndrome and the Griscelli syndrome.

Targeted mutation of the gene encoding prion protein in zebrafish reveals a conserved role in neuron excitability.

The function of the cellular prion protein (PrP(C)) in healthy brains remains poorly understood, in part because Prnp knockout mice are viable. On the other hand, transient knockdown of Prnp homologs in zebrafish (including two paralogs, prp1 and prp2) has suggested that PrP(C) is required for CNS development, cell adhesion, and neuroprotection. It has been argued that zebrafish Prp2 is most similar to mammalian PrP(C), yet it has remained intransigent to the most thorough confirmations of reagent specificity during knockdown. Thus we investigated the role of prp2 using targeted gene disruption via zinc finger nucleases. Prp2(-/-) zebrafish were viable and did not display overt developmental phenotypes. Back-crossing female prp2(-/-) fish ruled out a role for maternal mRNA contributions. Prp2(-/-) larvae were found to have increased seizure-like behavior following exposure to the convulsant pentylenetetrazol (PTZ), as compared to wild type fish. In situ recordings from intact hindbrains demonstrated that prp2 regulates closing of N-Methyl-d-aspartate (NMDA) receptors, concomitant with neuroprotection during glutamate excitotoxicity. Overall, the knockout of Prp2 function in zebrafish independently confirmed hypothesized roles for PrP, identifying deeply conserved functions in post-developmental regulation of neuron excitability that are consequential to the etiology of prion and Alzheimer diseases.

Parallel visual cycles in the zebrafish retina.

Vertebrate vision necessitates continuous recycling of the chromophore 11-cis retinal (RAL). The classical (or canonical) visual cycle employs a number of enzymes located in the photoreceptor outer segment and RPE (retinal pigment epithelium) of the retina to regenerate 11-cis RAL from all-trans RAL. Cone-dominant species are believed to utilize a second, intra-retinal, pathway for 11-cis RAL generation, involving retinal Müller glia cells. This review summarizes the efforts made in zebrafish to gain a better understanding of the role of these two visual cycles for rod and cone photoreceptor chromophore recycling.

Evaluating the Mutagenic Activity of Targeted Endonucleases Containing a Sharkey FokI Cleavage Domain Variant in Zebrafish

Synthetic targeted endonucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have recently emerged as powerful tools for targeted mutagenesis, especially in organisms that are not amenable to embryonic stem cell manipulation. Both ZFNs and TALENs consist of DNA-binding arrays that are fused to the nonspecific FokI nuclease domain. In an effort to improve targeted endonuclease mutagenesis efficiency, we enhanced their catalytic activity using the Sharkey FokI nuclease domain variant. All constructs tested display increased DNA cleavage activity in vitro. We demonstrate that one out of four ZFN arrays containing the Sharkey FokI variant exhibits a dramatic increase in mutagenesis frequency in vivo in zebrafish. The other three ZFNs exhibit no significant alteration of activity in vivo. Conversely, we demonstrate that TALENs containing the Sharkey FokI variant exhibit absent or severely reduced in vivo mutagenic activity in zebrafish. Notably, Sharkey ZFNs and TALENs do not generate increased toxicity-related defects or mortality. Our results present Sharkey ZFNs as an effective alternative to conventional ZFNs, but advise against the use of Sharkey TALENs.

Electroretinogram (ERG) Measurements in Larval Zebrafish

INTRODUCTIONThe electroretinogram (ERG) is an electrophysiological tool used to measure electrical activity originating in the outer retina in response to a light stimulus. Defects occurring at various levels of the retina can easily be detected by ERG measurements. Furthermore, the shape of the ERG response points toward the likely retinal cell type responsible for the deficit. Thus, this method is particularly useful for a rapid assessment of retinal function in genetically or pharmacologically manipulated animals. A typical ERG curve can be subdivided into three components: a small initial a-wave originating in photoreceptor activity, a large positive b-wave reflecting mainly ON bipolar cell depolarization, and a d-wave occurring at light offset. Here we present a noninvasive protocol for taking ERG measurements in larval zebrafish (4-7 days post-fertilization [dpf]). We use an extracellular recording electrode which is placed onto the surface of the cornea of the larva, and a light flash of a defined intensity and duration which is applied to evoke a response. In a typical larval ERG trace, we are able to record ERG a-, b-, and d-waves.