Why zebrafish model

Kalueff, A. Towards a comprehensive catalog of zebrafish behavior 1. Zebrafish 10, 70— Zebrafish as an emerging model for studying complex brain disorders. Trends Pharmacol. Karoglu, E. Aging alters the molecular dynamics of synapses in a sexually dimorphic pattern in zebrafish Danio rerio. Aging 54, 10— Cholinergic modulations of synaptic protein levels in male and female aged zebrafish. Kempermann, G. Experience-induced neurogenesis in the senescent dentate gyrus.

Kishi, S. The zebrafish as a vertebrate model of functional aging and very gradual senescence. Kitamura, T. Dietary restriction increases hippocampal neurogenesis by molecular mechanisms independent of NMDA receptors. Kizil, C. Adult neurogenesis and brain regeneration. Lee, J. Dietary restriction enhances neurotrophin expression and neurogenesis in the hippocampus of adult mice. Lin, S. Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration.

Luo, J. The aging neurogenic subventricular zone. Aging Cell 5, — Najafian, M. The effect of motion aftereffect on optomotor response in larva and adult zebrafish. Nam, R. NMDA receptor-dependent long-term potentiation in the telencephalon of the zebrafish. Neuhauss, S. Perry, M. Eker, A. Farrell, and C. Brauner London: Academic Press , 81— Google Scholar.

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Owsley, C. Vision and aging. Park, H. Neurogenic contributions made by dietary regulation to hippocampal neurogenesis. Park, J. Calorie restriction alleviates the age-related decrease in neural progenitor cell division in the aging brain. Parker, M. Developmental role of acetylcholinesterase in impulse control in zebrafish. Peters, A. Feature article: are neurons lost from the primate cerebral cortex during normal aging?

Cortex 8, — Poe, B. In vitro fertilization can be performed if necessary. The one-cell-stage fertilized eggs can be easily injected with DNA or RNA to permanently modify their genetic makeup in order to generate transgenic or knock-out zebrafish lines.

Working with mice in this way is much more complicated. Mouse embryos develop inside the mother, and to access and manipulate them the mother would have to be sacrificed. To keep the embryos alive after fertilizing or injecting them, they would need to be transplanted into another female mouse, as well. Zebrafish larva, the stage of development from between three and thirty days post-fertilization, grow in length from approximately 3.

Furthermore, zebrafish embryos are clear, which allows scientists to watch the fertilized eggs grow into fully formed baby fish under a microscope. Their transparency also enables the visualization of fluorescently labeled tissues in transgenic zebrafish embryos. Mouse embryos are not clear and develop inside the mother, so the observation of live embryo development like that in zebrafish is not possible. However, there is a limit on what types of diseases can be studied in zebrafish.

Human diseases caused by genes that do not exist in zebrafish require a different animal model. Additionally, zebrafish are not useful models for human diseases that mainly take place in a tissue type or body part that zebrafish do not have e. For instance, the structure of the muscle fibers can be examined for abnormalities under the microscope if the patient has a muscle disease. For a patient with a neurological disease, the neurons of knock-out embryos can be fluorescently labeled to see if they form incorrectly.

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Page-McCaw , W. Chen , and R. Leptin signaling regulates glucose homeostasis, but not adipostasis, in the zebrafish. Minchin ]]], J. Scahill , N. Staudt , E. Busch-Nentwich , and J. Deep phenotyping in zebrafish reveals genetic and diet-induced adiposity changes that may inform disease risk. Lipid Res. Miyares ]]], R. Zebrafish yolk lipid processing: a tractable tool for the study of vertebrate lipid transport and metabolism.

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Gibert , L. Berekelya , L. Bernard , F. Brunet , E. Guillot , J. Le Bail , J. Galzin , G. Triqueneaux , et al. Fasting induces CART down-regulation in the zebrafish nervous system in a cannabinoid receptor 1-dependent manner.

Oehlers ]]], S. Flores , K. Okuda , C. Hall , K. Crosier , and P. A chemical enterocolitis model in zebrafish larvae that is dependent on microbiota and responsive to pharmacological agents. Oka ]]], T. Nishimura , L. Zang , M. Hirano , Y. Shimada , Z. Wang , N. Umemoto , J. Kuroyanagi , N. Nishimura , and T. Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity. Olsen ]]], A. Sarras Jr. Leontovich , and R.

Heritable transmission of diabetic metabolic memory in zebrafish correlates with DNA hypomethylation and aberrant gene expression. Diabetes 61 : — Passeri ]]], M. Cinaroglu , C. Gao , and K. Hepatic steatosis in response to acute alcohol exposure in zebrafish requires sterol regulatory element binding protein activation. Hepatology 49 : — Poureetezadi ]]], S.

Cheng , J. Chambers , B. Drummond , and R. Prostaglandin signaling regulates nephron segment patterning of renal progenitors during zebrafish kidney development. Ribas ]]], L. The zebrafish Danio rerio as a model organism, with emphasis on applications for finfish aquaculture research.

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Evaluating the capacity of human gut microorganisms to colonize the zebrafish larvae Danio rerio. Wu ]]], R. Lam , H. Clay , D. Duong , R. For example, zebrafish mutants of stxbp1b , an ortholog of STXBP1 , which is associated with epileptic encephalopathy syndromes, display electrographic seizures at baseline, suggesting this may be a useful model for these syndromes Grone et al.

Zebrafish have also been used a genetic tool for rapidly screening the functionality of novel genes and rare variants identified in human genetics studies of epilepsy, ID and other neurodevelopmental disorders Table 3 , Bassuk et al. These studies assess the extent to which wild-type mRNA or mRNA carrying rare variants identified in affected individuals reverses morpholino-induced or CRISPR F0 phenotypes, providing an in vivo readout of the effect of the mutation on gene function.

For example, morpholino-induced knockdown of STX1B , which was identified by linkage analysis in large pedigrees as carrying damaging mutations in individuals with epilepsy, caused electrographic seizures Schubert et al. In addition, morpholino-induced knockdown of TRAPPC6B , which was identified as a risk gene by linkage analysis and homozygosity mapping in individuals with epilepsy, microcephaly, and ASD from consanguineous families, led to increased baseline neural activity and sensitivity to PTZ-induced seizures in zebrafish larvae Marin-Valencia et al.

Another approach to assess the functionality of newly identified human genes or rare variants is overexpression in zebrafish embryos. For example, overexpression of wild-type pk1a , the zebrafish ortholog of PK1A , caused a more severe phenotype than overexpressing mRNA carrying a mutation identified in individuals with progressive myoclonic epilepsy, suggesting that the mutation alters the in vivo function of this gene Bassuk et al.

In addition, overexpression of mRNA encoding human RHEB and versions of the gene containing two missense mutations identified in individuals with ID and macrocephaly, caused macrocephaly in zebrafish larvae, while F0 CRISPR mosaics of this gene displayed microcephaly, suggesting these variants may represent a gain-of-function Reijnders et al.

There are several points to consider in the use of zebrafish for screening variants identified in human genetics studies. First, while in vivo rescue or overexpression screens may be informative regarding the biological function of an identified variant, the identification of nonspecific neural phenotypes, particularly morpholino-based phenotypes, are not sufficient to establish causation of an identified gene or variant and should not be used a substitute for strong evidence from human genetic studies.

Second, while the presence of a phenotype in CRISPR F0 mosaics provides additional support for specificity, it is important to demonstrate in a stable mutant line that the phenotype results from loss of gene function and not nonspecific effects in F0-injected embryos. Schizophrenia is a psychotic disorder characterized by hallucinations, delusions and disorganized thought processes or behavior, as well as diminished affect, energy and motivation, which severely impacts overall functioning American Psychiatric Association, The genetics of schizophrenia are complex, with over common variants identified by genome-wide association studies GWAS; Schizophrenia Working Group of the Psychiatric Genomics Consortium, , and rare damaging variants and CNVs contributing to risk according to a polygenic model Walsh et al.

This genetic architecture complicates the functional analysis of risk variants in schizophrenia Fromer et al. Here, we discuss several studies that used zebrafish to analyze the function of schizophrenia-associated genes Table 4. While schizophrenia is highly polygenic, DISC1 is an example of a rare schizophrenia-associated gene, which was discovered in a large Scottish family where a balanced chromosomal translocation segregated with schizophrenia and other psychiatric disorders schizoaffective disorder, bipolar disorder, major depressive disorder; Millar et al.

Some studies have used zebrafish to investigate DISC1 function. For example, De Rienzo et al. In a subsequent study, Singh et al. Another study found evidence for altered hypothalamic development as well as stress responses in zebrafish disc1 mutants Eachus et al. Interestingly, this study found variable phenotypes in two disc1 mutant lines, such as differences in the time course of expression changes in markers of hypothalamic precursors, even though both lines carry mutations that induce early premature stop codons.

This suggests that the specific location of a mutation may alter the expression of a phenotype in mutants. Also, while this study used one of the same mutants as the previous study, no morphological defects were identified in disc1 homozygous mutants, suggesting that background variation may alter the expression of phenotypes in genetic mutants Eachus et al. Together, these studies highlight insights into DISC1 function gained from zebrafish models.

Additional studies have used zebrafish to rapidly assess the effect of changes in the expression of schizophrenia candidate genes implicated in human genetics studies.

By comparing risk variants identified by GWAS with RNA sequencing data from post-mortem brain samples from individuals with schizophrenia, Fromer et al.

By altering the expression of three implicated genes in zebrafish in the same direction as the RNA sequencing result from human brain tissue, this study found that morpholino-induced knockdown of the downregulated gene, furina , led to microcephaly and decreased cell proliferation, which was rescued by introducing human FURIN mRNA, while overexpression of the upregulated genes, tsnare and cntn4 , led to microcephaly and increased cell proliferation Fromer et al.

Morpholino-induced knockdown of mapk3 caused microcephaly, which was reversed by overexpression of human KCTD13 , another gene in the 16p As discussed earlier, while nonspecific neural phenotypes induced by morpholinos, overexpression, or CRISPR F0 mosaics, may be suggestive of a functional effect, replication of these findings in a stable mutant line is necessary for validation. Most studies of zebrafish models of neurodevelopmental disorders to date have focused primarily on early morphological and simple behavioral phenotypes.

However, recent advances in functional imaging are likely to transform these studies in the near future, allowing for the assessment of circuit-level phenotypes resulting from risk gene disruption. Progress in brain imaging is due in large part to the development of genetically-encoded calcium indicators GECIs , such as GCaMP, which provide a rapid readout of activity at the level of a single neuron Chen et al.

GCaMP can be expressed transgenically in a subset of neurons or throughout the brain of larval zebrafish, which is an ideal system for monitoring neural activity. By harnessing advances in imaging technologies, including two-photon and light-sheet microscopy, a number of studies are beginning to dissect neural circuit mechanisms in the developing zebrafish brain Ahrens et al.

Together with the transparency and relative simplicity of the larval zebrafish brain, these technologies are likely to have considerable translational potential for revealing mechanisms by which the disruption of risk genes leads to alterations in signaling networks in the developing vertebrate brain, resulting in simple behavioral phenotypes.

For example, several studies have used two-photon microscopy to record brain activity in response to visually-evoked stimuli. Two-photon microscopy is a point-scanning method that provides excellent spatial resolution, but is more limited in its imaging speed Keller and Ahrens, To image brain activity, zebrafish are immobilized in agarose, while visual stimuli are projected onto a screen to the side or below the fish while brain activity is recorded.

Portugues et al. By simultaneously recording brain activity, eye and tail movements during stimulus exposure, this study identified a stereotyped pattern of brain activity that occurs during the OKR, and found that activity in specific brain regions correlates with sensory or motor signals. In addition, Filosa et al. Interestingly, this study showed that hunger not only makes zebrafish more likely to pursue visual stimuli that resemble their food, but increases the responsiveness of specific cells in the optic tectum to these food-like stimuli, providing a neural correlate for the observed behavior.

Other studies have also used two-photon microscopy to examine behavioral circuits, such as those involved in prey capture, predator responses, responses to visual and olfactory stimuli, and the optomotor response, a reflexive behavior that occurs following a perceived change in whole-field motion Dreosti et al.

In addition, a growing number of studies have used light-sheet fluorescence microscopy for functional imaging of the zebrafish brain. Given its speed, light-sheet imaging was used to perform continuous whole-brain activity recordings at baseline, revealing functional networks of correlated activity in the zebrafish brain Ahrens et al.

Light-sheet imaging has also been used to study brain activity following exposure to various stimuli. For example, Thompson et al. Two-photon imaging offers an alternative in this regard, because it provides stimulation outside of the visible range of zebrafish.

Another approach is to position multiple light sheets to avoid direct retinal stimulation Vladimirov et al. Further, functional imaging in general generates considerably large datasets, which may be difficult to analyze, though computational algorithms have been developed to address this challenge Keller and Ahrens, At the same time, these functional imaging techniques are technically challenging, not high-throughput, and often require immobilizing the fish.

To address these limitations, Randlett et al. To identify regions of differential activity, images are mapped onto a zebrafish brain atlas Z-Brain. This approach can be used to obtain a readout of whole-brain activity in freely moving zebrafish either at baseline or in response to a stimulus or drug Randlett et al. Another method, developed by Lovett-Barron et al. By integrating the functional imaging and immunostained datasets, this method allows for the identification of the specific cell types that were active during a behavioral task.

Intriguingly, using this approach, Lovett-Barron et al. Therefore, findings in zebrafish models of neurodevelopmental disorders are likely to have translational relevance for understanding related circuits in mammals. Together, these technologies offer considerable promise for illuminating circuit-level mechanisms in zebrafish models of neurodevelopmental disorders.

Zebrafish have critical advantages as a model system for investigating the function of genes associated with neurodevelopmental disorders. A growing number of studies are beginning to capitalize on their unique features to illuminate neurobiological and pharmacological pathways underlying ASD, epilepsy, ID and schizophrenia. These studies have utilized the transparency, tractability and throughout of the zebrafish model to identify the effects of loss of risk gene function on the development of specific neuron populations, molecular pathways, and simple behaviors, all of which can be leveraged to screen for novel small molecule suppressors.

Because splice-site mutations may lead to alternative transcripts that reverse deleterious mutations Anderson et al. Assessing phenotypes over a developmental time course from embryonic to adult stages will likely provide key insights into when and where risk genes play important roles.

Moreover, future studies capitalizing on the strengths of zebrafish as a first-pass, high-throughput screening approach have the potential to reveal novel pharmacological candidates for further investigation in these disorders. Given the evidence for conservation of pharmacological and circuit-level pathways in zebrafish and mammals Rihel et al. Furthermore, advances in in vivo calcium imaging in zebrafish represent an exciting new avenue for investigating the circuit-level roles of risk genes with translational relevance.

Taken together, zebrafish represent a promising model system for the discovery of novel biological pathways, pharmacological candidates, and circuit mechanisms with relevance to neurodevelopmental disorders. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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