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BONY FISHES | Zebrafish
PHYSIOLOGICAL SPECIALIZATIONS OF DIFFERENT FISH GROUPS Bony Fishes Contents Zebrafish
Crucian Carp
Life in Hot Water: The Desert Pupfish
Blind Cavefish
Zebrafish JG Richards, University of British Columbia, Vancouver, BC, Canada ª 2011 Elsevier Inc. All rights reserved.
Introduction Appearance Taxonomy Distribution and Natural History
Glossary Forward genetics The induction of random mutations in an organism followed by examining of the resulting phenotype. Fusiform A body shape characterized by tapering at both the anterior and the posterior ends. Gastrulation Migration and rearrangement of cells after embryo undergoes proliferation to increase cell number (cleavage stage) to create three primary germ layers during early development.
Introduction The zebrafish, Danio rerio, is now one of the mostly widely used experimental animals in biological and biomedical research. This popularity is primarily due to their reasonably fast generation time and the vast array of genomic resources available, which allow for manipulations at the smallest scale. Furthermore, they are almost transparent during the majority of their embryonic development, making them amenable to direct visualization of developmental processes. Until
Lifecycle and Reproduction Zebrafish as a Model Organism for Scientific Research Further Reading
Insertional mutagenesis Insertion of one or more bases into the genome. Retrovirus An RNA virus that is replicated in a host cell via the enzyme reverse transcriptase to produce DNA from its RNA genome, which is then incorporated into the host’s genome. Reverse genetics Identification of gene function through selective manipulation of that gene followed by analysis of the arising phenotype.
recently, however, their use in physiological studies has been limited and, for the most part, the vast major ity of the published literature on these fish does not work toward understanding their physiology, but rather understanding basic biological processes. They are also extremely small when it comes to tissue sampling. With the incorporation of genomics tools into physio logical research, fish physiologists are now flocking to this small cyprinid to ask new and exciting questions about physiology, adaptation (see also Responses and Adaptations to the Environment: General Principles of
1819
1820 Bony Fishes | Zebrafish
Biochemical Adaptations), and how this animal responds to environmental challenge. This article, however, focuses primarily on the natural history, development, and genomic resources available in this important biolo gical model. Aspects of their physiology are covered elsewhere in this encyclopedia.
D. hikari D. kerri D. albolineatus D. kyathit D. rerio
Danio
D. nigrofasciatus D. choprae
Appearance The zebrafish is aptly named for the five uniform, hor izontal blue or black stripes that are located on the side of the body, all of which extend from the operculum through to the caudal fin (Figure 1). The fish shape is character ized by tapering at both the anterior and the posterior ends (commonly referred to as fusiform) and it is laterally compressed with an upward directed mouth. Zebrafish are sexually dimorphic, meaning males and females look different. Male zebrafish are thinner and more torpedo shaped, with yellow stripes between the blue/black stripes; female zebrafish are generally larger, with white bellies and silver stripes instead of yellow ones. Adult zebrafish have been recorded to grow up to �6.5 cm, but for the most part captive zebrafish do not exceed 4 cm in length.
D. dangila D. malabaricus D. aequipinnatus D. pathirana
Devario
D. devario D. shanensis Rasbora paviei Rasbora trilineata Pseudorasbora parva
Tanichthys albonubes albonubes
Figure 2 Phylogenetic relationship of 13 species of Danio and Davario based on maximum likelihood and Bayesian analysis of 12S and 16S ribosomal DNA sequence. Phylogeny has been modified from Parichy DM (2006) Evolution of Danio pigment pattern development. Heredity 97: 200–210.
showing the split in species is depicted in Figure 2 and was originally constructed in order to examine coloration patterns among different species.
Taxonomy Zebrafish are a tropical freshwater fish belonging to the family Cyprinidae from the taxonomic order Cypriniformes. The Cyprinidae is the largest family of freshwater fish with roughly 2500 species in just over 200 genera. Of particular note, this family includes the carps (e.g., the anoxia-tolerant Crucian carp (Carassius carassius; see also Bony Fishes: Crucian Carp) and the goldfish (Carassius auratus) as well as the true minnows. The genus Danio was at one point comprised of some where between 35 and 50 species, but this traditional grouping of all species under the genus Danio was split and now the species are divided among two different genera, Danio and Davario. An example phylogeny
Figure 1 Photo of a male Danio rerio.
Distribution and Natural History Zebrafish originate from the waters from the southeastern Himalayan region, but there is some controversy over their precise natural range. Recent analysis of collection records suggest that the natural geographic range of zebrafish extends from Pakistan to the western edge of Myanmar and from Nepal in the north to the Indian state of Karnataka in the south. They are primarily found in streams, canals, ditches, rice fields, and other slow-moving bodies of water with clay, silt, or cobble substrates. The waters are typically still and clear, roughly ranging in temperature between 27 and 34 � C and a slightly alkaline pH (7.9–8.5). In these environments, they mostly feed on mosquito larvae and other small insects, but they are omnivorous and have also been observed feeding on phytoplankton. They are unlikely to experience large variations in temperature within a single body of water, although the temperatures are warm and there is likely temperature variation over their natural range. The highly vegetated nature of their water and the fact that it can be stagnant likely means zebrafish experience large variations in dissolved oxygen on a daily basis (see also Hypoxia: The Expanding Hypoxic Environment). Oddly, despite the fact that hypoxia is likely prevalent in their natural environment, only a small amount of work has been done on their hypoxia tolerance. For more
Bony Fishes | Zebrafish
information on hypoxia response of zebrafish, the reader is referred to Hypoxia: Respiratory Responses to Hypoxia in Fishes.
Lifecycle and Reproduction The typical life span of zebrafish is roughly 2–3 years, but it can be up to 5 years under ideal conditions in captivity, but probably much shorter in the wild. The generation time from a fertilized egg to a reproductively active adult is between 3 and 4 months. Adult females are able to spawn every 2–3 days and each female can lay up to 200 eggs/ week, but ovulation and spawning only occur in the pre sence of a male. Zebrafish mate in the morning and it is well known that light is the primary cue for mating. Within 30 min of the lights being turned on (if in the laboratory) or sunrise (if in the natural environment), a female will some times lay hundreds of eggs, which are subsequently fertilized by a male. At high densities in aquarium-bred zebrafish, it is typical to observe group-spawning behavior where egg fertilization is done in an indiscriminate fashion. Upon the release of eggs, embryonic development begins immediately, but if not fertilized, growth is halted after the first few cell divisions and the eggs die or are eaten.
1821
Upon fertilization, the eggs almost immediately become transparent and development progresses quickly (see Figure 3 for an outline of the lifecycle of a zebrafish (see also Reproduction: The Diversity of Fish Reproduction: An Introduction). Within 4 h, the fertilized egg has gone from a single cell to �1000 cells. Shortly after that, the embryo undergoes gastrulation, which is the developmental stage associated with the formation of a double-walled embryo resulting from the invagination of the blastula, which is associated with the differentiation of the ectoderm into the mesoderm and endoderm. The development of discreet organs, referred to as organogen esis, begins between 16 and 24 h postfertilization (hpf) and the precursors to all major organs appear within 36 hpf. For example, the zebrafish heart starts off as a ‘heart tube’ which is formed by 18 hpf and rhythmic, peristaltic con tractions can be observed by 24 hpf (see also Circulation: The Circulatory System: An Introduction). Hatching Ultimately takes place 48–72 hpf, depending on the tem perature, and feeding and swimming begin at roughly 72 hpf. Within 2–3 months, the zebrafish have matured into adults and can then begin breeding. Although the reproductive behavior of captive zebrafish is well known, the reproductive behavior of wild zebrafish is largely unexplored. Wild-reared zebrafish
Thirty minutes after fertilization
2 hpf
60–90 dpf Adult
4 hpf Free swimming
6 hpf 2 dpf
8 hpf 1 dpf
16 hpf Figure 3 Lifecycle of the zebrafish. The development of zebrafish is rapid with the fertilized embryo transforming into a 1000-cell stage within 4 h postfertilization (hpf) and organs being visible at roughly 1 day postfertilization (dpf). Hatching occurs at 2 dpf and the zebrafish matures within 2–3 months. Image adapted from Wolpert L, Beddington R, Jessell T, et al. (2002) Principles of Development, 2nd edn. New York: Oxford University Press.
1822 Bony Fishes | Zebrafish
take up to 4–5 months to reach sexual maturity rather than the 2–3 months commonly observed in the labora tory. In one study, wild-caught zebrafish from India were observed to spawn in pairs rather than in groups as is common in aquaria. Furthermore, there was evidence for sexual selection and females taking an active role in choosing their mates. This discrimination may be primar ily mediated by density in that, at low densities as seen in the wild, there may be elevated levels of sexual selection and pair formation; however, at high densities, character istic of aquarium breeding, sexual selection and pair formation break down.
Zebrafish as a Model Organism for Scientific Research Zebrafish have long been an important model in devel opmental biology, but their rise as an important experimental system in biology emerged from several major advances that have occurred since the early 1990s. First, the most important advance came in the 1990s, where several research groups induced large-scale genetic mutations in the zebrafish and subsequently screened the mutants for identifiable phenotypes. This approach of inducing random mutations and then search ing for the genetic underpinnings is referred to as forward genetics. The second major advance was the start of a whole-genome sequencing project that was initiated in 2001 and is still underway. This large-scale sequencing project has facilitated the third major advance, reverse genetics, which involves modifying single genes and then examining the effects of these genetic changes on the whole-animal phenotype. Forward Genetics in Zebrafish The first step in the popularization of zebrafish as a model organism in biology was the mutagenesis projects initiated in the early 1990s. This work followed in the footsteps of earlier work performed on the fruit fly (Drosophila melanogaster) and the nematode (Caenorhabditis elegans). In 1996, two important works were published by Driever and Haffter and their respective co-workers that described two large-scale genetic mutation screenings, where a chemical mutagen, ethylnitrosourea, was used to induce random mutations in zebrafish sperm. Ethylnitrosourea induces point mutations in the sperm genome that could affect protein sequences or gene struc ture and ultimately cause, through random chance, a loss of some physiological or biochemical function. After breeding, the developing embryos were screened for visi ble abnormalities in muscle and other organs at several different time points and from these screens more than
6500 mutants were identified. It is important to note that the majority of the mutations were lethal if they appeared homozygous in the fish. At the same time as the chemical mutagenesis was underway, in a separate research laboratory Gaiano and co-workers used a retrovirus to perform insertional muta genesis, which refers to the insertion of one or more bases into the genome. The subsequent screening of these mutants resulted in the identification of roughly 500 mutations at the phenotypic level of which just over 300 have been explicitly linked to a specific genetic change. One important advantage of this technique over the che mical mutagenesis described above is that a portion of the retroviral gene sequence is inserted into the host zebrafish genome, thereby providing a genetic tag aiding in the identification of which gene is affected in the genome. The systemic issue with forward genetics is that it is resource intensive and the direct links between phenotype and specific mutations are difficult to establish (see also Cellular, Molecular, Genomics, and Biomedical Approaches: Fish as Model Organisms for Medical Research). Establishing these links is easier with insertional mutagenesis compared with chemical mutagenesis, but many of the direct links from these early studies have not yet been established. The genome sequencing project, started in 2001, has greatly enhanced the ability of researchers to identify where mutations have occurred and it plays an important role in advancing this field. The Zebrafish Genome Sequencing of the zebrafish genome was started in 2001 by the Sanger Institute and is due to be completed sometime in 2011. The zebrafish genome consists of 25 linkage groups, which are equivalent to chromosomes and the entire genome size is �1.4 gigabases. The genome is cur rently being annotated, which refers to the identification of genes and, once complete, this resource will be invaluable to researchers trying to understand the genetic basis of physiological and biochemical phenotypes. In addition, the genome sequencing has greatly facilitated the develop ment of reverse genetic approaches in zebrafish. Reverse Genetics in Zebrafish Reverse genetics refers to the identification of gene function through selective manipulation of the gene followed by analysis of the arising phenotype. This approach attempts to identify what phenotype arises as a result of a particular gene and, for the most part, this approach involves knocking down the expression of genes of interest with subsequent examination of phenotype. These types of studies are broadly referred to as loss-of-function studies. The most common approach for performing loss-of function studies in zebrafish is using antisense technology.
Bony Fishes | Zebrafish
The basic premise of this technique is that if the genetic sequence of a particular gene of interest is known, then researchers can synthesize a strand of RNA that will bind to the messenger RNA (mRNA) transcript produced by that gene. The synthetic RNA is called antisense because its nucleic acid sequence is complementary to the gene’s nor mal mRNA sequence, which is referred to as the ‘sense’ sequence. The binding of the synthetic antisense RNA to the sense mRNA results in a doubled-stranded RNA mole cule, which cannot be translated into protein. As a result, the incorporation of antisense RNA strands either limits or eliminates the protein coded for by the mRNA transcript. The most common antisense approach using zebrafish reverse genetics is morpholinos. Morpholinos are synthetic oligonucleotides, in which the normal ribose or deoxyribose sugars are replaced with a morpholine ring, which are con structed to be antisense to the mRNA of interest. Normally, morpholinos are �25 nucleotides long and are designed to bind to the native mRNA, usually near the protein transla tion start site. The advantage of incorporating a morpholine ring into the antisense sequence is that it makes the mor pholino resistant to the normal nucleases that are present in cells and would break down unwanted RNA. The binding of the morpholino to the native mRNA causes translation blockade, where the gene of interest is silenced and very little protein is translated. It is a typical practice in the zebrafish to inject the morpholinos into the freshly fertilized egg while it is still in the one-to-eight cell stage, after which the morpholino becomes more or less uniformly distributed through the cells of the developing embryo. After this point, the efficacy of the knockdown is assessed and the resultant effects of the knockdown on phenotype are typically assessed over the first 5 or so days postfertilization. Other commonly used methods of reverse genetics for loss-of-function studies in zebrafish are targeting-induced local lesions in genomes (TILLING) and zinc finger nucleases. TILLING combines forward genetic approaches with reverse genetics to identify specific mutations in genes of interest from a random mutagenesis screening. Specifically, this approach begins with the generation of a large number of mutated zebrafish, normally from a chemical mutagenesis screen as described earlier (see Section Forward Genetics in Zebrafish). DNA is then sampled from these fish and TILLING is performed. In the most basic sense, the purpose of TILLING is to look for specific mutations in specific genes in a collection of DNA samples prepared from chemically mutagenized individuals. Identification of these mutations is done using standard polymerase chain reaction (PCR) and sequencing of PCR products. Additional methods for identifying whether mutations have occurred in select areas can also be carried out to reduce the excessive sequencing costs associated with the TILLING approach. Zinc finger nucleases represent another method for reverse genetics. Zinc finger nucleases consist of several
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zinc-finger DNA-binding domains plus restriction enzymes that, when combined, can be engineered to target specific DNA sequences and cut the double-stranded DNA in intact cells. The normal cellular DNA repair mechan isms then repair the broken DNA strands, but often insert nucleotides during the repair process, thus altering the genetic sequence disrupting codon arrangement. Far less frequent are gain-of-function studies, which attempt to insert specific genes or alter genes in a way that could result in a new or enhanced phenotype. There are various techniques for inserting genes to examine gain of function, the simplest of which is microinjection of synthesized mRNA into the fertilized zebrafish eggs. These eggs then express, or, in some cases, overexpress the gene of interest and investigators can then examine the resulting phenotype. Other techniques are available for the expression or overexpression of genes in zebrafish and some of these techniques even allow researchers to target specific tissues or cells at precise developmental stages. Overall, there is an ever-expanding collection of cell biology and genomic techniques for the zebrafish that allow manipulation of proteins and development at the most basic level. Slowly, fish physiologists are adopting this model organism and its impressive array of tools to explicitly establish links between genes and physiological attributes. Consideration of the natural habitat and use of the wide array of species available will facilitate adoption of this model system to inves tigate environmental adaptation and evolutionary physiology. See also: Bony Fishes: Crucian Carp. Cellular, Molecular, Genomics, and Biomedical Approaches: Fish as Model Organisms for Medical Research. Circulation: The Circulatory System: An Introduction. Hypoxia: Respiratory Responses to Hypoxia in Fishes; The Expanding Hypoxic Environment. Reproduction: The Diversity of Fish Reproduction: An Introduction. Responses and Adaptations to the Environment: General Principles of Biochemical Adaptations.
Further Reading Driever W, SolnicaKrezel L, Schier AF, et al. (1996) A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123: 37–46. Gaiano N, Amsterdam A, Kawakami K, et al. (1996) Insertional mutagenesis and rapid cloning of essential genes in zebrafish. Nature 383: 829–832. Haffter P, Granato M, Brand M, et al. (1996) The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123: 1–36. Parichy DM (2006) Evolution of Danio pigment pattern development. Heredity 97: 200–210. Perry SF, Ekker M, Farrell AP, and Brauner CJ (2010) Zebrafish. Fish Physiology, vol. 29. San Diego, CA: Academic Press.
Crucian Carp
Life in Hot Water: The Desert Pupfish
Blind Cavefish
Zebrafish JG Richards, University of British Columbia, Vancouver, BC, Canada ª 2011 Elsevier Inc. All rights reserved.
Introduction Appearance Taxonomy Distribution and Natural History
Glossary Forward genetics The induction of random mutations in an organism followed by examining of the resulting phenotype. Fusiform A body shape characterized by tapering at both the anterior and the posterior ends. Gastrulation Migration and rearrangement of cells after embryo undergoes proliferation to increase cell number (cleavage stage) to create three primary germ layers during early development.
Introduction The zebrafish, Danio rerio, is now one of the mostly widely used experimental animals in biological and biomedical research. This popularity is primarily due to their reasonably fast generation time and the vast array of genomic resources available, which allow for manipulations at the smallest scale. Furthermore, they are almost transparent during the majority of their embryonic development, making them amenable to direct visualization of developmental processes. Until
Lifecycle and Reproduction Zebrafish as a Model Organism for Scientific Research Further Reading
Insertional mutagenesis Insertion of one or more bases into the genome. Retrovirus An RNA virus that is replicated in a host cell via the enzyme reverse transcriptase to produce DNA from its RNA genome, which is then incorporated into the host’s genome. Reverse genetics Identification of gene function through selective manipulation of that gene followed by analysis of the arising phenotype.
recently, however, their use in physiological studies has been limited and, for the most part, the vast major ity of the published literature on these fish does not work toward understanding their physiology, but rather understanding basic biological processes. They are also extremely small when it comes to tissue sampling. With the incorporation of genomics tools into physio logical research, fish physiologists are now flocking to this small cyprinid to ask new and exciting questions about physiology, adaptation (see also Responses and Adaptations to the Environment: General Principles of
1819
1820 Bony Fishes | Zebrafish
Biochemical Adaptations), and how this animal responds to environmental challenge. This article, however, focuses primarily on the natural history, development, and genomic resources available in this important biolo gical model. Aspects of their physiology are covered elsewhere in this encyclopedia.
D. hikari D. kerri D. albolineatus D. kyathit D. rerio
Danio
D. nigrofasciatus D. choprae
Appearance The zebrafish is aptly named for the five uniform, hor izontal blue or black stripes that are located on the side of the body, all of which extend from the operculum through to the caudal fin (Figure 1). The fish shape is character ized by tapering at both the anterior and the posterior ends (commonly referred to as fusiform) and it is laterally compressed with an upward directed mouth. Zebrafish are sexually dimorphic, meaning males and females look different. Male zebrafish are thinner and more torpedo shaped, with yellow stripes between the blue/black stripes; female zebrafish are generally larger, with white bellies and silver stripes instead of yellow ones. Adult zebrafish have been recorded to grow up to �6.5 cm, but for the most part captive zebrafish do not exceed 4 cm in length.
D. dangila D. malabaricus D. aequipinnatus D. pathirana
Devario
D. devario D. shanensis Rasbora paviei Rasbora trilineata Pseudorasbora parva
Tanichthys albonubes albonubes
Figure 2 Phylogenetic relationship of 13 species of Danio and Davario based on maximum likelihood and Bayesian analysis of 12S and 16S ribosomal DNA sequence. Phylogeny has been modified from Parichy DM (2006) Evolution of Danio pigment pattern development. Heredity 97: 200–210.
showing the split in species is depicted in Figure 2 and was originally constructed in order to examine coloration patterns among different species.
Taxonomy Zebrafish are a tropical freshwater fish belonging to the family Cyprinidae from the taxonomic order Cypriniformes. The Cyprinidae is the largest family of freshwater fish with roughly 2500 species in just over 200 genera. Of particular note, this family includes the carps (e.g., the anoxia-tolerant Crucian carp (Carassius carassius; see also Bony Fishes: Crucian Carp) and the goldfish (Carassius auratus) as well as the true minnows. The genus Danio was at one point comprised of some where between 35 and 50 species, but this traditional grouping of all species under the genus Danio was split and now the species are divided among two different genera, Danio and Davario. An example phylogeny
Figure 1 Photo of a male Danio rerio.
Distribution and Natural History Zebrafish originate from the waters from the southeastern Himalayan region, but there is some controversy over their precise natural range. Recent analysis of collection records suggest that the natural geographic range of zebrafish extends from Pakistan to the western edge of Myanmar and from Nepal in the north to the Indian state of Karnataka in the south. They are primarily found in streams, canals, ditches, rice fields, and other slow-moving bodies of water with clay, silt, or cobble substrates. The waters are typically still and clear, roughly ranging in temperature between 27 and 34 � C and a slightly alkaline pH (7.9–8.5). In these environments, they mostly feed on mosquito larvae and other small insects, but they are omnivorous and have also been observed feeding on phytoplankton. They are unlikely to experience large variations in temperature within a single body of water, although the temperatures are warm and there is likely temperature variation over their natural range. The highly vegetated nature of their water and the fact that it can be stagnant likely means zebrafish experience large variations in dissolved oxygen on a daily basis (see also Hypoxia: The Expanding Hypoxic Environment). Oddly, despite the fact that hypoxia is likely prevalent in their natural environment, only a small amount of work has been done on their hypoxia tolerance. For more
Bony Fishes | Zebrafish
information on hypoxia response of zebrafish, the reader is referred to Hypoxia: Respiratory Responses to Hypoxia in Fishes.
Lifecycle and Reproduction The typical life span of zebrafish is roughly 2–3 years, but it can be up to 5 years under ideal conditions in captivity, but probably much shorter in the wild. The generation time from a fertilized egg to a reproductively active adult is between 3 and 4 months. Adult females are able to spawn every 2–3 days and each female can lay up to 200 eggs/ week, but ovulation and spawning only occur in the pre sence of a male. Zebrafish mate in the morning and it is well known that light is the primary cue for mating. Within 30 min of the lights being turned on (if in the laboratory) or sunrise (if in the natural environment), a female will some times lay hundreds of eggs, which are subsequently fertilized by a male. At high densities in aquarium-bred zebrafish, it is typical to observe group-spawning behavior where egg fertilization is done in an indiscriminate fashion. Upon the release of eggs, embryonic development begins immediately, but if not fertilized, growth is halted after the first few cell divisions and the eggs die or are eaten.
1821
Upon fertilization, the eggs almost immediately become transparent and development progresses quickly (see Figure 3 for an outline of the lifecycle of a zebrafish (see also Reproduction: The Diversity of Fish Reproduction: An Introduction). Within 4 h, the fertilized egg has gone from a single cell to �1000 cells. Shortly after that, the embryo undergoes gastrulation, which is the developmental stage associated with the formation of a double-walled embryo resulting from the invagination of the blastula, which is associated with the differentiation of the ectoderm into the mesoderm and endoderm. The development of discreet organs, referred to as organogen esis, begins between 16 and 24 h postfertilization (hpf) and the precursors to all major organs appear within 36 hpf. For example, the zebrafish heart starts off as a ‘heart tube’ which is formed by 18 hpf and rhythmic, peristaltic con tractions can be observed by 24 hpf (see also Circulation: The Circulatory System: An Introduction). Hatching Ultimately takes place 48–72 hpf, depending on the tem perature, and feeding and swimming begin at roughly 72 hpf. Within 2–3 months, the zebrafish have matured into adults and can then begin breeding. Although the reproductive behavior of captive zebrafish is well known, the reproductive behavior of wild zebrafish is largely unexplored. Wild-reared zebrafish
Thirty minutes after fertilization
2 hpf
60–90 dpf Adult
4 hpf Free swimming
6 hpf 2 dpf
8 hpf 1 dpf
16 hpf Figure 3 Lifecycle of the zebrafish. The development of zebrafish is rapid with the fertilized embryo transforming into a 1000-cell stage within 4 h postfertilization (hpf) and organs being visible at roughly 1 day postfertilization (dpf). Hatching occurs at 2 dpf and the zebrafish matures within 2–3 months. Image adapted from Wolpert L, Beddington R, Jessell T, et al. (2002) Principles of Development, 2nd edn. New York: Oxford University Press.
1822 Bony Fishes | Zebrafish
take up to 4–5 months to reach sexual maturity rather than the 2–3 months commonly observed in the labora tory. In one study, wild-caught zebrafish from India were observed to spawn in pairs rather than in groups as is common in aquaria. Furthermore, there was evidence for sexual selection and females taking an active role in choosing their mates. This discrimination may be primar ily mediated by density in that, at low densities as seen in the wild, there may be elevated levels of sexual selection and pair formation; however, at high densities, character istic of aquarium breeding, sexual selection and pair formation break down.
Zebrafish as a Model Organism for Scientific Research Zebrafish have long been an important model in devel opmental biology, but their rise as an important experimental system in biology emerged from several major advances that have occurred since the early 1990s. First, the most important advance came in the 1990s, where several research groups induced large-scale genetic mutations in the zebrafish and subsequently screened the mutants for identifiable phenotypes. This approach of inducing random mutations and then search ing for the genetic underpinnings is referred to as forward genetics. The second major advance was the start of a whole-genome sequencing project that was initiated in 2001 and is still underway. This large-scale sequencing project has facilitated the third major advance, reverse genetics, which involves modifying single genes and then examining the effects of these genetic changes on the whole-animal phenotype. Forward Genetics in Zebrafish The first step in the popularization of zebrafish as a model organism in biology was the mutagenesis projects initiated in the early 1990s. This work followed in the footsteps of earlier work performed on the fruit fly (Drosophila melanogaster) and the nematode (Caenorhabditis elegans). In 1996, two important works were published by Driever and Haffter and their respective co-workers that described two large-scale genetic mutation screenings, where a chemical mutagen, ethylnitrosourea, was used to induce random mutations in zebrafish sperm. Ethylnitrosourea induces point mutations in the sperm genome that could affect protein sequences or gene struc ture and ultimately cause, through random chance, a loss of some physiological or biochemical function. After breeding, the developing embryos were screened for visi ble abnormalities in muscle and other organs at several different time points and from these screens more than
6500 mutants were identified. It is important to note that the majority of the mutations were lethal if they appeared homozygous in the fish. At the same time as the chemical mutagenesis was underway, in a separate research laboratory Gaiano and co-workers used a retrovirus to perform insertional muta genesis, which refers to the insertion of one or more bases into the genome. The subsequent screening of these mutants resulted in the identification of roughly 500 mutations at the phenotypic level of which just over 300 have been explicitly linked to a specific genetic change. One important advantage of this technique over the che mical mutagenesis described above is that a portion of the retroviral gene sequence is inserted into the host zebrafish genome, thereby providing a genetic tag aiding in the identification of which gene is affected in the genome. The systemic issue with forward genetics is that it is resource intensive and the direct links between phenotype and specific mutations are difficult to establish (see also Cellular, Molecular, Genomics, and Biomedical Approaches: Fish as Model Organisms for Medical Research). Establishing these links is easier with insertional mutagenesis compared with chemical mutagenesis, but many of the direct links from these early studies have not yet been established. The genome sequencing project, started in 2001, has greatly enhanced the ability of researchers to identify where mutations have occurred and it plays an important role in advancing this field. The Zebrafish Genome Sequencing of the zebrafish genome was started in 2001 by the Sanger Institute and is due to be completed sometime in 2011. The zebrafish genome consists of 25 linkage groups, which are equivalent to chromosomes and the entire genome size is �1.4 gigabases. The genome is cur rently being annotated, which refers to the identification of genes and, once complete, this resource will be invaluable to researchers trying to understand the genetic basis of physiological and biochemical phenotypes. In addition, the genome sequencing has greatly facilitated the develop ment of reverse genetic approaches in zebrafish. Reverse Genetics in Zebrafish Reverse genetics refers to the identification of gene function through selective manipulation of the gene followed by analysis of the arising phenotype. This approach attempts to identify what phenotype arises as a result of a particular gene and, for the most part, this approach involves knocking down the expression of genes of interest with subsequent examination of phenotype. These types of studies are broadly referred to as loss-of-function studies. The most common approach for performing loss-of function studies in zebrafish is using antisense technology.
Bony Fishes | Zebrafish
The basic premise of this technique is that if the genetic sequence of a particular gene of interest is known, then researchers can synthesize a strand of RNA that will bind to the messenger RNA (mRNA) transcript produced by that gene. The synthetic RNA is called antisense because its nucleic acid sequence is complementary to the gene’s nor mal mRNA sequence, which is referred to as the ‘sense’ sequence. The binding of the synthetic antisense RNA to the sense mRNA results in a doubled-stranded RNA mole cule, which cannot be translated into protein. As a result, the incorporation of antisense RNA strands either limits or eliminates the protein coded for by the mRNA transcript. The most common antisense approach using zebrafish reverse genetics is morpholinos. Morpholinos are synthetic oligonucleotides, in which the normal ribose or deoxyribose sugars are replaced with a morpholine ring, which are con structed to be antisense to the mRNA of interest. Normally, morpholinos are �25 nucleotides long and are designed to bind to the native mRNA, usually near the protein transla tion start site. The advantage of incorporating a morpholine ring into the antisense sequence is that it makes the mor pholino resistant to the normal nucleases that are present in cells and would break down unwanted RNA. The binding of the morpholino to the native mRNA causes translation blockade, where the gene of interest is silenced and very little protein is translated. It is a typical practice in the zebrafish to inject the morpholinos into the freshly fertilized egg while it is still in the one-to-eight cell stage, after which the morpholino becomes more or less uniformly distributed through the cells of the developing embryo. After this point, the efficacy of the knockdown is assessed and the resultant effects of the knockdown on phenotype are typically assessed over the first 5 or so days postfertilization. Other commonly used methods of reverse genetics for loss-of-function studies in zebrafish are targeting-induced local lesions in genomes (TILLING) and zinc finger nucleases. TILLING combines forward genetic approaches with reverse genetics to identify specific mutations in genes of interest from a random mutagenesis screening. Specifically, this approach begins with the generation of a large number of mutated zebrafish, normally from a chemical mutagenesis screen as described earlier (see Section Forward Genetics in Zebrafish). DNA is then sampled from these fish and TILLING is performed. In the most basic sense, the purpose of TILLING is to look for specific mutations in specific genes in a collection of DNA samples prepared from chemically mutagenized individuals. Identification of these mutations is done using standard polymerase chain reaction (PCR) and sequencing of PCR products. Additional methods for identifying whether mutations have occurred in select areas can also be carried out to reduce the excessive sequencing costs associated with the TILLING approach. Zinc finger nucleases represent another method for reverse genetics. Zinc finger nucleases consist of several
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zinc-finger DNA-binding domains plus restriction enzymes that, when combined, can be engineered to target specific DNA sequences and cut the double-stranded DNA in intact cells. The normal cellular DNA repair mechan isms then repair the broken DNA strands, but often insert nucleotides during the repair process, thus altering the genetic sequence disrupting codon arrangement. Far less frequent are gain-of-function studies, which attempt to insert specific genes or alter genes in a way that could result in a new or enhanced phenotype. There are various techniques for inserting genes to examine gain of function, the simplest of which is microinjection of synthesized mRNA into the fertilized zebrafish eggs. These eggs then express, or, in some cases, overexpress the gene of interest and investigators can then examine the resulting phenotype. Other techniques are available for the expression or overexpression of genes in zebrafish and some of these techniques even allow researchers to target specific tissues or cells at precise developmental stages. Overall, there is an ever-expanding collection of cell biology and genomic techniques for the zebrafish that allow manipulation of proteins and development at the most basic level. Slowly, fish physiologists are adopting this model organism and its impressive array of tools to explicitly establish links between genes and physiological attributes. Consideration of the natural habitat and use of the wide array of species available will facilitate adoption of this model system to inves tigate environmental adaptation and evolutionary physiology. See also: Bony Fishes: Crucian Carp. Cellular, Molecular, Genomics, and Biomedical Approaches: Fish as Model Organisms for Medical Research. Circulation: The Circulatory System: An Introduction. Hypoxia: Respiratory Responses to Hypoxia in Fishes; The Expanding Hypoxic Environment. Reproduction: The Diversity of Fish Reproduction: An Introduction. Responses and Adaptations to the Environment: General Principles of Biochemical Adaptations.
Further Reading Driever W, SolnicaKrezel L, Schier AF, et al. (1996) A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123: 37–46. Gaiano N, Amsterdam A, Kawakami K, et al. (1996) Insertional mutagenesis and rapid cloning of essential genes in zebrafish. Nature 383: 829–832. Haffter P, Granato M, Brand M, et al. (1996) The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123: 1–36. Parichy DM (2006) Evolution of Danio pigment pattern development. Heredity 97: 200–210. Perry SF, Ekker M, Farrell AP, and Brauner CJ (2010) Zebrafish. Fish Physiology, vol. 29. San Diego, CA: Academic Press.