DEV029249 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Transgene activation occurs throughout E18.5 lung epithelium and GFP expression does not alter lung morphology or differentiation. (A) An SPC-Cre driver was crossed to the Rosa26 reporter mouse to examine SPC-Cre transgene activity. β-gal staining (blue) was found throughout the epithelium of E18.5 lungs. Tissues outside the lung are not labeled. (B) Immunohistochemistry for GFP demonstrates that transgenic GFP expression is mosaic in SPC-Cre; NotchIC lung. Arrowheads indicate regions lacking GFP transgene activity and arrows indicate GFP-positive cysts. Notably, transgene activity is present proximally in the airways of the lung and distally in cystic regions. Moreover, transgene activation is associated with the formation of cystic dilations. (C-H′′) An SPC-Cre driver was crossed to a Rosa-lox-STOP-lox-GFP reporter mouse to examine GFP distribution and any phenotypic effects of GFP expression. Whole-mount florescence microscopy of E11.5 lungs reveals that the Cre-mediated recombination and GFP expression occur throughout the airway early enough to allow for GFP accumulation by E11.5. E18.5 immunohistochemistry (D-H) reveals GFP expression throughout the tracheal epithelium (D), the bronchiolar epithelium, and in a subset of distal alveolar cells (D′). GFP expression does not induce mucous metaplasia (red) (E). (F-H) E18.5 immunohistochemistry of Cre-negative GFP-negative lungs and Cre-positive GFP-positive lungs reveals no difference in expression patterns of differentiation markers. The distribution of type 2 cells, identified by SP-C staining, is unaltered between GFP-negative (F) and GFP-positive (F′,F′′) pups. The distribution of Clara cells, identified by CC10 staining, is unaltered between GFP-negative (G) and GFP-positive (G′,G′′) pups. The distribution of ciliated cells, identified by EphA7 staining, is unaltered between GFP-negative (H) and GFP-positive (H′,H′′) pups. Scale bars: 4.0 mm in A; 50 µm in B; 0.5 mm in C; 100 µm in D-H.

• Supplemental Figure S2 -

  Fig. S2. Clara cell patterning and numbers, and HNF-3β expression patterns are unchanged in NotchIC transgenic lungs. (A-D) CC10 patterning is unchanged in E18.5 NotchIC transgenic trachea. Immunohistochemistry of E18.5 trachea (A) demonstrates that GFP-positive NotchIC transgenic trachea (B) stain normally for the Clara cell marker CC10 (C). (D) Merge reveals that cells are double positive for both GFP and CC10. Inset in D is of control stain for CC10 in upper airway. Arrowheads indicate cartilaginous rings. (E-H) CC10 patterning is unchanged in airways near the BADJ. Immunohistochemistry of (E) E18.5 small airways adjacent to the BADJ (E) demonstrates that GFP-positive NotchIC transgenic airways (F) stained for CC10 in a normal pattern respecting the BADJ boundary (G). (H) Merge reveals that double-positive GFP and CC10 cells are present throughout the pre-BADJ epithelium. The inset shows normal control lung. A, airway; s, saccule. The arrow points to the BADJ. (I) Quantification of CC10-positive cells in E18.5 wild-type and transgenic lungs demonstrates that the proportions of Clara cells are unchanged in NotchIC transgenic airway epithelium. (J) Immunohistochemistry of E18.5 NotchIC transgenic airways shows that mucous cells co-stain for CC10. Arrows indicate double-positive cells. (K-L′′) HNF-3β expression is not reduced in metaplastic airways. E18.5 immunohistochemistry of wild-type (K) airways shows broad epithelial expression of HNF-3β and rare Muc5-positive cells. E18.5 immunohistochemistry of NotchIC transgenic airways reveals unchanged broad epithelial expression of HNF-3β (L) and increased expression of Muc5 (L′). The inset in L′ is a higher magnification view of the boxed region and depicts nuclear HNF-3β in airway epithelial cells. (L′′) Images merged. Scale bars: 100 µm.

• Supplemental Figure S3 -

  Fig. S3. Excess mucous cells express multiple markers of mucous cell differentiation. (A,B) Alcian Blue staining of E18.5 wild-type (A) and NotchIC transgenic (B) airway epithelium. Arrowheads indicate mucin-positive cells. (C-E) E18.5 immunohistochemistry of NotchIC airway staining of Muc5 (C) and Muc1 (D). Merged image (E) shows that mucous cells co-label for Muc5 and Muc1. Arrows indicate clusters of double-positive cells.