Synucleins are a family of small neuronal proteins, including α-, β-, and γ- synuclein (Lee and Trojanowski,2006). The function of synucleins remains a mystery, although there are suggestions that α-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 α-, β-, and γ-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 α-synuclein (α-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 α-syn (Polymeropoulos et al.,1997). Further studies identified additional families with other point mutations in α-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 α-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 α-syn was deleterious. But it was still unclear if α-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, α-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 α-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 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 α-, β-, or γ-synuclein. This search produced three unique uncharacterized zebrafish 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 β-synuclein (70% identical, 82% similar), whereas it was not clear which human proteins zSynA and zSynB corresponded to. Many zebrafish-human gene relationships can be inferred by synteny. Using human/zebrafish synteny maps, we determined that zSynA and zSynB likely represent duplicate copies of γ-synuclein (Fig. 1C). The human gene encoding γ-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 lobe-finned 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 α-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 α-synuclein from β- or γ-synucleins is the presence of a conserved stretch of 12 hydrophobic residues (VTGVTAVAQKTV; Giasson et al.,2001). Both pufferfish and stickleback α-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 α-synuclein. We were unable to identify a zebrafish α-synuclein in available EST or genomic sequence databases. Furthermore, using degenerate primers and low stringency polymerase chain reaction with reverse transcription (RT-PCR), we were unable to isolate an α-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 anti-sense riboprobe synthesis. We did not detect sncga (zebrafish γ-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 γ-synuclein gene, sncgb (zebrafish γ-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 β-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).
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. α-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 β- and γ-synucleins still remain a mystery. Emerging evidence indicates that the normal cellular function of α-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.
BLAST Searches and Multiple Sequence Alignments
We used protein sequences of human α-, β-, and γ-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.
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.,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).
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 University of Pennsylvania Alzheimer's Disease Core Center.