Fig 1.
Loss of Ext1 in the lung epithelium abrogates branching morphogenesis.
(A-F) The representative gross morphology of control lungs (A, C and E) and Ext1f/f; Shhcre mutant lungs (B, D and F) at the indicated developmental stages (E12.5, E14.5 and E18.5). The lungs from Ext1f/f; Shhcre mutant embryos show dilated branching tips and reduced branching number in all stages. The cranial (Cr), medial (Md), caudal (Cd), and accessory (Ac) lobes were seen in control lungs (A) at E12.5, while the right lung lobes were not properly patterned in Ext1f/f; Shhcre mutant lungs (B). The right lung lobes were specified at E14.5 and E18.5 but the Ac lobe was missing in Ext1f/f; Shhcre mutant lungs (C-F, arrows indicate Ac lobe). (G-L) The representative histology of control lungs (G, I and K) and Ext1f/f; Shhcre mutant lungs (H, J and L) at the indicated developmental stages (E12.5, E14.5 and E18.5). (M and N) Alcian Blue staining showing a failure of cartilage ring patterning of the trachea from Ext1f/f; Shhcre mutants.(O and P) The histological view of the Alcian Blue staining. Scale bars: A-F, 500μm; G-L, 200μm; M and N, 1mm;O and P, 100μm.
Fig 2.
Loss of Ext1 in the lung epithelium abrogates HS synthesis.
(A-H) Immunofluorescent staining for HS (3G10) and HS (10E4) in lungs at E12.5. Ext1 deletion abrogated the 3G10 signaling in the epithelium and basement membrane (A-D), and the 10E4 signaling in the epithelium (E-H). Scale bar: 50μm.
Fig 3.
Ext1 mutant lungs do not have epithelial differentiation and patterning defects but show maldevelopment of mesenchymal cells.
(A-D) Immunofluorescent staining for SOX2/SOX9 in control lungs and mutant lungs at E12.5 and E14.5. The proximal-distal patterning was maintained in Ext1f/f; Shhcre mutant lungs(A and B), but the mesenchymal SOX9+ progenitors in the tracheal region were less abundant (inserts in A and B), and SOX9+ mesenchymal cells were irregularly arranged at E14.5(C and D). (E and F) The α-SMA+ myoblast cells were seen in the bud stalk of control lungs, while they were restricted in a more proximal region in Ext1f/f; Shhcre lungs (arrow head in E and F). (G-N) Immunofluorescent staining for epithelial differentiation markers for Type I pneumocytes (T1α), Type II pneumocytes (SFTPC), Club cells (SCGB1A1), Ciliated cells (Acetylated-Tubulin, Ac-Tub), Mucus cells (MUC5AC) and basal cells (P63/K5) in control lungs and Ext1f/f; Shhcre lungs at E18.5. The epithelial differentiation was largely normal in Ext1f/f; Shhcre lungs. Scale bars: A and B, 200μm;C and D, 100μm; E andF, 200μm; G-N, 50μm.
Fig 4.
Ext1 mutant lungs exhibit a decrease in SHH-FGF10 signaling.
(A) qPCR analysis of key components of FGF signaling pathway in lungs from E14.5. The expression of Fgf10, Etv4, Spry2, Dusp6 and Bmp4 were increased, while the expression of Etv5 was decreased in Ext1f/f; Shhcre mutant lungs compared to control lungs. Data were presented as mean ± SEM. *p<0.05, n≥3. (B-E) Wholemount and section RNA ISH of Fgf10 in E12.5 lungs. The Fgf10 expression was increased in Ext1f/f; Shhcre lungs, and its expression domain was expanded instead of the split expression pattern seen in control lungs (arrowheads in B and D). (F and G) Immunofluorescent staining for phosphorylated -ERK in E12.5 lungs. The expression domain was expanded and the intensity was much stronger in Ext1f/f; Shhcre mutant lungs compared to control lungs, reflecting an increased FGF signaling activity. (H) qPCR analysis of key components of SHH signaling pathway in lungs from E14.5. The expression of Shh was not significantly changed while the SHH targets (Gli1, Ptch1 and Hhip1) were all decreased. Data were presented as mean ± SEM. *p<0.05, n≥3. (I-L) Wholemount and section RNA ISH of Shh in E12.5 lungs. The expression of Shh was not significantly altered in Ext1f/f; Shhcre mutant lungs. (M-P) Section RNA ISH of Ptch1 and Gli1 in E12.5 lungs. Ptch1 and Gli1 expression levels were significantly reduced. (Q-R) β-gal staining showing the expression of Gli1lacz allele in control lungs (Ext1f/w; Shhcre; Gli1lacz/+) and mutant lungs (Ext1f/f; Shhcre; Gli1lacz/+). The staining intensity was decreased in mutant lungs, indicating a decreased Gli1 expression. Scale bars: B, C, I and J, 500μm; D, E and K-R, 100μm; F and G, 50μm.
Fig 5.
Activation of SHH attenuates the increase of tip area and FGF signaling in Ext1 mutants.
(A-H) Representative images of explant cultures treated with DMSO or SAG for 48h. Treatment of Ext1f/f; Shhcre mutant lungs with SHH signaling agonist SAG rescued the tip dilation phenotype. The epithelium was outlined with grey line (E-H). (I -L) The corresponding enlarged view of the boxed area in (E-H). (M) Quantification of average tip area of the buds. Data were presented as mean ± SEM. *p<0.05, n = 3 for each group.(N-Q) Representative images of ISH showing Fgf10 expression of lung explant cultures treated with DMSO or SAG for 48h. Addition of SAG attenuated Fgf10 expression in Ext1f/f; Shhcre mutant lungs. (R-U) Representative images of pERK staining of lung explant cultures treated with DMSO or SAG for 48h, SAG treatment attenuated epithelial pERK staining in Ext1f/f; Shhcre mutant lungs, the epithelium was highlighted by white dashed lines. Scale bars: A-H, 500μm; N-Q,500μm; R-U, 50μm.
Fig 6.
Epithelial-derived HS is required for the production of biologically active form of SHH in the developing lung.
(A) Representative image of western blot analysis of SHH protein in E14.5 lungs. The protein level of SHH was not changed in Ext1f/f; Shhcre mutant lungs compared to control lungs. (B-G) Immunoflouorescent staining for SHH(5E1) and GFP in E12.5 lungs. The GFP expression is consistent with the Shh mRNA. The Ext1f/f; Shhcre mutant lungs presented less SHH particles in both the epithelium and mesenchyme. SHH particles were prone to localize on the basal side of the epithelium in control lungs, whereas the basal-localization of SHH was reduced in Ext1f/f; Shhcre mutant lungs.(H) Immunoprecipitation(IP) of E14.5 lung homogenates using 5E1 monoclonal antibody revealed reduced 5E1-immunoreactive SHH was produced in Ext1 mutant lungs compared with control lungs. The longer exposure of the 5E1 IP band was shown below the boxed area. C, Control; K, Ext1 mutant. (I) Model for the regulatory role of epithelial HS in lung branching morphogenesis. During normal lung branching, SHH produced by epithelium signals to mesenchyme to ensure a focal Fgf10 expression pattern, epithelial HS is required for the production of biologically active form (5E1 immunoreactive) of SHH ligand, possibly by controlling higher order processing. Loss of epithelial HS leads to a reduced production of biologically active form of SHH, thus causing a reduced SHH signaling and an elevated and expanded Fgf10 expression. Decreased SHH signaling and unrestricted FGF10 signaling lead to dilated branching tips and decreased branching number. Scale bar: B and E, 20μm.