NCI – Zebrafish Studies
Zebrafish Models of Cancer: Harnessing Genetics to Dissect Oncogenic Pathways and Identify
Therapeutic Vulnerabilities
Tom Look
Over the past ten years, the zebrafish has emerged as an invaluable model system for the study of human
cancers. Distinct advantages of the zebrafish arise from the evolutionary conservation of genetic pathways
implicated in cancer that are shared between fish and humans coupled to the unique attributes of zebrafish as
a tool for modeling human disease and analyzing the underlying cellular processes. Over the past few years, a
wide spectrum of zebrafish models of human cancer has been developed largely through well-established
transgenic methodologies to oncogenes active in human cancers and the disruption of tumor suppressor
genes throuth targeting with zinc finger, Talen and most recently CRISPR-cas guided endonucleases.
Zebrafish models of many types of human cancers are under intensive investigation in many laboratories,
including hematologic malignancies, melanoma, rhabdomyosarcoma, Ewing sarcoma, well-differentiated
liposarcoma, testicular cancer, neuroblastoma, neuroendocrine carcinoma, and glioblastoma.
The transparency of the zebrafish embryos and the recent development of the pigment-deficient “casper”
zebrafish line gives one the valuable capacity to observe directly cancer formation and progression in the living
animal. The optical clarity of zebrafish can be exploited further by the use of fluorescent tags to label specific
cell lineages to visualize tumor processes including initiation, progression, and regression. Recently, the
power of zebrafish has been underscored by the remarkable progress and utility of tumor transplantation
methodologies performed in this model organism. The zebrafish is experimentally amenable to transplantation
assays that test the serial passage and malignant potential of fluorescently-labeled tumor cells as well as their
capacity to disseminate and/or metastasize. Several groups have also applied xenotransplantation methods to
zebrafish for the study of the human cancer cell malignancy within the context of the whole organism. The
experimental repertoire of the zebrafish allows unprecedented inquiry into the in vivo processes involved in the
pathogenesis of malignancy.
Importantly, comparisons of cancer-associated gene expression profiles between zebrafish and human
cancers reveal a high degree of similarity in gene signatures. In fact, the genetics of human cancer have been
recapitulated in zebrafish transgenic lines through the overexpression of human wild-type or mutant oncogenes
and through the generation of mutant lines harboring inactivating mutations in orthologous tumor suppressor
genes. In addition, the resultant tumor histopathologies in these animals are strikingly similar to the human
diseases, further emphasizing the suitability of zebrafish for dissecting the molecular mechanisms that underlie
pathogenesis. Relevant pathways include those aberrantly affecting the cell cycle, genomic stability,
apoptosis, or normal development and differentiation. The zebrafish models of cancer established to date
have been highly informative with respect to the molecular basis of human tumorigenesis and progression.
Due to its fecundity and the optical clarity during embryonic development, the zebrafish has also proven to be
an excellent in vivo model system for high throughput drug screening, because it allows the visual assessment
of both drug efficacy and toxicity. Drug activities discovered in high throughput screens using zebrafish models
are now entering Phase I clinical trials in humans. Adult fish are also suitable for testing drugs that are
administered as water-soluble compounds, by simply adding them directly to the water for treatment. Taken
together, zebrafish represent a powerful vertebrate animal model both for dissecting the molecular pathways
underlying human cancer and chemical screening for anticancer drug development.
In summary, the zebrafish presents an exciting model organism system that is ideal for the in vivo analysis of
signaling pathways in malignancy and as a platform for the development of novel therapeutic strategies.
Zebrafish already play an important role in cancer research and the system is constantly evolving to
encompass novel experimental strategies that further empower its use for the study of carcinogenesis. For
example, there have been major recent advances in the development of transplantation techniques. These
transplantation strategies exploit the unique capability of zebrafish for in vivo imaging to address cancer cell
intravasation and metastasis, self-renewal, and host-tumor cell interactions using both zebrafish and human
cancer cells. Recent advances in zebrafish genetics further expand the cancer research tool box in this model
organism. The establishment of mosaic analysis in zebrafish, which permits rapid clonal analysis of gene
function and cell-lineage tracing via in vivo live imaging, introduces a valuable approach in zebrafish genetics
that can be utilized for the study of oncogene-expressing cells in the wild-type environment . The zebrafish is
also emerging as a powerful system for exploring the function of noncoding RNAs (microRNAs), a new class of
elegant molecular regulators, in cancer-relevant cellular processes. Understanding the specific regulatory
mechanisms of signaling pathways dysregulated in cancer by noncoding RNAs may provide further important
insights into the molecular basis of cancer initiation and progression.
NEI – Zebrafish Studies
Nicholas Katsanis Ph.D.
The National Eye Institute (NEI) has been a consistently strong supporter of zebrafish research. Although the
extramural research portfolio of the Institute has supported numerous diverse activities, there are three areas
that merit particular attention: a) understanding basic biology of the development, maintenance and function of
the organ; b) mechanisms of regeneration; c) modeling human genetic disorders of the eye
1. Basic biology of eye development, maintenance and function. Shared among the NIH institutes has been the
recognition that the speed, ease of access, translucency, and ability to manipulate embryos genetically renders
zebrafish as the premier tool to study development. In the context of the human eye, numerous intramural and
extramural investigators have used this system to understand basic developmental mechanisms of the eye, the
neural retina, cornea and lens. The field has grown robustly, evidenced by the large group of investigators
across the country who use the zebrafish model to study discrete pathways and processes and the
logarithmically expanding numbers of papers. In the past three years alone, the power of this model organism
has been used in studies that include forward genetic screens to identify new genes necessary for retinal
development (Lee et al, 2012); the study of transcription factors (Brown et al, 2009, Chen et al, 2012), secreted
molecules (Wan et al, 2012) and structural proteins (Clendenon et al, 2012) as they pertain to organogenesis;
and the dissection of mechanisms that regulate the development and maintenance of the retina at the level of
cell movements and migration (Kwan et al, 2012), axonal guidance (Xiao et al, 2012), and neural circuitry
integration (Stacher-Hörndli et al, 2012). This is a non-exhaustive list to illustrate the breadth of activity.
Crucially, much of this work is fully translatable in higher organisms, including humans, since many of the
fundamental processes discovered in the zebrafish developing retina have been implicated in human genetic
disorders, including not only genes relevant to tissue development and homeostasis, but also specialized
processes including phototransduction (e.g. Riazuddin S et al, 2012).
2. Zebrafish retina regeneration and injury repair. Similar tot he heart and the fin, the zebrafish retina has the
ability to regenerate (see Brockerhoff and Fadool, 2011, for a recent review). These observations hold the
promise of offering alternative treatments for degenerative disorders of the retina, a leading cause of blindness
worldwide. This is particularly important since even with promising advances in gene and stem cell therapy,
retinal disorders cause irreversible photoreceptor loss. Recent studies in the zebrafish photoreceptor have
begun to identify key pathways that regulate regeneration, including Wnt-b catenin and novel transcriptional
circuits (Ramachandran et al, 2011, 2012)
3. Modeling human genetic disorders. This is perhaps the newest facet of zebrafish research n the eye and is
coupled with the hype-acceleration of human genetics and genomics. A host of studies in patients and families
have identified firm or candidate genes for eye disorders; the zebrafish system has proven to be an invaluable
tool to complement these studies, both by providing in vivo experimental evidence for the pathogenic potential
of mutations found in patients (e.g. Riazuddin et al, 2012, Züchner et al, 2011) and for offering an inexpensive
and rapid means to obtaining an initial biological model (e.g. Peachy et al, 2012) as a means to study
pathomechanism (e.g. Bohnsack et al, 2012). Excitingly, these models are becoming useful in the study of
both rare and complex traits, as exemplified by the recent modeling of mutations in myopia (Veth et al, 2011),
corneal dystrophy (Riazuddin SA et al, 2010) and age-related macular degeneration (van de Ven et el, 2013).
References:
Bohnsack BL, Kasprick DS, Kish PE, Goldman D, Kahana A. A zebrafish model of axenfeld-rieger syndrome
reveals that pitx2 regulation by retinoic acid is essential for ocular and craniofacial development. Invest
Ophthalmol Vis Sci. 2012 Jan 3;53(1):7-22. doi: 10.1167/iovs.11-8494.
Brockerhoff SE, Fadool JM. Genetics of photoreceptor degeneration and regeneration in zebrafish. Cell Mol
Life Sci. 2011 Feb;68(4):651-9. doi: 10.1007/s00018-010-0563-8.
Brown, J. D et al. (2009) Expression profiling during ocular development identifies 2 Nlz genes with a critical
role in optic fissure closure. Proc Natl Acad Sci 106, 1462-7.
Chen Z, Li X, Desplan C. Deterministic or stochastic choices in retinal neuron specification. Neuron. 2012 Sep
6;75(5):739-42. doi: 10.1016/j.neuron.2012.08.008.
Clendenon SG, Sarmah S, Shah B, Liu Q, Marrs JA. Zebrafish cadherin-11 participates in retinal differentiation
and retinotectal axon projection during visual system development. Dev Dyn. 2012 Mar;241(3):442-54. doi:
10.1002/dvdy.23729.
Kwan KM, Otsuna H, Kidokoro H, Carney KR, Saijoh Y, Chien CB. A complex choreography of cell movements
shapes the vertebrate eye. Development. 2012 Jan;139(2):359-72. doi: 10.1242/dev.071407.
Lee J, Cox BD, Daly CM, Lee C, Nuckels RJ, Tittle RK, Uribe RA, Gross JM. An ENU mutagenesis screen
in zebrafish for visual system mutants identifies a novel splice-acceptor site mutation in patched2 that results in
Colobomas. Invest Ophthalmol Vis Sci. 2012 Dec 13;53(13):8214-21. doi: 10.1167/iovs.12-11061.
Peachey NS et al. GPR179 is required for depolarizing bipolar cell function and is mutated in autosomalrecessive complete congenital stationary night blindness. Am J Hum Genet. 2012 Feb 10;90(2):331-9. doi:
10.1016/j.ajhg.2011.12.006.
Ramachandran, R., Zhao, X-F. and Goldman, D. An Ascl1a/Dkk/beta-Catenin signaling pathway is necessary
and GSK-3beta inhibition is sufficient for zebrafish retina regeneration. Proc Natl Acad Sci USA, 2011;
108:15858-15863.
Ramachandran R, Zhao XF, Goldman D. Insm1a-mediated gene repression is essential for the formation and
differentiation of Müller glia-derived progenitors in the injured retina. Nat Cell Biol. 2012 Oct;14(10):1013-23.
doi: 10.1038/ncb2586.
Riazuddin SA, et al. Missense mutations in TCF8 cause late-onset Fuchs corneal dystrophy and interact with
FCD4 on chromosome 9p. Am J Hum Genet. 2010 Jan;86(1):45-53. doi: 10.1016/j.ajhg.2009.12.001.
Riazuddin S et al. Alterations of the CIB2 calcium- and integrin-binding protein cause Usher syndrome type 1J
and nonsyndromic deafness DFNB48. Nat Genet. 2012 Nov;44(11):1265-71. doi: 10.1038/ng.2426.
Stacher Hörndli C, Chien CB. Sonic hedgehog is indirectly required for intraretinal axon pathfinding by
regulating chemokine expression in the optic stalk. Development. 2012 Jul;139(14):2604-13. doi:
10.1242/dev.077594.
van de Ven et al. A functional variant in the CFI gene confers a high risk of age-related macular degeneration.
In Press Nat Genet (2013)
Veth KN, et al. Mutations in zebrafish lrp2 result in adult-onset ocular pathogenesis that models myopia and
other risk factors for glaucoma. PLoS Genet. 2011 Feb;7(2):e1001310. doi: 10.1371/journal.pgen.1001310.
Wan J, Ramachandran R, Goldman D. HB-EGF is necessary and sufficient for Müller glia dedifferentiation and
retina regeneration. Dev Cell. 2012 Feb 14;22(2):334-47. doi: 10.1016/j.devcel.2011.11.020.
Xiao T, Staub W, Robles E, Gosse NJ, Cole GJ, Baier H. Assembly of lamina-specific neuronal connections by
slit bound to type IV collagen. Cell. 2011 Jul 8;146(1):164-76. doi: 10.1016/j.cell.2011.06.016.
Züchner S, et al. Whole-exome sequencing links a variant in DHDDS to retinitis pigmentosa. Am J Hum Genet.
2011 Feb 11;88(2):201-6. doi: 10.1016/j.ajhg.2011.01.001. Epub 2011 Feb 3.
NHGRI – Zebrafish Studies
John Postlethwait
Shawn Burgess
Zebrafish research has added to our understanding of the evolution and function of vertebrate genomes, thus
contributing to the mission of the NHGRI: the advancement of health through genome research and the
expansion of our understanding of human biology.
The zebrafish genome provides several advantages for understanding the origin and function of the human
genome, including appropriate phylogenetic separation from the human genome; a genome duplication event
that provided zebrafish with two copies of many human genes that facilitates some analyses; and tractability
for mutagenesis and transgenesis that allows researchers insights into the functions of genes and regulatory
elements.
Defining the roles of conserved non-coding regions.
Many functional regions of the human genome have been strongly conserved even though they do not encode
a protein. The functions of most of these conserved noncoding elements (CNEs) is not well understood, but
according to evolutionary theory, they would have faded away if they were not preserved by selection.
Mammalian genomes are so closely related to the human genome that many sequences are similar due to
shared phylogeny rather than function. In contrast, the zebrafish genome is sufficiently diverged from the
human genome to identify CNEs that have been maintained due to conserved function. Many CNEs are
regulatory in nature (Woolfe, Goodson et al. 2005), and using zebrafish, it is straightforward to learn their
functions: simply excise the zebrafish or human CNE, place it upstream of a reporter gene like green
fluorescent protein driven by a basal promoter, inject the construct into one-cell stage zebrafish embryos, and
observe its expression pattern. For example, the PITX2 coding sequence is mutated in many cases of AxenfeldRieger syndrome, which causes ocular, dental, and umbilical abnormalities. The identification of CNEs around
the zebrafish ortholog of PITX2 and their functional testing revealed regulatory elements for tissues involved
in this syndrome and analysis of patient DNAs revealed deletions of some of these CNEs in the presence of
intact coding sequence, thus explaining the genomic origin of the disease (Volkmann, Zinkevich et al. 2011). A
similar strategy using zebrafish/human CNEs tested in zebrafish embryos identified a regulatory element
between the human fibrinogen alpha and beta genes that drives liver specific expression that is a candidate
for explaining genetic differences in fibrinogen production, a risk factor in cardiovascular disease (Fort, Fish et
al. 2011). In such zebrafish embryo assays, it is prudent to check both the human and the zebrafish element
(Ritter, Li et al. 2010). A genome-wide effort to identify CNEs shared by zebrafish and humans and a
systematic global investigation of their functions using zebrafish assays would help define functions for much
of the non-coding human genome, helping to fulfill the mission of the NHGRI.
The evolutionary dynamics involved in maintaining genomic regulatory blocks (gene syntenies).
Some genomic regions have remained intact with conserved syntenies shared from zebrafish to human and all
species in between. Functional genomics using zebrafish has identified the mechanisms that help retain these
genomic blocks. In a genomic regulatory block (GRB), enhancers residing in introns of one gene can control the
expression of other nearby genes; if chromosome rearrangements disrupt the positioning of the enhancer and
its target, then development becomes abnormal (Kikuta, Laplante et al. 2007; Navratilova, Fredman et al.
2009). This is important for understanding the function of the human genome because many single nucleotide
polymorphisms (SNPs) identified in genome wide association studies (GWAS) reside in introns or intergenic
regions and may be in linkage disequilibrium with enhancers that may be far from the genes they regulate and
thus difficult to understand. As an example, consider recent work that showed that IRX3, which resides in a
genomic regulatory block that includes two other genes and lies near SNPs identified by GWAS, is associated
with conserved elements that drive expression in pancreas, and that the knockdown of the zebrafish IRX3
ortholog decreased the number of insulin-producing beta-cells and glucagon-producing alpha-cells, thus
suggesting that the action of IRX3 may be related to both obesity and type 2 diabetes (Ragvin, Moro et al.
2010).
The role of gene duplication in subfunctionalization and neofunctionalization of genes in vertebrate evolution.
Two rounds of whole Genome Duplication preceded the radiation of vertebrates (VGD1 and VGD2) about 450
million years ago and these events shaped the human genome. Many, or maybe most, groups of paralogous
human genes come from these events, including the four HOX clusters, the four NOTCH genes, three DLX
clusters, three hedgehog paralogs (SHH, IHH, DHH) and many more. An additional round of genome
duplication occurred about 350 million years ago at the base of the teleost radiation (the TGD), resulting in
zebrafish having duplicate copies of about 25% of the homologous human genes (Postlethwait, Yan et al.
1998). The TGD serves as a model for evolutionary events occurring after the VGD1 and VGD2 events.
Investigation of the expression patterns of zebrafish gene duplicates revised our understanding of gene
evolution after genome duplication (Force, Lynch et al. 1999): most genes that are retained in duplicate copy
evolved by subfunctionalization – the partitioning of ancestral gene subfunctions between the two new gene
duplicates (Force, Lynch et al. 1999). The preservation of duplicate genes by subfunctionalization allows
formerly pleiotropic genes to become specialized for a subset of their original functions and in addition
preserves both copies for sufficient evolutionary time to increase the likelihood that they might assume new
functions by neofunctionalization.
Defining the function of all genes in the human genome via gene inactivation in the zebrafish model.
The zebrafish genome has become only the third vertebrate genome (after human and mouse) to obtain a
“finished” level of sequencing. As has been performed in most genetically tractable model organisms where
the transcriptome has been fully identified, the next step for zebrafish was to systematically knock out all
protein coding genes. The Zebrafish Mutation Project at the Sanger Institute has identified stop or essential
splice site mutations in 8530 (32%) of zebrafish genes, nearly a third of all genes in the animal (Consortium
2013). These are held as cryopreserved sperm and can be reconstituted as swimming fish available at ZIRC, the
Zebrafish International Resource Center. Parallel efforts at the National Human Genome Research Institute
intramural program performing mutagenesis by insertional mutations followed by rapid identification of
insertion sites has provided mutations in another 3,054 genes or another 12% of the genome (Burgess and
Hopkins 2000; Varshney et al. 2013). Each of these efforts has made the mutations available to the entire
research community without restrictions on use and both efforts are ongoing. Furthermore, the targeted
knockout of zebrafish genes by zinc finger nucleases (Ekker 2008; Foley, Yeh et al. 2009; Gupta, Meng et al.
2011; Reyon, Kirkpatrick et al. 2011; Sander, Dahlborg et al. 2011), TALENs (Huang, Xiao et al. 2011; Sander,
Cade et al. 2011; Moore, Reyon et al. 2012; Gupta, Hall et al. 2013) or, remarkably quickly and cheaply, by the
CRISPR-Cas system (Blackburn, Campbell et al. 2013; Chang, Sun et al. 2013; Hwang, Fu et al. 2013), makes it
realistic to think about a knockout in every zebrafish coding sequence. A knockout mutation in every gene in a
vertebrate would both teach us about what it takes to build and maintain function for vertebrates, and
provide “ready-made” models for human genetic diseases. These disease models provide both mechanistic
insights into the pathology of the diseases, and provides potential resources for screening or testing therapies
for the human diseases.
References:
Blackburn, P. R., J. M. Campbell, et al. (2013). "The CRISPR System-Keeping Zebrafish Gene Targeting Fresh."
Zebrafish.
Burgess, S. and N. Hopkins (2000). "Use of pseudotyped retroviruses in zebrafish as genetic tags." Methods
Enzymol 327: 145-161.
Chang, N., C. Sun, et al. (2013). "Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos." Cell
Res.
Consortium, Z. K. (2013). "Zebrafish mutation project: Knockouts for disease models." Retrieved 2013 03 31,
from http://www.sanger.ac.uk/Projects/D_rerio/zmp/.
Ekker, S. C. (2008). "Zinc finger-based knockout punches for zebrafish genes." Zebrafish 5(2): 121-123.
Foley, J. E., J. R. Yeh, et al. (2009). "Rapid mutation of endogenous zebrafish genes using zinc finger nucleases
made by Oligomerized Pool ENgineering (OPEN)." PLoS ONE 4(2): e4348.
Force, A., M. Lynch, et al. (1999). "Preservation of duplicate genes by complementary, degenerative
mutations." Genetics 151(4): 1531-1545.
Fort, A., R. J. Fish, et al. (2011). "A liver enhancer in the fibrinogen gene cluster." Blood 117(1): 276-282.
Gupta, A., V. L. Hall, et al. (2013). "Targeted Chromosomal Deletions and Inversions in Zebrafish." Genome
Res.
Gupta, A., X. Meng, et al. (2011). "Zinc finger protein-dependent and -independent contributions to the in vivo
off-target activity of zinc finger nucleases." Nucleic Acids Res 39(1): 381-392.
Huang, P., A. Xiao, et al. (2011). "Heritable gene targeting in zebrafish using customized TALENs." Nat
Biotechnol 29(8): 699-700.
Hwang, W. Y., Y. Fu, et al. (2013). "Efficient genome editing in zebrafish using a CRISPR-Cas system." Nat
Biotechnol 31(3): 227-229.
Kikuta, H., M. Laplante, et al. (2007). "Genomic regulatory blocks encompass multiple neighboring genes and
maintain conserved synteny in vertebrates." Genome Res 17(5): 545-555.
Moore, F. E., D. Reyon, et al. (2012). "Improved somatic mutagenesis in zebrafish using transcription activatorlike effector nucleases (TALENs)." PLoS ONE 7(5): e37877.
Navratilova, P., D. Fredman, et al. (2009). "Systematic human/zebrafish comparative identification of cisregulatory activity around vertebrate developmental transcription factor genes." Dev Biol 327(2): 526-540.
Postlethwait, J. H., Y.-L. Yan, et al. (1998). "Vertebrate genome evolution and the zebrafish gene map." Nat
Genet 18(4): 345-349.
Ragvin, A., E. Moro, et al. (2010). "Long-range gene regulation links genomic type 2 diabetes and obesity risk
regions to HHEX, SOX4, and IRX3." Proc Natl Acad Sci U S A 107(2): 775-780.
Reyon, D., J. R. Kirkpatrick, et al. (2011). "ZFNGenome: a comprehensive resource for locating zinc finger
nuclease target sites in model organisms." BMC Genomics 12: 83.
Ritter, D. I., Q. Li, et al. (2010). "The importance of being cis: evolution of orthologous fish and mammalian
enhancer activity." Mol Biol Evol 27(10): 2322-2332.
Sander, J. D., L. Cade, et al. (2011). "Targeted gene disruption in somatic zebrafish cells using engineered
TALENs." Nat Biotechnol 29(8): 697-698.
Sander, J. D., E. J. Dahlborg, et al. (2011). "Selection-free zinc-finger-nuclease engineering by contextdependent assembly (CoDA)." Nat Methods 8(1): 67-69.
Varshney, G. K., J. Liu, et. al. (2013) A large-scale zebrafish gene knockout resource for the genome-wide study
of gene function. Genome Res 23: 727-735.
Volkmann, B. A., N. S. Zinkevich, et al. (2011). "Potential novel mechanism for Axenfeld-Rieger syndrome:
deletion of a distant region containing regulatory elements of PITX2." Invest Ophthalmol Vis Sci 52(3): 14501459.
Woolfe, A., M. Goodson, et al. (2005). "Highly conserved non-coding sequences are associated with vertebrate
development." PLoS Biol 3(1): e7.
NHLBI – Zebrafish Studies
Nathan Lawson
Barry Paw
Debbie Yelon
Since developmental processes are conserved amongst animals and are often recapitulated in pathological
settings, the findings gained from zebrafish have yielded new insights into disease mechanisms. Nowhere has
this been more evident than in research related to the mission of the National Heart, Lung, and Blood Institute
(NHLBI), which promotes the prevention and treatment of cardiovascular diseases. The following are
examples from the past decade where the zebrafish has made major contributions to the NHLBI mission.
Hematopoietic Stem Cell (HSC) Biology. The transparency and external development of zebrafish embryos,
coupled with the use of transgenes expressing fluorescence reporters under tissue-specific promoters, has
allowed investigators to directly visualize HSCs in vivo, providing novel insights into the origin and homing of
HSCs [1, 2]. Zebrafish have also been used in screens to discover molecules that can modulate HSC
proliferation, leading to identification of new compounds that can expand HSC populations [3, 4]. This work has
set the stage for clinical trials to determine the efficacy of such compounds for HSC expansion prior to
transplantation in humans.
Iron and Heme Biology of Red Cells. The field of iron biology has undergone a renaissance based on
discoveries from zebrafish genetics. The major iron exporter, ferroportin, was discovered as the causative
gene responsible for the zebrafish mutant, weißherbst [5]. This gene is now known to be a regulator of iron
homeostasis in humans and is disrupted in patients with hemochromatosis type 4 [6]. Recent studies have
revealed additional genes regulating heme biogenesis and iron acquisition in zebrafish [7; 8], identifying
additional likely candidates for related human diseases.
Mechanisms of heart disease. Over the last ten years, zebrafish studies have revealed novel insights into
cardiac specification, morphogenesis, and physiology that are relevant to the etiologies of congenital heart
diseases in humans. Examples include the roles of the TGF-beta binding protein Ltbp3 in cardiac outflow tract
formation [9] and the sphingolipid transporter Spns2 in cardiac precursor cell migration [10, 11]. Researchers
have also employed zebrafish to reveal the genetic origins of human heart disease. Striking examples include
the use of zebrafish to implicate mutations in NEXN (encoding the Z-disk component Nexilin) as causative for
dilated cardiomyopathy [12] and the impact of the ciliopathy gene NPHP4 on cardiac laterality [13]. These
models have had tremendous impact on our understanding of disease mechanisms and have motivated new
therapeutic strategies.
Mechanisms of cardiac regeneration. The adult zebrafish heart has a remarkable ability to regenerate in
response to injury [14]. Investigations into this process have revealed new cellular and molecular mechanisms
underlying cardiac regeneration triggered by cardiac damage [15, 16], including roles for retinoic acid, FGF,
and PDGF pathways [17-19]. Continued pursuit of these mechanisms will provide a better comprehension of
the factors that limit the regenerative capacity of the human heart and inspire new therapeutic approaches for
stimulating cardiac repair in patients.
Basic mechanisms of blood vessel formation and function. The zebrafish has been an ideal model to study
vascular function and formation. Transgenic zebrafish embryos in which endothelial cells are fluorescently
labeled allow their direct visualization in vivo [20], revealing novel cellular mechanisms to describe sprouting
blood vessel growth [21] and how the nascent aorta forms [22, 23]. Transgenic zebrafish have also facilitated
genetic screens to reveal new genes required for vascular patterning [24], growth [25], and homeostasis [26].
Importantly, these highly conserved genes are considered therapeutic targets for manipulating blood vessel
growth in humans. The availability of such mutants allows detailed mechanistic studies to continue in parallel
to clinical efforts – an ideal platform to facilitate advances in translational medicine.
Models of vascular disease. The zebrafish is beginning to prove its worth in the study of vascular disease,
further demonstrating its potential in translational medicine. Recent work highlights the use of zebrafish to
study atherosclerosis [27] and reactive oxygen species (ROS) in blood vessel homeostasis [26]; both major
components of vascular disease. The use of zebrafish as a disease model has been aided by NHLBI-funded
research that led to breakthroughs in the development of targeted gene knockout approaches in zebrafish [28].
This technology now permits generation of zebrafish bearing mutations in genes known to be causative for
human disease (for example, [29]). Moving forward, application of this technology will enable the continued
development of zebrafish models of vascular diseases.
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NIA – Zebrafish Studies
Zebrafish for the Study of Biology and Biomedical Science on Aging
Shuji Kishi (Scripps) and Matthew Harris (Harvard)
A comprehensive understanding of the molecular mechanisms of aging in vertebrates is a major challenge of
modern biology and biomedical science. This is, in part, due to the complexity of the aging process and its
multi-factorial nature. Biomedical research into the pathophysiology and etiology of aging has excelled with the
use of animal models amenable for unbiased screens and experimental analysis of gene function in both early
and late life stages. The extension of research from invertebrate models has shown the conservation of
essential mechanisms of senescence and longevity in vertebrates. However there has not been a model within
vertebrates that could utilize an unbiased high-throughput genetic approach to expose vertebrate-specific
aspects of the molecular mechanisms of aging and the regulation of health span and longevity.
The zebrafish provides a powerful experimental model organism in which to systematically identify the genetic
regulation of aging and to analyze the physiological and anatomical changes occurring in normal aging, as well
as in the manifestation of aging-related disease. Age-associated phenotypes of zebrafish were first described
just about 10 years ago in 2003, and importantly, zebrafish exhibit gradual aging phenotypes similar to those
observed in humans (Kishi et al., 2003) (Gerhard, 2003). Furthermore, gene ablations or mutations associated
with premature aging in human progeroid syndromes lead to similar phenotypes in the zebrafish (Imamura and
Kishi, 2005) (Imamura et al., 2008) (Koshimizu et al., 2011) (Henriques et al., 2013). Research during the last
decade has further demonstrated the power and efficiency of the zebrafish model for analysis of vertebrate
aging and age-dependent pathophysiological changes in musculoskeletal and neuronal morphology, endocrine
factors, gene expression, regeneration, telomere metabolism, circadian clock, sleep and cognitive functions
(Kishi, 2006) (Tsai et al., 2007) (Zhdanova et al., 2008) (Yu et al., 2006) (Anchelin et al., 2011).
The zebrafish provides prominent experimental strengths in molecular genetics and genomics together with
optically anatomical transparency throughout the lifespan, having the entirely “see-through” casper and
absolute fish lines, which allow unprecedented analysis of gene function not only in early development but also
in adult tissues of live animals. Previous work on the zebrafish has focused on screens in early development to
permit identification of genes regulating senescence and aging (Kishi et al., 2008); these pioneering screens
clearly demonstrate the use of zebrafish as a tractable model system for the study of aging. Such vertebrate
models are of considerable importance, given the provocative evidence of common biochemical and functional
pathways modulating stress responses and lifespan as well as aging in a wide range of organisms. The
mutations identified by markers for embryonic and larval senescence indeed displayed accelerated aging
phenotypes in adult zebrafish, indicating that ‘senescence’ is detectable during development and future ‘aging’
can be predictable at least in part. Building on this work, one of current and future efforts is focusing on
identifications of rejuvenated “revertants” from accelerated aging and shortened longevity. These studies take
advantage of progeric zebrafish mutant lines to allow for systematic analysis of genes that can suppress the
expression of aging phenotypes. By use of such systematic, unbiased screens for stress response and
senescence-associated phenotypes, the zebrafish has the immediate potential to accelerate the discovery of
novel genes, chemicals and their new functions relevant for our understanding of aging processes common
among vertebrates. Such knowledge will be essential for the ultimate development of pharmacological,
nutritional, and behavioral interventions for the amelioration of age-associated diseases and disabilities in
humans.
An exciting prospect of the use of zebrafish in aging studies is the potential to quickly address the role of
particular genetic variants in the progression of aging and longevity. Given the power of genomic engineering
in zebrafish by using innovative reverse genetic approaches like TALEN and CRISPR/Cas systems (Cong et
al., 2013) (Bedell et al., 2012) (Dahlem et al., 2012), it is now possible, and efficient, to generate ‘humanized’
zebrafish harboring genetic variants identified in genome-wide association studies by creating mutations in
homologous genes in the fish; this allows for functional analysis of genetic variants on a scale unfeasible in
other vertebrate models. The zebrafish model is thus poised to provide fundamental insights into vertebrate
aging and the regulation of the onset and expressivity of human aging and age-associated disease.
References:
Anchelin, M., Murcia, L., Alcaraz-Perez, F., Garcia-Navarro, E.M., and Cayuela, M.L. (2011). Behaviour of
telomere and telomerase during aging and regeneration in zebrafish. PLoS One 6, e16955.
Bedell, V.M., Wang, Y., Campbell, J.M., Poshusta, T.L., Starker, C.G., Krug, R.G., 2nd, Tan, W., Penheiter,
S.G., Ma, A.C., Leung, A.Y., et al. (2012). In vivo genome editing using a high-efficiency TALEN system.
Nature 491, 114-118.
Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., et
al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823.
Dahlem, T.J., Hoshijima, K., Jurynec, M.J., Gunther, D., Starker, C.G., Locke, A.S., Weis, A.M., Voytas, D.F.,
and Grunwald, D.J. (2012). Simple methods for generating and detecting locus-specific mutations induced with
TALENs in the zebrafish genome. PLoS genetics 8, e1002861.
Gerhard, G.S. (2003). Comparative aspects of zebrafish (Danio rerio) as a model for aging research.
Experimental gerontology 38, 1333-1341.
Henriques, C.M., Carneiro, M.C., Tenente, I.M., Jacinto, A., and Ferreira, M.G. (2013). Telomerase is required
for zebrafish lifespan. PLoS genetics 9, e1003214.
Imamura, S., and Kishi, S. (2005). Molecular cloning and functional characterization of zebrafish ATM. The
international journal of biochemistry & cell biology 37, 1105-1116.
Imamura, S., Uchiyama, J., Koshimizu, E., Hanai, J., Raftopoulou, C., Murphey, R.D., Bayliss, P.E., Imai, Y.,
Burns, C.E., Masutomi, K., et al. (2008). A non-canonical function of zebrafish telomerase reverse
transcriptase is required for developmental hematopoiesis. PloS one 3, e3364.
Kishi, S. (2006). Zebrafish as Aging Models. Handbook of Models for Human Aging, 317-338.
Kishi, S., Bayliss, P.E., Uchiyama, J., Koshimizu, E., Qi, J., Nanjappa, P., Imamura, S., Islam, A., Neuberg, D.,
Amsterdam, A., et al. (2008). The identification of zebrafish mutants showing alterations in senescenceassociated biomarkers. PLoS genetics 4, e1000152.
Kishi, S., Uchiyama, J., Baughman, A.M., Goto, T., Lin, M.C., and Tsai, S.B. (2003). The zebrafish as a
vertebrate model of functional aging and very gradual senescence. Experimental gerontology 38, 777-786.
Tsai, S.B., Tucci, V., Uchiyama, J., Fabian, N.J., Lin, M.C., Bayliss, P.E., Neuberg, D.S., Zhdanova, I.V., and
Kishi, S. (2007). Differential effects of genotoxic stress on both concurrent body growth and gradual
senescence in the adult zebrafish. Aging cell 6, 209-224.
Yu, L., Tucci, V., Kishi, S., and Zhdanova, I.V. (2006). Cognitive aging in zebrafish. PloS one 1, e14.
Zhdanova, I.V., Yu, L., Lopez-Patino, M., Shang, E., Kishi, S., and Guelin, E. (2008). Aging of the circadian
system in zebrafish and the effects of melatonin on sleep and cognitive performance. Brain research bulletin
75, 433-441.
NiAAA – Zebrafish Studies
Eric Glasgow
Georgetown University Medical Center
An estimated 18 million Americans (8.5% of the population age 18 and older) suffer from alcohol use
disorders. This burdens U.S. taxpayers with an estimated $185 billion in annual costs. The NIAAA
supports research on the causes, consequences, prevention, and treatment of alcohol abuse and
related problems. Major challenges facing NIAAA’s goals of understanding mechanisms and
developing medications for alcohol use disorders are the large number of potential processes and
interactions responsible for the deleterious effects of alcohol, which depend on the concentration,
duration and life stage of exposure. Furthermore, alcohol and its metabolites can have differential
effects on different cells and tissues. Moreover, the causes and consequences of excessive alcohol
consumption are greatly influenced by genetic and environmental factors.
Zebrafish present an integrative, multi-organ, high-throughput vertebrate model for investigating
mechanisms of alcohol damage and developing medications for alcohol use disorders. Recent
research using zebrafish has demonstrated the relevance of alcohol studies in this organism to the
human condition. Zebrafish are being used to investigate an impressive array of alcohol-related
biological questions ranging from cellular consequences of alcohol exposure to behavioral outputs,
such as addiction. Zebrafish embryos exposed to various concentrations of alcohol show facial
dysmorphologies, defects in brain, somatic growth, and cardiac development, impaired endoplasmic
reticulum structure and function in hepatocytes, abnormalities in gene expression, and behavioral
abnormalities (startle reflex, learning and memory, and social behavior). The ease and accuracy of
dosing zebrafish embryos has resulted in the direct demonstration that the concentration and timing
of alcohol exposure is related to differential adverse developmental outcome. In addition, it has been
shown in zebrafish that specific neuronal cell-types are differentially sensitive to alcohol exposure
within identifiable developmental widows.
In adult zebrafish, acute alcohol exposure has been shown to result in significant changes in
dopamine, serotonin and related brain metabolites. Microarray analysis has been coupled with the
conditioned place preference assay to identify changes in behavior coupled to changes in gene
expression. Withdrawal after chronic alcohol exposure has been profiled in a novel tank diving test
and correlated with changes in whole body cortisol. In addition, different strains of zebrafish show
differences in behavioral and neurochemical responses while undergoing withdrawal.
Using zebrafish as a model system a number of molecules and pathways have been shown to modify
the detrimental effects of alcohol exposure including retinoic acid, FGF, sonic hedgehog, cAMP,
extracellular signal-regulated kinase, GSK3beta, and agrin. Furthermore, candidate molecules and
pathways that underlie neuro-adaptation to both ethanol and nicotine have been identified including,
glutamate receptors, benzodiazepine receptors and molecules associated with synaptic plasticity.
Alcohol elicits differential effects on multiple organ systems. Therefore, a distinct advantage of the
zebrafish embryo is its small size, which enables observation of all organ systems in the entire
animal. For example, the recent discovery of oxytocin induction in the hindbrain following alcohol
exposure has uncovered a potentially new neural pathway related to excessive alcohol consumption
and addiction.
References
Ali, S., Champagne, D.L., Alia, A., and Richardson, M.K. (2011). Large-scale analysis of acute ethanol
exposure in zebrafish development: a critical time window and resilience. PLoS One 6, e20037.
Buske, C., and Gerlai, R. (2011). Early embryonic ethanol exposure impairs shoaling and the dopaminergic
and serotoninergic systems in adult zebrafish. Neurotoxicol. Teratol.
Cachat, J., Canavello, P., Elegante, M., Bartels, B., Hart, P., Bergner, C., Egan, R., Duncan, A., Tien, D.,
Chung, A., et al. (2010). Modeling withdrawal syndrome in zebrafish. Behav. Brain Res. 208, 371-376.
Carvan, M., Loucks, E., Weber, D., and Williams, F. (2004). Ethanol effects on the developing zebrafish:
neurobehavior and skeletal morphogenesis. Neurotoxicol. Teratol. 26, 757-768.
Coffey, C.M., Solleveld, P.A., Fang, J., Roberts, A.K., Hong, S.K., Dawid, I.B., Laverriere, C.E., and Glasgow,
E. (2013). Novel oxytocin gene expression in the hindbrain is induced by alcohol exposure: transgenic
zebrafish enable visualization of sensitive neurons. PLoS One 8, e53991.
Dlugos, C.A., Brown, S.J., and Rabin, R.A. (2011). Gender differences in ethanol-induced behavioral sensitivity
in zebrafish. Alcohol 45, 11-18.
Egan, R.J., Bergner, C.L., Hart, P.C., Cachat, J.M., Canavello, P.R., Elegante, M.F., Elkhayat, S.I., Bartels,
B.K., Tien, A.K., Tien, D.H., et al. (2009). Understanding behavioral and physiological phenotypes of stress
and anxiety in zebrafish. Behav. Brain Res. 205, 38-44.
Fernandes, Y., and Gerlai, R. (2009). Long-Term Behavioral Changes in Response to Early Developmental
Exposure to Ethanol in Zebrafish. Alcoholism-Clinical and Experimental Research 33, 601-609.
Gerlai, R., Chatterjee, D., Pereira, T., Sawashima, T., and Krishnannair, R. (2009). Acute and chronic alcohol
dose: population differences in behavior and neurochemistry of zebrafish. Genes Brain and Behavior 8, 586599.
Howarth, D.L., Vacaru, A.M., Tsedensodnom, O., Mormone, E., Nieto, N., Costantini, L.M., Snapp, E.L., and
Sadler, K.C. (2012). Alcohol disrupts endoplasmic reticulum function and protein secretion in hepatocytes.
Alcohol. Clin. Exp. Res. 36, 14-23.
Li, Y., Yang, H., Zdanowicz, M., Sicklick, J.K., Qi, Y., Camp, T.J., and Diehl, A.M. (2007). Fetal alcohol
exposure impairs hedgehog cholesterol modification and signaling. Laboratory Investigation 87, 231-240.
Marrs, J.A., Clendenon, S.G., Ratcliffe, D.R., Fielding, S.M., Liu, Q., and Bosron, W.F. (2010). Zebrafish fetal
alcohol syndrome model: effects of ethanol are rescued by retinoic acid supplement. Alcohol 44, 707-715.
Miller, N., Greene, K., Dydinski, A., and Gerlai, R. (2013). Effects of nicotine and alcohol on zebrafish (Danio
rerio) shoaling. Behav. Brain Res. 240, 192-196.
Pan, Y., Kaiguo, M., Razak, Z., Westwood, J.T., and Gerlai, R. (2011). Chronic alcohol exposure induced gene
expression changes in the zebrafish brain. Behav. Brain Res. 216, 66-76.
Peng, J., Wagle, M., Mueller, T., Mathur, P., Lockwood, B.L., Bretaud, S., and Guo, S. (2009). Ethanolmodulated camouflage response screen in zebrafish uncovers a novel role for cAMP and extracellular signalregulated kinase signaling in behavioral sensitivity to ethanol. J. Neurosci. 29, 8408-8418.
Rosemberg, D.B., da Rocha, R.F., Rico, E.P., Zanotto-Filho, A., Dias, R.D., Bogo, M.R., Bonan, C.D., Moreira,
J.C., Klamt, F., and Souza, D.O. (2010). Taurine prevents enhancement of acetylcholinesterase activity
induced by acute ethanol exposure and decreases the level of markers of oxidative stress in zebrafish brain.
Neuroscience 171, 683-692.
Wagle, M., Mathur, P., and Guo, S. (2011). Corticotropin-releasing factor critical for zebrafish camouflage
behavior is regulated by light and sensitive to ethanol. J. Neurosci. 31, 214-224.
Yelin, R., Ben-Haroush Schyr, R., Kot, H., Zins, S., Frumkin, A., Pillemer, G., and Fainsod, A. (2005). Ethanol
exposure affects gene expression in the embryonic organizer and reduces retinoic acid levels. Dev. Biol. 279,
193-204.
Zhang, C., Ojiaku, P., and Cole, G.J. (2013). Forebrain and hindbrain development in zebrafish is sensitive to
ethanol exposure involving agrin, Fgf, and sonic hedgehog function. Birth Defects Res. A. Clin. Mol. Teratol.
97, 8-27.
NIAID – Zebrafish Studies
David Traver
Despite the zebrafish being only a relative newcomer to the immunological field, the past decade has seen a
wealth of novel findings arise in this model system that have proven complementary to those from studies in
standard rodent model systems. The proven power of the zebrafish in unbiased forward genetic approaches,
as well as in direct imaging modalities has yielded new insights into a variety of topics central to the mission of
the NIAID. These include insights into how a variety of infectious diseases are initiated, disseminated, and
propagated in the vertebrate animal, how each major immune cell lineage contributes to the immune cell
response, what the barriers are to immune cell transplantation, the ontogeny of immune cell subsets, and the
identification of novel small molecules able to modulate immunity and immune cell transformation.
The zebrafish provides unparalled ability to image the course of infection and its immune response in the
translucent embryo. For example, the dissemination of infectious Mycobacterium marinum bacteria, a close
relative of Mycobacterium tuberculosis, can be observed over days, weeks, and months following infection of
embryos. The development of fluorescent reporter lines that mark each immune cell lineage, coupled with the
labeling of M. marinum in complementary fluors has allowed the identification of the immune cell types that
both fight or foster infection. Unlike mouse models of M. tuberculosis infection, the zebrafish immune response
results in formation of robust granulomas, a hallmark of human disease. Formation of granulomas in the
zebrafish have been observed in real time in living embryos, and the cell types identified that spread infectious
particles to other tissues in the animal. Importantly, the facile nature of forward genetic and chemical screens
have also identified new pathways and drugs that have already proven useful in improved treatment of
tuberculosis patients.
The availability of a new vertebrate model for studies of inflammation and immunity provides the ability to
compare and contrast immune cell function over evolution. The zebrafish, like other teleosts, possesses all of
the major immune cell subsets found in mammals. Improvements in transgenic technology has resulted in the
creation of reporter lines for each of these major lineages in the zebrafish. For example, triple transgenic lines
where T lymphocytes, B lymphocytes, and dendritic cells are specifically labeled now allow the immune
response to be imaged in toto following experimental infection. Studies on natural killer cells have also shown
that a class of receptors similar to but distinct from human KIRs/LIRs is present in the zebrafish, enabling
comparative studies to better inform how our own cells function to control immunity and transplant tolerance or
rejection.
Developmental studies have demonstrated that immune cell precursors arise in three distinct waves during
embryogenesis. An emerging interest stemming from these developmental studies is the ontogeny and
function of dendritic cell subsets. It is widely appreciated that dendritic cells are the key coordinators of the
immune response, and recent studies have demonstrated that brain microglia are involved in the pathology of
a number of human neurodegenerative conditions. Creation of transgenic markers of microglia coupled with
brain inflammation models have positioned the zebrafish as a complementary system to study the interface of
immune cells with cells of the brain.
In closing, the availability of a new vertebrate model, with a rapidly increasing molecular toolbox, now enables
the study of self versus non-self recognition in a manner complementary to studies in rodent models and
human cell culture systems. In just a matter of years, discoveries made in the zebrafish have been translated
to clinical trials in humans. The exponential growth of investigators studying the immune response in zebrafish
bodes well for continued new discoveries relevant to human health.
Relevant Literature:
Traver D, Herbomel P, Patton EE, Murphey RD, Yoder JA, Litman GW, Catic A, Amemiya CT, Zon LI, Trede
NS. The zebrafish as a model organism to study development of the immune system. Adv Immunol.
2003;81:253-330. PMID: 14711058
Davis JM, Clay H, Lewis JL, Ghori N, Herbomel P, Ramakrishnan L. Real-time visualization of
mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos.
Immunity. 2002 Dec;17(6):693-702. PMID: 12479816.
Yoder JA, Litman RT, Mueller MG, Desai S, Dobrinski KP, Montgomery JS, Buzzeo MP, Ota T, Amemiya CT,
Trede NS, Wei S, Djeu JY, Humphray S, Jekosch K, Hernandez Prada JA, Ostrov DA, Litman GW.
Resolution of the novel immune-type receptor gene cluster in zebrafish. Proc Natl Acad Sci U S A. 2004
Nov 2;101(44):15706-11. Epub 2004 Oct 20. PMID: 15496470.
van der Sar AM, Stockhammer OW, van der Laan C, Spaink HP, Bitter W, Meijer AH. MyD88 innate immune
function in a zebrafish embryo infection model. Infect Immun. 2006 Apr;74(4):2436-41. PMID: 16552074.
Murayama E, Kissa K, Zapata A, Mordelet E, Briolat V, Lin HF, Handin RI, Herbomel P. Tracing hematopoietic
precursor migration to successive hematopoietic organs during zebrafish development. Immunity. 2006
Dec;25(6):963-75. Epub 2006 Dec 7. PMID: 17157041.
Li X, Wang S, Qi J, Echtenkamp SF, Chatterjee R, Wang M, Boons GJ, Dziarski R, Gupta D. Zebrafish
peptidoglycan recognition proteins are bactericidal amidases essential for defense against bacterial
infections. Immunity. 2007 Sep;27(3):518-29. PMID: 17892854.
North TE, Goessling W, Walkley CR, Lengerke C, Kopani KR, Lord AM, Weber GJ, Bowman TV, Jang IH,
Grosser T, Fitzgerald GA, Daley GQ, Orkin SH, Zon LI. Prostaglandin E2 regulates vertebrate
haematopoietic stem cell homeostasis. Nature. 2007 Jun 21;447(7147):1007-11. PMID: 17581586.
Bates JM, Akerlund J, Mittge E, Guillemin K. Intestinal alkaline phosphatase detoxifies lipopolysaccharide and
prevents inflammation in zebrafish in response to the gut microbiota. Cell Host Microbe. 2007 Dec
13;2(6):371-82. PMID: 18078689
Clay H, Davis JM, Beery D, Huttenlocher A, Lyons SE, Ramakrishnan L. Dichotomous role of the macrophage
in early Mycobacterium marinum infection of the zebrafish. Cell Host Microbe. 2007 Jul 12;2(1):29-39.
PMID: 18005715.
Laing KJ, Purcell MK, Winton JR, Hansen JD. A genomic view of the NOD-like receptor family in teleost fish:
identification of a novel NLR subfamily in zebrafish. BMC Evol Biol. 2008 Feb 6;8:42. doi: 10.1186/14712148-8-42. PMID: 18254971.
Boldajipour B, Mahabaleshwar H, Kardash E, Reichman-Fried M, Blaser H, Minina S, Wilson D, Xu Q, Raz E.
Control of chemokine-guided cell migration by ligand sequestration. Cell. 2008 Feb 8;132(3):463-73. doi:
10.1016/j.cell.2007.12.034. PMID: 18267076.
Cannon JP, Haire RN, Magis AT, Eason DD, Winfrey KN, Hernandez Prada JA, Bailey KM, Jakoncic J, Litman
GW, Ostrov DA. A bony fish immunological receptor of the NITR multigene family mediates allogeneic
recognition. Immunity. 2008 Aug 15;29(2):228-37. doi: 10.1016/j.immuni.2008.05.018. Epub 2008 Jul 31.
PMID: 18674935.
Miller EA, Ernst JD. Illuminating the black box of TNF action in tuberculous granulomas. Immunity. 2008 Aug
15;29(2):175-7. doi: 10.1016/j.immuni.2008.07.003. PMID: 18701080.
López-Muñoz A, Roca FJ, Meseguer J, Mulero V. New insights into the evolution of IFNs: zebrafish group II
IFNs induce a rapid and transient expression of IFN-dependent genes and display powerful antiviral
activities. J Immunol. 2009 Mar 15;182(6):3440-9. doi: 10.4049/jimmunol.0802528. PMID: 19265122.
Niethammer P, Grabher C, Look AT, Mitchison TJ. A tissue-scale gradient of hydrogen peroxide mediates
rapid wound detection in zebrafish. Nature. 2009 Jun 18;459(7249):996-9. doi: 10.1038/nature08119.
Epub 2009 Jun 3. PMID: 19494811.
Davis JM, Ramakrishnan L. The role of the granuloma in expansion and dissemination of early tuberculous
infection. Cell. 2009 Jan 9;136(1):37-49. doi: 10.1016/j.cell.2008.11.014. PMID: 19135887.
Volkman HE, Pozos TC, Zheng J, Davis JM, Rawls JF, Ramakrishnan L. Tuberculous granuloma induction via
interaction of a bacterial secreted protein with host epithelium. Science. 2010 Jan 22;327(5964):466-9. doi:
10.1126/science.1179663. Epub 2009 Dec 10. PMID: 20007864.
Hu YL, Xiang LX, Shao JZ. Identification and characterization of a novel immunoglobulin Z isotype in
zebrafish: implications for a distinct B cell receptor in lower vertebrates. Mol Immunol. 2010 Jan;47(4):73846. doi: 10.1016/j.molimm.2009.10.010. Epub 2009 Nov 20. PMID: 19931913.
Yoo SK, Deng Q, Cavnar PJ, Wu YI, Hahn KM, Huttenlocher A. Differential regulation of protrusion and
polarity by PI3K during neutrophil motility in live zebrafish. Dev Cell. 2010 Feb 16;18(2):226-36. doi:
10.1016/j.devcel.2009.11.015. Erratum in: Dev Cell. 2011 Aug 16;21(2):384. PMID: 20159593.
Lugo-Villarino G, Balla KM, Stachura DL, Bañuelos K, Werneck MB, Traver D. Identification of dendritic
antigen-presenting cells in the zebrafish. Proc Natl Acad Sci U S A. 2010 Sep 7;107(36):15850-5. doi:
10.1073/pnas.1000494107. Epub 2010 Aug 23. PMID: 20733076.
Balla KM, Lugo-Villarino G, Spitsbergen JM, Stachura DL, Hu Y, Bañuelos K, Romo-Fewell O, Aroian RV,
Traver D. Eosinophils in the zebrafish: prospective isolation, characterization, and eosinophilia induction
by helminth determinants. Blood. 2010 Nov 11;116(19):3944-54. doi: 10.1182/blood-2010-03-267419.
Epub 2010 Aug 16. PMID: 20713961.
Phennicie RT, Sullivan MJ, Singer JT, Yoder JA, Kim CH. Specific resistance to Pseudomonas aeruginosa
infection in zebrafish is mediated by the cystic fibrosis transmembrane conductance regulator. Infect
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Tobin DM, Vary JC Jr, Ray JP, Walsh GS, Dunstan SJ, Bang ND, Hagge DA, Khadge S, King MC, Hawn TR,
Moens CB, Ramakrishnan L. The lta4h locus modulates susceptibility to mycobacterial infection in
zebrafish and humans. Cell. 2010 Mar 5;140(5):717-30. doi: 10.1016/j.cell.2010.02.013. PMID: 20211140.
Feng Y, Santoriello C, Mione M, Hurlstone A, Martin P. Live imaging of innate immune cell sensing of
transformed cells in zebrafish larvae: parallels between tumor initiation and wound inflammation. PLoS
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Oehlers SH, Flores MV, Hall CJ, Swift S, Crosier KE, Crosier PS. The inflammatory bowel disease (IBD)
susceptibility genes NOD1 and NOD2 have conserved anti-bacterial roles in zebrafish. Dis Model Mech.
2011 Nov;4(6):832-41. doi: 10.1242/dmm.006122. Epub 2011 Jul 4. PMID: 21729873.
Deng Q, Yoo SK, Cavnar PJ, Green JM, Huttenlocher A. Dual roles for Rac2 in neutrophil motility and active
retention in zebrafish hematopoietic tissue. Dev Cell. 2011 Oct 18;21(4):735-45. doi:
10.1016/j.devcel.2011.07.013. PMID: 22014524.
Yoo SK, Starnes TW, Deng Q, Huttenlocher A. Lyn is a redox sensor that mediates leukocyte wound
attraction in vivo. Nature. 2011 Nov 20;480(7375):109-12. doi: 10.1038/nature10632. PMID: 22101434.
Adams KN, Takaki K, Connolly LE, Wiedenhoft H, Winglee K, Humbert O, Edelstein PH, Cosma CL,
Ramakrishnan L. Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux
mechanism. Cell. 2011 Apr 1;145(1):39-53. doi: 10.1016/j.cell.2011.02.022. Epub 2011 Mar 3. Erratum in:
Cell. 2011 Apr 1;145(1):159. PMID: 21376383.
de Jong JL, Burns CE, Chen AT, Pugach E, Mayhall EA, Smith AC, Feldman HA, Zhou Y, Zon LI.
Characterization of immune-matched hematopoietic transplantation in zebrafish. Blood. 2011 Apr
21;117(16):4234-42. doi: 10.1182/blood-2010-09-307488. Epub 2011 Feb 23. PMID: 21346254.
Wiemer AJ, Hegde S, Gumperz JE, Huttenlocher A. A live imaging cell motility screen identifies prostaglandin
E2 as a T cell stop signal antagonist. J Immunol. 2011 Oct 1;187(7):3663-70. doi:
10.4049/jimmunol.1100103. Epub 2011 Sep 7. PMID: 21900181.
Cusick MF, Libbey JE, Trede NS, Eckels DD, Fujinami RS. Human T cell expansion and experimental
autoimmune encephalomyelitis inhibited by Lenaldekar, a small molecule discovered in a zebrafish screen.
J Neuroimmunol. 2012 Mar;244(1-2):35-44. doi: 10.1016/j.jneuroim.2011.12.024. Epub 2012 Jan 14.
PMID: 22245285.
Hall CJ, Flores MV, Oehlers SH, Sanderson LE, Lam EY, Crosier KE, Crosier PS. Infection-responsive
expansion of the hematopoietic stem and progenitor cell compartment in zebrafish is dependent upon
inducible nitric oxide. Cell Stem Cell. 2012 Feb 3;10(2):198-209. doi: 10.1016/j.stem.2012.01.007. PMID:
22305569.
Hess I, Boehm T. Intravital imaging of thymopoiesis reveals dynamic lympho-epithelial interactions. Immunity.
2012 Feb 24;36(2):298-309. doi: 10.1016/j.immuni.2011.12.016. PMID: 22342843.
Kyritsis N, Kizil C, Zocher S, Kroehne V, Kaslin J, Freudenreich D, Iltzsche A, Brand M. Acute inflammation
initiates the regenerative response in the adult zebrafish brain. Science. 2012 Dec 7;338(6112):1353-6.
doi: 10.1126/science.1228773. Epub 2012 Nov 8. PMID: 23138980.
Zhu LY, Pan PP, Fang W, Shao JZ, Xiang LX. Essential role of IL-4 and IL-4Rα interaction in adaptive
immunity of zebrafish: insight into the origin of Th2-like regulatory mechanism in ancient vertebrates. J
Immunol. 2012 Jun 1;188(11):5571-84. doi: 10.4049/jimmunol.1102259. Epub 2012 Apr 30. PMID:
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absorption and metabolism of fatty acids in the zebrafish. Cell Host Microbe. 2012 Sep 13;12(3):277-88.
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Yang CT, Cambier CJ, Davis JM, Hall CJ, Crosier PS, Ramakrishnan L. Neutrophils exert protection in the
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NIAMS – Zebrafish Studies
Clarissa Henry and Sharon Amacher
ESOURCE ARTICLE
The goals
Zebrafish model of nemaline myopathy
of the National Institute for Arthritis
and Musculoskeletal and Skin Diseases (NIAMS) are to promote
research into the broad spectrum of diseases and syndromes that affect all components of the musculoskeletal
Fig. 3. Muscle contractions of neb larvae
system (including arthritis) and skin disorders.
generate less force than wild-type lar vae. Force
generation was measured during a bilat eral
contraction of whole neb and wild-type larvae at 3
dpf (n 5). (A) Representative force response from
neb and wild-type larvae. (B) Peak force normalized
to cross-sectional area (CSA) was significantly less
in neb larvae compared with wild t ype (4.0±2.0
mN/mm2 vs 20±1.7 mN/mm2, P<0.001). Data
expressed as mean ± s.e.m.
Zebrafish Shed Light on Muscle Disease: The unique and powerful aspects of the zebrafish system to
contribute to NIAMS’ mission are highlighted by the fact that distinct methodological approaches using the
zebrafish model have led to remarkable new insight into muscle diseases.
1: Technology Development. Morpholino-mediated antisense knockdown technology was first developed as
a tool for reverse “genetics” in zebrafish (Nasevicius and Ekker, 2000). NIH funded investigators then
pioneered the use of Morpholinos for exon skipping in zebrafish (Draper et al., 2001). Although the promise of
d neb zebrafish.exon
Isolated myofibers
were co-immunostained
with clustersDMD
of sarcomeric
material (Fig.had
5A). Higher
magnification of been described, the use of oligonucleotides that
skipping
as a potential
therapy
previously
tibodies to -actinin (Fig, 4B, red; marking the Z-band) and these regions revealed that these areas are mainly composed of
incorporate
a
modified
morpholino
ring
dramatically
delivery. Proof on concept trials using the
opomodulin (Fig, 4B, green; marking the tip of the thin filament). filamentous material (Fig. 5A, bottom panel) and improved
are thus
presentative images from control and neb myofibers are reminiscent in appearance to nemaline bodies (Schroder et al.,
morpholino
oligomer
AVI-4658
in
DMD
show
great
promise
(Kinali
et al., 2009).
2004). To further examine this, we used immunofluorescence on
esented in Fig. 4B.
Thin filament length was calculated both by measuring the
stance from Z-line to H-zone using electron micrographs and by
easuring the distance between red ( -actinin) and green
opomodulin) signals from confocal immunofluorescent images.
uantitative comparison between groups revealed that neb thin
aments were reduced in length by nearly 30% (Fig. 4C and data
t shown). These measurements are consistent with the
servations from myofibers of NEB patients (Ottenheijm et al.,
09). Of note, it is apparent both with electron microscopic
alysis and with immunostaining that some areas of the thinament–Z-band intersection are disorganized and/or poorly
marcated (arrow in Fig. 4B).
isolated myofibers with an antibody to -actinin. As with electron
microscopy, we saw
numerous
areas of accumulated
-actinin
2: Chemical Genetic Screening.
The
zebrafish
is a fantastic
system with which to do chemical genetic
staining in neb myofibers (Fig. 5B). Co-staining with phalloidin (Fig.
screens. The large clutch size
of
externally
fertilized
embryos
facilitates
automated, high-throughput, whole
5B, red) demonstrated that actin was also a component of these
aggregates (Fig. 5B, bottom panel, arrow). Such accumulations of
organism screens in multiwell
plates.
Recent
studies
funded
by
NIH
screened
for small molecules that prevent
-actinin have been reported in human NM myofibers (WallgrenPettersson et al.,
and, together withzebrafish.
the electron microscopy
and reverse muscle degeneration
in1995)
dystrophic
Multiple bioreactive, human-approved use
observations, are consistent with the presence of nemaline bodies
compounds that increasedinsurvival
and muscle.
reduced
degeneration were identified (Kawahara et al., 2011).
neb zebrafish skeletal
Of note, muscle
no such abnormalities
were detected in the skeletal muscle of control zebrafish.
3: Genetic Basis of Muscle
Disease. Technological advances in deep sequencing have facilitated
DISCUSSION
NM is a severe childhood-onset
without
treatment
identification of putative causative
mutationsmuscle
fordisease
many
diseases.
However, confirmation of a causative role for
bskeletal muscle contains nemaline bodies
or cure. Not only are there no current treatments for this condition,
particular
genes
is
usually
performed
using
animal
models.
The
facile
attributes of the zebrafish system, in
he defining histological feature of human NM is the presence of there are few viable therapeutic strategies in the research pipeline.
maline bodies in the muscle biopsy. We thus examined the The goal of the present study was to develop an in vivo reagent
combination
with
morpholino-mediated
knockdown,
have
greatly
assisted
in efforts to identify the genetic basis
eletal muscle of neb zebrafish for nemaline bodies by using both suitable for large-scale therapy development. In this vein, we have
rastructural and
immunohistological
analyses.
Using
electron
characterized
a
zebrafish
mutant
with
all
of
the
relevant
features
of orphan diseases (Manzini et al., 2012, Boyden et al., 2012)
croscopy, we identified numerous areas containing abnormal of NM. This mutant carries a recessive mutation in the nebulin
4: Disease Models and Mechanisms. A distinct advantage of the zebrafish system is the remarkable
conservation of phenotypic expression in disease models. Unlike the
murine DMD model, the zebrafish model of DMD reflects the severity
observed in human patients. Similarly, a NIAMS-funded investigator
generated the first vertebrate model that phenocopies the severity of
Ullrich congenital muscular dystrophy (Telfer et al., 2010). Using a
transgenic approach, the first vertebrate model of
facioscapulohumeral muscular dystrophy was recently generated
. 4. Altered thin filament length in skeletal muscle from neb embryos. (A) Ultrastructural analysis of neb embryos and wild-t ype clutchmates at 3 dpf. Thin
Altered
thin
filament
length
in a with wild t ype. Overall(Mitsuhashi
ments (I bands; marked with
r ed arrows) are
qualitatively
smaller in neb
embryos compared
sarcomere length is also reduced
(white 13). These models will be invaluable for
et al.,
ows). Scale bar: 500 nm. (B) Co-immunostaining of isolated myofibers with antibodies to -actinin (red), to mark the Z-lines, and tropomodulin (green), to
zebrafish
model
of
nemaline
investigating
the
cellular and molecular mechanisms of muscle
rk the ends of the thin filaments. Note that -actinin staining is somewhat diminished in neb embryos and that there are areas of disorganization of
pomodulin staining (arrow).
Scale bar: 10 m. (C)
Quantification
of thin filamentmutation
( TF) length. TF length was calculat ed using electron micrographs by
myopathy
due
to
nebulin
diseases
and
new routes to therapy development.
asuring from the Z-line to the beginning of the H zone. TF length in neb zebrafish was significantly reduced (in nm)
compared with controls
(CTL):identifying
794.3±5.7
(from Telfer et al., Disease Models
and Mechanisms 2012).
603.8±7.9, n 4 averaged measurements, P<0.0001.
2
Normal
control cells
(blue) and
dystroglycan
-deficient
cells (red)
transplanted into control embryos show
that the extracellular matrix
microenvironment plays a key role in
maintaining muscle attachment. (from
Goody et al., PLoS Biology 2012).
4: In vivodmm.biologists.org
Cell Biology and Adaptation. One approach to
understanding muscle disease is to elucidate the plethora of cell
adaptation mechanisms that promote muscle homeostasis. One
distinct advantage of the zebrafish system is the ability to integrate in
vivo cell biological analysis with genetics and physiology. Recent NIHfunded research identified an additional complex that mediates
adhesion of muscle cells to their extracellular matrix
microenvironment. Activation of the vitamin sensitive cell adhesion
pathway significantly reduced muscle degeneration and improved
swimming behavior in dystrophic zebrafish (Goody et al., 2012).
Despite the 500 million years of divergent evolution between zebrafish
and humans, this study has already led to the prescription of niacin
supplements for children with dystroglycanopathies (CA Henry,
personal communication).
Zebrafish Shed Light on Metastasis: Metastasis is responsible for the vast majority of cancer deaths, yet the
cellular and molecular basis of metastasis is incompletely understood. NIAMS-funded investigators have
exploited the zebrafish system to illuminate fundamental mechanisms of metastasis and tumorogenesis.
1. Tumor heterogeneity and metastasis in embryonal rhabdomyosarcoma: Embryonal rhabdomyosarcoma
(ERMS) is pediatric muscle sarcoma that is very aggressive. Remarkable live imaging experiments by a
NIAMS-funded investigator identified, for the first time, the tumor-propagating cells. This study also
demonstrated that the myf5+ tumor-propagating cells are distinct from the metastasizing cells. Thus, there is a
complex interplay between tumor- and non-tumor-propagating cells during metastasis that can now be
molecularly dissected in an in vivo model.
2. Transcriptional elongation and melanoma: Recent studies by a NIAMSfunded investigator integrated analyzed the transcriptional consequences of
expressing the most commonly mutated gene in melanoma, BRAF(V600E)
(White et al., 2011). Expression of human BRAF(V600E) in zebrafish
generated melanoma and resulted in upregulation of neural crest genes.
The investigators than undertook a chemical screen to identify small
molecule suppressors of neural crest development. The screen identified a
potent class of compounds (inhibitors of dihydroorotate dehydrogenase)
that not only inhibited neural crest development but also melanoma growth.
Thus, this study linked, for the first time, a developmental regulator of
transcriptional elongation with melanoma formation. A complementary
approach towards understanding melanoma progression was taken by
another NIAMS-funded investigator. In a screen for enhancers of BRAF(V600E) mediated
melanoma, a histone methyltransferase was identified as an oncogene in melanoma (Ceol et
al., 11). These studies exploited the advantages of the zebrafish system to provide dramatic
insight into potential new therapies for melanoma.
Cellular resolution of tumor
heterogeneity in late stage
emrbyonal rhabdomyosarcoma
(from Ignatius et al., Cancer Cell
2012).
Melanoma in
zebrafish
(from White
et al., Nature
2011)
Potential of the Zebrafish system for precision medicine: Clearly, the in vivo molecular,
cellular, and embryological tools available have enabled the zebrafish model to contribute
significantly to our understanding of multiple developmental and disease processes. One
technical limitation was the ability to generate targeted knockouts. This has recently been
overcome, and efficiency of genome editing improved with use of the Goldy TALEN
(transcription activator-like effector nucleases) modified scaffold (Bedell et al., 12). Incredibly, at some loci,
biallelic conversion in somatic tissues allows visualization of phenotypes. Recent data also indicate that
CRISPR-Cas-based RNA-guided endonucleases allow efficient genome editing, with the advantage being that
only one customized sgRNA is required for targeting. Therefore, the zebrafish system is now in a prime
position to facilitate rapid generation of models for precision medicine. This will be of especial
importance for generation of models for rare diseases and allelic series. Furthermore, one of the difficult
aspects of dealing with human diseases is the pleiotropic nature of phenotypic expression. This is especially
true for muscular dystrophies in general, where the age of onset and rate of progression are highly variable.
Importantly, extant zebrafish dystrophy models reflect clinical variations in phenotype. Taken together, the
above technological and conceptual advances highlight the success of NIH’s investment into zebrafish models,
and indicate that future endeavors will likewise lead to important breakthroughs.
NICHD – Zebrafish Studies
David Grunwald & Mary Mullins
Studies with the zebrafish have significantly advanced the stated mission of the National Institute of Child
Health and Human Development (NICHD): to promote “research on fertility, pregnancy, growth, development,
and medical rehabilitation [that] strives to ensure that every child is born healthy and wanted and grows up free
from disease and disability.” Work with zebrafish has been extraordinarily innovative, making cutting edge
contributions in at least seven different important fields, detailed below. Of importance, multiple insights
achieved in the zebrafish are distinct from what would have been gleaned using any other current experimental
system.
Intercellular signaling programs underlying birth defects can be elucidated in the zebrafish. Intercellular
signaling programs operate to designate embryonic cells as precursors destined to form specific tissues and to
organize the positions of tissue precursors so they can generate complex integrated body structures. Defects
in these intercellular signaling pathways cause common dysmorphologies affecting brain, craniofacial, limb,
and heart development, as well as overall stature and laterality patterning. Since genetic background,
environment, and medicines interact with or disrupt these signaling pathways, resulting in both birth defects
and cancers, understanding the detailed workings of these pathways is of utmost importance for guarding
healthy embryonic and childhood development. Due to the unique combination of genetic and optical tools
available in zebrafish, work with zebrafish has literally transformed our understanding of how signaling
molecules, called ‘morphogens’, move, behave, and interact with recipient cells within their native environment,
the intact embryo1,2. Furthermore, because of the ability to recognize in the embryo the most immediate and
direct consequences of aberrant intercellular signaling, large constellations of mutants that perturb individual
signaling pathways have been isolated in the zebrafish. The mutants have provided new insights into the
biochem-ical components that modify the transduction of signals and how these components work 3-7.
The zebrafish is providing unique insights into the functions and organization of the vertebrate egg.
Studies with zebrafish have made truly novel and profound contributions to our understanding of the formation
of the vertebrate egg and the components of the egg that guide normal embryonic development. Once again,
because the zebrafish affords a combination of genetic methods and accessibility of the egg and early embryo
that is unique among vertebrate experimental systems, it allowed for detection of the specific functions of
maternally supplied egg factors that contribute to normal development8-11
Rehabilitative and regenerative medicine studies in the zebrafish are transforming what is possible in
this field. Zebrafish researchers have identified resident cells capable of tissue and organ regeneration, as
well as molecules produced to either stimulate or dampen a regenerative response to injury12-18. Since the
zebrafish has remarkable capacity for regeneration, loss-of-function approaches can be applied to identify
specific factors needed for robust regeneration. This feature distinguishes the zebrafish from other existing
vertebrate experimental systems. As a result, work with the zebrafish has offered unique scientific insights.
Meaningful interpretation of variant human alleles will be revolutionized by zebrafish bioassay
systems. Assays of the ability of human alleles to complement and rescue the defects in existing loss-offunction mutants have already proven the value of zebrafish mutants for distinguishing human alleles of
wildtype function from those with severely diminished activity19-21. In the near future, genome sequencing of
individuals will confront us with a myriad of gene variants that may or may not be pathogenic. We will need
loss-of-function animal models to assay the activity of the human alleles and we will need to create animal
models that harbor precise recapitulations of the alleles to decipher the effects of these variants. New tools
have just been introduced in the zebrafish i) to knock out any gene of interest and ii) to introduce specific
variant alleles into the zebrafish genome22,23. These methods for manipulating the genome of the zebrafish will
revolutionize the ability of the zebrafish to contribute to our understanding of how human gene variants
contribute to development and disease processes.
The cellular and tissue basis of human structural birth defects can be dissected and analyzed in the
zebrafish. Many human structural birth defects arise from deficits in tissue migration and interactions that
normally occur during embryogenesis. Research with zebrafish has been at the vanguard of studies dissecting
the processes of tissue morphogenesis and cell migration as they occur in vivo 24-30.
Zebrafish research permits scientists to probe cell biological processes as they occur within the
complex environment of the intact embryo. Genetic and sub-cellular signaling and trafficking events can be
measured with single cell resolution, often in real time. Novel optical tools allow the study of how individual
cells process and transform information from their environment and how cells coordinate their responses to the
environment --- as one example, zebrafish studies have identified the specific cells required for heart
pacemaker activity24,31-37. The ongoing emergence of tools that allow genetic and experimental manipulations
and observations of selected cells within the zebrafish embryo will introduce a new level of understanding to
cell behavior and how to manipulate cell behavior during embryogenesis.
Zebrafish are easily amenable to high-throughput screens for drug compounds. Such studies permit the
identification of agents that ameliorate developmental malformations, augment the activity of selected signaling
pathways, or perturb gene expression38-40. New drugs can be combined with zebrafish models of disease
made possible by the recently developed methods for gene-targeting in the zebrafish, and will mean that
zebrafish will quickly become the experimental system of choice for finding drugs to promote the NICHD
mission that “every child is born healthy and wanted and grows up free from disease and disability”.
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NIDA – Zebrafish Studies
Su Guo
NIDA leads the nation in bringing the power of science to bear on drug abuse and addiction through support
and conduct of research across a broad range of disciplines and rapid and effective dissemination of research
results to improve prevention and treatment and to inform policy as it relates to drug abuse and addiction.
It has been well recognized that drug abuse and addiction is a brain disorder that is extremely complex and is
a result of gene-environment interactions. Moreover, the developing brain is particularly vulnerable to these
substances, as the brain is more impulsive at young ages. Involuntary exposure in utero is another threat to
the developing brain. Thus, questions of significance to understanding preventing and treating drug abuse and
addiction include: 1) fundamental understanding of neural circuit development that pertains to reward,
aversion, and stress. 2) How do drugs of abuse modify neural circuit development and function? 3) What are
the genes and pathways that regulate circuit development function and its response to drugs of abuse?
Zebrafish, with its easy accessibility to drug exposure from early development to adulthood, optically
transparent and relatively simple brains, amenability to genetic and chemical screening, represent a powerful
model system to address these questions.
Researchers have found that zebrafish display behavioral responses to addictive substances very much like
humans do. These include: 1) being stimulated by low doses of alcohol and sedated by high doses of alcohol
(Lockwood et al., 2004); 2) display preference for alcohol and drugs of abuse such as cocaine, morphine, and
amphetamine (Bretaud et al., 2007; Darland and Dowling, 2001; Lau et al., 2006; Mathur et al., 2011; Ninkovic
et al., 2006). 3) show an interaction of anxiety-like states with alcohol and drugs of abuse (López-Patiño et al.,
2008; Mathur and Guo, 2011). These findings indicate that molecular and cellular studies in zebrafish will
provide important insights into the etiology and pathogenesis mechanisms of drug abuse and addiction.
Researchers have also found that the zebrafish brain has conserved counterparts to that of humans, which
mediate reward, aversion, choice, and stress regulation (Lau et al., 2011; Rink and Wullimann, 2002; Wagle et
al., 2011).
Furthermore, researchers have also advanced technologies that permit the dissection of genetic pathways and
cellular circuitry underlying drugs of abuse and addiction. These include: 1) forward genetic screening (Clark
et al., 2011; Peng et al., 2009); 2) chemical genetic screening (Kokel et al., 2010; Rihel et al., 2010; Sun et al.,
2012); 3) reverse genetic technologies such as TALEN (Bedell et al., 2012; Cermak et al., 2011; Huang et al.,
2011; Sander et al., 2011) and RNAi (De Rienzo et al., 2012; Dong et al., 2009; Dong et al., 2013); 4) cellular
imaging (Ahrens et al., 2012; Naumann et al., 2010).
NIDA’s investment into zebrafish will continue to reward us with a better understanding of genetic and cellular
mechanisms underlying brain development and function, and how drugs of abuse modify the brain
development and function, ultimately leading to addiction. It is the time to continue and further strengthen the
support of the zebrafish model organism for drug abuse related basic brain research.
Reference cited:
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Bedell, V.M., Wang, Y., Campbell, J.M., Poshusta, T.L., Starker, C.G., Krug Ii, R.G., Tan, W., Penheiter, S.G.,
Ma, A.C., Leung, A.Y., et al. (2012). In vivo genome editing using a high-efficiency TALEN system. Nature
[Epub ahead of print].
Bretaud, S., Li, Q., Lockwood, L.L., Kobayashi, K., Lin, E., and Guo, S. (2007). A choice behavior for morphine
reveals experience-dependent drug preference and underlying neural substrates in developing larval zebrafish.
Neuroscience 146, 1109-1116.
Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J.A., Somia, N.V., Bogdanove,
A.J., and Voytas, D.F. (2011). Efficient design and assembly of custom TALEN and other TAL effector-based
constructs for DNA targeting. Nucleic Acids Res 39, e82.
Clark, K.J., Balciunas, D., Pogoda, H.M., Ding, Y., Westcot, S.E., Bedell, V.M., Greenwood, T.M., Urban, M.D.,
Skuster, K.J., Petzold, A.M., et al. (2011). In vivo protein trapping produces a functional expression codex of
the vertebrate proteome. Nat Methods 8, 506-515.
Darland, T., and Dowling, J.E. (2001). Behavioral screening for cocaine sensitivity in mutagenized zebrafish.
Proc Natl Acad USA 98, 11691-11696.
De Rienzo, G., Gutzman, J.H., and Sive, H. (2012). Efficient shRNA-Mediated Inhibition of Gene Expression in
Zebrafish. Zebrafish 9, 97-107.
Dong, M., Fu, Y.F., Du, T.T., Jing, C.B., Fu, C.T., Chen, Y., Jin, Y., Deng, M., and Liu, T.X. (2009). Heritable
and lineage-specific gene knockdown in zebrafish embryo. PLoS One 4, e6125.
Dong, Z., Peng, J., and Guo, S. (2013). Stable gene silencing in zebrafish with spatiotemporally targetable
RNA interference. Genetics In press.
Huang, P., Xiao, A., Zhou, M., Zhu, Z., Lin, S., and Zhang, B. (2011). Heritable gene targeting in zebrafish
using customized TALENs. Nat Biotechnol 29, 699-700.
Kokel, D., Bryan, J., Laggner, C., White, R., Cheung, C.Y., Mateus, R., Healey, D., Kim, S., Werdich, A.A.,
Haggarty, S.J., et al. (2010). Rapid behavior-based identification of neuroactive small molecules in the
zebrafish. Nat Chem Biol 6, 231-237.
Lau, B., Bretaud, S., Huang, Y., Lin, E., and Guo, S. (2006). Dissociation of food and opiate preference by a
genetic mutation in zebrafish. Genes Brain Behav 5, 497-505.
Lau, B.Y.B., Mathur, P., Gould, G.G., and Guo, S. (2011). Identification of a brain center whose activity
discriminates a choice behavior in zebrafish. Proc Natl Acad Sci U S A 108, 2581-2586.
Lockwood, B., Bjerke, S., Kobayashi, K., and Guo, S. (2004). Acute effects of alcohol on larval zebrafish: a
genetic system for large-scale screening. Pharm Biochem Behav 77, 647-654.
López-Patiño, M.A., Yu, L., Cabral, H., and Zhdanova, I.V. (2008). Anxiogenic effects of cocaine withdrawal in
zebrafish. Physiol Behav 93, 160-171.
Mathur, P., Berberoglu, M.A., and Guo, S. (2011). Preference for ethanol in zebrafish following a single
exposure. . Behav Brain Res 217, 128-133.
Mathur, P., and Guo, S. (2011). Differences of acute versus chronic ethanol exposure on anxiety-like
behavioral responses in zebrafish. Behav Brain Res 219, 234-239.
Naumann, E.A., Kampff, A.R., Prober, D.A., Schier, A.F., and Engert, F. (2010). Monitoring neural activity with
bioluminescence during natural behavior. Nat Neurosci 13, 513-520.
Ninkovic, J., Folchert, A., Makhankov, Y.V., Neuhauss, S.C., Sillaber, I., Straehle, U., and Bally-Cuif, L. (2006).
Genetic identification of AChE as a positive modulator of addiction to the psychostimulant D-amphetamine in
zebrafish. J Neurobiol 66, 463-475.
Peng, J., Wagle, M., Mueller, T., Mathur, P., Lockwood, B.L., Bretaud, S., and Guo, S. (2009). Ethanolmodulated camouflage response screen in zebrafish uncovers a novel role for cAMP and extracellular signalregulated kinase signaling in behavioral sensitivity to ethanol. . J Neurosci 29, 8408-8418.
Rihel, J., Prober, D.A., Arvanites, A., Lam, K., Zimmerman, S., Jang, S., Haggarty, S.J., Kokel, D., Rubin, L.L.,
Peterson, R.T., et al. (2010). Zebrafish behavioral profiling links drugs to biological targets and rest/wake
regulation. Science 327, 348-351.
Rink, E., and Wullimann, M.F. (2002). Connections of the ventral telencephalon and tyrosine hydroxylase
distribution in the zebrafish brain (Danio rerio) lead to identification of an ascending dopaminergic system in a
teleost. Brain Res Bull 57, 385-387.
Sander, J.D., Cade, L., Khayter, C., Reyon, D., Peterson, R.T., Joung, J.K., and Yeh, J.R. (2011). Targeted
gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotechnol 29, 697-698.
Sun, Y., Dong, Z., Khodabakhsh, H., Chatterjee, S., and Guo, S. (2012). Zebrafish chemical screening reveals
the impairment of dopaminergic neuronal survival by cardiac glycosides. PLoS One 7, e35645.
Wagle, M., Mathur, P., and Guo, S. (2011). Corticotropin-releasing factor critical for zebrafish camouflage
behavior is regulated by light and sensitive to ethanol. J Neurosci 31, 214-224.
NIDCD – Zebrafish Studies
Teresa Nicolson
David Raiable
Studies using zebrafish have made important contributions to understanding problems under the mission of
NIDCD. In particular the system has been successfully used to understand the function of mechanosensory
hair cells, which mediate the transduction of sound into electrical signals in the inner ear. We highlight here
three areas of study where significant contributions have been made.
Genetics of hearing loss: Mutations in hundreds of genes are associated with hearing loss, making genetic
forms of deafness one of the most common hereditary diseases in humans. Many of these deafness genes
cause similar if not identical phenotypes in animal models such as mice and zebrafish. Over the years, studies
of animal models have increased our general knowledge of the inner workings of the ear. However, some of
the major discoveries of how sensory hair cells transduce sound into electrical signals were only made within
the last decade, and the zebrafish model system was instrumental to these discoveries.
One of the very first components to be identified as playing a central role in mechanotransduction in sensory
hair cells was Cadherin 23 (Cdh23). It had been postulated for many years that extracellular filaments known
as tip links were the physical structure that mechanically gated channels in hair cells, yet the identity of the tip
link proteins remained elusive. The first studies that demonstrated the direct role of Cdh23 in transduction
included one performed in zebrafish (Söllner et al., 2003) and another done with mice (Siemens et al., 2004);
these studies were published back to back, illustrating the complementary nature of work done in different
model systems. It is now accepted that, based on the initial discoveries and subsequent studies, Cdh23 is one
of two proteins that comprise the tip link in hair cells. Further evidence that another novel cadherin,
Protocadherin 15 (Pcdh15) is the other half of the tip link emerged in another publication using zebrafish with
mutations in pcdh15 (Seiler et al., 2005). Both studies on zebrafish Cdh23 and Pcdh15 made a key
contribution to the field of hearing research—weaker alleles of either gene did not disrupt the morphology of
hair cells, yet mechanotransduction was defective. The hair cells of the mouse mutants available at the time
had severe morphological defects, confounding the conclusion that these cadherins played a direct role in
mechanotransduction. These studies demonstrate the value of complementarity of approaches in different
model systems to understand fundamental principles of hair cell function.
Mechanosensory hair cell death: Hair cell death due to aging and exposure to environmental insults is the
leading cause of hearing and balance disorders. Unfortunately hair cell death is a side effect of treatment with
some therapeutic drugs. Zebrafish hair cells show similar susceptibility to damage (Harris et al., 2003; Ou et
al., 2007), making the system a useful one for understanding the genetic and molecular mechanisms
underlying this process. Genetic screens have revealed loci that modulate sensitivity to toxic compounds
(Owens et al., 2008; Hailey et al., 2012). These genes have uncovered pathways regulating cell toxicity and
human orthologues may serve as candidate loci for modifiers of hair cell sensitivity to insult. The small size of
zebrafish larvae and their ready availability in large numbers makes them particularly amenable to small
molecule screening (Owens et al., 2008; Ou et al., 2009). Compounds identified in these screens have the
potential to be developed into the first drugs for prevention of hearing loss.
Hair cell regeneration: While human hair cells are permanently lost after damage, zebrafish hair cells are
capable of regeneration. Many aspects of hair cell development are conserved between zebrafish and other
species including mammals, however mammals are the exception in their failure hair cell regeneration.
Understanding regeneration in other species may reveal the distinct pathways that could be restored in
mammals to promote recovery from damage. The strengths of the zebrafish system in genetics and in vivo
imaging make this system well suited to identify regulators of regeneration. Studies in zebrafish have revealed
specific roles for Notch signaling in hair cell regeneration (Ma et al., 2008; Wibrowo et al., 2001), and recent
reports have suggested that manipulation of this pathway may promote some regeneration in mammals (Lin et
al., 2011; Mizutari et al., 2013).
With the NIH funding situation as it now stands, the competition for hearing research support has increased to
the point where a strong bias to perform studies only with ‘mammals’, i.e. mice, is increasingly having a
negative impact on laboratories that use zebrafish. Yet the studies mentioned above clearly demonstrate the
advantage and the need for work with zebrafish models of hearing loss.
References:
Hailey, D.W., Roberts, B., Owens, K.N., Stewart, A.K., Linbo, T., Pujol, R., Alper, S.L., Rubel, E.W, Raible,
D.W. (2012). Loss Slc4a1b Chloride/Bicarbonate Exchanger Function Protects Mechanosensory Hair Cells
from Aminoglycoside Damage in the Zebrafish Mutant persephone. PLoS Genetics, PLoS Genet 8(10):
e1002971. PMCID: PMC3469417
Harris, J.A., Cheng, A.G., Cunningham, L.L., MacDonald, G., Raible, D.W. and Rubel, E.W. (2003). Neomycininduced hair cell death in the lateral line of zebrafish (Danio rerio): a model system to study hair cell survival.
JARO 4: 219-234.
Lin V, Golub JS, Nguyen TB, Hume CR, Oesterle EC, Stone JS. (2011). Inhibition of Notch activity promotes
nonmitotic regeneration of hair cells in the adult mouse utricles. J Neurosci. Oct 26;31(43):15329-39. PMCID:
PMC3235543
Ma, E.Y., Rubel, E.W and Raible, D.W. (2008). Notch signaling regulates the extent of hair cell regeneration in
the zebrafish lateral line. J. Neurosci., 28: 2261-2273.
Mizutari K, Fujioka M, Hosoya M, Bramhall N, Okano HJ, Okano H, Edge AS. (2013). Notch inhibition induces
cochlear hair cell regeneration and recovery of hearing after acoustic trauma. Neuron. Jan 9;77(1):58-69.
Ou, H., Cunningham, L.L, Francis, S.P. Brandon, C.S.,Simon, J.A., Raible, D.W., and Rubel, E.W. (2009).
Identification of FDA-approved drugs and bioactives that protect hair cells in the zebrafish (Danio rerio) lateral
line and mouse (Mus musculus) utricle. JARO, 10:191-203. PMCID: PMC2674201
Ou, H.C, Raible, D.W. and Rubel, E.W (2007). Cisplatin-induced hair cell loss in zebrafish lateral line. Hearing
Res., 233:46-53. PMCID: PMC2080654
Owens, K.N., Santos, F., Roberts, B., Linbo, T., Knisely, A.J., Simon, J.A., Rubel, E.W and Raible, D.W.
(2008). Identification of genetic and chemical modulators of zebrafish mechanosensory hair cell death. PLoS
Genetics, 4: e1000020. PMCID: PMC2265478
Seiler, C., Finger-Baier, K., Rinner, O., Makhankov, Y., Schwarz, H., Neuhauss, S., and Nicolson, T. (2005)
Duplicated genes with split functions: independent roles of protocadherin 15 orthologues in zebrafish hearing
and vision. Development, 132: 615-623.
Siemens J, Lillo C, Dumont RA, Reynolds A, Williams DS, Gillespie PG, Müller U. (2004) Cadherin 23 is a
component of the tip link in hair-cell stereocilia. Nature 428(6986):950-5.
Söllner, C., Rauch, G., Siemens, J., Geisler, R., Schuster, S., Tübingen Screen Consortium, Müller, U., and
Nicolson, T. (2004) Mutations in cadherin 23 affect tip links in zebrafish sensory hair cells. Nature, 428: 955959.
Wibowo I, Pinto-Teixeira F, Satou C, Higashijima S, López-Schier H. (2011). Compartmentalized Notch
signaling sustains epithelial mirror symmetry. Development. Mar;138(6):1143-52.
NIDCR – Zebrafish Studies
Thomas Schilling
Research funded by the NIDCR aims to provide fundamental insights into oral and craniofacial health and to
develop new tools. The zebrafish is an ideal model system to contribute to this mission, because: 1) it is highly
accessible for embryological and genetic analysis, 2) its embryos and larvae form a relatively simple
craniofacial skeleton that can be imaged in vivo in the living animal, and 3) there are established fish models
for several common human craniofacial malformations and diseases, such as cleft palate. This list becomes
much longer if one considers the skeleton as a whole.
Some important recent contributions of zebrafish to oral and craniofacial biology include:
- genetic dissection of BMP, Endothelin1 (Et1), Notch, Fgf, Hh, Wnt, Pdgf and RA signaling. This has revealed
new roles for these pathways in neural crest (NC - which forms most of the skull and facial skeleton) and
craniofacial development. BMP and Et1 signaling induce the ventral pharyngeal skeleton, such as the lower
jaw, while the Notch ligand, Jagged, induces dorsal; Fgf induces pharyngeal pouches; Hh signaling from the
neural and non-neural ectoderm is critical for palate formation; Wnt signaling promotes both cranial NC
migration and pharyngeal pouch formation; Pdgf guides palate progenitor migration; RA signaling promotes
cranial NC segmentation and skeletal ossification.
- embryological and molecular analyses of zebrafish mutants have provided a framework for how signals and
transcription factors interact to regulate craniofacial development. Analysis of Et1, BMP and Notch signaling
has revealed a morphogen system in dorsal-ventral pharyngeal patterning. Novel roles for transcription factors
such as Tfap2, Foxd3, Prdm1, and Sox9 in skeletogenic NC cells have been explored through single and
double mutant analyses. Dlx genes specify locations of tooth primordia. Cranial myogenesis requires MyoD
and Myogenin function and Ret signals in some muscles.
- genetic/cellular analysis of both canonical and non-canonical Wnt signaling has shown requirements for these
pathways in cranial NC epithelial-mesenchymal transition (EMT) and subsequent migration.
- analysis of RA signaling has identified novel roles for this pathway, particularly RA degradation through
Cyp26 enzymes, in endochondral ossification and osteoblast activity at skull calvaria.
- analysis of zebrafish microRNAs has led to the discovery of mirn140 as a regulator of Pdgfra signaling and
palate morphogenesis.
This research has led to insights into human disease, birth defects and potential therapies, such as:
- Tfap2a mutations disrupt NC specification and survival and cause branchio-oculo-facial syndrome
- Hh pathway mutants disrupt NC differentiation into palatal cartilage and cause holoprosencephaly
- Cyp26b1 mutants, defective in RA degradation, cause craniosynostosis and osteoblast defects
- Sec23 mutants, defective in CopII complex protein transport, cause craniolenticulosutural dysplasia
- Et1 signaling mutants disrupt pharyngeal patterning and cause auriculocondylar syndrome
- Ext1/2 mutants in heparin sulphate synthesis cause multiple ostochondromas and tooth defects
Zebrafish research has also pioneered technological developments in craniofacial biology, including:
- large-scale genetic screens to identify mutations affecting craniofacial and oral development
- morpholinos to knockdown gene expresssion (many cause craniofacial defects – not listed here)
- TILLING to create an allelic series of mutations in any given gene
- Zinc Fingers and TALEN nucleases to perform targeted knockouts
- confocal imaging of fluorescent markers for high-resolution in vivo analysis of gene function
- transgenic lines for visualizing/fate mapping skeletal lineages in vivo (e.g. sox10:kaede; col2a1:gfp)
- small molecule screens to identify modulators of craniofacial development and possible therapeutics
References:
Alexander C, Zuniga E, Blitz IL, Wada N, Le Pabic P, Javidan Y, Zhang T, Cho KW, Crump JG and Schilling
TF (2011). Combinatorial roles for BMPs and Endothelin 1 in patterning the dorsal-ventral axis of the
craniofacial skeleton. Development 138, 5135Carmona-Fontaine C, Matthews HK, Kuriyama S, Moreno M, Dunn GA, Parsons M, Stern CD and Mayor R
(2008). Contact inhibition of locomotion in vivo controls neural crest directional migration. Nature 456, 957Choe CP, Collazo A, Trinh le A, Pan L, Moens CB and Crump JG (2013). Wnt-dependent epithelial transitions
drive pharyngeal pouch formation. Dev Cell 24, 296Clement A, Wiweger M, von der Hardt S, Rusch MA, Selleck SB, Chien CB and Roehl HH (2008). Regulation
of zebrafish skeletogenesis by ext2/dackel and papst1/pinscher. PLoS Genet 4(7):e1000136.
Crump JG, Maves L, Lawson ND, Weinstein BM and Kimmel CB (2004). An essential role for Fgfs in
endodermal pouch formation influences later craniofacial skeletal patterning. Development 131, 5703Dougherty M, Kamel G, Shubinets V, Hickey G, Grimaldi M and LIao EC (2012). Embryonic fate map of first
pharyngeal arch structures in the sox10:kaede zebrafish transgenic model. J Craniofac Surg 23, 1333Eberhart JK, Swartz ME, Crump JG and Kimmel CB (2006). Early Hedgehog signaling from neural to oral
epithelium organizes anterior craniofacial development. Development 133, 1069-1077.
Eberhart JK, He X, Swartz ME, Yan YL, Song H, Boling TC, Kunerth AK, Walker MB, Kimmel CB and
Postlethwait JH (2008). MicroRNA Mirn140 modulates Pdgf signaling during palatogenesis. Nat Genet 40,
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distinguish medial and lateral somitic, cranial and fin muscle fibre populations. Development 136, 403Hinits Y, Williams VC, Sweetman D, Donn TM, Ma TP, Moens CP and Hughes SM (2011). Defective cranial
skeletal development, larval lethality and haploinsufficiency in Myod mutant zebrafish. Dev Biol 358, 102Huitema LF, Apschner A, Logister I, Spoorendonk KM, Bussmann J, Hammond CL and Schulte-Merker S
(2012). Entpd5 is essential for skeletal mineralization and regulates phosphate homeostasis in zebrafish.
Proc Natl Acad Sci USA 109, 21372Jackman WR and Stock DW (2006). Transgenic analysis of Dlx regulation in fish tooth development reveals
evolutionary retention of enhancer function despite organ loss. Proc Natl Acad Sci USA 103, 19390Kague E, Gallagher M, Burke S, Parsons M, Franz-Odendaal T and Fisher S (2012). Skeletogenic fate of
zebrafish cranial and trunk neural crest. PLoS One 7(11):e47394
Knight RD, Nair S, Nelson SS, Afshar A, Javidan Y, Geisler R, Rauch G-J and Schilling TF (2003). lockjaw
encodes a zebrafish tfap2a required for early neural crest development. Development 130, 5755Knight RD and Schilling TF (2006). Cranial neural crest and development of the head skeleton. Adv Exp Med
Biol 589, 120Knight RD, Mebus K, d'Angelo A, Yokoya K, Heanue T; Tubingen 2000 Screen Consortium and Roehl HH
(2011). Ret signalling integrates a craniofacial muscle module during development. Development 138, 2015Lang MR, Lapierre LA, Frotscher M, Goldenring JR and Knapik EW (2006). Secretory COPII coat component
Sec23a is essential for craniofacial chondrocyte maturation. Nat Genet 38, 1198Laue K, Pogoda HM, Daniel PB, van Haeringen A, Alanay Y, von Ameln S, Rachwalski M, Morgan T, Gray MJ,
Breuning MH, Sawyer GM, Sutherland-Smith AJ, Nikkels PG, Kubisch C, Bloch W, Wollnik B,
Hammerschmidt M and Robertson SP (2011). Craniosynostosis and multiple skeletal anomalies in humans
and zebrafish result from a defect in the localized degradation of retinoic acid. Am J Hum Genet 89, 595Li N, Kelsh RN, Croucher P and Roehl HH (2010). Regulation of neural crest cell fate by the retinoic acid and
Pparg signalling pathways. Development 137, 389Mitchell RE, Huitema LF, Skinner RE, Brunt LH, Severn C, Schulte-Merker S and Hammond CL (2013). New
tools for studying osteoarthritis genetics in zebrafish. Osteoarthritis Cartilage 21, 269Wada N, Javidan Y, Nelson S, Carney TJ, Kelsh RN and Schilling TF (2005). Hedgehog signaling is required
for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.
Development 132, 3977Walker MB, Miller CT, Swartz ME, Eberhart JK and Kimmel CB (2007). phospholipase C, beta 3 is required for
endothelin1 regulation of pharyngeal arch patterning in zebrafish. Dev Biol 304, 194Wiweger MI, Zhao Z, van Merkesteyn RJ and Roehl HH (2012). HSPG-deficient zebrafish uncovers dental
aspects of multiple osteochondromas. PLoS One 7(1):e29734.
Yan YL, Willloughby J, Liu D, Crump JG, Wilson C, Miller CT, Singer A, Kimmel C, Westerfield M and
Postlethwait JH (2005). A pair of Sox: distinct and overlapping functions of zebrafish sox9 co-orthologs in
craniofacial and pectoral fin development. Development 132, 1069-
Zuniga E, Stellabotte F and Crump JG (2010). Jagged-Notch signaling ensures dorsal skeletal identity in the
vertebrate face. Development 137, 1843Zuniga E, Rippen M, Alexander C, Schilling TF and Crump JG (2011). Gremlin 2 regulates distinct roles of
BMP and Endothelin 1 signaling in dorsoventral patterning of the facial skeleton. Development 138, 5147-
NIDDK – Zebrafish Studies
Progress in modeling kidney and digestive system disease using the zebrafish
Iain Drumond, Friedhelm Hildebrandt, Wolfram Goessling
Over the past fifteen years, the zebrafish has been established as a powerful and relevant genetic and
developmental model of the diseases and organ systems in the interest areas of the NIDDK. Studies of the
zebrafish larval kidney, pancreas and gut have uncovered highly conserved and broadly significant
development and disease mechanisms that open the way to devising novel treatments of cystic kidney
disease, nephrotic syndrome, diabetes, and gastrointestinal disease.
The kidney
Nephrotic syndrome is characterized by a defective renal glomerular filtration barrier, podocyte dysfunction,
and proteinuria. Zebrafish glomerular podocytes and vasculature are essentially identical to human cells and
the human nephrotic syndrome genes Nephrin and Podocin function similarly in zebrafish 1. This lead to the
first high throughput assays for proteinuria in a model organism 1,2 and the identification novel human
proteinuria genes 3. Forward genetic approaches in zebrafish also identified novel pathways (the
crumbs/apical polarity pathway 1,4), not previously studied in podocytes, focusing attention on new candidate
human disease genes. Although rodent models of nephrotic syndrome have been utilized for a long time, it is
still very challenging to monitor glomerular function in vivo in rodents. Due to its simplicity and small size, the
zebrafish pronephric kidney serves as a useful bridge between in vitro and in vivo model systems currently in
use. New approaches to in vivo imaging of podocyte cell signaling using genetically encoded biosensors,
uniquely feasible in the transparent zebrafish, promise new routes for discovery in glomerular cell biology and
therapeutic testing.
Cystic kidney disease / nephronophthisis: Zebrafish mutant screens provided some of the first genetic
evidence implicating cilia in cystic disease and the spectrum of pathologies now classified as ciliopathies 5-7.
Zebrafish manifest phenotypes that resemble human disease phenotypes not only in the kidney, but also in
other organs affected by ciliopathies, such as brain, eyes, heart, and other visceral organs. The success in
using zebrafish to model human ciliopathies contributed directly to the identification of multiple mammalian
ciliopathy genes 8-12 to the extent that zebrafish knockdown or mutation studies are now a standard feature of
papers describing new ciliopathy genes.
Acute kidney injury, if not repaired, leads to chronic injury, fibrosis, nephron drop-out and eventually kidney
failure. Interventions that could prevent acute injury from progressing to chronic injury or restore kidney
function would have a broad impact on treatment of chronic kidney disease. The zebrafish mesonephros
continues to grow and add new nephrons throughout life. Acute injury triggers a synchronized, stem cellmediated nephrogenesis which accounts for the mesonephric regenerative capacity 13,14. Although this repair
mechanism has not been demonstrated in humans, we believe studying this process will help us understand
mechanisms controlling nephrogenesis and how new nephrons can be "plumbed in" to existing renal tubules in
the context of iPS cell therapies. Also, studies of zebrafish nephric precursor cells were among the first to
identify HDAC inhibitors as potential therapies for renal injury 15. Interestingly, zebrafish glomeruli seem to
undergo a repair process very similar to injured human podocytes making the zebrafish kidney a relevant
model system to study podocyte injury and regeneration 2.
The pancreas
Insulin producing beta cells of the zebrafish pancreas have been intensively studied as a model of type I
diabetes. Zebrafish have been a lead model for uncovering signaling systems essential for beta cell
differentiation and homeostasis 16-19. Novel in vivo reporter systems have allowed the use of whole zebrafish
larvae in high throughput screens for new diabetes therapeutics with notable recent success in discovering
broadly acting compounds that show effectiveness in mammalian models 20,21. Studies of zebrafish are now
yielding insights into heritable metabolic shifts relevant to hyperglycemia and type II diabetes 22. The unique
value of the zebrafish to model relevant human pathologies in a genetically manipulatable, rapidly developing
organism that is also amenable to in vivo high-throughput drug screening positions it as an essential tool for
reducing the health burden of this increasingly common human disease.
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filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM domain protein Mosaic eyes.
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Ebarasi, L. et al. A reverse genetic screen in the zebrafish identifies crb2b as a regulator of the
glomerular filtration barrier. Developmental biology 334, 1-9 (2009).
Sun, Z. et al. A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney.
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Drummond, I.A. et al. Early development of the zebrafish pronephros and analysis of mutations
affecting pronephric function. Development 125, 4655-67 (1998).
Pathak, N., Obara, T., Mangos, S., Liu, Y. & Drummond, I.A. The Zebrafish fleer Gene Encodes an
Essential Regulator of Cilia Tubulin Polyglutamylation. Mol Biol Cell 18, 4353-64 (2007).
Liu, S. et al. A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in
zebrafish. Development 129, 5839-46 (2002).
Chaki, M. et al. Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to
DNA damage response signaling. Cell 150, 533-48 (2012).
Sayer, J.A. et al. The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates
transcription factor ATF4. Nat Genet 38, 674-81 (2006).
Otto, E.A. et al. Mutations in INVS encoding inversin cause nephronophthisis type 2, linking renal cystic
disease to the function of primary cilia and left-right axis determination. Nat Genet 34, 413-20 (2003).
Panizzi, J.R. et al. CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of
ciliary dynein arms. Nature genetics 44, 714-9 (2012).
Diep, C.Q. et al. Identification of adult nephron progenitors capable of kidney regeneration in zebrafish.
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and differentiation in pancreatic endocrine progenitors. Development 139, 1557-67 (2012).
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reveals origins of adult insulin-producing beta-cells. Development 138, 609-17 (2011).
Andersson, O. et al. Adenosine signaling promotes regeneration of pancreatic beta cells in vivo. Cell
Metab 15, 885-94 (2012).
Rovira, M. et al. Chemical screen identifies FDA-approved drugs and target pathways that induce
precocious pancreatic endocrine differentiation. Proc Natl Acad Sci U S A 108, 19264-9 (2011).
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NIEHS – Zebrafish Studies
Harnessing the Power of Zebrafish to Unravel Gene Environmental Interactions
Robert L. Tanguay
Oregon State University
It is firmly established that human health is significantly impacted by lifetime interactions with environmental
factors. Broadly defined, environmental factors include the air we breathe, the food we eat, the water we drink,
the pharmaceuticals we ingest, and the daily stress we endure. Importantly, individual susceptibility to
environmental insults is modified by endogenous factors such as genetic variability and by age. The mission of
the National Institute of Environmental Health Sciences (NIEHS) is to discover how the environment affects
people in order to promote healthier lives. Therefore, the NIEHS has a unique mission that is focused on
prevention of disease rather than on treatment. The first theme in the current NIEHS strategic plan is focused
on fundamental research and there are exciting opportunities to utilize the advantages of zebrafish to discover
basic biological processes and pathways and to unravel how environmental stressors interact with them to
produce disease. A number of discoveries and advances have been made using zebrafish in the past decade,
laying the path for broader impacts moving forward. A few are described below:
Identifying Chemical Hazards and Fetal Origins of Human Diseases – It is clear that there is an association
between early life stage exposures and later life stages diseases (1). It is largely accepted that vertebrates are
more responsive to environmental insult at the earliest life stages. The probable molecular explanation for
increased embryonic susceptibility is that there is no other period of an animal’s life span when the full
repertoire of molecular signaling is necessary and active. Therefore, if a chemical is developmentally toxic, it
must interfere with, or modulate, a critical pathway. Numerous studies have demonstrated the sensitivity of the
embryonic model to discover phenotypes produced by exposure to broad chemical classes such as pesticides,
drugs of abuse, dioxins, PAHs, nanoparticles, and metals to list a few. The largest environmental health
challenge is identifying, from the sea of environmental chemicals, the ones that can interact with the normal
developmental plan to produce adverse health outcomes. This is a daunting challenge when one considers the
complexity of the human exposome (2). There are tens of thousands of man-made chemicals in the
environment, a rise in nanoparticle production, and enormous complexities in the environmental
transformations following release. The challenge is even more formidable when one considers that we are
exposed to these agents as complex mixtures; therefore, the diversity of chemical exposures is boundless.
The National Academy of Sciences recognized the shortcomings of traditional toxicological approaches and
challenged the field to obtain rapid, relevant toxicological data, anchored to relevant human exposures (3) in
order to predict and prevent human toxicity. Significant federal and commercial resources have been invested,
primarily in the development of high throughput cell culture systems to assess the inherent toxicity of
thousands of chemicals. Although there have been some successes; the principal challenge is that for most of
these chemicals there exists no whole animal phenotypes to anchor the in vitro results (4), this uncertainty is
paralyzing the Toxicity Testing for the 21st Century effort. Major advances in zebrafish high throughput
technology and assay development has the potential to provide critical in vivo links. For example, numerous
groups have automated the manipulation, handling, and imaging of early life stage zebrafish for unprecedented
rates of phenotype discovery (5-11). The development of these high throughput assays in zebrafish has put the
field in a position to greatly expand the in vivo testing of relevant environmental chemicals and mixtures. Proof
of concept studies completed using the 1100 EPA ToxCast Phase I and II chemicals reveal the power of
comparing zebrafish data to the available in vitro data (12, 13).
Identification and validation of biomarkers of exposure. Zebrafish transcriptomics, microRNAomics, proteomics
and metabolomics are practical and powerful means to facilitate the translation of zebrafish data across
species (14). Not surprisingly, combining omics approaches into a systems level analysis rapidly yields
plausible toxicity mechanisms and more informative comparative studies. A recent example in zebrafish
combined microarray transcriptome analysis of mercury hepatotoxicity, gene set enrichment analysis and data
mining comparison to reported human and mammalian data, phenotype anchoring, and gene expression
validation by real-time PCR, in one study (15). The result from this single study was a plausible in vivo
mechanism of mercury hepatotoxicity where none existed before (15). Other toxicogenomics studies in
zebrafish have significantly advanced our understanding of the transcriptional changes following AHR
activation and, in so doing, have provided much new knowledge about cardiac development and tissue
regeneration. Studies aimed at understanding the molecular mechanisms of AHR signaling that lead to
cardiovascular dysfunction have identified changes in genes important to xenobiotic metabolism, cell
proliferation, heart contractility and heart development (16). Gene expression studies performed in adult and
larval zebrafish regenerating fin tissue (an exception to the whole animal approach), after AHR activation by
TCDD, revealed a cluster of genes important for xenobiotic metabolism, cellular differentiation, signal
transduction and extracellular matrix composition (17).
In vivo evaluation of gene function. In the post genomic era, evaluation of the role of gene products in the
biological responses to environmental insult will be an important, but immense challenge. For example,
genome wide associations studies (GWAS) are rapidly being applied to determine areas of the genome that
alter susceptibility to pharmaceuticals (18) and environmental toxicants (19). Zebrafish offer a powerful means
to define the functions of the genes identified in GWAS. So called “humanized fish” have the potential to rapidly
close the loop between human hypothesis generation and functional confirmation because the role of genes
can be efficiently and cost effectively evaluated in zebrafish as part of an integrative applied research strategy.
Environmental influences on CNS development and function. A rapidly emerging area of concern is the impact
of low level human exposures to environmental contaminants on CNS development, function and
degeneration. The possibility that early life stage exposures may lead to persistent effects on learning,
memory, or behavior has not been adequately addressed using other vertebrate models. There are
tremendous opportunities to use zebrafish to fill this large information gap. Importantly assays and
instrumentation have been generated in the past decade to more rapidly evaluate zebrafish larval and adult
behaviors (20-25). The zebrafish model is now poised to identify the environmental agents that have the
potential to cause neurobehavioral effects in humans and provide a path towards therapeutic interventions.
References Cited:
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care. 2011;41(6):158-76. Epub 2011/06/21. doi: 10.1016/j.cppeds.2011.01.001. PubMed PMID: 21684471.
2. Wild CP. Complementing the genome with an "exposome": the outstanding challenge of environmental
exposure measurement in molecular epidemiology. Cancer Epidemiol Biomarkers Prev. 2005;14(8):184750. Epub 2005/08/17. doi: 10.1158/1055-9965.EPI-05-0456. PubMed PMID: 16103423.
3. Toxicity Testing in the 21st Century: A vision and a Strategy, National Research Council. Washington, DC:
Natl. Acad. Press; 2007.
4. Davis M, Boekelheide K, Boverhof DR, Eichenbaum G, Hartung T, Holsapple MP, et al. The new revolution
in toxicology: The good, the bad, and the ugly. Ann N Y Acad Sci. 2013;1278(1):11-24. Epub 2013/03/16.
doi: 10.1111/nyas.12086. PubMed PMID: 23488558.
5. Mandrell D, Truong L, Jephson C, Sarker MR, Moore A, Lang C, et al. Automated zebrafish chorion
removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens. J
Lab Autom. 2012;17(1):66-74. Epub 2012/02/24. doi: 10.1177/2211068211432197. PubMed PMID:
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6. Letamendia A, Quevedo C, Ibarbia I, Virto JM, Holgado O, Diez M, et al. Development and validation of an
automated high-throughput system for zebrafish in vivo screenings. PLoS ONE. 2012;7(5):e36690. Epub
2012/05/23. doi: 10.1371/journal.pone.0036690. PubMed PMID: 22615792; PubMed Central PMCID:
PMC3352927.
7. Pardo-Martin C, Chang TY, Koo BK, Gilleland CL, Wasserman SC, Yanik MF. High-throughput in vivo
vertebrate screening. Nature methods. 2010;7(8):634-6. Epub 2010/07/20. doi: 10.1038/nmeth.1481.
PubMed PMID: 20639868; PubMed Central PMCID: PMC2941625.
8. Graf SF, Hotzel S, Liebel U, Stemmer A, Knapp HF. Image-based fluidic sorting system for automated
Zebrafish egg sorting into multiwell plates. J Lab Autom. 2011;16(2):105-11. Epub 2011/05/26. doi: S15355535(10)00244-3 [pii]
10.1016/j.jala.2010.11.002. PubMed PMID: 21609691.
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10. Vogt A, Cholewinski A, Shen X, Nelson SG, Lazo JS, Tsang M, et al. Automated image-based phenotypic
analysis in zebrafish embryos. Dev Dyn. 2009;238(3):656-63. Epub 2009/02/25. doi: 10.1002/dvdy.21892.
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11. Vogt A, Codore H, Day BW, Hukriede NA, Tsang M. Development of automated imaging and analysis for
zebrafish chemical screens. J Vis Exp. 2010(40). Epub 2010/07/09. doi: 1900 [pii]
10.3791/1900. PubMed PMID: 20613708.
12. Truong L, Reif DM, Chalker L, Geier M, Tanguay RL. Large scale multi-dimensional in vivo hazard
assessment using zebrafish Environ Health Perspect. Submitted.
13. Padilla S, Corum D, Padnos B, Hunter DL, Beam A, Houck KA, et al. Zebrafish developmental screening of
the ToxCast Phase I chemical library. Reproductive toxicology (Elmsford, NY). 2012;33(2):174-87. Epub
2011/12/21. doi: 10.1016/j.reprotox.2011.10.018. PubMed PMID: 22182468.
14. Sukardi H, Ung CY, Gong Z, Lam SH. Incorporating zebrafish omics into chemical biology and toxicology.
zebrafish. 2010;7(1):41-52. Epub 2010/04/14. doi: 10.1089/zeb.2009.0636. PubMed PMID: 20384484.
15. Ung CY, Lam SH, Hlaing MM, Winata CL, Korzh S, Mathavan S, et al. Mercury-induced hepatotoxicity in
zebrafish: in vivo mechanistic insights from transcriptome analysis, phenotype anchoring and targeted
gene expression validation. BMC Genomics. 2010;11:212. Epub 2010/04/01. doi: 1471-2164-11-212 [pii]
10.1186/1471-2164-11-212. PubMed PMID: 20353558; PubMed Central PMCID: PMC2862047.
16. Carney SA, Chen J, Burns CG, Xiong KM, Peterson RE, Heideman W. AHR Activation Produces HeartSpecific Transcriptional and Toxic Responses in Developing Zebrafish. Mol Pharmacol. 2006. PubMed
PMID: 16714409.
17. Andreasen EA, Mathew LK, Tanguay RL. Regenerative growth is impacted by TCDD: gene expression
analysis reveals extracellular matrix modulation. Toxicol Sci. 2006;92(1):254-69. Epub 2006/01/31. doi:
10.1093/toxsci/kfj118. PubMed PMID: 16443690.
18. McBride JM, Jiang J, Abbas AR, Morimoto A, Li J, Maciuca R, et al. Safety and pharmacodynamics of
rontalizumab in patients with systemic lupus erythematosus: results of a phase I, placebo-controlled,
double-blind, dose-escalation study. Arthritis Rheum. 2012;64(11):3666-76. Epub 2012/07/27. doi:
10.1002/art.34632. PubMed PMID: 22833362.
19. Wei CY, Lee MT, Chen YT. Pharmacogenomics of adverse drug reactions: implementing personalized
medicine. Hum Mol Genet. 2012;21(R1):R58-65. Epub 2012/08/22. doi: 10.1093/hmg/dds341. PubMed
PMID: 22907657.
20. Ninkovic J, Bally-Cuif L. The zebrafish as a model system for assessing the reinforcing properties of drugs
of abuse. Methods. 2006;39(3):262-74. Epub 2006/07/01. doi: 10.1016/j.ymeth.2005.12.007. PubMed
PMID: 16809048.
21. Gerlai R. High-throughput behavioral screens: the first step towards finding genes involved in vertebrate
brain function using zebrafish. Molecules. 2010;15(4):2609-22. Epub 2010/04/30. doi:
10.3390/molecules15042609. PubMed PMID: 20428068.
22. Pelkowski SD, Kapoor M, Richendrfer HA, Wang X, Colwill RM, Creton R. A novel high-throughput imaging
system for automated analyses of avoidance behavior in zebrafish larvae. Behav Brain Res.
2011;223(1):135-44. Epub 2011/05/10. doi: 10.1016/j.bbr.2011.04.033. PubMed PMID: 21549762; PubMed
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23. Kokel D, Peterson RT. Using the zebrafish photomotor response for psychotropic drug screening. Methods
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24. Ahmad F, Richardson MK. Exploratory behaviour in the open field test adapted for larval zebrafish: impact
of environmental complexity. Behav Processes. 2013;92:88-98. Epub 2012/11/06. doi:
10.1016/j.beproc.2012.10.014. PubMed PMID: 23123970.
25. Bichara D, Calcaterra NB, Arranz S, Armas P, Simonetta SH. Set-up of an infrared fast behavioral assay
using zebrafish (Danio rerio) larvae, and its application in compound biotoxicity screening. J Appl Toxicol.
2013. Epub 2013/02/13. doi: 10.1002/jat.2856. PubMed PMID: 23401233.
NIGMS – Zebrafish Studies
Mary Mullins, Alexander Schier & Lila Solnica-Krezel
Research funded by the NIGMS aims to provide fundamental understanding of life processes and to develop
new tools and resources. The zebrafish has proven a powerful vertebrate model system in advancing this
mission, because it combines high-resolution phenotypic analysis with high-throughput genome manipulation.
Forward and reverse genetics studies in zebrafish advanced our understanding of cell signaling by
discovering new components and cellular mechanisms of signaling by the Nodal, FGF, BMP, Hedgehog, Wnt,
GPCR, and Notch pathways. For example, Scube solubilizes Hedgehog; the Sizzled protein Ogon is a
negative regulator of BMP signaling; the ubiquitin ligase Mind bomb is required for Delta/Notch signaling;
ligand heterodimers are required for BMP signaling.
Genetic and genomic studies in zebrafish have revealed the exquisite expression patterns and functions of
non-coding RNAs. For example, microRNA miR-430 is a key regulator of embryonic transitions, the long noncoding RNA megamind is involved in neural differentiation, and some microRNAs regulate translation rather
than RNA stability.
Molecular and imaging analyses of zebrafish mutants have uncovered the mechanisms underlying vertebrate
embryogenesis and organogenesis. For example, dorsal axis formation is globally repressed by GPCR
signaling; the Nodal/Lefty system constitutes a reaction-diffusion system, and BMP signaling drives temporally
progressive patterning. Large-scale genetic screens uncovered the function of hundreds of genes regulating
formation of heart, blood and lymphatic vessels, kidney, muscle, brain and other organs. For example, retinoic
acid signaling determines heart size, GPCRs and lipid signaling regulate heart fusion, and Notch signaling
synchronizes somite formation. Regeneration studies have revealed how adult zebrafish can regenerate heart,
kidney, eye, fins and brain. For example, FGF is a key regulator of many regenerative processes, and fin and
heart cell lineages retain fate restriction during regeneration.
Zebrafish has provided novel insights into cell motility and morphogenesis. Cells move through tissues
using filolamellipodia and/or blebbing locomotion, sort by modulation of cell adhesion and cortex tension, repel
each other through membrane depolarization, and extrude to retain epithelial cell number. Planar Cell Polarity
signaling coordinates morphogenetic behaviors of individual cells and tissues with embryonic polarity. The
Cxcr4/Ccr7 GPCR pair guides the migration of germ cells, neurons and many other cell types, fluid flow
regulates cardio-vascular development, and hydrogen peroxide attracts neutrophils to wound sites. Key genes
for the assembly of nuclei, cilia and other organelles have been isolated.
Zebrafish research has also pioneered technological developments and resources. For example, ingenious
genetic screens have identified mutations that regulate the exquisite architecture of oocytes, myelin or bones.
Knockout projects have mutated 9,000 genes, providing an unprecedented resource for the analysis of
vertebrate gene function. Retroviruses and transposable elements have been engineered and employed for
large-scale gene disruption. TALEN and CRISPR technology have been developed for rapid, efficient genome
editing. Antisense technologies have identified the causative genes for human diseases. Issues of gene
redundancy have been addressed by generating double and triple mutants and maternal-zygotic mutants.
Genomics studies have identified chromatin marks for enhancers and pluripotency. Small molecule screens
have identified drugs that modulate development, regeneration or behavior. New chemicals and optogenetic
tools have generated biosensors for organelles, cell signaling, cell cycle, cytoskeletal dynamics, protein
interactions, ligand diffusion, calcium dynamics, glycans, and mechanical forces. Dramatic progress in
microscopy and image acquisition has afforded in toto imaging of all cells through the entirety of
embryogenesis. Multicolor Brainbow imaging has revealed long-term lineage relationships. Light-regulated
proteins have allowed the manipulation of cell migration, transcription, and neural activity. The zebrafish field
has now reached a stage where any gene can be modified and its function analyzed at a resolution and
throughput not reached by any other vertebrate model system.
NIMH – Zebrafish Studies
Michael Granato
The major mission of the National Institute of Mental (NIMH) is to understand, treat, and prevent mental
illnesses through research on the brain and behavior. Over the past 10 years, the zebrafish has emerged as a
powerful system to study brain function and behavior (Berton et al, Science, 2012). Although traditionally
recognized for genetic and cellular studies of embryonic development and growth, the zebrafish is now one of
the leading systems in which to perform small molecule and genetic screens using diverse behavioral
paradigms affected by mental disorders. Importantly, the key neural substrates and pathways that mediate
behaviors affected in mental disorders are well conserved between the mammalian and zebrafish nervous
system (e.g. Burgess & Granato, 2008, J. Neuroscience).
Combined, small molecule and genetic screens in the zebrafish have been applied to the studies of various
behaviors, including wake/sleep, prepulse inhibition and learning/memory. For example, Rihel et al tested and
classified the effects of more than 5,500 neuroactive drugs on rest/wake behaviors (Rihel et al, Science, 2010),
and Kokel et al on simple motor behaviors (Kokel et al 2010, Nat. Chem. Bio.). This has led to the identification
of a novel small molecule that enables repeated photo activation of defined motor behaviors in both wild-type
zebrafish and mice (Kokel et al 2013, Nat. Chem. Bio.). Furthermore, using an automated high throughput
assay, Wolman et al performed the first comprehensive screen in vertebrates for modulators of memory
formation (Wolman et al, PNAS, 2011).
At the same time, the zebrafish has also made significant contributions to the understanding of the functional
relevance of neuronal cell types and brain regions. For example, the habenula, including its subdivision into
medial and lateral regions, is an evolutionarily conserved brain structure, however, its function has remained
ambiguous. Elegant studies using adult zebrafish have recently demonstrated that neural activity in the medial
habenula is important for controlling experience-dependent modification of fear responses (Agetsuma et al
Nature Neuroscience 2010). Finally, monitoring neuronal brain activity in freely behaving animals has been
possible for some time, however only at low spatial and temporal resolution. Recent spectacular work by Muto
et al reports on the visualization of neuronal brain activity of defined neurons in the brain of free behaving
zebrafish during prey capture (Muto et al, Current Biology, 2013).
Thus, by integrating technologies to visualize brain activity at the single cell level with refined behavioral
assays and combining this with small molecule and forward genetic screens, the young field of behavioral
genetics in zebrafish has already made several significant contributions to NIMH’s mission. By introducing the
concept of personalized medicine into the field, the zebrafish has a very bright future towards the goal of
treating mental illnesses. For example, by engineering zebrafish to express a mental disease causing gene in
the subset of relevant neurons, and then using such animals to screen tens of thousands of psychoactive
compounds using automated high throughput behavioral assays in a few months to identify ‘personalized’
drugs, is no longer wishful thinking but can readily be done.
NINDS – Zebrafish Studies
Florian Engert
Marnie Halpern
In support of its mission to reduce the burden of neurological disease, the National Institute of Neurological
Disorders and Stroke (NINDS) sponsors diverse research using the zebrafish model to elucidate biological
processes in neurons and glia and the genetic and cellular pathways that underlie devastating conditions such
as epilepsy, Parkinson’s disease and spinal muscular atrophy.
As many neurological disorders are developmental in origin, studies on zebrafish have provided valuable
insights into the bases of neuronal specification and connectivity. For example, a previously unsuspected
organization of neural networks in the hindbrain was discovered from the expression of transcription factors in
patterns of stripes. These stripes correlate with the neurotransmitter phenotypes and functional properties of
neuronal subtypes (Kinkhabwala et al., 2011). Even though pre-patterned neurons later reorganize to form
discrete nuclei, this early ground plan serves as the structural framework for the adult hindbrain, and,
importantly, presents a new mechanism for how sensory-motor circuits are established in the vertebrate brain.
With unprecedented cellular resolution, transgenic tools in the transparent zebrafish larva permit in vivo
visualization of neuronal subpopulations (Scott et al., 2007) and detailed analyses of axonal morphology during
innervation and following injury (Rosenberg et al., 2012). The zebrafish is ideally suited for studies on
neurodegeneration and on the capacity of neural stem cells in regeneration and repair. Recent work on postembryonic stages, for instance, reveals that Wnt signaling regulates the differentiation of neural progenitors in
the hypothalamus. New inroads are also being made to model hemorrhagic stroke (Butler et al., 2011) and
hypoxia induced brain damage in zebrafish (Yu and Li, 2011).
Cutting edge transgenic and imaging approaches have further enabled real-time mapping of neural activation
throughout the entire brain and changes in activity to be correlated with specific cognitive tasks (Ahrens et al.,
2012). Moreover, the small size of the larval brain makes it the only vertebrate preparation in which electron
microscopy datasets can be generated that encompass a whole brain connectome. Coupled with prior twophoton microscopy, nanoscale anatomical information can be integrated with micron-scale activity maps. Such
a technical tour de force, unique to the zebrafish, predicts an exciting future for the characterization of neural
circuit function during behavior in the brains of normal individuals or disease models.
On account of their amenability to large-scale genetic strategies and high throughput drug screening, and their
ability to mimic epileptic seizures, zebrafish larvae have been used to recover mutations that confer seizure
resistance (Hortopan et al., 2010) and to identify compounds with anti-convulsant properties (Baxendale et al.,
2012). Owing to the shortcomings of some disease models in rodents, as in experimental paradigms to
emulate multiple sclerosis (Rice, 2012), unbiased screening approaches afforded by zebrafish could also lead
to new avenues for myelin repair. Recent mutagenesis efforts supported by the NINDS have indeed implicated
unexpected proteins and signaling pathways in the myelination of peripheral nerves (Raphael and Talbot,
2011; Langworthy and Appel, 2012).
Despite the numerous contributions from research using zebrafish to a comprehensive understanding of neural
organization, function and dysfunction, the NIH Blueprint for Neuroscience Research has primarily focused on
mouse and primate models (Baughman et al., 2006). Through the commitment to their mission, the NINDS can
play a leading role in ensuring that critical insights into neurological diseases made possible by the special
attributes of the zebrafish system continue to accelerate.
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