Anxa4 mediated airway progenitor cell migration promotes distal epithelial cell fate specification

Abstract

Genetic studies have shown that FGF10/FGFR2 signaling is required for airway branching morphogenesis and FGF10 functions as a chemoattractant factor for distal epithelial cells during lung development. However, the detail downstream cellular and molecular mechanisms have not been fully characterized. Using live imaging of ex vivo cultured lungs, we found that tip airway epithelial progenitor cells migrate faster than cleft cells during airway bud formation and this migration process is controlled by FGFR2-mediated ERK1/2 signaling. Additionally, we found that airway progenitor cells that migrate faster tend to become distal airway progenitor cells. We identified that Anxa4 is a downstream target of ERK1/2 signaling. Anxa4^-/- airway epithelial cells exhibit a "lag-behind" behavior and tend to stay at the stalk airways. Moreover, we found that Anxa4-overexpressing cells tend to migrate to the bud tips. Finally, we demonstrated that Anxa4 functions redundantly with Anxa1 and Anxa6 in regulating endoderm budding process. Our study demonstrates that ERK1/2/Anxa4 signaling plays a role in promoting the migration of airway epithelial progenitor cells to distal airway tips and ensuring their distal cell fate.

Figure 1

Airway progenitor cells that migrate faster tend to commit to distal airway cell fate. (A–C) Pregnant females carrying Shh^CreER/+; Rosa26-mTmG embryos were treated with tamoxifen (TAM) at E9.5 and lungs were imaged at E12.5 (A). Bud tip of the first lateral branch in right caudal lobe (RCd.L1) was imaged. Finally, two new bud tips were formed while the middle cells between two bud tips formed the cleft (B). After five-hour imaging, the tip cells (one cell was highlighted by blue) directly moved out and formed a new bud tip, while the middle cells (one cell was highlighted by orange) moved slower and tended to lag behind (C). Scale bar: 50 μm. (D–G) Cell track plots of tip cells (24 cells) and cleft cells (17 cells) after aligning their starting positions (D), showing that tip cells can migrate longer than cleft cells (D,E); The migration velocity analysis showed that tip cells migrate faster than cleft cells (F); and the tip cells have longer apical-to-basal cell length (G). Data are presented as mean ± SEM; n = 3 live image samples; ***p < 0.001; Student’s t-test. (H) WT lung bud at E12.5 was analyzed by E-cadherin (red) and p-ERK1/2 (green) immunostaining. The bud tips showed higher p-ERK1/2 staining compared to cleft cells. Scale bar: 25 μm. (I–K) Pregnant females carrying Shh^CreER/+; Fgfr2^F/+; Rosa26-mTmG (control) and Shh^CreER/+; Fgfr2^F/F; Rosa26-mTmG (Fgfr2^cKO) embryos were treated with tamoxifen (TAM) at E9.5 and lungs were sampled at E15.5 (I). Immunostaining using antibodies against GFP and Sox9 showed that less GFP⁺Sox9⁺ cells were detected in Fgfr2^cKO lung as compared to control lung (J,K). Data are presented as mean ± SEM; ***p < 0.001; Student’s t-test. Scale bar: 25 μm.

Figure 2

ERK1/2 signaling regulates the expression of Anxa4. (A) Diagram illustrating the method for mesenchyme-free lung endoderm culture. Whole lung at E11.5 was digested with trypsin on ice and then the mesenchyme was removed using fine forceps to get intact lung endoderm. The distal lung endoderm buds were dissected out, embedded in Matrigel and cultured in serum-free medium containing recombinant FGF10. (B) Images of cultured lung endoderm taken at different time points. There is no obvious bud formation at 24 h after culture. By 48 h, the lung endoderm starts budding and forms many buds. Scale bar: 200 μm. (C) Western blot of cultured lung endoderm explants at 0 h, 24 h and 48 h using antibodies against p-ERK1/2, total ERK1/2 and reference Actin. The phosphorylation level of ERK1/2 was increased significantly at both 24 h and 48 h. Full-length blots are presented in Supplementary Fig. s6. (D) Bud of cultured lung endoderm were analyzed by E-cadherin (red) and p-ERK1/2 (green) immunostaining. The bud tips (arrowhead) showed higher p-ERK1/2 staining compared to middle cells. Scale bar: 25 μm. (E) Whole-mount and section in situ hybridization of Anxa4 in WT lungs at E12.5. Anxa4 was highly expressed in the bud tip epithelial cells adjacent to FGF10 expressing site. Scale bar: 200 μm. (F,G) The expression level of Anxa4 in cultured lung endoderm explants at 0 h, 24 h and 48 h. The expression level of Anxa4 was increased significantly at both 24 h and 48 h as compared to 0 h and its expression level was inhibited by MEK inhibitor, PD0325901 (F). The whole mount in situ hybridization of Anxa4 confirmed that PD0325901 treatment could decrease the expression of Anxa4 (G). Data are presented as mean ± SEM, n = 3; **p < 0.01; ***p < 0.001; Student’s t-test. Scale bar: 100 μm.

Figure 3

Anxa4 promotes airway epithelial progenitor cell migration. (A) Pregnant females carrying Shh^CreER/+; Anxa4^F/+; Rosa26-mTmG (control) and Shh^CreER/+; Anxa4^F/F; Rosa26-mTmG (Anxa4^cKO) embryos were treated with tamoxifen (TAM) at E9.5 and lungs were imaged at E12.5. (B) Representative images of mosaic labeled control and Anxa4^cKO airway bud formation. One tip cells in control and Anxa4^cKO RCd.L1 buds are highlighted by blue and orange, respectively. Scale bar: 50 μm. (C–F) Cell track plots of tip cells in control lung (25 cells) and Anxa4^cKO lungs (16 cells) after aligning their starting positions (C), showing that Anxa4^cKO tip cells had shorter migration tracks and migration displacement than that of control tip cells (C,D); The migration velocity analysis showed that the migration velocity of Anxa4^cKO cells was decreased significantly (E) and the apical-to-basal cell length of Anxa4^cKO cells was similar to that of control cells (F). Data are presented as mean ± SEM; n = 3 samples per genotype; n.s., not significant; ***p < 0.001; Student’s t-test.

Figure 4

Anxa4 promotes distal airway epithelial cell fate commitment. (A) Pregnant females carrying Shh^CreER/+; Anxa4^F/+; Rosa26-mTmG (control) and Shh^CreER/+; Anxa4^F/F; Rosa26-mTmG (Anxa4^cKO) embryos were treated with tamoxifen (TAM) at E9.5 and lungs were analyzed at different time point from E12.5 to E15.5. (B,C) Lungs at different time points from E12.5 to E15.5 were double stained for antibodies against GFP (green) and Sox9 (red), or GFP (green) and Sox2 (red) (B). Note that the proportion of GFP⁺ cells in distal airways (Sox9⁺) of Anxa4^cKO lungs decreased over time as compared to that of control lungs, while the proportion of GFP⁺ cells in stalk airways (Sox2⁺) of Anxa4^cKO lungs increased over time (C). Data are presented as mean ± S.E.M, n = 4. n.s., not significant; *p < 0.05; **p < 0.01; Student’s t-test. Scale bar: 25 μm. (D,E) GFP (green) and Sox2 (red) staining in cultured whole lung endoderm explants infected with vector or Anxa4^OE lentivirus (D). GFP is a reporter used to indicate lentivirus-infected epithelial cells. More GFP⁺ cells were located in the bud tips (Sox2⁻) in Anxa4^OE lung endoderm explants than in control explants (E). Data are presented as mean ± S.E.M, n = 3. **p < 0.01; Student’s t-test. Scale bar: 25 μm. (F) A model illustrating the effect of loss of Anxa4 on epithelial cell behavior and cell fate. At E11.5, in both control and Anxa4^cKO lung, mosaic labeled GFP cells are evenly distributed along the airways. Four days later (E15.5), in newly formed airways of control lung, GFP⁺ cells are still evenly distributed along the airways; however, in the newly formed airways of Anxa4^cKO lung, GFP⁺ (Anxa4^−/−) cells tend to lag behind and contribute to the stalk airways.

Figure 5

Anxa4 functions redundantly with Anxa1 and Anxa6 in regulating endoderm budding process. (A) Relative expression level of Anxa1, Anxa2, Anxa3, Anxa5, Anxa6, Anxa7 and Anxa11 in control and Shh^Cre/+; Anxa4^F/F lungs. (B) The relative expression level of Anxa1, Anxa4 and Anxa6 at 48 h in cultured control lung endoderm treated with Scramble shRNA, or shAnxa1, or shAnxa6. The knockdown efficiency of shAnxa1 or shAnxa6 in cultured lung endoderm explants at 48 h was about 40%. Data are presented as mean ± S.E.M, n = 3. ***p < 0.001; Student’s t-test. (C) Representative images of cultured control or Anxa4^−/− lung endoderm explants treated with Scramble shRNA, shAnxa1, or shAnxa6. Scale bar: 200 μm. (D) Quantification of bud number of different lung endoderm groups in (B). Data are presented as mean ± S.E.M, n = 12. n.s., not significant; *p < 0.05; ***p < 0.001; Student’s t-test.