Several gene ontology terms including transcription factor activity and biological process such as glucose metabolic process were enriched among these DETs. Pathways involved in response to handling and confinement stress were implicated by mapping the DETs to reference pathways in the KEGG database.Fish under intensive rearing conditions experience various stressors which have negative impacts on survival, growth, reproduction and fillet quality. Identifying and characterizing the molecular mechanisms underlying stress responses will facilitate the development of strategies that aim to improve animal welfare and aquaculture production efficiency. In this study, we used RNA-seq to identify transcripts which are differentially expressed in the rainbow trout liver in response to handling and confinement stress. These stressors were selected due to their relevance in aquaculture production. Total RNA was extracted from the livers of individual fish in five tanks having eight fish each, including three tanks of fish subjected to a 3 hour handling and confinement stress and two control tanks. Equal amount of total RNA of six individual fish was pooled by tank to create five RNA-seq libraries which were sequenced in one lane of Illumina HiSeq 2000. Three sequencing runs were conducted to obtain a total of 491,570,566 reads which were mapped onto the previously generated stress reference transcriptome to identify 316 differentially expressed transcripts (DETs). Twenty one DETs were selected for qPCR to validate the RNA-seq approach. The fold changes in gene expression identified by RNA-seq and qPCR were highly correlated (RRaw RNA-seq reads have been submitted to the NCBI Short Read Archive under accession number SRP022881.All customized scripts described in this paper are available from Dr. Guangtu Gao or the corresponding author. Oncorhynchus mykiss) is not only an important aquaculture species; it is also a primary research model for fish Aquaculture is the fastest-growing animal food producing sector of agriculture. Rainbow trout  axis and release of catecholamines from chromaffin cells that induce many biochemical and physiological changes Melanotaenia duboulayi) Ictalurus spp.) High-throughput next-generation DNA sequencing technologies have revolutionized analyses of genomes and transcriptomes. Transcriptome sequencing, or RNA-seq, overcomes many inherent limitations of microarray technologies, and has emerged recently as a powerful tool to characterize gene expression profiles in both model and non-model species et al.The experimental fish and the stress challenge have been reported in our previous publication Using the standard TRIzol protocol, total RNA was extracted from the liver of each individual fish in five tanks, including three challenged tanks and two control tanks. Equal amount of total RNA from six fish was pooled by tank, and the pooled RNA was used to prepare mRNA libraries for sequencing following the standard Illumina protocol. The five RNA-seq libraries were sequenced with a read length of 75 nucleotides in one lane of Illumina HiSeq 2000. To increase the read coverage and examine the technical sequencing variation, a total of three sequencing runs were performed.Software CD-HIT The GOSSIP st-strand cDNA synthesis kit following the manufacture's protocol . The cDNA was diluted and used for qPCR in a total reaction volume of 15 ul containing 7.5 ul SYBR green supermix, 4 ul cDNA template and an optimized amount of primers. The optimized reactions amplify a single product with a single peak for the melting curve and a single band on agarose gel. The PCRs were also optimized with an efficiency value between 1.85 and 2.15. After qPCR was optimized, the qPCR was performed in duplicate for each individual liver RNA samples used for RNA-seq. The housekeeping gene β-actin has been widely used as a control for qPCR in rainbow trout Twenty-one transcripts covering the full range of gene expression fold changes were selected for qPCR. The PCR primers designed with the software primer 3 are listed in In total, 491,570,566 reads were generated and have been archived in the NCBI's short read archive, accession number SRP022881 . About 7A total of 316 DETs  were ideGO terms were assigned to 158 out of the 190 DETs that were annotated by sequence similarity to well characterized genes in the NCBI database. For GO enrichment analysis, we focused on biological process and molecular function; 36 enriched GO terms were identified. Most of these were represented by a few DETs. GO terms represented by 10 or more DETs are shown in Using the web-based server KAAS, we were able to assign 78 k numbers to 91 DETs . Based op-value <0.0001). However, qPCR only identified significant differences in expression for 14 transcripts , both the direction and magnitude of gene expression changes were similar between those measured by qPCR and RNA-seq analysis . The traAs reported in Sanchez et al. RNA-seq has revolutionized transcriptome studies in both model and non-model organisms, with an emphasis in the latter for providing an efficient approach for gene discovery, expression analyses, and identification of sequence polymorphisms. We report the use of RNA-seq to identify 316 DETs that respond to handling and confinement stress in rainbow trout. The reliability and accuracy of RNA-seq analysis was demonstrated by the 0.88 coefficient of determination between RNA-seq and qPCR.Both known and novel stress response genes in fish were among the 316 DETs identified in this study. For example, Momoda et al. Among the 21 transcripts selected for qPCR, twenty transcripts had very similar fold changes in expression based on RNA-seq and qPCR, respectively. Only one transcript (primer pair RT43) had an opposite expression fold change based on RNA-seq and qPCR. This transcript was annotated as hsp70, whose expression in liver has been observed to increase in response to elevated plasma cortisol concentrations that are associated with some stress responses but not including handling and confinement in rainbow trout Also, the qPCR results were only determined to be statistically significant for 14 transcripts . This maActivation of HPI axis and catecholamine release leads to many changes at the biochemical and physiological level. Since liver is the major organ for metabolic adjustments, it is not surprising to observe enrichment of GO terms related to glucose metabolism and transcription factors in this study. Due to the limited annotation of DETs identified in this study and the paucity of well documented pathways in rainbow trout, it is challenging to describe all of the exact pathways involved in response to handling and confinement stress in rainbow trout. However, several pathways were implicated in response to handling and confinement stress in this study. Glucose is an important fuel that is oxidized to meet the energy demand associated with stress response. Elevation of plasma glucose concentration is a well-documented metabolic stress response in rainbow trout. Activation of the gluconeogenesis pathway in response to stress has been reported in several previous studies While cortisol is a key hormone associated with the stress response, other hormones such as insulin-like growth factor (IGF), insulin, and estrogen signaling may also play important roles. Similar to the increased expression of hepatic expression of IGF binding protein-1b (IGFBP-1b), -2a, and -3 during transport in zebrafish It is clear that the immune response was altered in rainbow trout in response to handling and confinement stress. Many important elements of innate and adaptive immunity such as cytokine receptors, antigen presenting and processing molecules, immunoglobins and transcriptional factors were among the DETs identified in this study. We observed that cytokine receptors such as c-x-c chemokine receptor type 4 (CXCR4), c-c chemokine receptor 9 (CCR9) and tumor necrosis factor receptor member 9 (SF9), were up-regulated. However, cytokine receptor KIT (proto-oncogene tyrosine protein kinase) was down-regulated. Transcription factor NF-κB might play critical roles in the regulation of diverse cellular responses including cell proliferation, survival and inflammation Even though induction of acute phase response in fish is often associated with the activation of the inflammatory response, acute phase response proteins (APPs) also play a role in response to confinement stress challenge in rainbow trout. Cairns et al. Deep sequencing of the rainbow trout liver transcriptome in response to handling and confinement stress identified 316 DETs. The reliability and accuracy of RNA-seq analysis was demonstrated by the 0.88 coefficient of determination between RNA-seq and qPCR. Several GO terms including transcription factor activity and -biological process such as glucose metabolic process were enriched among the 316 DETs. Pathways involved in response to handling and confinement stress were implicated by mapping the DETs to the reference pathways in KEGG database. The findings of this study provide a basis for further characterization of molecular response to handling and confinement stress in rainbow trout.Figure S1Comparison of normalized uniquely mapped reads among three runs. 1A, tank1; 1B, tank 2; 1C, tank 3, 1D, tank 4; and 1E, tank 5.(PPTX)Click here for additional data file.Table S1All 316 DETs in response to handling and confinement stress identified in this study.(XLSX)Click here for additional data file.Table S2Primers used for qPCR to validate transcript expression fold changes measured by RNA-seq.(XLSX)Click here for additional data file.


The ATP-binding cassette (ABC) transporter superfamily is the largest transporter gene family responsible for transporting specific molecules across lipid membranes in all living organisms. In insects, ABC transporters not only have important functions in molecule transport, but also play roles in insecticide resistance, metabolism and development.Bombyx mori, we have identified 51 putative ABC genes which are classified into eight subfamilies (A-H) by phylogenetic analysis. Gene duplication is very evident in the ABCC and ABCG subfamilies, whereas gene numbers and structures are well conserved in the ABCD, ABCE, ABCF, and ABCH subfamilies. Microarray analysis revealed that expression of 32 silkworm ABC genes can be detected in at least one tissue during different developmental stages, and the expression patterns of some of them were confirmed by quantitative real-time PCR. A large number of ABC genes were highly expressed in the testis compared to other tissues. One of the ABCG genes, BmABC002712, was exclusively and abundantly expressed in the Malpighian tubule implying that BmABC002712 plays a tissue-specific role. At least 5 ABCG genes, including BmABC005226, BmABC005203, BmABC005202, BmABC010555, and BmABC010557, were preferentially expressed in the midgut, showing similar developmental expression profiles to those of 20-hydroxyecdysone (20E)-response genes. 20E treatment induced the expression of these ABCG genes in the midgut and RNA interference-mediated knockdown of USP, a component of the 20E receptor, decreased their expression, indicating that these midgut-specific ABCG genes are 20E-responsive.From the genome of the silkworm, In this study, a genome-wide analysis of the silkworm ABC transporters has been conducted. A comparison of ABC transporters from 5 insect species provides an overview of this vital gene superfamily in insects. Moreover, tissue- and stage-specific expression data of the silkworm ABCG genes lay a foundation for future analysis of their physiological function and hormonal regulation. Drosophila melanogaster (Diptera) [Anopheles gambiae (Diptera), at the genome-wide level [The ATP-binding cassette (ABC) transporters form one of the largest family of membrane proteins . With 48Diptera) . In addiDiptera) , the ABCThe ABC transporters share highly conserved domains known as nucleotide binding domains (NBDs). Each NBD contains three characteristic motifs, including Walker A box, Walker B box, and ABC signature C which links the two Walker boxes . NBD binBombyx mori (Lepidoptera). One of the best studied insect ABC transporters is White, which is a typical ABCG transporter involved in pigment transport in insect eyes [According to their functions, the ABC proteins can be classified as importers, exporters and non-transport proteins . Importeect eyes ,12.In a microarray study published previously ,14, we dD. melanogaster ABC transporters were used as queries for BLASTP search against the two silkworm genome databases, SilkDB and KAIKObase. Each potential silkworm ABC transporter was validated by searching its known orthologs from the protein database of NCBI and further searching its NBD and TMD with the Pfam program. As a result, a total of 51 putative ABC genes were identified in the silkworm genome. A previous study has identified 56 ABC genes in D. melanogaster [Tribolium castaneum (Coleoptera), 52 from A. gambiae (8 more compared to 44 in [Apis mellifera (Hymenoptera)  and the smallest one (280 amino acids) are encoded by BmABC012789 and BmABC010825, respectively , we characterized each subfamily in the silkworm and four other insect species for comparison.BmABC007217, BmABC007218 and BmABC007221 are closely located on chromosome 17. The silkworm ABCA subfamily consists of 2 full transporters, 1 half transporter, and 3 incomplete ABC proteins containing one single NBD in each member. This subfamily includes two of the largest ABC transporters encoded by BmABC012789 (1794 amino acids) and BmABC007221 (1747 amino acids). In human, all ABCA transporters are full transporters [Arabidopsis thaliana, 11 are half transporters and only one is a full transporter [Saccharomyces cerevisiae [A. gambiae, are full transporters, whereas both full and half transporters exist in the ABCA subfamily of the four other insect species, indicating that the ABCA subfamily varies greatly during evolution in insects and other organisms. In addition, phylogenetic analysis reveals that 3 insect ABCA transporters, BmABC012789, AGAP010416 and TcABCA2, are closely related , implying that they are orthologs  phenotype to cancer, and later studies revealed several more MDRs in the ABCB subfamily . Based ochicines . Mdr49 iocarbons , but alsocarbons . The ABCtigation .T. castaneum. Interestingly, 8 of the 15 silkworm ABCC genes are located on chromosome 15 and 4 on chromosome 12. Phylogenetic analysis of the ABCC subfamily within the 5 insect species reveals that many ABCC genes belonging to the same insect species cluster together, indicating gene duplication is an active mechanism that generates high diversity in the ABCC subfamily in insects , are involved in ion transport, toxin secretion, signal transduction, and other physiological functions . It has d copper . AlthougThe ABCD transporters are located to the peroxisome at the subcellular level and involved in the import of fatty acids and/or fatty acyl-CoAs into this organelle . Each ofBmABC010129 which shows the highest expression level based on the EST information, is preferentially expressed in testis, ovary and fat body. However, RNA interference (RNAi)-mediated knockdown of BmABC010129 has no obvious effects on RNase L expression [The ABCE and ABCF proteins lack TMD and each member contains a pair of linked NBDs . Most eupression .GCN20 gene reduce eIF2α phosphorylation and thus translation in the ribosome [ABCF proteins function in ribosome biogenesis, translational control, and mRNA export, and are not involved in molecule transport . The besribosome . Later sribosome . Until nwhite, scarlet, and brown, in D. melanogaster [BmABC002922 (Bmwh3) encodes an ortholog of D. melanogaster white. Polyadenylated Bmwh3 transcript of about 2.7 kb length is detected in eggs, Malpighian tubule and pupal heads, but not in testes, posterior silk glands or fat body cells [Bmwh3 is responsible for the transportation of ommochrome precursors and uric acid into pigment granules and urate granules, respectively, and Bmwh3 mutations cause white eyes, white eggs, and translucent larval skin [BmABC002924 encodes an ortholog of D. melanogaster scarlet. In the silkworm, Scarlet forms a heterodimer with White to transport ommochrome precursors [BmABC002581 encodes an ortholog of D. melanogaster brown. In addition, phylogenetic analysis revealed that BmABC010557 is orthologous to D. melanogaster E23 (CG3327)  and the early pupal stage (60 hours after the wandering stage), when the 20E level is high. As determined by qRT-PCR, BmABC005226 is exclusively expressed in the midgut and highly expressed during the wandering and prepupal stages, when the 20E level is high . To this end, we investigated the spatial expression patterns of all the 13 silkworm ABCG genes using qRT-PCR. Besides BmABC005226, four other genes, including BmABC005203, BmABC005202, BmABC010555, and BmABC010557, are also preferentially expressed in the midgut. Moreover, we investigated their temporal expression patterns in the midgut, which revealed that the 4 ABCG genes are highly expressed during the wandering and prepupal stages as well. The qRT-PCR data  and BmABC012035 were highly expressed in brain, and the mRNA level of brown peaked during the feeding stage of the fifth instar  or USP dsRNA (10 μg). Twenty four hours after RNAi treatment, the larvae were sacrificed for qRT-PCR analysis [dsRNA was generated using the T7 RiboMAX™ Express RNAi system . At the initiation of the early wandering stage, each individual larva was injected with analysis ,14. ThirSL performed most of the experiments and analyzed the data. SZ, LT, EG helped with qRT-PCR, hormone treatment and RNAi. YL and JZ helped writing the paper and constructing the phylogenetic trees. SL designed the experiments, analyzed the data, wrote the paper, and coordinated the whole study. All authors approved the final manuscript.Figure S1. Phylogenetic tree of the ABC transporters from the honeybee, Apis mellifera.Click here for fileFigure S2. Phylogenetic tree of the ABC transporters from the mosquito, Anopheles gambiae.Click here for fileFigure S3. Phylogenetic tree of the ABC transporters from the flour beetle, Tribolium castaneum.Click here for fileFigure S4. Locations of the silkworm ABC genes on chromosomes. Genes in clusters, for example, BmABC000724 and BmABC000725 on Chromosome 1, are indicated by a vertical line.Click here for fileFigure S5. Phylogenetic tree of ABCA transporters from five insect spcies and the human. The phylogenetic tree was constructed using a neighbor-joining technique to analyze the amino acid sequences of the nucleotide binding domain (NBD). Analysis was performed with the program package MEGA4.0. The number at the branch point of the node represents the value resulting from 1000 replications and gaps were deleted with pairwise deletion method. Am, Apis mellifera; Ag, Anopheles gambiae; Bm, Bombyx mori; Dm, Drosophila melanogaster; Hs, Homo sapiens; Tc, Tribolium castaneum.Click here for fileFigure S6. Phylogenetic tree of ABCB transporters from five insect species and the human.Click here for fileFigure S7. Phylogenetic tree of ABCC transporters from five insect species and the human.Click here for fileFigure S8. Phylogenetic tree of ABCD transporters from five insect species and the human.Click here for fileFigure S9. Phylogenetic tree of ABCE and ABCF proteins from five insect species and the human.Click here for fileFigure S10. Phylogenetic tree of ABCG transporters from five insect species and the human.Click here for fileFigure S11. Phylogenetic tree of ABCH transporters from five insect species and the human.Click here for fileFigure S12. Tissue distribution and developmental expression profiles of the 2 silkworm ABCG genes, including BmABC002581and BmABC012035 in the brain from day 2 of 4th instar to day 2 of prepupae. M, molting; PP1, day 1 of prepupae; PP2, day 2 of prepupae.Click here for fileTable S1. A list of quantitative real-time PCR primers.Click here for file


This method paper describes a quantitative dual fluorescence approach to analyze protein–mRNA interactions in vivo. RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein–mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins , expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP . Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology. While CLama alpaca has been described (The green fluorescent protein (GFP) and spectral variants thereof are widely used tools to study protein localization and dynamics in living cells . In spitescribed . The GBPescribed . GBP wasescribed . Here, w32P-γ-ATP phosphate and T4 polynucleotide kinase, followed by electrophoretic separation of labeled complexes. Using fluorescently labeled fusion proteins in combination with UV cross-linking to RNA, stringent GBP immunoprecipitation and oligo(DT) hybridization 11, and random hexamers to detect polyadenylated RNA in samples from cells expressing the broad specificity RBP MOV10 fused to YFP (25 than with oligo(DT)11 and random hexamers (11 suggests that long poly(A)/oligo(DT) sequences are required for efficient hybridization under our experimental conditions. Such long poly(A) tracts are rarely found in internal regions of cellular RNAs, but are typically present at the 3′ end of polyadenylated RNAs. As an additional control, half of the samples were treated with RNases to assess whether the oligo(DT)-derived signal is RNase-sensitive. RNase treatment strongly diminished the Tred signal without affecting YFP fluorescence when an oligo(DT)25 probe was used -labeled oligo(DT)d to YFP . As a ned to YFP B,C. Nevehexamers B,C. The was used B,C. In ce probes B,C. Of re probes D,E, furte probes D,E and pNotably, the red/green ratio for MOV10 was about three times higher with GFP-Trap_A than with GFP-multiTrap 25 probe. The alexa Fluor 594 probe showed a modest improvement of the red/green fluorescence ratio of MOV10 and hnRNPC samples over RNase-treated controls, while the RNA-binding activity of FAM98A remained undetectable under these conditions in GFP-multiTrap assays (Family-with-sequence-similarity (FAM)98A has recently been identified and validated as an RBP in two independent studies . While Fp assays A,B. Strip assays A,B. This25 probes labeled with two molecules of ATTO647 or ATTO633. These fluorophores can be coupled simultaneously with the 5′ and 3′ ends of the DNA probe and display comparable absorption and emission wavelengths as well as extinction coefficients to WellRED D4. Since the two fluorophores are separated by 25 nt, quenching of the fluorescent signal is minimal. The optimal concentration of the Tred probes was determined to be 40 nM (data not shown). Because the optimal concentration of the doubly labeled probes may differ from that of the singly labeled ones, we conducted a titration experiment. The optimal red/green fluorescence ratio was obtained at 8 nM for all three far red probes, probably due to a lower basal red fluorescence in negative controls  probe. As the chemistry of WellRED D4 is incompatible with multiple labeling, we tested the performance of oligo(DT)controls C. The MOcontrols C,D. ATTOcontrols C,D. HoweThe dual fluorescence RNA-binding assay described here can be used to validate the in vivo RNA-binding activity of candidate RBPs identified by system-wide approaches . We assePABP interacts with poly(A) tracts by means of four RNA-recognition motifs (RRM) located in its N-terminal domain (NTD) A, whereaAnalysis of the HeLa mRNA interactome revealed that intrinsically disordered regions are overrepresented within RBPs and that their amino acids cluster into repetitive motifs. Combination of arginine (R) with glycine (G), referred to as RGG-box, is a classical RBP motif with a well-defined role as RNA-binding domain (RBD) . DiffereFurthermore, mRNA interactome capture had identified RBPs with long and repetitive basic patches rich in lysines (K), named K-boxes, which were found to be especially abundant among newly identified RBPs . Previouhttp://www.embl.de/mRNAinteractome)  network hypothesis , have been successfully applied to study RBPs. However, in a biological context the RNA-binding activity of RBPs is typically regulated by a plethora of mechanisms, including post-translational modifications , inducedractome) , suggestractome) . Phosphoractome) , 2011. Tpothesis  proposespothesis . In othepothesis . Interespothesis . Thus, tUsers of the dual fluorescent assay need to consider that the approach will fail to detect physiological RBP–RNA interactions when (i) protein-bound RNAs are not polyadenylated; (ii) the RBP fails to cross-link efficiently by either cCL or PAR-CL; (iii) the eGFP tag interferes with protein folding or RNA binding; (iv) target RNAs are expressed at low levels and the ectopically expressed RBP has to compete with its endogenous counterpart.Nevertheless, the method reported here has notable advantages over existing methodologies: (i) UV light is directly applied to monolayers of living cells, thus “freezing” physiological in vivo protein–RNA interactions; (ii) UV irradiation induces covalent bond formation between RBPs and RNA, but unlike chemical cross-linking does not promote protein–protein covalent bonds, allowing the specific capture of direct RNA binders following an immunoprecipitation procedure; (iii) GBP allows the high affinity and specificity isolation of eGFP- and YFP-tagged proteins, which is compatible with the use of high salt buffers (up to 1 M NaCl) and ionic detergents (up to 0.1% SDS), leading to efficient removal of noncovalently associated proteins; (iv) this assay is based on nonhazardous, rapid fluorescence measurements and the RNA-derived signal can be easily normalized to the fluorescence of immunoprecipitated GFP/YFP providing an accurate and reliable readout; (v) the dual fluorescence RNA-binding assay can be performed using commercially available GBP-coupled 96-well plates (GFP-multiTrap). GFP-multiTrap allows 96 immunoprecipitations simultaneously, decreasing the technical variability of the assay and enabling high-throughput applications. This important feature now allows studying the RNA-binding activity of a given RBP quantitatively under a dozen of different experimental conditions, even including technical and biological replicates as well as appropriate quality controls within the same plate.The dual fluorescence RNA-binding assay allows identification of RNA-binding domains and motifs. Here, we showed that the RBD of PABP is harbored within its RRM-containing NTD, whereas its CTD lacks RNA-binding activity. These in vivo data reflect previous structural information  and suppSaccharomyces cerevisiae, Caenorhabditis elegans, Drosophila embryos), providing a valuable resource to study RBP dynamics. Importantly, GFP-multiTrap allows highly parallel RNA-binding quantifications in a single experiment, offering the possibility to investigate the dynamics of RBPs under different environmental conditions. The method can also be used to map and characterize the RNA-binding architectures of RBPs in living cells, including disordered protein motifs of unknown function. The high-throughput potential of the dual-fluorescence RNA-binding assay may also help with exhaustive mutagenesis analyses to determine the role of individual amino acids  in RNA-binding or with small molecule screening projects.The dual fluorescence RNA-binding assay described in this report complements existing methodologies such as CLIP  or interChimeric cDNAs encoding the different eGFP- or YFP-tagged proteins were amplified by PCR from already established eGFP/YFP-containing plasmid libraries . Alterna2 UV light at 254 nm . Lysates were incubated for 10 min at 4°C, snap-frozen, and thawed, to accomplish complete cell lysis. Lysates were centrifuged for 10 min at 10,000 rpm and 4°C to pellet cellular debris, and the supernatants were collected and aliquoted into three tubes (100 μL per tube) for technical triplicates. Samples were mixed with 400 μL of dilution buffer , and then supplemented with 100 units/mL RNaseOUT, 1× protease inhibitor cocktail (Roche), and 10 μL of pre-equilibrated GFP-Trap_A (Chromotek). GFP-tagged proteins were immunoprecipitated for 2 h at 4°C with gentle rotation, subsequently washed with 500 μL of medium salt buffer , and incubated for 15 min at 4°C with 250 μL of blocking solution . Hybridization was performed by incubation with 250 μL of hybrydization buffer  supplemented with 40 nM of oligo(DT)25-Tred (Sigma) for 1 h at 4°C. Excess of fluorescent probe was removed by washing once with 500 μL of wash buffer 1  and twice with 500 μL of wash buffer 2 . GFP-Trap_A was resuspended in 100 μL of the latter buffer and transferred to a 96-well optical plate . All buffers indicated above were supplemented with 50 units/mL of RNaseOUT and 1× protease inhibitor cocktail. For control RNase treatments see below.For cCL, one 10-cm dish of HeLa cells at 40%–50% confluence was incubated for 16 h at 37°C with DMEM supplemented with 5% fetal calf serum (FCS) and 1 μg/mL tetracycline to induce the expression of the eGFP- or YFP-tagged proteins. Next, cells were washed twice with 5 mL of PBS. After complete removal of the PBS, cells were placed on ice and immediately irradiated with 0.15 J/cmt 254 nm . For PARescribed . Cells w25 Tred/8 nM of oligo(DT)25-WellRED for 1 h at 4°C. Excess of fluorescent probe was removed by washing once with wash buffer 1 and twice with wash buffer 2. Finally, 100 μL of wash buffer 2 was added to each well and fluorescence was immediately measured as described below.Cells were grown, UV-irradiated and recovered as described above, and then lysed into 300 μL of lysis buffer. Lysates were centrifuged for 10 min at 10,000 rpm and 4°C to pellet cellular debris. The supernatants were collected, their volumes adjusted to 400 µL with lysis buffer, and then supplemented with 100 μL of 5× dilution buffer  and 5 µL 5% SDS, and transferred to three wells (150 μL per well) of the GFP-mutiTrap (Chromotek) for technical triplicates. Immunoprecipitation was performed overnight at 4°C with agitation (800 rpm). We recommend distributing the technical replicates to distant positions from each other on the plate to control for reading and immunoprecipitation homogeneity. Samples were washed with 180 μL of medium salt buffer and then incubated for 15 min with 180 μL of blocking solution at 4°C. Hybridization was performed with 100 µL hybridization buffer supplemented with 40 nM oligo(DT)Fluorescence was measured in a TECAN Safire II microplate reader, using the following parameters: eGFP/YFP: the optimal excitation and emission are 475 nm and 509 nm, respectively, for eGFP and 514 nm and 527 nm for YFP. We determined that excitation 490 nm, emission 515 nm recovered comparable signals from both proteins. These parameters were used for experiments monitoring simultaneously both eGFP and YFP. For red and far red fluorophores we applied the following parameters: Tred and Alexa Fluor594 excitation 590 nm, emission 616 nm; WellRED and ATTO 647: excitation 650 nm, emission 670 nm. ATTO633: excitation 630 nm, emission 657 nm. In all cases bandwidth was restricted to 5–10 nm and gain was set using the positive controls as a reference. For higher reliability, we recommend measuring from the bottom of the plate (when this option is available).Cell lysis was performed in lysis buffer lacking RNase inhibitors . Lysates were incubated for 10 min at 4°C, snap-frozen, and thawed, to accomplish complete cell lysis. Lysates were centrifuged for 10 min at 10,000 rpm and 4°C to pellet cellular debris, and the supernatants were collected and supplemented with 350 units of RNase T1 and 1.5 units of RNase A. RNA digestion was performed for 30 min at 37°C with agitation. For DNase treatment, cells were lysed in 1× DNase buffer supplemented with 0.5% NP-40. After completing cell lysis and lysate pre-clearing (see above), samples were incubated with 2 μL of DNase I (Promega) for 1 h at 37°C.Positive  and negative controls  are well suited references to monitor the performance of the dual fluorescence RNA-binding assay as well as to define the dynamic range of the assay. RNase treatment can be used as an additional control.2 to maximize 4-SU-mediated protein–RNA covalent bonds.When working with RBPs with low cross-linking efficiency, the UV dosage can be optimized. Whereas RNA is damaged by exposure to high dosages of 254-nm UV irradiation, 365-nm UV light does not promote RNA breaks. Thus, 365-nm UV irradiation can be increased up to 0.9 J/cmAfter immunoprecipitation, we suggest to exclude all samples in which GFP-derived fluorescence does not exceed the background  by at least threefold. When the protein of interest is not well expressed, the number of cells used to prepare the lysate can be increased using the same experimental settings as described above. Three biological replicates and three technical replicates typically suffice for robust statistical analyses.2 dishes with 40%–50% cell confluence per condition are induced overnight and processed as described  following the manufacturer's recommendations.Supplemental material is available for this article.Ulrich Rothbauer is a shareholder of ChromoTek GmbH, Martinsried.


Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in  Caenorhabditis elegans to study how the VAB-1 Eph RTK regulates the growth cone structure for axon guidance. Genetic and molecular data show that VAB-1 regulates the conserved molecules NCK-1, WSP-1/N-WASP, and UNC-34/Ena. Our study provides a model of how the VAB-1 Eph RTK modulates the growth cone structure to inhibit axonal outgrowth. We show that activated VAB-1 can inhibit an NCK-1/UNC-34 interaction by binding to the NCK-1 SH2 domain. We also show that NCK-1 and WSP-1 can physically interact and that VAB-1/NCK-1 and WSP-1 form a complex in vitro. We suggest that the VAB-1 Eph RTK can contribute to the termination of axon outgrowth by two methods: 1) The VAB-1/NCK-1/WSP-1 complex activates ARP-2/3 to change the actin growth cone dynamics to that of a branched structure thus reducing the number of filopodia, and 2) VAB-1 inhibits axon extension by inhibiting UNC-34/Ena's function in actin polymerization.The correct wiring of the nervous system depends on the ability of axons to properly interpret extracellular cues that guide them to their targets. The Eph receptor tyrosine kinases (RTKs) have roles in guiding axons, but their signaling pathways are not completely understood. In this study, we used the nematode  Caenorhabditis elegans can simplify the analysis of the signal transduction events from the receptor. The C. elegans VAB-1 Eph RTK is required for various aspects of neuronal development, including neuroblast movements, and axon guidance During development, axons navigate to their final destination by interpreting extracellular guidance cues through their growth cone. The Eph receptor tyrosine kinases (RTKs) and their ephrin ligands are involved in directing axons to their proper location C. elegans has only been recently explored C. elegans NCK-1 is still unknown.The Nck adaptor proteins are known actin cytoskeleton regulators, and have been shown to function downstream of several axon guidance receptors including Robo, Dcc and the Eph RTKs C. elegans N-WASP homolog, WSP-1, functions in neuronal cell migration and axon guidance The WASP protein family (WASP and N-WASP) are scaffolds that integrate multiple signaling pathways, leading to the formation of short branched actin filaments through the activation of the Arp2/3 complex C. elegans, the Ena/VASP homolog UNC-34 is required for proper neuronal cell migration, axon guidance and filopodia formation The Ena/VASP proteins are involved in actin-dependent movements including neuronal migration and axon guidance, and are known for their role in promoting filopodia formation C. elegans, the Arp2/3 complex is required for axon guidance, and the initiation of growth cone filopodia downstream of an unidentified axon guidance signal The Arp2/3 complex is a conserved family of actin nucleators and when activated results in the formation of an elaborate network of branched actin filaments similar to those found in lamellipodia In this paper, we describe some of the molecular events that allow the VAB-1 Eph RTK to regulate actin dynamics for axon guidance. We provide genetic and biochemical evidence to show that VAB-1 signals through NCK-1 and WSP-1/N-WASP, and negatively regulates UNC-34/Ena. We propose a model for PLM  axon termination whereby the VAB-1 Eph RTK is able to prevent axon extension by inhibiting growth cone filopodia formation. This is accomplished by negatively regulating the activity of the filopodia elongator UNC-34/Ena, and simultaneously activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex.mec-4::myr-vab-1 (quIs5) which encodes a constitutively active VAB-1 tyrosine kinase (myristoylated-VAB-1 termed MYR-VAB-1) in the mechanosensory neurons myr-vab-1 caused neuronal defects in the mechanosensory neurons, in particular the premature termination of PLM axons  mutation partially suppressed the PLM axon premature termination  is predicted to be a null allele, thus all of our genetic work was carried out using the ok694 allele To identify VAB-1 Eph RTK effectors, we utilized transgenic animals carrying LM axons  [6]. Sinmination , indicatnck-1 loss-of-function mutation to have a phenotype similar to that of the vab-1 loss-of-function. Indeed, previous work showed that both vab-1 and nck-1 mutants have similar neuronal defects, including an overextension in PLM axons  (see Methods). Wild-type L1s had PLM axons that were 103–114 µm long, and terminated at a region anterior to the tip of the ALM cell body (93%) and is consistent with previous reports for L1 PLM lengths vab-1 and nck-1 animals had PLM axons that significantly overgrew beyond the wild-type termination region  (57%) (To determine if the PLM defects observed in n region . This inild-type . The PLM4) (57%) . These rWe previously showed that NCK-1 is expressed in various tissues including the nervous system In a parallel approach we used yeast two-hybrid screens to identify effectors of VAB-1/Eph RTK signaling and identified the full length NCK-1 as a binding partner of the VAB-1 intracellular kinase region. Yeast two-hybrid analysis showed that the NCK-1 SH2 domain is sufficient to bind VAB-1 and that VAB-1 tyrosine Y673 is crucial for the interaction with the NCK-1 SH2 domain .To further confirm the NCK-1/VAB-1 interaction we used GST-pull down assays. Deletion analyses confirmed that the SH2 domain is necessary and sufficient to bind VAB-1 . FurtherIn vitro binding assays with bacterially expressed NCK-1 and UNC-34 confirmed that both proteins do indeed physically interact ; unc-34(e566) double and found that nck-1 partially suppressed the unc-34 PLM termination defect, while unc-34 did not suppress the nck-1 overgrowth (unc-34 may negatively regulate nck-1. To provide further evidence for this genetic interaction we over expressed NCK-1 (mec-4::nck-1) in the PLM neurons of unc-34(e566) animals and this resulted in a synergistic enhancement of the unc-34 PLM termination phenotype  and it significantly reduced the MYR-VAB-1 PLM premature termination phenotype , so we used a mechanosensory specific unc-34 RNAi approach . The unc-34(RNAi) strain had PLM termination defects that were similar to unc-34(e566)  double showed that reducing the levels of unc-34 can rescue the PLM overextension defects seen in vab-1(dx31)  and it is expressed at high levels. When we introduce constitutively active VAB-1 only in the touch neurons (mec-4::myr-vab-1) it reduced the UNC-34::GFP levels significantly  . Analysi-1(dx31) , which i animals . To testficantly .In summary, our binding assays and genetic analyses show that VAB-1 activation results in binding NCK-1 which in turn blocks the UNC-34 binding to NCK-1, freeing NCK-1 from the negative influence of UNC-34 and in addition VAB-1 negatively regulates UNC-34 protein levels.wsp-1 mutants should suppress the PLM termination defect caused by MYR-VAB-1. Two wsp-1 alleles are predicted to affect the WSP-1 protein. The wsp-1(tm2299) is not well characterized, but is homozygous lethal and is predicted to be a null allele. The embryonic lethality is due to wsp-1 pleiotropy as WSP-1 is also required for cytokinesis during embryogenesis wsp-1(gm324) allele is a well characterized deletion that removes exons 2 and 3, furthermore, no WSP-1 protein nor mRNA can be detected, therefore wsp-1(gm324) is a strong loss-of-function allele wsp-1(gm324) displays some embryonic and larval lethality but can be maintained as a homozygote wsp-1(gm324) allele as it allowed us to bypass the embryonic lethality associated with the wsp-1 null allele. We found that wsp-1(gm324) could significantly suppress the MYR-VAB-1 PLM termination defect in young adults and L1s  had weak axon guidance defects, such as in the PDE and VD/DD neurons wsp-1(gm324) animals have overextended PLM defects in young adults, and 42% PLM axon overgrowth in L1s (wsp-1 PLM overextension frequency is much greater than vab-1 (vab-1(dx31);wsp-1(gm324) double mutants are synthetic lethal (data not shown), which is consistent with WSP-1 functioning in parallel pathways with VAB-1.If WSP-1 is an effector of VAB-1 signaling then we would expect to see neuronal defects similar to h in L1s . Since tan vab-1 , it implin vitrovcamec-4::wsp-1). The WSP-1VCA caused PLM premature termination defects that were very similar to MYR-VAB-1 ), a central section containing a basic region (BR), a GTPase binding domain (GBD) and a proline-rich region (PRO), and a C-terminal with two verprolin homology domains , a cofilin homology domain (C) and an acidic domain (A) YR-VAB-1 .wsp-1 can antagonize unc-34 function. Due to the synthetic lethality of wsp-1; unc-34 double mutants wsp-1; unc-34(RNAi) animals. Tissue specific unc-34 RNAi resulted in the partial suppression of PLM overextension defects caused by wsp-1(gm324)  , consistIn summary, we show that WSP-1 functions in PLM axon termination, through various signaling pathways, including the VAB-1 Eph RTK. Our results suggest that MYR-VAB-1 is exerting its effect by activating the Arp2/3 complex through WSP-1. We also suggest that WSP-1 can antagonize UNC-34 function by activating the Arp2/3 complex.in vitro binding assays to ask whether VAB-1, NCK-1 and WSP-1 could form a complex. WSP-1 was able to bind NCK-1   showing some filopodia structures (unc-34(e566) animals. We found that unc-34(e566) mutants, like myr-vab-1 animals, had growth cones void of filopodia structures with only 25% displaying filopodia structures . Our results show that activated VAB-1 can affect the PLM growth cone morphology by inhibiting filopodia formation.The VAB-1 RTK effectors NCK-1 and WSP-1 are known actin regulators and therefore implicate VAB-1 signaling in regulating actin cytoskeleton for axon guidance. To confirm this, we monitored the PLM growth cone of wild-type and in vitro interaction analysis identified NCK-1, WSP-1 and UNC-34 as molecules regulated by VAB-1 Eph RTK signaling. Our data supports a model in which VAB-1 suppresses axon extension by negatively regulating UNC-34, and activating the Arp2/3 complex through a VAB-1/NCK-1/WSP-1 complex. Furthermore, using time-lapse analysis we show that activation of VAB-1 inhibits filopodia formation in the PLM growth cone.We previously reported a functional role for VAB-1 as a receptor for a repellent or stop signal in PLM axon guidance C. elegans NCK-1 adaptor protein is an effector of the VAB-1 RTK signal in vivo. Several lines of evidence indicate that VAB-1 and NCK-1 act together to regulate axon guidance. First, nck-1 and vab-1 animals have similar neuronal defects. Second, NCK-1 and VAB-1 physically interact and co-localize in similar neuronal cells and axons. Finally, the nck-1 loss-of-function suppresses the defects caused by the constitutively active VAB-1. We found that NCK-1 binds the VAB-1 juxtamembrane tyrosine Y673 (YEDP) via its SH2 domain in a VAB-1 kinase dependent manner. This is consistent with the published binding specificity of the Nck SH2 domain, as well as reports of Nck1 binding to the second juxtamembrane tyrosine residue (YEDP) in EphA3 (Y602) and EphA2 (Y594) C. elegans provides relevant insight into how mammalian EphA receptors could regulate the actin cytoskeleton for axon guidance.Our results show that the C. elegans, UNC-34/Ena functions in neuronal cell migration, axon guidance and epithelial filopodia formation unc-34(e566) PLM axons terminated prematurely. The cause of early termination is likely due to a reduction of filopodia elongation in the growth cone, resulting in the persistence of more densely branched filaments that can slow axon migration. This is supported by the finding that unc-34 mutants have fewer filopodia structures on growth cones, and a reduced rate of growth cone migration The Ena/VASP protein family is required in processes that involve dynamic actin remodeling such as platelet shape change, axon guidance and Jurkat T cell polarization unc-34 resulted in PLM axon defects similar to the hyperactive MYR-VAB-1; 2) over expressing UNC-34 in the PLM partially suppressed the MYR-VAB-1 phenotype; 3) tissue specific unc-34 RNAi suppressed the vab-1 PLM overextension defects; and 4) over expressing VAB-1 reduced the UNC-34 protein levels. Although we do not know the mechanism of the reduction of the UNC-34 protein levels displayed in the hyperactive VAB-1, it is possible that UNC-34, when removed from its adaptor NCK-1, is more prone to degradation. In this case NCK-1 may play a dual role and may also promote UNC-34 function as well. It is also likely VAB-1 signaling could affect the unc-34 transcriptional level. Future experiments should resolve how VAB-1 regulates UNC-34 protein levels.We propose that VAB-1 negatively regulates UNC-34 for PLM termination. This is supported by our observations that: 1) the loss-of-function Our finding that VAB-1 negatively regulates UNC-34/Ena is different from a previous report that shows mammalian EphB4 as an activator of Ena/VASP vab-1, nck-1 and wsp-1 mutants share the same phenotype of PLM axon overextension, that both nck-1 and wsp-1 were able to partially suppress the MYR-VAB-1 PLM termination defect, that VAB-1, NCK-1 and WSP-1 are able to form a complex in vitro, and that the activation of the Arp2/3 complex via the WSP-1 VCA domain resulted in PLM termination defects similar to MYR-VAB-1. The role of N-WASP as a negative regulator of axon elongation has been shown by two separate reports, where the reduction of N-WASP resulted in the enhancement of axon elongation wsp-1 animals. There have been conflicting reports on the role of the Arp2/3 complex in axon elongation, where some reports suggest that the Arp2/3 complex acts as a negative regulator of axon elongation in vitroC. elegans epithelial cells unc-34 and wsp-1 function redundantly for epithelial cell migrations Our results provide evidence that VAB-1/Eph RTK can regulate the actin cytoskeleton through its interaction with NCK-1 and WSP-1. This is based on our observation that C. elegans intersectin (ITSN-1) is expressed in the nervous system, and it is enriched in presynaptic regions and has roles in neurotransmission N-WASP has been shown to interact in a complex with the mammalian EphB2, through the adaptor molecule intersectin unc-34 and nck-1 mutants have opposite PLM axon phenotypes, it suggests that nck-1's role in axon outgrowth is more dispensable or redundant than its role in axon termination. Once the VAB-1/Eph RTK receives the signal to inhibit axon extension, VAB-1 is autophosphorylated and provides a docking site (Y673) for NCK-1. The NCK-1-SH2 domain binds the activated VAB-1 receptor and this disrupts the interaction between NCK-1 and UNC-34 to release the inhibitory effect of UNC-34 on NCK-1. Through an unknown mechanism, we also show that VAB-1 negatively regulates the UNC-34/Ena protein levels. VAB-1/NCK-1 can now recruit and activate WSP-1 and all three proteins form a complex, which results in high levels of Arp2/3 activation, ultimately leading to a branched meshwork of actin filaments. The combined actions of VAB-1/Eph blocking UNC-34/Ena activity, while activating Arp2/3 through NCK-1/WSP-1 contributes to the molecular events required to stop the growth cone forward movement.We propose a model of how the proteins VAB-1, NCK-1, UNC-34, WSP-1 and Arp2/3 function in axon growth cones for extension and termination . During C. elegans strains were manipulated as described by Brenner zdIs5[mec-4::gfp]; LGII: vab-1(dx31), quIs5[mec-4::myr-vab-1]; LG IV: wsp-1(gm324), LG V: unc-34(e566); LGX:, quIs6[unc-34::unc-34::gfp]; Unmapped: quIs16[hs::myr-vab-1]quEx131[mec-4::nck-1A], quEx190[nck-1::nck-1A-gfp]quEx215[mec-4::unc-34::gfp], quEx281[mec-4::unc-34], quEx283 [mec-4::nck-1A::gfp]quEx321[mec-4::vca], quEx338[mec-4::unc-34 RNAi] (see tissue specific RNAi). Unless noted otherwise, all C. elegans strains were obtained from the C. elegans Genetics Stock Center, (U. of Minnesota).All mec-4 promoters on each side of a Multiple Cloning Site (MCS) such that the sense and antisense strands of an inserted cDNA would be transcribed. The mec-4::unc-34 RNAi construct (pIC727) was created by cloning an unc-34 cDNA fragment  into the pIC659 dual Pmec-4 RNAi cloning vector.To produce double stranded RNA (dsRNA) only in the mechanosensory neurons, we constructed a cloning vector (pIC659) with head to head Pmec-4::nck-1A construct (pIC313) was previously described in Mohamed and Chin-Sang (2011). The mec-4::unc-34 construct (pIC624) was generated by amplifying unc-34 cDNA and sub-cloning behind the mec-4 promoter. The same procedure was used to make the mec-4::unc-34::gfp (pIC540) construct, but unc-34 was fused to gfp amplified from pPD95.75. To create the mec-4::vca construct (pIC673), the VCA region of WSP-1  was amplified from genomic DNA and cloned behind the mec-4 promoter. The unc-34::unc-34::gfp translation reporter was generated by a PCR fusion approach unc-34, 2. Exons 2–7 were amplified from RB2 cDNA library, and 3. a 868 bp GFP fragment amplified from pPD95.75 (gift from Dr. Andrew Fire). The expression of the UNC-34::GFP rescued the unc-34(e566) uncoordinated phenotype. Details of plasmid/PCR constructs and primer sequences are available upon request.The unc-34::unc-34::gfp translational reporter was injected at a concentration of 20 ng/µL, and one of the unc-34 rescuing lines (quEx61) was integrated to create quIs6. The mec-4::unc-34 construct was injected at a concentration of 30 ng/µL into mec-4::gfp(zdIs5); mec-4::myr-vab-1(quIs5). The mec-4::unc-34::gfp construct was injected at a concentration of 30 ng/µL into N2. mec-4::unc-34RNAi, mec-4::vca and mec-4::nck-1 were injected into mec-4::gfp(zdIs5) at 30 ng/µL. mec-4::nck-1(quEx131) was later crossed into unc-34(e566), and mec-4::unc-34RNAi (quEx338) was crossed into vab-1(dx31) and wsp-1(gm324). Transgenic animals were identified by the co-injection marker pRF4/rol-6 (30 ng/µl), or odr-1::rfp (30 ng/µl) Transgenic animals were generated by germ-line transformation as previously described Mixed stage animals were fixed and stained as described in Chin-Sang et al. (1999) mec-4::gfp (zdIs5) reporter. Young adult animals were scored as having PLM axon overextension or premature termination as described previously (zdIs5) had L1 PLMs with an average PLM length of 108.5 (±5.5) microns with a PLM length/total body length (from head to tail) ratio of 0.48 (±0.04). L1 PLM axons were scored as overgrown if they were longer than 114 µm and had a PLM/total body length ratio of greater than 0.52. L1 PLM axons were scored as under grown if the PLMs were shorter than 103 µm and had a PLM length/total body length ratio of length less than 0.44. The L1 PLM axons were traced from photograph and measured in NIH Image J software. The wild-type neuron morphology was defined by analysis of neuronal GFP reporters and is consistent with the electron microscopic reconstruction of the C. elegans nervous system The mechanosensory neurons were visualized using the mec-4::gfp (zdIs5) reporter. Eggs were allowed to hatch for 5 minutes, and the newly hatched L1 animals were examined immediately on 3% agarose pads with a drop of 0.2% tricaine and 0.02% tetramisole in M9. PLM growth cones were imaged with a Zeiss LSM710 confocal microscope at intervals of 20–30 s. Axons were scored positive for filopodia if time-lapse movies revealed at least 2 protrusions, and there were dynamic movements (eg. growth and collapse) of the these structures within the 10–15 minutes of filming. See Videos S1 and S2 for examples.PLM growth cones were visualized using a vab-1 was cloned into pGBKT7 (pIC187) and used in a screen against the RB2 cDNA library (gift from Dr. R. Barstead), and about 600,000 colonies were screened and two independent nck-1 cDNA clones were isolated. Site directed mutagenesis  of pIC187 was used to change the juxtamembrane tyrosine 673 changed to glutamic acid (Y673E). The SH2 domains of NCK-1, MIG-10, SEM-5, ABL-1 and VAV-1 were cloned into the activation domain of the pGADT7 vector. Primer sequences and details of plasmid constructs are available upon request.Yeast cells were grown on standard and selective media as required st NCK-1 SH3 domain (1 aa–72 aa), pIC593 – 2nd NCK-1 SH3 domain (112 aa–186 aa), pIC309 – 3rd NCK-1 SH3 domain (198 aa–308 aa), pIC324 – full length (F.L.) NCK-1 (1 aa–397 aa), and pIC606 – F.L. UNC-34 (1 aa–454). The following constructs were created by cloning the desired cDNA fragment into Maltose Binding Protein : pIC225 – F. L. intracellular region of wild-type VAB-1 (581 aa–1117 aa), pIC119 – F. L. intracellular kinase deficient VAB-1 (G912E), pIC603 – UNC-34 RPO-EVH2 domain (128 aa–454 aa), pIC605 – F.L. UNC-34 (1 aa–454 aa), pIC671 – UNC-34 PRO domain (128 aa–274), pIC674 – UNC-34 EVH2 domain (246 aa–454 aa), pIC670 – WSP-1 VCA domain (334 aa–607 aa). pIC582 – His-6::VAB-1 (581 aa–1117 aa) was described in Brisbin et al (2009). All fusion constructs were expressed in E. coli Tuner (DE3). For stSH3), GST-NCK-1(2ndSH3), GST-NCK-1(3rdSH3), GST-NCK-1(SH2), His-6::VAB-1(581 aa–1117 aa) (pIC582) or GST-NCK-1 F.L. coexpressed with pIC582 bound to 50 µl glutathione sepharose beads . Unbound fractions were collected, protein bound to GST beads were washed four times , and a proportional loading of each sample was analyzed by standard SDS polyacrlyamide gel, followed by western blotting. All loads fused to MBP were detected using anti-MBP conjugated to HRP (New England Biolabs). His6-VAB-1 was detected using Rabbit anti-VAB-1 antibodies (antigen VAB-1-His6) : pIC282 – NCK-1 SH2 domain (298 aa–397 aa), pIC297 – all three NCK-1 SH3 domains (1 aa–308 aa), pIC308 – 1-1-His6) . GST andVideo S1PLM growth cone of wild-type animals. Time-lapse confocal imaging of wild-type (N2) PLM growth cone. Images were taken at 30 s intervals for 15 minutes.(MOV)Click here for additional data file.Video S2myr-vab-1) transgenic animals. Time-lapse confocal imaging of myr-vab-1 transgenic animals. Images were taken at 30 s intervals for 10 minutes.PLM growth cone of hyperactive VAB-1 ((MOV)Click here for additional data file.


Here, we adapted this glass slide-based MeFISH to the study of intact embryos, and established a method called whole-mount MeFISH. This method can be applied to any DNA sequences in theory and, as a proof-of-concept experiment, we examined the DNA methylation status of satellite repeats in developing mouse primordial germ cells, in which global DNA demethylation is known to take place, and obtained a result that was consistent with previous findings, thus validating the MeFISH method. We also succeeded in combining whole-mount MeFISH with immunostaining or RNA fluorescence in situ hybridization (RNA-FISH) techniques by adopting steps to retain signals of RNA-FISH or immunostaining after harsh denaturation step of MeFISH. The combined methods enabled the simultaneous visualization of DNA methylation and protein or RNA expression at single-cell resolution without destroying embryonic and nuclear structures. This whole-mount MeFISH technique should facilitate the study of the dynamics of DNA methylation status during embryonic development with unprecedented resolution.To understand the spatiotemporal changes in cellular status that occur during embryonic development, it is desirable to detect simultaneously the expression of genes, proteins, and epigenetic modifications in individual embryonic cells. A technique termed methylation-specific fluorescence  Despite its importance, methods of visualization of the epigenetic status at specific loci in individual cells have not been pursued extensively. DNA methylation is one of the most important epigenetic modifications that regulate gene expression in many organisms in situ analysis. Tanaka et al. in situ hybridization (MeFISH), for the detection of the DNA methylation status at specific sequences in individual cells . The sequence of the probe was 5′–catccacttgacgacttgaaaatgacBaaatcactaaaaaacgtg–3′ (B indicates a bipyridine-attached adenine derivative).The ICON probe for mouse major satellite repeats was the same as that used by Li et al. Blimp1-mRFP transgenic mouse line established by Sugimoto and Abe The Before conducting MeFISH, immunostaining or RNA-FISH, whole embryos or embryonic tissues were subjected to permeabilization and fixation as described previously As permeabilization is performed before fixation in the MeFISH protocol, leakage of target antigens from the cell samples may occur. Therefore, it is important to establish appropriate permeabilization conditions when immunocytochemistry is combined with MeFISH. For immunostaining of PGC precursors of 6.5 dpc embryos with an anti-RFP antibody, we were able to retain the immunofluorescence signal in the PGC precursors using the conditions described above.2OsO4•2H2O and a Tris-HCl solution (100 mM Tris-HCl (pH 7.4), 1 mM EDTA, 2 M NaCl, and 0.1% Triton X-100). Probes that were not cross-linked were removed by denaturation at 80°C for 15 min in a solution containing 90% formamide, 2xSSC and 0.1% Triton X-100, followed by washing in PBST. The biotinylated ICON probe was detected using the Tyramide Signal Amplification (TSA) Kit . After incubation of the samples in 3% BSA in PBST, endogenous peroxidase activity was quenched by treatment with 0.6% hydrogen peroxide for 10 min. After several washes in PBST, the samples were reacted with a streptavidin–HRP conjugate in 3% BSA in PBST for 30 min. The samples were washed three times in PBST for 5 min at 37°C, followed by incubation with a fluorescence-labeled tyramide solution (Alexa Fluor 488 or 546) with 0.0015% hydrogen peroxide. The tyramide solution used in this step had a different fluorescence wavelength from the one used in immunostaining or RNA-FISH. The samples were washed several times in PBST, and nuclear DNA was stained with TO-PRO-3 . Samples were imaged with a confocal fluorescence microscope .Samples processed as above were subjected to whole-mount MeFISH. Samples were incubated sequentially in 2xSSC with 0.1% Triton X-100 for 10 min, 2xSSC and 25% formamide with 0.1% Triton X-100 for 10 min, and 2xSSC and 50% formamide with 0.1% Triton X-100 for 10 min twice. After prehybridization of the samples in hybridization buffer , 0.1% Triton X-100, and 50% formamide) for 20 min, the samples were placed in a 1.5 ml plastic tube containing 30 µl of the hybridization buffer containing the major satellite ICON probe (0.1 ng/µl). After denaturation of genomic DNA by heating the samples at 98°C for 5 min, hybridization was performed overnight at room temperature (approximately 25°C). The samples were then washed three times in 2xSSC with 0.1% Triton X-100 at room temperature for 5 min, to remove unhybridized probe. To cross-link ICON probes with methylated target sequences, the samples were incubated in 100 µl of cross-link solution at 30°C for 10 min. The cross-link solution was made by mixing an equal amount of 25 mM KThe permeabilization and fixation of the samples were performed as described above. After incubation in blocking buffer (1% BSA in PBST) for 60 min, the samples were reacted with the primary antibody ; for OCT4, 1∶300 goat polyclonal anti-Oct-3/4 (N-19) ) in blocking buffer at 4°C overnight. When combining immunostaining with MeFISH, we used the TSA Kit for the detection of the target protein. Briefly, after PBST wash and quenching of endogenous peroxidase activity with 0.6% hydrogen peroxide, the samples were incubated with the secondary antibody conjugated with horseradish peroxidase (HRP) ; for OCT4, rabbit anti-goat IgG conjugated with HRP ) in blocking buffer at 4°C overnight. After rinsing with PBST, the samples were incubated with a fluorescence-labeled tyramide solution (Alexa Fluor 488 or 546) with 0.0015% hydrogen peroxide. After washing several times in PBST, the samples were then subjected to whole-mount MeFISH experiment.Xist RNA was prepared as follows. A single-stranded RNA corresponding to a part of the Xist cDNA  was synthesized by in vitro transcription using T7 RNA polymerase . Using the in vitro-transcribed RNA as a template, a biotinylated single-stranded DNA probe was generated by random-primed reverse transcription using SuperScript III reverse transcriptase . Samples subjected to RNA-FISH were processed using the same procedures as those described in the whole-mount MeFISH protocol, up to the prehybridization step. The biotinylated Xist probe dissolved in hybridization buffer was denatured by heating at 70°C for 10 min and the samples were added into the hybridization mixture. Hybridization was performed at 42°C overnight and the samples were washed twice with a solution containing 2xSSC, 50% formamide and 0.1% Triton X-100 for 5 min at 42°C, and twice with 2xSSC containing 0.1% Triton X-100 for 5 min at 42°C. For the fluorescence labeling of the probes and stabilization of the signals, TSA was performed as described above. After TSA procedures, the Endogenous Biotin-Blocking Kit  was used to block unreacted biotin in the probe with the streptavidin–HRP conjugate. The samples were then used in whole-mount MeFISH experiments.A strand-specific DNA probe for the detection of the The original MeFISH protocol was created for the analysis of either dissociated cells or frozen sections adhered to a glass slide Blimp1/Prdm1 in mouse 6.5 dpc embryos Blimp1-mRFP transgenic mice, in which PGC precursors can be detected by the expression of monomeric red fluorescent protein (mRFP) Next, we attempted to assess the methylation status of the satellite sequences in precursors of mouse PGCs using a combination of whole-mount MeFISH and immunocytochemistry. Global loss of DNA methylation in PGCs is observed at around 8.0 dpc To address whether these techniques can be applied to larger samples, such as pieces of embryonic tissues or organs from embryos at later developmental stages, and to determine if differences in the DNA methylation levels between distinct cell lineages can be detected, we performed a MeFISH analysis of both somatic cells and PGCs in the genital ridge of 12.5 dpc embryos; PGCs within the genital ridges of this stage should be globally demethylated in situ hybridization Xist RNA, which is a noncoding RNA that is essential for X chromosome inactivation Xist RNA covers the entire inactive X chromosome of female mammalian cells, forming an “Xist RNA cloud”, which is a hallmark of inactive X chromosomes Xist RNA-FISH on 6.5 dpc mouse embryos, followed by whole-mount MeFISH, as shown in Xist were detected clearly as clouds, and the MeFISH signals were also visible at the chromocenters, as observed in Finally, we challenged detection of DNA methylation signals and RNA expression simultaneously via a combination of the whole-mount MeFISH and RNA-FISH techniques. As RNA molecules are unstable compared with DNA, it is desirable to perform RNA-FISH before the MeFISH procedure. However, both the genomic DNA and non-cross-linked probe denaturation steps of the MeFISH protocol would dissociate the probe–target RNA hybrids generated by the RNA-FISH procedure. To overcome this problem, we used the tyramide signal amplification (TSA) technique . TSA is one of the signal amplification techniques that are used for immunostaining and Li et al. The methylation status of repeat DNA sequences has served as an important measure of the progress of epigenetic reprogramming Xist RNA expression in the same cell within mouse embryos. To our knowledge, this is the first report of the simultaneous detection of transcribed RNAs and specific DNA methylation at the single-cell level. This was enabled by the use of the TSA method, which is normally used for signal amplification enhancement DNA methylation dynamics has been studied to understand how developmental gene expression is regulated by epigenetic changes. However, there are no reports describing the relationship between the methylation of regulatory DNA sequences and gene expression with single-cell resolution, because no methods have been developed for the simultaneous detection of RNA expression and specific DNA methylation in the same cell. With this goal in mind, we combined whole-mount MeFISH and RNA-FISH, and successfully detected the methylation status of the satellite sequences and When MeFISH is combined with RNA or protein expression analysis, the harsh denaturation step of the MeFISH causes the loss of signals detected by RNA-FISH or immunostaining, making it difficult to perform such combined analysis. In particular, we experienced that RNA-FISH signals were almost completely lost during the denaturation step of the MeFISH procedure (data not shown). When RNA-FISH and DNA-FISH combination is conducted in samples fixed on a slide glass, the signals of RNA-FISH performed before DNA-FISH are imaged and the xy coordinates of the images on the glass are recorded. DNA-FISH images are then matched to the RNA-FISH signals according to the position information obtained previously, thus enabling detection of RNA- and DNA-FISH signals in the same cells in situ seems to be the number of fluorophores that associate with the target DNA region. In our preliminary experiments, MeFISH signals for mouse minor satellite sequences were detected without any signal amplification (data not shown). Mouse minor satellite repeats span a 300–600 kb sequence at the centromeric region of all chromosomes other than the Y chromosome and consist of a 120 bp unit In situ detection of chromosomal loci has been attempted using various fluorescent-labeling means In this study, MeFISH was performed using probes that targeted multicopy repeat sequences. As the ICON probe was originally developed to examine the CpG methylation status of any single-copy sequence using a PCR-based assay In conclusion, whole-mount MeFISH exhibited at least two advantages: 1) it provides spatiotemporal information on DNA methylation changes at specific loci and 2) theoretically, it can retrieve information on the DNA methylation of specific sequences, localization of specific proteins, and expression of specific RNA transcripts with single-cell resolution. Therefore, the whole-mount MeFISH technique should provide new opportunities for the study of the epigenetic basis of cellular dynamics during embryonic development.Figure S1Overview of MeFISH. After denaturation of genomic DNA in samples, labeled ICON probes are hybridized with target DNA. Hybridized ICON probes are cross-linked by osmium treatment when the target cytosine is methylated (or hydroxymethylated) (TIF)Click here for additional data file.Figure S2Overview of tyramide signal amplification (TSA) in RNA-FISH. (1) Biotinylated probes (arrows with white circle) are hybridized with nascent target RNA (wavy lines).  After binding of streptavidin–HRP conjugate (indicated as “HRP”) to biotin in the probes, fluorescence-labeled tyramide molecules  are activated by the conjugated HRP. (4) Activated tyramide molecules  bind covalently to proteins that are adjacent to RNA–probe hybrids. (5) The labeled tyramides with fluorescence labeling remain as RNA-FISH signals even after dissociation of the probes during MeFISH procedures (black stars indicate fluorescence labeling).(TIF)Click here for additional data file.Movie S1Movie of the optical serial sections shown in .(MOV)Click here for additional data file.Movie S2Movie of the 3D reconstructed images shown in .(MOV)Click here for additional data file.


Steady state dendritic cells (DC) found in non-lymphoid tissue sites under normal physiologic conditions play a pivotal role in triggering T cell responses upon immune provocation. CD11b+ and CD103+ DC have received considerable attention in this regard. However, still unknown is whether such CD11b+ and CD103+ DC even exist in the ocular mucosa, and if so, what functions they have in shaping immune responses. We herein identified in the ocular mucosa of normal wild-type (WT) and Flt3-/- mice the presence of a CD11b+ DC . CD103+ DC (i.e. CD11c+ MHCII+ CD11b low CD103+ CD8a+ DEC205+ Langerin+) were also present in WT, but not in Flt3-/- mice. These CD103+ DC expressed high levels of Id2 and Flt3 mRNA; whereas CD11b+ DC expressed high Irf4, Csfr, and Cx3cr1 mRNA. Additionally, the functions of these DC differed in response to allergic immune provocation. This was assessed utilizing a previously validated model, which includes transferring specific populations of exogenous DC into the ocular mucosa of ovalbumin (OVA)/alum-primed mice. Interestingly, in such mice, topical OVA instillation following engraftment of exogenous CD11b+ DC led to dominant allergic T cell responses and clinical signs of ocular allergy relative to those engrafted with CD103+ DC. Thus, although CD11b+ and CD103+ DC are both present in the normal ocular mucosa, the CD11b+ DC subset plays a dominant role in a mouse model of ocular allergy. As the most potent stimulators of T lymphocytes, dendritic cells (DC) are widely appreciated as a very unique subpopulation of antigen presenting cells and key in the generation, as well as the regulation, of adaptive immune responses. Much of their proficiency in T cell priming is attributed to high-level expression of MHC class II and costimulatory molecules such as CD80 and CD86. In addition, steady state DC, which take up residence in uninflamed non-lymphoid tissues such as the mucosa, are also highly efficient in mobilizing to lymphoid organs for Ag presentation to T cells.Now fully appreciated is the understanding that DC comprise multiple populations with differing ontogenies Considerable attention has also been focused on attempting to elucidate the precise functional roles of CD103+ and CD11b+ DC; however, many questions still remain. Indeed, numerous reports have converged on the importance of viral Ag cross presentation by CD103+ DC Many of the mucosal tissue sites  have already been probed for identification and function of CD11b+ and CD103+ DC, with an exception for the ocular mucosa  We therefore investigated this in the current study, and were able to determine that CD11b+ and CD103+ DC exist in the ocular mucosa of mice. Interestingly, use of a previously described model of ocular allergy, whereby exogenous DC  are engrafted into the ocular mucosa in topically challenged mice Steady state CD11b+ and CD103+ DC were previously identified in numerous tissues of the mouse, including the liver and lung Conclusive identification of CD103+ and CD11b+ DC in the ocular mucosa required further investigation, which we in part accomplished by querying phenotypic expressions unique to these DC subsets To further verify our identification of CD103+ and CD11b+ DC in the conj, we also examined unique transcriptional programs expressed by these subsets With the conclusive identification of CD103+ and CD11b+ DC in normal conj, we next moved on to assess the role of these respective DC subsets in a model of ocular allergy. We utilized our previously described model to accomplish this In the current study we utilized bone marrow (BM) precursors to differentiate CD11b+ DC via standard GMCSF conditioning We also examined the role of CD11b+ vs. CD103+ DC in mediating clinical signs of ocular allergy, which include conjunctival chemosis, hyperemia, lid edema, and tearing/discharge We next determined whether these results were relevant with responses generated by already differentiated CD11b+ and CD103+ nonlymphoid tissue DC. To accomplish this, we FACS sorted lung CD11b+ vs. CD103+ DC Fig. 5a Little is known about steady state DC subsets in the ocular mucosa, particularly regarding CD103+ and CD11b+ DC. One report by Ohbayashi et al identified via immunohistochemistry the presence of CD11b expressing DC in the normal mouse conjunctiva We were able to identify the presence of these DC in the ocular mucosa via direct comparison with CD103+ and CD11b+ DC previously described in the liver and lung To assess the functional role of these DC subsets in ocular allergy, we utilized our model previously described by Schlereth et al It is important to highlight, however, that our identification of a steady state CD11b+ DC dominance in the elicitation of allergic immune responses was only demonstrated here in a model of secondary immune responses. This is because CD11b+ or CD103+ DC were engrafted into mice adoptively transferred with OVA sensitized T cells, and consequent allergic responses to topical OVA challenges is predominantly due to reactivation of sensitized T cells Also requiring further investigation is the exact reasons by which CD11b+ DC are dominant in secondary allergic immune responses, as seen here and by others Also not offering a clear explanation as to the reasons for the dominant role for CD11b+ DC seen here, are other superior attributes previously described. CD11b+ DC are known to be proficient in phagocytosis, which could be advantageous with respect to allergen uptake In conclusion, we show here that steady state CD11b+ and CD103+ DC are present in the ocular mucosa; however, these DC have disparate roles in mediating adaptive immune responses. In a model of ocular allergy, wherein CD11b+ vs. CD103+ DC were engrafted into the ocular mucosa of mice adoptively transferred with sensitized T cells and challenged topically with allergen, revealed that CD11b+ DC are dominant in triggering secondary allergic immune responses relevant in ocular allergy. Future work is required to understand why CD11b+ DC are dominant in secondary allergic immune responses and whether such cells can be targeted therapeutically in treatment of atopic conditions such as allergic eye disease.Female C57BL/6 and Flt-3 knockout mice 8–12 wk old were purchased from Charles River Laboratories  and Jackson laboratories . Mice were housed in a specific pathogen-free environment at the Schepens Eye Research Institute animal facility. The Institutional Animal Care and Use Committee approved all procedures. All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Anesthesia was used for all surgical procedures with intraperitoneal administered ketamine/xylazine suspensions .For CD103 Ab we used clone 2E7 on PERCP-Cy5.5 (Biolegend). For CD11b Ab, we used clone M1/70 on PERCP-Cy5.5 (Biolegend), or PE (BD). For CD11c Ab, we used clone N418 on Alexa 488 or PE-Cy5 (Biolegend), or clone HL3 on PE-Cy7 (BD). For langerin (CD207) Ab we used clone eBioL31 on Alexa 647 (eBioscience). For CD45 Ab, we used clone 30-F11 on Alexa 488, PE, APC-Cy7 (BioLegend), or PE-Cy5 (BD). For DEC205 (CD205), we used clone NLDC-145 on PE-Cy7 (BioLegend). For F4/80 Ab, we used clone BM8 on APC-Cy7 (BioLegend). For I-A/I-E, we used clone M5/114.15.2 on PE-Cy7 or APC-Cy7 (BioLegend). For CD4, we used clone RM4-5 on PerCP-Cy5.5 (BioLegend); IL-4 on PE-Cy7 using clone 11B11 (BD Pharmingen); IL-5 on PE using clone TRFK5 (BioLegend); IL-13 on Alexa 647 using clone eBio13A (eBiosciences); and IFN-g on APC-Cy7 using clone XMG1.2 (BD Pharmingen). Cells were also stained with DAPI or fixable viability dye (eBioscience) for analysis of viable cells. All samples were pre-treated with purified Fc block (CD16/32) Ab clone 93 (eBioscience) according to manufacturer's instructions prior to staining. Aliquots were made for appropriate isotype controls.This procedure has been previously described Viable cells from single cell suspensions were enumerated via trypan blue exclusion assay, and subsequently stained for CD11c, I-A/E, CD11b, CD103 and live/dead, then washed thoroughly. CD11b+ DC were viability dye- CD11c+ I-A/E+ CD11b hi autofluorescence- , CD103-. In contrast, CD103+ DC were viability dye- CD11c+ I-A/E+ CD11b-/low autofluorescence- CD103+.Purified cells obtained via FACS sorting were used. The differential expression of selected genes was verified by using quantitative real-time PCR (qPCR) procedures. Total RNA was isolated using Trizol (Invitrogen) and RNeasy Microkit (Qiagen). The first strand complementary DNAs (cDNA) were transcribed by employing SuperScript III Reverse Transcriptase  and random hexamer primers (Invitrogen). Quantitative real-time polymerase chain reaction was performed in duplicates of the same concentration of cDNAs using Taqman Universal PCR Mastermix and FAM-MGB dye-labeled predesigned primers and probes  for Interferon regulatory factor 8 (Irf8) (Mm01250091_m1), Interferon regulatory factor 4 (Irf4) (Mm00516434_m1), Inhibitor of DNA binding 2 (ID2) (Mm00711781_m1), FMS-like tyrosine kinase 3 (Flt3) (Mm00439000_m1), Colony stimulating factor 3 receptor (granulocyte) (Csfr) (Mm01266656_g1), Chemokine (C-X3-C) receptor 1 (Cx3cr1) (Mm02620111_s1) and as the endogenous control, the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Mm99999915_g1). Differential gene expression was calculated according to theComparative Ct method outlined in Applied Biosystems User Bulletin 2 (updated 2001). The relative expression level of each sample was expressed as fold change from naïve control.BM derived DC were generated as previously described For BM-derived DC, cells were harvested on day 7 of cultures and prepared for magnetic sorting according to manufacturer's instructions . For CD11b+ DC, GM-CSF cultivated cells were MACS sorted using anti-CD11c beads alone, as all CD11c+ cells co-express CD11b+. For CD103+ DC, Flt-3 cultivated cells were sorted using anti-CD103 beads, as all CD103+ cells co-express CD11c+; <90% purity was achieved for both subsets. Purified cells were washed thoroughly and prepared for subconjunctival injection.Following enzymatic digestion, DC were first purified magnetically via anti-CD11c beads, according to manufacture's instructions (Miltenyi Biotec). Cells were then thoroughly washed and enumerated. Cells were then stained for FACS sorting as detailed above.This method in allergic eye disease has been previously described This model was previously described This procedure has been described previously Regional LN  were collected and pooled from freshly euthanized mice. Single-cell suspensions were prepared and enumerated via trypan blue exclusion assay. Cells were plated in round-bottom 96-wells at a concentration of 2.0×10ˆ6/ml in triplicate wells of RPMI (10% FBS) with OVA (1 mg/ml) for up to 72 hr and restimulated with PMA/Ionomycin with Golgiplug (BD Pharmingen) for up to 4 hours. Cells were harvested and stained for extracellular CD4, and intracellular IL-4, IL-5, IL-13, and IFN-g. Parallel cultures were established for ELISA analyses of culture supernatant, and thus restimulated with PMA/Ionomycin only. ELISAs (eBioscience) for IFN-g and IL-13 were analyzed.p-value <0.05 was considered statistically significant.Statistical analyses included 1-way ANOVA and Bonferroni's Multiple Comparison Test, in addition to two-tailed student's t-test. Standard error and standard deviation of the mean were calculated. A


Beet Necrotic Yellow Vein virus (BNYVV) is a member of the genus Benyvirus causing a worldwide sugar beet disease rhizomania. BNYVV contains four or five plus-sense single stranded RNAs. In altered selective conditions, multipartite RNA viruses of plant are prone to undergoing internal deletions, thus turning into Defective RNAs (D RNAs). Although several D RNAs have been reported in BNYVV infection, the spontaneous internal deletion mutants responsible for severe symptom in systemic host Nicotiana benthamiana (N. benthamiana) are not described so far.N. benthamiana was inoculated by Chinese BNYVV isolates. RT-PCR and Northern blot showed that the D RNAs forms of BNYVV RNA3 were present in the systemic infection of the N. benthamiana. Three distinct D-RNA3s, named as D-RNA 3α, D-RNA 3β and D-RNA 3γ, were made into infectious clones. When inoculated on the N. benthamiana, the in vitro transcripts of D forms exhibited more stable than that of wild-type RNA3 in systemic movement. Among the detected mutant, the p25 protein frame-shift mutant (D-RNA3α) induced obvious necrotic lesions on Tetragonia.expansa (T. expansa) and pronounced systemic symptom on the N. benthamiana. The D-RNA3α was further mutated artificially to pre-terminate the downstream N protein, leading to the abolishment of the pathogenicity, indicating the N protein was responsible for the necrotic symptom.Systemic host N. benthamiana. The internal deletions didn't affect the efficient replication of D-RNA3s, instead by improving the stability and pathogenicity of RNA3 in the systemic host N. benthamiana. Besides, our results also suggested the downstream N protein of RNA3, but not the upstream p25 protein, may play an important role in the systemic infection on N. benthamiana.Our studies demonstrated the internal deletion mutants of BNYVV-RNA3 were spontaneously generated in the systemic infection on  Defective RNAs (D RNAs), which have internal deletion of viral sequences, have been described for a variety of plant viruses ,2. Some Beet Necrotic Yellow Vein virus (BNYVV), transmitted by zoospores of Polymyxa betae, is a member of the genus Benyvirus causing a worldwide sugar beet disease rhizomania  were used. The PCR products were purified and cloned into the pMD19-T vector to obtain a full-length cDNA clone named pMDR3 and three D RNAs cDNA clones, pMDRNA3α, pMDRNA3β and pMDRNA3γ . The freshly synthesized transcriptions were mixed with total RNA of T. expansa infected by isolate BN12, the mixture supplemented with an equal volume of inoculation buffer  were rubbed onto T. expansa, C. amaranticolor or N. benthamiana leaves. Local lesions generally appeared at 5-8 days post inoculation, while systemic symptom of N. benthamiana appeared at 12-14 dpi.Infectious plasmids were linearized by T. expansa leaves and systemic infected N.benthamiana were harvested at 7 and 14 dpi, respectively. Total RNA was extracted for RT-PCR detection and Northern hybridization, as described previously [P-labeled cDNA specific for RNA1 (nt5815-6531), RNA2 (nt145-714), and RNA3 (nt445-1102) sequences, respectively.Inoculated eviously . Probes The following primers were used for RT-PCR: for detection of RNA 2, the forward primer BN81  and the reverse primer BN80 , for detection of RNA3, the forward primer BN78  and the reverse primer BN77 , for detection of RNA 4, the forward primer BN209  and the reverse primer BN210 (5'-CTAATCGTGATAAAAGACAAACCA-3'complementary to nt1205 to 1228) and for detection of RNA 5, the forward primer BN225  and the reverse primer BN226 .The authors declare that they have no competing interests.YW carried out most of the experiments and wrote the manuscript. HF anticipated the inoculation and the construction of D form RNA3s. XW and ML provided useful advice. CH, DL and JY conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.