Synucleins are small, vertebrate-specific proteins predominantly expressed in neurons. There are three closely related members in this gene family, including α-, β-, and γ-synuclein. The synucleins have received much attention because point mutations and multiplication of α-synuclein were associated with pathogenesis of Parkinson's disease (PD). A53T mutation of α-synuclein (SNCA) was firstly identified to cause familial PD (Maroteaux et al.,1988; Polymeropoulos et al.,1997; Abeliovich et al.,2000). Additional mutations (A30P and E46K) in α-synuclein as well as duplication and trisomy of α-synuclein genomic region were also identified to be associated with PD (Singleton et al.,2003; Chartier-Harlin et al.,2004; Ietsugu et al.,2004; Zarranz et al.,2004). Many studies suggest that α-synuclein inclusions cause dysfunction and degeneration of neurons, and the mutations and increased copies of α-synuclein may enhance the formation of fibrils and inclusions (Dawson and Dawson,2003; Cookson,2005). Therefore, α-synuclein has a dosage effect on the severity of PD phenotypes and the distribution of neural degeneration, probably because of excessive accumulation of this protein in neurons (Chartier-Harlin et al.,2004).
α-synuclein is the major component of Lewy bodies and cellular inclusions, which are pathological hallmarks of sporadic PD and dementia with Lewy bodies (DLB) (Golbe and Mouradian,2004). The β- and γ-synuclein proteins are not found in Lewy bodies, but they have been implicated in the pathology of hippocampal axon in PD and DLB (Galvin et al.,1999). In line with the above findings, transgenic mice expressing DLB-linked mutant β-synuclein developed progressive neurodegeneration. However, overexpression of wild type β-synuclein does not cause pathological changes in the nervous system; it in fact attenuates the symptom caused by overexpression of α-synuclein (Hashimoto et al.,2001; Fujita et al.,2010). Thus, β-synuclein seems to perform a protective role as it can inhibit α-synuclein aggregation, probably through the direct interaction between β-synuclein and Akt kinase (Hashimoto et al.,2001,2004). Similar to α-synuclein, mice expressing a high level of γ-synuclein develop severe and fatal neurological diseases because of the aggregation of γ-synuclein (Ninkina et al.,2009). Moreover, γ-synuclein is a marker for breast cancer progression and up-regulation of γ-synuclein has been observed in many types of cancer (Wu et al.,2003). γ-synuclein can also stimulate membrane-initiated estrogen signaling by chaperoning ER-α36 (Shi et al.,2010). Although synucleins are expressed in neurons, single and double α-, β-, and γ-synuclein knockout (KO) mice display no major phenotypes (Abeliovich et al.,2000; Ninkina et al.,2003; Chandra et al.,2004). α-, β-, and γ-synuclein triple KO mice showed defective synaptic structures and transmission, and age-dependent neuronal dysfunction (Greten-Harrison et al.,2010). These studies suggest that synucleins are important for the proper operation of the nervous system.
Although synuclein genes are highly related to neurodegenerative diseases, their cellular functions are still largely unknown. Several lines of study suggest α-synuclein is involved in the maintenance of synaptic vesicle pools and activity-dependent dopamine release (Abeliovich et al.,2000; Murphy et al.,2000; Cabin et al.,2002). α-synuclein is required for the repeated release of neurotransmitters by regulating the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes that drive vesicle fusion with the plasma membrane (Nemani et al.,2010). Triple KO mice lacking synucleins exhibited decreased SNARE-complex assembly, implying that synucleins may play roles in maintaining normal SNARE-complex assembly in a presynaptic terminal (Burre et al.,2010). However, even less is known about the functions of synucleins during early development of the neural system.
In this study, we identified three synucleins from Xenopus laevis (hereafter Xenopus) embryos, and examined their spatial and temporal expression patterns during early embryonic development. Our data set a stage for further studying the cellular functions of synucleins and their roles in nervous system development in Xenopus.
RESULTS AND DISCUSSION
Cloning of Xenopus laevis Synuclein Genes
In an attempt to understand the development of peripheral nervous system, we identified a gene encoding a protein of 129 amino acids by screening a cDNA library prepared with activin-treated Xenopus animal caps. It most closely corresponds to human γ-synuclein. We, therefore, named it Xenopus γ-synuclein (sncg, AY055119). Furthermore, α- and β-synuclein of Xenopus laevis were identified in GeneBank by using BLAST searching with human α- and β-synuclein (NP_000336, NP_003076), respectively. Protein sequence alignment indicated that Xenopus snca (α-synuclein, NP_001080623.1) shares 84% amino acid identity with its human homolog, and sncg is 74% identical to the human γ-synuclein. There were two homologs of human β-synuclein (SNCB) identified in Xenopus laevis, with the name of sncb (β-synuclein, NP_001088570.1) and MGC80089 (NP_001085449.1) in GeneBank. Their protein sequences share 82% and 88% identities with human β-synuclein, respectively. These two Xenopus proteins share 92% identity with each other. Since Xenopus laevis is a pseudotetraploid and each gene has two unidentical copies in its genome, we therefore thought these two genes were pseudoalleles of β-synuclein in Xenopus laevis. We named NM_ 001095101.1 β-synuclein a (sncba), and the NM_001091980 was named β-synuclein b (sncbb). The whole open reading frames (ORFs) of snca, sncbb, and sncg were obtained by polymerase chain reaction (PCR), and verified by further sequencing.
Similar to human synucleins, Xenopus synuclein proteins have a more conserved N-terminal domain than the acidic C terminus (Fig. 1A). The Xenopus synucleins also have seven imperfect repeats of amino acid residues with the consensus sequence of XKTKEGVXXXX, which is responsible for binding to acidic phospholipid surfaces (Fig. 1A) (Davidson et al.,1998). When compared with synucleins from other species, snca, sncbb, and sncg in Xenopus laevis were also found to share high homology with each other (see Supp. Fig. S1, which is available online). The sequence alignment also indicated that all α-synucleins contain a threonine at position 53, except that human α-synuclein has an alanine at the same position (Supp. Fig. S1A). This is rather surprising as the A53T mutation in human SNCA has been linked to Parkinson's disease, and such discrepancy may need to be further illustrated (Polymeropoulos et al.,1997). The phylogenetic tree illustrated the evolutionary distance of the synuclein family proteins in human, mouse, chick, zebrafish, and Xenopus laevis (Fig. 1B). The result shows that Xenopus sncbb is more closely related with human SNCB when compared with sncba.
Spatial and Temporal Expression of Xenopus Synuclein Genes
The expression of snca could not be detected by whole mount in situ hybridization in embryos prior to stage 23 (Fig. 2A). In tail bud stage embryos, snca was expressed in the olfactory placode, brain, otic vesicle, branchial arches, and somites (Fig. 2B,C,G,H,I). Weak expression of snca in notochord was observed in stage 28 embryos, but became indiscernible at later stages (Fig. 2B,C,E). At the tadpole stages, stronger signals were detected in various head regions including forebrain, midbrain, and hindbrain (Fig. 2D,F,G). The signals in neural tube were intense in the marginal and subventricular zones (Fig. 2J,K). snca expression was also detected in the ganglion cell layer (GCL) of the retina at tadpole stages (Fig. 2F,J). Arrow-shape signal strips of snca were observed in somites (Fig. 2H).
No obvious expression of sncbb was detected in embryos until stage 22. Weak signals appeared in the primordium of trigeminal nerve and spinal cord (Fig. 3A–C). sncbb signals were also observed in olfactory placode from stage 23, and then detected in the pineal gland similar to Nr2e3 (Fig. 3C–G′,L) (Martinez-De Luna and El-Hodiri,2007). Strong expression of sncbb was detected throughout the central nervous system, including forebrain, midbrain, hindbrain, and spinal cord at stage 35 (Fig. 3H,I,K). The midbrain expression region lay posterior to pineal gland and the expression in neural tube was localized in the marginal and subventricular zones (Fig. 3M–P). sncbb expression was also found in the trigeminal nerve and other cranial nerves including the vestibulocochlear nerve, cranial nerve IX, and cranial nerve X (Fig. 3F,G,I,J). Furthermore, sncbb expression was also detected in the developing retina (Fig. 3I,J,M).
sncg became detectable in the primordium of the trigeminal nerve at the early tail bud stages. Its signals can be observed in dorsal root ganglia and cranial sensory ganglia, forming two strips above the dorsal neural tube in later tail bud stages and tadpole stages (Fig. 4A–G, I, J). In addition, sncg transcripts were also detected in the pineal gland which is similar to its ortholog in zebrafish (Fig. 4D–H) (Sun and Gitler,2008). Similar to sncbb, sncg was also detected in the cranial nerve IX, cranial nerve X, and vestibulocochlear nerve (Fig. 4G). Thus, sncg expression is enriched in the peripheral nervous system.
The temporal expression pattern of Xenopus synuclein genes was examined using RT-PCR. The result showed that only sncg has a weak expression before the gastrula stage, and was detected at all the other stages examined (Fig. 5). The snca has a weak expression during gastrula stages, while sncbb remains undetectable (Fig. 5). The expressions of snca, sncbb, and sncg were weak at the neurula stages, and were gradually up-regulated during tail bud stages, which is in line with the results of whole mount in situ hybridization (Fig. 5). The expression level of snca at the tail bud stages was lower than sncbb and sncg.
In summary, we have identified Xenopus synuclein genes and examined their spatial and temporal expression patterns during early embryonic development. Our data indicate that all of them are expressed most intensely in the nervous system. snca and sncbb are both expressed in the brain and retina at the tadpole stages, and sncbb also showed strong expression in the spinal cord. The sncg is mainly expressed in the peripheral nervous system, overlapping with the expression of sncbb in the cranial nerves. Such distinct expression patterns suggest functional divergence of synucleins in embryonic development. The expression patterns of Xenopus synuclein genes are similar to their homologs in zebrafish and chicken, which implies that synucleins may have a conserved function in nervous system development. Our study will facilitate functional analysis of the synuclein family during embryonic development.
BLAST Searches and Phylogenetic Analysis
Human α- and β-synuclein were used to do BLAST (BLASTP) searches in the non-redundant protein sequence database at NCBI. Protein sequence alignments were performed using BioEdit v184.108.40.206. The dendrogram tree was constructed with the neighbor-joining method in Geneious v4.8.5.
Probes Preparation for Whole Mount In Situ Hybridization
The full length of sncg was obtained by large-scale screening of a cDNA library prepared by activin-treated animal caps. The open reading frames (ORFs) of Xenopus laevis snca and sncbb and partial sequence of Nr2e3 (accession number: CD328661) were amplified using RT-PCR. cDNA was synthesized by reverse transcription using total RNA from stage-35 embryos. The PCR products were subcloned into the pBluescript II KS (+) vector and verified by sequencing. Plasmids were linearized with XbaI and then used as templates for synthesis of digoxigenin-labeled antisense probes with T3 RNA polymerase (Roche, Indianapolis, IN).
Whole Mount In Situ Hybridization and Vibratome Sectioning
Xenopus laevis embryos were obtained by in vitro fertilization, and cultured in 0.2×MMR (20 mM NaCl, 0.4 mM KCl, 0.2 mM MgSO4, 0.4 mM CaCl2, 1 mM HEPES, 0.02 mM EDTA, pH7.8) at room temperature. Embryos were fixed in MEMFA (0.1 M HEPES, 2 mM EGTA, 1 mM MgSO4, 3.7% formaldehyde) for 1 hr and stored in absolute ethanol at −20°C (Kam et al.,2010).
Whole mount in situ hybridization was performed according to the standard protocol (Harland,1991). For vibratome sections, the embryos after in situ hybridization were embedded in a medium by mixing 2 ml of a solution (5 g/L gelatin, 380 g/L chick egg albumin, and 200 g/L sucrose in 0.1 M phosphate buffer, pH 7.4) with 0.2 ml 25% (v/v) glutaraldehyde and sectioned at a thickness of 50 μm. Hematoxylin and eosin staining was performed as we published previously (Zhao et al.,2001).
RNA Extraction and RT-PCR
Total RNA extraction and synthesis of cDNA were performed as previously described (Kam et al.,2010). The primers targeting the 3′ UTR of synucleins were designed using Primer 3. ornithine decarboxylase (ODC) was used as the loading control. The primer sequences for RT-PCR are listed as below.
snca Fw: AGACAGTAGAAGGAG CCG
snca Re: AGGTTGGAGTCATTG GGT
sncbb Fw: AGCAACATCAGCACA ACG
sncbb Re: GGGAGGGAAGCAGAA ATA
sncg Fw: ATTGAGCAGGTCGGT GAT
sncg Re: TCAGTAGTAGGTGCG TCA
ODC Fw: CAGCTAGCTGTGGTG TGG
ODC Re: CAACATGGAAACTCAC ACC.
This work is supported by a GRF grant from the Research Grants Council of Hong Kong (No. CUHK480709). W.C.D. is supported by a Graduate Studentship from the Chinese University of Hong Kong.