<i>Ext1</i> mutant lungs exhibit a decrease in SHH-FGF10 signaling.

<p>(A) qPCR analysis of key components of FGF signaling pathway in lungs from E14.5. The expression of <i>Fgf10</i>, <i>Etv4</i>, <i>Spry2</i>, <i>Dusp6</i> and <i>Bmp4</i> were increased, while the expression of <i>Etv5</i> was decreased in <i>Ext1</i>^<i>f/f</i><i>; Shh</i>^<i>cre</i> mutant lungs compared to control lungs. Data were presented as mean ± SEM. *<i>p</i><0.05, n≥3. (B-E) Wholemount and section RNA ISH of <i>Fgf10</i> in E12.5 lungs. The <i>Fgf10</i> expression was increased in <i>Ext1</i>^<i>f/f</i><i>; Shh</i>^<i>cre</i> 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 <i>Ext1</i>^<i>f/f</i><i>; Shh</i>^<i>cre</i> 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 <i>Shh</i> was not significantly changed while the SHH targets (<i>Gli1</i>, <i>Ptch1</i> and <i>Hhip1</i>) were all decreased. Data were presented as mean ± SEM. *<i>p</i><0.05, n≥3. (I-L) Wholemount and section RNA ISH of <i>Shh</i> in E12.5 lungs. The expression of <i>Shh</i> was not significantly altered in <i>Ext1</i>^<i>f/f</i><i>; Shh</i>^<i>cre</i> mutant lungs. (M-P) Section RNA ISH of <i>Ptch1</i> and <i>Gli1</i> in E12.5 lungs. <i>Ptch1</i> and <i>Gli1</i> expression levels were significantly reduced. (Q-R) β-gal staining showing the expression of <i>Gli1</i>^<i>lacz</i> allele in control lungs (<i>Ext1</i>^<i>f/w</i><i>; Shh</i>^<i>cre</i><i>; Gli1</i>^{<i>lacz/+</i>}) and mutant lungs (<i>Ext1</i>^<i>f/f</i><i>; Shh</i>^<i>cre</i><i>; Gli1</i>^{<i>lacz/+</i>}). The staining intensity was decreased in mutant lungs, indicating a decreased <i>Gli1</i> expression. Scale bars: B, C, I and J, 500μm; D, E and K-R, 100μm; F and G, 50μm.