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First published online 29 April 2009
doi: 10.1242/dev.034629

Development 136, 1899-1907 (2009)
Published by The Company of Biologists 2009
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Multiple roles for Sox2 in the developing and adult mouse trachea

Jianwen Que1, Xiaoyan Luo1, Robert J. Schwartz2 and Brigid L. M. Hogan1,*

1 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
2 Center for Molecular Development and Disease, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, TX 77030, USA.


Figure 1
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  		Fig. 1. Expression of Sox2 in the trachea and use of Nkx2.5-Cre to delete Sox2. (A-F) Sox2 expression in the developing and adult trachea. (A) Immunohistochemical localization of Sox2 protein in the esophagus and trachea at E13.5. Lower panels are magnified views of trachea stained with anti-Sox2 and anti-p63 antibodies. (B-D) Immunohistochemical localization of Sox2 in the trachea at E15.5 (B), P0 (C) and adult (D) stages. In D, p63 and Sox2 colocalize in basal cells. (E) Fluorescence microscopy of the trachea and lung at E15.5. (F) RT-PCR shows that Sox2 is exclusively expressed in the epithelium, and insert shows an E11.5 trachea immunostained with anti-Sox2 antibody. (G-I) Nkx2.5-Cre expression in the early foregut (stages E9.5-12.5) as detected in R26R embryos through X-gal staining. Left and right panels show the whole-mount X-gal staining and a section of these stained samples, respectively. The dashed line delineates where the section is cut. (J,K) Immunostaining with anti-Sox2 antibody on early foregut and its derivatives in Nkx2.5-Cre;Sox2+/COND (Sox2+/{Delta}) controls and Nkx2.5-Cre;Sox2COND/COND (Sox2 {Delta}/{Delta}) mutants. Nuclei are counterstained with DAPI (blue). Es, esophagus; Tr, trachea; Lu, lung; Fg, foregut; A, anterior; P, posterior; D, dorsal; V, ventral; M, mesenchyme; E, epithelium. Scale bars: 100 µm.

 

Figure 2
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  		Fig. 2. Abnormal development of larynx and trachea in Nkx2.5-Cre;Sox2COND/COND mutants. (A) Gross morphology of lung and trachea in control (left) and mutant (right) mice. (B,C) Short trachea (bracket) and long main bronchus in P0 mutants (n=5; *P<0.01). (D-F) Abnormal differentiation of mesenchymal cells in the mutant trachea. (D) Alcian blue staining reveals abnormal laryngeal and tracheal cartilage (bracket). Note the reduced number of incomplete cartilage rings in mutants (arrows). (E,F) Immunostaining with anti-SMA antibody to show ectopic expression of smooth muscle actin in the ventral side of the trachea in mutants. (G-I) Downregulated Shh signaling in the early foregut of mutants. (G,H) Immunohistochemical localization of Shh protein in the unseparated foregut. Nuclei are counterstained with DAPI (blue). (I) Semi-quantitative RT-PCR for expression in control and mutant tracheas of several genes involved in the development of laryngeal and tracheal cartilage. Th, thyroid; Cr, cricoid; Fg, foregut; Fp, floor plate; Nc, notochord; D, dorsal; V, ventral. Scale bar: 100 µm.

 

Figure 3
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  		Fig. 3. Decreased number of p63-positive cells in Nkx2.5-Cre;Sox2COND/COND mutants at different developmental stages. (A-D) Immunohistochemical staining with anti-p63 antibody in control (A,C) and mutant (B,D) tracheas at E15.5 (A,B) and E18.5 (C,D). Nuclei are counterstained with methyl green. (E,F) Immunostaining with anti-keratin 5 (green) and anti-p63 (red) antibodies in the P0 trachea. (G) Quantification of p63-positive cells in tracheas at E13.5, E15.5, E18.5 and P0. At each stage, three individual tracheas were included. Scale bars: 50 µm.

 

Figure 4
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  		Fig. 4. Abnormal differentiation of tracheal and lung epithelial cells at P0. (A,B) Decreased number of ciliated (acetylated {alpha}-tubulin-positive, green) and Clara (Scgb1a1-positive, red) cells in mutant trachea (B), compared with control (A). (C-H) Increased number of mucus-producing cells in mutant tracheas visualized by PAS (C,D), alcian blue (E,F) and anti-Muc5ac (G,H) staining. (I,J) Immunostaining with anti-Foxa2 antibody. (K) Quantification of the percentage of the different cell lineage to total epithelium in the trachea. Numbers are obtained from analyzing ten sections at the same level from each of three individual tracheas from both Nkx2.5-Cre;Sox2+/COND controls and Nkx2.5-Cre;Sox2COND/COND mutants. PAS, periodic acid-Schiff. Scale bar: 50 µm.

 

Figure 5
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  		Fig. 5. Sox2 regulates the proliferation of adult tracheal epithelium in culture. (A) Representative colonies present on culture day 10 express either p63 or Scgb1a1 (CC10; inserts). (B) Representative colony present on day 15 that co-expresses p63 and Sox2. (C-E) Efficient deletion of Sox2 in the adult trachea of CMV-CreER;Sox2COND/COND mutants. (C) Timeline of the Tamoxifen (Tmx) treatment protocol. (D,E) Immunohistochemistry of Sox2 in wild-type (D) and mutant (E) tracheas. (F-J) Sox2 deletion reduces proliferation of tracheal epithelium isolated from wild type (F,H) or conditional mutants (G,I) and cultured for 5 days. H and I are representative colonies from F and G, respectively. (J) Number of colonies per 35 mm culture dish. Data are from three independent experiments. (K-N) Basal cell colonies present in controls (K), but not in mutants (L), in 15-day cultures. M is a representative colony from K. N is from L, showing no surviving colony in this 35 mm culture dish. Insert in M shows representative colony stained with anti-p63. Colonies in F,G and K are stained with Wright blue. Scale bars: 50 µm.

 

Figure 6
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  		Fig. 6. Sox2 is required for homeostasis of the adult tracheal epithelium. (A) Timeline of the Tamoxifen treatment. (B,C) Immunohistochemistry with anti-Sox2 antibody. (D-E') Hematoxylin and eosin (H&E) staining of adult trachea. D' and E' are magnified views of boxed region in D and E, respectively. (F-G') Immunohistochemistry staining with anti-p63 antibody. (H,I) Immunostaining with anti-Scgb1a1 (red) and anti-acetylated {alpha}-tubulin (green) antibodies. (J-L) Decreased proliferation of tracheal epithelium after loss of Sox2 protein. (J,K) Immunohistochemistry staining with anti-Ki67 antibody. (L) Ratio of Ki67-positive epithelium along total tracheal epithelium. Four mutant and control tracheas are included. Scale bars: 100 µm.

 

Figure 7
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  		Fig. 7. Sox2 is required for proliferation and differentiation of the regenerating tracheal epithelium in a SO2-injured mouse model. (A) Timeline of the Tamoxifen and SO2 treatment protocol. (B-C'') H&E staining of regenerated trachea. B' (ventral), B'' (dorsal) and C' (ventral), C'' (dorsal) are magnified views of the boxed region in B and C, respectively. Arrowheads indicate epithelium. Note that in C there are numerous inflammatory cells within the lumen, asterisks in C indicate smooth muscles bulging into the lumen. (D-I) Immunohistochemistry of the regenerated epithelium with antibodies against Ki67 shows more proliferative cells (arrowheads) in control (D) versus mutant (E) tracheas. D' and E' are magnified views of the boxed region in D and E, respectively. (F,G) Anti-keratin 5 (green) and anti-p63 (red); (H,I) anti-Scgb1a1 (red) and anti-acetylated {alpha}-tubulin (green). D, dorsal; V, ventral. Scale bars: 50 µm.

 

Figure 8
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  		Fig. 8. Model for the roles of Sox2 in developing and adult trachea. At embryonic stage, Sox2 is required for the differentiation of tracheal mesenchyme and epithelium. Conditional deletion of Sox2 results in a decreased number of basal, Clara and ciliated cells, and more mucus-producing cells in the developing trachea. In the adult, Sox2 regulates both proliferation and differentiation at steady state and following injury. Sox2 deletion leads to reduced proliferation of tracheal epithelium both in vivo and in vitro. Loss of Sox2 also impairs the regeneration process of the trachea after inhaled SO2-induced injury.

 

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