Drosophila Tet Is Required for Maintaining Glial Homeostasis in Developing and Adult Fly Brains

dTet is required for Drosophila development and flies with dTet deficiency have severe locomotion defects

To investigate the role of dTet in the fly brain, we generated dTet-deficient flies by crossing Tet[MI03920] (DmelMI[MIC]TetMI03920) allele to either Tet[null] allele (Delatte et al., 2016; Tet[MI03920]/Tet[null]) or to Tet[Exel6091] (Df(3L)Exel6091) allele (Tet[MI03920]/Tet[Exel6091]). In addition, Tet[null] was crossed to itself and homozygous Tet[null] embryos were collected (Tet[null]/Tet[null]). Moreover, Tet[MI04973]-G4 flies, a Trojan-Gal4 line in which the Mi[MIC]MI04973 insertion has been replaced with a gene trap cassette containing a Trojan-Gal4 exon as well as an Hsp70 transcription termination signal, were either used heterozygous or crossed to Tet[null]. Mi[MIC]MI04973 is located in a coding intron of the four long dTet isoforms (Fig. 1A; Extended Data Fig. 1-1A). Because of lack of publicly available dTet antibody, we could not test for absence of dTet protein in the four different dTet mutants, but we tested three of the four mutants for levels of dTet mRNA expression as well as expression of directly adjacent genes using quantitative PCR (Extended Data Fig. 1-1B,D). While Tet[null]/Tet[null] embryos showed complete absence of dTet mRNA, combinations with the Tet[MI03920] allele showed about half the amount of dTet mRNA expression compared with wild-type. This is expected, since the MiMIC insertion contains stop codons in all three reading frames resulting in premature translation termination in the last coding exon of all six dTet isoforms without affecting transcription (Fig. 1A; Extended Data Fig. 1-1A–D). Survival rates of a wild-type control compared with different dTet-deficient allelic combinations showed that about half of Tet[MI03920]/Tet[null] and Tet[MI03920]/Tet[Exel6091] animals die during the pupal stage with 40–60% of flies eclosing. As reported previously (Zhang et al., 2015), none of the eclosed dTet-deficient flies survived more than 3 d posteclosion. Tet[null]/Tet[null] flies died as embryos or in early larval stages, and thus were not used for any additional experiments, since we focused our study on larval and adult stages (Fig. 1B). Tet[MI04973]-G4/TM3, Sb[1] Ser[1] heterozygous animals showed an early embryonic lethality (Fig. 1C). Tet[MI04973]-G4/Tet[MI04973]-G4 homozygous animals all died as embryos or in early larval stages (data not shown). Tet[MI04973]-G4/Tet[null] heterozygous animals showed a similar survival phenotype to the other Tet allele combinations with ∼50% of adults eclosing (Fig. 1D) that survived maximal 2 d posteclosion. The differences between survival rates in homozygous and heterozygous dTet allele combinations could either be because of a second (off-target) mutation on the Tet alleles, which make the homozygous alleles less fit, or can indicate only partial loss of function of some of the Tet mutant alleles. Interestingly, Tet[MI03920]/Tet[null] and Tet[MI03920]/Tet[Exel6091] third instar larvae, both displayed highly significant reduction in locomotion compared with wild-type control larvae as shown by crawling assays (Fig. 1E). This is in line with previously published knock-down experiments of dTet reporting locomotion defects (Wang et al., 2018; Ismail et al., 2019). Adult dTet-deficient flies cannot fly or climb and are prone to fall over while walking. In fact, even Tet[MI04973]-G4/TM3, Sb[1] Ser[1] animals that still have one dTet wild-type allele displayed impaired climbing activity compared with wild-type flies (Fig. 1F). Since dTet was reported to demethylate 6mA in genomic DNA (Yao et al., 2018; Zhang et al., 2015), we decided to measure the levels of 6mA in genomic DNA from wild-type versus dTet-deficient fly brains using dot blot and 6mA ELISA assay. As an additional control, we used genomic DNA from 6–8 h old wild-type embryos. With both assays, 6mA levels were higher in dTet-deficient brains compared with wild-type brains or embryos, further validating the function of dTet as a 6mA demethylase in fly brains (Fig. 1G,H).

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Figure 1.

dTet is required for Drosophila development and dTet-deficient flies have a severe locomotion phenotype. A, Schematic showing the cytogenetic location of Tet[MI03920] and Tet[MI04973]-G4 insertion, Tet[Df(3L)Excel6091] deficiency and Tet[null] deletion on Drosophila Chromosome 3 that were used to generate dTet-deficient flies. In order to generate dTet-deficient animals, Tet[MI03920] was either crossed to Tet[null] or to Tet[Df(3L)Excel6091]. In addition, Tet[null] was crossed to itself or to Tet[MI04973]. B, Kaplan–Meier survival curve of wild-type (n = 250), Tet[MI03920]/Tet[null] (n = 221), Tet[MI03920]/Tet[Df(3L)Excel6091] (n = 198), and Tet[null]/Tet[null] (n = 227) embryos is shown. Note that only 40–60% of dTet-deficient flies survived to the adult stage and those died within 2 d of eclosion. All Tet[null]/Tet[null] animals died as embryos or in early larval stages. C, Embryo survival assay of wild-type embryos (n = 218) compared with Tet[MI04973]-G4/TM3, Sb[1] Ser[1] heterozygous embryos (n = 159). The group of Tet[MI04973]-G4/TM3, Sb[1] Ser[1] heterozygous embryos showed an early lethality at the embryonic stage in >50% of observed animals. A chi-square test was used to determine the significance between the two groups (****p < 0.0001). D, Larval survival assay comparing Tet[MI04973]-G4/TM3, Sb[1] Ser[1] animals (n = 150) to Tet[MI04973]-G4/Tet[null] (n = 80) animals showed that only 50% of Tet[MI04973]-G4/Tet[null] animals eclose to adults, significantly less than in the control group. Statistical analysis was performed by a chi-square test (****p < 0.0001). E, Crawling assays with wandering third instar larvae on 0.4-cm grid paper showed that Tet[MI03920]/Tet[null] (n = 50) and Tet[MI03920]/Tet[Df(3L)Excel6091] animals (n = 50) have defective crawling activity. Statistical analysis was performed by ordinary one-way ANOVA and Sidak’s multiple comparison test (****p < 0.0001). At adult stages, both mutants are incapable to fly or climb. F, Climbing assay displaying the average number of adults that were able to reach a 6-cm mark within 10 s (n = 60). Heterozygous Tet[MI04973]-G4/TM3, Sb[1] Ser[1] flies showed significantly reduced mobility compared with wild-type flies. Statistical analysis was performed by a chi-square test (*p = 0.0107). G, Dot blot assay was performed on 200 ng of indicated genomic DNA samples using an anti-6mA antibody (up). Methylene blue staining was performed as DNA input control (down). H, Quantification of 6mA levels in total genomic DNA (300 ng) using a commercial m6A ELISA kit. Statistical analysis was performed by ordinary one-way ANOVA and Bonferroni’s multiple comparison test. [**p < 0.01 (n = 3); ns, p > 0.05 (n = 2)]. Genomic DNA samples are as follows: control E: 6- to 8-h-old wild-type embryos; control B: brains from wild-type third instar wandering larvae; Tet[MI03920]/Tet[null] B: brains from Tet[MI03920]/Tet[null] third instar wandering larvae and Tet[MI03920]/Tet[Df(3L)Excel6091] B: brains from Tet[MI03920]/Tet[Df(3L)Excel6091] third instar wandering larvae. See also Extended Data Figure 1-1.

dTet is expressed in most neurons and in specific glia populations in the optic lobe

To characterize which cells in the CNS show dTet reporter expression, we generated a dTet-Gal4 line, Tet[MI05009]-G4 (see Materials and Methods, Fly husbandry and generation of transgenic fly stocks; Fig. 2A, Extended Data Fig. 2-1A,B) and crossed it with either a nuclear or membrane targeted GFP reporter, nGFP and mCD8GFP, respectively. Moreover, we used a second, publicly available Trojan-Gal4 line, Tet[MI04973]-G4 to compare and validate expression patterns. Subsequently, we dissected third instar larvae and adult brains from Tet[MI05009]-G4>UAS-GFP animals as well as third instar larvae brains from Tet[MI04973]-G4>UAS-GFP animals and stained them with a range of neuronal and glia cell markers. Co-staining with the pan-neuronal marker Embryonic lethal abnormal vision (ELAV) showed that Tet[MI05009]-G4 is expressed in most neurons of the ventral nerve cord (VNC; L3) and adult central brain (Fig. 2B) similar what has been published for an endogenous dTet-GFP reporter (Ismail et al., 2019). Tet[MI04973]-G4 on the other side, showed less overlap with the neuronal marker ELAV (Fig. 2B). Co-staining with the homeodomain transcription factor Reversed polarity (Repo) marking all glial cells, except midline glia, revealed that while Tet[MI05009]-G4 is expressed in a considerable number of glial cells in the central brain, only few glial cells of the VNC display Tet[MI05009]-G4 expression (Fig. 2B). Interestingly, Tet[MI05009]-G4 is specifically expressed in a certain glia population of the optic lobe called giant glia of the inner optic chiasm (IOC; Fig. 2C; Extended Data Fig. 2-1C, white arrowheads) and in few cells that are part of the giant glia of the outer chiasm (Fig. 2C, empty arrowheads) from L3 onwards. Consistently, Tet[MI04973]-G4 is also expressed in a considerable number of glial cells in the central brain and in giant glia of the IOC. Contrary to Tet[MI05009]-G4, it is also expressed in many glial cells of the VNC (Fig. 2C). In fact, most of the Tet[MI04973]-G4 expression overlaps with the glial cell marker Repo. Studies have shown that chiasm glia are born early in larval life and thus may participate in the development of the optic lobe (Tix et al., 1997). During pupal development, optic chiasm glia gradually arrange into two rows: the outer chiasm glia are positioned in the periphery (Fig. 2C, lower panel, empty arrowheads) and the inner chiasm glia are found in the middle of the optic lobe (Fig. 2C, lower panel, white arrowheads). Each chiasm contains one stack of giant glia, enwrapping and enclosing a high density of axons (Extended Data Fig. 2-1C). This strategic location at fiber crossings implies that giant glia might be involved in neuronal pattern formation and provide cues for outgrowing axons (Tix et al., 1997). Overlapping expression in the midline glia (L3) was not observed when we co-stained Tet[MI05009]-G4>UAS-nGFP larvae with either of the two midline glia markers, slit or wrapper (Fig. 2D), but was observed with Tet[MI04973]-G4>UAS-nGFP larvae stained with slit. Besides, midline glia expression of dTet has previously been reported using an endogenous dTet-reporter (Ismail et al., 2019). Moreover, there was no overlap detected between Fas2 and dTet reporter expression in adult fly brains, whereas in larval stages, Tet[MI05009]-G4>mCD8GFP brains showed a faint signal in the mushroom body region (Fig. 2E). To summarize, three different dTet reporters exhibit clear variations in expression patterns possibly because of their different locations within the dTet locus. However, the expression of dTet in specific glia populations is further verified with expression of dTet in optic chiasm glia observed with three independent reporters and expression of dTet in midline glia observed with two reporters.

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Figure 2.

dTet is expressed in most neurons and in specific glia populations in the optic lobe of larval and adult brains. Genomic location of Gal4 cassette insertion site of Tet[MI05009]-G4 and Tet[MI04973]-G4 (A). Immunofluorescence co-staining of whole-mount Tet[MI05009]-G4> UAS-nuclear GFP/UAS-mCD8GFP or Tet[MI04973]-G4.UAS-nuclear GFP larval (L3) or 1-d-old adult (A) brains with five different markers including (B) anti-ELAV (neuronal cell marker), (C) anti-Repo (glial cell marker), (D) anti-slit (expressed in ventral midline, mushroom body, and others) and anti-wrapper (midline glia marker) as well as (E) anti-Fas2 antibody marking the mushroom body, longitudinal fascicles of the VNC and a subset of neurons. White arrowheads mark cells expressing both, dTet and the designated brain marker. See also Extended Data Figure 2-1. VNC: ventral nerve cord, CB: central brain, OL: optic lobe.

dTet-deficient larvae display distinct brain phenotypes

In order to further investigate the role of dTet in the brain, we dissected Tet[MI03920]/Tet[null] and Tet[MI03920]/Tet[Df(3L)Exel6091] brains of third instar larvae and compared them to wild-type control brains by immunofluorescence staining using a panel of markers including Repo, ELAV, and Robo-1, the later marking the neuropil region, as well as NC82, labeling Bruchpilot, a component of the presynaptic active zone that is essential for structural integrity and function of synaptic active zones (Wagh et al., 2006). Notably, panneuronal RNAi knock-down of Bruchpilot leads to locomotor inactivity and instable flight in flies (Wagh et al., 2006), a phenotype similar to what is observed for dTet-deficient flies. However, no obvious defects in Tet[MI03920]/Tet[null] or Tet[MI03920]/Tet[Df(3L)Exel6091] brains were detected (Fig. 3A). Furthermore, we examined markers for progenitor cell populations including homeodomain transcription factor Prospero (Pros) that is expressed in nuclei of ganglion mother cells (GMCs) as well as in undifferentiated neurons and Ase that is expressed in type 1 larval neuroblasts. There was no clear difference visible between control and Tet[MI03920]/Tet[null] and Tet[MI03920]/Tet[Df(3L)Exel6091] brains (Fig. 3B). We then assessed occurrence and distribution of cell proliferation and apoptosis, two important processes during development in dTet-deficient brains. For this, we checked cell proliferation by staining for phospho(Ser10)-H3, which detects mitotically active cells. As expected, the pS10-H3 signal was particularly strong in the outer and inner proliferation center of the optic lobes and the upper VNC. At low magnification, we observed more cell proliferation in Tet[MI03920]/Tet[null] and Tet[MI03920]/Tet[Df(3L)Exel6091] mutants compared with control brains (Fig. 3B). Staining for cleaved Death caspase-1 (Dcp-1) did not show any major differences for Tet[MI03920]/Tet[null] brains compared with control brains (Fig. 3B, lower panel) suggesting similar rates of apoptosis. Since 6mA in DNA was reported as the major substrate of dTet in flies (Zhang et al., 2015; Yao et al., 2018), we subsequently stained to detect 6mA levels in neurons and glia cells (Fig. 3C) and investigated how 6mA levels in glia cells are affected in Tet[MI03920]/Tet[null] brains compared with control brains. As seen by more quantitative assays including dot blot and ELISA (Fig. 1G,H), we detected an increase of 6mA signal in Tet[MI03920]/Tet[null] brains. Higher magnification images of the optic lobes showed that giant glia of the IOC and OC that specifically express dTet reporter (Fig. 2C, white arrowheads) possibly show lower levels of 6mA in control brains (Fig. 3D, white arrowheads) than in Tet[MI03920]/Tet[null] brains (Fig. 3D, empty arrowheads). Contrary, some glia cells in the periphery of the optic lobes that do not express dTet display more comparable 6mA signal in both, control and Tet[MI03920]/Tet[null] brains (Fig. 3D, gray arrowheads). Last, we checked whether Tet[MI03920]/Tet[null] brains of 1-d-old adults displayed any defects in neuron or glia populations compared with age matched control flies. We found that the glia population in the optic lobe between medulla and lobula that shows Tet[MI05009]-G4>nGFP expression (Fig. 2C, second panel) formed a straight line in control brains (white arrowhead), but appeared less organized in Tet[MI03920]/Tet[null] brains (Fig. 3E, open arrowhead).

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Figure 3.

dTet-deficient larvae display distinct brain phenotypes. Whole-mount control (wild-type w¹¹¹⁸), Tet[MI03920]/Tet[null] and Tet[MI03920]/Tet[Df(3L)Excel6091] third instar larval brains were dissected fixed and stained with (A) anti-ELAV (neuronal cell marker), anti-Repo (glial cell marker), anti-Robo1 (neuropile marker) and anti-NC82 (synaptic/active neuropile marker), (B) two neural progenitor markers, anti-Prospero (controls neuronal identity in a subset of neuroblast progeny and initiates the development of GMCs) and anti-Ase (marks embryonic/larval neuroblasts) as well as anti-phospho-Ser10 histone H3 (pS10-H3, mitotic marker) and anti-cleaved Death caspase-1 (Dcp-1, apoptosis marker). C, Whole-mount control (wild-type w¹¹¹⁸) brains were dissected fixed and stained with ELAV and 6mA (epigenetic DNA mark, possible dTet substrate) or Repo and 6mA to determine a rough 6mA profile for larval brains. D, Whole-mount control and Tet[MI03920]/Tet[null] third instar larval brains were dissected fixed and stained with anti-Repo and 6mA. In Tet[MI03920]/Tet[null] brains glial cells of the IOC display slightly increased 6mA signal (open arrowheads versus white arrowheads). Contrary, several glia in the periphery show comparable 6mA signal in control and Tet[MI03920]/Tet[null] brains (open arrowhead). E, Whole-mount control and Tet[MI03920]/Tet[null] adult brains were dissected fixed and stained with anti-ELAV or anti-Repo. In Repo staining, arrowheads mark the giant glia of the IOC. In dTet-deficient brains this glia population appeared scattered (open arrowhead) and not arranged in a row in the middle of the optic lobe as observed for control brains (white arrowhead). Note that maximum z-projections are displayed for all markers.

dTet-deficient brains display clear defects in midline glia organization and axon guidance

dTet was recently reported to be expressed in midline glia (Ismail et al., 2019), a highly-specialized cell type that is molecularly, functionally and developmentally distinct from other glia and corresponds to a structure in vertebrates called the floor plate (Jacobs, 2000). Here, we confirmed midline glia expression with another dTet reporter (Fig. 2D) and analyzed whether the midline glia organization is affected in dTet-deficient brains by staining for Slit protein, a repulsive signal secreted by midline glia (Battye et al., 1999), and the midline glia marker Wrapper (Noordermeer et al., 1998). We found that dTet-deficient brains exhibit defects in the midline glia structure characterized by at least one of the midline segments being disoriented or misaligned (Tet[MI03920]/Tet[null], empty arrowheads) in the normally straight midline (control, white arrowheads) for both analyzed markers (Fig. 4A,B), suggesting a disrupted migration of midline glia in the absence of dTet. Quantification of this midline phenotype showed that 60–80% of dTet-deficient brains display defects in the midline glia with 20–30% displaying more severe defects (more than two midline segments affected; Fig. 4A,B).

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Figure 4.

dTet-deficient larvae display clear defects in midline glia organization and axon guidance. Whole-mount control (wild-type w¹¹¹⁸), Tet[MI03920]/Tet[null] and Tet[MI03920]/Tet[Df(3L)Excel6091] larval brains were dissected fixed and stained with (A) anti-wrapper (midline glial cell marker). Higher magnification images (63× objective) of midline glia marked with wrapper showed that the organization of midline glia is disrupted in Tet[MI03920]/Tet[null] and Tet[MI03920]/Tet[Df(3L)Excel6091] larval brains (open arrowheads) compared with control brains (white arrowheads). For quantification of observed midline glia defects wrapper stains were scored blindly according to the indicated scoring system, where n corresponds to number of brains scored per group with control (n = 12), Tet[MI03920]/Tet[null] (n = 12) and Tet[MI03920]/Tet[Df(3L)Excel6091] (n = 8). B, Anti-slit (expressed in ventral midline and mushroom body among others). Higher magnification images (63× objective) of midline glia marked with slit showed that the organization of midline glia is disrupted in Tet[MI03920]/Tet[null] and Tet[MI03920]/Tet[Df(3L)Excel6091] larval brains (open arrowheads) compared with control brains (white arrowheads). For quantification of midline glia defects slit stains were scored blindly according to the indicated scoring system, where n corresponds to number of brains scored per group with control (n = 13), Tet[MI03920]/Tet[null] (n = 14) and Tet[MI03920]/Tet[Df(3L)Excel6091] (n = 9). C, Whole-mount control, Tet[MI03920]/Tet[null] and Tet[MI03920]/Tet[Df(3L)Excel6091] larval brains were dissected fixed and stained with anti-Fas3 (labeling one of the lateral axon tracts running lengthwise through the nerve cord and a band of axons that cross the midline in each segment). High-magnification images (63× objective) revealed that commissures crossing the midline appeared mostly discontinuous as if broken at the center (white stars) in the lower part of the VNC with some aberrant crossings in the upper part of the VNC (empty arrowhead). The graph below displays blinded scoring of disrupted horizontal commissure crossings according to the indicated scoring system, where n corresponds to number of brains scored per group with control (n = 13), Tet[MI03920]/Tet[null] (n = 14) and Tet[MI03920]/Tet[Df(3L)Excel6091] (n = 6). D, Whole-mount control and Tet[MI03920]/Tet[null] larval brains were dissected fixed and co-stained with anti-Fas2 antibody marking a subset of VNC axons and HRP (staining all VNC axons). In high-magnification images (63× objective, lower panel), Fas2 longitudinal tracts are designated by letters relative to their position in the dorsoventral (D, dorsal; C, central) and mediolateral (M, medial; I, intermediate; L, lateral) position. Cells of the midline (ML) are clearly visible with DAPI stain. Fas2-positive axons that cross the midline (ML) in Tet[MI03920]/Tet[null] mutants are marked by white stars. E, One-day-old adult control and Tet[MI03920]/Tet[null] Drosophila brains were dissected, fixed, and visualized using anti-Fas2 antibody that stains mushroom body axons. As seen in the control the mushroom body cells extend several axons bundles (so-called lobes) including dorsally projecting α-lobes, medially-projecting β- and γ-lobes. The expression of Fas2 in γ-lobes is weaker than in α- and β-lobes. The centrally located ellipsoid body is also visualized by Fas2 staining. Importantly, β-lobes of control flies usually terminate before the brain midline. The mushroom body axons of Tet[MI03920]/Tet[null] brains displayed multiple phenotypes including varying amounts of β-lobe mis-projection (open arrowheads) across the midline that was rarely observed in control brains (lower panel, left side) as well as frequently missing (white asterisk) or misdirected (open arrowheads) α- and/or β-lobes (lower panel, right side). Quantification of mushroom body phenotypes was done according to the indicated scoring systems. Maximum intensity z-stack projections of representative examples of each scored category are displayed.

Since midline glia play an important role in axon guidance by controlling repulsive and attractive signals during development, we wanted to investigate whether axon connections are affected in dTet-deficient mutants. Therefore, staining with Fasciclin 3 antibodies (Fas3) labeling one of the lateral axon tracts running lengthwise through the nerve cord and a band of axons that cross the midline in each segment, was conducted (Fig. 4C). As reported previously for tubulin and sim-Gal4-driven RNAi-mediated dTet knock-down (Ismail et al., 2019), dTet-deficient mutants displayed disruptions in axons and midline commissure organization in the VNC (Fig. 4C). The midline crossings appeared mostly discontinuous as if broken at the center (white stars) in the lower part of the VNC with some aberrant crossings in the upper part of the VNC (empty arrowhead). Blinded quantification of disrupted horizontal commissure crossings in larval brains detected at least one error in >80% of Tet[MI03920]/Tet[null] brains with >30% exhibiting two or more errors (Fig. 4C). Similar results were obtained for Tet[MI03920]/Tet[Df(3L)Exel6091] brains, where 100% of brains displayed at least one error and >30% showed more severe defects with two or more crossing errors (Fig. 4C). Next, co-staining with Fasciclin 2 antibody (Fas2) and HRP was conducted. Fas2 marks a subset of VNC axons including the longitudinal fascicles of the VNC that serve as a set of evenly distributed landmarks, since they remain comparatively constant between specimens and over developmental time (Landgraf et al., 2003). HRP, on the other side, stains all VNC axons. Fas2-positive axon pathways in Tet[MI03920]/Tet[null] brains were less organized compared with control, especially the longitudinal axons in the lower VNC are closer together and look more clustered as if they are fused together (Fig. 4D). In fact, it looked as if the midline (ML) of Tet[MI03920]/Tet[null] brains contains less cells compared with control brains when comparing the DAPI stains. Additionally, few Fas2-positive axons cross the midline (ML) in Tet[MI03920]/Tet[null] mutants (white stars). These midline crosses are completely absent in control brains. Subsequently, we analyzed the mushroom body morphology of 1-d-old adults by Fas2 antibody staining. Mushroom bodies are highly plastic brain regions essential for many forms of learning and memory (Heisenberg, 2003). Accordingly, defects in axonal guidance proteins often cause incompletely penetrant mushroom body phenotypes (Michel et al., 2004; Ng, 2012; Kelly et al., 2016). Fas2 is enriched on α- and β-axon branches and to a lesser extent on the γ-lobes and regions of the ellipsoid body (Fig. 4E). We identified two distinct mushroom body defects in adult Tet[MI03920]/Tet[null] brains including β-lobe midline crosses and missing or misdirected α- and/or β-lobe(s) (Fig. 4E). Both phenotypes categorize as incompletely penetrant mushroom body defects with 30% of Tet[MI03920]/Tet[null] brains displaying midline fusion defects including different stages ranging from mild over moderate to complete fusion (Fig. 4E, left panel, open arrowheads), while 60% of Tet[MI03920]/Tet[null] brains displayed missing (white asterisks) or misdirected (empty arrowheads) α- and/or β-lobes (Fig. 4E, right panel). To conclude, our data show that the deficiency of dTet results in midline glia defects in developing brains. This midline glia phenotype is accompanied by axon commissure defects as well as misdirected and fused Fas2-positive axon tracts. Furthermore, axon guidance defects in Tet[MI03920]/Tet[null] adult brains manifest in two distinct incompletely penetrant mushroom body phenotypes including β-lobe midline crosses and missing or misdirected α- and/or β-lobe(s).

dTet-deficient mutants display a highly significant increase in glial cell numbers in the brain lobes, accompanied by an increase in proliferating and apoptotic cells

Next, we conducted a more detailed analysis of glia cell numbers and analyzed whether the balance between proliferation and apoptosis is affected in Tet[MI03920]/Tet[null] brain lobes. Intriguingly, we found that Tet[MI03920]/Tet[null] brains displayed a sharp and highly significant increase in glia cell numbers in the focal plane that contains the optic chiasm glia (Fig. 5A). The increase in glial cells did not appear to arise from a specific glia population in the brain lobes and was accompanied by a distinct increase of mitotic cells particular concentrated in the laminar region (Fig. 5B). Moreover, analysis of double positive cells (Repo⁺, phH3⁺) showed that in almost half of the investigated Tet[MI03920]/Tet[null] mutants, glial cells were the major proliferating cell type (Fig. 5B, Tet[MI03920]/Tet[null] 2), which was not observed in control brains (Fig. 5B, control) or the other half of Tet[MI03920]/Tet[null] brains (Fig. 5B, Tet[MI03920]/Tet[null] 1), where on average one quarter of the detected mitotic cells were glial cells. Next, we investigated the number of apoptotic cells in brain lobes of Tet[MI03920]/Tet[null] brains and found a highly significant increase in apoptotic cells (Dcp-1⁺ cells) compared with control brains. This overall gain in apoptotic cell numbers can partly be explained by the rise in apoptotic glial cells (Repo⁺, Dcp-1⁺), but to some extent appears to be because of an increase of apoptosis in nonglial cells (Fig. 5C). To investigate whether the increase in glial cell numbers observed in larval stages persists to the adult stage, we counted number of glial cells in optic lobes of 1-d-old adult Tet[MI03920]/Tet[null] brains. Indeed, we observed a highly significant increase of glial cells in adult Tet[MI03920]/Tet[null] brains compared with an age matched control group (Fig. 5D). Since dTet reporter is specifically expressed in the giant glia of the IOC, we also counted cell numbers of this specific glia population. While the counts of giant glial cells of IOC were comparable in Tet[MI03920]/Tet[null] brains (averaged 27) and controls (averaged 26; Fig. 5D, right graph) and in a similar range to previous reports (averaged 31, ranging from 25 to 37; Tix et al., 1997), we noticed that the giant glia of the IOC in Tet[MI03920]/Tet[null] brains were scattered and not arranged in a row in the middle of the optic lobe as observed for control brains (Fig. 5D, white vs open arrowheads). Notably, the giant glia of the outer and inner chiasm migrate during pupal stages outwards into their final position and the scattered appearance of the inner chiasm glia in Tet[MI03920]/Tet[null] brains indicates a possible migration defect.

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Figure 5.

dTet-deficient mutants display a highly significant increase in glial cell numbers in the brain lobes, accompanied by an increase in proliferating and apoptotic cell numbers. Whole-mount control (wild-type w¹¹¹⁸) or Tet[MI03920]/Tet[null] larval brains were dissected fixed and stained with (A) anti-Repo (glial cell marker), (B) anti-Repo and anti-phospho-Ser10 histone H3 (pS10-H3; mitotic marker), or (C) anti-Repo and anti-cleaved Death caspase-1 (Dcp-1; apoptosis marker). Whole-mount brains of 1-d-old control or Tet[MI03920]/Tet[null] adult flies were dissected fixed and stained with anti-Repo (D). Tet[MI03920]/Tet[null] adult brains exhibited scattered giant glial cells of the IOC (open arrowheads) as compared with linear alignment in control brains (white arrowheads). Displayed images are maximum intensity projections of 6 (1 μm) z-stacks taken with 40× oil objective. Cell counts were conducted using ImageJ plug-in ITCN. Each dot corresponds to the average amount of cells detected per animal in one brain lobe, where n indicates the number of animals analyzed. Statistical significance was analyzed by unpaired Student’s t test and graphs generated using GraphPad Prism version 5.01 (****p < 0.0001; ns, not significant).

dTet-deficient mutants display a highly significant increase in glial cell numbers in the brain lobes that coincides with changes in hippo pathway activation

To elucidate the molecular basis for the increase in glial cells in optic lobes, we decided to investigate changes in the Hippo signaling pathway known to control tissue size by downregulating cell proliferation and upregulating apoptosis (Fig. 6A; Reddy and Irvine, 2011; Staley and Irvine, 2012). Expression analysis by qPCR indicated that hippo (hpo) and yorkie (yki) expression is significantly reduced in Tet[MI03920]/Tet[null] brains compared with wild-type control brains, whereas merlin (mer) and expanded (ex) levels, that act upstream of hpo, were not affected. We then examined the expression of several downstream targets of the hippo pathway, whose transcription is activated by the transcriptional co-activator yki. While divion abnormally delayed (dally) and Death-associated inhibitor of apoptosis 1 (Diap1) were significantly downregulated in Tet[MI03920]/Tet[null] brains, Cyclin E (CycE) and ex were not significantly downregulated in Tet[MI03920]/Tet[null] brains (Fig. 6B). Since the role of hippo signaling in glial cell proliferation was mainly studied in optic lobes and eye discs (Reddy and Irvine, 2011), we wanted to investigate how CycE protein levels are altered in optic lobes of Tet[MI03920]/Tet[null] brains. CycE is a downstream target of the hippo pathway and was reported to be the most important cyclin in G₁ to S phase transition, a critical point in the cell cycle, where the cell decides to either proliferate or differentiate (Richardson et al., 1993, 1995). Moreover, Yao and colleagues published that the genomic region of CycE exhibits two gain-of-6mA regions in dTet^null mutants relative to control flies (Fig. 6C) implicating that these regions may represent active 6mA demethylation loci in wild-type brains (Yao et al., 2018) that might be important for controlling CycE expression levels and thus cell proliferation in optic lobes. Intriguingly, CycE showed a slightly stronger signal in the lamina region of Tet[MI03920]/Tet[null] optic lobes that co-localized with the enhanced mitotic (pS10-H3) signal observed (Fig. 6D, white arrowheads vs empty arrowheads). The discrepancy of CycE quantities on mRNA and protein level might be because of the local increase of CycE protein levels in the laminar region of optic lobes that could not be detected in RNA extracted from whole brain.

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Figure 6.

dTet-deficient mutants display a highly significant increase in glial cell numbers in the brain lobes that coincides with changes in hippo pathway activation. A, Simplified schematic of the hippo signaling pathway, an intracellular kinase cascade that negatively regulates the transcriptional co-activator yki (yorkie), which in turn activates transcription of a wide range of downstream targets including mer (merlin), ex (expanded), diap1 (death-associated inhibitor of apoptosis 1), cycE (cyclin E), and dally (division abnormally delayed). Activation of the hippo pathway results in the downregulation of cell proliferation and upregulation of apoptosis. B, Relative expression of selected hippo pathway members and several downstream targets listed in A. Note that Tet[MI03920]/Tet[null] brains showed a 0.25-fold reduced transcription of hippo pathway member hpo that coincided with 0.4-fold reduced yki transcription. Additionally, downstream targets dally (0.35-fold) and diap1 (0.69-fold) were both significantly reduced in Tet[MI03920]/Tet[null] brains. Hippo pathway members mer and ex that are acting upstream of hpo showed no significant change in expression in Tet[MI03920]/Tet[null] brains. C, UCSC genome browser image showing two gain-of-6mA regions in the CycE genomic region in dTet-deficient brains relative to controls. These regions correspond to active 6mA demethylation loci in wild-type brains (Yao et al., 2018). D, Whole-mount control (wild-type w¹¹¹⁸) or Tet[MI03920]/Tet[null] larval brains were dissected fixed and stained with anti-CycE (Cyclin-E, control of cell cycle at G₁/S transition) and anti-phospho-Ser10 histone H3 (pS10-H3). In control brains, cells in the lamina region do not show much CycE or pS10-H3 signal (white arrowheads), while the corresponding cells in Tet[MI03920]/Tet[null] brains show an increase in CycE and pS10-H3 signal (open arrowheads). Note that maximum z-projections are displayed for all markers. Statistical significance was analyzed by unpaired Student’s t test and graphs generated using GraphPad Prism version 5.01 (***p < 0.001, **p < 0.01, *p < 0.05; ns, not significant).

In summary, Tet[MI03920]/Tet[null] brain lobes display a highly significant increase in glial cell numbers and the two mechanisms of cell survival and cell proliferation that usually balance cell numbers in the brain are both significantly altered in Tet[MI03920]/Tet[null] brains. Interestingly, in human brain tumors, reduction of hTETs has been observed to have an oncogenic effect (Orr et al., 2012). Additionally, lack of dTet resulted in disorganization of the giant glia of IOC reflected in their aberrant positioning rather than in altered cell quantity.

Glia-specific knock-down of dTet has no significant effect on survival or locomotion, but knock-down of dTet in chiasm glia has a negative effect on survival

To further validate the role of dTet in glial cells we knocked down dTet using Repo-Gal4 driver. As a random RNAi control, we used GFP RNAi. We monitored survival of wandering third instar larvae to adults and also performed crawling assays to account for effects of dTet knock-down on locomotion and number of body contractions. Knock-down of dTet in glia cells did not show any significant effect on motility or contractions (Fig. 7A,B). Next, we knocked down dTet in the outer optic chiasm glial cells using driver R25A01-Gal4 and performed survival assay on third instar larvae. Knock-down of dTet in the outer optic chiasm glial cells resulted in a moderate reduction in the number of eclosed adult flies compared with control flies. Interestingly, midline glia-specific knock-down of dTet (slit-Gal4/sim-Gal4) has been published to be associated with survival and locomotion defects as well as defects in axon patterning (Ismail et al., 2019). Therefore, dTet might not be required for locomotion in all glia as it has been reported for neurons (Wang et al., 2018), but rather is only crucial in certain glia populations such as the midline glia and optic chiasm glia, where dTet is expressed. Finally, we performed survival and locomotion assay on Tet[MI04973]-G4> dTet RNAi larvae that either co-expressed hTET3 or did not express hTET3 (control). We observed that additional hTET3 expression had a negative effect on survival with only 75% of flies eclosing compared with 94% without hTET3 expression (Fig. 7D). Locomotion assays showed that hTET3 was not able to rescue the locomotion defects observed in Tet[MI04973]-G4> dTet RNAi larvae (Fig. 7E). The expression of hTET3 transgene containing N-terminal HA tag through Tet[MI04973]-G4 driver alone and in combination with dTet RNAi was validated by staining of larval brains with HA antibody (Fig. 7F).

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Figure 7.

Glia-specific knock-down of dTet has no significant effect on survival or locomotion, but knock-down of dTet in chiasm glia has a negative effect on survival. A, Knock-down of dTet in glial cells (Repo-Gal4) does not affect the survival from third instar larvae to adult stage (n = 80). B, Crawling assays with wandering third instar larvae on 0.4-cm grid paper showed that knock-down of dTet in glial cells has no major effect on third instar larval locomotion or number of body contractions (n = 50). C, Survival assay on third instar larvae yielded a moderate reduction in the number of eclosed adult flies, when dTet is specifically knocked down in the outer optic chiasm glial cells using driver R25A01-Gal4 [*p = 0.0209 (n = 80)]. D, Survival assay on Tet[MI04973]-G4> dTet RNAi larvae that either co-expressed human TET3 or not, showed that simultaneous expression of hTET3 cannot rescue dTet knock-down. Larvae expressing dTet RNAi and hTET3 showed a significantly reduced survival rate [**p = 0.0011 (n = 80)]. E, Crawling assay on Tet[MI04973]-G4> dTet RNAi larvae that either co-express human TET3 or not, showed no significant changes between both groups (n = 50). GFP-RNAi was used as a control. Statistical analysis on survival assays was performed by a chi-square. Statistical analysis on crawling assays was performed by unpaired Student’s t test. F, Whole-mount Tet[MI04973]-G4> dTet RNAi, Tet[MI04973]-G4> hTET3 and Tet[MI04973]-G4> dTet RNAi; hTET3 larval brains were dissected fixed and stained with anti-HA antibody to validate expression of human TET3 transgene containing N-terminal Flag and HA tag (see Fig. 8A).

Expression of human TET3 in dTet-expressing cells results in an increase in glial cells in larval brain lobes as well as reduced life span and a circadian phenotype in adult flies

After establishing the effects of reducing Drosophila Tet on phenotype, survival, and glial cell proliferation status, we wanted to investigate the effects of overexpressing human TET in the fly (Fig. 8A,B). For this, we used human TET3, since its catalytic domain was proposed to be the closest to dTet’s catalytic domain in terms of structural similarity (Dunwell et al., 2013). Moreover, TET3 is the only mammalian TET homolog essential for embryonic development (Gu et al., 2011) and Tet3 transcript is the most abundant Tet transcript in the brain (Szwagierczak et al., 2010). First, we checked adult flies expressing TET3 through the Tet[MI05009]-G4 promoter for any obvious phenotypic alterations. We noticed two specific phenotypes that appeared in about half of TET3-expressing flies and were not observed in the driver control group (Fig. 8C). The first observed phenotype was incomplete fusion of the adult abdominal epidermis (49% of females and 33% of males), and the second was protrusion of the most posterior abdominal segments including the male genitalia that was male specific (29%, with 9% of males displaying both phenotypes; Fig. 8C). Next, we investigated the effects of expressing TET3 through Tet[MI05009]-G4 line on the glia population in larval brain lobes. For this, we analyzed (1) number of glial cells, (2) number of proliferating cells, (3) number of TET3-expressing cells, and (4) overlap between these three cell populations in brain lobes of third instar larvae. Surprisingly, we detected a highly significant increase in number of glial cells on TET3 expression accompanied by a slight increase in GFP-positive cells (Fig. 8D). Analysis of GFP positive and negative glial cell populations, which correspond to the glia population expressing TET3 and the glia population not expressing TET3, respectively, showed, that both glial cell populations (Repo⁺, GFP⁺) and (Repo⁺, GFP^-) are significantly increased in TET3-expressing larvae (Fig. 8D, lower left panel) indicating that TET3 has not only an autonomous, but also a nonautonomous effect on glial cell numbers. Notably, the increase in GFP⁺-glial cells was mainly observed in the medulla area, whereas the increase in GFP^--glial cells appeared to be mainly originating from the lamina region. Next, we checked whether the increase in glial cells coincides with an increase in proliferation by staining for the mitosis marker pS10-H3. However, we neither detected an overall increase in mitotic cells, nor an increase in mitotic glial cells, nor an increase in mitotic TET3-expressing cells (phH3⁺, GFP⁺; Fig. 8D, lower panel), suggesting that the observed increase may be because of factors other than proliferation such as changes in cell survival or differentiation.

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Figure 8.

Expression of human TET3 in dTet-expressing cells results in an increase in glial cells in larval brain lobes as well as a reduced life-span and a circadian phenotype in adult flies. A, Schematic representation of the human TET3 transgene with N-terminal Flag and HA tag used in the described experiments. Functional domains are indicated including a CXXC zinc finger, a Cys-rich domain and a double-stranded β helix (DSBH) domain containing iron (II) and 2-OG binding sites. B, The Gal4/UAS system was used for targeted expression of human TET3 (hTET3). The system is composed of two independent parent transgenic lines, the Tet[MI05009]-G4 driver line, in which the Gal4 gene is expressed in a dTet-specific pattern, and the hTET3 transgene containing line that contains the Gal4 DNA binding sequence UAS (upstream activating sequence) adjacent to the hTET3 gene. Mating of the described parental flies results in a F1 generation, where Gal4 is expressed in the transcriptional pattern of dTet and binds to the UAS to activate transcription of hTET3 in the same pattern. Here, the Gal4 driver was combined with a UAS-containing transgene to express nuclear GFP (nGFP) to visualize the cells/tissue that express hTET3 transgene. C, Light microscope images of control flies and Tet[MI05009]-G4> hTET3-expressing flies displaying incomplete fusion of the adult abdominal epidermis (open arrowhead, right panel) as well as protrusions of the most posterior abdominal segments including the male genitalia (open arrowhead, left panel). Approximately 50% of flies displayed either of the phenotypes or both as shown in the sex-specific quantification in the left graph. D, Whole-mount Tet[MI05009]-G4::UAS-nGFP> control or Tet[MI05009]-G4::UAS-nGFP> hTET3 larval brains were dissected fixed and double stained with anti-Repo and anti-pS10-H3. Displayed images are maximum z-projections of six (1 μm) z-stacks taken with 40× oil objective. Cell counts were conducted using ImageJ plug-in ITCN. Each dot corresponds to the average amount of cells detected per animal in one brain lobe, where n indicates the number of animals analyzed. Statistical significance was analyzed by unpaired Student’s t test and graphs generated using GraphPad Prism version 5.01 (**p < 0.005, *p < 0.05, ns, no statistical significance). E, Kaplan–Meier survival curve of male flies expressing either no transgene (driver control, n = 31) or hTET3 (n = 28) through Tet[MI05009]-G4. Statistical significance of difference between survival curves was determined using the Mantel–Haenszel test (p < 0.005). F, Activity graphs illustrating daily locomotor activities of flies over several days. For each group, the locomotor activity levels of individual flies (n ≅ 30) were measured in 5-min bins and then averaged to obtain a representative activity profile. Since locomotion is age-dependent, we subdivided flies in two age groups: “young”: 1–12 d old and “old”: 13–20 d old. Drosophila melanogaster generally exhibits two activity bouts one centered around ZT0 (morning peak) and the second around ZT12 (evening peak). Black arrows indicate the anticipatory increase in locomotor activity that occurs before light transition states. G, Graph showing average locomotor activity over 12-h intervals. Note that hTET3-expressing flies are significantly less active at night. Statistical significance was analyzed by unpaired Student’s t test (***p < 0.0005). H, Graph illustrating the wake activity in counts per min over 12-h intervals. Wake activity, is a measure of the activity rate when the flies are awake. Note that the wake activity is comparable between hTET3-expressing and control flies indicating that hTET3 flies are affected in sleep/rest behavior and not in locomotion. I, Graph showing the average of daily sleep minutes for all flies in one group for 12-h intervals (day: light on, night: light off) over 20 d. During day and night hTET3-expressing flies showed a significant increase in sleep time compared with control flies. Statistical significance was analyzed by unpaired Student’s t test (**p < 0.005, ***p < 0.0005). J, Graph indicating the mean rest bout length of each group in minutes for 12-h intervals. The mean rest bout length is a measure of how consolidated sleep is and was significantly higher for TET3-expressing flies during night time. Statistical significance was analyzed by unpaired Student’s t test (**p < 0.005). K, Graph illustrating the percent of time that flies spend sleeping over several days. For each group, the percent of flies sleeping was measured in 5-min bins and then averaged to obtain a representative sleep profile. Since sleep is age-dependent, we subdivided flies in two age groups as described above. ZT0 indicates morning peak and ZT12 the evening peak. Black arrows indicate the anticipatory phase occurring before light transition states. L, Graph showing the average number of rest bouts for all flies in one group for 12-h intervals over 20 d. During night hTET3-expressing flies showed significantly less rest bouts compared with control flies. Statistical significance was analyzed by unpaired Student’s t test (***p < 0.0005). ZT stands for Zeitgeber time. ZT0 indicates the beginning of the day (light phase) and ZT12 the beginning of the night (dark phase).

We next set out to determine effects of TET3 expression on adult life-span, changes in behavioral parameters including activity and sleep characteristics as well as changes in circadian rhythm. For this, a DAM system was used under a standard 12 h lights on/12 h lights off constant 25°C temperature regime. Flies expressing TET3 through Tet[MI05009]-G4 displayed significantly reduced life-span of maximal 23 d (median survival 8 d) compared with the control group, Tet[MI05009]-G4 flies crossed to wild-type flies, that lived for maximal 31 d (median survival 20 d; Fig. 8E). In addition, we analyzed circadian parameters by looking at average locomotor activities of two age groups, where 1- to 12-d-old flies are considered “young” and 13- to 20-d-old flies are considered “old.” Both, TET3-expressing and control flies, exhibited two major activity peaks, the first centered around Zeitgeber time 0 (ZT0), the beginning of the light phase, the so-called morning peak, and the second around Zeitgeber time 12 (ZT12), the beginning of the dark phase that is called the evening peak (Fig. 8F). Compared with control flies, TET3-expressing flies showed a lack of anticipatory increase in locomotor activity before the dark-to-light and light-to-dark transition indicating that TET3 expression may interfere with proper endogenous clock function. Interestingly, dTet has been previously linked to controlling the development of pigment-dispersing factor (PDF)-expressing neurons which are essential for the circadian rhythm, in particular the morning activity (Wang et al., 2018). While the total activity of TET3-expressing flies was significantly less during the night compared with control flies (Fig. 8G), the wake activity, which is a measure of the activity when flies are awake, was comparable between TET3-expressing and control flies (Fig. 8H). This indicates that TET3-expressing flies are actually affected in sleep/rest behavior and do not just display impaired locomotion. We found that the amount of sleep during light phase (day) and dark phase (night) was significantly higher in TET3-expressing flies (Fig. 8I,K), particularly in older flies (Fig. 8K). The mean rest bout length which is a measure of how consolidated the sleep is, was significantly longer for TET3-expressing flies at night, but not affected during the day (Fig. 8J). Accordingly, the number of rest bouts for TET3-expressing flies was significantly less at night (Fig. 8L), indicating that these flies display longer sleep phases.

Expression of human TET3 in Drosophila glial cells results in circadian phenotypes and decrease in mitotic cells

Since Tet[MI05009]-G4 drives expression in neurons and glia populations (Fig. 2A,B), we continued by expressing TET3 using the glia-specific driver Repo-Gal4 to understand which aspects of the observed phenotypes are glial specific. In addition, the Repo-Gal4 driver used was combined with UAS-mCD8-GFP, a membrane-targeted green fluorescent protein, to investigate whether TET3 would induce changes in cell migration or morphology (Fig. 9A). We also analyzed whether glial cell numbers, mitotic cell numbers or number of mitotic glial cells changed in amount, morphology, or position on Repo-Gal4-driven TET3 expression. The number of glial cells was unaltered in TET3-expressing larval brain lobes and no significant change was detected in mitotic glial cell numbers; however, there was a significant decrease in mitotic cells (Fig. 9B).

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Figure 9.

Expression of human TET3 in glial cells results in circadian phenotypes and decrease in mitotic cells. A, The Gal4/UAS system was used for targeted expression of human TET3. The system is composed of two independent parent transgenic lines, the Repo-Gal4 driver line, in which the Gal4 gene is expressed in a glia-specific pattern, and the hTET3 transgene containing line that contains the Gal4 DNA binding sequence UAS (upstream activating sequence) adjacent to the hTET3 gene. Mating of the described parental flies results in a F1 generation, where Gal4 is expressed in the transcriptional pattern of the glia marker Repo and binds to the UAS to activate transcription of hTET3 in the same pattern. Here, the Gal4 driver was combined with a UAS-containing transgene to express membrane-targeted GFP (mCD8-GFP) to visualize the cells/tissue that express hTET3 transgene. B, Whole-mount Repo-Gal4::UAS-mCD8GFP> control or Repo-Gal4::UAS-mCD8-GFP> hTET3 larval brains were dissected fixed and double stained with anti-Repo and anti-pS10-H3. Displayed images are maximum z-projections of six (1 μm) z-stacks taken with 40× oil objective. Cell counts were conducted using ImageJ plug-in ITCN. Each dot corresponds to the average amount of cells detected per animal in one brain lobe, where n indicates the number of animals analyzed. Statistical significance was analyzed by unpaired Student’s t test and graphs were generated using GraphPad Prism version 5.01 (*p < 0.05; ns, p > 0.05). C, Kaplan–Meier survival curve of male flies expressing either no transgene (driver control, n = 31) or hTET3 (n = 30) through Repo-Gal4. Statistical significance of difference between survival curves was determined using the Mantel–Haenszel test (p < 0.005). D, Activity graphs illustrating daily locomotor activities of flies over several days. For each group, the locomotor activity levels of individual flies (n ≅ 30) were measured in 5-min bins and then averaged to obtain a representative activity profile for two age groups as described above. Drosophila melanogaster generally exhibits two activity bouts one centered around ZT0 (morning peak) and the second around ZT12 (evening peak). Black arrows indicate the anticipatory increase in locomotor activity that occurs before light transition states. E, Graph showing average locomotor activity over 12-h intervals. Note that hTET3-expressing flies are significantly less active at night. Statistical significance was analyzed by unpaired Student’s t test (***p < 0.0005). F, Graph illustrating the wake activity counts per minute over 12-h intervals. G, Graph showing the average of daily sleep minutes for all flies in one group for 12-h intervals (day: light on, night: light off) over 20 d. During day and night hTET3-expressing flies showed a significant increase in sleep time compared with control flies. Statistical significance was analyzed by unpaired Student’s t test (**p < 0.005, ***p < 0.0005). H, Graph indicating the mean rest bout length of each group in minutes for 12-h intervals. The mean rest bout length is a measure of how consolidated sleep is and was significantly higher for TET3-expressing flies during night time. Statistical significance was analyzed by unpaired Student’s t test (**p < 0.005). I, Graph illustrating the percent of time that flies spend sleeping over several days. For each group, the percent of flies sleeping was measured in 5-min bins and then averaged to obtain a representative sleep profile. ZT0 indicates morning peak and ZT12 the evening peak. Black arrows indicate the anticipatory phase occurring before light transition states. J, Graph showing the average number of rest bouts for all flies in one group for 12-h intervals over 20 d. Note that the number of rest bouts are comparable between hTET3-expressing and control flies. ZT stands for Zeitgeber time. ZT0 indicates the beginning of the day (light phase) and ZT12 the beginning of the night (dark phase).

Adult flies expressing TET3 under the Repo-Gal4 promoter displayed no obvious phenotypic alterations (data not shown). As described above, a DAM system was used under the same conditions to determine effects on adult life-span and changes in behavioral parameters including activity and sleep characteristics as well as changes in circadian rhythm. Flies expressing TET3 under the Repo-Gal4 promoter appeared to die to an increased degree in the first 20 d, but the difference was not significant when compared with the control group. Moreover, both groups had few surviving flies after 35 d. Therefore, TET3 expression in all glial cells did not affect overall survival (Fig. 9C). Analysis of average locomotor activities of young (1–12 d) and old flies (13–20 d) showed that both, TET3-expressing and control flies, exhibited two major activity peaks around ZT0 and around ZT12 (Fig. 9D). As observed with Tet[MI05009]-G4-driven expression, glia-specific TET3 expression resulted in a lack of anticipatory increase in locomotor activity before the dark-to-light and light-to-dark transition indicating that TET3 expression in the glia population is sufficient to induce this circadian phenotype (Fig. 9D). While the total activity of TET3-expressing flies was significantly less during the day compared with control flies (Fig. 9E), the wake activity was not significantly reduced in TET3-expressing flies (Fig. 9F). Similar to Tet[MI05009]-G4-driven expression, the amount of sleep during light phase (day) was significantly higher in TET3-expressing flies (Fig. 9G,I), this effect was more obvious in older flies (Fig. 9I). However, neither the mean rest bout length (Fig. 9H) nor the amount of rest bouts (Fig. 9J) was affected in those flies, suggesting that both, Tet[MI05009]-G4 and glia-specific TET3 expression, affects the sleep/rest behavior, but to different degrees. To summarize, while glia-specific expression of TET3 had no effects on morphology and survival, we observed effects on the sleep/rest cycle and the anticipatory activity, indicating that this circadian phenotype might be caused by TET3 expression from the glia population.