Drug screening with zebrafish visual behavior identifies carvedilol as a potential treatment for an autosomal dominant form of retinitis pigmentosa.

Retinitis Pigmentosa (RP) is a mostly incurable inherited retinal degeneration affecting approximately 1 in 4000 individuals globally. The goal of this work was to identify drugs that can help patients suffering from the disease. To accomplish this, we screened drugs on a zebrafish autosomal dominant RP model. This model expresses a truncated human rhodopsin transgene (Q344X) causing significant rod degeneration by 7 days post-fertilization (dpf). Consequently, the larvae displayed a deficit in visual motor response (VMR) under scotopic condition. The diminished VMR was leveraged to screen an ENZO SCREEN-WELL REDOX library since oxidative stress is postulated to play a role in RP progression. Our screening identified a beta-blocker, carvedilol, that ameliorated the deficient VMR of the RP larvae and increased their rod number. Carvedilol may directly on rods as it affected the adrenergic pathway in the photoreceptor-like human Y79 cell line. Since carvedilol is an FDA-approved drug, our findings suggest that carvedilol can potentially be repurposed to treat autosomal dominant RP patients.

Rods Contribute to Visual Behavior in Larval Zebrafish.

Purpose: Although zebrafish rods begin to develop as early as 2 days postfertilization (dpf), they are not deemed anatomically mature and functional until 15 to 21 dpf. A recent study detected a small electroretinogram (ERG) from rods in a cone mutant called no optokinetic response f (nof) at 5 dpf, suggesting that young rods are functional. Whether they can mediate behavioral responses in larvae is unknown. Methods: We first confirmed rod function by measuring nof ERGs under photopic and scotopic illumination at 6 dpf. We evaluated the role of rods in visual behaviors using two different assays: the visual-motor response (VMR) and optokinetic response (OKR). We measured responses from wild-type (WT) larvae and nof mutants under photopic and scotopic illuminations at 6 dpf. Results: Nof mutants lacked a photopic ERG. However, after prolonged dark adaptation, they displayed scotopic ERGs. Compared with WT larvae, the nof mutants displayed reduced VMRs. The VMR difference during light onset gradually diminished with decreased illumination and became nearly identical at lower light intensities. Additionally, light-adapted nof mutants did not display an OKR, whereas dark-adapted nof mutants displayed scotopic OKRs. Conclusions: Because the nof mutants lacked a photopic ERG but displayed scotopic ERGs after dark adaptation, the mutants clearly had functional rods. WT larvae and the nof mutants displayed comparable scotopic light-On VMRs and scotopic OKRs after dark adaptation, suggesting that these responses were driven primarily by rods. Together, these observations indicate that rods contribute to zebrafish visual behaviors as early as 6 dpf.

A critical evaluation of TRPA1-mediated locomotor behavior in zebrafish as a screening tool for novel anti-nociceptive drug discovery.

Current medications inadequately treat the symptoms of chronic pain experienced by over 50 million people in the United States, and may come with substantial adverse effects signifying the need to find novel treatments. One novel therapeutic target is the Transient Receptor Potential A1 channel (TRPA1), an ion channel that mediates nociception through calcium influx of sensory neurons. Drug discovery still relies heavily on animal models, including zebrafish, a species in which TRPA1 activation produces hyperlocomotion. Here, we investigated if this hyperlocomotion follows zebrafish TRPA1 pharmacology and evaluated the strengths and limitations of using TRPA1-mediated hyperlocomotion as potential preclinical screening tool for drug discovery. To support face validity of the model, we pharmacologically characterized mouse and zebrafish TRPA1 in transfected HEK293 cells using calcium assays as well as in vivo. TRPA1 agonists and antagonists respectively activated or blocked TRPA1 activity in HEK293 cells, mice, and zebrafish in a dose-dependent manner. However, our results revealed complexities including partial agonist activity of TRPA1 antagonists, bidirectional locomotor activity, receptor desensitization, and off-target effects. We propose that TRPA1-mediated hyperlocomotion in zebrafish larvae has the potential to be used as in vivo screening tool for novel anti-nociceptive drugs but requires careful evaluation of the TRPA1 pharmacology.

Normalization of large-scale behavioural data collected from zebrafish.

Many contemporary neuroscience experiments utilize high-throughput approaches to simultaneously collect behavioural data from many animals. The resulting data are often complex in structure and are subjected to systematic biases, which require new approaches for analysis and normalization. This study addressed the normalization need by establishing an approach based on linear-regression modeling. The model was established using a dataset of visual motor response (VMR) obtained from several strains of wild-type (WT) zebrafish collected at multiple stages of development. The VMR is a locomotor response triggered by drastic light change, and is commonly measured repeatedly from multiple larvae arrayed in 96-well plates. This assay is subjected to several systematic variations. For example, the light emitted by the machine varies slightly from well to well. In addition to the light-intensity variation, biological replication also created batch-batch variation. These systematic variations may result in differences in the VMR and must be normalized. Our normalization approach explicitly modeled the effect of these systematic variations on VMR. It also normalized the activity profiles of different conditions to a common baseline. Our approach is versatile, as it can incorporate different normalization needs as separate factors. The versatility was demonstrated by an integrated normalization of three factors: light-intensity variation, batch-batch variation and baseline. After normalization, new biological insights were revealed from the data. For example, we found larvae of TL strain at 6 days post-fertilization (dpf) responded to light onset much stronger than the 9-dpf larvae, whereas previous analysis without normalization shows that their responses were relatively comparable. By removing systematic variations, our model-based normalization can facilitate downstream statistical comparisons and aid detecting true biological differences in high-throughput studies of neurobehaviour.

nox2/cybb Deficiency Affects Zebrafish Retinotectal Connectivity.

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) have been linked to neuronal polarity, axonal outgrowth, cerebellar development, regeneration of sensory axons, and neuroplasticity. However, the specific roles that individual Nox isoforms play during nervous system development in vivo remain unclear. To address this problem, we investigated the role of Nox activity in the development of retinotectal connections in zebrafish embryos. Zebrafish broadly express four nox genes (nox1, nox2/cybb, nox5, and duox) throughout the CNS during early development. Application of a pan-Nox inhibitor, celastrol, during the time of optic nerve (ON) outgrowth resulted in significant expansion of the ganglion cell layer (GCL), thinning of the ON, and a decrease in retinal axons reaching the optic tectum (OT). With the exception of GCL expansion, these effects were partially ameliorated by the addition of H2O2, a key ROS involved in Nox signaling. To address isoform-specific Nox functions, we used CRISPR/Cas9 to generate mutations in each zebrafish nox gene. We found that nox2/cybb chimeric mutants displayed ON thinning and decreased OT innervation. Furthermore, nox2/cybb homozygous mutants (nox2/cybb-/-) showed significant GCL expansion and mistargeted retinal axons in the OT. Neurite outgrowth from cultured zebrafish retinal ganglion cells was reduced by Nox inhibitors, suggesting a cell-autonomous role for Nox in these neurons. Collectively, our results show that Nox2/Cybb is important for retinotectal development in zebrafish.SIGNIFICANCE STATEMENT Most isoforms of NADPH oxidase (Nox) only produce reactive oxygen species (ROS) when activated by an upstream signal, making them ideal candidates for ROS signaling. Nox enzymes are present in neurons and their activity has been shown to be important for neuronal development and function largely by in vitro studies. However, whether Nox is involved in the development of axons and formation of neuronal connections in vivo has remained unclear. Using mutant zebrafish embryos, this study shows that a specific Nox isoform, Nox2/Cybb, is important for the establishment of axonal connections between retinal ganglion cells and the optic tectum.

Mutation of IPO13 causes recessive ocular coloboma, microphthalmia, and cataract.

Ocular coloboma is a developmental structural defect of the eye that often occurs as complex ocular anomalies. However, its genetic etiology remains largely unexplored. Here we report the identification of mutation (c.331C>T, p.R111C) in the IPO13 gene in a consanguineous family with ocular coloboma, microphthalmia, and cataract by a combination of whole-exome sequencing and homozygosity mapping. IPO13 encodes an importin-B family protein and has been proven to be associated with the pathogenesis of coloboma and microphthalmia. We found that Ipo13 was expressed in the cornea, sclera, lens, and retina in mice. Additionally, the mRNA expression level of Ipo13 decreased significantly in the patient compared with its expression in a healthy individual. Morpholino-oligonucleotide-induced knockdown of ipo13 in zebrafish caused dose-dependent microphthalmia and coloboma, which is highly similar to the ocular phenotypes in the patient. Moreover, both visual motor response and optokinetic response were impaired severely. Notably, these ocular phenotypes in ipo13-deficient zebrafish could be rescued remarkably by full-length ipo13 mRNA, suggesting that the phenotypes observed in zebrafish were due to insufficient ipo13 function. Altogether, our findings demonstrate, for the first time, a new role of IPO13 in eye morphogenesis and that loss of function of IPO13 could lead to ocular coloboma, microphthalmia, and cataract in humans and zebrafish.

Expression profiling of the retina of pde6c, a zebrafish model of retinal degeneration.

Retinal degeneration often affects the whole retina even though the disease-causing gene is specifically expressed in the light-sensitive photoreceptors. The molecular basis of the retinal defect can potentially be determined by gene-expression profiling of the whole retina. In this study, we measured the gene-expression profile of retinas microdissected from a zebrafish pde6cw59 (pde6c) mutant. This retinal-degeneration model not only displays cone degeneration caused by a cone-specific mutation, but also other secondary cellular changes starting from 4 days postfertilization (dpf). To capture the underlying molecular changes, we subjected pde6c and wild-type (WT) retinas at 5 dpf/ 120 h postfertilization (hpf) to RNA sequencing (RNA-Seq) on the Illumina HiSeq 2,000 platform. We also validated the RNA-Seq results by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) of seven phototransduction genes. Our analyses indicate that the RNA-Seq dataset was of high quality, and effectively captured the molecular changes in the whole pde6c retina. This dataset will facilitate the characterization of the molecular defects in the pde6c retina at the initial stage of retinal degeneration.

Statistical Analysis of Zebrafish Locomotor Behaviour by Generalized Linear Mixed Models.

Upon a drastic change in environmental illumination, zebrafish larvae display a rapid locomotor response. This response can be simultaneously tracked from larvae arranged in multi-well plates. The resulting data have provided new insights into neuro-behaviour. The features of these data, however, present a challenge to traditional statistical tests. For example, many larvae display little or no movement. Thus, the larval responses have many zero values and are imbalanced. These responses are also measured repeatedly from the same well, which results in correlated observations. These analytical issues were addressed in this study by the generalized linear mixed model (GLMM). This approach deals with binary responses and characterizes the correlation of observations in the same group. It was used to analyze a previously reported dataset. Before applying the GLMM, the activity values were transformed to binary responses (movement vs. no movement) to reduce data imbalance. Moreover, the GLMM estimated the variations among the effects of different well locations, which would eliminate the location effects when two biological groups or conditions were compared. By addressing the data-imbalance and location-correlation issues, the GLMM effectively quantified true biological effects on zebrafish locomotor response.

Utilizing Zebrafish Visual Behaviors in Drug Screening for Retinal Degeneration.

Zebrafish are a popular vertebrate model in drug discovery. They produce a large number of small and rapidly-developing embryos. These embryos display rich visual-behaviors that can be used to screen drugs for treating retinal degeneration (RD). RD comprises blinding diseases such as Retinitis Pigmentosa, which affects 1 in 4000 people. This disease has no definitive cure, emphasizing an urgency to identify new drugs. In this review, we will discuss advantages, challenges, and research developments in using zebrafish behaviors to screen drugs in vivo. We will specifically discuss a visual-motor response that can potentially expedite discovery of new RD drugs.

Molecular Evolution of MDM1, a "Duplication-Resistant" Gene in Vertebrates.

BACKGROUND: The mouse double minute 1 (Mdm1) gene was first reported and cloned in mouse tumor cell lines as an oncogene candidate. Later, it was found that mutation of Mdm1 might cause age-related retinal degeneration 2 in mice by genetic linkage analysis. Additionally, the MDM1 protein was found to be expressed in the centrosomes, cilia, and the nucleus of multiciliated tracheal epithelial cells in mice. These observations suggest that MDM1 may have some basal functions in cell physiology. However, the evolutionary history of this gene and its expression during embryonic development remain largely unexplored. RESULTS: Using molecular phylogenetic analysis, we found that the MDM1 gene encoded an evolutionarily conserved protein across all metazoans. We also found that the MDM1 gene was in a conserved synteny in vertebrates. In almost all the species that were analyzed, there was only one MDM1 gene based on current genome annotations. Since vertebrate genomes underwent two to three rounds of whole-genome duplications around the origin of the vertebrates, it is interesting that only one MDM1 ohnolog was retained. This observation implies that other MDM1 ohnologs were lost after the whole-genome duplications. Furthermore, using whole-mount in situ hybridization, we found that mdm1 was expressed in the forebrain, nephric ducts, and tail buds during zebrafish early embryonic development. CONCLUSION: MDM1 is an evolutionary conserved gene, and its homologous genes can be traced back to basal metazoan lineages. In vertebrates, the MDM1 gene is in a conserved synteny and there is only one MDM1 ohnolog suggesting it is a "duplication-resistant" gene. Its expression patterns in early zebrafish embryos indicate that mdm1 may play important roles in the development of the central nervous system, kidneys, and hematopoietic system.

Evolutionary and developmental analysis reveals KANK genes were co-opted for vertebrate vascular development.

Gene co-option, usually after gene duplication, in the evolution of development is found to contribute to vertebrate morphological innovations, including the endothelium-based vascular system. Recently, a zebrafish kank gene was found expressed in the vascular vessel primordium, suggesting KANK genes are a component of the developmental tool kit for the vertebrate vascular system. However, how the KANK gene family is involved in vascular vessel development during evolution remains largely unknown. First, we analyzed the molecular evolution of the KANK genes in metazoan, and found that KANK1, KANK2, KANK3 and KANK4 emerged in the lineage of vertebrate, consistent with the two rounds of vertebrate whole-genome duplications (WGD). Moreover, KANK genes were further duplicated in teleosts through the bony-fish specific WGD, while only kank1 and kank4 duplicates were retained in some of the examined fish species. We also found all zebrafish kank genes, except kank1b, are primarily expressed during embryonic vascular development. Compared to invertebrate KANK gene expression in the central nervous system, the vascular expression of zebrafish kank genes suggested KANK genes were co-opted for vertebrate vascular development. Given the cellular roles of KANK genes, our results suggest that this co-option may facilitate the evolutionary origin of vertebrate vascular vessels.

Correction: A Naturally-Derived Compound Schisandrin B Enhanced Light Sensation in the pde6c Zebrafish Model of Retinal Degeneration.

[This corrects the article DOI: 10.1371/journal.pone.0149663.].

A Naturally-Derived Compound Schisandrin B Enhanced Light Sensation in the pde6c Zebrafish Model of Retinal Degeneration.

Retinal degeneration is often progressive. This feature has provided a therapeutic window for intervention that may extend functional vision in patients. Even though this approach is feasible, few promising drug candidates are available. The scarcity of new drugs has motivated research to discover novel compounds through different sources. One such example is Schisandrin B (SchB), an active component isolated from the five-flavor fruit (Fructus Schisandrae) that is postulated in traditional Chinese medicines to exert prophylactic visual benefit. This SchB benefit was investigated in this study in pde6cw59, a zebrafish retinal-degeneration model. In this model, the pde6c gene (phosphodiesterase 6C, cGMP-specific, cone, alpha prime) carried a mutation which caused cone degeneration. This altered the local environment and caused the bystander rods to degenerate too. To test SchB on the pde6cw59 mutants, a treatment concentration was first determined that would not cause morphological defects, and would initiate known physiological response. Then, the mutants were treated with the optimized SchB concentration before the appearance of retinal degeneration at 3 days postfertilization (dpf). The light sensation of animals was evaluated at 6 dpf by the visual motor response (VMR), a visual startle that could be initiated by drastic light onset and offset. The results show that the VMR of pde6cw59 mutants towards light onset was enhanced by the SchB treatment, and that the initial phase of the enhancement was primarily mediated through the mutants' eyes. Further immunostaining analysis indicates that the treatment specifically reduced the size of the abnormally large rods. These observations implicate an interesting hypothesis: that the morphologically-improved rods drive the observed VMR enhancement. Together, these investigations have identified a possible visual benefit of SchB on retinal degeneration, a benefit that can potentially be further developed to extend functional vision in patients.

Computational classification of different wild-type zebrafish strains based on their variation in light-induced locomotor response.

Zebrafish larvae display a rapid and characteristic swimming behaviour after abrupt light onset or offset. This light-induced locomotor response (LLR) has been widely used for behavioural research and drug screening. However, the locomotor responses have long been shown to be different between different wild-type (WT) strains. Thus, it is critical to define the differences in the WT LLR to facilitate accurate interpretation of behavioural data. In this investigation, we used support vector machine (SVM) models to classify LLR data collected from three WT strains: AB, TL and TLAB (a hybrid of AB and TL), during early embryogenesis, from 3 to 9 days post-fertilisation (dpf). We analysed both the complete dataset and a subset of the data during the first 30after light change. This initial period of activity is substantially driven by vision, and is also known as the visual motor response (VMR). The analyses have resulted in three major conclusions: First, the LLR is different between the three WT strains, and at different developmental stages. Second, the distinguishable information in the VMR is comparable to, if not better than, the full dataset for classification purposes. Third, the distinguishable information of WT strains in the light-onset response differs from that in the light-offset response. While the classification accuracies were higher for the light-offset than light-onset response when using the complete LLR dataset, a reverse trend was observed when using a shorter VMR dataset. Together, our results indicate that one should use caution when extrapolating interpretations of LLR/VMR obtained from one WT strain to another.

Expression dynamics of NADPH oxidases during early zebrafish development.

Nicotinamide dinucleotide phosphate oxidases (NOX) control various cellular signaling cascades. In the nervous system, there is recent evidence that NOX-derived reactive oxygen species (ROS) regulate neurite outgrowth, regeneration, and stem cell proliferation; however, a comprehensive NOX gene expression analysis is missing for all major model systems. Zebrafish embryos provide an excellent model system to study neurodevelopment and regeneration because they develop quickly and are well suited for in vivo imaging and molecular approaches. Although the sequences of five NOX genes (nox1, nox2/cybb, nox4, nox5, and duox) have been identified in the zebrafish genome, nothing is known about their expression pattern. Here, we used quantitative polymerase chain reaction combined with in situ hybridization to develop a catalog of nox1, nox2/cybb, nox5, and duox expression in zebrafish during early nervous system development from 12 to 48 hours post fertilization. We found that expression levels of nox1, nox5, and duox are dynamic during the first 2 days of development, whereas nox2/cybb levels remain remarkably stable. By sectioning in situ hybridized embryos, we found a pattern of broad and overlapping NOX isoform expression at 1 and 1.5 days post fertilization. After 2 days of development, a few brain regions displayed increased NOX expression levels. Collectively, these results represent the first comprehensive analysis of NOX gene expression in the zebrafish and will provide a basis for future studies aimed at determining the functions of NOX enzymes in neurodevelopment and regeneration. J. Comp. Neurol. 524:2130-2141, 2016. © 2015 Wiley Periodicals, Inc.

Statistical Analysis of Zebrafish Locomotor Response.

Zebrafish larvae display rich locomotor behaviour upon external stimulation. The movement can be simultaneously tracked from many larvae arranged in multi-well plates. The resulting time-series locomotor data have been used to reveal new insights into neurobiology and pharmacology. However, the data are of large scale, and the corresponding locomotor behavior is affected by multiple factors. These issues pose a statistical challenge for comparing larval activities. To address this gap, this study has analyzed a visually-driven locomotor behaviour named the visual motor response (VMR) by the Hotelling's T-squared test. This test is congruent with comparing locomotor profiles from a time period. Different wild-type (WT) strains were compared using the test, which shows that they responded differently to light change at different developmental stages. The performance of this test was evaluated by a power analysis, which shows that the test was sensitive for detecting differences between experimental groups with sample numbers that were commonly used in various studies. In addition, this study investigated the effects of various factors that might affect the VMR by multivariate analysis of variance (MANOVA). The results indicate that the larval activity was generally affected by stage, light stimulus, their interaction, and location in the plate. Nonetheless, different factors affected larval activity differently over time, as indicated by a dynamical analysis of the activity at each second. Intriguingly, this analysis also shows that biological and technical repeats had negligible effect on larval activity. This finding is consistent with that from the Hotelling's T-squared test, and suggests that experimental repeats can be combined to enhance statistical power. Together, these investigations have established a statistical framework for analyzing VMR data, a framework that should be generally applicable to other locomotor data with similar structure.

The chromatin remodeler chd5 is necessary for proper head development during embryogenesis of Danio rerio.

The chromatin remodeler CHD5 plays a critical role in tumor suppression and neurogenesis in mammals. CHD5 contributes to gene expression during neurogenesis, but there is still much to learn regarding how this class of remodelers contributes to differentiation and development. CHD5 remodelers are vertebrate-specific, raising the prospect that CHD5 plays one or more conserved roles in this phylum. Expression of chd5 in adult fish closely mirrors expression of CHD5 in adult mammals. Knockdown of Chd5 during embryogenesis suggests new roles for CHD5 remodelers based on resulting defects in craniofacial development including reduced head and eye size as well as reduced cartilage formation in the head. In addition, knockdown of Chd5 results in altered expression of neural markers in the developing brain and eye as well as a profound defect in differentiation of dopaminergic amacrine cells. Recombinant zebrafish Chd5 protein exhibits nucleosome remodeling activity in vitro, suggesting that it is the loss of this activity that contributes to the observed phenotypes. Our studies indicate that zebrafish is an appropriate model for functional characterization of CHD5 remodelers in vertebrates and highlight the potential of this model for generating novel insights into the role of this vital class of remodelers.

Expression profiling of the RPE in zebrafish smarca4 mutant revealed altered signals that potentially affect RPE and retinal differentiation.

PURPOSE: The purpose of this study was to develop a framework for analyzing retinal pigment epithelium (RPE) expression profiles from zebrafish eye mutants. METHODS: The fish model we used was SWI/SNF-related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (smarca4), a retinal dystrophic mutant with a previously described retinal phenotype and expression profiles. Histological and Affymetrix GeneChip analyses were conducted to characterize the RPE defects and underlying differential expression, respectively. RESULTS: Histological analysis revealed that smarca4 RPE was formed, but its differentiation was abnormal. In particular, ultrastructural analysis of smarca4 RPE by transmission electron microscopy demonstrated several defects in melanogenesis. The nature of these defects also suggests that the cytoskeletal dynamics, which are tightly linked with melanogenesis, were impaired in smarca4 RPE. To compare the expression profile of normal wild-type (WT) and smarca4 RPE, the gene expression profiles of microdissected retinas and RPE-attached retinas were measured with Affymetrix GeneChip analysis. The RPE expression values were then estimated from these samples by subtracting the retinal expression values from the expression values of the RPE-attached retinas. A factorial analysis was conducted using the expression values of the RPE, retinal, and whole-embryo samples. Specific rules (contrasts) were built using the coefficients of the resulting fitted models to select for three groups of genes: 1) smarca4-regulated RPE genes, 2) smarca4-regulated retinal genes, and 3) smarca4-regulated RPE genes that are not differentially expressed in the retina. Interestingly, the third group consists of 39 genes that are highly related to cytoskeletal dynamics, melanogenesis, and paracrine and intracellular signal transduction. CONCLUSIONS: Our analytical framework provides an experimental approach to identify differentially-regulated genes in the retina and the RPE of zebrafish mutants in which both of these tissues are affected by the underlying mutation. Specifically, we have used the method to identify a group of 39 genes that can potentially explain the melanogenesis defect in the smarca4 RPE. In addition, several genes in this group are secreted signaling molecules. Thus, this observation further implicates that the smarca4 RPE might play a role in the retinal dystrophic phenotype in smarca4.

p35 promotes the differentiation of amacrine cell subtype in the zebrafish retina under the regulation of egr1.

BACKGROUND: Early growth response 1 (egr1) is a transcription factor (TF) for controlling the differentiation of Parvalbumin (Parv) -expressing amacrine cells (ACs) in zebrafish. However, the downstream factors of this process have not been identified. The purpose of this study was to investigate the role of p35, a neuronal-specific activator of cyclin-dependent kinase 5 (Cdk5) and a known in vitro target of egr1, in the differentiation of these ACs. RESULTS: In the p35-knockdown retinas, Parv+ but not islet1+ ACs were specifically reduced. This phenotype was highly similar to that in the Egr1-knockdown retinas. Furthermore, p35 expression was reduced in the Egr1-knockdown retinas, particularly in the AC region; while egr1 was only modestly reduced in this region in the p35-knockdown retinas. CONCLUSIONS: p35 likely acts downstream of egr1 to control the differentiation of Parv+ ACs.

A High-Throughput Zebrafish Screening Method for Visual Mutants by Light-Induced Locomotor Response.

Normal and visually-impaired zebrafish larvae have differentiable light-induced locomotor response (LLR), which is composed of visual and non-visual components. It is recently demonstrated that differences in the acute phase of the LLR, also known as the visual motor response (VMR), can be utilized to evaluate new eye drugs. However, most of the previous studies focused on the average LLR activity of a particular genotype, which left information that could address differences in individual zebrafish development unattended. In this study, machine learning techniques were employed to distinguish not only zebrafish larvae of different genotypes, but also different batches, based on their response to light stimuli. This approach allows us to perform efficient high-throughput zebrafish screening with relatively simple preparations. Following the general machine learning framework, some discriminative features were first extracted from the behavioral data. Both unsupervised and supervised learning algorithms were implemented for the classification of zebrafish of different genotypes and batches. The accuracy of the classification in genotype was over 80 percent and could achieve up to 95 percent in some cases. The results obtained shed light on the potential of using machine learning techniques for analyzing behavioral data of zebrafish, which may enhance the reliability of high-throughput drug screening.