Quantitative real-time RT-PCR analysis of the expression levels of E-cadherin

Transcription

1 Supplementary Information 1 Quantitative real-time RT-PCR analysis of the expression levels of E-cadherin and ribosomal protein L19 (RPL19) mrna in cleft and bud epithelial cells of embryonic salivary submandibular glands compared to adjacent mesenchyme. E-cadherin in clefts is 6-fold lower than in buds. 1

2 Supplementary Information 2 2

3 FN protein expression in isolated salivary epithelium during branching morphogenesis. Mesenchyme-free culture of epithelium was performed essentially as previously described. 1 After submandibular glands were isolated from 13.0-day embryos of ICR mice, the glands were treated with dispase (1000 protease units/ml), and epithelia were mechanically separated from mesenchyme using fine forceps. Epithelial explants were cultured on a Nuclepore membrane in a thin layer of growth factor-reduced Matrigel in serum-free DMEM/F12 medium with 100 ng/ml FGF7 and 1 ng/ml EGF for 0 h (a), 12 h (b), or 24 h (c). Scale bar, 100 µm. Local FN expression during branching morphogenesis of isolated mouse embryonic salivary epithelium after staining with anti-fn and imaging by confocal microscopy (d-o). Isolated epithelia of glands were cultured on membranes for 12 h. Triple-staining of the same confocal section with anti-fn (green; d, h, l), anti-e-cadherin (red; e, i, m) and SYBR green I (blue; f, j, n) is shown merged in panels g, k, and o. Open triangles indicate clefts, and filled triangles outline regions of FN expression in these single confocal sections. FN protein accumulates during cleft formation. Images h-k are enlarged images from d-g. Images l-o are from a later stage than h-k. Scale bar (g), 20 µm; scale bar (k, o), 10 µm. Phase-contrast images showing developing isolated epithelia of glands at 0 h (p, s), 12 h (q, t), and 24 h (r, u). Epithelia were treated with PBS (control, p, q, r) or 100 µg/ml cellular FN (s, t, u). Exogenously added cellular FN accelerated branching morphogenesis of isolated salivary epithelium in the absence of mesenchyme. Scale bar, 100 µm. 1. Morita, K. & Nogawa, H. EGF-dependent lobule formation and FGF7- dependent stalk elongation in branching morphogenesis of mouse salivary epithelium in vitro. Dev. Dyn. 215, (1999). 3

4 Supplementary Information 3 Partial rescue of branching after washing FN sirna-transfected salivary glands with fresh medium. Phase-contrast images show living glands at 10 h (a, d), 24 h (b, e), and 36 h (c, f). a-c, PBS-treated gland (control, see Figure 4) was washed with fresh culture medium after 5 h. d-f, FN sirna-treated gland (FN sirna, Figure 4) was washed with fresh medium after 5 h. All glands were cultured for 36 h. FN sirna inhibition was reversed by washing out the sirna (compare to the parallel sirna-treated culture in Figure 4). Scale bar, 100 µm. 4

5 Silencing of FN protein expression in salivary epithelial tissue is shown by confocal immunofluorescence microscopy imaging of staining for FN (g, k, o), E- cadherin (h, l, p), or nuclei (i, m, q). The three fluorescence images of the same confocal section are merged in j, n, and r. Open triangles indicate clefts, and closed triangles mark FN expression in clefts; note reduced FN expression in k. All sirna concentrations were 500 nm. Scale bar, 20 µm. 5

6 Exogenous fibronectin rescues sirna-suppressed glands. Phase-contrast images show salivary glands at 10 h (s, v), 24 h (t, w), and 36 h (u, x) cultured without (s-u) or with (v-x) added FN. Plasma FN (2 mg/ml) was added to FN sirna-treated glands, which were subsequently cultured for 36 h (s-x). Scale bar, 100 µm. 6

7 Supplementary Information 4 a, Effect of exogenous collagen III on branching morphogenesis compared to FN. b, Effect of TIMP1. The effects of collagen III and TIMP1 on branching were quantified by counting the number of buds per salivary gland at 10 h. There was no difference in branching after collagen III or TIMP1 treatment for 10 h, whereas FN (a, mg/ml; b, 8 mg/ml) stimulated branching. 7

8 Supplementary Information 5 FN protein expression during lung branching morphogenesis by staining with anti-fn and imaging by confocal microscopy. Open triangles indicate clefts, and 8

9 filled triangles outline regions of FN expression in these single confocal sections. FN staining accumulates at sites of epithelial indentation and cleft formation (a). Triple-staining of the same confocal section with anti-e-cadherin (red; b) and SYBR green I (blue; c) is shown merged in panel d. Scale bar, 20 µm. Inhibition of lung branching morphogenesis (l-r) by anti-fn antibody (e-k). Phase-contrast images of living E11.5 lungs cultured for 0 h (e, h), 24 h (f, i) and 48 h (g, j) after treatment with PBS control (e, f, g) or 1:50 diluted anti-fn R745 antibody (h, i, j). Effect of exogenous fibronectin on lung branching morphogenesis. Phasecontrast images show living lungs at 0 h (l, o), 24 h (m, p), and 48 h (n, q). Lung rudiments were cultured on Nuclepore membranes in serum-free DMEM/F12 medium as described for salivary glands and were treated with PBS (control, l, m, n) or 2 mg/ml fibronectin (+FN, o, p, q). Scale bar, 500 µm. Effects of anti-fn antibody (k), and FN (r) were quantified by counting numbers of buds per lung at the indicated times (0 h and 48 h) and normalizing to the initial number of buds at 0 h. k, Bar 1, PBS-treated glands (control); 2 and 3, R745-treated glands at: 2, 1:100 dilution; 3, 1:50; 4, 1:50 control rabbit serum. Bars indicate s.e. Data in (k) were analyzed by Bonferroni s multiple comparison test., P <0.05 and, P <0.001 versus controls. The differences between control and FN in (r) were significant at p < 0.05 ( ) by unpaired two-tailed t-test. Six lungs were analyzed for each condition. The experiment was repeated three times, with data shown from a representative experiment. 9

10 FN protein expression during branching morphogenesis of ureteric bud of mouse kidney by staining with anti-fn and imaging with confocal microscopy. Open triangle indicates cleft, and filled triangles outline regions of FN expression in these single confocal sections. FN staining is particularly prominent near epithelium undergoing cleft formation or apparent constriction (s, w, a ). Triplestaining of the same confocal section with anti-e-cadherin (red; t, x, b ) and SYBR green I (blue; u, y, c ) is shown merged in panels v, z, d. Scale bar, 20 µm. 10

11 Inhibition of kidney branching morphogenesis by anti-fn antibody. E11.5 kidneys were cultured on Nuclepore membranes in serum-free DMEM/F12 medium for 48 h (e -j ) after treatment with PBS as the control (e ), 1:50 diluted anti-fn R745 antibody (f ), or 1:50 diluted control rabbit serum (g ) as described for salivary glands (see Figure 3). Inhibition by FN sirna of kidney branching morphogenesis. Kidneys were cultured after treatment with 1 µm control FN scrambled sirna (h ) or 1 µm FN sirna (i ) as described for salivary glands (see Figure 4). Effect of exogenous fibronectin on kidney branching morphogenesis. Kidneys were cultured for 48 h in the presence of 2 mg/ml of plasma fibronectin (+FN, j ). The branching ureteric buds were visualized with anti-e-cadherin antibody as described in Figure 2. Each image is a maximum projection of a 20- image z-series obtained using a Zeiss LSM 510 laser scanning confocal microscope on an Axiovert 200 microscope using a 10X 0.3 NA Plan-Neofluar objective. Scale bar, 100 µm. 11

12 Supplementary Information 6 Local suppression of cadherin localization by exogenous cellular fibronectin in human salivary gland cell (HSG) cells. HSG cells were cultured with 25 µg/ml pre-aggregated chick cellular fibronectin for 24 h, then fixed with methanolacetone (1:1) for 5 min at 4 C, then 20 min at -20 C. Samples were rehydrated in Dulbecco s PBS and stained with anti-pan-cadherin antibody (a) or antifibronectin antibody (b) and examined with a Zeiss Axiophot fluorescence microscope with a Photometrics CH350 chilled CCD camera. The two images in panels a and b were merged and pseudo-colored using MetaMorph software (c). The same local reductions of cadherin staining in membranes of epithelial cells immediately adjacent to FN fibrils were observed using these alternative fixation and microscopy conditions compared to those used for Figure 5. Scale bar, 20 µm. 12