In an effort to identify distinct morphological subtypes of glial cells in the mature Drosophila
brain, we used the MARCM system (Lee and Luo 2001
) to generate small clones of glial cells labeled with membrane tethered GFP. Larvae containing a hs-flipase
allele and a wild type chromosome arm for recombination were subjected to a short heat shock (37°C) and glial cells within clones were visualized by use of the pan-glial driver, repo-Gal4
. Our analysis of glial subtype morphology is focused mainly on the adult antennal lobe region (cartoon schematic depicted in ) owing to its well-defined histology and accessibility to genetic manipulation. In the adult brain we identified clones resembling each of the three main types of glial cells found in embryos and larvae: (1) cortex glia, which resided outside the neuropil in regions housing neuronal cell bodies, ramified dramatically to surround individual cell bodies (); (2) surface glia, which appeared as large flat cells enveloping the surface of the brain, did not extend any processes into the brain (not shown); and (3) neuropil glia, which were closely associated with the neuropil, extended membranes into synaptic regions, and surrounded large bundles of axons (). As in the embryo and larva, glial cell bodies were not found within the neuropil, rather they resided at the edge of the neuropil (neuropil glia), in the cortex (cortex glia) or at the surface of the brain (surface glia).
Identification of morphologically distinct subtypes of glial cells in the adult Drosophila brain
Interestingly, the single-cell resolution provided by MARCM analysis allowed us to further subdivide neuropil glia into two distinct morphological classes, “ensheathing glia” and “astrocytic glia”. Ensheathing glia () appeared as flattened cells that lined the borders of the neuropil and subdivided regions of the brain by isolating neuropil from the surrounding cortex. Within the antennal lobe, ensheathing glial membranes surrounded individual glomeruli (the functional units of the antennal lobe) but did not extend into the synaptic regions of the glomeruli. In addition, we identified an astrocyte-like cell type () that extended membrane processes deeply into the neuropil and ramified profusely in synaptic-rich regions. This latter cell type we refer to as the fly “astrocyte,” based on its striking morphological similarity to mammalian astrocytes, as well as the conserved expression of a number of molecular markers used to identify astrocytes in the mammalian brain. Mammalian astrocytes remove excess amounts of extracellular glutamate through the high-affinity excitatory amino acid transporters (EAAT), GLAST and GLT-1, which transports the glutamate into glial cells where it is then converted into glutamine by glutamine synthetase (Chaudhry et al. 1995
; Levy et al. 1995; Rival et al. 2004
). We found that Drosophila astrocytes also express the transporter EAAT1 (Supplemental Fig. 1
). While we used the adult antennal lobe as our primary model tissue in this study, we observed the morphological glial subtypes described above in all brain regions examined, suggesting that our results are generally applicable to glial populations throughout the adult Drosophila
brain (Supplemental Fig. 2
We next sought to identify Gal4 driver lines that would allow us to uniquely label and manipulate these glial populations, with our major focus being to genetically subdivide neuropil glia (i.e. ensheathing glia versus astrocytes). To accomplish this, we crossed UAS-mCD8::GFP
to a previously described collection of embryonic and larval glial drivers (Ito et al. 1995
), as well as a number of drivers generated in our own laboratory. We then looked in the adult antennal lobe to examine the morphology and spatial distribution of cell types marked by these drivers in a background with glial nuclei (α-Repo) and the neuropil (α-nc82) also labeled. The repo-Gal4
driver labeled all Repo+
glial subtypes in the adult brain, as evidence by α-Repo immunostaining in the nuclei of GFP+
cells (). Membrane processes from Repo+
cells are found throughout the adult brain, and together they constitute the diverse collection of glial subtypes identified in our single-cell MARCM analysis. Upon examination of a single glomerulus within the antennal lobe we found that membranes from Repo+
cells both surround and invade glomeruli (). All GFP expression in the adult brain driven by the repo-Gal4
driver can be suppressed by co-expression of Gal80 (a Gal4 inhibitor) under control of the repo promoter (repo-Gal80
) (), arguing that repo-Gal80
can efficiently block Gal4-mediated activation of UAS-reporters in all adult brain glia.
Characterization of Gal4 drivers that uniquely label astrocytes and ensheathing glial subtypes
Two drivers, mz0709-Gal4 and alrm-Gal4, appeared to show very specific expression in ensheathing glia and astrocytes, respectively (). Glial processes labeled by mz0709-Gal4 were found at the edge of the antennal lobe and extended deeply into the neuropil region (). These flattened glial processes surrounded, but did not invade, individual glomeruli (), and did not extend into the cortex region. With the exception of variable expression in a small number of neurons, all mz0709-Gal4-induced expression was suppressed by repo-Gal80, indicating that mz0709-Gal4 is largely specific to ensheathing glia. The generation of MARCM clones labeled with the mz0709-Gal4 driver resulted in the consistent labeling of ensheathing glia, but not astrocytes, within the antennal lobes. Reciprocally, alrm-Gal4 was found to be expressed exclusively in astrocytes (). All cellular processes from cells labeled with alrm-Gal4 extended into the neuropil (), showed a highly branched or tufted morphology, invaded individual glomeruli (), and all alrm-Gal4-driven expression was suppressed by repo-Gal80 (). Additionally, we found that single cell MARCM clones labeled with the alrm-Gal4 driver resulted in the consistent labeling of astrocytes, but not ensheathing glia. Together, these drivers are excellent tools to manipulate and functionally distinguish different subtypes of glia in the adult Drosophila brain.
Ensheathing glia, but not astrocytes, express the engulfment receptor Draper
What are the functional roles for each glial subtype in the adult brain? Is each subtype responsible for a unique collection of tasks, or are all glial subtypes functionally equivalent? As a first step to determining the in vivo
functional differences between adult brain glial subtypes we explored the cell autonomy of glial phagocytic function. Severing olfactory receptor neuron (ORN) axons by surgical ablation of maxillary palps leads to axon degeneration (termed Wallerian degeneration), recruitment of glial membranes to fragmenting axons, and glial engulfment of axonal debris. These glial responses are mediated by Draper, the Drosophila
ortholog of the C. elegans
cell corpse engulfment receptor CED-1. In draper
null mutants, glia fail to extend membranes to degenerating ORN axons and axonal debris is not removed from the CNS (MacDonald et al. 2006
). Thus, Draper function should be autonomously required in phagocytic glial subtypes and Draper expression is predicted to act as a molecular marker for glial cells capable of performing engulfment functions.
To define the precise cell types that express Draper, we first labeled all glial membranes with mCD8::GFP driven by repo-Gal4
, stained with α-Draper antibodies, and assayed for colocalization of Draper and GFP (). As previously reported, we found extensive overlap of Draper and GFP in this background (MacDonald et al. 2006
). Draper and GFP signals overlapped at the edge of the neuropil, in membranes surrounding antennal lobe glomeruli, and in all cortex glia (). This labeling was specific to Draper since expression of a UAS-draperRNAi
construct with repo-Gal4
led to the elimination of all Draper immunoreactivity in the adult brain (). Thus, the entire population of cortex glia appear to express Draper and are likely to be phagocytic. However, cortex glia do not extend membranes into the antennal lobe neuropil, even after ORN axon injury (data not shown). Therefore, cortex glia are not likely responsible for clearing severed ORN axonal debris from the antennal lobe neuropil.
The engulfment receptor Draper is expressed in ensheathing and cortex glia but not in astrocytes
Interestingly, when mCD8::GFP was driven by mz0709-Gal4 we observed extensive overlap of Draper and GFP in neuropil-associated ensheathing glia (). A high magnification view of the antennal lobe revealed Draper and mz0709-Gal4 labeled membranes colocalizing and surrounding, but not innervating individual glomeruli (). Moreover, expression of UAS-draperRNAi in ensheathing glia with mz0709-Gal4 led to a dramatic reduction in Draper immunoreactivity in the neuropil, but the weaker Draper immunoreactivity in the cortex remained unchanged (). Conversely, we observed no overlap between Draper and GFP when we labeled astrocytic membranes using the alrm-Gal4 driver (). Furthermore, driving the expression of UAS-draperRNAi in astrocytes had no obvious effect on Draper expression in the brain (). These results indicate that Draper is expressed in cortex glia and ensheathing glia but not in astrocytes.
Ensheathing glia use Draper to extend membranes to degenerating axons and engulf axonal debris
The specific expression of Draper in antennal lobe ensheathing glia suggests that this glial subset is the phagocytic cell type responsible for engulfing degenerating axonal debris after ORN axotomy. To explore this possibility, we asked whether ensheathing glia or astrocytes extend membranes to severed axons after injury, and in which cell type Draper was required for clearance of axonal debris from the CNS. To assay extension of glial membranes to severed axons, we labeled glial membranes with mCD8::GFP, severed maxillary palp axons, and assayed for colocalization of Draper and GFP in glomeruli housing severed ORN axons. Within one day after injury, Repo+ glial membranes were found to localize to glomeruli housing severed maxillary palp axons and these membranes were decorated with Draper immunoreactivity (). Similarly, we found that mz0709+ glial membranes also localized to severed axons and colocalized with intense Draper immunoreactivity 1 day after injury (). Knockdown of Draper with UAS-draperRNAi using repo-Gal4 or mz0709-Gal4 completely suppressed the recruitment of both Draper and glial membranes to severed axons (). In contrast, when astrocyte membranes were labeled with GFP we did not observe colocalization of GFP and Draper immunoreactivity 1 day after axotomy (). In addition, knockdown of Draper in astrocytes with UAS-draperRNAi did not suppress the recruitment of Draper to severed axons (). In an effort to identify any indirect role for astrocytes during the injury response, we examined the morphology of astrocytes both before and after injury to determine whether they exhibited any overt changes in morphology or retracted their membranes from the site of injury to accommodate the recruitment of ensheathing glial membranes. However, we did not detect any obvious changes in morphology or in the positions of the astrocyte glial cells in response to axon injury. Together, these data indicate that Draper is required in ensheathing glia for recruitment of glial membranes and accumulation of Draper on severed ORN axons, and suggest that Drosophila astrocytic glia do not undergo any dramatic changes in morphology in response to ORN axotomy.
Ensheathing glia express Draper, are recruited to severed ORN axons, and phagocytose degenerating axonal debris
From the above data we predicted that ensheathing glia would act as phagocytes to engulf degenerating ORN axonal debris from the CNS. To test this we labeled a subset of maxillary palp ORN axons with mCD8::GFP using the OR85e-mCD8::GFP transgene, knocked down Draper function in glial subsets using our subset-specific driver lines, severed maxillary palp ORN axons, and assayed clearance of axons 5 days after injury. We first severed GFP-labeled axons in control animals with each driver and found that GFP+ axonal debris was efficiently cleared from the CNS within 5 days after injury (), confirming that glial phagocytic function is not affected in the driver lines. Strikingly, RNAi knockdown of Draper using UAS-draperRNAi in a background with repo-Gal4 or mz0709-Gal4 completely blocked clearance of GFP labeled axonal debris from the CNS (), while RNAi knockdown of Draper in astrocytes with alrm-Gal4 had no effect on axon clearance (). Thus, Draper is required autonomously in ensheathing glia for the clearance of degenerating ORN axonal debris from the CNS. In addition, knockdown of Draper in ensheathing glia with mz0709-Gal4 had no measureable effect on Draper expression in cortex glia (see ), arguing that cortex glia are not capable of compensating for the loss of phagocytic activity in ensheathing glia during the clearance of axonal debris from the antennal lobe neuropil after axotomy. From these data on morphogenic responses to injury and phagocytic function, we conclude that astrocytic, cortex, and ensheathing glia represent functionally distinct subsets of glial cells in the adult Drosophila brain.
Shark, a Src-family kinase acting downstream of Draper, is required in ensheathing glia for clearance of degenerating axons
We have recently shown that Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70, is part of the Draper signaling cascade and is essential to initiate phagocytic signaling events downstream of Draper during the engulfment of degenerating axons (Ziegenfuss et al. 2008
). Our model that Draper functions exclusively in ensheathing glia for clearance of degenerating ORN axons predicts that Shark and other components of the Draper signaling cascade would also function in ensheathing glia. To determine whether Shark function is required in ensheathing glia, we knocked down Shark in glial subsets and assayed the recruitment of Draper to severed ORN axons 1 day after injury, and the clearance of degenerating axonal debris from the CNS 5 days after axotomy. Consistent with a role for Shark in ensheathing glia, we found that knockdown of Shark with UAS-SharkRNAi
(Ziegenfuss et al. 2008
) driven by repo-Gal4
strongly suppressed both the recruitment of mCD8::GFP-labeled glial membranes and Draper to severed axons (), as well as the clearance of degenerating axonal debris from the CNS (). However, knockdown of Shark with alrm-Gal4
had no effect on the recruitment of Draper or GFP-labeled glial membranes to degenerating axons (), nor did it inhibit the clearance of axonal debris from the CNS (). Thus, Shark, like Draper, is required in ensheathing glia for efficient extension of glial membranes to degenerating axons and clearance of degenerating axonal debris from the CNS.
The non-receptor tyrosine kinase Shark functions in ensheathing glia to drive engulfment of ORN axonal debris
dCed-6 is expressed in ensheathing and cortex glia, and is required for glial clearance of degenerating axons
In C. elegans
the PTB domain adaptor protein CED-6 acts genetically downstream of CED-1 during engulfment of apoptotic cells (Liu and Hengartner 1998
) and, during Drosophila
metamorphosis, RNAi knockdown of dCed-6 has been shown to partially suppress glial engulfment of pruned axon arbors during remodeling of larval mushroom body γ neurons (Awasaki et al. 2006
). We asked whether dCed-6 was involved in glial reponses to axon injury in the adult brain. First, to determine where dCed-6 is expressed in the adult CNS we stained control animals with Draper and dCed-6 antibodies and found that Draper and dCed-6 immunoreactivity perfectly overlapped throughout the adult brain (). Since we have shown that Draper is expressed in cortex and ensheathing glia, we conclude that dCed-6 is also expressed in these glial subtypes.
Next we asked if dCed-6 was recruited to degenerating ORN axons. We performed maxillary palp or antennal ablations and compared dCed-6 and Draper recruitment 1 day after injury. We consistently found that dCed-6 colocalized with Draper at sites of severed axons after maxillary palp () and antennal injury (), which is consistent with dCed-6 being expressed in ensheathing glia and possibly functioning downstream of Draper.
dCed-6 is recruited to severed ORN axons after injury and genetically interacts with Draper
To obtain genetic evidence that dCed-6 was essential for glial clearance of degenerating axonal debris, we used the Df(2R)w73−1 deletion chromosome, which harbors a small deletion that removes the dCed-6 gene, and assayed for genetic interactions between this dCed-6 deletion chromosome and draper mutants. In control animals, GFP-labeled ORN axons are largely cleared 3 days after injury (). Interestingly, we found that while the majority of axons were cleared at this time point in draperΔ5/+ or Df(2R)w73−1/+ animals (), a significant number of axons remained in Df(2R)w73−1/+; draperΔ5/+ trans-heterozygous animals. () These data are consistent with draper and ced-6 exhibiting strong genetic interactions during glial clearance of degenerating axons.
The expression pattern of dCed-6 in the adult brain suggests that it is acting in ensheathing and/or cortex glia to mediate Draper-dependent glial engulfment functions. To determine the autonomy of dCed-6 function in the adult brain, we knocked down dCed-6 in different glial subsets using a UAS-dced-6RNAi construct and assayed Draper recruitment to severed axons and clearance of degenerating axonal debris (). Knockdown of dCed-6 in all glial cells using repo-Gal4 suppressed both the recruitment of Draper to the site of injury () as well as the clearance of degenerating GFP+ axon material five days after injury as compared to control animals (). Based on our findings using DraperRNAi and SharkRNAi, we expected Ced-6RNAi treatment using the mz0709-Gal4 driver to also suppress glial responses to axon injury. However, we found normal levels of Draper recruited to severed axons one day after injury () and efficient clearance of axon material five days after injury (). To assay the level of dCed-6 knockdown by RNAi, we stained brains with α-dCed-6 antibodies five days after maxillary palp ablation and found moderate levels of dCed-6 staining when UAS-dced-6RNAi was driven by mz0709-Gal4 or alrm-Gal4, but not when driven by repo-Gal4 (). Thus, mz0709-Gal4 does not appear to provide a complete knockdown of dCed-6 in ensheathing glia. Nevertheless, based on the colocalization of Draper, and dCed-6 in ensheathing glial subtypes in the adult brain, we propose that dCed-6 acts in ensheathing glia to promote Draper-dependent recruitment of glial membranes to severed axons and clearance of degenerating axonal debris.
Glial-specific knockdown of dCed-6 suppresses clearance of degenerating ORN axonal debris
Blocking endocytosis in ensheathing glia suppresses glial clearance of severed axons
Our analyses of Draper, Shark, and dCed-6 indicate that ensheathing glia express all components of the engulfment machinery and that the Draper signaling pathway is essential in ensheathing glia for efficient clearance of degenerating axons in the adult brain. These findings argue that ensheathing glia are the primary phagocytic cell type in the adult brain neuropil. Nevertheless, to further exclude any possible role for astrocytic glia in engulfing degenerating axons, we used the dominant temperature-sensitive shibirets molecule (UAS-shibirets) to conditionally block endocytosis in either astrocytes or ensheathing glia, and subsequently assayed glial responses to axon injury.
Interestingly, when we raised animals expressing shibirets under the control of repo-Gal4 at 18°C and then subsequently shifted the adult animals to the restrictive temperature of 30°C, we observed 100% lethality within 3 days after temperature shift. Thus, suppressing glial endocytic function in all glia results in rapid adult lethality, indicating that in the healthy adult Drosophila brain glial cells likely perform a high level of endocytic events that are essential for viability.
We next drove shibirets in ensheathing or astrocytic glia and assayed recruitment of Draper to severed axons 1 day after injury. At the permissive temperature of 18°C, expression of Shits in ensheathing glia had no effect on Draper recruitment to severed axons. However, shifting animals to the restrictive temperature strongly suppressed this response (). In contrast, expression of Shits in astrocytes had no effect on Draper recruitment to severed axons at either permissive or restrictive temperatures (). Moreover, we found that blocking endocytic function in ensheathing glia with Shits (at restrictive temperature) strongly suppressed glial clearance of degenerating axons from the brain, while the same treatment of astrocytes had no effect on axon clearance (). These data provide additional compelling evidence that ensheathing glia are the primary phagocytic cell type responsible for engulfing degenerating ORN axons. We also note, since blocking endocytic function in astrocytes did not affect glial clearance of severed axons (), these data further argue that neither phagocytic activity nor signaling events involving endocytosis in astrocytes are essential for efficient clearance of degenerating axonal debris from the CNS.
Endocytic function is required in ensheathing glia, but not astrocytes, for glial clearance of degenerating ORN axons