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Files in this Data Supplement:
Fig. S1. Wnt5a mis/overexpression does not affect epithelial cellular differentiation but alters smooth muscle of large vessels. (A) Nkx2.1 (a lung epithelial marker) is normally expressed in the lung epithelium at E16 (arrow) and in the budding epithelium (arrowhead). (B) At the same stage in Wnt5a-mis/overexpressed lungs, Nkx2.1 is expressed in the proximal epithelium but is decreased in the distal budding epithelium budding (arrowhead). (C-F) Surfactant Protein (SPB, normally expressed in differentiated alveolocytes) and Clara Cell protein 16 (CC16, expressed in differentiated proximal epithelial cells) expression at E20 is normal in the Wnt5a-mis/overexpressing lungs. (G-L) Actin smooth muscle (ASM) is expressed in both control and Wnt5a-mis/overexpressing atrial muscle in the airways (red arrow; G,H,J,L) and interstitial vasculature (red arrowhead; G,H,I,K). Wild-type pulmonary arterioles are well muscularized (arrowhead; G,I). Actin smooth muscle expression highlights the abnormally thin and abnormally developed muscularized vessel walls with mis/overexpression of Wnt5a (arrowhead; H,K). Actin smooth muscle localization shows larger atrial muscle bundles around the airways in Wnt5a-mis/overexpressing lungs than in wild-type lungs (red arrow; J,L).
Fig. S2. Wnt5a affects lung growth via inhibiting cell proliferation. (A-D) PCNA immunohistochemistry at E14 and E20 shows that the majority of proliferating cells are in the epithelium and the mesenchyme surrounding the airways (red square; A,C) in wild-type (WT) chick lungs. At E14, PCNA expression is reduced in RCAS-Wnt5a-infected lungs in both the epithelium and the mesenchyme (red square; B). At E20, PCNA expression in the RCAS-Wnt5a-infected lungs is detected only in the proximal epithelium and not in the distal epithelium (D). (E,F) To visualize PCNA (red) endothelial-positive cells, double staining with elderberry bark lectin (green) was performed (arrows). (G) Quantification of the number of PCNA-positive cells of the Wnt5a-mis/overexpressing lung compared with control at E14. Five photomicrographs were captured at 20× in both wild type and RCAS-Wnt5a-infected lungs and total numbers of cells in the field were counted. Student’s t-test using the mean of PCNA-positive cells over 100 total cells, with P<0.05 considered significant, error bars represent s.e.m.
Fig. S3. Expression of Bmp4, Fgf10 and Nkx2.1 in Wnt5a-mis/overexpressing lungs at E10. (A) In wild-type (WT) lungs, Bmp4 is detected in the distal epithelium (arrow) and its surrounding mesenchyme (arrowhead). (B) In RCAS-Wnt5a-injected lungs, Bmp4 is not detected in the pulmonary epithelium (arrow), but is widely expressed in the mesenchyme (arrowhead). (C) Wild-type lungs strongly express Fgf10 in the interstitial mesenchyme. (D) In Wnt5a mis/overexpressing-lungs, Fgf10 expression is markedly decreased. (E) Nkx2.1 is expressed in the pulmonary epithelium in wild type. (F) No changes in Nkx2.1 expression are detected in RCAS- wnt5a-injected lungs.
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