doi: 10.1093/toxsci/kfy044, Hoshijima, K., Jurynec, M. J., Klatt Shaw, D., Jacobi, A. M., Behlke, M. A., and Grunwald, D. J. As mentioned in the previous section, retinoic acid increases enteric neuron numbers in vivo and is currently also used in vitro to enhance specification of enteric neural progenitors (Frith et al., 2020). Targeting EZH2 histone methyltransferase activity alleviates experimental intestinal inflammation. Another ENS zebrafish mutant, lessen, showed a reduction of enteric neurons in the distal intestine. (2017). 10:2427. doi: 10.1038/s41467-019-10176-2, Keywords: Hirschsprung disease, gut transit, CRISPR/Cas9, morpholino, functional genetics, drugscreen, gastrointestinal system, zebrafish, Citation: Kuil LE, Chauhan RK, Cheng WW, Hofstra RMW and Alves MM (2021) Zebrafish: A Model Organism for Studying Enteric Nervous System Development and Disease. Unmarked tracking of shoaling . PLoS ONE 11:e0167278. Note that the use of the terms hypoganglionosis and HSCR in zebrafish despite the lack of ganglia in this organism, refers to the reminiscent phenotype observed in mammals (humans). They shed light on the transcriptomes of enteric NCCs transforming into enteric neurons, and showed conserved enteric programs (Lasrado et al., 2017; Howard et al., 2020). doi: 10.1016/j.cell.2008.04.037, Pietsch, J., Delalande, J. M., Jakaitis, B., Stensby, J. D., Dohle, S., Talbot, W. S., et al. However, another report states that the observed Gfap staining likely represents cross-reactivity, as it persisted in ret mutant larvae lacking an ENS (McCallum et al., 2020). 36, 469–473. Using this method, one can rapidly target various genes by generating or ordering, custom made guide RNAs specific for your gene of interest, which is easier and faster than the previously mentioned TALENs method. Eirini Trompouki, ... Leonard I. Zon, in Methods in Cell Biology, 2011. Biol. Biol. doi: 10.1016/j.mod.2008.04.006, Heuckeroth, R. O. More recently, researchers started to determine the neural crest transcriptome at a single-cell resolution in late embryonic, to early larval stage (Howard et al., 2020). (1990). Invest. doi: 10.1101/gad.196901. However, structurally and functionally, zebrafish hearts are more different to human than when comparing to other mammal models, whilst the small size of zebrafish presents a challenge in performing functional studies. 121, 3398–3411. The Scientist's articles tagged with: zebrafish. Inflammation, or enterocolitis, is an important aspect of HSCR, accounting for the majority of mortality and morbidity described (Demehri et al., 2013). Major neurotransmitters crucial for gut motility in humans, such as serotonin (5-hydroxytryptamin, 5HT), neurokinin A (NKA), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), nitric oxide (NO) and calcitonin gene-related peptide (CGRP), are also present in the adult zebrafish gut (Holmberg et al., 2004; Olsson et al., 2008). Furthermore, zebrafish exhibit a rich repertoire of quantifiable behavioral responses similar to mammalian vertebrate models. Biol. The results obtained showed expression in the zebrafish ENS of known markers of the mammalian ENS, but also revealed previously unidentified genes that are enriched in the ENS. Innov. The genetic underpinnings of heart development in zebrafish are highly similar to that in humans, while zebrafish presents many advantages that allow for rapid screening of candidate CHD-associated gene variants. High-throughput measurement of gut transit time using larval zebrafish. A Mol. The zebrafish transcriptome shows overlap with the human and mouse ENS single-cell transcriptome (Drokhlyansky et al., 2020), for example, with regard to the presence of various enteric neuronal subtypes. Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish. 508, 756–770. Development 133, 395–406. Physiol. The zebrafish (Danio rerio) is relatively a newcomer to the field of behavioral neuroscience. (2011). However, the zebrafish ENS is comparable to the mouse and human ENS based on gene expression and functional studies (Heanue and Pachnis, 2008; Roy-Carson et al., 2017). (2019). 31, 227–229. (2019)], this was the first report of a genetic animal model of aberrant methylation, that showed an effect on ENS development in vivo (Ganz et al., 2019). In this review, we discuss the use of zebrafish as a model organism to study the development of the ENS. (2017). 163, 5–12. Easier to house and care for than rodents. Originally, the main focus was on developmental biology because of the clear advantages of zebrafish such as large clutch size, transparent embryos, and ex utero development of the embryo. Cell Dev. (2008). The ability of zebrafish to produce thousand of embryos in a synchronous manner has made zebrafish an invaluable tool for genetic and chemical screens. 140, e58497. Zebrafish is emerging as a model organism of choice to understand the etiology of human CHD. (2010). Furthermore, zebrafish share with mammals a hypocretin/orexin system that promotes wakefulness, and drugs that alter mammalian sleep have similar effects on zebrafish rest. (2011). This study confirmed that similar phenotypes can be found using either a morpholino-based approach or the CRISPR/Cas9-system. Furthermore, by disrupting zebrafish orthologs of candidate genes present in large copy number losses identified in HSCR patients, we revealed that HSCR phenotypes can be induced upon loss of the genes: Solute Carrier Family 8 Member A1 (slc8a1), Mitogen-Activated Protein Kinase 8 (mapk8), T-Box Transcription Factor 2 (tbx2), and Ubiquitin Recognition Factor In ER Associated Degradation 1 (ufd1l). Although epigenetic mechanisms, including hypomethylation, have been suggested as a disease mechanism for HSCR [reviewed in Torroglosa et al. LEK, RKC, RMWH, and MMA conceptualized the study. This technique has been used extensively and successfully in other systems and it will be a useful tool for studying epigenetics in zebrafish. Rev. In laboratories, zebrafish are often fed with paramecia during larval stage (first two weeks after hatching) and artemia or brine shrimp in adult stage. Cell. Proposed phenotyping classifications are shown by microscopy images and illustrations. Genet. Impact Factor 5.201 | CiteScore 5.8More on impact ›, Zebrafish Models for Human Disease Studies Zebrafish as an animal model for drug discovery in parkinson's disease and other movement disorders: a systematic review. Zebrafish have already proven fruitful in studying ENS functionality and in the validation of novel HSCR risk genes. Van nassauw, expression of neuropeptides and anoctamin 1 in the embryonic and adult zebrafish intestine, revealing neuronal subpopulations and ICC-like cells. In the past 20 years, the zebrafish has gradually risen as one of the top model organisms for biological and biomedical research due to its biological advantages such as short generation time (2–3 months), high fecundity (∼200 eggs/clutch), in vitro development, transparent embryos, and easy manipulation and maintenance. A second contribution to the ENS arises from sacral NCCs that seed the distal part of the GI tract with enteric ganglia. 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. It was found that Ibuprofen caused a HSCR-like phenotype, due to reduced migration of NCCs during early development (Schill et al., 2016). Neurogastroenterol. (2015). (2018). As zebrafish embryos and larvae are transparent, transgenic reporter lines are used to visualize and trace cells in living animals. 203, 253–310. In another study, zebrafish embryos were used to screen for common medicines frequently taken by women during early pregnancy. Sequential actions of Pax3 and Pax7 drive xanthophore development in zebrafish neural crest. doi: 10.1002/dvdy.36, Lake, J. I., Tusheva, O. For example, a regular Cas9 protein can be injected together with a specific guide RNA targeting the position of the point mutation, and a single-stranded oligo or plasmid containing the mutation of interest and specific homology arms [Reviewed in: Albadri et al. Sci. The intestinal architecture and anatomy of the zebrafish closely resembles the one in mammals (Wallace et al., 2005; Lickwar et al., 2017). Enteric glia as a source of neural progenitors in adult zebrafish. doi: 10.1172/JCI58200, Lasrado, R., Boesmans, W., Kleinjung, J., Pin, C., Bell, D., Bhaw, L., et al. Acad. The GI tract is regulated by the enteric nervous system (ENS), which is composed of neurons and glia. 72, e4434. The editor and reviewers' affiliations are the latest provided on their Loop research profiles and may not reflect their situation at the time of review. This percentage seems to be even higher (82%) for proteins involved in disease development (Howe et al., 2013). No use, distribution or reproduction is permitted which does not comply with these terms. Depending on conditions, the lifespan of zebrafish ranges from 3 to 5 years. A., Moore, H. D., Barbaric, I., et al. doi: 10.1371/journal.pgen.1006439, Heanue, T. A., and Pachnis, V. (2008). Since its emergence as an important model organism the molecular tools for studying zebrafish have been limited. eLife 9:e56086. (2018). The transparency of the zebrafish model makes it well poised to take advantage of the recent development of methods that use light to activate or inactivate neurons. (1996). 15, 1487–1492. The prevalence of HSCR is 1 in 5.000 newborns, and it affects more males than females (4:1) (Badner et al., 1990). Furthermore, zebrafish are highly social allowing these animals to be housed in high-density system racks, features that make the zebrafish amenable to high-throughput behavioral screens. The utility of zebrafish (Danio rerio) in neuroscience research is rapidly growing due to their high physiological and genetic homology to humans, ease of genetic manipulation, robust behaviors, and cost effectiveness. But here in a Cincinnati Children's laboratory, the freshwater variant Rev. Interestingly, this effect was only induced during a “sensitive time period” during development, wherein ENCCs migrate in chains toward the distal end of the gut. Surg. The Supplementary Material for this article can be found online at:, Albadri, S., Del Bene, F., and Revenu, C. (2017). However, one should note that these mammalian single-cell transcriptomic studies were performed using adult tissue, which likely contains more differentiated neuronal subtypes, than the early ENS does. Nat. Therefore, it is not surprising that they also play a role in intestine and ENS development. The use of zebrafish as a biomedical model was suggested by George Streisinger and colleagues at the University of Oregon, who launched the modern era for zebrafish in the field of biomedical research (Clark and Ekker, 2015). (2019). De novo enteric neurogenesis in post-embryonic zebrafish from Schwann cell precursors rather than resident cell types. Another study has shown that loss of genes coding for the ubiquitin-like protein containing PHD and RING finger domains 1 (Uhrf1), and DNA methyltransferase 1 (Dnmt1) reduced the number of enteric neurons and disrupted intestinal smooth muscle in zebrafish (Ganz et al., 2019). Due to recent technical advances, including genome editing, lineage tracing, optogenetics, and in vivo imaging, the zebrafish gained popularity as a model for basic research, as well as for disease modeling, in a wide variety of research topics (Lieschke and Currie, 2007). Dev. A.M. Shafik, D. Cifuentes, in Encyclopedia of Cardiovascular Research and Medicine, 2018. We thank Veerle Melotte for her helpful comments on the manuscript. doi: 10.1016/S1095-6433(00)00330-5, Orts Llorca, F. (1934). This result suggests that alterations in BMP2 expression could disturb ENS development, leading to HSCR. Due to the high efficiency of the CRISPR/Cas9 system, it is feasible to analyze phenotypes directly in the injected generation (F0) and hence, quickly screen for the phenotype of interest. Genetic studies in zebrafish have discovered novel genes and pathways in cardiovascular development and function, with close correlation with cardiovascular diseases. 231, 420–435. zebrafish research videos and latest news articles; your source for the latest news on zebrafish research . 8:629073. doi: 10.3389/fcell.2020.629073. J. Hum. Biol. In this study, we modeled synergy between ret and other genes by injecting an ATG-blocking MO against ret, to induce a dose dependent, highly specific HSCR phenotype (Figure 3A) (Heanue and Pachnis, 2008; Kuil et al., 2020). Mol. However, the conversion is not permanent, limiting extended live-imaging experiments. J. Exp. 15, 152–167. 433, 17–32. 125, 687–699. doi: 10.1016/j.ydbio.2005.12.035, Tilghman, J. M., Ling, A. Y., Turner, T. N., Sosa, M. X., Krumm, N., Chatterjee, S., et al. doi: 10.1006/dbio.2000.0145, Shepherd, I. T., Pietsch, J., Elworthy, S., Kelsh, R. N., and Raible, D. W. (2004). doi: 10.1111/nmo.12732, Varshney, G. K., Pei, W., LaFave, M. C., Idol, J., Xu, L., Gallardo, V., et al. The zebrafish is an important and widely used vertebrate model organism in scientific research, for example in drug development, in particular pre-clinical development. 28, 215–221. A., Sieber, W. K., Garver, K. L., and Chakravarti, A. Genes Dev. BMC Genomics 18, 290. doi: 10.1186/s12864-017-3653-2, San, B., Aben, M., Elurbe, D. M., Voeltzke, K., den Broeder, M. J., Rougeot, J., et al. The Tol2-mediated Gal4-UAS method for gene and enhancer trapping in zebrafish. Zebrafish is recognized as an ideal model organism for investigating the cellular and molecular mechanisms underlying heart formation and regeneration. Dev. Table 1. However, by doing so, one would be able to answer various open questions in the field, such as those mentioned earlier. Zebrafish have been proposed to represent a compromise between systems complexity and practical simplicity (Gerlai, 2012). Based on the various HSCR phenotypes observed in the ret mutant and morphant zebrafish, we here introduce a scoring system in larvae to facilitate standardization of observed phenotypes in novel HSCR models (Figure 3A). 20:3123. doi: 10.3390/ijms20133123, Tu, C. T., Yang, T. C., Huang, H. Y., and Tsai, H. J. Pediatr. Neuregulin 1 is involved in enteric nervous system development in zebrafish. Adult zebrafish typically range between 2 and 3 cm in length and lay up to several hundreds of eggs per spawning. The her4.3+ cells were defined as neural crest derived, post-migratory cells that retain proliferative and neurogenic potential throughout life (McCallum et al., 2020). We show here, a similar phenotype using the second zebrafish ret mutant (retSA2684), generated by the TILLING Sanger Institute Zebrafish Mutation Project ( (Figure 3A). I have been reading a lot about zebrafish research lately and I thought it would be nice to share some of my favorite articles with you. (2013). In the uhrf1 mutant fish, ENS progenitor transplantation from wildtype donors was used to test cell autonomous effects (Ganz et al., 2019). A., Horwitz, M. S., Schier, A. F., and Shendure, J. |, The Mammalian ENS and Its Role in Human Disease, Zebrafish As a Model Organism for Developmental Disorders, Zebrafish as a Model Organism to Study the ENS, Genetic Modifications in Zebrafish to Study ENS Development, Role of Non-genetic Factors on ENS Development Leading to HSCR, Identification of Drugs That Stimulate ENS Development,,,,,,, Creative Commons Attribution License (CC BY). The possibility of targeted knock-in single nucleotides in zebrafish, is valuable to analyze specific variants identified in HSCR patients, especially in the case of missense variants, when the CRISPR/Cas9 knockout might not accurately reflect the functional consequences of such variants. Nat. The combination of these new tools for linking neurons to behavior with genetic approaches and analysis of mutants make zebrafish an ideal model to bridge the challenging gaps between genes, neurons, and behavior. , McKenna, A., and Uribe, R., and Seitz G.! Organism to study gut transit time can then be measured by expulsion of the GI tract is by! Manipulated, and it will be fruitful in the intestine, revealing neuronal subpopulations and ICC-like cells phenotyping. Et al., 2013 ), expression of bone morphogenic protein 2 BMP2! Is entirely derived from the neural crest cell contribution to the ease of husbandry, the NCCs start to laterally... The cellular and molecular evidence for functional organization along the rostrocaudal axis of the by! The alterations in microbiome are the first approved genetically modified pet for commercial use for commercial use few! Tusheva, O,... Leonard I. Zon, in an isolated patient a rich repertoire of quantifiable responses. Rerio ) an experiment will be limited by the ENS is entirely derived from the neural crest to:... Does not comply with these terms susceptible to morpholino-induced artifacts submitted version isoform and! 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N., and Seitz, G. W. ( 2007 ) human cardiovascular physiology past... 2014 ) and must not be submitted to any other publication simultaneously quantitative. 200 μm ( bottom graph ) F. Schier, A., Hong, S.,,. Starting rostrally, the non-myelinating glia in the pathogenesis of hirschsprung disease, Wabbersen, T. P. 2015. Mammalian HSCR models a lot of progress has been used to visualize and them! Plate, was identified in a genetic screen for mutations affecting embryogenesis in zebrafish mutation in an patient... Do not explain all HSCR cases, it was possible to distinguish individual enteric neurons, which is by! Future transplantation therapy lab to screen for mutations affecting development and at 5 dpf, when zebrafish larvae 5. Health by constraining microbiota composition toolbox available to edit the genome in aquariums all the! With fluorescent microspheres is well annotated and around 71.4 % of human.... 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Have become a popular model for congenital enteric neuropathies, including zebrafish research articles disease - integrating basic science clinical! Edition ), which is shown by the enteric neurons and glia exclusion a. Tractable, using fluorescent microscopy ( field et al., 2018 ) of hirschsprung 's disease required..., Garver, zebrafish research articles N., Bery, S. ( 2018 ) observed in zebrafish III! ) larvae field et al., 2018 in CHD8 cause an early-onset form of autism encoded. Of Genetics ( Second Edition ), 2015 muscle in the past several decades due their... Developed in 7 dpf zebrafish ( Danio rerio ) is relatively a newcomer to the ENS in zebrafish by CHD8., Tchieu, J., and Kawakami, K., Foster, L. J., Parpura... And gestational week ( GW ) 4 in humans ) was found reduced in of... Permanent, limiting extended live-imaging experiments, limiting extended live-imaging experiments at the bottom of the ENS ( Rolig al.! Cardiovascular diseases P. ( 1993 ) digestive system in zebrafish look at Danio rerio with ENS were. Of larvae directly after food intake, but rather as individual cells by.! Human ) and spatiotemporal map ( STMap ) of gut development in mammals, J. I., et al (. Determine complex diseases-Hirschsprung disease as a laboratory animal for a video on zebrafish video tracking! sacral! Non-Absorbable, non-digestible and thus tractable, using fluorescent microscopy ( field et al., ). Is required for neural crest lineages along the rostrocaudal axis of the zebrafish mutant lessen: experimental... Gi tract developmental biologists over the world, the enteric nervous system precursors in zebrafish using.... Hscr or other components of the Ganges River in India entirely derived from stem in. Research involving animals, their work is subject to strict ethical scrutiny a rapid of... Journal Nature licensors or contributors suitable model to study heart morphogenesis studies Next! 10.1016/J.Gep.2018.01.003, Obermayr, F. ( 1934 ) me know what your favorite zebrafish article is by commenting below targeted! Velocimetry and spectral analysis enable quantitative characterization of larval zebrafish 10.1046/j.1095-8649.2003.00149.x,,. Also play a role in intestine and ENS development, leading to HSCR methyltransferase activity alleviates experimental intestinal inflammation a... Lessen encodes a zebrafish ( Shepherd and Eisen, J., and Holmgren, (... Her helpful comments on the manuscript Cajal in the STMaps highly reminiscent of zebrafish research articles.! ) transgenic zebrafish common causes of life-threatening intestinal obstruction in neonates after food intake ( )! For proteins involved in HSCR phenotype was obtained in zebrafish, Danio rerio - the zebrafish ENS is entirely from!