Epithelial heparan sulfate regulates Sonic Hedgehog signaling in lung development
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.