Zebrafish

C H A P T E R

7

Zebrafish Edward A. Burton Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh, Pittsburgh, PA, USA; Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA; Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, USA; Geriatric Research Education and Clinical Center, Pittsburgh VA Healthcare System, Pittsburgh, PA, USA; Department of Neurology, Pittsburgh VA Healthcare System, Pittsburgh, PA, USA O U T L I N E 7.1 Introduction

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7.2 Why Zebrafish Models Are Useful: Screens, In Vivo Imaging, and Genetic Tools

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7.3 Zebrafish Genes, Cells, and Circuits Relevant to Studying Human Motor System Diseases 119 7.3.1 Phylogenetic Conservation of Genes Implicated in Human Neurological Disease 119 7.3.2 Neuroanatomical Considerations and Neural Circuits Implicated in Motor Control 122 7.3.3 Neuronal and Glial Populations of Relevance to Human Disease 123 7.4 Zebrafish Genetic Methods: Knockouts and Transgenic Lines 7.4.1  Gene Knockouts and Knockdowns

7.4.2  Transgenic Zebrafish

126 126

127

7.4.2.1  Transient Transgene Expression 7.4.2.2  Stable Transgenesis

128 128

7.5 Neurobehavioral Testing in Zebrafish 7.5.1  Manual Observation of Motor Function 7.5.2  Automated Analysis of Motor Function

130 131 131

7.6  Chemical Screening

133

7.7 Conclusions

134

References135 125 125

7.1 INTRODUCTION Zebrafish were initially introduced into b ­iomedical research as a model for studying vertebrate ­development. Zebrafish embryos develop externally so that embryogenesis and larval development can be visualized directly with microscopy; morphological observations are further facilitated by the optical translucency of zebrafish during their first few days of life. Large numbers of zebrafish can be housed in a manageable space, allowing generation of extensive collections of mutants, by N-ethyl-N-nitrosourea (ENU) or retroviral mutagenesis. By screening these mutant collections against phenotypic assays, genetic mutations that disrupt specific stages of embryogenesis have been

Movement Disorders, Second Edition http://dx.doi.org/10.1016/B978-0-12-405195-9.00007-X

7.4.1.1  Transient Gene Knockdown 7.4.1.2  Stable Gene Knockout

identified. This has allowed elucidation of numerous conserved molecular mechanisms underlying early vertebrate development (Haffter et al., 1996; Mullins et al., 1994). The rapid adoption of zebrafish as a powerful tool for developmental biology over the past three decades has been accompanied by major technological advances that now allow automated phenotypic assays, direct observation of cell biological processes through deployment of fluorescent reporter proteins, and facile techniques for transgenesis and (more recently) targeted mutagenesis. The possibility that this extensive toolbox of powerful methodologies could be deployed to elucidate the molecular basis of human disease has only recently been considered and, consequently, the generation of zebrafish models of neurological

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© 2015 Elsevier Inc. All rights reserved.

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disease and methods for their analysis is relatively new. This chapter describes molecular and neurobiological properties of zebrafish, concentrating on aspects that make zebrafish both suitable and potentially very useful as a model to study diseases of the human motor system. Methods for genetic and environmental manipulation of zebrafish, and techniques for analysis of motor behavior and neural circuits underlying motor behavior, are reviewed to provide the reader with essential background information for approaching chapters in which zebrafish models of specific diseases are considered.

7.2  WHY ZEBRAFISH MODELS ARE USEFUL: SCREENS, IN VIVO IMAGING, AND GENETIC TOOLS The zebrafish is a vertebrate and consequently shares basic nervous system organization with other vertebrates, including humans (Wullimann et al., 1996). As discussed in following sections, there are significant differences of scale and complexity between the zebrafish and human nervous systems, but (similar to rodent and primate models) conservation of critical structural and molecular features suggests that zebrafish could be used to study how cellular and mole­ cular disturbances cause the death of specific neuronal populations or dysfunction of neural circuits that underlie human movement disorders. The trade-off in terms of phylogenetic distance from humans is compensated by methods that, uniquely among vertebrates, could be applied to analyze zebrafish models:   

• Z  ebrafish produce large clutches of offspring, and extensive collections of mutants can be housed practicably. By using a similar approach to the classic developmental biology screens performed in zebrafish, phenotype-based mutagenesis screens for genetic modifiers of pathogenesis in a disease model could be used to identify novel genes underlying pathophysiology. This could potentially provide highly valuable information relevant to human disease, such as new information on genetic modifiers, elucidation of pathogenic pathways, or identification of therapeutic targets. • Larval zebrafish can be housed in 96-well plates, where they can be exposed to small molecule libraries (Zon and Peterson, 2005). By using this approach in a zebrafish model of human neurological disease, it may be possible to discover novel chemical modifiers of pathogenesis that could form the basis for development of new and effective drugs. The same methodology should also be amenable to a complementary approach—identification of environmental factors that can provoke pathology on

a susceptible genetic background, by adding putative environmental toxins to the medium. For both types of chemical screening applications, the 96-well plate format commonly used to house and analyze larval zebrafish should allow easy determination of dose– response relationships in statistically robust samples, in addition to primary screens. • Zebrafish are amenable to in vivo imaging modalities (Peri and Nusslein-Volhard, 2008). Although embryos and larvae are naturally translucent during early development, the practical time window for in vivo imaging of the zebrafish nervous system has been extended considerably by mutant lines that lack pigment (White et al., 2008). This allows imaging of neurons directly in vivo in the brain and spinal cord after central nervous system (CNS) structure is formed and differentiation of neuronal and glial subtypes is complete. The use of fluorescent reporter proteins whose expression can be targeted to specific neuronal (Bai and Burton, 2009; Fujimoto et al., 2011) or glial (Bernardos and Raymond, 2006; Munzel et al., 2012; Peri and Nusslein-Volhard, 2008) populations allows in vivo evaluation of structure and viability of target cell groups. Other genetic reporters have been used to probe cell biological functions, such as mitochondrial transport (Plucinska et al., 2012) or apoptosis (van Ham et al., 2010). These methods provide unique opportunities to address biochemical questions in disease-relevant populations of neurons in vivo, potentially a critical capability in seeking to understand the molecular basis for cellular specificity in neurodegenerative diseases.   

Importantly, both genetic and chemical screening approaches could use phenotypic assays to identify novel molecular modifiers from large numbers of randomly induced mutations or from a structurally diverse chemical library. The use of phenotypic assays that do not rely on preconceptions about pathogenic mechanisms could allow for discovery of previously unsuspected disease mechanisms and novel candidate therapeutics. Furthermore, because these experiments are carried out in the intact organism, phenotypes relevant to motor disorders, such as abnormalities in movement assays or loss of disease-relevant populations of neurons, could be used as assay outputs. This would potentially allow discovery of modifiers that act on neural circuits or cell nonautonomous mechanisms, which could not be isolated from traditional screening assays using cultured cells or biochemical targets. Finally, a wealth of molecular and physiological ­methods can be deployed in zebrafish to analyze nervous system function in vivo using more traditional hypothesis-driven approaches. In this regard, the zebrafish

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7.3  Zebrafish Genes, Cells, and Circuits Relevant to Studying Human Motor System Diseases

provides a genetically tractable and readily manipulated system for experiments that aim to understand how molecular perturbations alter the functions of motor circuits or the survival of neuronal groups. Facile generation of double and triple transgenic animals, conditionally expressed transgenes, and gene knockouts—combined with straightforward manipulation of environmental factors—allows multifactorial paradigms to be modeled faithfully. Methods for motor behavioral analysis (Cario et al., 2011), electrophysiological analysis (Lambert et al., 2012), and standard histochemical and biochemical (Bai et al., 2011) methods can be applied to determine the functional and molecular properties of these interactions, allowing rapid hypothesis testing in disease-relevant neuronal populations and circuits in vivo. Because the zebrafish is a relatively new model, many of these more complex analyses have not yet been accomplished. For example, small molecule screens in the zebrafish CNS have so far been limited to the identification of neuropharmacologically active compounds through their effects on behavior (Rihel et al., 2010). Furthermore, the first transgenic (Bai et al., 2007) and gene knockout (Schmid et al., 2013) zebrafish models of human neurological disease have only recently been developed. However, the zebrafish model holds great promise as a means to accelerate understanding of the neurobiology of motor system disorders, and there is consequently considerable excitement regarding its development.

7.3  ZEBRAFISH GENES, CELLS, AND CIRCUITS RELEVANT TO STUDYING HUMAN MOTOR SYSTEM DISEASES The relevance of the zebrafish model to studying human pathophysiology is a key consideration since there is significant evolutionary distance between the two organisms. Zebrafish and humans diverged approximately 450 million years ago compared with 112 million years ago for mice and humans (Kumar and Hedges, 1998). Three lines of evidence suggest that zebrafish are a valid and suitable model system for studying human neurological diseases:   

• E  xtensive phylogenetic conservation of key genes implicated in human neurological diseases suggests that molecular pathways underlying human disease could be recapitulated in zebrafish models. • The presence of differentiated populations of neurons and glial cells analogous to those involved in human diseases suggests that cell type–specific and cell nonautonomous pathologies that occur in human neurological diseases could be recapitulated in the zebrafish nervous system.

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• C  onservation of vertebrate CNS organization, neurochemical systems, and circuits may allow insights into the role of specific neuronal groups and neurotransmitters in particular types of motor behavior and movement abnormalities.

7.3.1  Phylogenetic Conservation of Genes Implicated in Human Neurological Disease The zebrafish has a diploid genome that shares considerable phylogenetic conservation with respect to the human genome. There are, however, two major differences between zebrafish and human genetics:   

• T  he zebrafish has 25 chromosomes and, unlike mammals, there are no X and Y heteromorphic sex chromosomes. The role of genetics in determining the sex of a zebrafish is incompletely resolved. Genetic loci linked to sex specification in zebrafish have been reported on chromosomes 5 and 16 (Bradley et al., 2011). These loci contain strong candidates for involvement in sex determination, including dmrt1 and cyp21a2. DMRT1, the human orthologue of dmrt1, operates downstream of SRY in human sex determination; haploinsufficiency of DMRT1 results in feminization of genetically XY individuals (Muroya et al., 2000). CYP21A2, the human orthologue of cyp21a2, encodes 21-hydroxylase involved in steroid biosynthesis; CYP21A2 mutations can result in virilization of genetically XX individuals. However, these two loci together account for only 16% of trait variance, suggesting that sex determination is complex in zebrafish and that other genetic and nongenetic factors are important. Environmental factors such as temperature and housing density can also contribute to sex determination in zebrafish. The importance of our limited understanding of zebrafish sex determination to modeling human disease is currently unclear. Some human neurological diseases are known to be more prevalent in one or the other sex. The underlying reasons are unknown and could reflect many factors ranging from sex-specific biochemistry to societal factors dictating likelihood of exposure to etiological agents. Enhanced understanding of the biological basis of sex in zebrafish would increase the usefulness of the model for exploring sex differences in disease susceptibility. • A whole genome duplication event occurred during the evolution of teleosts (Amores et al., 1998); many of the duplicated genes were lost during subsequent evolution, and it has been argued that the extreme diversity of fish species is partly attributable to retention of different combinations of duplicated genes. As a consequence, there are

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numerous instances in which the zebrafish genome contains two homologues of a human gene. In some cases, there is evidence that the functions of the parent gene have become divided between dual homologues. In other instances, it appears that paralogues show redundant biochemical functions but discreet expression patterns. Although instances in which dual paralogues have discreet functions or expression patterns provide opportunities to determine component properties of the relevant human gene, it is possible that gene duplication has also allowed acquisition of new functions by the zebrafish genes. This possibility has not been fully explored, and its importance remains uncertain.   

Many genes that have been implicated in human movement disorders have highly conserved zebra­ fish orthologues. A selection of these genes is shown in Table 7.1. In many instances, studies have shown that the products have key functions in motor system development (Milanese et al., 2012), neuronal survival (Flinn et al., 2009), or susceptibility to toxins (Sallinen et al., 2010), which are thought to be highly relevant to understanding the functions of the homologous human genes. These data lend strong support to the idea that zebrafish are a valid model for gene function studies in neurological disease and form a solid foundation for the construction of disease models. However, there are several differences between key human genes involved

TABLE 7.1  Phylogenetic Conservation of Human Genes Implicated in Movement Disorders and Their Zebrafish Orthologues

Disease

Protein

Amino Acid Identity/ Similarity between Human Protein and Zebrafish Orthologue

Parkinson disease

α-synuclein

No known orthologue

DJ-1

83/89%

Parkin

62/75%

PINK-1

54/62%

LRRK2

38/50%

Progressive supranuclear palsy

Microtubuleassociated protein Tau

33/43% (mapta)

Huntington disease

Huntingtin

70/81%

Spinocerebellar Ataxia

Ataxin 1

32/43% (atxn1a)

41/51% (maptb)

42/53% (atxn1b) Ataxin 7

51/66%

Primary dystonia TorsinA

59/78%

in human motor disorders and their zebrafish homologues that merit further discussion. Synucleins are a family of vertebrate-specific presynaptic proteins of uncertain function. Mammals express α-, β-, and γ-synucleins from separate SNCA, SNCB, and SNCG genes. α-Synuclein has been heavily implicated in human neurological disease. Genetic mutations causing overexpression of α-synuclein (Singleton et al., 2003), or missense mutations altering the protein’s biophysical properties (Polymeropoulos et al., 1997), are a rare cause of familial parkinsonism. However, genome-wide association studies implicate SNCA gene variants in determining risk of developing the common sporadic form of Parkinson disease (PD) (Satake et al., 2009; ­Simon-­Sanchez et al., 2009), and insoluble deposits of α-synuclein are a prominent pathological feature of PD, dementia with Lewy bodies, and multiple system atrophy (MSA) (­Spillantini et al., 1997; Tu et al., 1998). These data suggest that α-synuclein has critical functions in human neurodegenerative disease. Zebrafish also express synucleins. However, the SNCA locus was lost during zebrafish evolution (Milanese et al., 2012) and an ancestral SNCG locus was duplicated (Sun and Gitler, 2008); consequently, zebrafish do not express α-synuclein but rather express β-synuclein and two different γ-synucleins, termed γ1-synuclein and γ2-synuclein. Both β- and γ1-synuclein are expressed strongly in the zebrafish CNS (Sun and Gitler, 2008) and are important for early development of motor function and the dopamine system (Milanese et al., 2012). Importantly, human α-synuclein was able to complement loss of β- and γ1-synucleins in zebrafish embryos, suggesting that there is functional redundancy between human and zebrafish synucleins and that the zebrafish will be a valid model for investigating synuclein functions (Milanese et al., 2012). However, it has been suggested that the involvement of α-synuclein in neurodegeneration is attributable to gain of a novel toxic function. It is conceivable that the relevant property is specific to human α-synuclein, in which case full recapitulation of pathogenesis in zebrafish models of PD might depend on generation of transgenic zebrafish expressing human α-synuclein. This is an important priority that is currently being pursued by several groups. The microtubule-associated protein Tau (tau; MAPT) is a neuronal protein with functions in microtubule stabilization and regulation of axonal transport. Mutations in MAPT cause frontotemporal dementia with parkinsonism, an uncommon familial motor and cognitive disorder (Hutton et al., 1998). However, tau is also implicated in more common movement disorders. A haplotype of markers at the MAPT locus is strongly associated with the neurodegenerative movement disorders progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) (Baker et al., 1999), compatible with the prominent deposits of insoluble forms of tau found

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7.3  Zebrafish Genes, Cells, and Circuits Relevant to Studying Human Motor System Diseases

neuropathologically in these diseases (Arai et al., 2001). Human MAPT is alternatively spliced to generate a series of six protein isoforms in the CNS (reviewed in Bai and Burton, 2011). The isoforms are split into two groups according the inclusion or exclusion of exon 10, resulting in proteins with three or four microtubule-binding repeats; PSP and CBD are predominantly associated with deposition of 4R Tau, whereas the dementia Pick disease is associated with deposition of 3R Tau. Importantly, mutations that disrupt regulation of exon 10 splicing, altering the balance of 4R and 3R isoforms also result in neurodegeneration. In zebrafish, two paralogues of human MAPT, termed mapta and maptb, were reported (Chen et al., 2009a). Analysis of genomic structure, sequence conservation, and synteny suggested that these genes arose from duplication of an ancestral MAPT. Both mapta and maptb were shown to be alternatively spliced in zebrafish embryos at 24 and 48 hours postfertilization (hpf) (Chen et al., 2009a). Splice variants encoding four, five, or six microtubule-binding repeats were expressed from mapta, whereas maptb gave rise only to three-repeat isoforms. Both genes were expressed in the developing CNS. The expression and splicing of these genes later in development, or in the adult zebrafish, have not been reported. There are currently no functional data, or lossof-function phenotypes, reported for these genes. It has been suggested that segregation of human tau functions between the proteins encoded by mapta and maptb might provide opportunities to determine the specific roles of three- and four-repeat tau isoforms in the CNS (Chen et al., 2009a). In view of data showing that the emergence of neurodegenerative phenotypes in transgenic tau mice was enhanced in some instances by loss of endogenous murine tau (Andorfer et al., 2003), the development of stable null alleles for mapta and maptb may be of importance to the generation of zebrafish tauopathy models. TorsinA is an endoplasmic reticulum chaperone of the AAA+ family, which has been implicated in the etiology of DYT1 dystonia (Ozelius et al., 1997). Patients with DYT1 dystonia harbor an in-frame trinucleotide deletion within TOR1A, resulting in loss of a glutamic acid residue from the C-terminus of torsinA. In mammals, the two closely related torsin1 family genes TOR1A and TOR1B are located on chromosome 9, adjacent to one another, and inverted with respect to each other. The duplication of an ancestral torsin1 gene that gave rise to TOR1A and TOR1B occurred at the root of the tetrapod lineage (Sager et al., 2012). Consequently, zebrafish have a single tor1 gene that is homologous to both human TOR1A and TOR1B. Zebrafish tor1 is alternatively spliced to give rise to transcripts encoding protein isoforms with different N-termini. Transient loss of zebrafish tor1 early in development did not disrupt motor function or formation of the dopaminergic system, suggesting that tor1 is nonessential for early development in zebrafish (Sager et al., 2012).

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In addition to tor1, the zebrafish genome contains four additional genes encoding torsin1-like proteins. These are more distantly related than tor1 to human TOR1A, and their expression patterns and roles in development have not yet been evaluated. Tyrosine hydroxylase (TH) catalyzes the rate-­limiting step in the biosynthesis of dopamine. Because the enzyme is a critical component of the dopaminergic neurochemical phenotype, antibodies to TH (and cRNA probes to th mRNA) are frequently used as a marker to identify dopamine neurons in tissue sections and whole mount samples. Similar to other fish species, birds, and amphibians, the zebrafish genome contains two putative th genes, termed th1 and th2 (Candy and Collet, 2005). Comparison of syntenic relationships and genomic organization between different species suggests that th1 and th2 are derived through duplication of a common ancestor, occurring before the divergence of jawed vertebrates (this event is distinct from the whole genome duplication that occurred at the root of the teleost lineage) (Yamamoto et al., 2010). The th2 gene was subsequently lost in placental mammals. The zebra­fish th1 gene is most closely related to mammalian TH and encodes a protein with 60% identity to human TH, whereas the protein encoded by th2 shows around 53% identity with human TH. Zebrafish th genes show distinct expression patterns; in adult tissues, th1 mRNA was more abundant than th2 mRNA in the brain and eyes, whereas th2 was more abundant in liver, kidney, heart, and gills (Chen et al., 2009b). In the zebrafish CNS, very few neuronal groups show overlapping expression of the two transcripts (Chen et al., 2009b; Filippi et al., 2010) and it was initially thought that a combination of th1 and th2 or another marker, for example slc6a3 encoding the dopamine transporter (Holzschuh et al., 2001), would be necessary to identify and evaluate all groups of dopaminergic neurons in zebrafish models of Parkinsonian movement disorders. However, a recent report showed that th2 has tryptophan hydroxylase activity but no tyrosine hydroxylase activity (Ren et al., 2013). Furthermore, th2 expression was found in similar brain regions to serotonin, suggesting that th2 labels a subset of serotonergic neurons. Consequently, th1 alone is likely adequate as a marker for dopaminergic neurons. Despite these illustrative differences in detail, the extensive phylogenetic conservation between zebrafish and human genes suggests that zebrafish neurons should be able to mediate many of the biochemical events that underlie neurological disease in humans. As with any model system, a key to effective deployment is awareness of its limitations. The differences between zebrafish and human genes highlighted in the preceding paragraphs do not present insurmountable obstacles to the exploitation of zebrafish models but will need to be accounted for the interpretation of data from these models.

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7.3.2  Neuroanatomical Considerations and Neural Circuits Implicated in Motor Control Despite the obvious and highly significant difference in scale between zebrafish and humans, the anatomical organization of their nervous systems follows the same basic vertebrate plan (Figure 7.1). In both organisms, the nervous system can be divided into the peripheral nervous system (PNS) and CNS: the PNS includes motor, sensory, autonomic, and enteric components, and the CNS includes the spinal cord and brain, with the latter divided into forebrain, midbrain, and hindbrain. Furthermore, the basic organization of specialized sensory organs such as the eye, olfactory system, and ear and the arrangement of the cranial nerves are similar between humans and zebrafish (Wullimann et al., 1996). Because zebrafish are aquatic organisms, their movement repertoire differs substantially from that of terrestrial mammals. Underwater propulsion of zebrafish is driven by tail-beating movements that result from phasic rhythmic contraction of contralateral trunk muscles. Interestingly, the neural pathways mediating these movements may share commonalities with the systems mediating locomotion in mammals. These shared mechanisms include spinal pattern generators (Wiggin et al., 2012), brainstem centers regulating locomotor activity, and regulation of spontaneous movement by dopamine (Anichtchik et al., 2004; Farrell et al., 2011). In zebrafish, similar to mammals, abnormalities of α-motor neurons and their axons (Gros-Louis et al., 2008; Paquet et al., 2009), neuromuscular junction (Saint-Amant et al., 2008), and muscle (Telfer et al., 2010) cause locomotor deficits. Likewise, spinal cord transsection in zebrafish causes motor paresis (Becker et al., 2004). The roles of other parts of the CNS in movement are less certain and many systems are still being analyzed. For example, the structure of the cerebellum shows marked similarities between zebrafish and mammals, with a laminar cortex containing granule cell, Purkinje cell, and molecular layers (Bae et al., 2009). However, it is not currently clear how loss of

cerebellar function alters motor function in zebrafish. An ablation study suggested that learned motor responses to a conditioned stimulus depend on cerebellar function (Aizenberg and Schuman, 2011), but there have been no other detailed studies on the functional consequences of cerebellar lesions on zebrafish movement. The basal ganglia are of central importance in human motor systems disorders. Conservation of relevant basal ganglia circuits in the zebrafish CNS is therefore of considerable interest with respect to the development of zebrafish models of motor system disorders. Interpretation of anatomical homology between zebrafish and human forebrains is complicated, because the zebrafish telencephalon undergoes eversion during development, in contrast to the evagination that occurs in mammals, resulting in significant structural divergence between zebrafish and mammals. Furthermore, relative to the remainder of the CNS, the zebrafish forebrain is strikingly smaller and less complex than the mammalian forebrain. However, areas of the zebrafish telencephalon that are thought to be homologous to regions of the human basal ganglia involved in motor disorders have been identified, through homology of gene expression, neurochemistry, and axonal projections (Rink and ­Wullimann, 2001, 2004; Wullimann and Rink, 2002). The dorsal nucleus of the ventral telencephalic area, arising from the embryonic subpallium, is thought to be the zebrafish homologue of the mammalian striatum (Rink and Wullimann, 2004). Similar to the projection neurons of the mammalian striatum, neurons of the fish ventral telencephalic area are GABAergic, and cells in the region express substance P (Sharma et al., 1989), enkephalin (Reiner and Northcutt, 1992), and D1 (Kapsimali et al., 2000) and D2 (Boehmler et al., 2004) dopamine receptors in a variety of different fish species. Furthermore, this area is rich in dopaminergic nerve terminals, derived from both an ascending dopaminergic projection from the diencephalon and intrinsic telencephalic dopaminergic neurons (see later), further corroborating the proposed homology of the region to the striatum. Another division of the dorsal telencephalon contains choline acetyltransferase–expressing neurons that project

FIGURE

7.1  Comparative neuroanatomy of human and zebrafish brains. The illustrations are not drawn to scale to allow comparison of the structural organization of the human and zebrafish central nervous system. Regions that are thought homologous between the two different species are shaded in the same color. Despite the smaller size and reduced complexity, the zebrafish brain shares the same basic architecture as other vertebrate brains.

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7.3  Zebrafish Genes, Cells, and Circuits Relevant to Studying Human Motor System Diseases

to the dorsal telencephalic area and is a proposed homologue of the nucleus basalis of Meynert (Mueller et al., 2004; Rink and Wullimann, 2004). There is no substantia nigra in the zebrafish CNS and no midbrain dopaminergic neurons. Well-defined groups of dopaminergic neurons are situated in the olfactory bulb, telencephalon, pretectal region, and ventral diencephalon (Ma, 2003). Although dopamine neurons of the posterior tuberculum give rise to an ascending dopaminergic projection to the ventral telencephalon (Rink and Wullimann, 2001), the possibility that this may be the major dopaminergic input to the forebrain has been revised following recent work using transgenic zebrafish to define the dopaminergic ‘projectome’ (Tay et al., 2011). This demonstrated that diencephalic neurons contributing to the ascending pathway also give rise to descending axons that innervate the spinal cord and that there are relatively few ascending diencephalotelencephalic dopaminergic axons, at least in larval zebrafish. It is possible that the majority of dopaminergic innervation of the ventral telencephalon arises from intrinsic telencephalic neurons, although this is currently uncertain. It is well established that dopaminergic function is involved in the regulation of movement in both larval and adult zebrafish. Ablation of dopaminergic neurons by toxins such as MPP+ (Farrell et al., 2011; Sallinen et al., 2009) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Lam et al., 2005; McKinley et al., 2005; Sallinen et al., 2009) caused reduced dopamine levels and hypokinesia. Similarly, drugs that block dopamine receptors such as haloperidol and chlorpromazine cause hypokinesia (Farrell et al., 2011; Giacomini et al., 2006; Irons et al., 2013) (Figure 7.2). Conversely, dopamine receptor agonists have been reported to increase movement or to cause erratic bursting movements (Farrell et al., 2011; Irons et al., 2013). The populations of dopamine neurons responsible for controlling motor responses are currently unclear. The toxins and drugs described here do not target

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particular subpopulations of dopamine neurons. However, the recent development of chemogenetic ablation and optogenetic techniques in zebrafish coupled with increasingly large collections of cell type–specific transgenic driver lines (see later) should allow this question to be addressed through targeted inactivation of individual groups of neurons to determine their functional roles in movement. A recent study used this approach to determine that descending spinal projections from diencephalic dopamine neurons, homologous to the A11 group implicated in restless leg syndrome in humans, are involved in the developmental switch in motor behavior that occurs between 3 and 4 days postfertilization (dpf) (Lambert et al., 2012). It will be of considerable interest to determine how other groups of CNS dopamine neurons influence specific aspects of locomotion. In addition to the dopaminergic system, numerous other neurochemical systems of potential relevance to motor physiology have been characterized in zebrafish, including the serotonergic system and raphe nuclei and noradrenergic, histaminergic, and cholinergic systems. A detailed description of these is beyond the scope of this chapter, but the reader is referred to an excellent review (Panula et al., 2010) for further details.

7.3.3  Neuronal and Glial Populations of Relevance to Human Disease Animal models of disease present opportunities to determine the basis for vulnerability of specific cell groups to disease, in addition to the possibility to explore cell nonautonomous disease mechanisms. Consideration of whether the zebrafish CNS contains representative cellular populations for studies of disease pathogenesis is thus central to the question of whether zebrafish models can contribute to this aspect of understanding neurological disease.

FIGURE 7.2  Dopamine is involved in the regulation of spontaneous movement in zebrafish. The figure shows a color scale intensity map depicting the spontaneous movements made by 48 zebrafish larvae at 7 days postfertilization. Each animal is shown on a separate row, and the amount of movement in each successive time bin is indicated by the color scale shown to the right of the figure. The top 16 rows show animals treated with apomorphine, a dopamine receptor agonist; the middle 16 rows show control animals not exposed to drug; the bottom 16 rows show animals treated with chlorpromazine, a dopamine receptor antagonist. The control animals show continuous slow movements, which are reduced significantly by addition of the dopamine receptor antagonist. The addition of a dopamine receptor agonist induces bursts of high-velocity movement.

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The zebrafish CNS contains many populations of neurons, such as α-motor neurons and cerebellar Purkinje cells, which are easily recognizable as homologous to their human counterparts, through their anatomical location, morphology, gene expression patterns, and connectivity (Figure 7.3). However, there are some notable exceptions to the phylogenetic conservation of disease-relevant neuroanatomy. For example, although the zebrafish CNS is rich in dopamine neurons, which are present in a number of well-defined locations, there are no dopamine neurons in the zebrafish midbrain and no structural homologue of the substantia nigra. It is currently unclear whether this will present an impediment to modeling cellular degeneration or dysfunction relevant to parkinsonism. For example, Parkinson disease pathology affects substantia nigra dopamine neurons but spares adjacent ventral tegmental–area dopamine neurons, suggesting that factors other than neurochemistry are responsible for selective cellular vulnerability. It is unknown whether these factors are cell-autonomous, perhaps relating to differences in the expression of genes other than those specifying dopaminergic neurochemistry, or whether the responsible factors are extrinsic and related to aspects of the local brain environment such as glial cell abundance or vascularity, or even connectivity with other brain regions. Other, more phylogenetically distant organisms show specific loss of dopamine neurons in response to systemic triggers relevant to PD, such as α-synuclein expression in Drosophila

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(Feany and Bender, 2000) or rotenone exposure in snails (Vehovszky et al., 2007), suggesting that cell type–specific pathology is not necessarily dependent on the presence of an anatomically homologous brain region. However, it will be of great interest to determine experimentally whether aspects of PD pathology can be replicated in zebrafish, because differences in vulnerability of dopamine neuron populations could be informative for understanding pathogenic mechanisms. In addition to differentiated neuronal populations of relevance to motor disorders, the zebrafish CNS is rich in glial cells. Specialized populations of microglia, astroglia, and oligodendroglia are unique to vertebrates and have been implicated in the pathogenesis of motor system disorders in humans. For example, neuroinflammation, manifest as increased numbers of activated microglia, is increasingly thought to be central to neurodegenerative disease progression (Hong, 2005). The zebrafish CNS contains resident macrophage lineage cells with morphological features reminiscent of microglia (Peri and Nusslein-Volhard, 2008). Zebrafish microglia have been shown by live cell imaging to clear degenerating neurons via phagocytosis, but their role in zebrafish models of neurological disease has been relatively unexplored. This may provide a fruitful area for future studies, because transgenic lines and live imaging modalities are available to monitor cellular behavior of zebrafish microglia in vivo. The zebrafish CNS

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FIGURE 7.3  Zebrafish circuits and neurochemical systems of relevance to human movement disorders. (A) Whole mount antibody labeling of a zebrafish larva at 96 hour postfertilization using an antibody to tyrosine hydroxylase (TH) shows the developing dopaminergic system. A dorsal view is shown, TH immunoreactivity has been pseudocolored green. TH-expressing cells are evident in the olfactory bulbs, retina, and diencephalon. (B) The same TH antibody was used to stain a parasagittal section of the adult central nervous system (CNS). The photomicrograph shows the diencephalon indicating the different groups of dopaminergic neurons in this part of the brain (PT, pretectal nucleus; PP, preoptic nuclei; TP, posterior tuberal nuclei. The inset panel shows the olfactory bulb (OB). (C) Parasagittal section of the cerebellum labeled with an antibody to IP3 receptor 1 (IP3R1, red) to show cerebellar Purkinje cells. The molecular (M), Purkinje cell (P), and granule cell (G) layers are indicated. Purkinje cell bodies are shown with arrows; fibrillar labeling in the molecular layer represents Purkinje cell dendrites. The functional organization of the cerebellum is very similar to mammals, except that there are no deep nuclei. Instead, the output cells, eurydendroid cells, are present in the Purkinje cell layer. (D) Whole mount in situ hybridization analysis of a 72-h larva, using a probe for the chat transcript encoding choline acetyltransferase. This marker selectively labels developing cholinergic neurons (purple), which are shown within the spinal cord, retina, and brainstem at this developmental point. (E) Whole mount in situ hybridization analysis of a 72-h larva, using a probe for the gad transcript encoding glutamic acid decarboxylase. This marker selectively labels developing GABAergic neurons (purple), which are shown throughout the CNS at this developmental point. Particularly prominent labeling is apparent within the developing cerebellum.

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7.4  Zebrafish Genetic Methods: Knockouts and Transgenic Lines

also contains glial cells that express an orthologue of the intermediate filament protein glial fibrillary acidic protein (GFAP), a hallmark of astrocytes in the human brain (Tomizawa et al., 2000). However, the morphology of these cells differs between the mammalian and zebrafish nervous systems; in the zebrafish, the cell bodies are located adjacent to the ependymal surface of the CNS and long radial processes pass through the underlying neural substance to reach the pial surface of the brain. GFAP immunoreactive structures with stellate morphology typical of mammalian astrocytes have not been described in the zebrafish brain (Grupp et al., 2010). However, GFAP-expressing star-shaped cells were reported in the zebrafish spinal cord in one study (Kawai et al., 2001), and it has been further suggested that there may be cytokeratin-immunoreactive glia in the optic nerve that do not express GFAP (Conrad et al., 1998). This raises the possibility that there may be important differences between the human and zebrafish CNS in astroglial cell gene expression. Oligodendrocytes have been implicated heavily in pathogenesis of the parkinsonian movement disorders PSP and MSA, where the typical pathological features include coiled bodies (oligodendroglial accumulations of phospho-Tau found in PSP) and glial cytoplasmic inclusions (oligodendroglial inclusions of α-synuclein typical of MSA) (Nishimura et al., 1995; Papp and Lantos, 1994). The zebrafish CNS is rich in oligodendrocytes that generate and maintain myelin sheaths around axons, suggesting that the role of these cells in neurological disease could be studied productively using zebrafish models. Zebrafish express orthologues of mammalian genes involved in myelin formation, such as myelin basic protein (Avila et al., 2007; Brösamle and Halpern, 2002) and proteolipoprotein (PLP) (Brösamle and Halpern, 2002; Schweitzer et al., 2006), in addition to molecules known to determine oligodendrocyte proliferation and survival, sites and timing of myelination, and functional organization of axonal domains, such as notch1 (Milan et al., 2006), neuregulin (Milan et al., 2006), erbB (Lyons et al., 2005), jagged1 (Zecchin et al., 2005), and neural cell adhesion molecule (Mizuno et al., 2001). Although zebrafish CNS myelin shows a similar ultrastructure to mammalian CNS myelin, there are several differences in its protein composition, and it is unclear whether this is important with respect to modeling the role of oligodendrocytes in neurological disease. In mammals, myelin P0 expression is restricted to Schwann cells of the PNS, whereas the zebrafish P0 orthologue is expressed in the adult CNS and PNS (Bai et al., 2011; Brösamle and Halpern, 2002; Schweitzer et al., 2003). The displacement of P0-like proteins from CNS myelin by PLP is specific to mammals and is postulated to allow more compact myelin formation (Schweitzer et al., 2006). Another difference between zebrafish and mammalian

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oligodendrocytes is provided by 2′-3′-cyclic-nucleotide3′-phosphodiesterase (CNPase), which is expressed in mammalian oligodendrocytes and is essential for axonal survival in mice (Lappe-Siefke et al., 2003). A Zebrafish CNPase orthologue is expressed in retinal ganglion cells (Ballestero et al., 1999), but its expression in CNS oligodendrocytes has not been reported. It is unclear at present whether these differences from mammalian oligodendrocytes will prove to be important in modeling neurodegenerative disease in zebrafish. Together, these observations demonstrate an unexpectedly striking degree of phylogenetic conservation between the structure and function of the zebrafish and the human CNS, which extends from the molecular level through to the cellular and systems levels. These considerations suggest that the zebrafish CNS could provide a valid setting in which to model human motor systems disorders experimentally, potentially recapitulating biochemical and physiological abnormalities underlying disease in an experimentally-tractable system.

7.4  ZEBRAFISH GENETIC METHODS: KNOCKOUTS AND TRANSGENIC LINES Many recent advances in understanding human motor system disorders have arisen through defining the genetic basis of diseases, either by studying families with Mendelian disorders or by genome-wide association studies in common sporadic diseases. Facile methods in the zebrafish allow endogenous genes to be down-­ regulated or knocked out, or exogenous genes introduced. This allows convenient determination of the behavioral (Prober et al., 2006), neurophysiological (Mongeon et al., 2008), molecular (McCollum et al., 2007), cellular (Guo et al., 1999), and morphological (Reifers et al., 1998) consequences of altered gene function in the intact nervous system. This is potentially a very powerful tool with which to study the functions of genes implicated in disease. Current methods for genetic modification of zebrafish are summarized in the following paragraphs.

7.4.1  Gene Knockouts and Knockdowns The physiological functions of proteins in vivo have traditionally been determined by studying loss-offunction mutants; the phenotypes of animals lacking defined proteins are frequently informative for understanding the normal roles of the missing proteins. Lossof-function mutations underlie a number of genetic movement disorders, in particular those with autosomal recessive pattern of inheritance, and reduced gene function may also be central to other sporadic diseases. The zebrafish model provides opportunities for both transient and stable gene targeting.

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7.4.1.1  Transient Gene Knockdown Transient gene knockdown in zebrafish larvae is readily accomplished through the deployment of morpholino oligonucleotides (MOs) (Bill et al., 2009). MOs can be targeted to block translation of a target mRNA, by hybridization to the ATG initiation codon, or to prevent correct splicing by annealing to exon-intron splice signals in the pre-mRNA (Eisen and Smith, 2008) (Figure 7.4). MOs are injected into embryos at the one- to four-cell stage, immediately after fertilization, and can cause highly efficient gene knockdown in the early stages of development, but the effect quickly resolves and gene knockdown rarely persists beyond 5 dpf. MOs directed against splice sites allow rapid evaluation of knockdown by reverse transcription–polymerase chain reaction analysis, using primers that span the region of altered splicing. In contrast, translation-inhibiting MOs must be verified by detecting the protein, which depends on the availability of effective and specific antibodies. MOs provide a rapid and potentially high-throughput methodology, but there are some commonly encountered problems. Observed phenotypes may be caused by off-target effects attributable to unintended alteration of expression of another gene. Furthermore, abnormalities including CNS necrosis can be caused by MO-dependent induction of p53-dependent cell death, attributable to increased transcription of a truncated p53 isoform from an internal p53 promoter (Robu et al., 2007). Consequently, controls for MO experiments are essential to ensure that observed phenotypes are caused by loss-of-function of the targeted gene. The most convincing controls are (1) showing that similar phenotypes arise from two different MOs targeting the same gene and (2) demonstrating rescue of MO-induced phenotypes by coinjection of mRNA encoding the protein of interest (the synthetic mRNA is

modified so that it is not a target for the MO) (Bandmann and Burton, 2010). However, these controls are not always possible, and alternatives that are commonly used include nontargeting MOs to show that phenotypes do not arise nonspecifically from MOs at equivalent dose and coinjection of anti-p53 MO to prevent nonspecific p53-depedent toxicity. Despite these controls, even the most carefully executed MO experiments can yield findings that differ from those seen in stable null mutants of the same gene and, given the recent availability of methodology to target genes efficiently, the field is starting to move away from using MOs. However, there are several applications for which MOs remain useful. First, MOs can target translation of zygotic mRNA, which could still be transcribed in stable knockouts if the mother is only heterozygous for the mutation, as is often the case. Second, MOs are relatively rapid to deploy compared with the lengthy process of generating stable null alleles, so it is possible that medium-throughput applications (e.g., rapid testing of multiple hypotheses such as potential modifier genes) could be carried out using MOs. Finally, by adjusting the dose of MO it should be possible to dictate the expression level of a gene, whereas the effects of a mutation are relatively fixed. Consequently, MOs continue to be used, but gene-targeting techniques are becoming increasing widely deployed for inducing lossof-function mutations, particularly in situations where the anticipated phenotype arises after 5 dpf. 7.4.1.2  Stable Gene Knockout The zebrafish was initially used as a powerful developmental genetic model by exploiting chemical mutagenesis, using the alkylating agent ENU, to induce random point mutations throughout the genome. Subsequent screens for mutants with altered phenotypes, followed

FIGURE 7.4  Methods for transient knockdown and stable inactivation of zebrafish genes. The major methods for transient gene knockdown (morpholino oligonucleotides) and stable gene inactivation (transcription activator like effector nucleases, TALENs) in zebrafish, described in detail in the text, are depicted schematically. The TALEN method is relatively new, and is potentially very powerful, allowing routine generation of stable gene knockout and knockin lines. IHC, immunohistochemistry.

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7.4  Zebrafish Genetic Methods: Knockouts and Transgenic Lines

by genetic mapping—forward genetics—allowed identification of genes contributing to developmental processes of interest. The induction of mutations in specific target genes followed by phenotypic characterization of the resulting mutants—reverse genetics—has been a more recent development in zebrafish. Although it is possible to screen large numbers of zebrafish to identify ENU-induced mutations in genes of interest using a strategy called targeting-induced local lesions in genomes (Stemple, 2004), this is laborious and relatively inefficient. Homologous recombination in ES cells, the strategy commonly used for targeted gene inactivation in mice, has not been deployed in zebrafish. However, the development of nucleases that can be engineered to target specific sites in zebrafish genes has provided a novel and very powerful tool for targeted gene inactivation. In this approach, a chimeric protein is constructed containing a DNA-binding domain and an endonuclease domain. The DNA-binding domain is designed to specifically recognize the gene of interest (GOI); binding to the cognate DNA motif allows the nuclease domain to induce double-stranded DNA breaks in the target sequence. The breaks are repaired by error-prone mechanisms including nonhomologous end-joining; this frequently results in insertions or deletions of several bases (‘IN/DEL’ mutations) at the site of cleavage. If the mutation occurs within the open reading frame and the number of bases lost or gained is not a multiple of 3, a frameshift mutation occurs. This often results in a premature stop codon, which may result in production of a nonfunctional truncated protein or loss of mRNA by nonsense-mediated decay, with either outcome resulting in loss of gene function and a null allele. The major difficulty with this approach has been the design and construction of proteins that predictably bind to DNA in a sequence-specific manner. Two different approaches have been used successfully in zebrafish:   

• Z  inc finger nucleases (ZFN): Zing fingers (ZFs) are DNA-binding protein motifs commonly found in transcription factors. ZFs can be engineered to bind to specific DNA sequences (Miller et al., 2007). ZFNs contain a three-ZF tail to direct binding to 18–base pair (bp) sequences within the GOI. For genetargeting applications in zebrafish, pairs of ZFNs are deployed that recognize either the sense or antisense strand of the target gene. The head part of the ZFN contains a FokI endonuclease, which is activated when both ZFNs bind to the GOI with overlapping heads. ZFNs allowed effective gene targeting for the first time in zebrafish (Doyon et al., 2008; Meng et al., 2008b), but the rules linking the amino acid sequence of the ZF to the cognate DNA sequence are complex and not well understood, meaning that synthesis is complicated because empiric data are necessary to

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direct their design. Although studies using ZFNinduced mutations have yielded important data concerning gene function (Schmid et al., 2013), the difficulty in designing ZFs to target specific genes, and the lack of suitable target sites in many genes, has led the field to move away from ZFNs in the last 1–2 years. • Transcription activator–like effector nucleases (TALENs): TAL effector DNA-binding domains consist of repeated arrays of 34 amino acid units, each of which binds to a single nucleotide in doublestranded DNA. A simple code links the target DNA sequence with a two–amino acid motif embedded in each repeat (A]NI; C]HD; G]NN; T]NG) (Boch et al., 2009; Moscou and Bogdanove, 2009), allowing the design and construction of sequencespecific TAL effector DNA-binding domains by a facile process based on modular assembly. TALENs are fusion proteins containing TAL effector DNAbinding domains coupled to a nonspecific FokI endonuclease. Pairs of sequence-specific TALENs that recognize two 16- to 18-bp DNA motifs flanking a target site are deployed, enhancing specificity. In zebrafish embryos, transient expression of TALENs during early development frequently causes IN/DEL mutations in the target gene; the induction of mutations has been reported to be as high as 30% of genomes (Huang et al., 2011; Sander et al., 2011) and has been shown to cause heritable frameshift mutations in the encoded genes (Figure 7.4).   

The design and construction of TALENs and ZFNs have been greatly facilitated by open access design software (see http://zifit.partners.org/ZiFiT/) and freely available reagents for their construction. The recent availability of simple cost-effective systems for deploying gene-targeting approaches in zebrafish will likely greatly expand the usefulness of this model for investigating gene function. This may be particularly true for the study of motor disorders, where gene products may have critical functions in juvenile or adult neurons and circuits, which would not be amenable to investigation using the transient knockdown approaches traditionally used in zebrafish.

7.4.2  Transgenic Zebrafish Expression of exogenous transgenes in the zebrafish CNS could potentially be used to model a number of human neurological diseases associated with movement disorders. Diseases caused by genetic mutations that cause toxic gain of function in the relevant proteins or that are associated with protein overexpression could be modeled using this strategy; examples include Huntington disease and the tauopathies. Furthermore, transgenesis

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may be essential to enable modeling of some diseases in zebrafish. For example, the zebrafish genome does not contain a gene encoding α-synuclein; given the proposed central importance of this protein in PD, it could be argued that full recapitulation of underlying pathogenic events in zebrafish might necessitate transgenic expression of human α-synuclein. Finally, a number of fluorescent reporter proteins have been described that could be useful for elucidating pathogenic mechanisms in vivo using live imaging; expression of these in particular cells or at defined developmental points is dependent on methods for transgenesis. Several approaches have been described, allowing either transient or stable transgene expression. 7.4.2.1  Transient Transgene Expression Transgenes can be expressed in zebrafish during development by direct injection of mRNA into single-cell embryos. This gives rise to transient ubiquitous expression and is a simple and commonly deployed technique. Use of a DNA construct, in which expression is driven by a tissue-specific regulatory element, allows restriction of transgene expression to cell types of interest, although transient expression from DNA constructs usually gives rise to significant mosaicism. Transient mRNA expression is often used as a control to show specificity in MO experiments and to determine the role of genes early in development. Expression rarely persists beyond a few days, so stable transgene expression is used when long-term or tissuespecific effects of gene expression are being investigated. 7.4.2.2  Stable Transgenesis Microinjection of linearized plasmid DNA into the cytoplasm of single-cell embryos (Stuart et al., 1988) results in the formation of multicopy DNA concatemers, which are distributed unevenly during early cell divisions and inefficiently integrated later. This results in significant mosaicism and rare integration events, such that large numbers of injected fish must be screened to identify germline transgenic founders. Refinements to zebrafish transgenic methodology have substantially increased the efficiency of this process. Two methods are currently routinely deployed to introduce transgenes into the zebrafish genome (Figure 7.5):   

• M  eganuclease-mediated transgenesis: I-sceI, a nuclease derived from Saccharomyces cerevisiae, cleaves DNA at a specific 18-bp recognition site that is not present within the zebrafish genome (Jacquier and Dujon, 1985). By generating a plasmid in which the transgene expression cassette is flanked by I-sceI sites and then coinjecting the plasmid with I-sceI enzyme into single-cell zebrafish embryos, efficient transgene expression with substantially reduced mosaicism is seen compared with linearized DNA injection (Thermes et al., 2002). The underlying mechanisms

FIGURE 7.5  Methods for generation of transgenic zebrafish. The two major methods for generation of transgenic zebrafish, described in detail in the text, are depicted schematically. The transposon method in particular has greatly enhanced the efficiency of zebrafish transgenesis and the construction of transgenic lines is now straightforward and convenient.

are uncertain, but the technique is efficient enough to facilitate meaningful transient assays and substantially improves the rate of transgene transmission for the establishment of stable lines (Grabher et al., 2004). This method most frequently results in the integration of multiple tandem copies of the transgene at a single site in the genome. • T  ransposon-mediated transgenesis: The Tol2 transposon was initially discovered in the genome of the freshwater fish Medaka (Koga et al., 1996). Deletion of the transposase open reading frame from Tol2 yielded a nonautonomous element, which could efficiently insert into the zebrafish genome when the transposase was supplied in trans (Kawakami et al., 2000). By flanking a transgene expression cassette with the nonautonomous Tol2 elements and coinjecting the plasmid and transposase mRNA into single-cell embryos, single-copy integration events occur with high efficiency, such that a relatively small number of animals has to be screened to identify stable transgenic lines (Kawakami, 2004). Tol2-mediated transgenesis has shown particular use for deployment of the Gal4/upstream activating sequence (UAS) conditional expression system described later. The efficiency of transgenesis has allowed isolation of tissue-specific Gal4 driver lines by random insertion adjacent to endogenous regulatory elements. In addition, single-copy insertions reduce the likelihood of epigenetic inactivation of UAS constructs. The efficiency of the Tol2 method can result in multiple

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7.4  Zebrafish Genetic Methods: Knockouts and Transgenic Lines

single-copy transgene insertions, so that care must be taken during subsequent breeding to identify and propagate individual transgene alleles.   

Expression of transgenes depends on appropriate cisacting regulatory elements to drive transgene expression in a suitable temporal and spatial expression pattern. In the case of transgenic models of neurological disease, it is also necessary to express the transgene at sufficient levels to provoke pathology. The desired transgene expression pattern will likely differ by application, and a complete list of available promoter and enhancer elements is beyond the scope of this chapter; however, some examples are illustrative. Strong regulatory elements derived from neuronal ‘housekeeping’ genes have been used to express transgenes widely in neurons. These cis-regulatory elements, derived from the gene encoding the neuronal RNA-binding protein HuC/D (Kim et al., 1996; Park et al., 2000) or the neuronal γ-enolase gene (Bai et al., 2007), allow transgene expression in neurons throughout the neuraxis and may be useful in applications such as determination of cell-specific vulnerability to ubiquitous transgene expression or for modeling diseases with widespread pathology. The cis-regulatory elements derived from these genes are relatively small (12 kb or less), allowing convenient generation of plasmid transgene constructs. In some situations, it will be necessary to target transgene expression to specific groups of neurons or glial cells, such as to model diseases where pathology occurs specifically in certain cell types or to determine the role of cell-autonomous gene functions, in which case regulatory elements that direct expression in a more specific spatiotemporal pattern will be necessary. A variety of other constructs have been described, including those that direct transgene expression to oligodendrocytes (Munzel et al., 2012) or radial glia (Bernardos and Raymond, 2006). With respect to modeling movement disorders, the dopaminergic system is of considerable interest and several groups have developed approaches to directing transgene expression to the zebrafish dopaminergic system. An obvious approach is to isolate small regulatory sequences derived from genes specifying the dopaminergic neurochemical phenotype, such as genes whose products are involved in dopamine biosynthesis, reuptake, or vesicular storage. However, deployment of small regulatory elements derived from genes such as th and sl6a3/dat has been only partially effective in driving expression in dopamine neurons, suggesting that endogenous expression of these genes depends on coordinate activity of widely distributed cisacting elements (Bai and Burton, 2009; Gao et al., 2005; Meng et al., 2008a). This has led other groups to use larger genomic fragments for transgene expression, such as a 35-kb fragment of the slc6a3 gene encoding the dopamine transporter (Xi et al., 2011) or a genomic bacterial

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artificial chromosome containing a large fragment of the th1 gene (Tay et al., 2011). Similar to studies in mice, use of larger constructs containing an entire gene with all of its associated regulatory elements is more likely to drive transgene expression in a pattern that replicates the endogenous zebrafish gene (Chen et al., 2007; Kirby et al., 2006; McGraw et al., 2008; Peri and Nusslein-­ Volhard, 2008; Sato et al., 2007; Yang et al., 2007). However, the manipulation of large constructs is unwieldy and the generation of transgenic animals using large constructs is inefficient using present methods for transgenesis. Another approach based on the binary Gal4-UAS conditional expression system, which has been successfully used in Drosophila, was recently described in zebrafish and may solve many of these problems (Scheer and C ­ ampos-Ortega, 1999). The principle of this system is that the transgene of interest is expressed under control of a minimal promoter and an enhancer element called the UAS, which is derived from a yeast gene sequence (­Figure 7.6). In a wild-type zebrafish, the UAS sequence is not recognized by any endogenous zebrafish transcription factors, so the UAS:transgene construct is not expressed. Gal4 is the yeast transcription factor that naturally and specifically binds to the UAS sequence. Crossing the Tg (UAS:transgene) zebrafish with another transgenic zebrafish expressing Gal4 gives rise to progeny that harbor both Gal4 and the UAS:transgene construct; in this case Gal4 trans-activates the UAS enhancer and causes transgene expression. This conditional system has three potential advantages over constitutive transgene expression:   

• C  onditional expression should allow the establishment of stable transgenic lines in the absence of transgene expression, until the

FIGURE 7.6  Gal4-upstream activating sequence (UAS) genetics for conditional transgene expression in zebrafish. The figure shows the principle behind the application of Gal4-UAS in zebrafish, and the breeding scheme that results in transgene expression. Although this method is attractive for a number of reasons, its implementation in zebrafish has presented a number of problems that have been addressed by modifications to both the Gal4 and UAS components previously used successfully in Drosophila.

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transgene is trans-activated by Gal4. This could be advantageous if the transgene causes severe phenotypes that prevent reproduction; in this case, the resulting disease models might be very useful, but stable lines with constitutive expression could not be established, because the neurological phenotypes would impair the viability and reproductive potential of transgenic founders. • Transactivation of the UAS enhancer by Gal4 might give rise to transcriptional amplification, potentially allowing higher expression levels of the transgene than would be possible by driving expression directly using a tissue-specific regulatory construct. • Because the UAS construct is very small, generation of transgene constructs is simple, and transgenesis using the Tol2 system is straightforward and efficient. The issue of finding appropriate regulatory elements then applies to the Gal4 driver line. However, because driver lines only have to be constructed once, after which they can be applied to multiple different transgenic models, generation of well-characterized and specific transgenic drivers, even with very large or complex cis-regulatory elements, is easier to justify.   

A variety of Gal4 transgenic driver lines has already been constructed, including those expressing Gal4 under transcriptional control of smaller cis-regulatory elements previously used for constitutive transgene expression, in addition to much larger constructs expressing Gal4 using large genomic fragments contained in bacterial artificial chromosomes. Because transposon-mediated transgenesis is very efficient, it has also been possible to express Gal4 from endogenous genomic regulatory sequences by randomly inserting a Gal4 enhancer trap construct in the genome using the Tol2 method, and then isolating driver lines with expression patterns that appear useful after crossing them with UAS reporter lines (Scott et al., 2007). This latter approach seems very promising, because Gal4 may be expressed physiologically in response to endogenous signals and consequently may show highly specific expression patterns mimicking endogenous genes. Assembly of a large collection of validated and characterized Gal4 driver lines is an ongoing community effort but likely to be extremely useful, because tissue-specific transgene expression in the future will involve generating a novel UAS responder line and then selecting a characterized Gal4 driver line to express the transgene in the desired pattern. Despite this early promise, there have been some significant problems with the Gal4-UAS system in zebrafish:

However, although low expression levels of Gal4-VP16 are not problematic in zebrafish, more robust expression levels appear toxic, especially in neurons, preventing establishment of stable driver lines. Consequently, modified Gal4-VP16 derivatives that express truncated forms of the VP16 domain have been developed; these seem to show similar trans-activating potential to Gal4-VP16 but attenuated toxicity (Asakawa et al., 2008; Distel et al., 2009). • In Drosophila, a 14-copy UAS repeat was used as an enhancer element, allowing robust transactivation of gene expression by binding of multiple Gal4 molecules to the enhancer. However, the 14× UAS enhancer becomes methylated in zebrafish, resulting in epigenetic silencing of gene expression. This manifests as variegated transgene expression, subMendelian ratios of transgene-expressing animals, or complete loss of gene expression over several generations. A 5× UAS enhancer was found to be less susceptible to epigenetic silencing in zebrafish, without drastically reducing gene expression levels (Akitake et al., 2011; Asakawa et al., 2008; Distel et al., 2009). However, even the 5× UAS is silenced when transgenic lines are made using the I-sceI technique. Apparently, the integration of multiple tandem transgene copies results in methylation of the 5× UAS construct, similar to single integrations of the 14× UAS. Consequently, construction of effective UAS responder transgenic lines requires both a 5× UAS element and Tol2 transgenesis to allow single-copy integration.   

Further developments in Gal4-UAS for zebrafish have included the construction of synthetic UAS elements that are more resistant to epigenetic silencing (Akitake et al., 2011). Although the field continues to refine this methodology for conditional transgene expression, the tools developed so far are powerful and useful. Construction of transgenic animals is now routine and efficient, and well-characterized driver lines are becoming available for tissue-specific transgene expression in a number of cell types. These resources are expected to greatly facilitate the production of transgenic and gene knockout models of human motor system disorders.

7.5  NEUROBEHAVIORAL TESTING IN ZEBRAFISH

  

• In Drosophila, the transcriptional activating effect of Gal4 was increased by fusing the UAS-specific Gal4 DNA-binding domain to the transactivation domain of a herpes simplex virus transcription factor, VP16, which is a potent activator of transcription.

An essential prerequisite for the development of useful zebrafish models of motor disorders is the availability of reliable methods for measuring motor function. Because zebrafish are aquatic organisms, handling―often part of the motor evaluation in murine models—is not part of

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7.5  Neurobehavioral Testing in Zebrafish

the behavioral evaluation. Consequently, motor testing in zebrafish is observational. Furthermore, common motor tests used in rodents, which evaluate antigravity support, balance, and limb dexterity, are clearly not applicable in zebrafish. However, a variety of motor behavioral assays have been described in zebrafish. Some of these rely on observations of spontaneous movement, while others depend on measuring evoked responses to sensory stimuli. Zebrafish larvae show stereotyped development of movement (Drapeau et al., 2002; Saint-Amant and Drapeau, 1998). Spontaneous coiling movements start at 18 hpf. This is followed by propulsive movements that can be evoked from 28 hpf, although spontaneous propulsive movements usually commence at the time of hatching, around 48 hpf. During later larval development, more mature swimming patterns and more complex behaviors such as visually guided predatory behavior develop, and movements can be evoked by mechanical, acoustic, or visual stimuli. The zebrafish has been considered an attractive model for studying the neural basis for these movements, because the motor repertoire during early development is composed of tail-beating movements—mediating forward propulsion—and several discrete types of trunkcurling movement that mediate turns, escape responses, and prey-capture orienting responses. These curling movements are named C-bends, O-bends, and J-turns, according to the shape the zebrafish adopts when viewed from above during the curling movement (Borla et al., 2002; ­Burgess and Granato, 2007; Kimmel et al., 1974). The neural pathways for some of these movement types have been determined by using laser ablation, chemogenetic approaches, genetic mutants, and electrophysiology (Gahtan et al., 2005; McLean and Fetcho, 2008). Later in development, zebrafish show more complex behaviors, including reproductive behavior and the development of a diurnal pattern of activity that resembles the sleep–wake cycles in mammals (Prober et al., 2006). A complete discussion of the behavioral repertoire of zebrafish is beyond the scope of this chapter; many of the types of behavior are not directly relevant to studying human motor system disorders, and current models of movement disorders have used a few basic assays. Experimental systems for evaluating zebrafish movement vary greatly in complexity and in details of the types of movements they can measure and whether they are best suited to adult or larval assays.

7.5.1  Manual Observation of Motor Function In some models, there is clear motor paralysis or other abnormalities of movement that are obvious or that can be evoked or detected by a trained observer.

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In these cases, detailed quantitative analysis may be unnecessary. More quantitative methods for manual motor evaluation include an observer counting the number of times an animal crosses a series of marks on the bottom of the tank to measure activity or measuring the proportion of time an animal spends in each part of a behavioral field for a place-preference test. These methods are not suited to analyzing multiple animals simultaneously to determine quantitative differences or changes in behavioral patterns and are difficult to apply to analysis of larval zebrafish, because of the rapidity with which many of the movements occur. However, in some situations, they are appropriate and require minimal investment in equipment. A refinement of manual observation uses high frame-rate video recordings, taken using a macro lens or through a microscope, which are analyzed offline by a trained observer. The basic motor repertoire of larval zebrafish was largely defined by this method; magnification of the larvae and frame-by-frame analysis using this method allowed manual verification of categories of movement and measurements of angles of body curvature. However, as discussed earlier, one major advantage of zebrafish for movement disorders research is the potential to develop high-throughput applications, and manual analyses are not suitable for high-throughput screens unless the phenotype is severe and unmistakable and can be determined very quickly by inspection. Our experience in zebrafish models suggests that even for hypokinesia induced by severe loss of dopaminergic function, motor deficits are quantitative and not obvious with the naked eye, suggesting that meaningful analysis of these models will require more objective methods.

7.5.2  Automated Analysis of Motor Function Several automated methods are available for quantifying motor behavior, each allowing simultaneous analysis of multiple animals; these techniques are especially suited to larvae and to high-throughput applications but have also been used for adult zebrafish. Each of the methods is based on analysis of video recordings of animals moving, usually filmed from above or below such that only movements in the horizontal plane can be analyzed. This is reasonable for analysis of propulsion, reorienting movements and responses that depend on place preference. However, most of the current methods do not analyze the depth at which an animal is swimming, which may be a fruitful area for future studies. Furthermore, current methods generally lack the spatial resolution to allow easy determination of the movements and functions of the pectoral fins, so analysis is restricted to measuring displacement of the animal or movements mediated by trunk muscles.

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FIGURE 7.7  Methods for measuring zebrafish movement. The top row of images shows a video recording of a zebrafish larva at 7 days postfertilization making a routine turn in the well of a 96-well plate. Consecutive frames at 30 frames per second are shown, with time annotated above each frame. In the next row, the video was analyzed using a freeware program for measuring zebrafish movement (LSRtrack, Cario et al., 2011). The well margins are colored red, the zebrafish colored blue, and the larval centroid is marked with a yellow circle. In the lower row of frames, pixels that changed from the previous video frame were colored black and the well margins colored blue using MATLAB®, to illustrate the pixel quantification method. The same movement is shown quantitatively in the lower graphs, measured using each of the methods for comparison.

There are three basic approaches for analyzing video recordings of zebrafish for movement (Figure 7.7). These are described next in order of increasing complexity:   

• P  ixel quantification: In each field of measurement, a simple count is made of the number of pixels whose grayscale value (0–255) changes in every successive frame of the video. This measure can be reported as pixels per second or the percent time that the value exceeds a predetermined threshold, to give an index of motor activity. One advantage of this method is that multiple animals can be analyzed in the same behavioral field, because it is not necessary to establish the identities of individual objects. This method is also sensitive to nonpropulsive movements, such as changes in body shape, which are not accompanied by a change in the location of the animal. The disadvantages of this method are the heavy dependence of the results on details of the methodology (magnification, illumination, fish size, and pigmentation), lack of positional information, and lack of detail on the nature of any movement abnormalities detected. For example, slowed movements and reduced numbers of movement events could give the same results using the pixel quantification method. This methodology

is especially applicable to measuring activity levels in populations of animals and has been deployed successfully in a chemical screen to discover compounds with neuropharmacological actions that alter activity levels during the circadian cycle (Prober et al., 2006; Rihel et al., 2010). Pixel quantification is sensitive to movement early in development and can be used to monitor activity in groups of developing larvae that have undergone experimental manipulations. These properties allowed its use to demonstrate a hypokinetic phenotype in zebrafish transiently lacking β- and γ1-synucleins during early development, pointing to a role for these key proteins in the formation of relevant motor circuitry (Milanese et al., 2012). • C  entroid location: The position of an animal, usually calculated as the geometric centroid of its silhouette, is located in each frame of a video (Cario et al., 2011). This allows calculation of the distance moved during the recording, number of movement events, peak and mean velocity, acceleration, and the amount of time each movement lasts. In addition, other parameters can be calculated, such as positional place preference of the animal in different phases of the experiment and details about the direction

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and radius of turns. One potential advantage of this method is that, because the results are expressed in physical units such as mm/s that relate directly to the initial observations, the data output should be more dependent on the properties of the zebrafish than the recording methodology, potentially facilitating comparison of data sets from different sources. The disadvantage of this method is that identifying an object and calculating its geometric centroid is more computationally intensive than the algorithms used for pixel quantification. Furthermore, present centroid tracking technologies do not allow for tracking multiple objects reliably in the same behavioral testing arena, because collision events (e.g., where one fish swims over another) require a means to unequivocally determine the identity of each animal afterward. Because this problem has not yet been fully resolved, most tracking paradigms house each animal individually, such as within the wells of a 96-well plate. Although this allows for testing multiple different animals simultaneously, it precludes analysis of behaviors requiring direct contact between animals, potentially altering the types of responses that can be detected. In addition, this also limits the size of testing arena that can be practicably used for a given sample size, presenting an increasing concern with larval age that the physical constraints of larvae swimming in the wells of multiwell plates will reduce measured motor function, thereby introducing artifacts (Farrell et al., 2011). • Determination of body shape: In this approach, in addition to identifying each zebrafish and calculating its location, the software also determines the shape of the zebrafish by segmenting the zebrafish outline and calculating the angle of curvature of the zebrafish along its long axis. Automated systems can automatically assign trunk-bending movements to different categories (e.g., O-bend, C-bend) and allow determination of the rate of change of trunkbend angle during tail-beating movements involved in propulsive swimming (Burgess and Granato, 2007). Although this method clearly gives the most information about zebrafish movement, there are some limitations with current methodology. First, the high-resolution images and high frame-rate video recordings necessary to capture the movements in sufficient fidelity to identify and calculate these parameters are associated with very large files. This restricts the length of time that recordings can be carried out, limiting experimental throughput. Consequently, many of the reported analyses using this method are limited to evoked responses with high frame-rate recordings for a second or longer after each stimulus. This approach has been helpful

for analyzing some types of behavioral paradigm; however, it is still unresolved how best to evaluate the neural pathways of interest for the study of human movement disorders using evoked stimuli.

  

As a consequence of continuing dramatic improvements in the specifications and performance of computer hardware and the ongoing development of high-­ resolution cameras and methods for high-speed data transfer, automated recording and analysis of zebrafish movement are rapidly evolving. Presently, both commercial and open source (Burgess and Granato, 2007; Cario et al., 2011) zebrafish behavioral applications are available, and the capabilities of both are continually expanding. Currently available methods have provided some notable insights into zebrafish motor function, as related to models of human motor system diseases. First, simple activity measurements have allowed proof-of-concept experiments to show that medium-throughput automated screens for small molecules with neuropharmacological actions are possible, using practicable animal sample sizes and experimental resources (Rihel et al., 2010). Second, basic manual tests, such as the escape response following mechanical stimulus, have allowed detection of motor phenotypes in some models that show robust abnormalities, such as a transgenic tau model with impaired motor neuron development (Paquet et al., 2009). Third, quantification of simple metrics such as mean velocity (distance moved/time of assay) allowed detection of functional abnormalities of the dopamine system, caused by the toxin MPP+ or dopamine receptor antagonist drugs (Farrell et al., 2011). These assays, based on centroid tracking in 96-well plates, were quantitative and reproducible, allowing reliable detection of partial phenotypic rescue after loss of dopaminergic function. This has intriguing implications regarding the deployment of zebrafish parkinsonism models for drug discovery applications. Finally, the availability of simple and robust motor assays allows determination of the nervous system functions of genes, when loss of gene function does not result in morphological abnormalities. For example, we have used pixel quantification methods to show that synucleins are required for early development of motor function in larval zebrafish (Milanese et al., 2012) but that the zebrafish DYT1 dystonia gene homologue tor1 is not (Sager et al., 2012).

7.6  CHEMICAL SCREENING Unlike many other models that are traditionally used for high-throughput discovery applications, zebrafish embryos readily absorb small molecules from their surrounding medium, greatly simplifying drug administration and dosing (Peterson and Macrae, 2012). Embryos and larvae can be housed in 96-well plates, allowing

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their individual exposure to large collections of chemical compounds. Together, these factors provide a convenient means for screening large libraries of molecules against whole organism phenotypes. Furthermore, embryos are dimethyl sulfoxide tolerant, allowing even relatively hydrophobic molecules to be dissolved in aqueous medium for testing. After the first few days of development, additional factors determine distribution of small molecules into the zebrafish CNS. First, after early larval development, absorption of many small molecules likely depends on transport across the gastrointestinal tract, rather than direct diffusion into the embryo; this results in some selectivity in the range of compounds that are absorbed (Umans and Taylor, 2012). Second, after 3  dpf, the zebrafish CNS vasculature develops a blood–brain barrier (BBB) that is not permeable to circulating proteins and that regulates the passage of small molecules from the circulation into the CNS (Jeong et al., 2008; Watanabe et al., 2012; Xie et al., 2010). Zebrafish brain endothelia express orthologues of transporter proteins expressed in mammals, including MDR1, involved in excluding compounds from the CNS (Umans and Taylor, 2012), and GLUT1, involved in cerebral glucose transport and often considered a canonical marker of the BBB (Zheng et al., 2010). Furthermore, zebrafish brain endothelia also express Claudin-5, implicated in formation of tight junctions (Xie et al., 2010). Because the zebrafish BBB has at least some functional properties in common with the mammalian BBB, it is possible that screens based on neurological phenotypes of interest will select for compounds that are likely to be orally bioavailable and to cross the BBB, in addition to interacting with the pharmacological target of interest. This could potentially increase the likelihood of small-molecule ‘hits’ giving rise to a useful starting point for the development of drug therapies, especially since the whole-organism phenotypic approach may also exclude compounds that show acute toxicity. The application of chemical screens in zebrafish models of motor disorders could fulfill several functions dependent on the phenotypic end point assay used. For example, in vivo imaging could allow isolation of compounds that prevent cell death, such as loss of neurons in models of neurodegenerative diseases associated with movement disorders. However, the development of automated high-throughput assays provides another possibility that could be applicable to diseases, such as primary dystonia, where there may be minimal or no neurodegeneration. In this situation, it may be possible to use zebrafish models to isolate small molecules that alter abnormal motor responses, with potential applications for the development of drugs that alleviate motor symptoms in movement disorders. Although chemical screens for compounds with neuropharmacological

actions have been described, a zebrafish model and accompanying assays suitable for drug discovery to ameliorate pathology in human neurological disease have not yet been described. This is currently a priority for the field, because this is an area where zebrafish models have potential to make a unique impact.

7.7 CONCLUSIONS The idea that zebrafish could be used to model human movement disorders is attractive, potentially building on a rapidly expanding arsenal of powerful experimental techniques and resources arising from use of zebrafish models in developmental biology. Early results are promising, suggesting that abnormalities representative of human motor systems disorders could be replicated in zebrafish, although many key experimental resources and phenotypic assays are still under development and the full potential of this model has not yet been realized. The major advantages of zebrafish models over other vertebrate models may be applications such as chemical and genetic modifier screens that cannot practicably be executed in mammalian models and the unique applicability of in vivo imaging in live intact organisms. Other considerations include the ease of making transgenic lines and gene knockouts and practical advantages related to housing large colonies of animals. These considerations are offset against the phylogenetic distance that separates humans from zebrafish, compared with commonly deployed rodent and primate models, and differences in the details of movement control that result from comparison of aquatic and terrestrial organisms. The importance of these considerations is currently unclear; because basic CNS structure and organization are conserved among vertebrates, along with many of the cell types, neurochemical systems, genes, and biochemical pathways implicated in human disease, it seems likely that the zebrafish CNS provides an appropriate setting in which to model human disease. Conservation of vertebrate genetics and CNS structure provides the key advantage of zebrafish models compared with ­Drosophila and Caenorhabditis elegans models, in addition to the ease with which zebrafish can be exposed to chemical libraries. However, methodologies for transgenesis, gene knockouts, and genetic screens are currently better characterized and less laborious in these classic invertebrate models, which have provided several key insights into the biochemistry of neurodegeneration. It seems likely that the zebrafish model will occupy a key niche in the repertoire of available models, providing unique opportunities to carry out high-throughput experiments traditionally limited to invertebrates, in a setting where vertebrate genetics and CNS organization are important.

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References

Drug discovery for motor diseases is one obvious application, but there are many others, such as determination of the mechanisms for cell-type specificity, investigation of the role of glia and cell nonautonomous mechanisms in disease pathogenesis, and deployment of in vivo imaging modalities for unique structural and biochemical insights into neurobiology of disease. Future work will concentrate on development and characterization of representative zebrafish models of motor disorders, before these methods can be exploited to provide mechanistic insights into pathogenesis and to allow the discovery of novel therapeutic agents based on their phenotypic effects in vivo.

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