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Fig. 5. FGF9 induces sub-mesothelial mesenchymal proliferation independent of SHH signaling. (A-C) Two histologically distinct regions of mesenchymal cells are evident at E13.5. A population of sub-epithelial mesenchymal cells (SEM) wrap around the epithelial ducts, and a loose network of non-oriented sub-mesothelial mesenchymal cells (SMM) inhabit the area between the mesothelium and the SEM (A). In Fgf9^–/– lungs, the SMM is largely absent (B), whereas a large expanse of SMM is present in Fgf9^dox(48) lungs (C). (D-G) Whole-mount immunohistochemistry with anti-phosphohistone H3 (PH3) identified an increase in PH3-labeled cells in E11.5 lung explants incubated with FGF9 for 24 hours (E), compared with BSA-treated controls (D). Cyclopamine-only treated explants showed decreased overall proliferation (G). Explants incubated with both FGF9 and cyclopamine (F) appeared similar to FGF9-only treated explants (E) at low magnification. At higher magnification, cyclopamine-treated cultures (F',G') showed reduced cell proliferation in the SEM, whereas FGF9 increased proliferation in the SMM (E',F'). Explants treated with both cyclopamine and FGF9 (Cy/F9) have a combined effect, with increased PH3 labeling in the SMM and decreased labeling in the SEM (F9). (H-K) Staining for active caspase 3 demonstrates high levels of apoptosis in cyclopamine-treated explants (K), compared with relatively few labeled cells in BSA or FGF9-treated explants (H,I). A moderate amount of active caspase 3 staining is evident in the SEM of Cy/F9-treated explants, indicating a partial rescue of apoptosis by FGF9 (J). (L,M) Quantification of cell proliferation (PH3 labeled nuclei/unit area) in explants treated with FGF9 and cyclopamine (Cy) for 24 (L) and 48 (M) hours. Student t-test, ^***P<0.0001; ^**P<0.001; ^*P<0.01.

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