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First published online 27 July 2004
doi: 10.1242/dev.01273

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Interactions between ID and OLIG proteins mediate the inhibitory effects of BMP4 on oligodendroglial differentiation

Northwestern University's Feinberg School of Medicine, Department of Neurology, Chicago, IL 60611, USA

View larger version (97K): [in a new window]   Fig. 1. BMP4 inhibits oligodendrocyte (OL) lineage commitment by neural progenitor cells. (A) EGF-generated neurosphere cells from E17 mice (A,B) were grown for 3 days in 10 ng/ml each of NT3, PDGF-AA and EGF. Cells were then dissociated and plated in media containing 2 ng/ml EGF in absence (A, parts a-c) or presence of 20 ng/ml BMP4 (A, parts d-f). Cells were stained for CNPase (A, parts a,d), MBP (A, parts b,e) and GFAP (A, parts c,f). (B) Cells were immunostained with OLIG1, NG2, CNPase, MBP, GFAP, and ß-tubulinIII, and the mean numbers of immunoreactive cells were determined. BMP4 treatment significantly reduced the number of all OL species while increasing the number of astrocytes. Scale bars represent means±s.e.m. of four independent experiments. Anova was done to determine significance (*P<0.05).

View larger version (48K): [in a new window]   Fig. 2. BMP4 decreases expression of OL transcription factors and increases expression of ID genes. (A) Expression of mRNAs for Olig1, Olig2, Nkx2.2 and Sox10 was analyzed by quantitative RT-PCR performed with mRNA extracted from E17 progenitor cells grown in absence (blue) or presence (red) of 20 ng/ml BMP4 for 2, 8 and 35 hours. BMP4 treatment did not reduce levels of the transcripts until 8 hours. However, by 35 hours all transcripts were reduced by as much as 60-fold. (B) Expression of Id2 and Id4 mRNAs was analyzed by quantitative RT-PCR performed with mRNA extracted from E17 progenitor cells grown in absence (blue) or presence (red) of 20 ng/ml BMP4 for 2, 8 and 24 hours. Id4 and Id2 transcripts were both induced by BMP4 at 8 hours and subsequent time points. Graphs shown are representative of experiments done in duplicate three times. (C) Western blot analyses were performed to detect ID1-4 proteins using cell lysates from E17 progenitor cells grown in the absence (control) or presence of BMP4 for 12 hours. BMP4 treatment increased the expression of all the ID proteins.

View larger version (85K): [in a new window]   Fig. 3. BMP4 acts via ID4 and ID2 to inhibit oligodendrogenesis. (A) Western blot analyses of ID2 and ID4 proteins was done in E17 progenitor cells infected with lentivirus containing empty vector (lenti-gfp) or Id2 or Id4 (lenti-Id). (B) E17 progenitor cells were infected for 2 days with lentivirus containing empty vector (parts a-f), Id2 (parts g-i), Id4 (parts j-l) or Id2+Id4 (parts m-o). Cells infected with empty vector were treated with 20 ng/ml BMP4 in parts d-f. Immunofluorescence was done for CNPase (parts a,d,g,j,m), MBP (parts b,e,h,k,n) and GFAP (parts c,f,i,l,o). DAPI was used to stain the nuclei. (C) Quantification for CNPase, MBP, GFAP and ß-tubulinIII shows that ID overexpression has effects similar to those of BMP4 treatment, i.e. reduced numbers of OLs and increased numbers of astrocytes. Scale bars represent means±s.e.m. of four independent experiments. ANOVA was done to determine significance (P<0.05). The control group (vector alone) differed significantly from other groups in numbers of CNPase, MBP and GFAP immunoreactive cells. There were no significant differences among ß-tubulinIII groups.

View larger version (50K): [in a new window]   Fig. 4. Inhibition of ID4 prevents the effects of BMP4 on glial lineage commitment. The effectiveness of inhibition of Id4 expression by RNAi was confirmed by RT-PCR (A) and quantitative RT-PCR (B) using mRNA extracted from E17 progenitor cells treated with BMP4 (20 ng/ml) for 12 hours and infected with lentivirus containing empty vector (green in B) or Id4-RNAi constructs (blue in B). The graphs shown are representative of experiments done in duplicate three times. (C) E17 progenitor cells infected with lentivirus containing empty vector (parts a-f) or Id4-RNAi (parts g-l) were grown with (parts d-f,j-l) or without (parts a-c,g-i) BMP4 (20 ng/ml) for 2 days. Cells were stained for CNPase, MBP and GFAP. DAPI was used as a nuclear stain and virus-infected cells were identified by staining for GFP. (D) Quantification of CNPase+, GFAP+ and ß-tubulinIII+ cells shows increased numbers of OLs with Id4-RNAi. Scale bars represent means±s.e.m. of four independent experiments. ANOVA was done to determine significance (P<0.05). The control (vector alone) group differed significantly from both BMP4-treated and Id4-RNAi groups with respect to CNPase and GFAP, but did not differ from the group treated with both BMP4 and Id4-RNAi. The BMP4-treated and Id4-RNAi-treated groups each differed from all other groups with respect to CNPase and GFAP. There were no significant differences among groups with respect to ß-tubulinIII.

View larger version (59K): [in a new window]   Fig. 5. OLIG proteins interact with ID2 and ID4 in HEK293T cells. (A) ID1-4 and OLIG1-2 proteins were overexpressed in HEK293T cells. (B) Coimmunoprecipitations were done with protein lysates from HEK293T cells transfected with Olig1/2-myc-his6 and Id1-4. OLIG proteins were pulled down with anti-his6 antibody and immunoblotted with anti-myc antibody, whereas specific ID antibodies were used to pull down or immunoblot IDs.

View larger version (29K): [in a new window]   Fig. 6. OLIG proteins co-localize and interact with ID2 and ID4 in neural progenitor cells. (A) E17 neural progenitor cells were grown for 7 days in the presence of 10 ng/ml EGF (parts a,d), 3 days with 10 ng/ml PDGF-AA+NT3+EGF (parts b,e) and 12 hours with 20 ng/ml BMP4 (parts c,f-h). Cells were plated and stained for OLIG1 and ID2 (part g), OLIG1 and ID4 (part h), OLIG2 and ID2 (parts a-c) and OLIG2 and ID4 (parts d-f). Nuclei were counterstained with DAPI. In the absence of BMP4, both OLIG1 and OLIG2 were localized primarily in the nucleus. However, after BMP4 treatment both OLIG1 and OLIG2 were colocalized with ID2 and ID4 in the cytoplasm. (B,C) Co-immunoprecipitations were done with protein lysates from cultured E17 neural progenitor cells treated with BMP4 for 12 hours. OLIG1 (B) and OLIG2 (C) interact with both ID2 and ID4.

View larger version (63K): [in a new window]   Fig. 7. OLIG1 and OLIG2 interact with E2A proteins E12 and E47. Coimmunoprecipitations were done with protein lysates from HEK293T cells transfected with Olig1/2-myc-his6 and E12 or E47. OLIG proteins were pulled down with his6 antibody and immunoblotted with myc antibody, whereas specific E2A antibodies were used to pull down or immunoblot E2A proteins. OLIG1 (A) and OLIG2 (B) interact with both E12 and E47.

View larger version (56K): [in a new window]   Fig. 8. OLIG proteins partially overcome BMP4 effects. (A) Cultured E17 progenitor cells were infected for 2 days with lentivirus containing Olig1 or Olig2 (parts a-l) along with Id4 (parts g-i) or Id4-RNAi (parts j-l). Cells were treated with 20 ng/ml BMP4 in parts d-f and parts j-l. Immunofluorescence was done for CNPase (parts a,d,g,j), MBP (parts b,e,h,k) and GFAP (parts c,f,i,l). DAPI was used to stain the nuclei. (B,C) Quantification for CNPase, GFAP and ß-tubulinIII shows that OLIG proteins can partially rescue the effects of BMP4 treatment or ID4 overexpression on oligodendroglia. The rescue is complete with Id4-RNAi (C). Scale bars represent means±s.e.m. of four independent experiments. ANOVA was done to determine significance (P<0.05).

View larger version (36K): [in a new window]   Fig. 9. Model of the role of ID proteins in mediating effects of BMP signaling. OLIG proteins bind to promoter regions after heterodimerizing with the E2A proteins, resulting in transcription of their downstream targets. In the presence of BMP4, all four ID proteins are induced, resulting in sequestration of OLIG 1/2 by ID2 and ID4 and of E2A proteins by all the IDs in the cytoplasm of progenitor cells. This inhibits OLIG binding to promoters of target genes, resulting in inhibition of OL development and enhancement of commitment to the astrocytic fate.