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First published online August 26, 2004
doi: 10.1242/10.1242/jcs.01291

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TGFß/BMP activate the smooth muscle/bone differentiation programs in mesoangioblasts

Enrico Tagliafico^1,*, Silvia Brunelli^2,*, Anna Bergamaschi¹, Luciana De Angelis^3,4, Raffaella Scardigli^3,4, Daniela Galli², Renata Battini¹, Paolo Bianco^3,5, Sergio Ferrari¹, Giulio Cossu^2,3,4, and Stefano Ferrari^1,

¹ Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, Via G. Campi 287, 41100 Modena, Italy
² Stem Cell Research Institute, Dibit, H. San Raffaele, Via Olgettina 58, 20132 Milano, Italy
³ Institute of Cell Biology and Tissue Engineering, San Raffaele Science Park, Via Castel Romano 100, 00128 Roma, Italy
⁴ Dipartimento di Istologia ed Embriologia, Università di Roma `La Sapienza', Via A. Scarpa 14, 00161 Roma, Italy
<sup>5</sup> Dipartimento di Medicina Sperimentale, Università di Roma `La Sapienza', Via A. Scarpa 14, 00161 Roma, Italy

View larger version (24K): [in a new window]   Fig. 1. Absolute analysis of RNA transcripts in mesoangioblasts and 10T1/2 cells. The Venn diagram shows the overall number of sequences expressed by all mesoangioblast clones (9233) and 10T1/2 cells (11861), as well as the number of sequences expressed by both mesoangioblasts and 10T1/2 cells (8674), and specifically by mesoangioblasts (203) or 10T1/2 cells (580).

View larger version (40K): [in a new window]   Fig. 2. Clustering analysis of functionally selected genes specifically expressed by mesoangioblasts. The figure shows a combination of two hierarchical clustering analyses (gene tree and condition tree) performed on functionally selected genes, grouped in the following categories: (A) DNA binding/transcription factors/transcriptional regulators; (B) Integral membrane proteins/membrane receptors; (C) Cytoskeleton/cell adhesion and/or motility; (D) Defence response/immune response/inflammatory response. The gene tree is shown on the left, the condition tree on the top of each panel. Gene colouring was based on normalized signals as shown in the colour bar.

View larger version (74K): [in a new window]   Fig. 3. Abortive neural differentiation of mesoangioblasts. (a) Immunofluorescence analysis of a co-culture of GFP expressing mesoangioblasts with differentiating cells from E10 embryonic neural tube, under neural stem cell differentiation conditions. Cultures were stained with TuJ1, an antibody against ß III-tubulin (red). GFP-positive cells appear green mainly in the nucleus where strong staining for ß III-tubulin is not present. (b) Immunofluorescence analysis of mesoangioblasts, cultured under neural stem cell differentiation conditions and stained with anti-GFAP (red); nuclei were stained with DAPI (blue). (c,d) Whole-mount fluorescence analysis of an E5 chick embryo brain injected with GFP-labelled mesoangioblasts. Clusters of GFP-positive cells can be detected both in forebrain (fb) and hindbrain where they appear to be restricted to specific rhombomeres (rh). Scale bar in c: 50 µm for a; 75 µm for b; 600 µm for c and d.

View larger version (53K): [in a new window]   Fig. 4. TGFß, BMP and insulin mediated differentiation of mesoangioblasts. (a,b) Schematic representation of the TGFß/BMP (a) and insulin (b) signalling pathways in mesoangioblasts: the colour codes indicate the abundance of the different pathway components, ranging from red (highly expressed) to pale red to orange to yellow (poorly expressed); white: not in the array. (c) Western blot analysis of cell lysates from mesoangioblasts treated or untreated (C) with TGFß1, BMP2, or insulin; antibodies against TGFßRI, TGFßRII, BMPR1A, Smad1, Smad2/3, or IGF1Rß were used. (d) Histogram representing the percentage of mesoangioblasts staining Red Oil positive, after treatment with insulin-containing medium. (e-g) Immunofluorescence analysis of mesoangioblasts treated with TGFß1 and stained with antibodies against smooth muscle -actin (SMA; e), SM22 (f), calponin 1 (g) and smooth muscle myosin (SM-MyoHC; h) (all in green), merged with DAPI-stained nuclei (blue). (i) AP and Von Kossa staining of mesoangioblasts treated with BMP2 under osteogenic-promoting condition. Arrows point to calcified nodules. (j) Red Oil staining of mesoangioblasts treated with insulin under adipogenic-promoting condition. Scale bar in j: 50 µm for e-h; 200 µm for i; 150 µm for j.

View larger version (50K): [in a new window]   Fig. 5. Inhibition of the TGFß and BMP2 differentiation pathway. (a-c) Immunofluorescence analysis of control mesoangioblasts (a), mesoangioblasts treated with TGFß1 (b), or treated with the neutralizing anti-TGFbRII antibody prior to the addition of TGFß1 (c). The antibody to the smooth muscle marker Calponin 1 was used throughout. (d) Histogram representing the percentage of mesoangioblasts expressing Calponin 1 in the indicated culture conditions. (e-g) AP staining of control mesoangioblasts (e), mesoangioblasts treated with BMP2 (f), or transfected with noggin expression vector prior to the addition of BMP2 (g). (h) Histogram representing the percentage of AP-positive mesoangioblasts in the indicated culture conditions. Scale bar in e: 150 µm for a-c; 200 µm for d-f.

View larger version (32K): [in a new window]   Fig. 6. Wnt pathway and proliferation in mesoangioblasts. (a) Schematic representation of the Wnt signalling pathway in mesoangioblasts; colour codes as in Fig. 4. (b) Western blot analysis of cell lysates from the mesoangioblast clones A2 and A4, using antibodies against Wnt5a, Frizzled and ß-catenin. (c) Western blot analysis of concentrated culture medium from Frzb1-HA transfected 293T cells, using anti-HA antibody. (d) Growth curve of mesoangioblasts exposed to different concentrations of Frzb1 (pink and yellow lines, respectively, representing 50% and 100% of added medium from Frzb1-HA transfected 293T cells) and in control culture conditions (blue line).