Conditional overexpression of ewg reduces synaptic growth. (a) Western blot of EWG in larval brains upon induced expression. EWG levels in larval brains were compared between elav-GS-GAL4 UAS-ewg animals fed with RU486 (1.2, 4.8 and 19.2 μg/10 ml food, lanes 3-5) and uninduced animals (lane 2), and to adult heads (lane 1). Note that a two-fold overexpression of EWG is effective to reduce synaptic growth (lane 3) compared to wild type (lane 2). (b,c) NMJs of control and EWG overexpressing animals. Shown are NMJs at muscle 13 of uninduced (b) or induced (c, 1.2 μg RU486/10 ml food) elav-GS-GAL4 UAS-ewg animals stained with anti-HRP antibodies. The scale bar in (b) is 20 μm. (d) Quantification of synaptic growth defects with excess EWG. Shown are means of bouton numbers (type 1b at muscle 13) with standard errors (n = 12-15) of uninduced (b) or induced (c, 1.2 μg RU486/10 ml food) elav-GS-GAL4 UAS-ewg animals. Statistically significant differences are indicated by asterisks (***p < 0.0001).

Genes differentially regulated in late embryos of ewg^l1mutants. (a,b) Functional classification of genes differentially regulated in late embryos of ewg^l1mutants. Down-regulated (a) and up-regulated (b) genes were classified according to Gene Ontology processes. (c,d) List of genes differentially regulated in late embryos of ewg^l1mutants, giving functional classification and nervous system expression. Hierarchical clustering of normalized expression levels of down-regulated (c) and up-regulated (d) genes in ewg^l1embryos compared with wild type and ewg^l1embryos rescued with elav-EWG. Gene names and functional categories are shown to the right together with nervous system expression data determined by RNA in situ hybridization (+, expressed in the nervous system; -, not expressed in the nervous system; n.s., no staining; n.d., not determined). Differential expression is visualized by blue (down-regulation) and red (up-regulation). (e) Quantitative RT-PCR of selected genes differentially regulated in ewg^l1mutants. PCR products using ³²P labeled forward primers from genes up-regulated in ewg^l1mutants Ac3, CG7646, CG1909 and Srca (cycles 28, 24, 28 and 28), and from genes down-regulated in ewg^l1mutants Pepck, osi14, CG5171 and CG10585 (cycles 26) were analyzed on 6% polyacrylamide gels. elav: control (cycle 28).

Validation of functional relationships of ewg co-regulated genes by genetic interactions. (a-d) Top view of head and thorax of wild type, gro and Ac3 mutants, and gro Ac3 double mutants. Note the strong overproliferation of frontal bristle on the head and humeral bristles on the thorax of gro Ac3 double mutants compared to gro mutants (arrowheads). Some Ac3 mutants, as shown in (d), have a reduced number of frontal bristles (arrowhead). Deficiencies used are listed in Table S2 in Additional data file 1. The scale bar in (a) represents 100 μm. (e) Analysis of frontal bristle numbers in single and double mutants of genes down-regulated in ewg^l1mutants with a synaptic overgrowth phenotype. Note that in all double mutants tested the frontal bristle phenotype is either enhanced or suppressed. Deficiencies used are listed in Table S2 in Additional data file 1. (f) Quantitative RT-PCR of genes down-regulated in ewg^l1mutants with a synaptic overgrowth phenotype. PCR products using ³²P labeled forward primers from cycle 26 were analyzed on 6% polyacrylamide gels. elav: control (cycle 28).

Erect wing restricts synaptic growth at third instar neuromuscular junctions. (a) Schematic of the eFeG construct used for clonal analysis of ewg^l1, an embryonic lethal allele. (b,c) FLP/FRT mediated recombination in photoreceptor neurons in third instar larval eye disc. Note that CD8::GFP is expressed (c) in the ewg^l1clone (b). The scale bar in (c) is 25 μm. (d-i) NMJs of control and ewg^l1clones in third instar larvae. Clones of controls (d-f, in ewg^l1eFeG/+; hs-flp/+ UAS-CD8::GFP/+ females, rec. control in (j)) and of ewg^l1(g-i, in ewg^l1eFeG/Y; hs-flp/+ UAS-CD8::GFP/+ males) were stained with anti-SYT or with anti-CD8 antibodies to visualize synaptic growth defects of type 1b boutons at muscle 13. The scale bar in (i) is 20 μm. (j) Quantification of synaptic growth defects in ewg^l1mutant neurons. Shown are means of bouton numbers (type 1b at muscle 13) with standard errors (n = 21-35). Rec. control refers to clones made in the presence of one copy of ewg⁺ as in ewg^l1eFeG/+; hs-flp/+ UAS-CD8::GFP/+ females. Tetanus toxin was expressed from a UAS transgene in clones by the recombined eFeG construct. Statistical significance of differences from comparisons with wild type is shown on top of bars (***p < 0.0001, **p < 0.001, n.s. for non significant). Other relevant comparisons are marked by horizontal bars with the statistical significance indicated on the side. (k-n) Distribution of synaptic markers is normal at ewg^l1NMJs. Active zones were stained with anti-Nc82 at wild type (k) or ewg^l1NMJs (m) and periactive zones were stained with anti-Highwire at wild type (l) or ewg^l1NMJs (n). The scale bar in (n) is 1 μm.

Erect wing rescues viability and synaptic growth defects of elav mutants. (a) Rescue of viability of elav mutants by ewg transgenes. An elav-EWG transgene fully rescues viability of a temperature sensitive elav allele (elav^ts1transheterozygous for the elav^e5, a null allele); when early functions in neuronal differentiation are provided by rearing flies at the permissive temperature (three days at 18°C and then at 25°C, n = 250-350 animals per genotype). (b-f) Rescue of synaptic growth defects of elav mutants by EWG. Synaptic growth in elav^ts1/elav^e5mutants (c,f) is significantly reduced (p ≤ 0.0001) when reared at the restrictive temperature during larval life compared to wild type (b,f) and is rescued by elav-EWG (d,f) and elav-ELAV (e,f). Bouton numbers (type 1b at muscle 13) in (f) are shown as means with standard errors (n = 21-35). Statistical significance of differences from comparisons with wild type is shown on top of bars (***p < 0.0001, n.s. for non significant). Other relevant comparisons are marked by horizontal bars with the statistical significance indicated on the side. The scale bar in (b) is 20 μm.

Analysis of synaptic growth in mutants of genes differentially regulated in ewg^l1mutants. (a,b) Quantification of synaptic growth in mutants of genes differentially regulated in ewg^l1mutants. Type 1b boutons at muscle 13 were quantified and are shown as means with standard error (n = 18-22) in transheterozygotes for either a chromosomal deficiency or a second allele from mutants of genes down-regulated (a) or up-regulated (b) in ewg^l1mutants that were significantly different (^ap ≤ 0.0001, ^bp ≤ 0.001, ^cp ≤ 0.05) compared to controls (y w and y w transheterozygous for the corresponding chromosomal deficiency) except for genes involved in basal metabolism. Detailed genotypes with corresponding bouton numbers are listed in Table S2 in Additional data file 1. Genes are clustered according to their function with the color codes used in Figure 4. Note that genes down-regulated in ewg^l1mutants are highly enriched for expression in the nervous system (90%) while only a minor portion of genes up-regulated in ewg^l1mutants is expressed in the nervous system (36%). (c) Summary of mutants in genes differentially regulated in ewg^l1mutants with synaptic growth defects and model of EWG regulation of these genes and their roles in synaptic growth regulation. CNS, central nervous system; PNS, peripheral nervous system.

erect wing integrates cellular signaling to regulate synaptic growth. (a-d) Genetic interaction of ewg with known signaling pathways in synaptic growth regulation. Synaptic growth was analyzed at third instar NMJs by quantifying type 1b boutons at muscle 13 of ewg^l1eFeG clones (white bars and '-' at the bottom of the column) or of ewg^l1eFeG clones in the presence of an ewg wild-type copy (grey bars and '+' at the bottom of the column) in combination with either transgenes for UAS constructs or transheterozygous combinations of mutant alleles (wit^A12/wit^B11) as described for Figure 1. Wild type (bar 1), ewg loss of function (LOF, bar 2) and ewg gain of function (GOF, black bars, bar 3) were compared to LOF and GOF mutants in Notch (a), Wingless (b), AP-1 (c) and TGF-β/BMP (d) pathways. Overexpression of N is shown in the presence of both ewg copies ('++' at the bottom of the column), as one ewg copy in the presence of excess N does not result in a significant increase of bouton numbers compared to wild type. For both N and tkvA, two copies of UAS transgenes were used; a single copy did not significantly alter bouton numbers. Shown are means of bouton numbers with standard errors (n = 11-23, numbers at the bottom of bars). Bars are numbered below the x-axis. Statistical significance of differences from comparisons with wild type is shown on top of bars (***p < 0.0001, ** p < 0.005, n.s. for non significant). Other relevant comparisons are marked by horizontal bars with the statistical significance indicated on the side.