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FGF9 and FGF10 activate distinct signaling pathways to direct lung epithelial specification and branching

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Science Signaling  03 Mar 2020:
Vol. 13, Issue 621, eaay4353
DOI: 10.1126/scisignal.aay4353
  • Fig. 1 FGF9 signals to the lung epithelium in the absence of functional mesenchymal FGFRs.

    (A) Quantification of bud number in response to bovine serum albumin (BSA) or FGF9 in lung explants from E11.5 embryos of the indicated genotypes and wild-type controls cultured for 48 hours. (B to E) Explants from wild-type control (B), Dermo1-βCat-KO (C), Dermo1-FGFR1/2-KO (D), and Fgf9−/− (E) mice cultured in media with BSA or FGF9. Two-way ANOVA, Sidak’s multiple comparisons test, n = 4 to 5 explants per genotype and condition. *P < 0.01; **P < 0.02; ***P < 0.0001. Scale bar, 200 μm.

  • Fig. 2 FGF9 signals to the lung epithelium through FGFR3.

    (A) Isolated E11.5 distal lung epithelium from Fgfr3−/+ and Fgfr3−/− embryos cultured in Matrigel in the presence or absence of FGF9 for the indicated periods of time. (B) Quantification of lung epithelial diameter in tissue from (A) at 48 hours. ANOVA with Tukey’s multiple comparison test. n = 3 epithelial explants per genotype. (C) Anterior views of gross dissections of Fgfr3−/+ and Fgfr3−/− lungs at E11.5. (D) Quantification of duct length and bud number in Fgfr3−/+ and Fgfr3−/− lungs at E11.5. Duct lengths and number of buds were counted in middle (M), caudal (C), and left (L) lobe regions. (E) Anterior views of gross dissections of Fgfr3−/+ and Fgfr3−/− lungs at E12.5. (F) Quantification of duct length and bud number in Fgfr3−/+ and Fgfr3−/− lungs at E12.5. The caudal lobe and left lobe duct lengths and bud numbers were counted. Bud length was analyzed using the Wilcoxon rank sum test, and bud number was analyzed using the Student’s t test. n = 9 to 10 lungs per genotype at E11.5 and 8 lungs per genotype at E12.5. *P < 0.05; ***P < 0.001. Scale bars, 100 (A) and 200 μm (C and E).

  • Fig. 3 FGF9 directs distal epithelial specification and differentiation in the pseudoglandular-stage lung.

    (A) Immunofluorescence imaging showing SOX2 and SOX9 distribution in whole E13.5 Fgf9−/+ and Fgf9−/− lungs. (B) Diagram of the distribution patterns shown in (A). (C and D) Immunostaining for SOX2 and SOX9 (C) and SFTPC (D) in control (Sftpc-rtTA) and SPC-FGF9-OE lung sections that were induced with doxycycline from E10.5 to E14.5 and harvested at E14.5. (E) Comparison of Sftpc expression (in situ hybridization) in Fgf9−/+ and Fgf9−/− lungs at E12.5. All images are representative of at least three embryos. Tr, trachea. Scale bars, 200 (A and E) and 100 μm (C and D).

  • Fig. 4 FGF9 can reverse epithelial specification without affecting epithelial identity.

    (A to E) SPC-FGF9-OE mice and Sftpc-rtTA control mice were induced with doxycycline (Dox) from E16.5 to E18.5, and lung sections were immunostained for SOX2 (A and B), SOX9 (C), NKX2-1 (D), and TRP63 (E). Additional controls included SPC-FGF9-OE mice that were not induced with doxycycline. All images are representative of at least three embryos. Tr, trachea. Scale bars, 100 μm.

  • Fig. 5 FGF9 and FGF10 have distinct effects on pseudoglandular-stage lung epithelium.

    (A) Anterior views of whole E12.5 lungs from mice expressing control (Sftpc-rtTA), FGF10 overexpression (SPC-FGF10-OE), or FGF9 overexpression (SPC-FGF9-OE) transgenes. All embryos were induced with doxycycline from E10.5 to E12.5. (B) Immunofluorescence showing SOX2 and SOX9 distribution. (C) Immunostaining showing SFTPC distribution. All images are representative of at least three embryos. Scale bars, 200 (A), 100 (B), and 50 μm (C).

  • Fig. 6 Conditional epithelial inactivation of Fgfr2 shows similar phenotypes to Fgf9 overexpression.

    (A) Experimental plan. All embryos were induced with tamoxifen (intraperitoneal injection of dam) at E10.5 and harvested at E13.5. (B) Frozen section of a lung from ShhCreER; ROSAtdTomato embryo showing lineage-labeled cells in the epithelial tips. (C to F) Anterior views of E13.5 whole control lungs [ShhCreER (C and D) or ShhCreER; Fgfr2f/+ (E and F)] and lungs lacking epithelial FGFR2 (SHH-FGFR2-CKO). Bright-field images (C) and immunostaining for SOX2 (D), SOX9 (E), and SFTPC (F) are shown. All images are representative of at least three embryos. Scale bars, 100 (B), 50 (C), and 200 μm (D to F).

  • Fig. 7 Preferential use of distinct cellular signaling pathways by FGF9 and FGF10.

    (A to C) E11.5 wild-type lung explants cultured for 48 hours in media alone, with FGF9 or with FGF10 as indicated, in the presence of dimethyl sulfoxide (DMSO) (A), the MEK inhibitor U01206 (B), or the PI3K inhibitor Ly294002 (C). (D to F) E11.5 wild-type lung explants cultured for 48 hours and treated with U01206 and Ly294002 (D and F) or DMSO (E). Bright-field image of whole-lung explant (D) and TUNEL-stained sections (E and F) are shown. All images are representative of at least three embryos. Scale bars, 200 μm (A to D) and 100 μm (E and F).

  • Fig. 8 Activation of AKT and ERK signaling by FGF9 and FGF10 in lung explants lacking mesenchymal FGFR1 and FGFR2.

    (A) Images of E11.5 whole-lung explants from Dermo1-FGFR1/2-KO mice cultured for 48 hours and treated with BSA, FGF9, or FGF10. (B and C) Sections of lung explants treated as in (A) immunostained for doubly phosphorylated (active) ERK (dpERK) (B) and phosphorylated (active) AKT (pAKT) (C). All images are representative of at least three embryos. Dashed lines outline ductal lumens. Scale bars, 200 (A) and 100 μm (B and C).

  • Fig. 9 Model for FGF9 and FGF10 signaling during pseudoglandular-stage lung development.

    FGF10 signaling through FGFR2b preferentially activates signaling through the MAPK ERK and is in balance with FGF9 signaling through FGFR3, which preferentially activates PI3K.

  • Table 1 Cell death and cell proliferation in lung epithelium.

    ATUNEL+ cells per 1000 epithelial cells
    E10.5E11.5E12.5
    Control10.6* ± 6.70.0 ± 00.6 ± 0.5
    Fgf9−/−1.7 ± 2.90.8 ± 1.40.9 ± 0.7
    P0.10.40.7
    BPHH3+ cells per 1000 epithelial cells
    E10.5E11.5E12.5
    Control65.8 ± 29.044.5 ± 32.752.6 ± 23.2
    Fgf9−/−81.1 ± 26.073.6 ± 8.931.2 ± 12.0
    P0.50.20.2

    *Means ± SD. Three control and three Fgf9−/− lungs were sectioned, and all epithelial cells in all sections from each lung were counted (294 to 1411 epithelial cells per lung).

    Supplementary Materials

    • stke.sciencemag.org/cgi/content/full/13/621/eaay4353/DC1

      Fig. S1. FGFR2 and FGFR3 are present in embryonic airway epithelium.

      Fig. S2. The absence of FGFR3 partially rescues the suppression of branching induced by FGF9.

      Fig. S3. Loss of FGF9 does not affect epithelial cell death or proliferation.

      Fig. S4. Overexpression of FGF9 or FGF10 in early-stage lung development does not affect epithelial cell identity.

      Fig. S5. FGFR3 directs distal epithelial specification and differentiation in pseudoglandular-stage lung.

      Fig. S6. Overexpression of FGF9 in late-stage lung development does not affect epithelial cell identity.

      Fig. S7. Inhibition of STAT3 has minimal effects on FGF9 and FGF10 signaling.

    • This PDF file includes:

      • Fig. S1. FGFR2 and FGFR3 are present in embryonic airway epithelium.
      • Fig. S2. The absence of FGFR3 partially rescues the suppression of branching induced by FGF9.
      • Fig. S3. Loss of FGF9 does not affect epithelial cell death or proliferation.
      • Fig. S4. Overexpression of FGF9 or FGF10 in early-stage lung development does not affect epithelial cell identity.
      • Fig. S5. FGFR3 directs distal epithelial specification and differentiation in pseudoglandular-stage lung.
      • Fig. S6. Overexpression of FGF9 in late-stage lung development does not affect epithelial cell identity.
      • Fig. S7. Inhibition of STAT3 has minimal effects on FGF9 and FGF10 signaling.

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