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First published online 6 January 2004
doi: 10.1242/jcs.00867

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Murine tenascin-W: a novel mammalian tenascin expressed in kidney and at sites of bone and smooth muscle development

¹ Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Maulbeerstrasse 66, CH-4058 Basel, Switzerland
² Department of Cell Biology and Human Anatomy, University of California at Davis, Davis, CA 95616, USA

View larger version (56K): [in a new window]   Fig. 1. Primary structure of the mouse tenascin-W protein. (A) Mouse tenascin-W amino acid sequence translated from the DNA database entry (accession number AJ580920). The signal peptide is underlined (dashed). Amino acids 99-132 represent the heptad repeats flanked by cysteines, amino acids 155-259 the EGF repeats, amino acids 260-1056 the FNIII repeats and amino acids 1057-1296 the fibrinogen globe. The RGD sequence in the second FNIII repeat is boxed (grey), the IDG motifs are double underlined and BBXB sites are underlined. (B) Relationship between FNIII repeats created by the program pileup of the GCG software package. (C) Schematic representation of the domain structure of mouse and zebrafish tenascin-W depicting an N-terminal domain (wedge), heptad repeats (wavy line), EGF-like repeats (diamonds), FNIII repeats (squares; central highly similar repeats shaded in black) and fibrinogen globe (circle). Homologous FNIII repeats between mouse and zebrafish tenascin-W are interconnected by dashed lines.

View larger version (73K): [in a new window]   Fig. 2. Purification of tenascin-W. (A) Protein gels of purified tenascin-W (W) in comparison to tenascin-C (C) reveals that both proteins form high molecular weight disulphide-linked oligomers (arrow), as can be seen by their slow migration in the absence of reducing agent (-BME). Migration under reducing condition (+BME) reveals for tenascin-W a Mr of 180 kDa. An immunoblot with anti-tenascin-W antibody (anti-TNW) reveals that this antiserum specifically reacts with tenascin-W, but not with tenascin-C. (B) Electron microscopy after rotary shadowing of recombinant mouse tenascin-W reveals that it forms hexameric oligomers. Scale bar, 50 nm.

View larger version (77K): [in a new window]   Fig. 3. Tenascin-W is first expressed in maxillary processes of mouse embryos. Immunohistochemistry of tenascin-W (A), tenascin-C (B) and a merged picture (C) in a section through an E11.5 mouse embryo reveals co-expression in the maxillary process (mp). Tenascin-W is still expressed in the mp at E14.5 (D). In situ hybridization analysis of E12.5 mouse embryos with probes against tenascin-W reveals expression in `moustache-like' structures corresponding to the mp when viewed from below (arrows, E) and in whole embryos (arrow, F). Embryos incubated with sense control probes were unlabeled (not shown). Scale bars, 100 µm (A-D), 1 mm (E,F).

View larger version (161K): [in a new window]   Fig. 4. Similar but distinct expression patterns of tenascin-W and tenascin-C in mouse embryos. Immunohistochemistry of tenascin-W and tenascin-C (as indicated) at E14.5 (A,B,E,F,I,J), E15.5 (C,D,G,H,K,L) and E16.5 (M-P). At E16.5 (M-P), double labelling is shown with tenascin-W in green, tenascin-C in red and overlap in yellow. Tenascin-W is expressed in the circular muscular layer (cml) of the stomach at E14.5 (A) and in the muscularis mucosa (mm) and muscularis externa (me) at E15.5 (C). At E14.5, tenascin-C is expressed in both the cml and longitudinal muscular layer (lml) of the stomach (B). At E15.5, tenascin-C is found in the mm, but staining in the me (i.e. combined cml and lml) is faint. Tenascin-C is expressed in the developing liver (l) at both E14.5 (B) and E15.5 (D), but tenascin-W is not. Tenascin-W is expressed in the cml of the ileum at E14.5 (E) and in both the me and mm at E15.5 (G). It is not seen in the colon (c). Tenascin-C is not detected in the ileum at either E14.5 (F) or E15.5 (H) but, unlike tenascin-W, it is expressed in the developing musculature of the colon at E15.5 (H). At E14.5, tenascin-W is not present in the cartilaginous vertebral arches (va, I), but expression appears at E15.5 in the periosteum (po) around the ribs (K). Tenascin-C was expressed at both embryonic stages in the cartilaginous vertebral arches (J) and rib cartilage (ca, L). At E16.5, tenascin-W was expressed in mandible (m, M), palate (p, N) and molars (mo, O), mostly as a subset of tenascin-C expression (e.g. arrow, O). At the same stage, only tenascin-W was expressed in the matrix of the masseter (ma, P). sn, spinal nerve; oc, oral cavity; g, salivary gland. Scale bar, 200 µm.

View larger version (155K): [in a new window]   Fig. 5. Expression of tenascin-W and tenascin-C in the adult mouse. Each figure shows double staining of tenascin-W (green) and tenascin-C (red), with the overlap being yellow. Tenascin-C immunoreactivity is found in the molecular layer (ml) of the cerebellum (A). Tenascin-W was not detected here or elsewhere in the adult central nervous system. Tenascin-W and tenascin-C co-localize in the matrix of kidney tubules (B). Tenascin-C is found in the matrix of major blood vessels but tenascin-W is expressed only at the base of the cusps (c) of the aortic (arrow, C) and pulmonary valves. Tenascin-W is also expressed as a subset of the tenascin-C expression pattern in the corneal limbus (lm, D) and the periosteum (po) of the rib (r) (E). Tenascin-C is seen in the matrix of adult stomach (F) and intestine (G), but tenascin-W is not. c, valve cusp; cb, ciliary body; co, cornea; g, glands; icm, intercostal muscle; me, muscularis externa; ml, molecular layer; mm, muscularis mucosa; Pcl, Purkinje cell layer; sm, submucosa; t, tendon; v, villus. Scale bar, 200 µm.

View larger version (35K): [in a new window]   Fig. 6. Identification of endogenous tenascin-W. (A) Western blot analysis with an anti-tenascin-W antibody of extracts from E10.5 and E12.5 whole embryos, intestine and liver of E18.5 embryos, periosteum and kidney of adult mice and of embryonic stem (ES) cells conditioned medium. All of the samples reveal a single tenascin-W band of 180 kDa except for liver, which is negative for tenascin-W. (B) Agarose gel of nested-PCR products amplified from an E18.5 whole embryo mouse library using primers covering the second to the ninth fibronectin type III repeats of mouse TN-W, with (a) and without (b) template.

View larger version (42K): [in a new window]   Fig. 7. C2C12 osteoblasts express tenascin-W and adhere to a tenascin-W coated substratum. (A) Tenascin-W accumulates in the conditioned medium of BMP2-treated C2C12 cells (+) but not in untreated cultures (-) collected after 2, 4 and 6 days of treatment as revealed on the immunoblot with anti-tenascin-W antibody. The C2C12-produced tenascin-W migrates with the purified recombinant tenascin-W loaded as control (TNW). (B) Adhesion assay of C2C12 cells after culturing for 6 days with or without BMP2 on fibronectin. (C) Adhesion assay of C2C12 cells after culturing for 6 days with or without BMP2 on tenascin-W. Treated and untreated cells adhered equally well to fibronectin but BMP2 treatment increased the adhesivity of the cells to tenascin-W. (D,E) Photographs of the C2C12 cells of the adhesion assay shown in (B,C). Cells without BMP treatment on fibronectin (D), on tenascin-W (E) and BMP2-treated cells on tenascin-W (F). The cell morphology of treated and untreated cells remains the same. Scale bar, 20 µm (D-F).

View larger version (35K): [in a new window]   Fig. 8. Adhesion to tenascin-W is 8ß1-integrin dependent. (A) Adhesion assay of T98G glioblastoma cells on tenascin-W coated at 20 µg ml-1. Function-blocking antibodies against specific integrin subunits were included at 10 µg ml-1 as indicated. None of the -specific antibodies tested inhibited adhesion, but the anti-ß1 antibody reduced the adhesion to the level of the BSA coating. (B) Adhesion assay of SW480 colon carcinoma cells transfected with 9 integrin (SW480-9) or with the empty plasmid (SW480-mock) on tenascin-W (TN-W), fibronectin (FN) or collagen (COLL). (C) Adhesion assays with 8-integrin- or mock-transfected K562 leukaemia cells on increasing concentrations of tenascin-W reveals that 8-transfected cells acquire adhesiveness to tenascin-W. (D) Adhesion of T98G cells to tenascin-W (TN-W, coated at 20 µg ml-1) and fibronectin (FN; coated at 20 µg ml-1) in the presence or absence of PBS, 100 µg ml-1 and 500 µg ml-1 of GRGDS or 500 µg ml-1 SDGRG peptides as indicated.

View larger version (81K): [in a new window]   Fig. 9. Overexpression of 8 integrin increases adhesion of C2C12 cells to tenascin-W. Phalloidin staining of C2C12 cells after 1 hour of adhesion on fibronectin (A) reveals stress fibre formation. Phalloidin staining of C2C12 cells reveals the extension of actin-rich processes in cells adherent to tenascin-W (B). C2C12 cells were transfected with an 8 expression plasmid alone (C,D) together with an EGFP plasmid (E-G) or with the EGFP plasmid alone (H-J) and, 2 days later, replated for 1 hour on tenascin-W-coated wells. After fixation, cells shown in (C,D) were stained with anti-8 antiserum (C) and phalloidin (D). Cells overexpressing 8 are more spread and contain stress fibres, whereas the untransfected cell to the right (arrow) starts to make actin-rich processes. Cells transfected with 8 are more spread and have longer processes (E-G) than EGFP-only transfected cells (H-J). Scale bar, 25 µm (A,B,E-J), 10 µm (C,D).

View larger version (110K): [in a new window]   Fig. 10. Co-localization of 8 integrin with tenascin-W. Immunostaining of tenascin-W (red, A,C) and 8 integrin (green, B,D) in sections of an E16.5 mouse vertebra (A,B) and of an adult mouse kidney (C,D). Scale bar, 200 µm.