Haas M , Gómez Vázquez JL , Sun DI , Tran HT , Brislinger M , Tasca A , Shomroni O , Vleminckx K , Walentek P .

K99 HL127275 NHLBI NIH HHS

             lung basal cell differentiation

	Graphical Abstract
	Figure 1. Wnt/β-Catenin Signaling Is Active in MCCs and Basal Progenitors (A) Analysis of Wnt/β-catenin activity in the X. laevis mucociliary epidermis using the pbin7LEF:dGFP reporter line (green). Nuclei are stained by DAPI (blue). Red arrowheads indicate GFP-positive cells in the outer epithelial layer. Dashed lines outline the epidermal layers. Embryonic stages (st. 8–33) are indicated. (B) Analysis of Wnt/β-catenin activity in the mouse developing airway mucociliary epithelium using the TCF/LEF:H2B-GFP reporter line (green). Nuclei are stained by DAPI (blue) and MCCs are marked by acetylated-α-tubulin (Ac.-α-tubulin, magenta) staining. Dashed lines outline the epithelium. Embryonic (E14.5–18.5) and post-natal (P1–7) stages are indicated. (C) MTEC ALI cultures generated from the TCF/LEF:H2B-GFP reporter line (green) and cultured up to 21 days (D21) revealed Wnt signaling activity throughout the different stages. n = 3 cultures per time point. MTECs were stained for Ac.-α-tubulin (blue) and CC10 (magenta). Only primary cilia were present at days 1 and 4 (D1 and 4), and MCCs could be detected from day 7 (D7) onward. (D) En-face imaging of the mature Xenopus epidermis at st. 33 shows elevated signaling levels (green) in MCCs (Ac.-α-tubulin, blue). SSCs (5HT, blue). Cell membranes are visualized by actin staining (magenta). (E) Immunostaining for Ac.-α-tubulin (magenta) and nuclei (DAPI, blue) shows high levels of Wnt signaling (green) in cells with BC morphology and intermediate signaling levels in differentiating MCCs. (F) Single confocal sections of MTECs at ALI day 7 (D7) show MCCs (Ac.-α-tubulin, blue) with variably elevated signaling levels (green), Club cells (CC10, magenta) without Wnt activity, and non-MCC/non-Club cells with elevated Wnt signaling levels at the base. Related to Figure S1.
	Figure 2. Wnt/β-Catenin Regulates MCC Numbers and DeltaN-tp63 Expression (A) Morpholino-oligonucleotide (MO) knockdown of β-catenin in Xenopus increases MCC numbers (Ac.-α-tubulin, green), but MCCs present fewer and shorter cilia than controls (ctrl.). Actin staining (magenta). Insets indicate locations of magnified areas I–IV. (B) Quantification of results depicted in (A). Mann Whitney test, ∗∗∗ p ≤ 0.001. (C) ISH reveals increased MCC numbers (foxj+ cells) after unilateral knockdown of β-catenin (β-catenin MO). Controls (n = 13); β-catenin MO (n = 22). (D) ISH reveals decreased DeltaN-tp63 expression after β-catenin MO. Controls (n = 27); β-catenin MO (n = 37). In (B) and (C), the injected side is indicated by red arrowheads. Dorsal views are shown in upper panels, lateral views are shown in the lower panels, and magnified views are depicted in white boxes.
	Figure 3. ΔN-tp63 Mediates Wnt/β-Catenin-Induced Inhibition of Cell Fate Specification in Xenopus (A–D) BIO treatments from st. 8–17 or st. 8–30. DMSO was used as vehicle control. (A) Confocal imaging shows Wnt/β-catenin signaling activation (green) and thickening of the epidermis in BIO-treated embryos. Nuclei (DAPI, blue). DMSO (n = 2); BIO (n = 2). (B) In situ hybridization (ISH) shows increased intensity and thickness of the ΔN-tp63 expression domain in BIO-treated whole mounts (upper row) and transversal sections (bottom row). (C) BIO treatment reduces MCC (Ac.-α-tubulin, green), Ionocyte (no staining, black), and SSC (large vesicles, peanut agglutinin [PNA] staining, magenta) numbers in confocal micrographs. Actin staining (green). ΔN-tp63 MO in controls leads to increased MCCs and Ionocytes, while ΔN-tp63 MO in BIO-treated embryos rescues MCC and Ionocyte formation. (D) ISH reveals reduced MCC numbers (foxj+ cells) after BIO treatment, while unilateral knockdown of ΔN-tp63 in control treated embryos leads to more foxj+ cells and rescues foxj+ cell numbers in BIO-treated embryos. DMSO (n = 5); BIO (n = 7); DMSO+ΔN-tp63 MO (n = 5); BIO+ΔN-tp63 MO (n = 4). Injected side is indicated by red arrowhead. Dashed lines indicate epidermal area in BIO-treated embryos. (E) Overexpression of gfp-ΔN-tp63 mRNA leads to nuclear localization of the protein (green) and reduced MCC (Ac.-α-tubulin, blue) numbers at st. 30. Actin staining (magenta). Differentiated MCCs in injected specimens show no nuclear GFP signal (asterisks), indicating that they were not targeted. Magnified areas I–II are indicated. (F) ISH for foxj1 at st. 17 shows reduced MCCs in ΔN-tp63 mRNA injected embryos. Related to Figures S2 and S3.
	Figure 4. Wnt/β-Catenin Signaling Inhibits Differentiation and Promotes Stemness in Human BCs (A–F) Human immortalized BC (BCIs) kept in air-liquid interface (ALI) culture for up to 4 weeks. Human recombinant R-spondin2 (RSPO2) was used to activate Wnt/β-catenin signaling starting at ALI day 7 (D7). n = 3 cultures per time point and treatment. (A) Confocal imaging of specimens stained for Ac.-α-tubulin (MCCs, blue), CC10 (Club cells, green), and Actin (cell membranes, magenta) show reduced MCC and Club cell numbers in RSPO2-treated cultures. (B) RSPO2 does not reduce the number of ΔN-TP63+ (green) cells. Nuclei (DAPI, blue). (C) Quantification from (A) and (B). Mann Whitney test, not significant, ns (p > 0.05); ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. (D) Quantitative real-time PCR. Expression levels are depicted relative to stage controls. RSPO2 reduces expression of MCC (FOXJ1, MCIDAS) and Club cell (SCGB1A1) markers but increases expression of BC markers (ΔN-TP63, KRT5). Student’s t test, not significant, ns (p > 0.05); ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. (E and F) Optical orthogonal sections of confocal images. RSPO2-treated cultures display increased epithelial thickness and cells staining for BC markers ΔN-TP63 (green, in E) and KRT5 (green, in F). Related to Figure S4.
	Figure 5. Knockdown of ΔN-tp63 Stimulates MCC and Ionocyte Specification at the Expense of BC and SSCs in Xenopus (A–D) Analysis of cell type composition by confocal microscopy and staining for MCCs (Ac.-α-tubulin, green), Ionocytes (no staining, black), SSCs (large vesicles, PNA staining, magenta), and Goblet (outer-layer) cells (small granules, PNA staining, magenta) at st. 34 (A) and st. 30 (B). Actin staining (green). (A) ΔN-tp63 MO increases MCC and Ionocyte numbers but reduces numbers of SSCs. (B) BIO application from st. 11 does not affect MCC and Ionocyte numbers but prevents specification of SSCs. (C and D) Quantification from (A) and (B), respectively. Mann Whitney test, not significant, ns (p > 0.05); ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. (E) RNA sequencing at st. 10, 17, and 25 on Xenopus mucociliary organoids comparing controls to ΔN-tp63 MO injected. n = 3 per stage and treatment. Heatmap and hierarchical clustering of log2 fold changes (fcs) in cell type gene expression in ΔN-tp63 morphants relative to controls. “Upregulated” clusters (red); “downregulated” cluster (blue). Related to Figure S5.
	Figure 6. Inhibition of Wnt/β-Catenin Signaling Transiently Reduces Stemness and Promotes Differentiation in Human BCs (A–F) BCIs in ALI culture for up to 4 weeks. Human recombinant DKK1 (DKK1) was used to inhibit Wnt/β-catenin signaling starting at ALI day 7 (D7). (A) Confocal imaging of specimens stained for Ac.-α-tubulin (MCCs, blue), CC10 (Club cells, green), and Actin (cell membranes, magenta) shows moderately increased MCC and Club cell numbers after DKK1 treatment. (B) DKK1 leads to a transient decrease in BCs but does not lead to loss of ΔN-TP63+ (green) cells. Nuclei (DAPI, blue). (C) Quantification from (A) and (B), respectively. Mann Whitney test, not significant, ns (p > 0.05); ∗∗∗p ≤ 0.001. (D) Quantitative real-time PCR expression levels are depicted relative to stage controls. DKK1 increases expression of MCC (FOXJ1, MCIDAS) and to a lesser extent Club cell (SCGB1A1) markers but without reduction of BC markers (ΔN-TP63, KRT5). Student’s t test, not significant, ns (p > 0.05); ∗p ≤ 0.05; ∗∗p ≤ 0.01. (E and F) Optical orthogonal sections of confocal images after staining for BC markers ΔN-TP63 (green, in E) and KRT5 (green, in F). Related to Figure S5.
	Figure 7. Wnt/β-Catenin-Induced Increase in BCs and Loss of Epithelial Differentiation Are Reversible (A and B) In Xenopus, BIO treatments from st. 8–17 or st. 8–30 inhibit differentiation as compared to DMSO treated controls, but the epithelium can regenerate after removal of BIO and recovery until st. 33 (A) or st. 25 (B). (A) BIO treatment reduces MCC (Ac.-α-tubulin, green), Ionocyte (no staining, black), and SSC (large vesicles, PNA staining, magenta) numbers in confocal micrographs at st. 28, which recover after regeneration until st. 33. Actin staining (green). (B) ISH shows reduction in foxj1 expressing cells and an increase in ΔN-tp63 expression in BIO-treated whole mounts (upper row) and transversal sections (bottom row) at st. 17, which both recover after regeneration until st. 25. DMSO (n = 5); BIO st. 17 (n = 5); DMSO st. 25 (n = 5); BIO+recovery st. 25 (n = 5). (C) Confocal imaging of specimens stained for Ac.-α-tubulin (MCCs, blue), CC10 (Club cells, green), and Actin (cell membranes, magenta) show reduced MCC and Club cell numbers in RSPO2-treated cultures from ALI D7 to D21 but regeneration of MCCs and Club cells at ALI D28. n = 3 cultures per time point and treatment (upper panels). No loss of ΔN-TP63+ (green) BCs is observed in these experiments (bottom panels). Nuclei (DAPI, blue). (D and E) Optical orthogonal sections of confocal images. RSPO2-treated cultures display normalized epithelial thickness and staining for BC markers ΔN-TP63 (green, in D) and KRT5 (green, in E) after regeneration at ALI D28.
	Supplemental Figure S1: Wnt/β-Catenin signaling in the Xenopus mucociliary epidermis and in airway cells of the mouse, Related to Figure 1 (A-B) Reconstructed confocal tile-scans of sectioned Xenopus and mice tissues. (A) Analysis of Wnt/β-Catenin activity in X. laevis whole embryo sections using the pbin7LEF:dGFP reporter line (green; signal was enhanced by anti-GFP immunofluorescent staining). Nuclei are stained by DAPI (blue). Embryonic stages (st. 8-33) are indicated. Orientation of embryos is indicated in the upper-right corner of color-merged micrographs. an = animal; veg = vegetal; d = dorsal; v = ventral; l = left; r = right. Embryonic structures are indicated. DL = dorsal blastopore lip; PSM = presomitic mesoderm; BP = blastopore; SM = somatic mesoderm; NOT = notochord; M = muscles; E = eye; CG = cement gland; NT = neural tube. Magnified areas are highlighted in color-merged micrographs. (B) Analysis of Wnt/β-Cateninactivity in the developing mouse airway mucociliary epithelium using the TCF/LEF:H2B-GFP reporter line (green). MCCs are marked by Acetylated-alpha-tubulin (Ac.-alpha-tubulin, blue) and Club cells are marked by CC10 (magenta) staining. Dashed lines outline the epithelium. Embryonic (E14.5-18.5) / post-natal (P1-7) stages are indicated. Magnified areas are highlighted. Note that GFP signals are largely restricted to epithelia of bronchi and bronchioles. (C) En-face imaging of the Xenopus epidermis at st. 25 shows elevated signaling levels (green) in intercalating MCCs (Ac.-alpha-tubulin, blue) and Ionocytes, but not in outer layer-derived Goblet cells. Cell membranes are visualized by Actin staining (magenta). (D) Magnified view of a P1 Wnt-reporter (green) bronchiole stained for Ac.-alpha-tubulin (magenta) and DAPI (blue) depicting multiple differentiating MCCs with elevated Wnt signaling. Related to Figure 1E. (E) MTEC ALI cultures generated from the TCF/LEF:H2B-GFP reporter line (green) and cultured up to 21 days (D21) revealed Wnt signaling activity throughout the different stages. N=3 cultures per timepoint. Optical orthogonal sections of confocal images of Wnt reporter (green) MTECs at the indicated ALI day stained for Actin (magenta), nuclei (DAPI, blue).
	Splemental Figure S2: Analysis and quantification of gain of Wnt/β-Catenin and δN-tp63 functions in Xenopus, Related to Figure 3. (A) Quantification of results depicted in Figure 2B. (B,C) Quantification of results depicted in Figure 2C. In (A,C) Mann Whitney test, not significant, ns = P>0.05; * = P≤0.05; ** = P≤0.01; *** = P≤0.001. (D) ISH for cell type markers foxj (MCCs), foxi1 (Ionocytes) and foxa1 (SSCs) in embryos at after BIO incubation during st. 8-17, DMSO control treatment, δN-tp63 MO injections, and BIO + δN-tp63 MO. All embryos are depicted in ventral view with anterior up. Related to Figure 2D. (E) Quantification of ISH results from (D). Chi-squared test, not significant, ns = P>0.05; * = P≤0.05; ** = P≤0.01; *** = P≤0.001.
	Supplemental Figure S3: Analysis and quantification of δN-tp63 gain-of-function effects in Xenopus, Related to Figure 3 (A) Quantification of results depicted in Figure 2E. Mann Whitney test, *** = P≤0.001. (B) ISH for cell type markers foxj (MCCs), foxi1 (Ionocytes) and foxa1 (SSCs) in embryos injected unilaterally with δN-tp63 mRNA. Injected side is indicated by red arrowheads. Dorsal views are shown in upper panels, lateral views are shown in the lower panels. (C) Quantification of results depicted in (B). Chi-squared test, *** = P≤0.001. (D-G) Confocal images of embryos injected with GFP- δN-tp63 -GR mRNA (green) and stained for MCCs (Ac.-α-tubulin, blue) and cell membranes (Actin, magenta). (D) Only Dex-activated GFP-δN-tp63-GR localizes to the nucleus. (E) Activation of the construct at st. 9 inhibits MCC formation, but not activation at st. 24 or vehicle treatment (Ethanol). (F) Quantification of results shown in (E). Mann Whitney test, not significant, ns = P>0.05; *** = P≤0.001. (G) Magnified views of fully differentiated MCC and Ionocyte with nuclear GFP-δN--tp63-GR after activation at st. 24.
	Supplemental Figure S4: Analysis and quantification of gain of Wnt/β-Catenin effects in BCIs, Related to Figure 4. (A-H) Human BCIs cultured in ALI conditions for up to 4 weeks. RSPO2 was used to activate Wnt/β-Catenin signaling starting at ALI day 7 (D7). N=3 cultures per timepoint and treatment. (A,B) Optical orthogonal sections of confocal images. RSPO2-treated cultures display increased epithelial thickness and cells staining for BC markers δN-TP63 (green, in A) and KRT5 (green, in B) over time of regeneration. Actin (magenta) staining reveals membranes and nuclei are stained with DAPI (blue). Related to Figure 3E and F. (C) Ki67 (green) staining reveals proliferating cells. Nuclei are stained with DAPI (blue). (D) Quantification of results depicted in (C). (E) Quantification of epithelial cell numbers. Related to Figure 3A. Mann Whitney test, not significant, ns = P>0.05; * = P≤0.05. (F, G) qPCR expression levels are depicted relative to stage controls. RSPO2 reduces expression of Goblet cell marker MUC5A/C, but increase expression of the Goblet marker MUC5B. Student’s t-test, not significant, ns = P>0.05; * = P≤0.05; ** = P≤0.01; ***= P≤0.001. Related to Figure 3D. (H) MUC5B (green) staining reveals no increase in Goblet cell numbers in the epithelium of RSPO2 treated cultures. Actin (cell membranes, magenta).
	Supplemental Figure S5: RNA-sequencing in Xenopus mucociliary organoids and analysis of loss of Wnt/β-Catenin effects in BCIs, Related to Figure 5 and 6 (A) MA-plots generated by DEseq2 analysis of differential expression changes in RNA-sequencing data at st.10, 17 and 25 on Xenopus mucociliary organoids comparing controls to δN-tp63 MO injected. N=3 per stage and treatment. Stage and number of differentially expressed genes are indicated. Red dots represent differentially expressed genes, black dots are not significantly differently expressed. (B) GO-term analysis of differentially upregulated (red box) and downregulated (blue boxes) genes at st. 25. Significantly enriched terms are depicted, including fold enrichment of term representation, P value, and false discovery rate (FDR). (C-H) Human BCIs cultured in ALI conditions for up to 4 weeks. DKK1 was used to inhibit Wnt/β-Catenin signaling starting at ALI day 7 (D7). N cultures per timepoint and treatment are indicated. (C) Ki67 (green) staining reveals proliferating cells. Nuclei are stained with DAPI (blue). (D) Quantification of results depicted in (C). Mann Whitney test, not significant, ns = P>0.05. (E) Quantification of epithelial cell numbers. Related to Figure 5A. Mann Whitney test, not significant, ns = P>0.05. (F) MUC5B (green) staining reveals no increase or loss of Goblet cell numbers in the epithelium of DKK1-treated cultures. Actin (cell membranes, magenta). (G,H) qPCR expression levels are depicted relative to stage controls. DKK1 increases expression of Goblet cell marker MUC5A/C (G), but has no effect on expression of the Goblet marker MUC5B (H).Student’s t-test, not significant, ns = P>0.05; *** = P≤0.001. Related to Figure 3D.
	Supplemental Figure S6: Analysis and quantification of regeneration experiments in Xenopus, Related to Figure 7 (A) Quantification of results depicted in Figure 6A. (B) Quantification of results depicted in Figure 6B. In (A,B) Mann Whitney test, ns = P>0.05; * = P≤0.05; ** = P≤0.01; *** = P≤0.001. (C) ISH for cell type markers foxj (MCCs), foxi1 (Ionocytes) and foxa1 (SSCs) in embryos after BIO incubation during st. 8-17 or DMSO control treatment at st. 17, and at st. 25 after recovery during st. 17-25. St. 17 embryos are depicted in ventral view with anterior up, st. 25 embryos are depicted in lateral view with anterior to the left. Related to Figure 6B. (D) Quantification of results depicted in (C). Chi-squared test, ns = P>0.05; * = P≤0.05; ** = P≤0.01; *** = P≤0.001.
	Supplemental Figure S7: Analysis and quantification of regeneration experiments in BCIs, Related to Figure 7 (A-F) Human BCIs cultured in ALI conditions for up to 4 weeks. RSPO2 was used to activate Wnt/β-Catenin signaling starting at ALI day 7 (D7) until ALI D21 (D21). After treatment, RSPO2 was removed from the medium and cultures were allowed to recover for 7 days. Number of cultures per treatment are indicated. (A) Quantification of results depicted in Figure 6C. (B) qPCR expression levels are depicted relative to stage controls. Student’s t-test, not significant, ns = P>0.05; * = P≤0.05; *** = P≤0.001. Related to Figure 6C. (C) Ki67 (green) staining reveals proliferating cells. Nuclei are stained with DAPI (blue). (D) Quantification of results depicted in (C). Mann Whitney test, ns = P>0.05. (E) Quantification of epithelial cell numbers. Related to Figure 6C. (F) MUC5B (green) staining reveals Goblet cells in the epithelium. Actin (cell membranes, magenta).

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