A model view of the embryonic <i>Drosophila</i> NMJ.

<p>In late <i>Drosophila</i> embryos, presynaptic motorneuronal boutons (blue) are attached with half of their surfaces to muscles (beige), and synapses (dashed ellipse) are assembled at these neuromuscular cell-cell contacts. Neuromuscular synapses contain presynaptic active zones with key components such as the scaffolding protein Bruchpilot (Brp) or the Cacophony (Cac) calcium channel including its associated subunit Straightjacket (Stj) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Owald1" target="_blank">[25]</a>. Postsynaptically, neuromuscular synapses contain clusters of GluRs composed of the three obligatory C, D and E subunits and the variable A and B subunits. For most CAMs, such as Leukocyte-antigen-related-like (Lar) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Johnson1" target="_blank">[107]</a>, Neuroligins (Nlg) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Sun2" target="_blank">[110]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Banovic1" target="_blank">[111]</a>, Neurexins (Nrx; as mentioned in text), classical cadherins (CadN; as mentioned in text), it remains to be clarified whether they localise within synapses or extra-synaptically; for Fasciclin2 (Fas2) peri-synaptic localisation has already been reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Sone1" target="_blank">[112]</a>. All these components are interlinked through intracellular scaffolds. Discs large (Dlg) selectively stabilises GluRB receptors at the synapse, but also anchors Shaker potassium channels (Sh) or Fas2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Thomas1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Chen2" target="_blank">[113]</a>. The band 4.1 superfamily protein Coracle (Cora) interacts with the carboxy-terminus of GluRIIA but not GluRIIB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Chen3" target="_blank">[114]</a>, but has likewise been shown to interact with Nrx-IV in other cellular contexts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Lamb1" target="_blank">[115]</a>. Links of the Lar-associated scaffold protein Liprin-α to Brp, or of Nrx-IV to Brp have been explained elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Owald1" target="_blank">[25]</a>. Many more interactions with further scaffold proteins on both sides of the junction are to be expected. The glycocalyx (stippled area) within the synaptic cleft forms a third scaffold established through the linkage of carbohydrate-side chains, often mediated through lectins, such as Mind-the-gap (Mtg). BM links in a Laminin A-dependent manner to cell surfaces through yet unidentified receptors (?), although PS-integrin-mediated Laminin A-independent adhesion at focal contacts has been described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Prokop3" target="_blank">[28]</a>. BM is likely to compete with motorneuronal terminals for muscle surface, and BM adhesion needs to be excluded from neuromuscular adhesions (blue T) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Prokop5" target="_blank">[116]</a>. Proteins downstream of the Mef2 transcription factor are likely to contribute to this process, as is suggested by complete loss of NMJ adhesion in <i>mef2</i> mutant embryos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Prokop4" target="_blank">[70]</a>.</p

Quantifications of ultrastructural NMJ phenotypes.

<p>Embryonic NMJ boutons displaying active zones (arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g001" target="_blank">Figs. 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g005" target="_blank">5</a>) were measured. Genotypes are grouped into combinations of cadherins and neurexins (<b>A</b>), Laminin-deficient conditions (<b>B</b>), combinations of <i>lanA^9.32</i> with loss of cadherins (<b>C</b>), loss of other potential adhesion factors (as explained below) in combination with <i>lanA^9.32</i> (<b>D</b>), loss of classical laminin receptors (<b>E</b>), and collagen type IV-deficient conditions (<b>F</b>). The following parameters were analysed: <i>“adhesion index”</i>, the percentage of the circumference of active zone-bearing boutons that is in contact with muscle membrane (between curved arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g001" target="_blank">Figs. 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g005" target="_blank">5</a>); “<i>synapse length</i>”, mean length of electron dense cleft material known to indicate synapse diameter (between double chevrons in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g001" target="_blank">Figs. 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g005" target="_blank">5</a>); “<i>cleft width</i>”, mean distance between pre- and postsynaptic membranes at synapses. <i>Bars</i> represent mean ± standard error of the mean; <i>n</i>, number of assessed NMJ boutons sampled from at least 5 embryos, respectively; <i>asterisks</i> indicate statistical significances as compared to wt (black asterisks) or lanA (grey asterisks; *, P≤0.1; **, P≤0.01; ***, P≤0.001; ****, P≤0.0001 according to Mann Whitney tests). Additional information on included CAMs not explained in the main text: the immunoglobulin adhesion receptor Klingon is suggested to express potential synaptic functions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Butler1" target="_blank">[88]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Matsuno1" target="_blank">[89]</a>; the immunoglobulin adhesion receptor Turtle acts as a homophilic adhesion factor in S2 cell assays which has demonstrated neuronal phenotypes <i>in vivo </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-AlAnzi1" target="_blank">[91]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Long1" target="_blank">[92]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Ferguson1" target="_blank">[104]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Bodily2" target="_blank">[105]</a>; the transmembrane heparan sulfate proteoglycan Syndecan (Sdc) might act as a CAM by serving as a ligand for the motorneuronal receptor Lar (Leukocyte-antigen-related-like, a close homolog of avian protein tyrosin phosphatase ó) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Fox1" target="_blank">[106]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Aricescu1" target="_blank">[108]</a>.</p

Examples of ultrastructural phenotypes of doubly or multiply mutant embryos.

<p>Images of neuromuscular bouton profiles (A–H) and close-ups of their respective synapses (A′–H′) in late stage 17 embryos of wildtype (<b>A</b>) or animals carrying the following mutant allele combinations: <i>CadN-CadN2(ΔN14)</i>, <i>stan¹⁹²</i> in homozygosis (<b>B</b>), <i>CadN-CadN2(Δ14)</i>, <i>stan¹⁹²</i>; <i>Nrx-1^Δ83</i>, <i>Nrx-IV⁴³⁰⁴</i> in homozygosis (<b>C</b>), <i>lanA^9.32</i>/<i>lanA^9.32</i> (<b>D</b>), <i>lanA^9.32</i>/<i>Df(3L)Excel8101</i> (<b>E</b>), <i>lanB1^DEF</i>/<i>lanB1^DEF</i> (<b>F</b>), <i>CadN-CadN2(ΔN14)</i>, <i>stan¹⁹²; lanA^9.32</i> in homozygosis (<b>G</b>), <i>GluRIIC¹; lanA^9.32</i> in homozygosis (<b>H</b>; see further info on GluRIIC in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g002" target="_blank">Fig. 2</a>). Symbols and abbreviations are consistently used for all micrographs throughout this manuscript: Bo, presynaptic bouton; Mu, postsynaptic muscle; Hl, haemolymph; black arrows, active zones; arrow heads, BMs; curved arrows, demarcate neuromuscular contacts; double chevrons, demarcate synapses; white arrow heads, cell surfaces lacking BMs. No changes in adhesion index or synaptic structure were detected in A–C, whereas the adhesion index in D–H was changed from ∼50% to ∼25% in the absence of any further structural changes (quantified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g002" target="_blank">Fig. 2A–D</a>). Scale bar in A represents 500 nm in A–H and 200 nm in A′–H′.</p

Exploring molecular mechanisms of Laminin A-dependent BM attachment.

<p>Images of neuromuscular bouton profiles (A–C) in late stage 17 embryos carrying the following mutant allele combinations: <i>mys^XG43; ßInt-v¹</i> in homozygosis (<b>A</b>), <i>Sdc⁹⁷</i>/<i>Sdc²³</i> (<b>B</b>), <i>Dg⁰⁴³</i>/<i>Dg⁰⁸⁶</i> (<b>C</b>); no changes in adhesion indices were detected (statistical validation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g002" target="_blank">Fig. 2E</a>); white arrows indicate pseudo-cell contacts separated by BMs, all other symbols as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g001" target="_blank">Fig. 1</a>. <b>D–E′</b>) Tissues of late stage 17 wildtype (left) or <i>lanA^9.32</i> mutant embryos (right): D–I) show flat-dissected whole body preparations (insets show the ventro-longitudinal muscles VL1-4) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Bate1" target="_blank">[109]</a>; D′–H′) show isolated CNSs; I′ shows a close up of a flat dissected embryo; preparations are immuno-stained against Laminin, Nidogen or Perlecan (in green; as indicated on the left) in combination with anti-HRP labelling neuronal tissues (magenta). Perlecan and Nidogen are still present within fragmented BMs of <i>lanA^9.32</i>-mutant embryos. Scale bar in A represents 600 nm in A, 200 nm in B and C, 80 µm in D–I (insets 2.5 fold enhanced), and 30 µm D–I′.</p

Protein levels of WAVE/SCAR complex subunits in different mutant contexts

<p><b>Copyright information:</b></p><p>Taken from "HSPC300 and its role in neuronal connectivity"</p><p>http://www.neuraldevelopment.com/content/2/1/18</p><p>Neural Development 2007;2():18-18.</p><p>Published online 25 Sep 2007</p><p>PMCID:PMC2098765.</p><p></p> Immunoprecipitation experiments in S2 cytoplasmic cell extracts using the anti-HSPC300 antibody. From left to right: anti-HSPC300 immunoprecipitation, IgG immunoprecipitation, input (cytoplasmic extract). Proteins are indicated to the right, corresponding molecular weights to the left. Quantitative analysis of CYFIP, Kette, SCAR, Abi and HSPC300 protein levels by western blot on third instar larval extracts of the following genotypes: wild type (WT); zygotic null; zygotic null and maternal hypomorph; zygotic null. Proteins are indicated to the right, corresponding molecular weights to the left. β-tubulin represents a loading control

The Epigenetic Regulator G9a Mediates Tolerance to RNA Virus Infection in <i>Drosophila</i>

<div><p>Little is known about the tolerance mechanisms that reduce the negative effects of microbial infection on host fitness. Here, we demonstrate that the histone H3 lysine 9 methyltransferase <i>G9a</i> regulates tolerance to virus infection by shaping the response of the evolutionary conserved Jak-Stat pathway in <i>Drosophila</i>. <i>G9a</i>-deficient mutants are more sensitive to RNA virus infection and succumb faster to infection than wild-type controls, which was associated with strongly increased Jak-Stat dependent responses, but not with major differences in viral load. Genetic experiments indicate that hyperactivated Jak-Stat responses are associated with early lethality in virus-infected flies. Our results identify an essential epigenetic mechanism underlying tolerance to virus infection.</p></div

Loss of <i>G9a</i> does not affect viral loads upon DCV infection.

<p>(<b>A,B</b>) Wild-type or <i>G9a</i> mutant flies were inoculated with DCV and viral titers were determined over time in (<b>A</b>) whole flies, and (<b>B</b>) dissected fat bodies. Data represent means and s.d of three independent experiments. Each experiment contained three biological replicates of 5 female flies (<b>A</b>), or 10 fat bodies (<b>B</b>) per replicate for each genotype. (<b>C</b>,<b>D</b>) DCV RNA levels over the course of 3 days post-infection analyzed by RT-qPCR in (<b>C</b>) whole flies or (<b>D</b>) fat bodies of wild-type and <i>G9a</i> mutant flies. DCV RNA levels were normalized to transcript levels of the housekeeping gene <i>Ribosomal Protein 49</i> and are calculated relative to the viral RNA levels in flies harvested immediately after inoculation (t₀). Data represent means and s.d. of three biological replicates of 5 female flies (<b>C</b>) or 10 fat bodies (<b>D</b>) per replicate for each genotype. Data in panel <b>C</b> and <b>D</b> are from one experiment representative of 2 independent experiments. *<i>P</i> < 0.05 (Student’s t-test).</p

Hyperactivation of the Jak-Stat pathway renders flies hypersensitive to virus infection.

<p>(<b>A</b>) Experimental set-up. Expression of the <i>Upd</i> transgene was induced specifically in adult flies using the <i>Gal4/Gal80ts</i> system. <i>Gal80ts</i> is a temperature-sensitive allele of the Gal80 inhibitor that binds Gal4 to prevent activation of gene expression at 20°C. At 29°C, Gal80ts is degraded, allowing Gal4 to bind to the Upstream Activating Sequences (UAS) to induce gene expression. Flies were reared at 20°C, and 0 to 3-day-old adults were conditioned at 29°C for 3 days prior to viral challenge. (<b>B</b>) Expression levels by RT-qPCR of <i>Upd</i> and <i>TotA</i> in flies carrying the temperature-dependent <i>Upd</i> overexpression system (<i>UAS-Upd; tubulin-Gal4/Gal80ts</i>) after 3 days conditioning at 29°C. The <i>Gal4</i> and <i>Gal80ts</i> transgenes were combined with the <i>UAS-Upd</i> by standard genetic crosses at 20°C and 0 to 3-day-old adult offspring was cultured for 3 days at 20°C or at 29°C before RNA levels were analyzed by RT-qPCR. Transcript levels of <i>Upd</i> and <i>TotA</i> were normalized to RNA levels of the housekeeping gene <i>Ribosomal Protein 49</i>, and expressed as fold change relative to control flies carrying only the <i>UAS-Upd</i> transgene. (<b>C</b>) Survival of flies carrying the temperature-dependent <i>Upd</i> overexpression system (<i>UAS-Upd; tubulin-Gal4/Gal80ts</i>) and genetic control flies upon DCV infection (1,000 TCID₅₀ = units) at 29°C. Data are means and s.d. of three independent pools of at least 10 male flies for each genotype. Data in (<b>C</b>) are from one experiment representative of 2 independent experiments. Differences in expression of <i>Upd</i> and <i>TotA</i> were evaluated with a Student’s t-test (*<i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001).</p

Genetic interaction between <i>G9a</i> and the Jak-Stat pathway.

<p>(<b>A,B</b>) Survival upon DCV infection (1,000 TCID₅₀ units) of wild-type or <i>G9a</i> mutant and wild-type flies overexpressing (<b>A</b>) a dominant negative version of the <i>domeless</i> receptor (dome^ΔCyt), or (<b>B</b>) the negative regulator of Jak-Stat signaling <i>Socs36E</i>. The UAS/Gal4 system was used to drive transgene expression. Gal4 is expressed under control of the actin promoter (<i>Act-Gal4</i>) to drive ubiquitous expression of the <i>UAS-dome</i>^ΔCyt and <i>UAS-Socs36E</i> transgenes. Control flies expressing only the <i>Act-Gal4</i>, the <i>UAS-dome</i>^ΔCyt, or the <i>UAS-Socs36E</i> transgenes were included as controls (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.s012" target="_blank">S5A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.s012" target="_blank">S5B</a> Dataset). Mock infections where performed along the experiments and are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.s006" target="_blank">S6A and S6B Fig</a>. (<b>C,D</b>) Expression of <i>TotA</i> and <i>vir-1</i> upon DCV infection of wild-type or <i>G9a</i> mutant flies, expressing (<b>C</b>) dome^ΔCyt, or (<b>D</b>) <i>Socs36E</i>. Expression of the gene of interest (by RT-qPCR) was normalized to transcript levels of the housekeeping gene <i>Ribosomal Protein 49</i> and expressed as fold change relative to mock infection (Tris buffer). Data are means and s.d. of three independent pools of at least 15 male flies for each genotype. (<b>A</b>,<b>B</b>) A representative experiment of two independent experiments is shown. Differences in expression of <i>TotA</i> and <i>vir-1</i> were evaluated with a Student’s t-test (*<i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001).</p

<i>G9a</i> targets genes of the Jak-Stat pathway.

<p>(<b>A</b>) Expression levels of <i>domeless</i>, <i>dPIAS</i>, and <i>Socs36E</i> at 24 hpi in fat bodies of 3 to 5-day-old female wild-type or <i>G9a</i> mutant flies challenged with DCV (10,000 TCID₅₀ units). Data are expressed as fold change relative to mock infection (Tris buffer). (<b>B</b>) Basal expression levels of Jak-Stat genes measured by RT-qPCR on fat bodies of 3 to 5-day-old unchallenged female wild-type and <i>G9a</i> mutant flies. Basal expression is presented as dCt (difference between Ct of the gene of interest and the Ct of <i>Ribosomal Protein 49)</i>. (<b>C</b>) Representative example of a <i>G9a</i> target locus within the <i>domeless</i> gene, defined as a genomic region in which the H3K9me2 mark is present in wild-type flies, but not in <i>G9a</i> mutants, in a previous study [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.ref020" target="_blank">20</a>]. Blue and red plots represent sequence reads in H3K9me2 ChIP-seq analyses of wild-type and <i>G9a</i> mutants, respectively [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.ref020" target="_blank">20</a>]. Gene structure is indicated with boxes for exons, lines for introns, and gray boxes for untranslated regions. The arrow represents the position of the amplicon generated by qPCR after Chromatin-Immunoprecipitation (ChIP-qPCR). (<b>D</b>) H3K9me2 ChIP-qPCR on fat bodies of wild-type or <i>G9a</i> mutant flies. Fold enrichment is the percentage of input of the gene of interest normalized to that of a reference gene with very low H3K9me2 marks (<i>moca</i>). Specificity control experiments for ChIP-qPCR experiments are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.s005" target="_blank">S5E–S5J Fig</a>. Data are means and s.d. of (<b>A,B</b>) three independent pools of at least 10 fat bodies, or (<b>D</b>) three independent pools of 80 female fat bodies, for each genotype. Data are from one experiment representative of 2 (<b>A</b>,<b>B</b>) or 6 (<b>D</b>) independent experiments. *<i>P</i> < 0.05 (Student’s t-test).</p