Characterization of three synuclein genes in Xenopus laevis

The synuclein family consists of three small intracellular proteins mainly expressed in neural tissues, and has been associated with human neurodegenerative diseases. We have examined the spatial and temporal expression patterns of three synuclein genes during embryogenesis of Xenopus laevis. The Xenopus synucleins were firstly expressed in the developing nervous system at the tail bud stages. At tadpole stages, Xenopus snca was expressed in the brain, branchial arch and somite, and sncbb signals were detected in entire brain and spinal cord. However, sncg was only expressed in the peripheral nervous system including trigeminal nerve and dorsal root ganglion. RT‐PCR indicated that expression of synucleins was up‐regulated at the end of neurulation, and then maintained at later examined stages. Our study provides the spatiotemporal expression patterns of the synuclein family genes in Xenopus embryos, and forms a basis for further functional analysis of synucleins. Developmental Dynamics 240:2028–2033, 2011. © 2011 Wiley‐Liss, Inc.     

Organization, expression and polymorphism of the human persyn gene

Persyn is a recently identified member of the synuclein family with a distinct pattern of expression during pre- and postnatal development of the mouse peripheral and central nervous systems. As with other synucleins, persyn is believed to be involved in the pathogenesis of human neurodegenerative diseases. However, in contrast to other synucleins, high levels of persyn mRNA expression were also found in advanced breast carcinomas, suggesting an involvement of the encoded protein in breast tumour progression. Here we have used an antibody specific to human persyn to demonstrate that the level of this protein is increased in ageing cerebral cortex and in breast tumours. We cloned, characterized and sequenced the human persyn genomic locus and localized it to the long arm of chromosome 10 in the q23.2-q23.3 region. Sequence information was used to search for specific mutations in the protein coding regions of persyn mRNA and the persyn gene in breast tumours and tumour cell lines. No tumour-specific mutations were found, but two linked polymorphisms in the coding region were detected, both in mRNA and exons III and IV of the gene. These results suggest that development of breast tumours correlates with overexpression of the wild-type persyn protein. Detailed characterization of the human persyn locus is important for further studies of the involvement of persyn in neurodegeneration and malignancy.      

Identification and expression of voltage‐gated calcium channel β subunits in Zebrafish

Voltage‐gated calcium channels (VGCC) play important roles in electrically excitable cells and embryonic development. The VGCC β subunits are essential for membrane localization of the channel and exert modulatory effects on channel functions. In mammals, the VGCC β subunit gene family contains four members. In zebrafish, there appear to be seven VGCC β subunits including the previously identified β1 subunit. cDNAs for six additional VGCC β subunit homologs were identified in zebrafish, their chromosomal locations determined and their expression patterns characterized during embryonic development. These six genes are primarily expressed in the nervous system with cacnb4a also expressed in the developing heart. Sequence homology, genomic synteny and expression patterns suggest that there are three pairs of duplicate genes for β2, β3, and β4 in zebrafish with distinct expression patterns during embryonic development. Developmental Dynamics 237:3842–3852, 2008. © 2008 Wiley‐Liss, Inc.     

Combinatorial expression patterns of heparan sulfate sulfotransferases in zebrafish: I. The 3-O-sulfotransferase family.

Heparan sulfate (HS) is an unbranched chain of repetitive disaccharides, which specifically binds ligands when attached to the cell surface or secreted extracellularly. HS chains contain sulfated domains termed the HS fine structure, which gives HS specific binding affinities for extracellular ligands. HS 3-O-sulfotransferases (3-OST) catalyze the transfer of sulfate groups to the 3-O position of glucosamine residues of HS, a rare, but essential HS chain modification required for HS fine structure. We report here the first characterization and developmental expression analysis of the 3-OST gene family in a vertebrate. There are eight 3-OST genes in zebrafish: seven genes with homology to known 3-OST genes in mouse and human, as well as a novel, 3-OST-7. A phylogenetic comparison of human, mouse, and zebrafish indicates the 3-OST family can be subdivided into two distinct subgroups. We examined the mRNA expression patterns in several tissues/organs throughout early zebrafish development, including early cleavage stages, somites, brain, internal body organ primordial, and pectoral fin development. The 3-OST gene family has both specifically expressed and ubiquitously expressed genes, suggesting in vivo functional differences exist between members of this family.     

Expression of sox11 gene duplicates in zebrafish suggests the reciprocal loss of ancestral gene expression patterns in development.

To investigate the role of sox genes in vertebrate development, we have isolated sox11 from zebrafish (Danio rerio). Two distinct classes of sox11-related cDNAs were identified, sox11a and sox11b. The predicted protein sequences shared 75% identity. In a gene phylogeny, both sox11a and sox11b cluster with human, mouse, chick, and Xenopus Sox11, indicating that zebrafish, like Xenopus, has two orthologues of tetrapod Sox11. The work reported here investigates the evolutionary origin of these two gene duplicates and the consequences of their duplication for development. The sox11a and sox11b genes map to linkage groups 17 and 20, respectively, together with other loci whose orthologues are syntenic with human SOX11, suggesting that during the fish lineage, a large chromosome region sharing conserved syntenies with mammals has become duplicated. Studies in mouse and chick have shown that Sox11 is expressed in the central nervous system during development. Expression patterns of zebrafish sox11a and sox11b confirm that they are expressed in the developing nervous system, including the forebrain, midbrain, hindbrain, eyes, and ears from an early stage. Other sites of expression include the fin buds and somites. The two sox genes, sox11a and sox11b, are expressed in both overlapping and distinct sites. Their expression patterns suggest that sox11a and sox11b may share the developmental domains of the single Sox11 gene present in mouse and chick. For example, zebrafish sox11a is expressed in the anterior somites, and zebrafish sox11b is expressed in the posterior somites, but the single Sox11 gene of mouse is expressed in all the somites. Thus, the zebrafish duplicate genes appear to have reciprocally lost expression domains present in the sox11 gene of the last common ancestor of tetrapods and zebrafish. This splitting of the roles of Sox11 between two paralogues suggests that regulatory elements governing the expression of the sox11 gene in the common ancestor of zebrafish and tetrapods may have been reciprocally mutated in the zebrafish gene duplicates. This is consistent with duplicate gene evolution via a duplication-degeneration-complementation process.     

Protein Partners of α‐Synuclein in Health and Disease

α‐synuclein is normally situated in the nerve terminal but it accumulates and aggregates in axons and cell bodies in synucleinopathies such as Parkinson's disease. The conformational changes occurring during α‐synucleins aggregation process affects its interactions with other proteins and its subcellular localization. This review focuses on interaction partners of α‐synuclein within different compartments of the cell with a focus on those preferentially binding aggregated α‐synuclein. The aggregation state of α‐synuclein also affects its catabolism and we hypothesize impaired macroautophagy is involved neuronal excretion of α‐synuclein species responsible for the prion‐like spreading of α‐synuclein pathology.     

Fine epitope mapping of monoclonal antibodies specific to human alpha-synuclein

The neuronal phosphoprotein alpha-synuclein has been increasingly implicated in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative diseases; however, the exact function of alpha-synuclein still remains illusive. Suitable antibodies (Abs) specific for the gene of interest are indispensable for studying biological and immunological properties of the target gene. Here, we report not only the generation and characterization of monoclonal Abs, Syn-1 and Syn-17, against human alpha-synuclein, but also the epitope mapping by using recombinant synuclein family proteins and various GST fusion proteins of human alpha-synuclein domains. Syn-17 recognizes human and rodent alpha-synuclein, and its epitope is localized within residues 97-99 and 101 of alpha-synuclein. In contrast, the Syn-1 epitope is localized in residues 121 and 122 of human alpha-synuclein, and Syn-1 recognizes only human but not rodent alpha-synuclein, indicating that it can be utilized as a useful reagent for studying human alpha-synuclein transgenic mouse and zebrafish lines.     

Molecular characterization of the zebrafish P2X receptor subunit gene family

P2X receptors are non-selective cation channels gated by extracellular ATP and are encoded by a family of seven subunit genes in mammals. These receptors exhibit high permeabilities to calcium and in the mammalian nervous system they have been linked to modulation of neurotransmitter release. Previously, three complementary DNAs (cDNAs) encoding members of the zebrafish gene family have been described. We report here the cloning and characterization of an additional six genes of this family. Sequence analysis of all nine genes suggests that six are orthologs of mammalian genes, two are paralogs of previously described zebrafish subunits, and one remains unclassified. All nine subunits were physically mapped onto the zebrafish genome using radiation hybrid analysis. Of the nine gene products, seven give functional homo-oligomeric receptors when recombinantly expressed in human embryonic kidney cell line 293 cells. In addition, these subunits can form hetero-oligomeric receptors with phenotypes distinct from the parent subunits. Analysis of gene expression patterns was carried out using in situ hybridization, and seven of the nine genes were found to be expressed in embryos at 24 and 48 h post-fertilization. Of the seven that were expressed, six were present in the nervous system and four of these demonstrated considerable overlap in cells present in the sensory nervous system. These results suggest that P2X receptors might play a role in the early development and/or function of the sensory nervous system in vertebrates.     

Six cadm/SynCAM genes are expressed in the nervous system of developing zebrafish.

The Cadm (cell adhesion molecule) family of cell adhesion molecules (also known as IGSF4, SynCAM, Necl and TSLC) has been implicated in a multitude of physiological and pathological processes, such as spermatogenesis, synapse formation and lung cancer. The precise mechanisms by which these adhesion molecules mediate these diverse functions remain unknown. To investigate mechanisms of action of these molecules during development, we have identified zebrafish orthologs of Cadm family members and have examined their expression patterns during development and in the adult. Zebrafish possess six cadm genes. Sequence comparisons and phylogenetic analysis suggest that four of the zebrafish cadm genes represent duplicates of two tetrapod Cadm genes, whereas the other two cadm genes are single orthologs of tetrapod Cadm genes. All six zebrafish cadms are expressed throughout the nervous system both during development and in the adult. The spatial and temporal patterns of expression suggest multiple roles for Cadms during nervous system development.     

Comparison of Iroquois gene expression in limbs/fins of vertebrate embryos

In Drosophila, Iroquois (Irx) genes have various functions including the specification of the identity of wing veins. Vertebrate Iroquois (Irx) genes have been reported to be expressed in the developing digits of mouse limbs. Here we carry out a phylogenetic analysis of vertebrate Irx genes and compare expression in developing limbs of mouse, chick and human embryos and in zebrafish pectoral fin buds. We confirm that the six Irx gene families in vertebrates are well defined and that Clusters A and B are duplicates; in contrast, Irx1 and 3, Irx2 and 5, and Irx4 and 6 are paralogs. All Irx genes in mouse and chick are expressed in developing limbs. Detailed comparison of the expression patterns in mouse and chick shows that expression patterns of genes in the same cluster are generally similar but paralogous genes have different expression patterns. Mouse and chick Irx1 are expressed in digit condensations, whereas mouse and chick Irx6 are expressed interdigitally. The timing of Irx1 expression in individual digits in mouse and chick is different. Irx1 is also expressed in digit condensations in developing human limbs, thus showing conservation of expression of this gene in higher vertebrates. In zebrafish, Irx genes of all but six of the families are expressed in early stage pectoral fin buds but not at later stages, suggesting that these genes are not involved in patterning distal structures in zebrafish fins.     

The Zebrafish Annexin Gene Family

The Annexins (ANXs) are a family of calcium- and phospholipid-binding proteins that have been implicated in many cellular processes, including channel formation, membrane fusion, vesicle transport, and regulation of phospholipase A₂ activity. As a first step toward understanding in vivo function, we have cloned 11 zebrafish anx genes. Four genes (anx1a, anx2a, anx5,and anx11a) were identified by screening a zebrafish cDNA library with a Xenopus anx2 fragment. For these genes, full-length cDNA sequences were used to cluster 212 EST sequences generated by the Zebrafish Genome Resources Project. The EST analysis revealed seven additional anx genes that were subsequently cloned. The genetic map positions of all 11 genes were determined by using a zebrafish radiation hybrid panel. Sequence and syntenic relationships between zebrafish and human genes indicate that the 11 genes represent orthologs of human anx1,2,4,5,6,11,13,and suggest that several zebrafish anx genes resulted from duplications that arose after divergence of the zebrafish and mammalian genomes. Zebrafish anx genes are expressed in a wide range of tissues during embryonic and larval stages. Analysis of the expression patterns of duplicated genes revealed both redundancy and divergence, with the most similar genes having almost identical tissue-specific patterns of expression and with less similar duplicates showing no overlap. The differences in gene expression of recently duplicated anx genes could explain why highly related paralogs were maintained in the genome and did not rapidly become pseudogenes.     

猫α-synuclein蛋白的表达纯化及生物学特征分析

目的对猫突触核蛋白(α-synuclein)进行克隆、表达及纯化,并探讨其生物信息学特征。方法在Genebank中α-synuclein基因的保守区域内设计引物,从猫脑c DNA文库中PCR扩增得到猫的α-synuclein基因,再将此基因双酶切后克隆到p ET28a原核表达载体中,构建重组质粒,测序正确的重组质粒转化BL21(DE3)大肠杆菌,采用IPTG诱导表达。然后对猫α-synuclein氨基酸的同源性和疏水性进行分析。结果实验成功从猫脑c DNA文库中扩增出α-synuclein基因,基因全长381个碱基,编码126个氨基酸。获得的全长基因成功克隆进入p ET28a,最后转染大肠杆菌BL21(DE3),获得可溶性表达的α-synuclein蛋白质,蛋白质分子量为13.12k D,与预期分子量一致。生物信息学分析显示猫α-synuclein蛋白与人源及鼠源α-synuclein氨基酸具有很高的同源性,分别为87.35%和83.15%,但是与鼠和人的氨基酸序列比较,猫α-synuclein氨基酸缺失41~54位氨基酸。蛋白质结构预测显示猫α-synuclein具有很好的疏水性,有助于诱导表达时形成可溶性蛋白,这一结果在本研究中得到证实。结论本研究首次克隆了猫α-synuclein基因,并在大肠杆菌中实施了可溶性表达,为后期研究α-synuclein的进化、蛋白晶体结构、生物学功能和帕金森动物模型的构建奠定了一定的基础。     

Specific activation of mammalian Hox promoters in mosaic transgenic zebrafish.

Homeo box-containing genes (Hox) are expressed in restricted regions of vertebrate embryos and may specify positional information. The organization and expression patterns of these genes are highly conserved among different species, suggesting that their regulation may also have been conserved. We developed a transient expression system, using mosaically transgenic zebrafish, which allows rapid analysis of transgene expression, and examined the activities of two mammalian Hox genes, mouse Hox-1.1 and human HOX-3.3. We found that these Hox promoters are activated in specific regions and tissues of developing zebrafish embryos and that this specificity depends upon the same regulatory elements within the promoters that specify the spatial expression of these genes in mice. Our results suggest that the promoter activities have been remarkably conserved from fish to mammals. To study the regulation of Hox expression in the developing nervous system, we analyzed the promoter activities in spt-1 mutants that have a mesodermal deficiency. Our results suggest that interactions, probably with the paraxial mesoderm, differentially regulate the activities of Hox promoters in the developing nervous system.     

Expression of the eight AMPA receptor subunit genes in the developing central nervous system and sensory organs of zebrafish.

The AMPA type glutamate receptors mediate the majority of fast synaptic transmission in the vertebrate nervous system. Whereas mammals have four subunit genes, Gria1-4, zebrafish has retained a duplicated set of eight genes named gria1-4a and b. We give here a detailed overview of the expression patterns of all eight zebrafish subunits within the developing central nervous system and sensory organs at 24, 48, and 72 hr after fertilization. Expression domains include distinct neuronal subsets in the developing forebrain, midbrain, hindbrain, and spinal cord, as well as in the ganglion- and inner nuclear layers of the retina. As a general rule, each pair of duplicated gria genes is differentially expressed, indicating subfunctionalization of AMPA receptor subunit expression in the teleost lineage. Our findings suggest that zebrafish can serve as a useful model system to investigate the role of AMPA receptors and their differential expression in the vertebrate nervous system.     

Two Frodo/Dapper homologs are expressed in the developing brain and mesoderm of zebrafish.

Members of the Wnt family of extracellular proteins play essential roles during many phases of vertebrate embryonic development. The molecular mechanism of their action involves a complex cascade of intracellular signaling events, which remains to be understood completely. Recently, two novel cytoplasmic modulators of Wnt signaling, Frodo and Dapper, were identified in Xenopus. We report isolation of their homologs in zebrafish, and show that these genes, frd1 and frd2, are expressed in restricted domains during embryogenesis. Both genes are expressed during early gastrulation in the future mesendoderm, and continue to be expressed in distinct patterns in the forming neurectoderm and mesoderm. Comparative sequence analysis and similar expression patterns argue that frd1 is the zebrafish ortholog of Frodo and Dapper, whereas frd2 is a more divergent member of the same family. Our data suggest important roles for zebrafish frd1 and frd2 in patterning the neural plate and several mesodermal derivatives.     

Impairment of the tRNA-splicing endonuclease subunit 54 (tsen54) gene causes neurological abnormalities and larval death in zebrafish models of pontocerebellar hypoplasia

Pontocerebellar hypoplasia (PCH) represents a group (PCH1-6) of neurodegenerative autosomal recessive disorders characterized by hypoplasia and/or atrophy of the cerebellum, hypoplasia of the ventral pons, progressive microcephaly and variable neocortical atrophy. The majority of PCH2 and PCH4 cases are caused by mutations in the TSEN54 gene; one of the four subunits comprising the tRNA-splicing endonuclease (TSEN) complex. We hypothesized that TSEN54 mutations act through a loss of function mechanism. At 8 weeks of gestation, human TSEN54 is expressed ubiquitously in the brain, yet strong expression is seen within the telencephalon and metencephalon. Comparable expression patterns for tsen54 are observed in zebrafish embryos. Morpholino (MO) knockdown of tsen54 in zebrafish embryos results in loss of structural definition in the brain. This phenotype was partially rescued by co-injecting the MO with human TSEN54 mRNA. A developmental patterning defect was not associated with tsen54 knockdown; however, an increase in cell death within the brain was observed, thus bearing resemblance to PCH pathophysiology. Additionally, N-methyl-N-nitrosourea mutant zebrafish homozygous for a tsen54 premature stop-codon mutation die within 9 days post-fertilization. To determine whether a common disease pathway exists between TSEN54 and other PCH-related genes, we also monitored the effects of mitochondrial arginyl-tRNA synthetase (rars2; PCH1?and PCH6) knockdown in zebrafish. Comparable brain phenotypes were observed following the inhibition of both genes. These data strongly support the hypothesis that TSEN54 mutations cause PCH through a loss of function mechanism. Also we suggest that a common disease pathway may exist between TSEN54- and RARS2-related PCH, which may involve a tRNA processing-related mechanism.     

α-synuclein-pEGFP真核表达载体的构建及其在SH-SY5Y细胞中的表达

为了构建 α-synuclein-p EGFP真核表达载体 ,检测其在 SH-SY5 Y细胞内的表达 ,本研究应用下述方法 :PCR扩增 α-synuclein基因并消除终止密码子 ;PCR产物连入 p GEM T-easy载体 ,经测序确认无误后 ,亚克隆入 p EGFP-N1,构建α-synucle-in-p EGF P真核表达载体 ;Lipofect AMINE法转染 SH-SY5 Y细胞 ;荧光显微镜检测报告基因表达产物 EGFP,原位杂交和免疫荧光细胞化学法检测α-synuclein m RNA及其蛋白表达。结果显示 ,该载体转染 SH-SY5 Y细胞后 ,可在细胞内观察到报告基因和目的基因的表达产物。结论 :α-synuclein-p EGFP真核表达载体构建成功 ,并可在细胞内表达。本工作为今后动态观察研究α-synu-clein致 Parkinson病多巴胺能神经细胞损伤机制奠定基础