| Danio rerio | |
|---|---|
 | |
| An adult female zebrafish | |
Scientific classification | |
| Kingdom: | Animalia |
| Phylum: | Chordata |
| Class: | Actinopterygii |
| Order: | Cypriniformes |
| Family: | Danionidae |
| Subfamily: | Danioninae |
| Genus: | Danio |
| Species: | D. rerio |
| Binomial name | |
| Danio rerio (F. Hamilton, 1822) | |
| Synonyms[2] | |
| |
Thezebrafish (Danio rerio) is a species of freshwaterray-finned fish belonging to thefamilyDanionidae of theorderCypriniformes. Native to South Asia,[3] it is a popularaquarium fish, frequently sold under the trade namezebra danio[4] (and thus often called a "tropical fish" although it is both tropical andsubtropical).
The zebrafish is an important and widely usedvertebratemodel organism in scientific research, particularlydevelopmental biology, but also gene function,oncology,teratology, anddrug development, in particularpre-clinical development, due to its scalability from high numbers of offspring and ease of drug delivery through water into the gills.[5] It is also notable for itsregenerative abilities,[6] and has beenmodified by researchers to produce manytransgenic strains.[7][8][9]
The zebrafish is aderived member of the genusBrachydanio, of the familyCyprinidae.[10] It has asister-group relationship withDanio aesculapii.[11] Zebrafish are also closely related to the genusDevario, as demonstrated by aphylogenetic tree of close species.[12]
The zebrafish is native to freshwater habitats in South Asia where it is found in India, Pakistan, Bangladesh, Nepal and Bhutan.[1][13][14][15] The northern limit is in the SouthHimalayas, ranging from theSutlej river basin in the Pakistan–India border region to the state ofArunachal Pradesh in northeast India.[1][14] Its range is concentrated in theGanges andBrahmaputra River basins,[10] and the species was first described fromKosi River (lower Ganges basin) of India. Its range further south is more local, with scattered records from theWestern andEastern Ghats regions.[15][16] It has frequently been said to occur in Myanmar (Burma), but this is entirely based on pre-1930 records and likely refers to close relatives only described later, notablyDanio quagga andDanio kyathit.[15][17][18][19][20] Likewise, old[clarification needed] records from Sri Lanka are highly questionable and remain unconfirmed.[17]
Zebrafish have beenintroduced to a variety of places outside their natural range,[10][15] including California, Connecticut, Florida and New Mexico in the United States, presumably by deliberate release by aquarists or by escape fromfish farms. The New Mexico population had been extirpated by 2003 and it is unclear if the others survive, as the last published records were decades ago.[21] Elsewhere the species has been introduced to Colombia and Malaysia.[14][22]
Zebrafish typically inhabit moderately flowing tostagnant clear water of quite shallow depth in streams, canals, ditches,oxbow lakes, ponds andrice paddies.[15][22][23][10] There is usually some vegetation, either submerged or overhanging from the banks, and the bottom is sandy, muddy or silty, often mixed with pebbles or gravel. In surveys of zebrafish locations throughout much of its Bangladeshi and Indian distribution, the water had a near-neutral to somewhat basicpH and mostly ranged from 16.5 to 34 °C (61.7–93.2 °F) in temperature.[15][24] One unusually cold site was only 12.3 °C (54.1 °F) and another unusually warm site was 38.6 °C (101.5 °F), but the zebrafish still appeared healthy. The unusually cold temperature was at one of the highest known zebrafish locations at 1,576 m (5,171 ft) above sea level, although the species has been recorded to 1,795 m (5,889 ft).[15]
The zebrafish is named for the five uniform, pigmented, horizontal, blue stripes on the side of the body, which are reminiscent of a zebra's stripes, and which extend to the end of thecaudal fin.[23] Its shape isfusiform and laterally compressed, with its mouth directed upwards. The male istorpedo-shaped, with gold stripes between the blue stripes; the female has a larger, whitish belly and silver stripes instead of gold. Adult females exhibit a smallgenital papilla in front of theanal fin origin. The zebrafish can reach up to 4–5 cm (1.6–2.0 in) in length,[18] although they typically are 1.8–3.7 cm (0.7–1.5 in) in the wild with some variations depending on location.[25] Its lifespan in captivity is around two to three years, although in ideal conditions, this may be extended to over five years.[23][26] In the wild it is typically an annual species.[1]
In 2015, a study was published about zebrafishes' capacity forepisodic memory. The individuals showed a capacity to remember context with respect to objects, locations and occasions (what, when, where). Episodic memory is a capacity of explicit memory systems, typically associated withconscious experience.[27]
Zebrafish are social animals as adults, existing in groups that exhibit shoaling, schooling and escape behaviors. Social preference emerges around 3 weeks of life, when juvenile zebrafish begin to prefer compartments that place them within view of other zebrafish. Other social behaviors include recognizing conspecifics, members of the same species, same-sex aggression, and mating.[28]
TheMauthner cells integrate a wide array of sensory stimuli to produce theescape reflex. Those stimuli are found to include thelateral line signals by McHenry et al. 2009 andvisual signals consistent with looming objects by Temizer et al. 2015, Dunn et al. 2016, and Yao et al. 2016.[29]
The approximategeneration time forDanio rerio is three months. A male must be present forovulation andspawning to occur. Zebrafish are asynchronous spawners[30] and under optimal conditions (such as food availability and favorable water parameters) can spawn successfully frequently, even on a daily basis.[31] Females are able to spawn at intervals of two to three days, laying hundreds of eggs in eachclutch. Upon release, embryonic development begins; in absence of sperm, growth stops after the first few cell divisions. Fertilized eggs almost immediately become transparent, a characteristic that makesD. rerio a convenient researchmodel species.[23] Sex determination of common laboratory strains was shown to be a complex genetic trait, rather than to follow a simple ZW or XY system.[32]
The zebrafish embryo develops rapidly, with precursors to all major organs appearing within 36 hours of fertilization. The embryo begins as a yolk with a single enormous cell on top (see image, 0 h panel), which divides into two (0.75 h panel) and continues dividing until there are thousands of small cells (3.25 h panel). The cells then migrate down the sides of the yolk (8 h panel) and begin forming a head and tail (16 h panel). The tail then grows and separates from the body (24 h panel). The yolk shrinks over time because the fish uses it for food as it matures during the first few days (72 h panel). After a few months, the adult fish reaches reproductive maturity (bottom panel).
To encourage the fish to spawn, some researchers use a fish tank with a sliding bottom insert, which reduces the depth of the pool to simulate the shore of a river. Zebrafish spawn best in the morning due to theirCircadian rhythms. Researchers have been able to collect 10,000 embryos in 10 minutes using this method.[33] In particular, one pair of adult fish is capable of laying 200–300 eggs in one morning in approximately 5 to 10 at time.[34] Male zebrafish are furthermore known to respond to more pronounced markings on females, i.e., "good stripes", but in a group, males will mate with whichever females they can find. What attracts females is not currently understood. The presence of plants, even plastic plants, also apparently encourages spawning.[33]
Exposure to environmentally relevant concentrations ofdiisononyl phthalate (DINP), commonly used in a large variety of plastic items, disrupt theendocannabinoid system and thereby affect reproduction in a sex-specific manner.[35]
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Zebrafish feeding practices vary significantly across different developmental stages, reflecting their changing nutritional needs. For newly hatched larvae, which begin feeding at approximately 5 days post-fertilization (dpf), small live prey such asParamecium or rotifers are commonly used until they reach 9–15 dpf.[36] This early diet is crucial for their growth and survival, as these small organisms provide essential nutrients. As the larvae develop, from 15 dpf onwards, they are typically transitioned to a diet that includes brine shrimp nauplii and dry feeds, which are more nutritionally balanced and easier to manage in laboratory settings. For larvae aged 25 dpf, feeding rates can range from 50% to 300% of their body weight (BW) per day, depending on their size and growth requirements.[37] As zebrafish grow into juveniles (30–90 dpf), the recommended feeding rate decreases to about 6–8% of their BW per day, with a focus on high-quality dry feeds that meet their protein and energy needs. Upon reaching adulthood (over 90 dpf), zebrafish typically require a feeding rate of around 5% of their BW per day. Throughout these stages, it is essential to adjust the particle size of the feed: less than 100 μm for newly hatched larvae, 100–200 μm for those between 16 and 30 dpf, and larger particles for juveniles and adults. This structured approach to feeding not only supports optimal growth and health but also enhances the reliability of experimental outcomes in research settings.[38]
Zebrafish are hardy fish and considered good for beginner aquarists. Their enduring popularity can be attributed to their playful disposition,[39] as well as their rapid breeding, aesthetics, cheap price and broad availability. They also do well in schools orshoals of six or more, and interact well with other fish species in the aquarium. However, they are susceptible toOodinium or velvet disease,microsporidia (Pseudoloma neurophilia), andMycobacterium species. Given the opportunity, adults eat hatchlings, which may be protected by separating the two groups with a net, breeding box or separate tank.In captivity, zebrafish live approximately forty-two months. Some captive zebrafish can develop a curved spine.[40]
The zebra danio was also used to make genetically modified fish and were the first species to be sold asGloFish (fluorescent colored fish).
In late 2003,transgenic zebrafish that expressgreen, red, andyellow fluorescent proteins became commercially available in the United States. The fluorescent strains are trade-namedGloFish; other cultivated varieties include "golden", "sandy", "longfin" and "leopard".
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The leopard danio, previously known asDanio frankei, is a spotted colourmorph of the zebrafish which arose due to a pigment mutation.[41]Xanthistic forms of both the zebra and leopard pattern, along with long-finned strains, have been obtained via selective breeding programs for the aquarium trade.[42]
Various transgenic and mutant strains of zebrafish were stored at theChina Zebrafish Resource Center (CZRC), a non-profit organization, which was jointly supported by theMinistry of Science and Technology of China and theChinese Academy of Sciences.[43]
TheZebrafish Information Network (ZFIN) provides up-to-date information about current knownwild-type (WT) strains ofD. rerio, some of which are listed below.[44]
Hybrids between differentDanio species may be fertile: for example, betweenD. rerio andD. nigrofasciatus.[12]
D. rerio is a common and useful scientificmodel organism for studies ofvertebrate development andgene function. Its use as a laboratory animal was pioneered by the Americanmolecular biologistGeorge Streisinger and his colleagues at theUniversity of Oregon in the 1970s and 1980s; Streisinger's zebrafishclones were among the earliest successful vertebrate clones created.[45] Its importance has been consolidated by successful large-scale forwardgenetic screens (commonly referred to as the Tübingen/Boston screens). The fish has a dedicated online database of genetic, genomic, and developmental information, theZebrafish Information Network (ZFIN). The Zebrafish International Resource Center (ZIRC) is a genetic resource repository with 29,250alleles available for distribution to the research community.D. rerio is also one of the few fish speciesto have been sent into space.
Research withD. rerio has yielded advances in the fields ofdevelopmental biology,oncology,[46]toxicology,[34][47][48] reproductive studies,teratology,genetics,neurobiology,environmental sciences,stem cell research,regenerative medicine,[49][50]muscular dystrophies[51] andevolutionary theory.[12]
As a modelbiological system, the zebrafish possesses numerous advantages for scientists. Itsgenome has beenfully sequenced at ~1.4 millionbase pairs,[52] and it has well-understood, easily observable and testable developmental behaviors. Itsembryonic development is very rapid, and its embryos are relatively large, robust, and transparent, and able to develop outside their mother.[53] Furthermore, well-characterized mutant strains are readily available.
Other advantages include the species' nearly constant size during early development, which enables simplestaining techniques to be used, and the fact that its two-celled embryo can be fused into a single cell to create ahomozygous embryo. The zebrafish embryos are transparent and they develop outside of the uterus, which allows scientists to study the details of development starting from fertilization and continuing throughout development. The zebrafish is also demonstrably similar to mammalian models and humans in toxicity testing, and exhibits a diurnal sleep cycle with similarities to mammalian sleep behavior.[54] However, zebrafish are not a universally ideal research model; there are a number of disadvantages to their scientific use, such as the absence of a standard diet[55] and the presence of small but important differences between zebrafish and mammals in the roles of some genes related to human disorders.[56][57]
Zebrafish have the ability toregenerate their heart andlateral linehair cells during their larval stages.[58][59] The cardiac regenerative process likely involves signaling pathways such asNotch andWnt; hemodynamic changes in the damaged heart are sensed by ventricularendothelial cells and their associated cardiac cilia by way of the mechanosensitive ion channelTRPV4, subsequently facilitating theNotch signaling pathway viaKLF2 and activating various downstream effectors such asBMP-2 andHER2/neu.[60] In 2011, theBritish Heart Foundation ran an advertising campaign publicising its intention to study the applicability of this ability to humans, stating that it aimed to raise £50 million in research funding.[61][62]
Zebrafish have also been found to regeneratephotoreceptor cells andretinal neurons following injury, which has been shown to be mediated by the dedifferentiation and proliferation ofMüller glia.[63] Researchers frequentlyamputate the dorsal and ventral tail fins and analyze their regrowth to test for mutations. It has been found thathistone demethylation occurs at the site of the amputation, switching the zebrafish's cells to an "active", regenerative, stem cell-like state.[64][65] In 2012, Australian scientists published a study revealing that zebrafish use a specialisedprotein, known asfibroblast growth factor, to ensure theirspinal cords heal withoutglial scarring after injury.[6][66] In addition,hair cells of the posteriorlateral line have also been found to regenerate following damage or developmental disruption.[59][67] Study of gene expression during regeneration has allowed for the identification of several important signaling pathways involved in the process, such asWnt signaling andFibroblast growth factor.[67][68]
In probing disorders of the nervous system, including neurodegenerative diseases, movement disorders, psychiatric disorders and deafness, researchers are using the zebrafish to understand how the genetic defects underlying these conditions cause functional abnormalities in the human brain, spinal cord and sensory organs.[69][70][71][72] Researchers have also studied the zebrafish to gain new insights into the complexities of human musculoskeletal diseases, such asmuscular dystrophy.[73] Another focus of zebrafish research is to understand how a gene calledHedgehog, a biological signal that underlies a number of human cancers, controls cell growth.
Inbred strains and traditional outbred stocks have not been developed for laboratory zebrafish, and the genetic variability of wild-type lines among institutions may contribute to thereplication crisis in biomedical research.[74] Genetic differences in wild-type lines among populations maintained at different research institutions have been demonstrated using bothSingle-nucleotide polymorphisms[75] andmicrosatellite analysis.[76]
Due to their fast and short life cycles and relatively large clutch sizes,D. rerio or zebrafish are a useful model for genetic studies. A commonreverse genetics technique is toreduce gene expression or modifysplicing usingMorpholinoantisense technology. Morpholinooligonucleotides (MO) are stable, syntheticmacromolecules that contain the samebases as DNA or RNA; by binding to complementary RNA sequences, they can reduce theexpression of specific genes or block other processes from occurring on RNA. MO can be injected into one cell of an embryo after the 32-cell stage, reducing gene expression in only cells descended from that cell. However, cells in the early embryo (less than 32 cells) are permeable to large molecules,[77][78] allowing diffusion between cells. Guidelines for using Morpholinos in zebrafish describe appropriate control strategies.[79] Morpholinos are commonlymicroinjected in 500pL directly into 1–2 cell stage zebrafish embryos. The morpholino is able to integrate into most cells of the embryo.[80]
A known problem with gene knockdowns is that, because the genome underwent aduplication after the divergence ofray-finned fishes andlobe-finned fishes, it is not always easy to silence the activity of one of the two geneparalogs reliably due tocomplementation by the other paralog.[81] Despite the complications of the zebrafishgenome, a number of commercially available global platforms exist for analysis of both gene expression bymicroarrays and promoter regulation usingChIP-on-chip.[82]
TheWellcome Trust Sanger Institute started the zebrafish genome sequencing project in 2001, and the full genome sequence of the Tuebingen reference strain is publicly available at theNational Center for Biotechnology Information (NCBI)'sZebrafish Genome Page. The zebrafish reference genome sequence is annotated as part of theEnsemblproject, and is maintained by theGenome Reference Consortium.[83]
In 2009, researchers at theInstitute of Genomics and Integrative Biology in Delhi, India, announced the sequencing of the genome of a wild zebrafish strain, containing an estimated 1.7 billion genetic letters.[84][85] The genome of the wild zebrafish was sequenced at 39-fold coverage. Comparative analysis with the zebrafish reference genome revealed over 5 million single nucleotide variations and over 1.6 million insertion deletion variations. The zebrafish reference genome sequence of 1.4GB and over 26,000 protein coding genes was published by Kerstin Howeet al. in 2013.[86]
In October 2001, researchers from theUniversity of Oklahoma publishedD. rerio's completemitochondrial DNA sequence.[87] Its length is 16,596 base pairs. This is within 100 base pairs of other related species of fish, and it is notably only 18 pairs longer than the goldfish (Carassius auratus) and 21 longer than thecarp (Cyprinus carpio). Its gene order and content are identical to the commonvertebrate form of mitochondrial DNA. It contains 13protein-coding genes and a noncoding control region containing theorigin of replication for the heavy strand. In between a grouping of fivetRNA genes, a sequence resembling vertebrate origin of light strand replication is found. It is difficult to draw evolutionary conclusions because it is difficult to determine whether base pair changes have adaptive significance via comparisons with other vertebrates'nucleotide sequences.[87]
T-boxes andhomeoboxes are vital inDanio similarly to other vertebrates.[88][89] The Bruce et al. team are known for this area, and in Bruce et al. 2003 & Bruce et al. 2005 uncover the role of two of these elements inoocytes of this species.[88][89] By interfering via adominant nonfunctionalallele and amorpholino they find the T-box transcription activatorEomesodermin and its targetmtx2 – atranscription factor – are vital toepiboly.[88][89] (In Bruce et al. 2003 they failed to support the possibility that Eomesodermin behaves likeVegt.[88] Neither they nor anyone else has been able to locate anymutation which – in the mother – will prevent initiation of themesoderm orendoderm development processes in this species.)[88]
In 1999, thenacre mutation was identified in the zebrafish ortholog of the mammalianMITF transcription factor.[90] Mutations in humanMITF result in eye defects and loss of pigment, a type ofWaardenburg Syndrome. In December 2005, a study of thegolden strain identified the gene responsible for its unusual pigmentation asSLC24A5, asolute carrier that appeared to be required formelanin production, and confirmed its function with a Morpholino knockdown. Theorthologous gene was then characterized in humans and a one base pair difference was found to strongly segregate fair-skinned Europeans and dark-skinned Africans.[91] Zebrafish with thenacre mutation have since been bred with fish with aroy orbison (roy) mutation to make Casper strain fish that have no melanophores or iridophores, and are transparent into adulthood. These fish are characterized by uniformly pigmented eyes and translucent skin.[8][92]
Transgenesis is a popular approach to study the function of genes in zebrafish. Construction of transgenic zebrafish is rather easy by a method using theTol2 transposon system.Tol2 element which encodes a gene for a fully functional transposase capable of catalyzing transposition in the zebrafish germ lineage.Tol2 is the only natural DNA transposable element in vertebrates from which an autonomous member has been identified.[93][94] Examples include the artificial interaction produced betweenLEF1 andCatenin beta-1/β-catenin/CTNNB1. Dorsky et al. 2002 investigated the developmental role ofWnt by transgenically expressing a Lef1/β-catenin reporter.[95] The Tol2 transposon system was used to develop transgenic zebrafish as sensitive biosensors for heavy metal detection. This involved creating a transgenic zebrafish line expressing a fluorescent protein under the control of a heavy metal-responsive promoter, enabling the detection of low concentrations of cadmium (Cd2+) and zinc (Zn2+).[96]
There are well-established protocols for editing zebrafish genes usingCRISPR-Cas9[97] and this tool has been used to generate genetically modified models.
In 2008, researchers atBoston Children's Hospital developed a new strain of zebrafish, named Casper, whose adult bodies had transparent skin.[8] This allows for detailed visualization of cellular activity, circulation,metastasis and many other phenomena.[8] In 2019 researchers published a crossing of aprkdc-/- and aIL2rga-/- strain that produced transparent, immunodeficient offspring, lackingnatural killer cells as well asB- andT-cells. This strain can be adapted to 37 °C (99 °F) warm water and the absence of an immune system makes the use of patient derivedxenografts possible.[98] In January 2013, Japanese scientists genetically modified a transparent zebrafish specimen to produce a visible glow during periods of intense brain activity.[9]
In January 2007, Chinese researchers atFudan University genetically modified zebrafish to detectoestrogen pollution in lakes and rivers, which is linked to male infertility. The researchers cloned oestrogen-sensitive genes and injected them into the fertile eggs of zebrafish. The modified fish turned green if placed into water that was polluted by oestrogen.[7]
In 2015, researchers atBrown University discovered that 10% of zebrafish genes do not need to rely on theU2AF2protein to initiateRNA splicing. These genes have the DNA base pairs AC and TG as repeated sequences at the ends of eachintron. On the 3'ss (3' splicing site), the base pairsadenine andcytosine alternate and repeat, and on the 5'ss (5' splicing site), their complementsthymine andguanine alternate and repeat as well. They found that there was less reliance on U2AF2 protein than in humans, in which the protein is required for the splicing process to occur. The pattern of repeating base pairs around introns that alters RNAsecondary structure was found in otherteleosts, but not intetrapods. This indicates that an evolutionary change in tetrapods may have led to humans relying on the U2AF2 protein for RNA splicing while these genes in zebrafish undergo splicing regardless of the presence of the protein.[99]
D. rerio has threetransferrins, all of which cluster closely with othervertebrates.[100]
When close relatives mate, progeny may exhibit the detrimental effects ofinbreeding depression. Inbreeding depression is predominantly caused by thehomozygous expression of recessive deleterious alleles.[101] For zebrafish, inbreeding depression might be expected to be more severe in stressful environments, including those caused byanthropogenic pollution. Exposure of zebrafish to environmental stress induced by the chemical clotrimazole, an imidazole fungicide used in agriculture and in veterinary and human medicine, amplified the effects of inbreeding on key reproductive traits.[102] Embryo viability was significantly reduced in inbred exposed fish and there was a tendency for inbred males to sire fewer offspring.
Zebrafish are common models for research intofish farming, includingpathogens[103][104][105] andparasites[103][105] causing yield loss or spreading to adjacent wild populations.
This usefulness is less than it might be due toDanio'staxonomic distance from the most common aquaculture species.[104] Because the most common aresalmonids andcod in theProtacanthopterygii andsea bass,sea bream,tilapia, andflatfish, in thePercomorpha, zebrafish results may not be perfectly applicable.[104] Various othermodels – Goldfish (Carassius auratus), Medaka (Oryzias latipes), Stickleback (Gasterosteus aculeatus), Roach (Rutilus rutilus), Pufferfish (Takifugu rubripes), Swordtail (Xiphophorus hellerii) – are less used normally but would be closer to particular target species.[105]
The only exception are theCarp (including Grass Carp,Ctenopharyngodon idella)[104] and Milkfish (Chanos chanos)[105] which are quite close, both being in theCyprinidae. However it should also be noted thatDanio consistently proves to be a useful model for mammals in many cases and there is dramatically moregenetic distance between them than betweenDanio and any farmed fish.[104]
In aglucocorticoid receptor-defective mutant with reducedexploratory behavior,fluoxetine rescued the normal exploratory behavior.[106] This demonstrates relationships between glucocorticoids, fluoxetine, and exploration in this fish.[106]
Zebrafish have been used as a model for studying DNA repair pathways.[107] Embryos of externally fertilized fish species, such as zebrafish during their development, are directly exposed to environmental conditions such as pollutants andreactive oxygen species that may causedamage to their DNA.[107] To cope with such DNA damages, a variety of differentDNA repair pathways are expressed during development.[107] Zebrafish have, in recent years, proven to be a useful model for assessing environmental pollutants that might cause DNA damage.[108]
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The zebrafish and zebrafish larva is a suitable model organism for drug discovery and development. As a vertebrate with 70% genetic homology with humans,[86] it can be predictive of human health and disease, while its small size and fast development facilitates experiments on a larger and quicker scale than with more traditionalin vivo studies, including the development of higher-throughput, automated investigative tools.[109][110] As demonstrated through ongoing research programmes, the zebrafish model enables researchers not only to identify genes that might underlie human disease, but also to develop novel therapeutic agents in drug discovery programmes.[111] Zebrafish embryos have proven to be a rapid, cost-efficient, and reliableteratology assay model.[112]
Drug screens in zebrafish can be used to identify novel classes of compounds with biological effects, or to repurpose existing drugs for novel uses; an example of the latter would be a screen which found that a commonly used statin (rosuvastatin) can suppress the growth ofprostate cancer.[113] To date, 65 small-molecule screens have been carried out and at least one has led to clinical trials.[114] Within these screens, many technical challenges remain to be resolved, including differing rates of drug absorption resulting in levels of internal exposure that cannot be extrapolated from the water concentration, and high levels of natural variation between individual animals.[114]
To understand drug effects, the internal drug exposure is essential, as this drives the pharmacological effect. Translating experimental results from zebrafish to higher vertebrates (like humans) requires concentration-effect relationships, which can be derived frompharmacokinetic andpharmacodynamic analysis.[5]Because of its small size, however, it is very challenging to quantify the internal drug exposure. Traditionally multiple blood samples would be drawn to characterize the drug concentration profile over time, but this technique remains to be developed. To date, only a single pharmacokinetic model forparacetamol has been developed in zebrafish larvae.[115]
Using smart data analysis methods, pathophysiological and pharmacological processes can be understood and subsequently translated to higher vertebrates, including humans.[5][116] An example is the use ofsystems pharmacology, which is the integration ofsystems biology andpharmacometrics. Systems biology characterizes (part of) an organism by a mathematical description of all relevant processes. These can be for example different signal transduction pathways that upon a specific signal lead to a certain response. By quantifying these processes, their behaviour in healthy and diseased situation can be understood and predicted. Pharmacometrics uses data from preclinical experiments andclinical trials to characterize the pharmacological processes that are underlying the relation between the drug dose and its response or clinical outcome. These can be for example the drugabsorption in orclearance from the body, or its interaction with the target to achieve a certain effect. By quantifying these processes, their behaviour after different doses or in different patients can be understood and predicted to new doses or patients.By integrating these two fields, systems pharmacology has the potential to improve the understanding of the interaction of the drug with the biological system by mathematical quantification and subsequent prediction to new situations, like new drugs or new organisms or patients.Using these computational methods, the previously mentioned analysis of paracetamol internal exposure in zebrafish larvae showed reasonable correlation between paracetamol clearance in zebrafish with that of higher vertebrates, including humans.[115]
Zebrafish have been used to make several transgenic models of cancer, includingmelanoma,leukemia,pancreatic cancer andhepatocellular carcinoma.[117][118] Zebrafish expressing mutated forms of either the BRAF or NRASoncogenes develop melanoma when placed onto a p53 deficient background.Histologically, these tumors strongly resemble the human disease, are fully transplantable, and exhibit large-scale genomic alterations. The BRAF melanoma model was utilized as a platform for two screens published in March 2011 in the journalNature. In one study, the model was used as a tool to understand the functional importance of genes known to be amplified and overexpressed in human melanoma.[119] One gene, SETDB1, markedly accelerated tumor formation in the zebrafish system, demonstrating its importance as a new melanoma oncogene. This was particularly significant because SETDB1 is known to be involved in the epigenetic regulation that is increasingly appreciated to be central to tumor cell biology.
In another study, an effort was made to therapeutically target the genetic program present in the tumor's originneural crest cell using a chemical screening approach.[120] This revealed that an inhibition of the DHODH protein (by a small molecule called leflunomide) prevented development of the neural crest stem cells which ultimately give rise to melanoma via interference with the process oftranscriptional elongation. Because this approach would aim to target the "identity" of the melanoma cell rather than a single genetic mutation, leflunomide may have utility in treating human melanoma.[121]
In cardiovascular research, the zebrafish has been used to model human myocardial infarction model. The zebrafish heart completely regenerates after about 2 months of injury without any scar formation.[122] TheAlpha-1 adrenergic signalling mechanism involved in this process was identified in a 2023 study.[123] Zebrafish is also used as a model forblood clotting,blood vessel development, andcongenital heart and kidney disease.[124]
In programmes of research into acuteinflammation, a major underpinning process in many diseases, researchers have established a zebrafish model of inflammation, and its resolution. This approach allows detailed study of the genetic controls of inflammation and the possibility of identifying potential new drugs.[125]
Zebrafish has been extensively used as a model organism to study vertebrate innate immunity. The innate immune system is capable of phagocytic activity by 28 to 30 h postfertilization (hpf)[126] while adaptive immunity is not functionally mature until at least 4 weeks postfertilization.[127]
As the immune system is relatively conserved between zebrafish and humans, many human infectious diseases can be modeled in zebrafish.[128][129][130][131] The transparent early life stages are well suited forin vivo imaging and genetic dissection of host-pathogen interactions.[132][133][134][135] Zebrafish models for a wide range of bacterial, viral and parasitic pathogens have already been established; for example, the zebrafish model for tuberculosis provides fundamental insights into the mechanisms of pathogenesis of mycobacteria.[136][137][138][139] Other bacteria commonly studied using zebrafish models includeClostridioides difficile,Staphylococcus aureus, andPseudomonas aeruginosa.[140] Furthermore, robotic technology has been developed for high-throughput antimicrobial drug screening using zebrafish infection models.[141][142]
Another notable characteristic of the zebrafish is that it possesses four types ofcone cell, withultraviolet-sensitive cells supplementing the red, green and blue cone cell subtypes found in humans. Zebrafish can thus observe a very wide spectrum of colours. The species is also studied to better understand the development of the retina; in particular, how the cone cells of the retina become arranged into the so-called 'cone mosaic'. Zebrafish, in addition to certain otherteleost fish, are particularly noted for having extreme precision of cone cell arrangement.[143]
This study of the zebrafish's retinal characteristics has also extrapolated into medical enquiry. In 2007, researchers atUniversity College London grew a type of zebrafish adultstem cell found in the eyes of fish and mammals that develops intoneurons in the retina. These could be injected into the eye to treat diseases that damage retinal neurons—nearly every disease of the eye, includingmacular degeneration,glaucoma, anddiabetes-related blindness. The researchers studied Müllerglial cells in the eyes of humans aged from 18 months to 91 years, and were able to develop them into all types of retinal neurons. They were also able to grow them easily in the lab. The stem cells successfully migrated into diseased rats' retinas, and took on the characteristics of the surrounding neurons. The team stated that they intended to develop the same approach in humans.[144][145]
Muscular dystrophies (MD) are a heterogeneous group of genetic disorders that cause muscle weakness, abnormal contractions and muscle wasting, often leading to premature death. Zebrafish is widely used as model organism to study muscular dystrophies.[51] For example, thesapje (sap) mutant is the zebrafish orthologue of humanDuchenne muscular dystrophy (DMD).[146] The Machuca-Tzili and co-workers applied zebrafish to determine the role of alternative splicing factor, MBNL, inmyotonic dystrophy type 1 (DM1) pathogenesis.[147] More recently, Todd et al. described a new zebrafish model designed to explore the impact of CUG repeat expression during early development in DM1 disease.[148] Zebrafish is also an excellent animal model to study congenital muscular dystrophies including CMD Type 1 A (CMD 1A) caused by mutation in the human laminin α2 (LAMA2) gene.[149] The zebrafish, because of its advantages discussed above, and in particular the ability of zebrafish embryos to absorb chemicals, has become a model of choice in screening and testing new drugs against muscular dystrophies.[150]
Zebrafish have been used as model organisms for bone metabolism, tissue turnover, and resorbing activity. These processes are largely evolutionary conserved. They have been used to study osteogenesis (bone formation), evaluating differentiation, matrix deposition activity, and cross-talk of skeletal cells, to create and isolate mutants modeling human bone diseases, and test new chemical compounds for the ability to revert bone defects.[151][152] The larvae can be used to follow new (de novo) osteoblast formation during bone development. They start mineralising bone elements as early as 4 days post fertilisation. Recently, adult zebrafish are being used to study complex age related bone diseases such asosteoporosis andosteogenesis imperfecta.[153] The (elasmoid)scales of zebrafish function as a protective external layer and are little bony plates made by osteoblasts. These exoskeletal structures are formed by bone matrix depositing osteoblasts and are remodeled by osteoclasts. The scales also act as the main calcium storage of the fish. They can be cultured ex-vivo (kept alive outside of the organism) in a multi-well plate, which allows manipulation with drugs and even screening for new drugs that could change bone metabolism (between osteoblasts and osteoclasts).[153][154][155]
Zebrafish pancreas development is very homologous to mammals, such as mice. The signaling mechanisms and way the pancreas functions are very similar. The pancreas has an endocrine compartment, which contains a variety of cells. Pancreatic PP cells that produce polypeptides, and β-cells that produce insulin are two examples of those such cells. This structure of the pancreas, along with the glucose homeostasis system, are helpful in studying diseases, such as diabetes, that are related to the pancreas. Models for pancreas function, such as fluorescent staining of proteins, are useful in determining the processes of glucose homeostasis and the development of the pancreas. Glucose tolerance tests have been developed using zebrafish, and can now be used to test for glucose intolerance or diabetes in humans. The function of insulin are also being tested in zebrafish, which will further contribute to human medicine. The majority of work done surrounding knowledge on glucose homeostasis has come from work on zebrafish transferred to humans.[156]
Zebrafish have been used as a model system to study obesity, with research into both genetic obesity and over-nutrition induced obesity. Obese zebrafish, similar to obese mammals, show dysregulation of lipid controlling metabolic pathways, which leads to weight gain without normal lipid metabolism.[156] Also like mammals, zebrafish store excess lipids in visceral, intramuscular, and subcutaneous adipose deposits. These reasons and others make zebrafish good models for studying obesity in humans and other species. Genetic obesity is usually studied in transgenic or mutated zebrafish with obesogenic genes. As an example, transgenic zebrafish with overexpressed AgRP, an endogenous melanocortin antagonist, showed increased body weight and adipose deposition during growth.[156] Though zebrafish genes may not be the exact same as human genes, these tests could provide important insight into possible genetic causes and treatments for human genetic obesity.[156] Diet-induced obesity zebrafish models are useful, as diet can be modified from a very early age. High fat diets and general overfeeding diets both show rapid increases in adipose deposition, increased BMI, hepatosteatosis, and hypertriglyceridemia.[156] However, the normal fat, overfed specimens are still metabolically healthy, while high-fat diet specimens are not.[156] Understanding differences between types of feeding-induced obesity could prove useful in human treatment of obesity and related health conditions.[156]
Zebrafish have been used as a model system inenvironmental toxicology studies.[34]
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The combination of transparent zebrafish larva,light sheet fluorescence microscopy, andoptical calcium indicators such asGCaMP, allow the monitoring of all neurons in an awake, behaving animal.[157]
Zebrafish have been used as a model system to study epilepsy. Mammalian seizures can be recapitulated molecularly, behaviorally, and electrophysiologically, using a fraction of the resources required for experiments in mammals.[158]
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