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Rapid clearance of epigenetic protein reporters from wound edge cells in Drosophila larvae does not depend on the JNK or PDGFR/VEGFR signaling pathways.

Anderson AE, Galko MJ - Regeneration (Oxf) (2014)

Bottom Line: Three downregulated proteins, Osa, Kismet, and Spt6, are generally associated with active chromatin, while four others, Sin3A, Sap130, Mi-2, and Mip120, are associated with repressed chromatin.In all cases reporter down regulation was independent of the Jun N-terminal Kinase and Pvr pathways, suggesting that novel signals control reporter clearance.Taken together, our results suggest that clearance of chromatin modifying factors may enable wound edge cells to rapidly and comprehensively change their transcriptional state following tissue damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Unit 1000, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

ABSTRACT
The drastic cellular changes required for epidermal cells to dedifferentiate and become motile during wound closure are accompanied by changes in gene transcription, suggesting corresponding alterations in chromatin. However, the epigenetic changes that underlie wound-induced transcriptional programs remain poorly understood partly because a comprehensive study of epigenetic factor expression during wound healing has not been practical. To determine which chromatin modifying factors might contribute to wound healing, we screened publicly available fluorescently-tagged reporter lines in Drosophila for altered expression at the wound periphery during healing. Thirteen reporters tagging seven different proteins showed strongly diminished expression at the wound edge. Three downregulated proteins, Osa, Kismet, and Spt6, are generally associated with active chromatin, while four others, Sin3A, Sap130, Mi-2, and Mip120, are associated with repressed chromatin. In all cases reporter down regulation was independent of the Jun N-terminal Kinase and Pvr pathways, suggesting that novel signals control reporter clearance. Taken together, our results suggest that clearance of chromatin modifying factors may enable wound edge cells to rapidly and comprehensively change their transcriptional state following tissue damage.

No MeSH data available.


Related in: MedlinePlus

Wound reporter expression is not under the control of JNK. (A)−(G*) Dissected epidermal whole mounts of larvae heterozygous for UAS‐DsRed2Nuc, an epidermal Gal4 driver, the indicated reporter transgene and either bskRNAix2 or w1118 were immunostained for Fasciclin III at 4 h post‐wounding. One example for each protein is shown. Specific reporter lines and epidermal Gal4 drivers are as indicated; see Table 1. Note that in each case diminished reporter expression can be observed at the wound edge (A′′−G′′ and A′′′−G′′′), similar to staining control when the UAS‐bskRNAix2 transgenes are absent (A*−G*). Arrows indicate examples of epidermal nuclei with absent or reduced reporter expression. Scale bar 200 μm.
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reg212-fig-0004: Wound reporter expression is not under the control of JNK. (A)−(G*) Dissected epidermal whole mounts of larvae heterozygous for UAS‐DsRed2Nuc, an epidermal Gal4 driver, the indicated reporter transgene and either bskRNAix2 or w1118 were immunostained for Fasciclin III at 4 h post‐wounding. One example for each protein is shown. Specific reporter lines and epidermal Gal4 drivers are as indicated; see Table 1. Note that in each case diminished reporter expression can be observed at the wound edge (A′′−G′′ and A′′′−G′′′), similar to staining control when the UAS‐bskRNAix2 transgenes are absent (A*−G*). Arrows indicate examples of epidermal nuclei with absent or reduced reporter expression. Scale bar 200 μm.

Mentions: We asked whether diminished reporter expression is controlled by known wound healing pathways. Previous work by our laboratory and others has shown that the conserved JNK and Pvr pathways are required for wound closure in the fly larva (Galko and Krasnow 2004; Wu et al. 2009). We combined each epigenetic reporter line with one of two epidermal Gal4 lines (either A58‐Gal4 or e22c‐Gal4; Galko and Krasnow 2004) driving nuclear DsRed (UAS‐DsRed2Nuc) to ensure the presence of the reporter and labeling of epidermal nuclei as in our previous experiments. We then crossed these animals to potent UAS‐RNAi lines targeting Pvr and JNK that had been shown previously to block wound closure (Wu et al. 2009; Lesch et al. 2010; Brock et al. 2012). This allowed us to assess, in progeny larvae bearing the appropriate transgenes, whether reporter expression is still diminished 4 h after wounding. For all seven tagged proteins, blocking JNK signaling via expression of UAS‐bskRNAi had no obvious effect on loss of reporter expression in wound edge cells for any of the reporters examined (Fig. 4). This result was consistent across all additional reporter lines for Sin3A, Osa, and Mi‐2 (Fig. S5). Next, we blocked Pvr signaling via epidermal expression of UAS‐PvrRNAi. As with loss of JNK signaling, loss of Pvr signaling had no effect on wound edge reporter clearance in any of the 13 reporters examined (Figs. 5 and S6).


Rapid clearance of epigenetic protein reporters from wound edge cells in Drosophila larvae does not depend on the JNK or PDGFR/VEGFR signaling pathways.

Anderson AE, Galko MJ - Regeneration (Oxf) (2014)

Wound reporter expression is not under the control of JNK. (A)−(G*) Dissected epidermal whole mounts of larvae heterozygous for UAS‐DsRed2Nuc, an epidermal Gal4 driver, the indicated reporter transgene and either bskRNAix2 or w1118 were immunostained for Fasciclin III at 4 h post‐wounding. One example for each protein is shown. Specific reporter lines and epidermal Gal4 drivers are as indicated; see Table 1. Note that in each case diminished reporter expression can be observed at the wound edge (A′′−G′′ and A′′′−G′′′), similar to staining control when the UAS‐bskRNAix2 transgenes are absent (A*−G*). Arrows indicate examples of epidermal nuclei with absent or reduced reporter expression. Scale bar 200 μm.
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reg212-fig-0004: Wound reporter expression is not under the control of JNK. (A)−(G*) Dissected epidermal whole mounts of larvae heterozygous for UAS‐DsRed2Nuc, an epidermal Gal4 driver, the indicated reporter transgene and either bskRNAix2 or w1118 were immunostained for Fasciclin III at 4 h post‐wounding. One example for each protein is shown. Specific reporter lines and epidermal Gal4 drivers are as indicated; see Table 1. Note that in each case diminished reporter expression can be observed at the wound edge (A′′−G′′ and A′′′−G′′′), similar to staining control when the UAS‐bskRNAix2 transgenes are absent (A*−G*). Arrows indicate examples of epidermal nuclei with absent or reduced reporter expression. Scale bar 200 μm.
Mentions: We asked whether diminished reporter expression is controlled by known wound healing pathways. Previous work by our laboratory and others has shown that the conserved JNK and Pvr pathways are required for wound closure in the fly larva (Galko and Krasnow 2004; Wu et al. 2009). We combined each epigenetic reporter line with one of two epidermal Gal4 lines (either A58‐Gal4 or e22c‐Gal4; Galko and Krasnow 2004) driving nuclear DsRed (UAS‐DsRed2Nuc) to ensure the presence of the reporter and labeling of epidermal nuclei as in our previous experiments. We then crossed these animals to potent UAS‐RNAi lines targeting Pvr and JNK that had been shown previously to block wound closure (Wu et al. 2009; Lesch et al. 2010; Brock et al. 2012). This allowed us to assess, in progeny larvae bearing the appropriate transgenes, whether reporter expression is still diminished 4 h after wounding. For all seven tagged proteins, blocking JNK signaling via expression of UAS‐bskRNAi had no obvious effect on loss of reporter expression in wound edge cells for any of the reporters examined (Fig. 4). This result was consistent across all additional reporter lines for Sin3A, Osa, and Mi‐2 (Fig. S5). Next, we blocked Pvr signaling via epidermal expression of UAS‐PvrRNAi. As with loss of JNK signaling, loss of Pvr signaling had no effect on wound edge reporter clearance in any of the 13 reporters examined (Figs. 5 and S6).

Bottom Line: Three downregulated proteins, Osa, Kismet, and Spt6, are generally associated with active chromatin, while four others, Sin3A, Sap130, Mi-2, and Mip120, are associated with repressed chromatin.In all cases reporter down regulation was independent of the Jun N-terminal Kinase and Pvr pathways, suggesting that novel signals control reporter clearance.Taken together, our results suggest that clearance of chromatin modifying factors may enable wound edge cells to rapidly and comprehensively change their transcriptional state following tissue damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Unit 1000, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

ABSTRACT
The drastic cellular changes required for epidermal cells to dedifferentiate and become motile during wound closure are accompanied by changes in gene transcription, suggesting corresponding alterations in chromatin. However, the epigenetic changes that underlie wound-induced transcriptional programs remain poorly understood partly because a comprehensive study of epigenetic factor expression during wound healing has not been practical. To determine which chromatin modifying factors might contribute to wound healing, we screened publicly available fluorescently-tagged reporter lines in Drosophila for altered expression at the wound periphery during healing. Thirteen reporters tagging seven different proteins showed strongly diminished expression at the wound edge. Three downregulated proteins, Osa, Kismet, and Spt6, are generally associated with active chromatin, while four others, Sin3A, Sap130, Mi-2, and Mip120, are associated with repressed chromatin. In all cases reporter down regulation was independent of the Jun N-terminal Kinase and Pvr pathways, suggesting that novel signals control reporter clearance. Taken together, our results suggest that clearance of chromatin modifying factors may enable wound edge cells to rapidly and comprehensively change their transcriptional state following tissue damage.

No MeSH data available.


Related in: MedlinePlus