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. 2008 Apr 17;9(4):R73.
doi: 10.1186/gb-2008-9-4-r73.

Erect Wing Regulates Synaptic Growth in Drosophila by Integration of Multiple Signaling Pathways

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Free PMC article

Erect Wing Regulates Synaptic Growth in Drosophila by Integration of Multiple Signaling Pathways

Irmgard U Haussmann et al. Genome Biol. .
Free PMC article

Abstract

Background: Formation of synaptic connections is a dynamic and highly regulated process. Little is known about the gene networks that regulate synaptic growth and how they balance stimulatory and restrictive signals.

Results: Here we show that the neuronally expressed transcription factor gene erect wing (ewg) is a major target of the RNA binding protein ELAV and that EWG restricts synaptic growth at neuromuscular junctions. Using a functional genomics approach we demonstrate that EWG acts primarily through increasing mRNA levels of genes involved in transcriptional and post-transcriptional regulation of gene expression, while genes at the end of the regulatory expression hierarchy (effector genes) represent only a minor portion, indicating an extensive regulatory network. Among EWG-regulated genes are components of Wingless and Notch signaling pathways. In a clonal analysis we demonstrate that EWG genetically interacts with Wingless and Notch, and also with TGF-beta and AP-1 pathways in the regulation of synaptic growth.

Conclusion: Our results show that EWG restricts synaptic growth by integrating multiple cellular signaling pathways into an extensive regulatory gene expression network.

Figures

Figure 1
Figure 1
Erect wing restricts synaptic growth at third instar neuromuscular junctions. (a) Schematic of the eFeG construct used for clonal analysis of ewgl1, 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 ewgl1clone (b). The scale bar in (c) is 25 μm. (d-i) NMJs of control and ewgl1clones in third instar larvae. Clones of controls (d-f, in ewgl1eFeG/+; hs-flp/+ UAS-CD8::GFP/+ females, rec. control in (j)) and of ewgl1(g-i, in ewgl1eFeG/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 ewgl1mutant 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 ewgl1eFeG/+; 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 ewgl1NMJs. Active zones were stained with anti-Nc82 at wild type (k) or ewgl1NMJs (m) and periactive zones were stained with anti-Highwire at wild type (l) or ewgl1NMJs (n). The scale bar in (n) is 1 μm.
Figure 2
Figure 2
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).
Figure 3
Figure 3
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 (elavts1transheterozygous for the elave5, 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 elavts1/elave5mutants (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.
Figure 4
Figure 4
Genes differentially regulated in late embryos of ewgl1mutants. (a,b) Functional classification of genes differentially regulated in late embryos of ewgl1mutants. 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 ewgl1mutants, giving functional classification and nervous system expression. Hierarchical clustering of normalized expression levels of down-regulated (c) and up-regulated (d) genes in ewgl1embryos compared with wild type and ewgl1embryos 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 ewgl1mutants. PCR products using 32P labeled forward primers from genes up-regulated in ewgl1mutants Ac3, CG7646, CG1909 and Srca (cycles 28, 24, 28 and 28), and from genes down-regulated in ewgl1mutants Pepck, osi14, CG5171 and CG10585 (cycles 26) were analyzed on 6% polyacrylamide gels. elav: control (cycle 28).
Figure 5
Figure 5
Analysis of synaptic growth in mutants of genes differentially regulated in ewgl1mutants. (a,b) Quantification of synaptic growth in mutants of genes differentially regulated in ewgl1mutants. 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 ewgl1mutants 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 ewgl1mutants are highly enriched for expression in the nervous system (90%) while only a minor portion of genes up-regulated in ewgl1mutants is expressed in the nervous system (36%). (c) Summary of mutants in genes differentially regulated in ewgl1mutants 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.
Figure 6
Figure 6
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 ewgl1mutants 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 ewgl1mutants 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).
Figure 7
Figure 7
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 ewgl1eFeG clones (white bars and '-' at the bottom of the column) or of ewgl1eFeG 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 (witA12/witB11) 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.

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