The organization and evolution of the dipteran and hymenopteran Down syndrome cell adhesion molecule (Dscam) genes

The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor and can generate 38,016 different isoforms via the alternative splicing of 95 variable exons. Dscam contains 10 immunoglobulin (Ig), six Fibronectin type III, a transmembrane (TM), and cytoplasmic domains. The different Dscam isoforms vary in the amino acid sequence of three of the Ig domains and the TM domain. Here, we have compared the organization of the Dscam gene from three members of the Drosophila subgenus (D. melanogaster, D. pseudoobscura, and D. virilis), the mosquito Anopheles gambiae, and the honeybee Apis mellifera. Each of these organisms contains numerous alternative exons and can potentially synthesize tens of thousands of isoforms. Interestingly, most of the alternative exons in one species are more similar to one another than to the corresponding alternative exons in the other species. These observations provide strong evidence that many of the alternative exons have arisen by reiterative exon duplication and deletion events. In addition, these findings suggest that the expression of a large Dscam repertoire is more important for the development and function of the insect nervous system than the actual sequence of each isoform.     

Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies

Dscam is an immunoglobulin (Ig) superfamily member that regulates axon guidance and targeting in Drosophila. Alternative splicing potentially generates 38,016 isoforms differing in their extracellular Ig and transmembrane domains. We demonstrate that Dscam mediates the sorting of axons in the developing mushroom body (MB). This correlates with the precise spatiotemporal pattern of Dscam protein expression. We demonstrate that MB neurons express different arrays of Dscam isoforms and that single MB neurons express multiple isoforms. Two different Dscam isoforms differing in their extracellular domains introduced as transgenes into single mutant cells partially rescued the mutant phenotype. Expression of one isoform of Dscam in a cohort of MB neurons induced dominant phenotypes, while expression of a single isoform in a single cell did not. We propose that different extracellular domains of Dscam share a common function and that differences in isoforms expressed on the surface of neighboring axons influence interactions between them.     

Complementary chimeric isoforms reveal Dscam1 binding specificity in vivo

Dscam1 potentially encodes 19,008 ectodomains of a cell recognition molecule of the immunoglobulin (Ig) superfamily through alternative splicing. Each ectodomain, comprising a unique combination of three variable (Ig) domains, exhibits isoform-specific homophilic binding in?vitro. Although we have proposed that the ability of Dscam1 isoforms to distinguish between one another is crucial for neural circuit assembly, via a process called self-avoidance, whether recognition specificity is essential in?vivo has not been addressed. Here we tackle this issue by assessing the function of Dscam1 isoforms with altered binding specificities. We generated pairs of chimeric isoforms that bind to each other (heterophilic) but not to themselves (homophilic). These isoforms failed to support self-avoidance or did so poorly. By contrast, coexpression of complementary isoforms within the same neuron restored self-avoidance. These data establish that recognition between Dscam1 isoforms on neurites of the same cell provides the molecular basis for self-avoidance.     

A double S shape provides the structural basis for the extraordinary binding specificity of Dscam isoforms

Drosophila Dscam encodes a vast family of immunoglobulin (Ig)-containing proteins that exhibit isoform-specific homophilic binding. This diversity is essential for cell recognition events required for wiring the brain. Each isoform binds to itself but rarely to other isoforms. Specificity is determined by "matching" of three variable Ig domains within an approximately 220 kD ectodomain. Here, we present the structure of the homophilic binding region of Dscam, comprising the eight N-terminal Ig domains (Dscam(1-8)). Dscam(1-8) forms a symmetric homodimer of S-shaped molecules. This conformation, comprising two reverse turns, allows each pair of the three variable domains to "match" in an antiparallel fashion. Structural, genetic, and biochemical studies demonstrate that, in addition to variable domain "matching," intramolecular interactions between constant domains promote homophilic binding. These studies provide insight into how "matching" at all three pairs of variable domains in Dscam mediates isoform-specific recognition.     

Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity

A Drosophila homolog of human Down syndrome cell adhesion molecule (DSCAM), an immunoglobulin superfamily member, was isolated by its affinity to Dock, an SH3/SH2 adaptor protein required for axon guidance. Dscam binds directly to both Dock's SH2 and SH3 domains. Genetic studies revealed that Dscam, Dock and Pak, a serine/threonine kinase, act together to direct pathfinding of Bolwig's nerve, containing a subclass of sensory axons, to an intermediate target in the embryo. Dscam also is required for the formation of axon pathways in the embryonic central nervous system. cDNA and genomic analyses reveal the existence of multiple forms of Dscam with a conserved architecture containing variable Ig and transmembrane domains. Alternative splicing can potentially generate more than 38,000 Dscam isoforms. This molecular diversity may contribute to the specificity of neuronal connectivity.     

Drosophila sensory neurons require Dscam for dendritic self-avoidance and proper dendritic field organization

A neuron's dendrites typically do not cross one another. This intrinsic self-avoidance mechanism ensures unambiguous processing of sensory or synaptic inputs. Moreover, some neurons respect the territory of others of the same type, a phenomenon known as tiling. Different types of neurons, however, often have overlapping dendritic fields. We found that Down's syndrome Cell Adhesion Molecule (Dscam) is required for dendritic self-avoidance of all four classes of Drosophila dendritic arborization (da) neurons. However, neighboring mutant class IV da neurons still exhibited tiling, suggesting that self-avoidance and tiling differ in their recognition and repulsion mechanisms. Introducing 1 of the 38,016 Dscam isoforms to da neurons in Dscam mutants was sufficient to significantly restore self-avoidance. Remarkably, expression of a common Dscam isoform in da neurons of different classes prevented their dendrites from sharing the same territory, suggesting that coexistence of dendritic fields of different neuronal classes requires divergent expression of Dscam isoforms.     

Quantitative Profiling of Drosophila melanogaster Dscam1 Isoforms Reveals No Changes in Splicing after Bacterial Exposure

The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, we for the first time, use a long read RNA sequencing method to directly investigate the Dscam1 splicing pattern after exposing adult Drosophila melanogaster and a S2 cell line to live Escherichia coli. After bacterial exposure both models showed increased expression of immune-related genes, indicating that the immune system had been activated. However there were no changes in total Dscam1 mRNA expression. RNA sequencing further showed that there were no significant changes in individual exon expression and no changes in isoform splicing patterns in response to bacterial exposure. Therefore our studies do not support a change of D. melanogaster Dscam1 isoform diversity in response to live E. coli. Nevertheless, in future this approach could be used to identify potentially immune-related Dscam1 splicing regulation in other host species or in response to other pathogens.     

Alternative splicing of Drosophila Dscam generates axon guidance receptors that exhibit isoform-specific homophilic binding

Dscam is an immunoglobulin (Ig) superfamily protein required for the formation of neuronal connections in Drosophila. Through alternative splicing, Dscam potentially gives rise to 19,008 different extracellular domains linked to one of two alternative transmembrane segments, resulting in 38,016 isoforms. All isoforms share the same domain structure but contain variable amino acid sequences within three Ig domains in the extracellular region. We demonstrate that different isoforms exhibit different binding specificity. Each isoform binds to itself but does not bind or binds poorly to other isoforms. The amino acid sequences of all three variable Ig domains determine binding specificity. Even closely related isoforms sharing nearly identical amino acid sequences exhibit isoform-specific binding. We propose that this preferential homophilic binding specificity regulates interactions between cells and contributes to the formation of complex patterns of neuronal connections.     

Alternative splicing of the Drosophila Dscam pre-mRNA is both temporally and spatially regulated

The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor that can express 38,016 different mRNAs by virtue of alternative splicing. The Dscam gene contains 95 alternative exons that are organized into four clusters of 12, 48, 33, and 2 exons each. Although numerous Dscam mRNA isoforms can be synthesized, it remains to be determined whether different Dscam isoforms are synthesized at different times in development or in different tissues. We have investigated the alternative splicing of the Dscam exon 4 cluster, which contains 12 mutually exclusive alternative exons, and found that Dscam exon 4 alternative splicing is developmentally regulated. The most highly regulated exon, 4.2, is infrequently used in early embryos but is the predominant exon 4 variant used in adults. Moreover, the developmental regulation of exon 4.2 alternative splicing is conserved in D. yakuba. In addition, different adult tissues express distinct collections of Dscam mRNA isoforms. Given the role of Dscam in neural development, these results suggest that the regulation of alternative splicing plays an important role in determining the specificity of neuronal wiring. In addition, this work provides a framework to determine the mechanisms by which complex alternative splicing events are regulated.     

Human down syndrome cell adhesion molecules (DSCAMs) are functionally conserved with Drosophila Dscam[TM1] isoforms in controlling neurodevelopment

Drosophila Down syndrome cell adhesion molecule (Dscam) potentially produces more than 150,000 cell adhesion molecules that share two alternative transmembrane/juxtamembrane (TM) domains, which dictate the dendrite versus axon subcellular distribution and function of different Dscam isoforms. Vertebrate genomes contain two closely related genes, DSCAM and DSCAM-Like1 (DSCAML1), which do not have extensive alternative splicing. We investigated the functional conservation between invertebrate Dscams and vertebrate DSCAMs by cross-species rescue assays and found that human DSCAM and DSCAML1 partially, but substantially, rescued the larval lethality of Drosophila Dscam mutants. Interestingly, both human DSCAM and DSCAML1 were targeted to the dendrites in Drosophila neurons, had synergistic rescue effects with Drosophila Dscam[TM2], and preferentially rescued the dendrite defects of Drosophila Dscam mutant neurons. Therefore, human DSCAM and DSCAML1 are functionally conserved with Drosophila Dscam[TM1] isoforms.     

Transmembrane/juxtamembrane domain-dependent Dscam distribution and function during mushroom body neuronal morphogenesis

Besides 19,008 possible ectodomains, Drosophila Dscam contains two alternative transmembrane/juxtamembrane segments, respectively, derived from exon 17.1 and exon 17.2. We wondered whether specific Dscam isoforms mediate formation and segregation of axonal branches in the Drosophila mushroom bodies (MBs). Removal of various subsets of the 12 exon 4s does not affect MB neuronal morphogenesis, while expression of a Dscam transgene only partially rescues Dscam mutant phenotypes. Interestingly, differential rescuing effects are observed between two Dscam transgenes that each possesses one of the two possible exon 17s. Axon bifurcation/segregation abnormalities are better rescued by the exon 17.2-containing transgene, but coexpression of both transgenes is required for rescuing mutant viability. Meanwhile, exon 17.1 targets ectopically expressed Dscam-GFP to dendrites while Dscam[exon 17.2]-GFP is enriched in axons; only Dscam[exon 17.2] affects MB axons. These results suggest that exon 17.1 is minimally involved in axonal morphogenesis and that morphogenesis of MB axons probably involves multiple distinct exon 17.2-containing Dscam isoforms.     

Silencing VP28 Gene of White Spot Syndrome Virus of Shrimp by Bacterially Expressed dsRNA

An in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria to provide a practical control of white spot syndrome virus (WSSV) in shrimp was developed. The bacterially synthesized dsRNA specific to VP28 gene of WSSV promoted gene-specific interference with the WSSV infection in shrimp. Virus infectivity was significantly reduced in WSSV-challenged shrimp injected with VP28-dsRNA and 100% survival was recorded. The inhibition of the expression of WSSV VP28 gene in experimentally challenged animals by VP28-dsRNA was confirmed by RT-PCR and Western blot analyses. Furthermore, we have demonstrated the efficacy of bacterially expressed VP28-dsRNA to silence VP28 gene expression in SISK cell line transfected with eukaryotic expression vector (pcDNA3.1) inserted with VP28 gene of WSSV. The expression level of VP28 gene in SISK cells was determined by fluorescent microscopy and ELISA. The results showed that the expression was significantly reduced in cells transfected with VP28dsRNA, whereas the cells transected with pcDNA-VP28 alone showed higher expression. The in vivo production of dsRNA using prokaryotic expression system could be an alternative to in vitro method for large-scale production of dsRNA corresponding to VP28 gene of WSSV for practical application to control the WSSV in shrimp farming.     

The molecular diversity of Dscam is functionally required for neuronal wiring specificity in Drosophila

Alternative splicing of Dscam generates an enormous molecular diversity with maximally 38,016 different receptors. Whether this large diversity is required in vivo is currently unclear. We examined the role of Dscam in neuron-target recognition of single mechanosensory neurons, which connect with different target cells through multiple axonal branches. Analysis of Dscam null neurons demonstrated an essential role of Dscam for growth and directed extension of axon branches. Expression of randomly chosen single isoforms could not rescue connectivity but did restore basic axonal extension and rudimentary branching. Moreover, two Dscam alleles were generated that each reduced the maximally possible Dscam diversity to 22,176 isoforms. Reduction of Dscam diversity resulted in specific connectivity defects of mechanosensory neurons. Furthermore, the observed allele-specific phenotypes suggest functional differences among isoforms. Our findings provide evidence that a very large number of structurally unique receptor isoforms is required to ensure fidelity and precision of neuronal connectivity.     

Penaeus monodon Dscam (PmDscam) has a highly diverse cytoplasmic tail and is the first membrane-bound shrimp Dscam to be reported

Down syndrome cell adhesion molecule (Dscam) seems likely to play a key role in the "alternative adaptive immunity" that has been reported in invertebrates. Dscam consists of a cytoplasmic tail that is involved in signal transduction and a hypervariable extracellular region that might use a pathogen recognition mechanism similar to that used by the vertebrate antibodies. In our previous paper, we isolated a unique tail-less form of Dscam from Litopenaeus vannamei. In this study, we report the first membrane-bound form of shrimp Dscam: PmDscam was isolated from Penaeus monodon, and it occurred in both membrane-bound and tail-less forms. Phylogenetic analysis showed that while the crustacean Dscams from shrimp and water flea did not share a single subclade, they were distinct from the invertebrate Dscam-like molecules and from the insecta Dscams. In the extracellular region, the variable regions of PmDscam were located in N-terminal Ig2, N-terminal Ig3 and the entire Ig7 domain. The PmDscam extracellular variants and transmembrane domain variants were produced by mutually exclusive alternative splicing events. The cytoplasmic tail variants were produced by exon inclusion/exclusion. Based on the genomic organization of Daphnia Dscam's cytoplasmic tail, we propose a model of how the shrimp Dscam genomic locus might use Type III polyadenylation to generate both the tail-less and membrane-bound forms.     

对虾白斑综合症病毒囊膜蛋白VP19的融合表达及其抗病毒感染作用

根据GenBank上WSSV囊膜蛋白基因vp19的序列,设计并合成引物,PCR扩增得到vp19基因并克隆到pGEM‐T载体中,经过BamHⅠ/HindⅢ酶切、连接并将vp19插入到pET32b表达载体中。用重组质粒pET32b-vp19转化大肠杆菌Origam(iDE3)pLysS,在IPTG诱导下,融合蛋白Trx-VP19以可溶性的形式得到表达,经SDS-PAGE和Western-blot检测显示其分子量与预期的大小相符合。目的蛋白经Ni2 柱纯化并定量后分别直接注射鳌虾和包被饲料投喂鳌虾。实验结果表明注射Trx-VP19可以提高鳌虾个体抗WSSV感染力的作用。     

Shrimp vaccination trials with the VP292 protein of white spot syndrome virus

AIMS: Construction of a recombinant vector that expresses VP292 protein of white spot syndrome virus (WSSV) and to exploit the possibility of obtaining the vaccine conferring protection against WSSV infection in shrimps. METHODS AND RESULTS: VP292 protein of WSSV was amplified from WSSV genomic DNA by PCR. The target 814 bp amplified product specific for VP292 protein was inserted in to pQE30 expression vector. The recombinant plasmid of VP292 protein was transformed and expressed in Escherichia coli under induction of isopropyl-1-1-thio-beta-D-galactoside (IPTG) and the immunoreactivity of the fusion protein was detected by Western blot. Shrimp were vaccinated by intramuscular injection of the purified protein VP292 of WSSV and challenged for 0-30 days. Vaccination trial experiments show that two injections with recombinant VP292 (rVP292) protein induced a higher resistance, with 52% relative percentage survival value, in the shrimp at the 30th day postvaccination. CONCLUSIONS: The expression system of protein VP292 of WSSV with a high efficiency has been successfully constructed. Vaccination trials show significant resistance in the shrimp vaccinated twice with recombinant VP292. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study prosper the development of WSSV protein vaccine against WSSV infection in shrimps.     

多重RT-PCR同时检测鉴别三种对虾病毒的研究与应用

根据基因库中对虾桃拉综合征病毒（TSV）、白斑综合征病毒（WSSV）、传染性皮下和造血器官坏死病毒（IHHNV）的基因序列,分别设计了三对特异性引物,通过对多重RT—PCR扩增条件的优化,研究建立了可同时检测鉴别TSV、WSSV和IHHNV的多重RT—PCR。该技术对同一样品中的TSV RNA、WSSV DNA和IHHNV DNA模板进行扩增,结果均同时得到3条大小与实验设计相符的231bp（TSV）、593bp（WSSV）和356bp（IHHNV）的特异性多重RT—PCR扩增带,对其它对虾病原核酸的扩增结果为阴性。敏感性试验结果表明,该技术最低能检测到10pgTSV RNA、100pg WSSV DNA和100pg IHHNV DNA。临床检测试验结果表明,该技术对TSV、WSSV和I—HHNV的检出率明显高于传统的临床症状观察和组织病理学检查,提示该技术适用于这三种病毒的临床快速检测和鉴别诊断。