DEV034629 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Expression pattern of Sox2, p63 Nkx2.5-Cre in the early trachea and lung. (A) Immunohistochemistry of Sox2 and p63 in E15.5 trachea. Note the colocalization of p63 and Sox2 in the bottom layer of the pseudostratified epithelium. (B) Immunohistochemistry of Sox2 in the adult trachea. Note the different level of Sox2 protein in the epithelial cells. (C) Immunohistochemistry of Sox2 and p63 in the adult trachea. Sox2 is expressed in most but not all (arrowhead) p63-positive basal cells. (D) Nkx2.5-Cre expression in both trachea and lung of Nkx2.5-Cre;R26R as detected by X-gal staining at E16.5. Left panel is a whole-mount view; middle and right panel are sections. D, dorsal; V, ventral. Scale bars: 50 µm.

• Supplemental Figure S2 -

  Fig. S2. Abnormal patterning of the anterior foregut and its derivatives after Sox2 deletion. (A,B) Laryngeal and tracheal cartilage visualized by alcian blue staining. (B) EA/TEF formation in 10% of the P0 Nkx2.5-Cre;Sox2^COND/COND mutants. Arrowhead indicates EA and arrow indicates the site where the TEF joins the abnormal trachea. Note the abnormal cartilages in the mutant. (C,D) Immunohistochemistry of sections of E15.5 trachea with antibodies against smooth muscle actin (SMA) and Sox9. (E,F) Immunohistochemistry of FoxA2. (G) Costaining mutant trachea with FoxA2 and alcian blue. (H) Mucin-containing vesicles in tracheal epithelium visualized by transmission electron microscopy. Note that the vesicles are present at the apical region of the epithelium in insert. The insert is a magnified view of the boxed region, and the asterisks indicate mucus-producing cells. (I,J) Immunostaining with antibody against acetylated tubulin (green) and Scgb1a1 (red) in P0 lung. Th, thyroid; Cr, cricoid; Tr, trachea; Es, esophagus; EA, esophageal atresia; TEF, tracheoesophageal fistula; V, ventral; D, dorsal. Scale bars: 100 µm in D, 50 µm in F,G,J.

• Supplemental Figure S3 -

  Fig. S3. Unaltered epithelial proliferation after Sox2 deletion in the trachea at E18.5 and P0. (A-D) Representatives of immunohistochemical localization of BrdU in Nkx2.5-Cre;Sox2^+/COND controls (A,C) and Nkx2.5-Cre;Sox2^COND/COND mutants (B,D). Nuclei are counterstained with hematoxylin. Scale bars: 100 µm. (E) Quantification of proliferation index (PI; BrdU-positive epithelium/total epithelium). Each bar represents the average of the PI obtained from three independent samples.

• Supplemental Figure S4 -

  Fig. S4. In vitro culture of the adult tracheal epithelium with or without the Sox2 gene. (A-C) Representative colonies formed in 5-day culture of wild-type tracheal epithelium. Colonies are immunostained with p63 (red), Scgb1a1 (green) and with DAPI (blue). (D) Timeline of the Tamoxifen treatment. (E) Section of X-gal-stained CMV-CreER;R26R trachea after Tmx injection. (F,G) Immunohistochemistry staining with anti-p63 antibody; (H,I) Immunostaining with Scgb1a1 (red) and acetylated tubulin (green) antibodies. (J,K) Immunostaining with phosphorylated Histone H3 antibody. (L-O) Conditional deletion of Sox2 decreases proliferation of tracheal epithelium indicated by immunohistochemistry of BrdU (L,M). Each bar in O represents the average of the proliferation index obtained from three independent samples. Scale bars: 50 µm.