Synergistic interactions in <i>Drosophila</i> between <i>shibire</i> and <i>alpha spectrin</i>, the orthologues of ASD-candidate genes from a <i>de novo</i> gain CNV 12235_chr9_gain_129907917_l.

<p><b>A.</b> The Locus of the CNV with mapped <i>Drosophila</i> orthologues (Target, green; control, red). <b>B.</b> Synaptic alterations were characterised by NMJ type IB bouton number. Individual heterozygous mutants of candidate gene orthologues gave no significant change in NMJ morphology over <i>w</i>^<i>1118</i> controls. However, <i>Shibire</i> and <i>alpha-spectrin</i> double over expressers display reduced bouton numbers (using 1032-GAL4; UAS-Dynamin/UAS-alpha-spectrin; n>20, Kruskal-Wallis test, * P<0.05). Non-candidate gene controls <i>Su(P)</i> (using <i>Su(P)</i>^{<i>EY13245</i>}) and <i>CG14104</i> (using <i>CG14104</i>^{<i>f07593</i>}) selected from genes found within CNV gave no significant NMJ phenotype singularly or when crossed to form transheterozygotes with candidate genes. <b>C.</b> Circadian rhythm analysis of candidate genes. Negative controls and candidate gene orthologue overexpression of <i>alpha-spectrin</i> displayed normal light/dark differences in sleeping patterns singularly or when crossed. However, <i>Shibire</i> overexpression, and co-overexpression with <i>alpha-spectrin</i> lost the dark bias, and displayed no significant difference between light/dark sleeping patterns (<b>t</b>).</p

Selective genetic interactions observed between the <i>Drosophila</i> orthologues of ASD candidate genes and Neurexin IV.

<p>A sensitised background of Neurexin IV (<i>NrxIV/+</i>), the orthologue of the autism gene <i>CTNAP2</i>, was used to look for interactions between <i>NrxIV</i> and the ASD candidate gene orthologues <i>dlg, pak, p120ctn, pasha</i>, and <i>org-1</i>. <b>A</b>. <i>dlg/+, pak/+</i> and <i>NrxIV/+</i> heterozygous mutants have no significant change in NMJ morphology over <i>w</i>^<i>1118</i> controls. However, <i>dlg</i>/<i>NrxIV</i> and <i>pak</i>/<i>NrxIV</i> crosses both displayed reduced bouton numbers (n>20 Kruskal-Wallis test, ** P<0.01). <b>B.</b> Significant NMJ morphology changes are also seen for the <i>NrxIV</i> cross with the homozygous <i>p120ctn</i> mutant cross, when compared to the homozygous <i>p120ctn</i> mutant cross alone (n>20 Kruskal-Wallis test, ** P<0.01). <b>C.</b> No NMJ morphology changes were observed when <i>pasha</i> and <i>org-1</i>, both from human <i>de novo</i> loss 12239_chr22_loss_17249508_l, were crossed to <i>NrxIV/+</i>. <b>D. to F.</b> Circadian rhythm analyses of models in Panels A, B and C support observed genetic interactions: d<i>lg</i>/<i>NrxIV, pak</i>/<i>NrxIV</i> transheterozygotes and the <i>NrxIV</i> cross with the homozygous <i>p120ctn</i> mutant flies lost the dark bias, and displayed no significant difference between light/dark sleeping patterns (<b>t</b>), while <i>pasha</i>/<i>NrxIV</i> and <i>org-1</i>/<i>NrxIV</i> transheterozygotes displayed no abnormal circadian phenotype.</p

<i>Drosophila</i> screening strategy to detect interactions between the orthologues of genes simultaneously affected by a de novo CNVs identified in individuals with ASD (see Methods).

<p><i>Drosophila</i> screening strategy to detect interactions between the orthologues of genes simultaneously affected by a de novo CNVs identified in individuals with ASD (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004998#sec010" target="_blank">Methods</a>).</p

Different genetic interactions effect distinct synaptic defects suggesting that distinct molecular aetiologies underlie ASD.

<p><b>A.</b> The <i>Drosophila</i> NMJ contains presynaptic active zones (labelled by Bruchpilot, BRP) and postsynaptic glutamate receptor (labelled by GluRIIA). <b>B.</b> Representative images of BRP staining demonstrate a reduced number of active zone (BRP) puncta in the transheterozygotes <i>dlg/pak, NrxIV/dlg</i> and <i>NrxIV/pak</i> as compared to control (<i>w</i>^<i>1118</i>), <b>C.</b> The number of active zone (BRP) puncta (normalised to bouton size) were significantly reduced in <i>dlg/pak, NrxIV/Dlg</i> and <i>NrxIV/Pak</i> transheterozygotes. <b>D.</b> The fluorescence of the post-synaptic glutamate receptors were scored and normalised to bouton size HRP levels. <i>Pasha/Sep4</i> transheterozygotes were the only genotype to demonstrate decreased glutamate receptor abundance. <b>E.</b> Representative images of GluRIIA staining demonstrating the reduced fluorescence in the transheterozygote <i>Pasha/Sep4</i> when compared to control. <b>F.</b> A schematic showing the sub-types of genetic interactions supporting distinct molecular aetiologies underlying ASD that converge to yield defects at the synapse.</p

Synergistic interaction in <i>Drosophila</i> between <i>Dlg</i> and <i>Pak</i>, the orthologues of ASD-candidate genes from a <i>de novo</i> loss CNV 11079_chr3_197208363.

<p><b>A.</b> The Locus of the CNV with mapped <i>Drosophila</i> orthologues (Candidates, green; controls, red). <b>B.</b> Representative pictures of NMJs from <i>dlg</i>/+ (using <i>dlg</i>^<i>1</i>), <i>pak</i>/+ (using <i>pak</i>^<i>6</i>), and <i>dlg/pak</i> 3^rd instar larvae; Scale bar = 20μm. <b>C.</b> Synaptic alterations were characterised by NMJ bouton number. Individual heterozygous mutants of candidate gene orthologues <i>dlg</i> and <i>pak</i> (<i>dlg/+</i> and <i>pak/+)</i> gave no significant change in NMJ morphology over <i>w</i>^<i>1118</i> controls. However, <i>dlg</i>/<i>pak</i> transheterozygotes have reduced bouton numbers. (n>20, Kruskal-Wallis test, ** P<0.01). <b>D.</b> Non-candidate gene controls <i>fsn</i> (using <i>Fsn</i>^{<i>KG08128</i>}) and CG5359 (using <i>CG5359</i>^{<i>e03976</i>}) selected from genes found within CNV gave no significant NMJ phenotype singularly or when crossed to form transheterozygotes with <i>dlg</i> or <i>pak</i>. <b>E.</b> and <b>F.</b> Circadian rhythm analysis of candidate genes. All negative control <b>F.</b> and single mutants displayed normal light/dark differences in sleeping patterns. However, transheterozygote <i>dlg</i>/<i>pak</i> flies lost the dark bias, and displayed no significant difference between light/dark sleeping patterns (<b>t</b>).</p

<i>Myc</i> overexpression is sufficient to induce cytoophidia formation in Stage 10b follicle cells.

(A-E) In this stage-10b egg chamber, non-clonal cells (cells that are lack of GFP in B) have low levels of Myc (D) and only have very short cytoophidia, as indicated by an antibody against CTPsyn (C). UAS-Myc overexpression clones (i.e. Myc overexpression) marked with GFP (B, outlined in yellow in A-E) have increased levels of Myc (D) and much longer cytoophidia than non-clonal (nc, i.e. non-green) cells (C). DNA staining show that the nuclei in clones (green cells in E) are larger than those of nc cells (non-green cells in D). (F) Quantification shows that cytoophidia in Myc overexpression (UAS-Myc) cells are significantly increased in length, compared to those in nc cells. ***P<0.001. Error bars show SEM.</p

Cytoophidium formation correlates with Myc expression in <i>Drosophila</i> follicle cells.

(A) Schematic representation of the stages of Drosophila oogenesis within an ovariole. (B-H) Immunostaining of Drosophila follicle cells at different stages with antibodies against Myc and CTPsyn. Myc expression is observed at Region 2 of the germarium (B) and is continuously expressed at early- (C) and mid-oogenesis (D, E) until Stage 10a (F). Myc expression in follicle cells drops from Stage 10b (G) to late-stage egg chambers (H). The appearance of cytoophidia correlates with Myc expression. Cytoophidia are detectable from Region 2 to stage 10a (B-F). (B) Cytoophidia are first observed at region 2 of the germarium concomitant with high Myc expression. (C) Two egg chambers at stages 2–5. Note large filamentous structures are macro-cytoophidia in germline cells. (D) An egg chamber at Stage 7. Note large filamentous structures are macro-cytoophidia in germline cells. (E) Follicle cells of a Stage-9 egg chamber. (F) Follicle cells of a Stage-10a egg chamber. (G) Follicle cells of a Stage-10b egg chamber. In stage 10b follicle cells, Myc expression is low and cytoophidia are hardly detectable. (H) Follicle cells of a Stage-12 egg chamber. In stage-12 follicle cells, Myc expression is low and no cytoophidia are detectable. (I) Quantification of follicle cell cytoophidia lengths at various stages of oogenesis. Quantification represents the mean cytoophidium length from > 50 cells in > 3 egg chambers at each stage in w1118 flies. See S1–S7 Figs for individual channels of images shown.</p

Synergistic Interactions between <i>Drosophila</i> Orthologues of Genes Spanned by <i>De Novo</i> Human CNVs Support Multiple-Hit Models of Autism

<div><p>Autism spectrum disorders (ASDs) are highly heritable and characterised by deficits in social interaction and communication, as well as restricted and repetitive behaviours. Although a number of highly penetrant ASD gene variants have been identified, there is growing evidence to support a causal role for combinatorial effects arising from the contributions of multiple loci. By examining synaptic and circadian neurological phenotypes resulting from the dosage variants of unique human:fly orthologues in <i>Drosophila</i>, we observe numerous synergistic interactions between pairs of informatically-identified candidate genes whose orthologues are jointly affected by large <i>de novo</i> copy number variants (CNVs). These CNVs were found in the genomes of individuals with autism, including a patient carrying a 22q11.2 deletion. We first demonstrate that dosage alterations of the unique <i>Drosophila</i> orthologues of candidate genes from <i>de novo</i> CNVs that harbour only a single candidate gene display neurological defects similar to those previously reported in <i>Drosophila</i> models of ASD-associated variants. We then considered pairwise dosage changes within the set of orthologues of candidate genes that were affected by the same single human <i>de novo</i> CNV. For three of four CNVs with complete orthologous relationships, we observed significant synergistic effects following the simultaneous dosage change of gene pairs drawn from a single CNV. The phenotypic variation observed at the <i>Drosophila</i> synapse that results from these interacting genetic variants supports a concordant phenotypic outcome across all interacting gene pairs following the direction of human gene copy number change. We observe both specificity and transitivity between interactors, both within and between CNV candidate gene sets, supporting shared and distinct genetic aetiologies. We then show that different interactions affect divergent synaptic processes, demonstrating distinct molecular aetiologies. Our study illustrates mechanisms through which synergistic effects resulting from large structural variation can contribute to human disease.</p></div

<i>Myc</i> overexpression promotes cytoophidium formation in follicle cells.

<p>(<b>A-D</b>) In stage-8 egg chambers, <i>UAS</i>-<i>Myc</i> overexpression clones marked with GFP (<b>B</b>, outlined in yellow in <b>A-D</b>) have longer cytoophidia, as indicated by CTPsyn staining (<b>C</b>), than non-GFP cells. DNA staining shows that the nuclei in clones (green cells in <b>D</b>) are larger than those of neighbouring cells (non-green cells in <b>D</b>). (<b>E</b>) Cytoophidia in <i>Myc</i> overexpression (<i>UAS-Myc</i>) cells increase significantly in length, compared with those in neighbouring cells. ***P<0.001. Error bars show SEM.</p

Bouton number at the <i>Drosophila</i> NMJ following the overexpression and mutation of the <i>Drosophila</i> unique orthologues of candidate genes identified from human autism-associated copy number variants (CNVs).

<p><b>A.</b> Representative pictures of NMJs from <i>NrxIV/NrxIV</i> (using <i>Nrx-IV</i>^<i>4304</i>), Notch overexpessing (1032-GAL4, UAS-Notch), and <i>p120ctn/p120ctn</i> (using <i>p120ctn</i>^<i>308</i>) 3^rd instar larvae; Scale bar = 20μm. <b>B.</b> Homozygous disruption of <i>NrxIV</i>, the orthologue of the autism gene <i>CTNAP2</i>, provided a positive control and displayed a reduced NMJ bouton number as described previously. Heterozygous mutants of <i>NrxIV</i> yielded no bouton number reduction. Overexpression of <i>Notch</i> (1032-GAL4; UAS-Notch-full), whose human orthologue is duplicated in the <i>de novo</i> gain CNV_946_301_chr9_gain_138505259 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004998#pgen.1004998.t001" target="_blank">Table 1</a>), gave reduced bouton numbers (n>20, Kruskal-Wallis test, ** P<0.01). <b>C.</b> Homozygous disruption of <i>p120ctn</i>, that is affected by the <i>de novo</i> loss CNVs 12289_chr5_loss_1140362 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004998#pgen.1004998.t001" target="_blank">Table 1</a>), yields reduced bouton numbers. (n>20, Kruskal-Wallis test, ** P<0.01). Heterozygous mutants of <i>p120ctn</i> have no significant change on NMJ morphology. <b>D. and E.</b> Circadian sleep/rest rhythm analysis of candidate genes from the CNV sets. 1032-GAL4, UAS-Notch, and <i>p120ctn/p120ctn</i> flies lost the dark bias, displaying no significant difference between light/dark sleeping patterns (<b>t,</b> representing the crosses where no light/dark sleep/rest bias was observed). Light/dark sleeping bias was measured using student’s t-tests.</p