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DEVELOPMENTAL DYNAMICS 237:2490 –2495, 2008

PATTERNS & PHENOTYPES

Discovery and Characterization of Three Novel Synuclein Genes in Zebraﬁsh Zhihui Sun and Aaron D. Gitler*

The neuronal protein ␣-synuclein has been linked to the pathogenesis of synucleinopathies, a collection of neurodegenerative disorders, including Parkinson’s disease. ␣-Synuclein belongs to a family of synuclein genes that includes ␤- and ␥-synuclein. However, despite being associated with several fatal human neurodegenerative diseases, little is known about the normal function of synucleins. Here we report the cloning and characterization of three synucleins from zebraﬁsh, sncga, sncgb, and sncb. The sequences of these zebraﬁsh synucleins are very similar to those of the human proteins. We used whole-mount in situ hybridization to analyze their spatial and temporal expression patterns during development. sncgb was expressed exclusively in the notochord, while sncga and sncb were expressed strongly in the nervous system. Our identiﬁcation of synuclein genes in zebraﬁsh and the characterization of their expression patterns will facilitate future experiments aimed at assessing their functions in normal physiology as well as their role in pathophysiology. Developmental Dynamics 237:2490 –2495, 2008. © 2008 Wiley-Liss, Inc. Key words: zebraﬁsh; synuclein; expression pattern; Parkinson’s disease Accepted 8 April 2008

INTRODUCTION Synucleins are a family of small neuronal proteins, including ␣-, ␤-, and ␥synuclein (Lee and Trojanowski, 2006). The function of synucleins remains a mystery, although there are suggestions that ␣-synuclein might play a role in maintenance of synaptic vesicle pools (Murphy et al., 2000), vesicle priming at the synapse (Larsen et al., 2006), activity-dependent dopamine release (Abeliovich et al., 2000), or as a kind of chaperone for SNARE complex assembly (Chandra et al., 2005). Single and double knockouts of ␣-, ␤-, and ␥-synuclein are viable and exhibit very little (if any) phenotype under basal conditions. However, in certain contexts it has been shown recently that synuclein

proteins might have an essential function (Bonini and Giasson, 2005; Chandra et al., 2005). Increased attention was focused on ␣-synuclein (␣-syn) when it was discovered that a family with an early onset form of Parkinson’s disease (PD) harbored a point mutation in the gene encoding ␣-syn (Polymeropoulos et al., 1997). Further studies identiﬁed additional families with other point mutations in ␣-syn that also led to early onset PD (Kruger et al., 1998; Zarranz et al., 2004). And recently, duplication or triplication of the wild-type ␣-syn locus was shown to decrease the age of PD onset (Singleton et al., 2003; Chartier-Harlin et al., 2004; Ibanez et al., 2004), suggesting that abnormal accumulation of even wild-type ␣-syn

was deleterious. But it was still unclear if ␣-syn was only important for the familial forms of the disease or if it actually played a more general role in the far more common sporadic cases. Thus, the subsequent ﬁnding that, even in the sporadic forms, ␣-syn is the most abundant protein aggregated in Lewy Bodies, the pathological hallmark of the disease (Spillantini et al., 1997) was truly remarkable, and strongly suggested that ␣-syn was a key player in the pathogenesis of PD. Since then, intense effort has been aimed at understanding the normal function of the synuclein gene family. The zebraﬁsh, Danio rerio, is a powerful vertebrate genetic model organism for the study of many complicated developmental processes and human

Additional Supporting Information may be found in the online version of this article. Department of Cell and Developmental Biology, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Grant sponsor: the University of Pennsylvania Alzheimer’s Disease Core Center. *Correspondence to: Aaron D. Gitler, 1109 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104. E-mail: [email protected] DOI 10.1002/dvdy.21569 Published online 3 June 2008 in Wiley InterScience (www.interscience.wiley.com).

© 2008 Wiley-Liss, Inc.

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Fig. 1. Identiﬁcation of three zebraﬁsh synuclein genes. A: Multiple sequence alignment compares human ␣-, ␤-, and ␥-synuclein to the predicted zebraﬁsh proteins (zSynA, zSynB, zSynC) identiﬁed from searching available zebraﬁsh expressed sequence tag (EST) and genome sequence databases. Predicted protein sequences for stickleback and pufferﬁsh ␣-synuclein are also included. The gray bar indicates a conserved stretch of 12 hydrophobic residues (VTGVTAVAQKTV) that distinguishes ␣-synuclein from ␤- and ␥-synuclein. This motif is conserved in stickleback and pufferﬁsh ␣-synuclein but not in zSynA, zSynB, or zSynC. B: Phylogenetic tree representation of the comparison between the zebraﬁsh and human synucleins. C: Syntenic relationships between zSynA, zSynB, and human SNCG (encodes ␥-synuclein) indicate that zSynA and zSynB are likely duplicates of ␥-synuclein, because both genes are located between genes encoding bone morphogenetic protein receptor 1A and glutamate dehydrogenase.

diseases (Grunwald and Eisen, 2002). Zebraﬁsh are well suited for the study of the nervous system. The embryos (0 –5 days old) acquire simple sensory and locomotor activities and the larvae (5 days to 2 weeks old) exhibit many patterns of behavior relating to hunting for food and escaping from predators (Guo, 2004). Both embryo and larvae are optically transparent, enabling the visualization of complex neuronal circuitry with newly developed ﬂuorescent probes and imaging methods (Hatta et al., 2006). In the present study, we have identiﬁed three synuclein genes from zebraﬁsh and characterized their expression patterns during development. Of interest, while these three synuclein genes share a high degree of sequence similarity,

their expression patterns are quite distinct. The identiﬁcation and spatiotemporal expression analysis of zebraﬁsh synuclein genes will facilitate future functional studies.

RESULTS AND DISCUSSION Cloning of Zebraﬁsh Synuclein Genes To explore the possibility of using zebraﬁsh as a model to deﬁne the normal functions of synucleins, we ﬁrst asked if zebraﬁsh contained synuclein-related genes. We used a bioinformatics-based screen for sequences in the zebraﬁsh expressed sequence tag (EST) database that resembled human ␣-, ␤-, or ␥-synuclein. This search produced three unique uncharacterized zebra-

ﬁsh cDNAs (zgc:112131, zgc:110133, zgc:66343), which we referred to initially as zSynA, zSynB, zSynC. The predicted protein products of these cDNAs shared a high degree of sequence similarity to human synucleins (Fig. 1A and Supplementary Figure S1, which can be viewed at http:// www.interscience.wiley.com/jpages/ 1058-8388/suppmat). Thus, zebraﬁsh contain a family of synuclein genes. We constructed a phylogenetic tree to determine relationships between the zebraﬁsh and human synucleins (Fig. 1B). One of the zebraﬁsh synucleins, zSynC, was most closely related to human ␤-synuclein (70% identical, 82% similar), whereas it was not clear which human proteins zSynA and zSynB corresponded to. Many zebraﬁsh-human gene relation-

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ships can be inferred by synteny. Using human/zebraﬁsh synteny maps, we determined that zSynA and zSynB likely represent duplicate copies of ␥-synuclein (Fig. 1C). The human gene encoding ␥-synuclein (SNCG) is ﬂanked by genes encoding a type 1 bone morphogenetic protein receptor and glutamate dehydrogenase. Similar genes in zebraﬁsh ﬂank both zSynA and zSynB. Gene duplication is common in zebraﬁsh because of a chromosome doubling event, probably by whole genome duplication, after the divergence of ray-ﬁnned and lobeﬁnned ﬁshes (Amores et al., 1998). Thus, based on standard naming conventions, we now refer to zSynA, zSynB, and zSynC as sncga, sncgb, and sncb, respectively. Because our initial bioinformatics screen only identiﬁed three zebraﬁsh synuclein genes, and did not include a clear ␣-synuclein orthologue, we expanded our search to include cDNAs from all teleost ﬁshes (e.g., pufferﬁsh, salmon, stickleback, trout). This revealed the presence of a fourth synuclein-like cDNA sequence in many of these ﬁshes, also in agreement with a recent biochemical study on synucleins from pufferﬁsh (Yoshida et al., 2006). A feature that distinguishes human ␣-synuclein from ␤- or ␥-synucleins is the presence of a conserved stretch of 12 hydrophobic residues (VTGVTAVAQKTV; Giasson et al., 2001). Both pufferﬁsh and stickleback ␣-synuclein proteins contain this stretch (100% identical in pufferﬁsh and 92% identical in stickleback, Fig. 1A). This motif is not present in zebraﬁsh synucleins A, B, or C, further evidence that these zebraﬁsh synucleins do not correspond to ␣-synuclein. We were unable to identify a zebraﬁsh ␣-synuclein in available EST or genomic sequence databases. Furthermore, using degenerate primers and low stringency polymerase chain reaction with reverse transcription (RTPCR), we were unable to isolate an ␣-synuclein from zebraﬁsh cDNA libraries (Z.S. and A.D.G. unpublished observations). When sequencing of the zebraﬁsh genome is completed, we will be able to determine whether there is indeed a fourth synuclein gene present.

Spatial and Temporal Expression Patterns of Zebraﬁsh Synuclein Genes sncga, sncgb, and sncb To deﬁne when and where each of the zebraﬁsh synuclein genes was expressed during development, we performed whole-mount in situ hybridization on staged embryos. Because of the high degree of sequence similarity between the three genes, we used 3⬘ UTR sequences as templates for antisense riboprobe synthesis. We did not detect sncga (zebraﬁsh ␥-synuclein A) expression at early developmental stages but its expression initiated in the nervous system beginning at 26 hours postfertilization (hpf). At this stage, sncga was expressed in spinal cord neurons and the pineal gland (epiphysis; Fig. 2A). At 38 hpf, sncga expression was detected in hindbrain neurons (Fig. 2B,C). Two days postfertilization (dpf), sncga expression in the brain and cranial ganglia was much more prominent (Fig. 2D,E) and by 3 dpf, its expression became restricted to the brain and retina (Fig. 2F,G). Expression of the other zebraﬁsh ␥-synuclein gene, sncgb (zebraﬁsh ␥-synuclein B) initiated earlier than sncga, beginning at the 12-somite stage. However, its expression remained restricted to the notochord throughout embryogenesis and by 2 dpf began to diminish (Fig. 3). We were unable to detect sncgb transcripts by in situ hybridization later than 2 dpf (Fig. 3H, and data not shown). sncb (zebraﬁsh ␤-synuclein) expression initiated the earliest of the three synuclein genes. Beginning at the 8-somite stage and continuing through the 16-somite stage, sncb expression was restricted to the trigeminal placode (Fig. 4A–E). By the 20-somite stage, sncb expression expanded to the ventral diencephalon, olfactory placode, ventral tegmentum, and spinal cord neurons (Fig. 4F–I). At later stages, beginning at approximately 45 hpf, sncb expression became restricted to the brain and retina (Fig. J,K), where its expression continued past 3 dpf (Fig. 4L–N).

Summary We have isolated three zebraﬁsh synuclein genes and characterized their expression patterns during development. Despite having remarkable sequence similarity, each of the synuclein genes displayed strikingly different spatial and temporal patterns of expression. ␣-Synuclein has been causally linked to the pathogenesis of Parkinson’s disease (Lee and Trojanowski, 2006), but, despite over a decade of investigation, the normal functions of that protein or those of the related ␤- and ␥-synucleins still remain a mystery. Emerging evidence indicates that the normal cellular function of ␣-synuclein may in fact be related to its later role in disease pathogenesis (Gitler and Shorter, 2007), reinforcing the notion that understanding the role of synuclein genes in normal biology may provide insight into their later role in disease. Our identiﬁcation and expression studies of synuclein genes in zebraﬁsh will help guide future loss-of-function experiments. We anticipate that the zebraﬁsh model system, with its simpler vertebrate nervous system and genetic tractability, will be an excellent platform for exploring synuclein gene function and may provide novel insights for follow-up experiments in mouse.

EXPERIMENTAL PROCEDURES BLAST Searches and Multiple Sequence Alignments We used protein sequences of human ␣-, ␤-, and ␥-synuclein to perform translated BLAST (tblastn) searches against the zebraﬁsh EST database at NCBI. We used the Clustal-W algorithm in MacVector v9.5.2 to align human and zebraﬁsh synucleins.

Zebraﬁsh Embryos All embryos used were derived from the wild-type Tuebingen or (AB) strain. Embryos were incubated in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 0.1% Methylene Blue) at 28.5°C, and staged according to (Kimmel et al.,

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Fig. 2. Expression of sncga. sncga is not expressed at early developmental stages but is expressed in the nervous system beginning at 26 hours postfertilization (hpf). A: At 26 hpf, lateral view, arrowhead indicates epiphysis; arrows indicate spinal cord neurons. B: At 38 hpf, lateral view, arrowhead point to epiphysis; *indicates hindbrain neurons. C: At 38 hpf, dorsal view, *indicates hindbrain neurons. D: At 2 days postfertilization (dpf), lateral view, arrow indicates cranial ganglia. E: At 2 dpf, dorsal view, arrows indicate cranial ganglia. F: At 3 dpf, dorsal view. G: At 3 dpf, lateral view. Fig. 3. Expression of sncgb. A: sncgb expression initiates earlier than sncga (beginning at the 13-somite stage), but its expression is restricted to the notochord throughout embryogenesis. B: At the 14-somite stage. C: At the 16-somite stage. D: At the 18-somite stage. E: At the 20-somite stage. F: At the 25-somite stage. G: At 1 day postfertilization (dpf), crosssection shown as inset. nt, neural tube; nc, notochord. H: By 48 dpf, sncgb expression begins to diminish. hpf, hours postfertilization; dpf, days postfertilization.

Fig. 2.

Fig. 3.

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Fig. 4. Expression of sncb. sncb expression comes on the earliest of the three synuclein genes, beginning at the eight-somite stage. A: At the 10-somite stage. B: At the 13-somite stage, arrows indicate trigeminal placode. C: At the 14-somite stage, lateral view. D: At the 14-somite stage, dorsal view, arrows indicate trigeminal placode. E: At the 16-somite stage. F: At the 20-somite stage. G: At the 22-somite stage. H: At the 25-somite stage. vd, ventral diencephalon; vt, ventral tegmentum; op, olfactory placode. I: A the 25-somite stage. cg, cranial ganglia. J: At 45 hours postfertilization (hpf), lateral view. K: At 45 hpf, dorsal view. L: At 2 days postfertilization (dpf). M: At 3 dpf, lateral view. N: At 3 dpf, dorsal view.

1995). To suppress pigmentation, embryos at later stages (from 26 to 72 hpf) were incubated with 0.003% phenylthiourea (PTU). Embryos at appropriate stages were ﬁxed with 4% paraformaldehyde in phosphate buffered saline and processed for in situ hybridization.

In Situ Probes To generate in situ probes, we used PCR to amplify 3⬘ untranslated region sequences from each zebraﬁsh synuclein gene. PCR products were cloned into the PCR II-TOPO vector

(Invitrogen). For sncga and sncb, we generated digoxigenin-UTP-labeled antisense RNA probes by linearization with BamHI digestion, followed by transcription with T7 polymerase. For sncgb, we generated the digoxigenin-UTP-labeled antisense RNA probe by linearization with XhoI digestion, followed by transcription with Sp6 polymerase. Complete sequences for each synuclein cDNA, including sequences for in situ probe templates are shown in Supplementary Figure S1. Primer sequences are available upon request.

Whole-Mount In Situ Hybridization Whole-mount in situ hybridizations were performed according to standard protocols (Westerﬁeld, 1995).

ACKNOWLEDGMENTS We thank the Penn CDB Zebraﬁsh Core Facility, especially Jie He, for providing embryos. We also thank Lisa Chang, Michael Pack, Mary Mullins, Michael Granato, and Lili Jing for helpful suggestions. A.D.G. was funded by a Pilot grant from the Uni-

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versity of Pennsylvania Alzheimer’s Disease Core Center.

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