PI3K Signaling and Stat92E Converge to Modulate Glial Responsiveness to Axonal Injury

Activation of glial cells following axon injury is mediated by a positive feedback loop downstream of the glial phagocytic receptor Draper, allowing the strength of the response to match the severity of injury.


Introduction
Glial cells are extraordinarily sensitive to disruptions in central nervous system (CNS) homeostasis and exhibit an impressive ability to respond to a diversity of neural injuries including hypoxia, chemical insults, and mechanical injury (e.g., axotomy or traumatic brain injury) [1][2][3]. During injury responses, glia exhibit robust changes in gene expression, migrate or extend membranes to sites of trauma, and phagocytose degenerating neuronal debris. Glial reactive responses after injury can be beneficial and promote recovery. For example, glial clearance of degenerating neuronal debris is thought to suppress nervous system inflammation and facilitate remyelination [4][5][6][7][8]. However, glia can also exacerbate damage in the CNS by driving inflammation through the release of pro-inflammatory cytokines and actively destroying healthy cells [9][10][11][12][13][14]. Whether reactive gliosis is ultimately more beneficial or harmful to the nervous system remains an open question and is likely context dependent. However, mounting evidence points to reactive glial cells as an excellent target for therapeutically modifying CNS disorders [15].
Reactive glial cells have been studied in a variety of neurodegenerative diseases and injury models but surprisingly little is known about how glial sensitivity to neuronal health is initially established in the healthy brain, or how glial perception of neuron-derived ''injury signals'' is translated into dynamic reactive responses after trauma. A wealth of studies indicate that reactive gliosis is not an all-or-none response, but rather includes a wide range of graded responses that correlate with the severity of nervous system injury [15]. The ability of glia to ''measure'' the severity of trauma in the nervous system and respond accordingly implies a tight functional coupling between signaling pathways sensing neural injury and those executing glial reactive responses [16]. In some cases reactive glia can modulate the intensity of reactive gliosis in an autocrine way. For example, focal demyelination leads to activation and release of endothilin-1 (ET-1) from astrocytes, which in turn can signal back to astrocytes (through ET-1 receptors) and further enhance astrocyte cellular hypertrophy, proliferation, and GFAP expression [17][18][19][20][21][22]. However, the initial injury signal which activates ET-1 expression remains to be identified. An intriguing possibility is that so-called ''eat me'' cues on degenerating neuronal debris directly activate receptors on glial cells, which in turn modulate initiation of reactive gliosis, but evidence for such molecular regulation is lacking.
We have previously shown that Draper, a glial-expressed immunoreceptor-like molecule, is a key surface receptor required for glial engulfment of degenerating axons after axotomy in the adult Drosophila brain [23][24][25]. Draper is the Drosophila ortholog of CED-1, an engulfment receptor that is essential for engulfment of cell corpses in Caenorhabditis elegans [26]. Within hours after axotomy of Drosophila antennal olfactory receptor neuron (ORN) axons, Draper protein, and mRNA levels are dramatically increased in glia surrounding degenerating axons. Glial mem-branes are then recruited to severed axons, glia phagocytose axonal debris, and ultimately glia return to a resting state [24,27]. Loss of Draper function blocks all glial morphological and molecular responses to axonal injury and axonal debris lingers in the brain for the life of the animal. Similar phenotypes have been observed when the components of the Src-family kinase signaling cascade that acts downstream of Draper (i.e., Shark and Src42a) are eliminated specifically from glial cells [25]. These data argue that Draper acts very early in the activation of Drosophila glia after axonal injury, perhaps even in the recognition of cues presented by engulfment targets like degenerating axons. Recently the mammalian orthologs of CED-1/Draper, MEGF10, and Jedi, have been implicated in satellite glial engulfment of neuronal cell corpses in developing mouse dorsal root ganglia [28] and MEGF10 has been shown to engulf pruned synapses in the postnatal dorsal lateral geniculate nucleus during activity-dependent synaptic refinement [29]. Thus, Draper/MEGF10/Jedi engulfment signaling appears to be a conserved feature of glial cells in evolutionarily distant species.
To further explore the molecular mechanisms by which glial cells establish competence to respond to axonal injury and dynamically regulate reactive responses, we performed an in vivo RNAi screen for novel signaling molecules required for glial engulfment of degenerating ORN axons in Drosophila. We identified PI3K signaling and the Stat92E transcription factor as important regulators of glial Draper expression. Interestingly, both of these pathways were necessary for the expression of Draper in the resting, uninjured brain. However, while PI3K signaling was dispensable for injury-induced up-regulation of Draper, we found that Stat92E was necessary in glial cells for both injury-induced up-regulation of Draper and clearance of degenerating axonal debris. Surprisingly, we find that Stat92E acts downstream of Draper to activate transcription. We propose a simple model for glial activation after axotomy whereby Draper signaling is stimulated in a graded fashion according to the level of axonal debris (i.e., the severity of axonal injury), which in turn promotes Stat92E-dependent changes in glial gene expression in a way that is proportional to the strength of Draper pathway signaling. Such a mechanism places levels of glial activation directly downstream of the total amount of axonal debris present in the adult brain.

Glial PI3K Signaling Modulates Levels of the Engulfment Receptor Draper in the Healthy Brain
In order to identify new glial engulfment genes we performed an in vivo RNAi screen using a previously established assay [24]. Briefly, ,300 candidate engulfment genes were knocked down specifically in glia by driving UAS-regulated RNAi constructs with the glial specific repo-Gal4 driver. To assay the ability of glia to clear degenerating axonal debris we labeled a subset of maxillary palp ORNs with green fluorescent protein (GFP), severed the axons by surgically removing the maxillary palps, and assayed for clearance of GFP-labeled ORN axonal debris at various timepoints. Interestingly, we found glial-specific knockdown of pi3k92e, raptor, or pdk-1-key components of the phosphoinositide 3-kinase (PI3K) signaling pathway-led to a decrease in glial clearance of axonal debris 5 days after axotomy ( Figure 1A and 1B; Data S1). Similar results were found when we drove glial expression of a dominant-negative version of PI3K92e (UAS-pi3k92e DN ; Figure S1; Data S2). We note that while axon clearance was delayed in these backgrounds, ultimately all axonal debris was cleared from the brain 7-10 days after axotomy (unpublished data), indicating that glia exhibited a delay in clearance rather than a blockade.
To further explore the cellular basis for this delayed axon clearance phenotype, we assayed glial expression of the engulfment receptor Draper. Surprisingly, glial knockdown of pi3k92e, raptor, or pdk1, or over expression of pi3k92e DN reduced Draper expression significantly in the uninjured brain (Figures 1C and S1; Data S3). Reductions in Draper protein levels were confirmed and quantified on Western blots ( Figures 1D, 1E, and S1; Data S3). Reciprocally, we found that over-expression of a constitutively activate version of PI3K92E (UAS-pi3k92e CAAX ) in glia led to a dramatic increase in Draper levels in uninjured brains (Figure 1C-1E; Data S3). Thus, Draper expression was tightly correlated with glial PI3K signaling levels in the healthy uninjured brain. To our knowledge this is the first pathway shown to modulate the establishment of Draper expression levels in glia.
While maxillary palps house ,60 ORN cell bodies and ablation of maxillary palps results in a modest increase in Draper protein expression, more severe injury of ORN axons by removal of antennae (which house ,600 ORN cell bodies) results in a dramatic up-regulation of Draper protein and mRNA levels [24,27]. To determine whether this response was normal when the PI3K pathway was compromised, we ablated antennae in control, pi3k92e RNAi , raptor RNAi , and pdk1 RNAi backgrounds. Despite the fact that knockdown of pi3k92e, raptor, or pdk1 significantly reduced Draper levels in the brains of uninjured control animals, we found that antennal ORN axotomy induced significant upregulation of Draper levels in glia surrounding the antennal lobe ( Figure 1C). While it is possible that this injury-induced Draper up-regulation could be the result of incomplete RNAi mediated knockdown of components of the PI3K signaling pathway, on the

Author Summary
Acute injuries of the central nervous system (CNS) trigger a robust reaction from glial cells-a non-neuronal population of cells that regulate and support neural development and physiology. Although this process occurs after all types of CNS trauma in mammals, how it is activated and its precise role in recovery remain poorly understood. Using the fruit fly Drosophila melanogaster as a model, we previously identified a cell surface receptor called Draper, which is required for the activation of glia after local axon injury (''axotomy'') and for the removal of degenerating axonal debris by phagocytosis. Here, we show that regulation of Draper protein levels and glial activation through the Draper signaling pathway are mediated by the well-conserved PI3K and signal transducer and activator of transcription (STAT) signaling cascades. We find that STAT transcriptional activity is activated in glia in response to axotomy, and identify an injury-responsive regulatory element within the draper gene that appears to be directly modulated by STAT. Interestingly, the intensity of STAT activity in glial cells after axotomy correlates tightly with the number of local severed axons, indicating that Drosophila glia are able to fine-tune their response to neuronal injury according to its severity. In summary, we propose that the initial phagocytic competence of glia is regulated by setting Draper baseline levels (via PI3K), whereas injury-activated glial phagocytic activity is modulated through a positive feedback loop that requires STAT-dependent activation of draper. We speculate that the level of activation of this cascade is determined by glial cell recognition of Draper ligands present on degenerating axon material, thereby matching the levels of glial reactivity to the amount of injured axonal material.
basis of the consistency in results among the different components of the pathway we favor the notion that basal levels of Draper (i.e., those present in the healthy brain) are regulated by PI3K signaling and injury-induced up-regulation of Draper is regulated by alternate signaling pathways.

The Draper Locus Contains an Enhancer Element That Is Responsive to Axonal Injury through Stat92E
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 59, intronic, or 39 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. repo-Gal4 was used to drive UAS-pdk1 RNAi , UAS-pi3k92e RNAi , and UAS-raptor RNAi in a background containing the temperature sensitive Gal4 repressor, Gal80 ts ; Uninjured, 3 days, and 5 days after maxillary palp ablation are shown. Dotted lines indicate 85e + glomerulus used for quantification of GFP remaining. (B) Quantification of GFP intensities in 85e + glomeruli from (A), p-values were calculated using one-way ANOVA and Fisher's LSD post-hoc analysis. Error bars represent standard error of the mean (SEM). (C) Single slice confocal images of adult brains; Draper antibody stains in control (OR85e-GFP,Gal80 ts /+; repo-gal4/+) and RNAi backgrounds (OR85e-GFP,Gal80 ts /+; repo-gal4/UAS-pdk1 RNAi ; UAS-pi3k92e RNAi , OR85e-GFP,Gal80 ts /+; repo-gal4/+; and OR85e-GFP,Gal80 ts /UAS-raptor RNAi ; repo-gal4/+). Uninjured and one day after antennal ablation are shown. (D) Western blot analysis of Draper protein levels in ,3 adult central brain regions of control (OR85e-GFP,Gal80 ts /+; repo-gal4/+), pi3k92e RNAi (UAS-pi3k92e RNAi , OR85e-GFP,Gal80 ts / +; repo-gal4/+), and pi3k92e CAAX (UAS-pi3k92e CAAX , OR85e-GFP,Gal80 ts /+; repo-gal4/+) genotypes. a-Tubulin was used as a loading control. Full blot located in Figure S1. (E) Quantification of Draper protein levels shown on Western blot after normalization to Tubulin, n = 3, p-values were calculated using one-way ANOVA followed by Tukey's post hoc test. doi:10.1371/journal.pbio.1001985.g001 To determine whether any of these potential DEEs were responsive to axotomy, we ablated antennae or maxillary palps and assayed reporter activation in glia one day after injury. We did not observe glial expression after axonal injury with dee2-6or dee8-10-Gal4 lines (unpublished data). However, one day after antennal ORN axotomy, we observed a striking increase in glial expression of mCD8::GFP in the dee7-Gal4 reporter background, and GFP levels were further increased four days after axon injury ( Figure 2B). We note that in uninjured animals we observed low level expression of this element in astrocyte-like glia, which were randomly distributed in the neuropil ( Figure 2B) but it did not drive GFP expression prior to axon injury in ensheathing or cortex glia, those adult brain glia which normally express Draper [23]. Interestingly, not only was injury-induced reporter expression strong in ensheathing glia surrounding the antennal lobe-those glia that normally engulf degenerating ORN axons [23]-but the reporter expression also increased robustly in cortex glia throughout the brain ( Figure 2B and 2D). This widespread activation of the reporter supports the notion that glia, even at locations distant from the injury site, can respond molecularly to axonal damage. Notably, we found the activation of the dee7-Gal4 element appeared to scale with the severity of the axonal injury: when we ablated one maxillary palp (,60 ORNs), two maxillary palps (,120 ORNs), one antenna (,600 ORNs), or both antennal (,1,200 ORNs), we observed a correlated increase in dee7-Gal4driven mCD8::GFP ( Figure 2D). We further note that ablation of maxillary palps, whose axons are found in the maxillary nerve and ventro-medial regions of the antennal lobe, resulted in a much reduced and more localized increase in dee7-Gal4-driven mCD8::GFP in cortex glia located in the ventral region of the antennal lobe ( Figure 2D).
Sequence analysis of the 2619 bp dee7 element led to the discovery of three consensus Stat92E binding sites (TTC3n/ 4nGAA) [31], the sole member of the signal transducer and activator of transcription (STAT) family of molecules in Drosophila [31,32]. Of these three Stat92E sites, two were also present in dee7-Gal4 or dee7 MUT -Gal4 driving two copies of UAS-mCD8::GFP (dee7.2XmCD8::GFP, dee7 MUT .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 a-GFP and a-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. doi:10.1371/journal.pbio.1001985.g002 PI3K and Stat92E Regulate Draper and Glial Activation PLOS Biology | www.plosbiology.org dee6-Gal4, which was not responsive to axonal injury ( Figure 2A). We mutated the Stat92E binding site specific to the dee7-Gal4 element, integrated this dee7 MUT -Gal4 construct into the same genomic location used for the previously generated reporter lines, and examined its responsiveness to axonal injury. While baseline levels of mCD8::GFP expression were similar to dee7-Gal4, dee7 MUT -Gal4 exhibited an ,30%-40% decrease in the injuryinduced expression of mCD8::GFP at both 1 and 4 days after axotomy ( Figure 2B and 2C; Data S4). In contrast, simultaneous mutation of the two other Stat92E binding sites within the dee7 element had no effect on transcriptional activation of the dee7-Gal4 reporter (Figure S2A-S2C; Data S5). From these data we conclude that dee7 contains a glial transcriptional regulatory element that is responsive to axonal injury, and our data suggest that at least one Stat92E binding site is required for maximal activation of this element after axotomy.

Glial Loss of STAT92E Suppresses Clearance of Axonal Debris and Eliminates Expression of Draper
We next sought to determine whether Stat92E was required for modulating glial responses to axonal injury and clearance of degenerating axonal debris. Stat92E function was knocked down specifically in glia by expressing a UAS-stat92e RNAi construct with the pan-glial repo-Gal4 driver. In controls the majority of GFP + maxillary palp ORN axon material was cleared from the brain 5 days after injury (Figure 3A-3C; Data S6). However, in stat92e RNAi animals, GFP + axonal debris persisted 5 days after injury (Figure 3A-3C; Data S6). In contrast to depletion of the PI3K signaling cascade, glial stat92e RNAi suppressed clearance of antennal ORN axons even 15 days after injury ( Figure S3A), indicating an essential requirement for STAT92E in glial clearance of axonal debris. We were able to confirm the UAS-Stat92E RNAi line efficiently targets stat92e as glial co-expression of a GFP-tagged stat92e molecule with the stat92e RNAi construct eliminated all Stat92E-GFP expression compared to controls ( Figure S3B). These observations argue that Stat92E is an important regulator of glial engulfment activity in the adult brain.
On the basis of our identification of a Stat92E-dependent injury-responsive element in the draper gene, we predicted Stat92E would modulate glial phagocytic activity by regulating draper expression after axotomy. Draper is normally expressed in ensheathing and cortex glia throughout the uninjured brain and is up-regulated around the antennal lobe after antennal ORN axotomy ( Figure 3D). Glial-specific knockdown of Stat92E led to nearly undetectable levels of Draper expression in the adult brain even prior to injury ( Figure 3D and 3E; Data S7). We confirmed this widespread loss of Draper by performing Western blots on dissected adult brains from control, stat92e RNAi , and draper RNAi animals ( Figures 3F, 3G, and S3; Data S8). In contrast to loss of PI3K signaling, knockdown of Stat92E in glia was also sufficient to potently suppress glial activation of draper after axotomy: stat92e RNAi animals exhibited no detectable increase in Draper levels after antennal ablation compared to controls. In addition, while Draper was localized specifically to severed ORN axons after maxillary palp ablation in controls, we found no detectable Draper localization to severed axons in stat92e RNAi animals ( Figure 3D and 3E; Data S7). These data suggest that Stat92E is required to establish normal basal levels of Draper expression in glia, and dynamically regulates increased Draper levels as glia respond to axotomy.
STAT signaling is involved in multiple cellular processes including cell survival, differentiation, motility, and immunity [33][34][35][36][37][38][39][40][41][42]. To exclude the possibility that the defects we observed in stat92e RNAi animals were the result of abnormalities in glial cell development we used the conditional Gal80 ts system to specifically activate stat92e RNAi at adult stages. When temperature sensitive stat92e RNAi animals were raised and tested at 18uC, we found that glia efficiently cleared axonal debris and expressed normal levels of Draper ( Figure S4; Data S9). However, when they were shifted to and tested at the restrictive temperature during adult stages (thereby activating the RNAi construct only after development was complete), we found that stat92e RNAi animals exhibited reduced expression of Draper and failed to clear degenerating axons ( Figure S4; Data S9). Glial cell morphology (visualized with membrane-tethered GFP) and numbers (counted with a-Repo antibody nuclear staining) appear grossly normal in these animals, arguing that these phenotypes are not the result of glial cell loss in stat92e RNAi backgrounds ( Figure S5A and S5B). Moreover, we found that adult-specific activation of the RNAi was reversible, as shifting these animals back to 18uC (thereby turning the RNAi off) re-established normal levels of Draper and initiated clearance of axonal debris ( Figure S4; Data S9). Thus Stat92E functions in adult brain glia, where it modulates Draper expression and glial phagocytosis of degenerating axons.
Draper and the PTB domain-containing protein dCed-6 are both required for glial engulfment of degenerating axons and are expressed in glial cells in the adult brain [23]. To determine whether Stat92E broadly regulates engulfment gene expression we assayed dCed-6 levels in the adult brain in animals expressing stat92e RNAi in glia and found that dCed-6 was still present at high levels throughout the brain ( Figure S5C and S5D). Interestingly, while dCed-6 levels appeared grossly normal in a stat92e RNAi background, dCed-6 was not recruited to severed maxillary palp axons 1 day after axotomy ( Figure S5C). Thus, while Stat92E is necessary for dCed-6 recruitment to severed axons (i.e., glial responses to injury), basal levels of dCed-6 do not appear to be regulated by a Stat92E dependent mechanism.
Finally, we sought to determine whether Draper levels were regulated transcriptionally by STAT92E and/or PI3K signaling. We performed quantitative real-time PCR to measure draper transcript levels in dissected brains from control animals and animals expressing glial draper RNAi , pi3k92e CAAX (gain-of-function), pi3k92e RNAi , or stat92e RNAi . Consistent with STAT92E regulating draper at the transcriptional level, we found that glial RNAi for stat92e severely reduced draper transcripts to a level comparable to depletion with draper RNAi ( Figure 3H; Data S10). In contrast, neither loss-or gain-of-function manipulation of PI3K92E resulted in a statistically significant difference in draper transcript levels. While this argues for a post-transcriptional mechanism of Draper regulation by PI3K signaling, we cannot rule out the possibility that PI3K at least partially regulates draper at the transcriptional level considering loss of PI3K resulted in a trend of decreased (,40%) draper mRNA ( Figure 3H; Data S10).

Stat92E Transcriptional Activity Is Transiently Upregulated in Glia after Axonal Injury
To explore the dynamics of Stat92E signaling in adult brain glia we examined the expression patterns of transcriptional reporters for Stat92E activity [43]. These reporters have been previously shown to accurately reflect Stat92E transcriptional activity during development as well as in the adult [43][44][45][46]. We first used the 10XStat92E-GFP, which harbors ten Stat92E binding sites driving expression of enhanced GFP. In co-stains with a-Draper and a-Repo (a glial nuclear marker) we observed quite specific glial expression of the Stat92E reporter in uninjured controls ( Figure 4A). Moreover, after ablation of antennae we found strong GFP labeling of antennal lobe glia, and the GFP signal completely overlapped with Draper ( Figure 4A). After ablation of maxillary palps we found GFP signals co-localized with Draper in glomeruli housing severed axons ( Figure 4A). These data argue that Stat92E is active at a transcriptional level in adult brain glia.
The GFP driven by the 10XStat92E-GFP reporter is quite stable and can perdure in cells for ,20 hours after activation, which precludes our use of this construct to examine dynamic changes in Stat92E transcriptional activity. We therefore used a second reporter, 10XStat92E-dGFP, which drives the expression of a rapidly degraded, destabilized GFP (dGFP), thereby allowing for increased temporal resolution of Stat92E activity. Prior to injury, we were unable to detect any activation of this Stat92E transcriptional reporter in adult brains ( Figure 4B). However, beginning ,16 hours after antennal ablation we detected 10XStat92E-dGFP expression in cells surrounding the antennal lobe ( Figure 4B, arrows). GFP intensity peaked at ,24 hours after antennal ablation and disappeared by 48 hours after axotomy ( Figure S6).
To confirm that activation of the 10XStat92E-dGFP reporter after axotomy is Stat92E-dependent and glial specific, we knocked down Stat92e specifically in glia, severed axons, and assayed 10XStat92E-dGFP activity. We found that glial-specific knockdown of Stat92E completely suppressed the axotomy-induced activation of the 10XStat92E-dGFP transcriptional reporter ( Figure 4B). Consistent with our observations of widespread activation of the dee7-Gal4 driver in glia throughout the brain after injury, we also found that axonal injury led to broad Uninjured, one day after maxillary palp injury and one day after antennal ablation are shown. Dotted circles and boxes represent regions used for quantification of Draper immunoreactivity. (E) Quantification of data from (D), p-values were calculated using one-way ANOVA followed by Tukey's post hoc test, n.s., not significant. Error bars represent SEM. (F) Western blot analysis of Draper protein levels in ,3 adult central brain regions of control (repo-gal4/+), stat92e RNAi (UAS-stat92e RNAi /+; repo-gal4/+), and draper RNAi (draper RNAi /+;repo-gal4/+) genotypes. a-Tubulin was used as a loading control. Full blot located in Figure S3. (G) Quantification of Draper protein levels shown on Western blot after normalization to a-Tubulin, n = 3, pvalues were calculated using one-way ANOVA followed by Tukey's post hoc test. (H) Real-time PCR analysis of draper mRNA levels in adult central brain region of control (repo-gal4/+), drpr RNAi (draper RNAi /+;repo-gal4/+), pi3k92e CAAX (UAS-pi3k92e CAAX /+;repo-gal4/+), pi3k92e RNAi (UAS-pi3k92e RNAi / +;repo-gal4/+), and stat92e RNAi (stat92e RNAi /+;repo-gal4/+) genotypes. Draper Ct values were normalized to ribosomal protein L32 and results are presented as fold induction relative to control. Values represent mean 6 SEM from three independent RNA isolations. p-Values were calculated using one-way ANOVA and Dunnett's post hoc test. doi:10.1371/journal.pbio.1001985.g003 activation of the 10XStat92E-dGFP reporter in glial cells ( Figure 4C) and expression of these two reporters colocalize in glial cells after axotomy ( Figure 4D), indicating they are active in the same cells. Together these data indicate that Stat92E can transiently increase the transcriptional activation of target genes in glial cells throughout the brain after axonal injury, with the strongest increases in target gene activation occurring adjacent to injury sites.

Stat92E-Dependent Activation of Draper Does Not Require Components of the Canonical JAK/STAT Signaling Pathway
STAT activity is generally regulated by the JAK signaling platform, and this pathway is conserved in all higher metazoans. The Drosophila JAK/STAT signaling pathway consists of a single JAK molecule, Hopscotch (hop) [47], and the cytokine like receptor Domeless (Dome) [48]. To determine if Stat92Edependent activation of draper is mediated through canonical JAK/STAT signaling we drove RNAi constructs targeted against hop, and a dominant negative Domeless molecule, Domeless DCYT [49], in glial cells, and assayed Draper expression and clearance of severed axons. Surprisingly, in each of these backgrounds we found Draper levels were similar to control animals and axons were efficiently cleared 5 days after injury ( Figure S7A and S7B; Data S11). Reciprocally, we found a gain-of-function allele of hop, hop TUM , which has been shown in numerous assays to activate Stat92E transcriptional activity [43,50,51], failed to activate the10XStat92E-dGFP reporter in adult brain glia ( Figure S7C). Notably, expression of an activated version of Stat92E, Sta-t92E DNDC [52], led to strong activation of the 10XStat92E-dGFP reporter ( Figure S7C). Stat92E DNDC has previously been shown to require phosphorylation at Y711 for activation [52]. We therefore expressed a version of Stat92E DNDC with a Y711F mutation in glia, and found it was insufficient for activation of the 10XStat92E-dGFP reporter ( Figure S7C). Together these data argue that while phosphorylated Stat92E mediates activation of Stat92E reporters in glia, canonical JAK/STAT signaling is neither necessary nor sufficient to activate Stat92E-dependent glial responses to axon injury.

Signaling through the Draper Receptor Activates Stat92E-Dependent Transcriptional Up-regulation of draper
To date Domeless is the only receptor known to positively regulate Stat92E signaling under normal physiological conditions [48,53,54]. Considering we were unable to demonstrate a role for Domeless signaling in activation of Draper after injury, we sought to determine whether Draper itself might have a role in modulating glial gene expression. We assayed 10XStat92E-dGFP activation after axonal injury in draper D5 null mutants and, intriguingly, loss of Draper resulted in a complete lack of 10XStat92E-dGFP activation after axotomy ( Figure 5A). Consistent with a direct requirement for Draper signaling in STAT92E-dependent activation of draper after axotomy, we also found a lack of activation of the dee7-Gal4 reporter after axonal injury in draper D5 animals ( Figure 5B). These data demonstrate a direct role for the Draper receptor in modulating Stat92E-dependent changes in glial gene expression following local axonal injury.
In summary, our data suggest that axonal injury leads to Stat92E-dependent transcriptional up-regulation of draper through a Draper/Src42a/Shark/Rac1-dependent signaling cascade. While activation of Stat92E, Src42A, or Rac1 led to robust activation of the 10XStat92E reporter, none were sufficient to increase basal Draper levels (unpublished data), suggesting additional transcriptional inputs are required for injury-induced transcriptional activation of draper.

Draper Is a Primary Target for Stat92E during Glial Clearance of Degenerating Axons
Together our data argue that draper transcription is regulated by Stat92E after axotomy. Stat92E may regulate many genes after axotomy, or only a few critical targets essential for engulfment. To further explore the relationship between Stat92E and the draper gene, we over-expressed Draper in a stat92e RNAi background to determine whether resupplying Draper was sufficient to overcome the engulfment deficit observed in Stat92E knockdown animals. Remarkably, over-expression of Draper in stat92e RNAi animals led to a near complete rescue of the engulfment defect ( Figure 6A and 6B; Data S12). As an alternate method to increase Draper levels we also drove glial expression of the activated PI3K molecule, PI3K92e CAAX , in the presence of stat92e RNAi or control draper RNAi animals. We found that activation of PI3K signaling was sufficient to increase Draper levels ( Figures 6E, 6F, and S9; Data S13) and rescue engulfment defects in stat92E RNAi backgrounds ( Figure 6C and 6D; Data S14).
Consistent with the ability of PI3K signaling to drive Draper expression independently of Stat92E, we also found that activated PI3K signaling was not sufficient to activate expression of the 10XStat92edGFP reporter ( Figures 6E, 6F, and S9). However, PI3K signaling appeared to sensitize brain glia to injury as glial PI3K92e CAAX enhanced 10XStat92E-dGFP activation after antennal ablation compared to controls ( Figure S9), perhaps through driving increased Draper levels. From these data we conclude that draper is a critical target of Stat92E during glial responses to axonal injury. In addition, our data argue that activated PI3K signaling results in Stat92E-independent increases in Draper levels.

PI3K Signaling and Stat92E Converge to Regulate Draper Expression in the Adult Brain
Our work identifies two new signaling pathways important for regulating glial engulfment function in vivo. First, we show that the PI3K signaling pathway modulates Draper levels in the healthy, uninjured brain as reduced PI3K signaling leads to dramatically decreased glial Draper and constitutive activation of PI3K signaling leads to Draper up-regulation. However, depletion of PI3K signaling components delays but does not completely block the ability of glial cells to up-regulate Draper levels or clear axonal debris in response to axotomy, perhaps due to positive signaling through the small amount of Draper that remains under these conditions. Second, we identify Stat92E as a potent regulator of both basal and injury induced Draper levels in adult brain glia. Loss of Stat92E in mature glia results in significantly decreased draper transcript levels and a near complete loss of Draper protein in the uninjured brain.
What is the relationship between PI3K signaling and Stat92E in regulating basal levels of Draper in the healthy brain? On the basis of our analysis of draper mRNA levels we speculate that STAT92E regulates draper at least in part at the transcriptional level. It appears unlikely that Stat92E functions downstream of PI3K signaling since loss of Stat92E in a constitutively activated PI3K background did not suppress PI3K-dependent increases in Draper levels, and gain-of-function PI3K was sufficient to rescue reduced Draper levels and engulfment defects in stat92e RNAi animals. STAT molecules have been shown to be capable of acting as adaptor molecules for receptors that ultimately lead to activation of PI3K signaling [57][58][59], therefore Stat92E could function in adult brain glia upstream of PI3K signaling in a nontranscriptional manner to regulate basal levels of Draper. Finally, Stat92E might transcriptionally regulate key molecules required for activation or execution of PI3K signaling. In such a situation Stat92E and PI3K signaling could modulate Draper levels through parallel mechanisms, but both would be required for expression of appropriate levels of Draper in the adult brain.
A role for PI3K signaling in phagocytic function appears to be conserved from Drosophila to mammals. Activation of PI3K signaling occurs downstream of the Fcc receptor [60][61][62][63]. This finding is intriguing in light of the fact that Draper appears to act as an ancient immunoreceptor, activating a Src family kinase signaling cascade through ITAM/ITIM-dependent mechanisms [25,27]. PI3K signaling is required for efficient formation of the phagocytic cup in macrophages [61,64,65], and loss of PI3K has been reported to lead to a delay in clearance of cell corpses and myelin [62,66,67]. This latter finding argues for conservation of a requirement for PI3K signaling even among glial cell types in Drosophila and mammals. In our study we also find a delay of axonal clearance when PI3K signaling is suppressed, but ultimately axons are cleared. We speculate this phenotype is a result of decreased expression of Draper since we can fully rescue loss of PI3K signaling phenotypes by resupplying Draper. This observation argues strongly that a key role for PI3K signaling in phagocytic function is the regulation of engulfment factors, and in particular Draper.

Axonal Injury Causes Widespread Activation of Glial Cells throughout the Drosophila Brain
Ablation of Drosophila third antennal segments leads to axotomy of all antennal ORNs and Wallerian degeneration of ,1,200 ORN axons and their synapses within the antennal lobe of the brain. We previously observed a robust increase in Draper levels in glia surrounding the antennal lobe after antennal injury (but not elsewhere) and proposed that Draper increases were only local [23,24]. Using the dee7-Gal4 and 10XStat92E-dGFP reporters we have now shown that severe axonal injury (i.e., axotomy of nearly all ORNs) in the antennal lobe is sufficient to induce a transcriptional response in glial cells throughout the entire Drosophila brain (Figures 2B-2D, 4B-4D, 5A, and 5B). How can glial cells in distant parts of the brain receive signals that an injury has occurred? We can envision at least two scenarios to explain this observation. First, severed axons could release signals that act at a long distance to activate glia. If so, the reception of this axon-derived signal by glia would be Draper-dependent, since we show that Draper signaling is required for activation of both the dee7-Gal4 and 10XStat92E-dGFP reporters in glia after injury. Alternatively, spreading of an injury signal throughout the brain could be accomplished by glial to glial signaling. Astrocytes are indeed heavily coupled in mammals [68] and the same could be true in Drosophila adult brain glia where signals could spread through gap junctiondependent mechanisms. In the future it will be very exciting to define the molecules that regulate the spreading of the injury signal to distant glial subtypes, and determine their functional role in brain recovery from trauma.

A DraperRStat92Erdraper Gene Auto-Regulatory Loop Increases Glial Engulfment Activity after Axotomy
We propose a novel injury-induced auto-regulatory loop whereby activation of Draper in glial cells responding to axonal injury leads to downstream signaling through Src42a/Shark/ Rac1, and in turn Stat92E-dependent transcriptional activation of the draper locus. We provide multiple lines of evidence that activation of glial cells after local axotomy involves Stat92Edependent transcriptional activation of draper: RNAi knockdown of Stat92E leads to a loss of injury-induced Draper expression, mutation of a single Stat92E binding site in the dee7 enhancer element leads to ,40%-50% reduction in dee7-Gal4 activity upon injury, the 10XStat92E-dGFP transcriptional reporter is activated in glial cells upon injury, and activation of both 10XStat92E-dGFP and the dee7-Gal4 reporter are lost in draper null animals. We provide compelling evidence that the dee7 reporter activity resembles injury induced transcriptional activation of Draper: it expresses in the subtype of glia known to respond to injury and only after axotomy; it correlates with our data showing a larger increase in overall Draper up-regulation in the brain during the more severe antennal ablation; expression of the reporter is lost in drpr mutant animals; and its expression overlaps with the 10XStatDGFP reporter activity. As with all transcriptional reporters it remains to be determined whether it fully recapitulates endogenous draper regulation after injury, nevertheless, the dee7-Gal4 element will be an extremely useful tool with which to dissect pathways mediating axon injury-induced molecular changes in glial transcription. Finally, based on the presence of STAT92E binding sites in the draper locus, and the requirement of these for maximal activation of the injury-responsive dee7-Gal4 element, we suspect the draper gene may be a direct transcriptional target of STAT92E after axotomy.
Since mutation of the STAT92E binding sites in the dee7 element only led to a partial inhibition of dee7 activity after injury, it seems likely that Stat92E acts in cooperation with other factors. Evidence from mammals suggests that based on their weak transcriptional activation, STAT proteins often act in combination with other transcription factors for maximal target gene activation. Interestingly, we recently showed JNK signaling is required in glial cells for up-regulation of Draper after axotomy and that transcriptional reporters for JNK pathway signaling to dAP-1 increased in glia after axonal injury [69]. Mammalian STAT and AP-1 act together in reactive microglial cells to mediate the upregulation of inflammatory proteins [70,71]. It therefore remains an interesting possibility that combinations of Stat92E binding sites, AP-1 binding sites, and binding sites for other unidentified co-factors are involved in precisely modulating levels of Draper expression in the context of injury.
Our work has revealed a novel role for engulfment signaling pathways (i.e., Draper signaling) in the regulation of glial transcription after local axon injury, and revealed that Stat92E acts downstream. Despite the fact that Stat92E signaling is essential for a wide range of developmental processes in Drosophila, Draper is only the second receptor implicated in positively regulating the ability of Stat92E to activate transcriptional targets in vivo [48,53,54]. At the same time our work identifies the draper gene as a novel target for Stat92E-only three other genes, eve, crb, and dome, have been shown by in vivo analyses to be directly regulated by Stat92E [72][73][74]. Our work therefore enriches our understanding of the physiological roles for engulfment receptors and Stat92E signaling in the modulation of glial responsiveness to axonal injury.
We anticipate a similar role for mammalian engulfment receptors and STATs in reactive gliosis. Indeed, activation of STAT molecules in mammalian glial cells (determined primarily by using phospho-STAT-specific antibodies) has been widely reported in response to focal brain lesion [75,76], traumatic brain injury [77,78], and spinal cord injury (SCI) [79]. STAT3 is a critical modulator of reactive gliosis: loss of STAT3 from astrocytes in the context of SCI has been shown to lead to attenuated activation of GFAP expression, a lack of astrocyte hypertrophy, and reduced formation of the glial scar. This in turn results in increased demyelination, enhanced inflammation, and less efficient motor axon recovery [80,81]. IL-6 has been shown to act upstream of STAT3-dependent GFAP induction in Schwann cells during peripheral nerve regeneration, but both the source of IL-6 in vivo and how STATs are activated in glia after injury remains unclear [82]. Based on this study we speculate mammalian STATs may be activated in reactive glia by the Draper orthologs MEGF10 or Jedi.

Fly Strains and Molecular Biology
The The dee7-Gal4 construct was made by PCR amplification of the 2,619 bp fragment from the Draper BACR17K18 clone using the following primers: forward 59caccagacctactcttagctctgatggagg-39, reverse 59-gtttgtgtttccatggattcaggcttggg-39. The PCR product was purified using a Qiagen Gel Purification kit, directionally cloned into the Invitrogen pENTR/D-TOPO vector and transformed into One Shot Competent Cells using the pENTR/D-TOPO Cloning Kit (Invitrogen catalog number K2400-20). Colonies were prepped using the Qiagen miniprep kit. The dee7 fragment was then shuttled into the pBGUw destination vector [15] using the Invitrogen Gateway LR Clonase Enzyme and transformed into heat shock competent DH5a cells. The construct was sequence verified and transgenic flies were generated by Best Gene Inc. using the PhiC31 targeted integration system.
To generate the dee7 MUT -GAL4 construct, the dee7/TOPO construct was used as a template and PCR was carried out using Invitrogen Quick Change II Site-Directed Mutagenesis kit (catalog number 200523) with the following primers (mutation sites underlined): forward-59 CTG TGC CGA ACA CGT TAA CCA TTG AAA AAT CTC GC 39, reverse-59 GCG AGA TTT TTC AAT GGT TAA CGT GTT CGG CAC AG 39. A DpnI digestion was performed and DNA was transformed into XL-1 Blue super competent cells and plated. Colonies were prepped using the Qiagen miniprep kit and the mutations were verified by sequencing (Genewiz). The dee7 MUT enhancer fragment was then shuttled into the pBGUw vector using methods described above and transgenic flies were generated by Best Gene Inc. using PhiC3 targeted integration.

Olfactory Neuron Injury Protocol, Immunohistochemistry, and Confocal Microscopy
Maxillary palp and third antennal segment ablations, adult brain dissections, and antibody stainings were performed using previously described methods [24,85]. Samples were mounted in Vectashield (Vector Laboratories) antifade reagent and viewed on a Zeiss LSM5 Pascal confocal microscope. In all experiments, laser settings were kept identical for all brains imaged as part of that experiment. For Figure 2C, single slice confocal images of approximately the same depth in the brain were identified and total intensity of GFP in the brain was measured. The minimum threshold was set at 3 to eliminate most background and the maximum threshold was set at the maximum value of 255. Measures of GFP intensity within maxillary palp glomeruli were performed as previously described [24]. For Figure 3E, Draper expression after maxillary palp injury was measured from single confocal slices at the depth of the OR85einnervated glomerulus. A circle was drawn around the area of the OR85e + innervated glomerulus and total intensity of Draper was measured. Draper expression after antennal injury was measured from single confocal slices about half way through the antennal lobe. A fixed area rectangle at the edge of the antennal lobe was used to measure total intensity of Draper. All quantification of measurements was performed using Image J software. The following antibodies were used: 1:200 mouse anti-GFP (Invitrogen), 1:500 rabbit anti-Draper [86], 1:500 rat anti-dCed6 [87], 1:200 FITC anti-mouse IgG, 1:200 Cy3 anti-rabbit IgG, 1:200 Cy3 anti-rat IgG (Jackson ImmunoResearch). Flies were raised and maintained at 25uC unless otherwise noted. In experiments utilizing Gal80 ts flies were raised at 18uC and shifted to 30uC for at least 7 days prior to injury or dissection.

Real-time PCR
Drosophila central brain regions were dissected in Jan's saline (1.8 mM Ca 2+ ) and immediately frozen on dry ice. Total RNA was extracted in Trizol and the aqueous phase was passed over an Omega Bio-Tek E.Z.N.A MicroElute RNA Clean-Up Column with an on-column DNAase I treatment (Omega). RNA concentration was determined on a Nanodrop 2000c spectrometer (Thermo Scientific). RNA was diluted to equal concentration and 125 ng of total RNA was reverse-transcribed with the SuperScript VILO cDNA synthesis Kit for 2 h at 42uC.Relative quantification of gene expression was carried out on an ABI 7000 Real-Time PCR machine. The following Taqman assays (Applied Biosystems) were used: (i) Ribosomal protein L32 (ABI pre-made assay Dm02151827_g1) (ii) Draper-I custom assay, F-primer, TGTGATCATGGTTACGGAGGAC; R-primer, CAGCCG-GGTGGGCAA; probe, CGCCTGCGATATAA [27]. Assay efficiencies were experimentally determined (RpL32, 102%; Draper-I, 103%; using a 5-point dilution series of cDNA spanning a 20-fold range in concentration. The raw threshold cycle (Ct) of the normalization control (RpL32) did not vary by more than 0.5 cycles across all time points analyzed. Statistical analysis (ANOVA, with Dunnett's Multiple Comparisons Test) was performed on 2 2DCt values. Draper Ct values were normalized to ribosomal protein L32 and results are presented as fold induction relative to uninjured levels.  Figure S4 Stat92E is required in the adult brain for draper expression and glial engulfment function. (A) Z-stack confocal images of OR85e-GFP axons, single slice confocal images of Draper antibody stain; UAS-stat92e RNAi was driven in all glia using repo-Gal4 in a background containing the temperature sensitive Gal4 repressor, Gal80 ts . Axons (OR85e-GFP, green) and Draper levels (red) are shown in control (OR85e-GFP,Gal80 ts / +;repo-gal4/+) or stat92e RNAi knockdown (OR85e-GFP,Gal80 ts / stat92e RNAi ;repo-Gal4/+) animals. Temperature shifts were performed as follows: 18uC indicates that flies were raised and kept at 18uC throughout experiment. 18uC-30uC indicates that flies were raised at 18uC and then shifted to 30uC as adults for 7 days prior to and 5 days following maxillary palp ablation. 18uC-30uC-18uC indicates that flies were raised at 18uC, shifted to 30uC as adults for 7 days, and then shifted back to 18uC for 7 days prior to and 5 days following maxillary palp ablation. (B) Quantification of GFP intensities in 85e + glomeruli from (A). Error bars represent SEM, n$10.

(XLS)
Data S12 Excel spreadsheet containing the raw numerical data and statistical analyses for Figure 6A and 6B. (XLSX) Data S13 Excel spreadsheet containing the raw numerical data and statistical analyses for Figure 6E and 6F. (XLSX) Data S14 Excel spreadsheet containing the raw numerical data and statistical analyses for Figure 6C and 6D. (XLSX) Methods S1 Supporting methods and references. (DOC)