Hypokinesia and Reduced Dopamine Levels in Zebrafish Lacking β- and γ1-Synucleins*

Background: Synucleins are implicated in Parkinson disease pathogenesis, but their functions are incompletely understood. Results: Zebrafish lacking β- and γ1-synucleins showed hypokinesia and decreased dopamine levels. These abnormalities were rescued by human α-synuclein. Conclusion: Synucleins are necessary for spontaneous movement and dopaminergic function. Significance: A key functional role for synucleins in dopamine neurons may be relevant to Parkinson disease pathogenesis. α-Synuclein is strongly implicated in the pathogenesis of Parkinson disease. However, the normal functions of synucleins and how these relate to disease pathogenesis are uncertain. We characterized endogenous zebrafish synucleins in order to develop tractable models to elucidate the physiological roles of synucleins in neurons in vivo. Three zebrafish genes, sncb, sncg1, and sncg2 (encoding β-, γ1-, and γ2-synucleins respectively), show extensive phylogenetic conservation with respect to their human paralogues. A zebrafish α-synuclein orthologue was not found. Abundant 1.45-kb sncb and 2.7-kb sncg1 mRNAs were detected in the CNS from early development through adulthood and showed overlapping but distinct expression patterns. Both transcripts were detected in catecholaminergic neurons throughout the CNS. Zebrafish lacking β-, γ1-, or both synucleins during early development showed normal CNS and body morphology but exhibited decreased spontaneous motor activity that resolved as gene expression recovered. Zebrafish lacking both β- and γ1-synucleins were more severely hypokinetic than animals lacking one or the other synuclein and showed delayed differentiation of dopaminergic neurons and reduced dopamine levels. Phenotypic abnormalities resulting from loss of endogenous zebrafish synucleins were rescued by expression of human α-synuclein. These data demonstrate that synucleins have essential phylogenetically conserved neuronal functions that regulate dopamine homeostasis and spontaneous motor behavior. Zebrafish models will allow further elucidation of the molecular physiology and pathophysiology of synucleins in vivo.

␣-Synuclein is strongly implicated in the pathogenesis of Parkinson disease. However, the normal functions of synucleins and how these relate to disease pathogenesis are uncertain. We characterized endogenous zebrafish synucleins in order to develop tractable models to elucidate the physiological roles of synucleins in neurons in vivo. Three zebrafish genes, sncb, sncg1, and sncg2 (encoding ␤-, ␥1-, and ␥2-synucleins respectively), show extensive phylogenetic conservation with respect to their human paralogues. A zebrafish ␣-synuclein orthologue was not found. Abundant 1.45-kb sncb and 2.7-kb sncg1 mRNAs were detected in the CNS from early development through adulthood and showed overlapping but distinct expression patterns. Both transcripts were detected in catecholaminergic neurons throughout the CNS. Zebrafish lacking ␤-, ␥1-, or both synucleins during early development showed normal CNS and body morphology but exhibited decreased spontaneous motor activity that resolved as gene expression recovered. Zebrafish lacking both ␤and ␥1-synucleins were more severely hypokinetic than animals lacking one or the other synuclein and showed delayed differentiation of dopaminergic neurons and reduced dopamine levels. Phenotypic abnormalities resulting from loss of endogenous zebrafish synucleins were rescued by expression of human ␣-synuclein. These data demonstrate that synucleins have essential phylogenetically conserved neuronal functions that regulate dopamine homeostasis and spontaneous motor behavior. Zebrafish models will allow further elucidation of the molecular physiology and pathophysiology of synucleins in vivo.
Synucleins are small proteins of 100 -140 amino acids that are expressed abundantly in neuronal presynaptic terminals (reviewed in Ref. 1). The three members of the synuclein family, ␣-, ␤-, and ␥-synuclein, are encoded by separate genes and are widely conserved throughout the vertebrate subphylum. Pathogenic missense (2)(3)(4) and gene dose (5,6) mutations in the SNCA gene encoding ␣-synuclein are an uncommon cause of familial parkinsonism. Convergent lines of evidence suggest that ␣-synuclein is also centrally involved in the pathogenesis of the common sporadic form of Parkinson disease (PD). 3 The pathological hallmark intraneuronal inclusions of sporadic PD, Lewy bodies, contain insoluble fibrillar aggregates of ␣-synuclein (7). Furthermore, genome-wide association studies (8 -10) show an association between genetic variants at the SNCA locus and risk of developing PD. Recent studies support the possibility that SNCA variants influencing PD risk may alter ␣-synuclein expression (11), suggesting that alterations in ␣-synuclein levels may predispose susceptible neuronal groups to develop pathology in PD. It is currently unclear whether pathogenic involvement of ␣-synuclein in PD reflects quantitative changes in the activity of its normal cellular functions or the emergence of new pathological functions as its cellular abundance changes. Understanding the normal functions of synucleins is thus an important goal, because this might elucidate aspects of pathogenesis and facilitate identification of therapeutic targets in PD.
Prominent localization of synucleins at presynaptic terminals suggests that they play an important role in synaptic transmission. Mice lacking ␣-synuclein (12)(13)(14)(15)(16), ␤-synuclein (17), or ␥-synuclein (18) show normal development, survival, CNS morphology, synaptic protein expression, synaptic density, and behavior. Changes in the number of dopaminergic neurons (19), striatal dopamine levels and dopamine release (14), and synaptic vesicle pools (16) have been reported in individual lines of SNCA Ϫ/Ϫ animals and may reflect variations in genetic background. Synucleins show overlapping expression patterns, raising the possibility that minor abnormalities in single knockout animals are attributable to functional compensation by other synucleins. Mild dopaminergic abnormalities and up-regulation of ␤-synuclein were found in animals lacking both ␣and ␥-synucleins (19). Loss of ␣and ␤-synucleins together caused changes in the expression of presynaptic proteins, including up-regulation of ␥-synuclein, and a modest reduction in striatal dopamine levels (17). Loss of all three synucleins gave rise to progressive motor impairment; reduced survival; changes in synaptic morphology, physiology, and protein expression; and reduced SNARE complex formation (20,21) in addition to reduced striatal dopamine content and altered dopamine release (22). These studies show that synucleins are essential for long term maintenance of synapses in the vertebrate CNS, although the molecular basis of these observations and their relevance to Parkinson disease remain undefined. An accessible model allowing mechanistic studies in the intact vertebrate nervous system in vivo might be valuable, allowing further definition of the role of synucleins in the molecular physiology of presynaptic terminals.
The zebrafish is a powerful model for studying the vertebrate nervous system. Larvae can be manipulated to be optically transparent, enabling direct visualization of neurons (23,24), neural circuitry formation (25), and neuronal activity (26,27) in vivo, using transgenically expressed reporter proteins. Zebrafish may also be a useful model in which to study the pathogenesis of neurological disease (reviewed in Refs. 28 -30). Importantly, and uniquely among vertebrate models, zebrafish can be subjected to unbiased phenotype-based genetic (31,32) and chemical (33,34) screening approaches in order to discover novel molecular insights. The purpose of the present study was to identify and characterize endogenous zebrafish synucleins to allow studies of their functions in vivo and to facilitate the development of zebrafish models of Parkinson disease. Here we report that zebrafish do not express ␣-synuclein. Zebrafish ␤and ␥1-synucleins are expressed widely in CNS neurons, including dopamine neurons, and are necessary for the development of normal motor and dopaminergic function in zebrafish larvae.

EXPERIMENTAL PROCEDURES
Zebrafish-Experiments were carried out in accordance with National Institutes of Health guidelines and Institutional Animal Care and Use Committee approval. Adult strain AB zebrafish were maintained at 28.5°C and euthanized by deep tricaine anesthesia followed by exposure to ice-cold water. Embryos were raised in E3 buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 ), supplemented where necessary with 0.003% 1-phenyl-2-thiourea to inhibit pigmentation.
Synteny Analysis-Online tools (NCBI genome viewer and Cinteny (35), both available on the World Wide Web) were used to evaluate syntenic relationships. Proposed orthologues were verified by multiple sequence alignments, and synteny maps were drawn manually.
RT-PCR and 5Ј-RACE-First strand cDNA was generated by oligo(dT)-primed reverse transcription of total RNA from whole adult zebrafish brains or pooled embryo lysate (Super-Script III, Invitrogen). Nested 5Ј-RACE was carried out (RLM-RACE, Ambion, Austin, TX) as described previously (24,36,37). Primer sequences are listed in supplemental Table 1.
Morpholino Oligonucleotides-Antisense morpholino oligonucleotides (MOs; Gene Tools LLC, Philomath, OR) were designed to inhibit zebrafish synuclein gene expression by targeting splice signals in the primary transcripts. Expression of sncb was disrupted by MOs targeted against splice donor sites of exons 2 and 3 (sncb E2/I2, aatgttgcatgacttacCCACAAAC; sncb E3/I3, ttgcagaatcaccttacCTGTTGCA; sequence complementary to the 5Ј-end of each exon is shown in capital letters). Expression of sncg1 was disrupted by MOs targeted against the splice donor sites of exons 1 and 3 (sncg1 E1/I1, tatacatcagcact-tacCCACATAC; sncg1 E3/I3, ctgattgctattcactcacCGGGT). Control MOs (random MO (N 25 ) and non-targeting MO CCTCTTACCTCAGTTACAATTTATA) were used to exclude nonspecific effects arising from morpholino exposure. mRNA Rescue-A NotI/XhoI fragment of pIRES2-EGFP (Clontech, Mountain View, CA) was inserted into the XbaI/ XhoI sites of pCS2 ϩ (40) to generate plasmid pCS-IG. Human ␣-synuclein cDNA was amplified from NT2 cells by RT-PCR, sequenced, and inserted into the XhoI/XmaI sites of pCS-IG to generate pCS-syn-IG. pCS-IG and pCS-syn-IG were linearized at the NotI site 3Ј to the poly(A) signal, and mRNA was transcribed from the Sp6 promoter (mMessage Machine, Ambion, Austin, TX). Intracellular injection of 1-1.25 nl of mRNA solution (250 pg/nl mRNA, 0.25% phenol red, 120 mM KCl, 20 mM HEPES, pH 7.4) was carried out at the single cell stage, followed by MO injection into the yolk sac as above.
Motor Analysis-Larval movement was analyzed using a proprietary system (Zebralab; ViewPoint Life Sciences, Montreal, Canada) as reported previously (34). Zebralab software was used in "quantization mode" with detection sensitivity set to 16 and with burst and freezing activity thresholds set to 10 and 2, respectively. Groups of 15 embryos were analyzed in 12-well plates containing 3 ml of E3 buffer/well. Spontaneous activity was measured for an 8-h period daily, from 24 h postfertilization (hpf) to 7 days postfertilization (dpf).
Neurochemical Measurements-Neurochemical measurements were performed by adapting a previously reported method (41). 30 larvae were sonicated in 150 l of 4 M perchloric acid, 0.1% Na 2 S 2 O 5 , 0.1% EDTA at 4°C. The sample was centrifuged at 17,000 ϫ g at 4°C, and the supernatant was filtered through a 0.22-m nylon membrane (Spin-X, Corning Glass). 25 l was loaded into a Waters 2695 HPLC separation module (Waters, Milford, MA). The HPLC mobile phase consisted of 0.06 M sodium phosphate monobasic, 0.03 M citric acid, 8% methanol, 1.1 mM 1-octanesulfonic acid, 0.1 mM EDTA, 2 mM sodium chloride, pH 3.5. The flow rate was 1.0 ml/min. Neurotransmitters were separated on a Waters XBridge C18 4.6 ϫ 150-mm column, particle size 3.5 m, at 34°C, and detected using a Waters 2465 electrochemical detector with a glassy carbon electrode set at 750 mV, referenced to an ISAAC electrode. Neurotransmitters were quantified as pg/larva, using a standard curve generated from injection of high purity standards. Each data point was then normalized to the value for wild type animals in the same experiment.
Statistical Analysis-Quantitative data were shown to be normally distributed by Pearson's and D'Agostino's tests and were analyzed using parametric statistical tests. All experiments were repeated at least in triplicate. Graphs show means Ϯ S.E. One-way ANOVA was carried out after confirming the assumption of equal variance using Bartlett's test and was followed by Dunnett's post hoc test to evaluate statistically significant differences between test groups.

RESULTS
Zebrafish Synucleins-Identification of zebrafish synucleins was recently reported by two other groups (42,43). We independently cloned the same genes at the start of this study (not shown). sncb encodes the zebrafish orthologue of human ␤-synuclein, and sncg1 and sncg2 encode dual paralogues of human ␥-synuclein. Zebrafish synucleins show extensive sequence similarity to human synucleins, except that the C termini of the two zebrafish ␥-synucleins are relatively divergent (Fig. 1). We did not identify an orthologue of human ␣-synuclein in zebrafish genomic, mRNA, or expressed sequence tag databases, which was unexpected because ␣-synuclein orthologues have been identified in mammals, birds, reptiles, amphibians, and other fish species. Despite employing a variety of RT-PCR and RACE strategies to amplify a putative snca transcript (supplemental Table 4), we did not find evidence of a zebrafish ␣-synuclein orthologue expressed in the CNS.
In order to further understand how human and zebrafish synuclein genes are related, we constructed synteny maps (Fig.  2). Two of the genes flanking the sncb gene on zebrafish chromosome 13 are orthologues of genes flanking the human SNCB gene on chromosome 5 ( Fig. 2A), suggesting that the genes are derived from a common ancestral locus. A large genomic region surrounding zebrafish sncg1 and sncg2 is duplicated and is syntenic with the human genomic region containing SNCG (Fig. 2B). These observations suggest that sncg1 and sncg2 are derived from duplication of an ancestral locus shared with human SNCG. Zebrafish orthologues of genes immediately flanking human SNCA were not found in the available zebrafish genome sequence, whereas the wider genomic region in the human showed two blocks of genes that were syntenic to different regions of the zebrafish genome (Fig. 2C). It is possible that the region between these syntenic blocks was deleted from an ancestral chromosome during evolution, accounting for the lack of zebrafish genomic or mRNA sequences with homology to human SNCA.
mRNA Transcripts and Genomic Organization of Zebrafish Synucleins-Zebrafish sncb and sncg1 transcripts were detected abundantly in adult brain by Northern blot (Fig. 3A). Each gene gave rise to a single mRNA species of 1.45 kb (sncb) or 2.7 kb (sncg1). sncg2 was alternatively spliced to yield two transcripts encoding isoforms with divergent C termini; although both transcript variants were amplified from CNS by PCR (Fig. 3B), we were unable to identify sncg2 transcripts in the CNS by Northern blot hybridization or RNA in situ hybridization and conclude that they are present at low abundance. The remainder of this study focused on sncb and sncg1, encoding the predominant CNS synucleins in zebrafish.
5Ј-RACE (Fig. 3C), using the tobacco acid pyrophosphatase (TAP) method (as described previously (24,36,37)), was employed to map the 5Ј-ends of the sncb and sncg1 transcripts. TAP Ϫ and RT-PCR controls verified that the boundary between the RACE adapter and the cDNA sequence in the RACE products occurred at the mRNA cap site. Both sncb and sncg1 promoter regions lack canonical TATA motifs and show multiple transcriptional start sites from motifs with homology to the initiator consensus 5Ј-YYA ϩ1 NWYY-3Ј (44) (Fig. 3, D and E). We next examined the genomic organization of sncb, sncg1, and sncg2 by comparing cDNA sequences with the zebrafish genomic sequence (Fig. 3, F-H, and supplemental Tables 2 and 3). Like its human orthologue, sncb contains six exons, the first of which is non-coding. Similar to their human paralogue, both sncg1 and sncg2 contain five exons. The positions and phases of the splice boundaries in sncb, sncg1, and sncg2 are conserved with respect to their human paralogues, providing further evidence of a common origin (supplemental material).
Expression Patterns of Zebrafish Synucleins-The expression patterns of zebrafish synucleins were determined by RNA in situ hybridization. As reported previously during early development (42,45), sncb and sncg1 were expressed in the CNS, whereas sncg2 was found in the notochord (Fig. 4, A-C). In the adult zebrafish CNS, both sncb and sncg1 were expressed in cells with neuronal morphology, widely distributed throughout gray matter of the brain and spinal cord. However, their topographical expression patterns differed significantly; sncb was more robustly expressed rostrally, whereas sncg1 expression was more prominent caudally (Fig. 4, D and E). Differences in the expression patterns of sncb and sncg1 in specific brain regions are illustrated in Fig. 4F. Whereas prominent sncb expression was detected in the olfactory bulb and dorsal telencephalon, sncg1 was not expressed at high levels in these regions. Conversely, robust sncg1 expression in the medulla oblongata and habenula was accompanied by weak expression of sncb in these areas. Even within some brain regions where there was overlap between sncb and sncg1 expression, the two genes were expressed in different cellular populations. For example, within the cerebellum, sncb was expressed within granule cells, whereas sncg1 was expressed most prominently in the Purkinje cell layer (Fig. 4G).
We next examined expression of sncb and sncg1 in catecholaminergic neurons in the zebrafish CNS. In the absence of available antibodies to zebrafish ␤or ␥1-synucleins, we used a double label ISH/IHC technique, allowing us to detect sncb or sncg1 and tyrosine hydroxylase simultaneously in the same tissue sections. Examples are shown in Fig. 4H; dopaminergic neurons of the periventricular hypothalamus were identified by their morphology and by robust tyrosine hydroxylase immunoreactivity in their processes. Strong expression of sncb (Fig. 4H, left) and sncg1 (right) was also evident in the perinuclear cytoplasm. Using this method, we examined each of the major catecholaminergic cell groups within the olfactory bulb, telencephalon, diencephalon, and hindbrain. With the exception that sncg1 expression was relatively weak in the olfactory bulb, all tyrosine hydroxylaseimmunoreactive groups also expressed both sncb and sncg1. These data suggest that sncb and sncg1 may be co-expressed in catecholaminergic neurons.
␤and ␥1-Synucleins Are Not Required for Early Embryogenesis or CNS Morphogenesis-We next asked whether perturbing expression of sncb or sncg1 using antisense MOs would result in a neurodevelopmental phenotype that might be  to the right. B, total RNA derived from adult brain or embryos at 48 hpf was subjected to RT-PCR using primers complementary to exons 3 and 5 of sncg2. The picture shows an ethidium-stained agarose gel under UV illumination following electrophoretic separation of the products. Bands corresponding to sncg2 tv1 (lacking exon 4) and sncg2 tv2 (containing exon 4) are labeled to the right of the picture. C, 5Ј-RACE was used to map the transcriptional start sites of sncb (lanes 1, 3, 5, and 7) and sncg1 (lanes 2, 4, 6, and 8). The picture shows an ethidium-stained agarose gel under UV illumination following electrophoretic separation of PCR products. 5Ј-RACE (lanes 1-4) and RT-PCR (lanes 5-8) were carried out using cDNA template derived from samples in which RNA was either treated with tobacco acid pyrophosphatase (TAP ϩ ; lanes 1, 2, 5, and 6 informative regarding the normal functions of ␤and ␥1-synucleins. Antibodies to zebrafish synucleins are not available, so verification of translation-inhibiting MOs was not possible; consequently, we inhibited gene expression by targeting pre-mRNA splice sites because splicing could be verified by RT-PCR. Pairs of MOs targeting two splice boundaries in each transcript greatly reduced expression, such that sncb or sncg1 mRNA could not be detected by RT-PCR up to 72 hpf (Fig. 5, A  and B). We refer to the pair of MOs targeting sncb as ␤ MO and the pair of MOs targeting sncg1 as ␥1 MO. As a control for nonspecific effects of MOs, we used both non-targeting and random MOs (termed Ctrl MO in subsequent figures) at the same concentrations. These did not affect expression of either sncb or sncg1 (Fig. 5, A and B).
Zebrafish lacking ␤-, ␥1-, or both synucleins were viable and showed normal development of the basic body plan, internal organs, and circulation (Fig. 5C). In order to examine the morphology of the nervous system in more detail, we employed Tg(eno2:egfp) zebrafish, which express GFP in a pan-neuronal pattern (24), allowing delineation of CNS structure and exam- A-C, whole mount RNA in situ hybridization was carried out in zebrafish embryos at 24 hpf using cRNA probes to sncb (A), sncg1 (B), or sncg2 (C). Hybridized probe was revealed using a chromogenic substrate with a purple/blue reaction product. Structures expressing each transcript are labeled. FB, forebrain; HB, hindbrain; SC, spinal cord; NC, notochord. D and E, RNA in situ hybridization was carried out on parasagittal sections from fixed adult brains using cRNA probes specific to sncb (D) and sncg1 (E). Hybridized probe was revealed using a chromogenic substrate with a purple/blue reaction product. The images show low magnification micrographs to illustrate the topographical expression patterns of the transcripts. The scale for both images is shown to the right of D. Structures expressing each transcript are labeled in each image. OB, olfactory bulb; D, dorsal telencephalon; TeO, optic tectum; Ha, habenula; P, cerebellar Purkinje cell layer; IRF, inferior reticular formation. F and G, adult brain sections processed identically to those shown in D and E are shown at higher magnification in order to illustrate regions in which sncb (top row) and sncg1 (bottom row) showed different expression patterns. The anatomical region is labeled above each pair of images, and the scale for both micrographs is shown in the upper of the two. In G, the cerebellum is shown at two different magnifications. M, molecular layer; P, Purkinje cell layer; G, granule cell layer. H, two-color RNA in situ hybridization/immunohistochemistry (38) was used to examine expression of synuclein mRNAs in dopaminergic neurons. In each image, RNA in situ hybridization was carried out using cRNA probes complementary to sncb (left) or sncg1 (right), and hybridized probe was detected using a chromogenic reaction with a blue/purple product. The sections were then labeled using an antibody to tyrosine hydroxylase (64), and bound antibody was detected using a second chromogenic reaction with a red product. Examples are shown of dopaminergic neurons in the periventricular hypothalamus that showed expression of sncb or sncg1 mRNA in their cell bodies (purple arrows) and tyrosine hydroxylase (TH) in their processes (red arrows). The scale for both images is shown in the lower right corner of the left panel. ination of spinal morphology and nerve roots. Compared with controls, we did not detect any abnormalities of CNS or PNS structure in Tg(eno2:egfp) larvae lacking ␤-, ␥1-, or both synucleins. In particular, the CNS appeared structurally unremarkable at 4 dpf, the time point at which the most robust neurobehavioral abnormalities were found (see below). These data show that ␤and ␥1-synucleins are not necessary for development of normal CNS morphology.
␤and ␥1-Synucleins Are Required for Normal Development of Motor Function-To evaluate the role of ␤and ␥1synycleins in neuronal function, we employed an assay of spontaneous larval movement. A live video stream showing zebrafish larvae housed in multiwell plates was analyzed for movement by quantifying the changes in pixel gray scale values that accompany larval displacement from frame to frame. In order to detect changes in the frequency with which spontaneous movements were executed, we measured the proportion of video frames in which pixel gray scale changes exceeded the threshold for detection of movement above stochastic pixel noise. To confirm that this method could quantify spontaneous movement under the conditions used for our study, we housed 0, 2, 4, 8, or 16 zebrafish in each well of a 12-well plate and quantified motor activity at 2, 3, and 7 dpf (Fig. 6A). No movement was detected in empty wells, and the relationship between the number of larvae in a well and measured motor activity was linear.
We next analyzed movement of groups of 15 larvae from each experimental group (WT, buffer-injected, Ctrl MO, ␤ MO, ␥1 MO, or ␤ ϩ ␥1 MO). Six independent experimental replicates were carried out at each time point between 2 and 7 dpf (Fig. 6B). In uninjected larvae, little movement was detected at early developmental points; however, motor activity increased sharply at 4 dpf and remained at approximately the same level until 7 dpf. At each time point, there were no statistically significant differences between uninjected larvae, larvae injected with buffer only, and larvae injected with control MOs at the highest doses used in gene targeting experiments. Thus, neither the mechanical consequences of microinjection nor nonspecific toxic effects of exposure to MOs induced changes in spontaneous movement in this assay. At 2 dpf, small statistically significant reductions in activity were apparent between controls and larvae lacking ␤-, ␥1-, or both synucleins, but these changes resolved by 3 dpf. Between 3 and 4 dpf, larvae lacking ␤or ␥1-synucleins showed only a modest increase in activity, compared with the large increase seen in controls. Consequently, at 4 dpf, larvae lacking ␤or ␥1synucleins showed a robust reduction in spontaneous movement compared with controls. Loss of motor activity was more severe in larvae lacking both ␤and ␥1-synucleins than in larvae lacking one or other synuclein. As gene expression recovered later in development, the hypokinetic phenotype started to resolve. By 5 dpf, spontaneous movement in synuclein knockdown animals had almost returned to control levels, and only animals injected with ␤ ϩ ␥1 MO showed statistically significant differences from controls. By 7 dpf, the groups were statistically indistinguishable, although measured motor activity remained lower in larvae exposed to ␤ ϩ ␥1 MO than in the other experimental groups.
In order to firmly establish whether these motor abnormalities were attributable to loss of synucleins, we next asked whether this hypokinetic phenotype could be rescued by an exogenous synuclein. Human ␣-synuclein was expressed in zebrafish embryos by microinjecting a bicistronic mRNA encoding both ␣-synuclein and GFP. mRNA encoding GFP alone was used as a control. Because the IRES element functions weakly in zebrafish (not shown), human ␣-synuclein expression was verified by other methods. Western blot showed a single 16-kDa ␣-synuclein immunoreactive band specific to microinjected embryos; immunofluorescence demonstrated robust ␣-synuclein expression throughout microinjected embryos, including the neural tube and developing retina (Fig. 6C). Expression of either GFP or human ␣-synuclein did not alter WT larval motor activity (Fig. 6D). Reduced motor activity at 4 dpf was seen in GFP-expressing larvae lacking ␤-, ␥1-, or ␤and ␥1-synucleins, similar to results in larvae expressing no exogenous mRNA. In contrast, animals expressing human ␣-synuclein showed normal levels of motor activity in the absence of ␤or ␥1-synuclein. In the absence of both ␤and ␥1-synucleins, it was necessary to express a higher level of human ␣-synuclein in order to prevent loss of spontaneous movement. These data show that the hypokinetic abnormality observed after loss of ␤-, ␥1-, or ␤and ␥1-synucleins is attributable to loss of synucleins rather than an artifact of the experimental system. Together, these findings demonstrate that ␤and ␥1-synuclein expression in neurons is essential for physiological functions that underlie normal motor behavior during development.
␤and ␥1-Synucleins Are Required for Development of the Dopamine System-We next addressed the hypothesis that the motor phenotype resulting from loss of ␤or ␥1-synuclein was attributable to abnormalities of the dopamine system. We first asked whether development of dopaminergic neurons differed between controls and zebrafish lacking ␤-, ␥1-, or both synucleins. RNA in situ hybridization was carried out at 48, 72, and 96 hpf, using a cRNA probe to slc6a3 (dopamine transporter, dat), which is specifically expressed in dopamine neurons (38,46) (Fig. 7A). No qualitative differences were observed in the distribution or staining intensity of slc6a3-expressing cells between controls and larvae lacking ␤-, ␥1-, or both synucleins. However, quantification by an observer blinded to MO exposure showed a modest reduction in the number of slc6a3 ϩ cells in larvae lacking both ␤and ␥1-synucleins at 48 hpf (Fig. 7B). This abnormality had recovered by 72 hpf, such that there was no difference in the number of slc6a3 ϩ cells between WT larvae and those exposed to ␤ ϩ ␥1 MO (Fig. 7C). Expression of GFP or human ␣-synuclein did not alter the number of slc6a3 ϩ neurons in control larvae (Fig. 7D). However, expression of human ␣-synuclein was sufficient to prevent a reduction in the number of slc6a3 ϩ cells in the absence of ␤and ␥1-synucleins, whereas GFP expression did not rescue this phenotype. These data show that both ␤and ␥1-synuclein are necessary for early development or differentiation of dopamine neurons. However, there was a normal complement of dopamine neurons by 4 dpf, when the hypokinetic phenotype in zebrafish lacking synucleins was most prominent.
Finally, we asked whether abnormalities of monoaminergic neurochemistry might explain the hypokinesia resulting from loss of ␤or ␥1-synucleins. We examined steady-state whole larval dopamine levels at 7 dpf, which was the earliest time point at which we could measure neurotransmitters with sufficiently low variability to enable detection of differences between multiple experimental groups. Although gene expression had recovered by this time point, zebrafish lacking both ␤-and ␥1-synucleins earlier in development showed significantly reduced dopamine levels at 7 dpf compared with Ctrl MO-injected animals (Fig. 7E). None of the other experimental groups showed significant differences from controls. Transient expression of GFP or human ␣-synuclein early in development did not change steady state levels of dopamine in WT larvae at 7 dpf. However, ␣-synuclein but not GFP prevented the loss of dopamine that occurred in the absence of zebrafish ␤and ␥1-sy- nucleins (Fig. 7F). These data show that early developmental expression of both ␤and ␥1-synucleins is necessary for establishment of normal dopamine levels. Interestingly, 5-hydroxytryptamine levels were not reduced in larvae lacking synucleins (Fig. 7G), suggesting that the relevant function of synucleins in this context may be specific to dopaminergic neurons.

DISCUSSION
A genomic duplication occurred early in the evolution of ray-finned fish (47) and probably accounts for the presence of two sncg genes in zebrafish, similar to fugu, medaka, salmon, and stickleback (48). In some cases, duplicated genes have complementary roles, sharing multiple functions of the ancestral gene (49). The dual zebrafish paralogues of ␥-synuclein show distinct temporal and spatial expression patterns; this may have allowed segregation of neural and non-neural functions of ␥-sy-nuclein into separate proteins and provided evolutionary pressure to retain both genes. Conversely, our data suggest that an ancestral snca gene and its flanking genes were lost during zebrafish evolution. The putative deletion appears to have occurred more recently than the duplication of sncg, because a number of other fish species, including salmon and fugu, harbor duplicate sncg genes in addition to snca and sncb. A precedent for spontaneous loss of snca has been described in a subpopulation of the C57BL/6J strain of mice that developed a de novo deletion of the SNCA locus (15). It is likely that other synuclein family members provide compensatory functions accommodating the loss of a single synuclein gene; mice lacking ␣-, ␤-, and ␥-synucleins (20 -22) showed more severe phenotypes than animals lacking one or two synucleins (17,18), and abnormalities in ␣-, ␤-, and ␥-synuclein null mice were mitigated by expression of human ␣-synuclein (21). Here we provide further evidence of cross-functionality between dif- ferent synucleins by showing that loss of zebrafish ␤and ␥1-synucleins gave rise to a more severe phenotype than loss of one or other synuclein and that abnormalities arising from loss of either protein were rescued by expression of human ␣-synuclein.
By using an assay sensitive to motor function early in development, we demonstrated that transient abrogation of ␤or ␥1-synuclein expression suppressed normal spontaneous motor activity in larval zebrafish. There was no accompanying alteration in the structure of the body or CNS, and we conclude that the mechanisms underlying the observed hypokinesia are likely to involve a disturbance of neuronal function. Resolution of hypokinesia was temporally correlated with the gradual recovery of gene expression after 72-96 hpf, suggesting that synucleins dynamically regulate aspects of neuronal function governing initiation of spontaneous movement. Abnormalities of synaptic transmission have been demonstrated in murine synuclein knock-out models (20,21), and it is possible that similar defects arise in circuits mediating initiation of spontaneous movement in zebrafish lacking ␤or ␥1-synucleins. Given the importance of dopaminergic mechanisms in human hypokinetic movement disorders associated with synucleinopathy and the possibility that synucleins have important specific functions in dopamine neurons (22), we evaluated the role of ␤and ␥1-synucleins in the dopaminergic system. Both proteins were expressed in dopamine neurons throughout the CNS, and loss of both proteins delayed the appearance of a full complement of slc6a3 ϩ -expressing neurons in the developing brain. Although the number of slc6a3 ϩ -neurons was normal at 4 dpf, it is possible that delayed differentiation of dopaminergic neurons might contribute to later abnormalities of motility, by affecting the formation of motor circuits. Even in the presence of a normal number of dopamine neurons, we found evidence of disturbed dopaminergic function; at 7 dpf, zebrafish exposed to ␤ ϩ ␥1 MOs showed decreased dopamine levels, similar to previous studies in synuclein gene knock-out mice (17,22). Because reproducible measurements of dopamine levels were not technically feasible at earlier time points, we did not exclude the possibility that reduced dopamine levels were also present at the time points where the most prominent hypokinesia was observed. The development of zebrafish lines harboring stable null alleles of sncb and sncg1 will allow clarification of the relationship between synucleins, dopamine levels, and motor phenotypes later in development when neurochemical measurements are more robust. Stable sncb Ϫ/Ϫ and sncg1 Ϫ/Ϫ lines will also allow unbiased screening approaches to isolate modifiers of the hypokinetic synuclein-null phenotype that might be informative concerning genetic interactions of synucleins in vertebrate neurons in vivo. Previous screens for genetic modifiers have been carried out in yeast expressing human ␣-synuclein (50 -52), and the role of a subset of identified modifiers was confirmed in fly (53) and worm (52) models. Although these studies provide information about the pathways involved in cellular responses to ectopic ␣-synuclein overexpression, the physiological roles of synucleins may depend on specific protein interactions absent from organisms lacking synucleins natively. Consequently, zebrafish models may con-tribute important new insights into synuclein function at the vertebrate presynaptic terminal.
There has been increasing interest in the development of zebrafish models of human neurological disease for therapeutic target identification and drug discovery in vivo (28). Transient knockdown of a number of different zebrafish orthologues of human genes implicated in parkinsonism resulted in dopaminergic phenotypes, including loss of neurons (54 -56), altered function (56), or susceptibility to toxins (55,57). Furthermore, zebrafish exposed to toxins implicated in PD pathogenesis (41, 58 -61) showed loss of dopamine neurons, dopamine content, or dopaminergic function. Together, these data have provided encouragement that it might be possible to recapitulate aspects of PD pathogenesis accurately in zebrafish, an essential prerequisite for the development of useful models. We report here that zebrafish lack endogenous ␣-synuclein, which may cast doubt on whether key aspects of PD pathogenesis can be reproduced in a zebrafish model. ␣-Synuclein pathology is not prominent in some types of PD (62,63), and it is currently unclear whether ␣-synuclein is necessary for PD pathogenesis in all cases. However, we consider the construction of transgenic zebrafish lines expressing physiological levels of human or fish ␣-synucleins an important priority. Because the zebrafish natively presents an snca-null background, comparison of ␣-synuclein-expressing transgenic lines with controls should clarify the role of ␣-synuclein in neuronal loss caused by PD-relevant environmental and genetic triggers.