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doi: 10.1242/10.1242/dev.00468

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Drosophila homeodomain protein REPO controls glial differentiation by cooperating with ETS and BTB transcription factors

¹ Division of Neuroanatomy (D12), Department of Neuroscience, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
² Department of Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
³ Department of Genetics, The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan
⁴ Department of Molecular Neurobiology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan
⁵ Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
⁶ Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan
⁷ Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi, 332-0012 Japan

View larger version (19K): [in a new window]   Fig. 1. REPO is a transcriptional activator. (A) REPO activates transcription through a homeodomain-binding site. The effector constructs express REPO (pACT-repo) or REPO lacking its homeodomain (REPObox, pACT-repobox), driven by the Drosophila Act5C promoter. The luciferase reporter contains two copies of CAATTA (CAATTA-luc) or CAGTTA (CAGTTA-luc) motifs placed upstream of the hsp70 minimal promoter. The CaSpeR-luc reporter has a luciferase gene with the hsp70 minimal promoter alone. Three luciferase reporter plasmids were each transfected into S2 cells with pACT-repo (black column), pACT-repobox (white column) or vector (pACT) alone (gray column). The amount of luciferase activity generated by the co-transfection of CaSpeR-luc and pACT was defined as 1. (B) REPO contains multiple transcriptional activation domains. Various regions of REPO were fused to the GAL4 DNA-binding domain (DBD) and tagged with three copies of the Myc epitope at their C terminus. The reporter was the luciferase gene placed downstream of the hsp70 TATA and five copies of UAS (GAL4-binding sites). The luciferase activity obtained with each effector is presented as the relative value compared with the value obtained with GAL4-DBD alone. The expression of the GAL4 fusion proteins was confirmed by western blot analysis of whole-cell extracts using a monoclonal antibody directed against the Myc-tag. H, homeodomain; M, Myc tag.

View larger version (145K): [in a new window]   Fig. 2. REPO activates the expression of the ftz HDS reporter gene in glial cells. (A,D) Wild-type embryos; (B,E) repo mutant embryos; (C,F) ectopic expression of repo in scabrous-GAL4/UAS-repo. (A-C) Stage 16 embryonic PNS preparations. (A) In wild-type embryos, expression of the ftz HDS reporter gene is detected in glial cells of the peripheral nervous system, including the support cells of the bipolar dendritic neuron, peripheral glia, ligament cells of the chordotonal organ and exit glia. (B) In the repo mutant, the expression of the ftz HDS reporter gene in the PNS was absent or dramatically reduced. (C) In response to the ectopic expression of REPO, many cells in the dorsal epidermis expressed the ftz HDS reporter. (D-F) Dissected stage 16 embryonic CNS preparations. (D) In the wild-type CNS, the ftz HDS reporter gene is expressed in a subset of longitudinal glia, the A glia, B glia and intersegmental nerve root glia. (E) In the repo mutant, the expression of the ftz HDS reporter gene in the CNS glia was greatly reduced, as it was in the PNS. (F) Ectopic expression of REPO did not induce ectopic expression of the ftz HDS reporter gene within the CNS, although many cells showed ectopic expression in the periphery. Anterior is leftwards.

View larger version (100K): [in a new window]   Fig. 3. repo, pointed and ttk are expressed independent of each other. (A-C) The expression of repo was demonstrated using a REPO antibody. (A) In wild-type embryos, REPO is expressed in all CNS glia except the midline glia. REPO-positive glial cells were still present in pntD88 (B) or ttk1e11 (C) mutant embryos. (D) A pointed enhancer-trap strain (pntrM254) expressed ß-galactosidase in CNS glia that expressed REPO. (E) pntrM254 was expressed in CNS glia in repo mutant embryos. (F) A ttk enhancer-trap strain (ttk0219) expressed ß-galactosidase in all CNS glia, in wild-type (F) as well as repo (G) mutant embryos. Anterior is upwards in A-C and leftwards in D-G.

View larger version (155K): [in a new window]   Fig. 4. Cooperative action of REPO and TTK69 activates the glial expression of the M84 marker. (A-D) CNS of stage 16 embryos carrying the M84 glial marker. (A) In wild-type embryos, M84 ß-galactosidase expression could be detected in the subperineurial glia and channel glia. (B) In repo mutant embryos, M84 expression was absent or dramatically reduced. (C) pointed mutant embryos expressed the M84 marker at normal levels, although the arrangement of the glial cells was irregular. (D) In ttk mutant embryos, the expression level of M84 was lower than in wild type. (E-H) Ventral views of stage 12 embryos carrying the M84 glial marker. In wild-type embryos, few cells expressed the M84 marker at stage 13 (E). Ectopic expression of REPO induced additional cells to express the M84 marker (F), whereas TTK69 had no effect (G). Co-expression of REPO and TTK69 had a synergistic effect on the activation of the M84 marker (H). (F, inset) Supernumerary M84-positive cells induced by the ectopic expression of REPO also expressed endogenous TTK69. The ectopically expressed M84 marker is shown in magenta and TTK69 is shown in green. An overlay of both colors appears as white. The majority of ectopic M84-positive cells were located in the epidermis, which normally do not express this marker. The number of M84-positive epidermal cells per hemisegment were 10.2±5.8 for REPO misexpression and 32.6±9.7 for co-expression of REPO and TTK69 (n=23). Anterior is leftwards.

View larger version (70K): [in a new window]   Fig. 5. REPO and PNTP1 cooperate to activate the transcription of loco. (A-C) Whole-mount in situ hybridization of stage 16 embryos using the loco-c1 probe. Ventral views. Wild-type embryos (A) express loco-c1 mRNA in two rows of longitudinal glial cells (arrowheads). loco-c1 mRNA expression in longitudinal glia was undetectable in repo mutant embryos (B) or in pointed mutant embryos (C). Anterior is leftwards. (D) Structure of the loco-c1 promoter-luciferase reporter genes used for transfection assays. AEE-luc carries a 0.7 kb loco promoter fragment, which contain two CAATTA motifs (square). Single base changes (asterisk) were introduced in both motifs in AEE*-luc reporter. AES-luc has a 1.4 kb loco fragment, which includes an Ets-binding site (triangle) and a GCM-binding site (oval), both identified by Granderath et al. (Granderath et al., 2000). The glial enhancer fragment (Rrk) used by Granderath et al. (Granderath et al., 2000) is shown above the map. The exact position of the transcriptional start site is not known. (E) REPO activates transcription through the CAATTA motif in the loco-c1 promoter. S2 cells were transfected with AEE-luc (black) or AEE*-luc (gray) reporter and REPO-expressing plasmid (REPO) or the empty vector (ACT). Luciferase activity obtained after the transfection of the effector constructs was normalized to the activity of the AEE-luc reporter co-transfected with the empty vector. (F) REPO and PNTP1 cooperates on the expression of the loco promoter. S2 cells were co-transfected with the AES-luc reporter gene and effector constructs that expressed GCM (column 2), REPO (column 3), PNTP1 (column 4) or REPO and PNTP1 (column 5). Luciferase activity was normalized to the value obtained with the empty vector (column 1). (G-L) REPO and PNTP1 has synergistic effects on loco expression. (G-K) lacZ expression of rC56, an enhancer trap insertion into the loco locus. The following transgenes were misexpressed in the entire neuroectoderm using the scabrous-GAL4 strain: (G) none; (H) GCM; (I) REPO; (J) PNTP1; (K) REPO and PNTP1. (L) Number of rC56-positive cells upon misexpression using the engrailed-GAL4 driver. The number of rC56-positive cells in the engrailed-positive region in each segment were scored (n=15).

View larger version (74K): [in a new window]   Fig. 6. REPO and TTK69 cooperate to suppress neuronal development. The expression of the neuron-specific protein ELAV in stage 13 embryos. (A) Wild type. (B-E) Ectopic expression using the scabrous GAL4 driver. (B) Ectopic expression of GCM reduced the number of ELAV-expressing cells. Ectopic expression of REPO (C) or TTK69 (D) caused, respectively, little or a modest reduction in the number of ELAV-expressing cells. When REPO and TTK69 were co-expressed, they synergistically reduced the number of ELAV-positive cells (E). Anterior is leftwards.

View larger version (68K): [in a new window]   Fig. 7. REPO is necessary for the inhibition of neuronal differentiation by GCM. (A-D,I) The axonal scaffold was labeled using mAb BP102 (brown). (A-D) Glial cells were labeled by ß-galactosidase expression from the glial marker M84 (black). (E-H,J) All neurons in the CNS were labeled using the ELAV antibody. (A,E) Wild type. (B,F) Ectopic expression of GCM expression using the scabrous GAL4 driver. Ectopic expression of GCM in the repo mutant background (C,G), pointed mutant background (D,H) or ttk mutant background (I,J). Ectopic expression of GCM caused a reduction in the number of ELAV-positive neurons and axonal extension, as well as an increased number of cells that expressed the M84 marker (B,F). Removal of repo function resulted in a dramatic restoration of ELAV-positive cells and axonal development (C,G). The effect of removing pointed (D,H) or ttk (I,J) function was, respectively, undetectable or minor (compare with B,F). Note that the ttk mutant (I,J) is labeled for axons (I; mAB BP102) and neurons (J; ELAV); this animal did not carry the glial marker M84 (I,J). All embryos were stage 15. Anterior is upwards.

View larger version (20K): [in a new window]   Fig. 8. Model of glial development. Glial cell development involves two processes that both depend on REPO: the activation of glial differentiation and the inhibition of neuronal differentiation.

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