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First published online August 14, 2006
doi: 10.1242/10.1242/dev.02522

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An oligodendrocyte-specific zinc-finger transcription regulator cooperates with Olig2 to promote oligodendrocyte differentiation

Center for Developmental Biology and Kent Waldrep Foundation Center for Basic Neuroscience Research on Nerve Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

View larger version (60K): [in a new window]   Fig. 1. Identification, predicted primary sequence, homology and expression of Zfp488 mRNA transcript in the brain. (A) Differential gene expression between wild-type and Olig1-null (KO) optic nerves was examined by mRNA differential display with duplicate samples. Arrows indicate the differentially expressed genes between wild-type and Olig1-null optic nerves. Red arrow indicates the position for Zfp488. (B) Zfp488 expression in the brain of wild-type and Olig1-null mice at P14. Upper panel, a northern blot of RNA extracted from brain tissues of wild-type and Olig1 mutant mice was probed with 32P-labeled Zfp488, revealing a 3.5 kb mRNA transcript in the wild-type brain and its absence in the Olig1-null brain. Lower panel shows expression of the housekeeping gene Gapdh examined by semi-quantitative RT-PCR (20 cycles) as a loading control. (C) Predicted amino acid sequence of mouse Zfp488 protein, showing C2H2 type (boxed) zinc-finger domains in the C-terminal region. Potential nuclear localization signals are underlined. (D) Alignment of evolutionarily conserved zinc finger domains of Zfp488 among invertebrates and vertebrates.

View larger version (120K): [in a new window]   Fig. 2. Expression ofZfp488 coincides with that of differentiated oligodendrocyte markers Mbp and Plp1/DM20 in the embryonic spinal cord. In situ hybridization on transverse spinal cord sections from E12.5 (A-C), E14.5 (D-F) and E18.5 (G-I) with probes to murine Zfp488, Pdgfra, Mbp and Plp1/DM20. (A-C) At E12.5, Pdgfra is expressed in the ventral spinal cord (arrow) but Zfp488 and Mbp are not. (D-F) Expression of Zfp488, Mbp and Plp1/DM20 is initially detected at E14.5 in the ventral ventricular domain (D-F, red arrows) and Zfp488 is absent in the dorsal root ganglia (blue arrows). (G-I) At E18.5, expression of Zfp488, Mbp and Plp1/DM20 occurs in a similar distribution of cells in the ventral lateral white matter of the spinal cord (arrows).

View larger version (122K): [in a new window]   Fig. 3. Zfp488 expression in oligodendrocytes of the postnatal CNS. In situ hybridization of transverse sections of postnatal cerebral cortex, cerebellum and longitudinal sections of optic nerves. (A) At the neonatal stage P7, Zfp488 expression (red arrows) is mainly confined to the spinal white matter. (B-D) Expression of Zfp488 (red arrows) is highly enriched in the white matter of the cerebral cortex (B), and the cerebellum (C) and the optic nerve (D) at P14. A small population of Zfp488-expressing cells in the gray matter of the spinal cord and the forebrain is also evident (A and B, black arrows). (E) Double in situ hybridization for Zfp488 (blue color) and Plp1 (brown color) shows that Zfp488-expressing cells (arrows) are co-labeled with Plp1 in the corpus callosum. Small arrowheads indicate a linear array of interfascicular oligodendrocytes expressing both Zfp488 and Plp1. (F) Double in situ hybridization for Zfp488 and Pdgfra in the P14 cerebral cortex shows that Zfp488-expressing cells (purple) are not co-labeled with Pdgfra (brown) as indicated by purple and brown arrows, respectively. SC, spinal cord; Fbn, forebrain; Cbm, cerebellum; OP, optic nerve; CC, corpus callosum; Ctx, cortex.

View larger version (74K): [in a new window]   Fig. 4. Zfp488 expression is absent throughout the CNS in the Olig1-null mice. (A-F) Expression of Zfp488 mRNA was analyzed in situ on the spinal cord, the corpus callosum and the cerebellum taken from wild-type (A,C,E) or Olig1KO (B,D,E) mice at E18.5 and P14 as indicated. (B,D,F) Absence of Zfp488 expression in the spinal cord (SC), the corpus callosum (CC) and cerebellum (CBM) was observed, indicating that Zfp488 expression requires Olig1. Arrows indicates Zfp488 expression in the white matter of SC (A), CC (C) and CBM (E), respectively. (G) Luciferase reporter activity driven by a 3.2 kb Zfp488 upstream regulatory region in the pGL3 vector indicates that transfection of Olig1 activates Zfp488 expression by approximately fourfold (P<0.01). By contrast, Olig2 activity on the putative promoter is statistically insignificant (P>0.05) using the Student's t-test. Data are derived from at least three independent experiments and presented as the mean±standard deviation.

View larger version (43K): [in a new window]   Fig. 5. Zfp488 exhibits nuclear localization and has transcriptional repression activity. Cells transfected with expression vectors (pCS2) for Myc-tagged Zfp488 and its various mutants were assessed for nuclear localization defined by DAPI staining and for Zfp488 transcription activity in an in vitro assay. (A,B) Nuclear localization of Myc-tag staining (arrows) is detected in both COS7 and NIH3T3 cells when transfected with Myc-Zfp488 and analyzed by indirect immunofluorescence using anti-Myc antibody (red) together with DAPI (blue) to delineate the nucleus. (C-F) COS7 cells transfected with expression vectors for Myc-tagged proteins of Zfp488 full-length (C), Zfp488F2 (D), Zfp488F12 (E) and ZF12 (F) domain only were examined for Myc (red) and DAPI (blue) immunofluorescence. Arrows indicate expression of Myc-Zfp488 and its derivatives. (G) A schematic diagram shows the in vitro assay for Zfp488 transcription activity. (H) NIH3T3 cells were transiently transfected with L8G5-luc reporter (Lu et al., 1999) and expression vectors encoding LexA-VP16, GAL4-Zfp488 or its truncated forms, as indicated. Transfection of GAL4-Zfp488 resulted in repression of luciferase reporter activity induced by LexA-VP16 in this assay. The Zfp488 construct lacking both zinc-finger motifs (Zfp488ZF12) and its N-terminal fragments 1-184 (Zfp488N184) and 1-69 (Zfp488N69), as well as its zinc-finger domain only segment (Zfp488ZF12), were fused in-frame with GAL4 in an expression vector. The relative activity of GAL4-Zfp488 truncated derivatives was normalized to that of LexA-VP16 activation. At least three independent transfection experiments were performed and data are presented as the mean±s.d.

View larger version (103K): [in a new window]   Fig. 6. Zfp488 induces ectopic oligodendrocyte precursor markers in the presence of Notch signaling activation. Chick neural tubes at the stage HH14 (E2.5) stage were electroporated with expression vectors for Zfp488 (A,B), NICD (C,D) or both constructs (E-H), and harvested 3 days later at E5.5. Sections of chick neural tube were hybridized with the probes as indicated. Transgene (Tg) was detected by Zfp488 (A,E) and NICD (C). Overexpression of neither Zfp488 (A,B) nor NICD (C,D) alone could induce ectopic oligodendrocyte marker expression. The combination of NICD and Zfp488 induces expression of ectopic oligodendroglial markers Sox10 (F, arrow) and Pdgfra (G, arrow), but not Mbp (H).

View larger version (92K): [in a new window]   Fig. 7. Zfp488 cooperates withOlig2 to promote ectopic and precocious oligodendrocyte differentiation. E2.5 chick embryos were electroporated with expression vectors for Olig2 (A,B), Zfp488/Olig2 (C-F,I-J) or Zfp488/Nkx2.2 (G,H), and harvested 3 days later at E5.5. In situ hybridization of the neural tube was performed with the probes as indicated in panels. (A,B) Misexpression of the Olig2 transgene (Tg) alone did not induce oligodendrocyte markers in the dorsal region (red arrow in B), while a small number of Sox10+ cells were detected in the ventral domain (black arrowhead). (C-F) Co-electroporation of Zfp488/Olig2 induced robust ectopic expression of Sox10 (D, arrows), Pdgfra (E, arrows) and Mbp (F, arrows) in the electroporated side of the neural tube. (G,H) Co-electroporation of Zfp488/Nkx2.2 did not induce ectopic Sox10 expression in the transgenic side of the chick neural tube. (I,J) Dorsally confined misexpression of Zfp488 and Olig2 resulted in ectopic Sox10 expression in the dorsal region of the spinal cord (J, red arrow) but not in the ventral region (J, black arrowhead). (K,L) Double in situ labeling for chick ortholog of Zfp488 (purple) and immunostaining of Olig2 (brown) were performed in the chick spinal cord at E18. Co-expression of chick Zfp488 and Olig2 was detected in the spinal cord. L is a high magnification of an area outlined in K, showing the co-labeling of Zfp488 and Olig2 in the same cells of the chick spinal cord (red arrows). White arrowheads in L indicate the cells that express only Olig2. (M) Physical interaction between Zfp488 and Olig2. Vectors expressing Myc-tagged Zfp488 and Flag-tagged Olig2 were co-transfected into Cos7 cells. Co-immunoprecipitation (IP) of cell lysates (600 µg total) 48-hours post-transfection was performed with anti-Flag antibody. Western blot was carried out to detect the input proteins for Zfp488 (upper panel) and Olig2 (middle panel). The immunoprecipitated Myc-Zfp488 was detected by anti-Flag (lower panel, arrow). (N) Absence of Zfp488 and Nkx2.2 interaction. Vectors expressing Myc-tagged Zfp488 and Nkx2.2 were co-transfected into COS7 cells. Co-immunoprecipitation was performed with anti-Myc antibody. Western blot was performed to detect the input proteins for Myc-Zfp488 (upper panel) and Nkx2.2 (middle panel). The co-immunoprecipitated complex was detected by anti-Nkx2.2 (lower panel). Star indicates the prospective Nkx2.2 position.

View larger version (26K): [in a new window]   Fig. 8. Expression of Zfp488 is correlated with oligodendrocyte differentiation. (A) Expression of Zfp488 increased as oligodendroglial cells underwent differentiation. Primary rat OPCs were isolated from neonate rat at P2 and cultured in growth medium. Total RNA was harvested from cell lysates before and after switching to differentiation medium at the time indicated. Real-time RT-PCR was performed to determine the relative amounts of Zfp488, Cnp, Mbp and Nkx2.2 expression. (B) CG4 cells were analogously treated and analyzed by real-time RT-PCR as described above in A. (C) Cultured CG4 cells in growth medium were transfected with control GFP siRNA and Zfp488 siRNA (100 nM) for 48 hours. Total RNAs were isolated before and after siRNA transfection. Real-time PCR was performed to determine the expression level of Zfp488, Cnp, Mbp and Gapdh. All data were derived from three independent experiments and shown as mean±s.d. Gapdh gene expression was used as the internal control.