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First published online November 3, 2003
doi: 10.1242/10.1242/dev.00834

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Functional Notch signaling is required for BMP4-induced inhibition of myogenic differentiation

Camilla Dahlqvist1, Andries Blokzijl2, Gavin Chapman1, Anna Falk1, Karin Dannaeus1, Carlos F. Ibâñez2 and Urban Lendahl1,*

1 Department of Cell and Molecular Biology, Karolinska Institute, SE-171 77 Stockholm, Sweden
2 Department of Neuroscience, Medical Nobel Institute, Karolinska Institute, SE-171 77 Stockholm, Sweden



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  		Fig. 1. Effects of BMP4 and Notch signaling on C2C12 differentiation. (A) C2C12 cells were analyzed 6 days post-addition of differentiation medium, and after BMP4 (50 ng/ml) and/or {gamma}-secretase inhibitor (L-685,458; 1 µM) treatment. Myocyte differentiation was visualized by immunostaining for MHC (red; left panel) and DAPI staining (blue; right panel) was used to identify cell nuclei. Note that a considerable proportion of the cells were MHC-positive under normal differentiation conditions (no addition), and that the addition of BMP4 strongly reduced differentiation (BMP4). Treatment with L-685,458 (L-685,458) led to an increase in the number of differentiated cells compared with treatment with differentiation medium alone, and the combined treatment with BMP4 and L-685,458 (L-685,458 + BMP4) resulted in an increase in the number of MHC-positive cells, as compared with treatment with BMP4 alone. (B) C2C12 cells were analyzed for MHC expression 2 days post-addition of differentiation medium in the presence of BMP4 (50 ng/ml), and after transfection of CSL R218H (upper panel) or ß-gal (lower panel).

 


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  		Fig. 2. (Top) Analysis of differentiation of C2C12 and satellite cells. C2C12 and satellite cells were differentiated in the presence and absence of BMP4 and/or L-685,458. The number of MHC-positive cells and total number of DAPI-positive cell nuclei were counted in randomly selected microscope fields after immunostaining. The percentage of MHC-positive cells compared with the number of DAPI-stained nuclei was calculated. To the right are examples of multinucleated C2C12 and satellite cells stained for MHC. (A-H) Analysis of Hes1 and Hey1 mRNA expression in response to BMP4 and Notch signaling. The amount of Hes1 (A) and Hey1 (B) mRNA, as measured by quantitative PCR, in C2C12 cells after addition of L-685,458 and/or BMP4. (C) An experiment similar to that in A and B, but measured at both 0 and 5 days after induction of differentiation. (D) Comparison of changes in expression of Hey1 and Runx2 in response to BMP4 and/or L-685,458. (E) Western blot analysis of MHC and MyoD protein expression at various time points after induction of C2C12 differentiation. Below is a quantitative PCR experiment demonstrating changes in MyoD expression in response to BMP4 and/or L-685,458. (F,G) The amount of Hey1 (F) and Hes1 (G) mRNA, as measured by quantitative PCR, in satellite cells after addition of L-685,458 and/or BMP4. (H) The amount of Hey1 mRNA, as measured by quantitative PCR, in neural stem cells cultured as neurospheres under normal conditions or after addition of BMP4.

 


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  		Fig. 3. The effect of Notch and BMP cross-talk is direct and requires cell-cell contact. (A) Quantitative PCR of Hey1 mRNA expression in C2C12 cells exposed to BMP4 and/or cycloheximide (Chx). (B) Quantitative PCR of Hey1 mRNA expression in C2C12 cells cultured at different densities (20, 40 or 80% confluent) and exposed to various concentrations of BMP4 [25 (+) or 50 (++) ng/ml BMP4]. (C) The amount of Dll1 and Serrate 1 mRNA, as measured by quantitative PCR, in C2C12 cells after addition BMP4.

 


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  		Fig. 4. BMP4 and Notch 1 ICD synergistically activate the Hey1 promoter. (A) Transfection of C2C12 cells with SMAD1 in the absence (–) or presence (+) of exogenous BMP4 (50 ng/ml), measured by activation of a Hey1-luciferase reporter construct. Note the induction when SMAD1-transfected cells are exposed to BMP4. (B) Transfection with Notch 1 ICD combined with BMP4 stimulation (50 ng/ml). Note the effect of BMP4 on Notch 1 ICD-transfected cells. (C) Co-transfection with Notch 1 ICD and SMAD1, in the absence (white bars) or presence (black bars) of exogenous BMP4 (50 ng/ml). (D) Transfection of Cos-7 cells with Notch 1 ICD, combined with BMP4 stimulation (50 ng/ml) or transfection with Alk6CA.

 


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  		Fig. 5. The synergistic effect from Notch 1 ICD and SMAD1 is dose-dependent and partially CSL-dependent. (A) Transfection of Notch 1 ICD and variable amounts (100, 200 and 300 ng) of SMAD1 in COS-7 cells, in the absence (white bars) or presence (black bars) of exogenous BMP4 (50 ng/ml), measured by activation of a Hey1-luciferase reporter construct. (B) An experiment similar to that in A, carried out in the absence of exogenous BMP4, with 100 and 200 ng SMAD1. Shown below is a western blot for Notch 1 ICD and SMAD1. (C) Transfection of Notch 1 ICD (67 ng), SMAD1 (67 ng) and/or R218H CSL (67 ng) in COS-7 cells, in the absence (white bars) or presence (black bars) of exogenous BMP4 (50 ng/ml), measured by activation of a Hey1-luciferase reporter construct.

 


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  		Fig. 6. Analysis of Notch 1 ICD and SMAD1-responsive elements in the Hey1 promoter. (A) Schematic picture of the Hey1 reporter construct with CSL-binding sites and the GC-rich domain (Hey1-luciferase), and the two mutants containing only the GC-rich domain (GC-luciferase) and comprising the Hey1 promoter lacking the GC-rich domain (Hey1-{Delta}GC). (B) Transfection of Notch 1 ICD (67 ng), SMAD1 (67 ng) and/or R218H CSL (67 ng) in COS-7 cells, in the absence (white bars) or presence (black bars) of exogenous BMP4 (50 ng/ml), measured by activation of the GC-luciferase reporter construct. (C) Transfection of Notch 1 ICD (100 ng) and SMAD1 (100 ng) in COS-7 cells, in the absence (left) or presence (right) of exogenous (50 ng/ml) BMP4, measured by activation of Hey1-luciferase (white bars) or Hey1-{Delta}GC (black bars) reporter constructs.

 


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  		Fig. 7. SMAD1 potentiates Notch 1 ICD activation from promoters lacking SMAD1-binding sites. (A) Transfection of Gal4-Notch 1 ICD (100 ng) and SMAD1 (100 ng) in COS-7 cells, in the absence (white bars) or presence (black bars) of exogenous BMP4 (50 ng/ml), measured by activation of the UAS-luc reporter construct. (B) Transfection of Notch 1 ICD (100 ng) and SMAD1 (100 ng) in COS-7 cells, in the absence (white bars) or presence (black bars) of exogenous BMP4 (50 ng/ml), measured by activation of the 12xCSL-luc reporter construct.

 


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  		Fig. 8. Interaction between Notch 1 ICD and SMAD1. (A) Western blot of a pull-down with a GST-Notch 1 ICD fusion protein from lysates of cells transfected with CSL or SMAD1 proteins. The precipitated SMAD1 protein was visualized using a Flag antibody (the two lanes to the left), and the CSL protein using a Myc antibody (the two lanes to the right). Note that a small amount of SMAD1 was precipitated by GST-Notch 1 ICD, but not by GST. (B) Immunoprecipitation from COS-7 cells transfected with Flag-tagged SMAD1 or Flag-tagged P/CAF in the presence of Myc-tagged Notch 1 ICD. Immunoprecipitation with an anti-Flag-antibody was followed by analysis by western blot using an anti-Myc antibody.

 

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© The Company of Biologists Ltd 2003