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PI3K signaling and Stat92E converge to modulate glial responsiveness to axonal injury.

Doherty J, Sheehan AE, Bradshaw R, Fox AN, Lu TY, Freeman MR - PLoS Biol. (2014)

Bottom Line: Surprisingly, canonical JAK/STAT signaling does not regulate draper expression.Rather, we find injury-induced draper activation is downstream of the Draper/Src42a/Shark/Rac1 engulfment signaling pathway.We provide evidence for a positive auto-regulatory mechanism whereby signaling through the injury-responsive Draper receptor leads to Stat92E-dependent, transcriptional activation of the draper gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Glial cells are exquisitely sensitive to neuronal injury but mechanisms by which glia establish competence to respond to injury, continuously gauge neuronal health, and rapidly activate reactive responses remain poorly defined. Here, we show glial PI3K signaling in the uninjured brain regulates baseline levels of Draper, a receptor essential for Drosophila glia to sense and respond to axonal injury. After injury, Draper levels are up-regulated through a Stat92E-modulated, injury-responsive enhancer element within the draper gene. Surprisingly, canonical JAK/STAT signaling does not regulate draper expression. Rather, we find injury-induced draper activation is downstream of the Draper/Src42a/Shark/Rac1 engulfment signaling pathway. Thus, PI3K signaling and Stat92E are critical in vivo regulators of glial responsiveness to axonal injury. We provide evidence for a positive auto-regulatory mechanism whereby signaling through the injury-responsive Draper receptor leads to Stat92E-dependent, transcriptional activation of the draper gene. We propose that Drosophila glia use this auto-regulatory loop as a mechanism to adjust their reactive state following injury.

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The draper locus contains a Stat92E-regulated injury responsive element.(A) Schematic representation of the draper locus and regions used to generate DEEs 2–10. Blue box highlights dee7 region located in the first intron of the draper gene. Red lines indicate Stat92E binding sites in dee7. Asterisk indicates binding site in dee7MUT containing two point mutations. (B) Single slice confocal images of antennal lobe regions; dee7-Gal4 or dee7MUT-Gal4 driving two copies of UAS-mCD8::GFP (dee7>2XmCD8::GFP, dee7MUT>2XmCD8::GFP). Uninjured, one day after antennal ablation and four days after antennal ablation are shown. Dashed circles outline antennal lobes, the site of injury. Asterisk indicates cortex glia responding to axotomy. (C) Quantification for (B), p-values were calculated using Student's t test, n.s., not significant. Error bars represent SEM. (D) Single slice confocal images of antennal lobe regions stained with α-GFP and α-Repo; dee7-Gal4 was used to drive two copies of UAS-mCD8::GFP. Removal of one maxillary palp, two maxillary palps, one antenna, and two antennae are shown one day after injury.
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pbio-1001985-g002: The draper locus contains a Stat92E-regulated injury responsive element.(A) Schematic representation of the draper locus and regions used to generate DEEs 2–10. Blue box highlights dee7 region located in the first intron of the draper gene. Red lines indicate Stat92E binding sites in dee7. Asterisk indicates binding site in dee7MUT containing two point mutations. (B) Single slice confocal images of antennal lobe regions; dee7-Gal4 or dee7MUT-Gal4 driving two copies of UAS-mCD8::GFP (dee7>2XmCD8::GFP, dee7MUT>2XmCD8::GFP). Uninjured, one day after antennal ablation and four days after antennal ablation are shown. Dashed circles outline antennal lobes, the site of injury. Asterisk indicates cortex glia responding to axotomy. (C) Quantification for (B), p-values were calculated using Student's t test, n.s., not significant. Error bars represent SEM. (D) Single slice confocal images of antennal lobe regions stained with α-GFP and α-Repo; dee7-Gal4 was used to drive two copies of UAS-mCD8::GFP. Removal of one maxillary palp, two maxillary palps, one antenna, and two antennae are shown one day after injury.

Mentions: Our observations that basal and injury induced Draper expression were controlled by distinct molecular pathways prompted us to attempt to identify draper gene enhancer elements responsible for establishing basal levels of draper expression in adult brain glia, and/or increasing draper expression specifically after ORN axotomy. We focused our search on an ∼40 kb region centered around the draper locus (Figure 2A). We cloned nine different potential draper enhancer elements (termed dee2-dee10) from 5′, intronic, or 3′ regions of the draper gene into the Gal4-based pBGW vector [30] and inserted these elements into identical genomic locations (Figure 2A). Each dee-Gal4 line was then used to drive two copies of UAS-mCD8::GFP in vivo and expression patterns were examined in the adult brain before and after injury. No expression in ensheathing or cortex glia was observed with any of the enhancer element lines in the healthy, uninjured brain (unpublished data). We therefore failed to identify any single enhancer element that was capable of driving glial expression of reporters in the adult brain in a pattern similar to endogenous Draper protein. This observation suggests that PI3K-dependent regulation of Draper levels might be governed by an enhancer element some distance from the draper gene, requires the convergent activity of multiple enhancers along the draper gene, or could be controlled through post-transcriptional mechanisms.


PI3K signaling and Stat92E converge to modulate glial responsiveness to axonal injury.

Doherty J, Sheehan AE, Bradshaw R, Fox AN, Lu TY, Freeman MR - PLoS Biol. (2014)

The draper locus contains a Stat92E-regulated injury responsive element.(A) Schematic representation of the draper locus and regions used to generate DEEs 2–10. Blue box highlights dee7 region located in the first intron of the draper gene. Red lines indicate Stat92E binding sites in dee7. Asterisk indicates binding site in dee7MUT containing two point mutations. (B) Single slice confocal images of antennal lobe regions; dee7-Gal4 or dee7MUT-Gal4 driving two copies of UAS-mCD8::GFP (dee7>2XmCD8::GFP, dee7MUT>2XmCD8::GFP). Uninjured, one day after antennal ablation and four days after antennal ablation are shown. Dashed circles outline antennal lobes, the site of injury. Asterisk indicates cortex glia responding to axotomy. (C) Quantification for (B), p-values were calculated using Student's t test, n.s., not significant. Error bars represent SEM. (D) Single slice confocal images of antennal lobe regions stained with α-GFP and α-Repo; dee7-Gal4 was used to drive two copies of UAS-mCD8::GFP. Removal of one maxillary palp, two maxillary palps, one antenna, and two antennae are shown one day after injury.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219656&req=5

pbio-1001985-g002: The draper locus contains a Stat92E-regulated injury responsive element.(A) Schematic representation of the draper locus and regions used to generate DEEs 2–10. Blue box highlights dee7 region located in the first intron of the draper gene. Red lines indicate Stat92E binding sites in dee7. Asterisk indicates binding site in dee7MUT containing two point mutations. (B) Single slice confocal images of antennal lobe regions; dee7-Gal4 or dee7MUT-Gal4 driving two copies of UAS-mCD8::GFP (dee7>2XmCD8::GFP, dee7MUT>2XmCD8::GFP). Uninjured, one day after antennal ablation and four days after antennal ablation are shown. Dashed circles outline antennal lobes, the site of injury. Asterisk indicates cortex glia responding to axotomy. (C) Quantification for (B), p-values were calculated using Student's t test, n.s., not significant. Error bars represent SEM. (D) Single slice confocal images of antennal lobe regions stained with α-GFP and α-Repo; dee7-Gal4 was used to drive two copies of UAS-mCD8::GFP. Removal of one maxillary palp, two maxillary palps, one antenna, and two antennae are shown one day after injury.
Mentions: Our observations that basal and injury induced Draper expression were controlled by distinct molecular pathways prompted us to attempt to identify draper gene enhancer elements responsible for establishing basal levels of draper expression in adult brain glia, and/or increasing draper expression specifically after ORN axotomy. We focused our search on an ∼40 kb region centered around the draper locus (Figure 2A). We cloned nine different potential draper enhancer elements (termed dee2-dee10) from 5′, intronic, or 3′ regions of the draper gene into the Gal4-based pBGW vector [30] and inserted these elements into identical genomic locations (Figure 2A). Each dee-Gal4 line was then used to drive two copies of UAS-mCD8::GFP in vivo and expression patterns were examined in the adult brain before and after injury. No expression in ensheathing or cortex glia was observed with any of the enhancer element lines in the healthy, uninjured brain (unpublished data). We therefore failed to identify any single enhancer element that was capable of driving glial expression of reporters in the adult brain in a pattern similar to endogenous Draper protein. This observation suggests that PI3K-dependent regulation of Draper levels might be governed by an enhancer element some distance from the draper gene, requires the convergent activity of multiple enhancers along the draper gene, or could be controlled through post-transcriptional mechanisms.

Bottom Line: Surprisingly, canonical JAK/STAT signaling does not regulate draper expression.Rather, we find injury-induced draper activation is downstream of the Draper/Src42a/Shark/Rac1 engulfment signaling pathway.We provide evidence for a positive auto-regulatory mechanism whereby signaling through the injury-responsive Draper receptor leads to Stat92E-dependent, transcriptional activation of the draper gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Glial cells are exquisitely sensitive to neuronal injury but mechanisms by which glia establish competence to respond to injury, continuously gauge neuronal health, and rapidly activate reactive responses remain poorly defined. Here, we show glial PI3K signaling in the uninjured brain regulates baseline levels of Draper, a receptor essential for Drosophila glia to sense and respond to axonal injury. After injury, Draper levels are up-regulated through a Stat92E-modulated, injury-responsive enhancer element within the draper gene. Surprisingly, canonical JAK/STAT signaling does not regulate draper expression. Rather, we find injury-induced draper activation is downstream of the Draper/Src42a/Shark/Rac1 engulfment signaling pathway. Thus, PI3K signaling and Stat92E are critical in vivo regulators of glial responsiveness to axonal injury. We provide evidence for a positive auto-regulatory mechanism whereby signaling through the injury-responsive Draper receptor leads to Stat92E-dependent, transcriptional activation of the draper gene. We propose that Drosophila glia use this auto-regulatory loop as a mechanism to adjust their reactive state following injury.

Show MeSH
Related in: MedlinePlus