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Erect wing regulates synaptic growth in Drosophila by integration of multiple signaling pathways.

Haussmann IU, White K, Soller M - Genome Biol. (2008)

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

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

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.

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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).
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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).

Mentions: Next, we validated functional connections among genes down-regulated in ewgl1mutants with a synaptic overgrowth phenotype in another assay. One of the genes in this class is gro, for which hypomorphic alleles are known that are associated with an overproliferation of frontal bristles on the head (Figure 6b). gro has been described as a transcriptional co-repressor that interacts with a subset of negative transcriptional regulators [38]. We therefore tested a representative number of mutants in genes downregulated in ewgl1mutants in combination with gro for a change of the gro bristle phenotype. All mutants tested genetically interacted with gro, resulting in either an enhancement or suppression of the gro bristle phenotype (Figure 6e). None of the single mutants had more bristles, but some had less (Figure 6d), suggesting that the gro phenotype can be suppressed (for example, CG8924 and Bcl7-like), indicated by several roles of gro in peripheral nervous system specification [39]. With quantitative RT-PCR, we verified differential expression of these genes in ewgl1mutants and rescue to wild-type expression levels by the presence of an elav-EWG transgene in ewgl1mutants (Figure 6f). The genes are also expressed predominantly in the ventral nerve cord (Figure S3 in Additional data file 1; Figure 4c) [40]. Taken together, these data strongly suggest that these genes down-regulated in ewgl1mutants are functionally connected and operate in a common pathway.


Erect wing regulates synaptic growth in Drosophila by integration of multiple signaling pathways.

Haussmann IU, White K, Soller M - Genome Biol. (2008)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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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).
Mentions: Next, we validated functional connections among genes down-regulated in ewgl1mutants with a synaptic overgrowth phenotype in another assay. One of the genes in this class is gro, for which hypomorphic alleles are known that are associated with an overproliferation of frontal bristles on the head (Figure 6b). gro has been described as a transcriptional co-repressor that interacts with a subset of negative transcriptional regulators [38]. We therefore tested a representative number of mutants in genes downregulated in ewgl1mutants in combination with gro for a change of the gro bristle phenotype. All mutants tested genetically interacted with gro, resulting in either an enhancement or suppression of the gro bristle phenotype (Figure 6e). None of the single mutants had more bristles, but some had less (Figure 6d), suggesting that the gro phenotype can be suppressed (for example, CG8924 and Bcl7-like), indicated by several roles of gro in peripheral nervous system specification [39]. With quantitative RT-PCR, we verified differential expression of these genes in ewgl1mutants and rescue to wild-type expression levels by the presence of an elav-EWG transgene in ewgl1mutants (Figure 6f). The genes are also expressed predominantly in the ventral nerve cord (Figure S3 in Additional data file 1; Figure 4c) [40]. Taken together, these data strongly suggest that these genes down-regulated in ewgl1mutants are functionally connected and operate in a common pathway.

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

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

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.

Show MeSH
Related in: MedlinePlus