Emergence of a Wave of Wnt Signaling that Regulates Lung Alveologenesis by Controlling Epithelial Self-Renewal and Differentiation

Abstract

Alveologenesis is the culmination of lung development and involves the correct temporal and spatial signals to generate the delicate gas exchange interface required for respiration. Using a Wnt-signaling reporter system, we demonstrate the emergence of a Wnt-responsive alveolar epithelial cell sublineage, which arises during alveologenesis, called the axin2+ alveolar type 2 cell, or AT2^Axin2. The number of AT2^Axin2 cells increases substantially during late lung development, correlating with a wave of Wnt signaling during alveologenesis. Transcriptome analysis, in vivo clonal analysis, and ex vivo lung organoid assays reveal that AT2s^Axin2 promote enhanced AT2 cell growth during generation of the alveolus. Activating Wnt signaling results in the expansion of AT2s, whereas inhibition of Wnt signaling inhibits AT2 cell development and shunts alveolar epithelial development toward the alveolar type 1 cell lineage. These findings reveal a wave of Wnt-dependent AT2 expansion required for lung alveologenesis and maturation.

Keywords: Wnt; alveolus; cell fate; lung.

Figure 1. A novel reporter mouse defines Wnt-responsiveness in the developing lung

(A and B) The Axin2^{CreERT2-TdTom} allele produces TdTomato expression in Wnt-responsive (Axin2+) cells located in the mesenchyme and proximal and distal Nkx2.1+ epithelium (A) during branching morphogenesis with (B) showing the same image with RFP and DAPI staining only. Dashed white lines outline distal and solid lines outline proximal airways, respectively. (C) Induction of recombination at E10.5 in Axin2^{CreERT2-TdTom}:R26R^EYFP mice followed by a four-day chase demonstrates that Axin2+ cells give rise to both proximal and distal Nkx2.1+ lung epithelium, with a larger number of cells labeled in the distal region (yellow bracket). (D and E) From E15.5-E16.5, lineage labeled Axin2+ cells become restricted to distal Sox2− (D), Sox9+ (E) lung epithelium. (F-I) At E17.5-E18, Axin2+ lung epithelial cells are sparsely located throughout distal bronchiolar and alveolar epithelium. Bronchiolar Axin2+ cells are predominantly Scgb1a1 secretory cells (F) with very rare Tubb4+ ciliated cells (G). In the alveolar region, there is a small number of Pdpn+ AT1s (H) and Sftpc+ AT2s (I). At P4, Axin2+ secretory (J) and ciliated (K) bronchiolar cells and AT1s (L) become very rare. However, there is a dramatic increase in Axin2+ AT2 (AT2^Axin2) alveolar cells (M). (N) Quantification of Axin2+ secretory (Scgb1a1+) and ciliated (Tubb4+) bronchiolar epithelium, AT2s (Sftpc+), and AT1s (Pdpn+) reveals significant changes in lineage specific Axin2+ Wnt responsive lung epithelium. (O) At E18, a minority of the Axin2+ Wnt responsive epithelium is Sftpc+. However, by P4 more than 97% of the total Axin2+ epithelium is a sublineage of the total Sftpc+ AT2 lineage i.e. AT2^Axin2s. Arrows in F-M indicate examples of a positive cell and insets are higher magnification of Axin2+ cells. D=distal lung endoderm/epithelium, P=proximal lung endoderm/epithelium. Quantification of cell numbers represented as mean ± SEM. Two-tailed student's t-test: *P < 0.05, n.s = non-significant; n=3 for each group. Scale bars = 50 μm.

Figure 2. A wave of Wnt signaling occurs in AT2s during alveologenesis

(A) Graphic model illustrates the dynamic changes in Axin2+ Wnt-responsive lung epithelium during lung development. Late lung development is marked by restriction of Axin2+ epithelial cells to the distal alveolus and a significant increase in AT2^Axin2 cells during alveologenesis. While there are few AT2^Axin2 cells at E17.5-E18.0 (B and C), their numbers increase significantly by P1 (D and E) and P4 (F and G). AT2^Axin2 cells continue to be observed at P30 (H and I). Dotted lines surround Sftpc+ AT2 and AT2^Axin2 cells in insets C, E, G, I. (J-L) Lineage traced AT2^Axin2 cells from E18.5 to P10 (J) and P4 to P10 (K) show a marked increase in AT2^Axin2 cell expansion between these time points. Arrows indicate positive cells, which are quantified in (L). (M) Total AT2s isolated by FACS following a 24-hour tamoxifen induction. (N) Expression of Axin2 mRNA at P4 compared to E18.5 in AT2s showing a two-fold increase in expression. (O and P) Axin2+ lung epithelial cells are isolated using negative selection for CD31 and CD45 (upper FACS plot) and positive selection for TdTomato and EpCAM (lower FACS plot). (P) By flow cytometry, isolated Axin2+ lung epithelial cells are composed predominantly of AT2s^Axin2 as measured by intracellular Sftpc and extracellular Pdpn staining. Quantification of cell numbers, qPCR data, and cell type percentage represented as mean ± SEM. Two-tailed student's t-test: P < 0.05; n=3-4 for each group. Scale bars: B, D, F, H, J, K = 50 μm; C, E, G, I = 10 μm; J, K = 5 μm.

Figure 3. AT2s^Axin2 have increased clonal growth potential

(A and B) Clonal expansion is enhanced in AT2s^Axin2 during alveologenesis. Following lineage tracing with the R26R^Br2.1 reporter, AT2s^Axin2 can expand clonally to a greater extent than the AT2 lineage in general. Arrows indicate single cell clones, dashed lined circles indicate two cell clones, and solid lined circles indicate three cell clones. Note the three cell clone in the AT2^Axin2 lineage trace versus the two cell clone in the Sftpc+ lineage trace. (C) Quantitatively, the average AT2^Axin2 clone size is larger than AT2s in general. (D) The distribution of clone size exhibits a shift towards more two and three cell clones in AT2s^Axin2 compared to the total AT2 population. (E-H) Assessment of proliferation by EdU incorporation at P4 shows that the majority of AT2s^Axin2 are actively proliferating compared to a minority of Axin2- AT2s. Arrows represent EdU+ AT2s^Axin2, and arrowheads indicate EdU+ Axin2− AT2s. (I) Quantification of average clone size and percent of EdU+ AT2s are represented as mean ± SEM, and size distribution is represented % of total clones. Two-tailed student's t-test: *P < 0.05 and Fisher's Exact Test: **P < 0.007, n=5 (58 clones) for AT2s^Axin2, n=4 (191 clones) for total AT2s; Two-tailed student's t-test: ***P < 0.05, n=3 with 10 independent fields of view per mouse. Scale bars: A and B = 50 μm, E-H = 5 μm.

Figure 4. The AT2^Axin2 sublineage is highly enriched in genes important for alveologenesis

(A) Principal component analysis illustrates unique segregation of the AT2^Axin2 sublineage from the total AT2 population. (B) Microarray analysis comparing the total AT2 population versus the AT2^Axin2 sublineage demonstrates enrichment of up-regulated genes for cell cycle, vascular development, and extracellular matrix interactions and production and down-regulation of genes associated with metabolism and lipid metabolism in the AT2^Axin2 sublineage. (C-L) qPCR analysis on genes important in alveolar growth and differentiation confirms microarray findings on selected genes. Quantification of qPCR data is represented as mean ± SEM. Two-tailed student's t-test: *P < 0.05, n=4 for each group.

Figure 5. AT2s^Axin2 exhibit enhanced ex vivo lung organoid formation

(A-C) AT2s^Axin2 exhibit an increase in colony forming efficiency for lung alveolar organoid formation compared to total AT2s at passage 0 (P0) and passage 1 (P1). By passage 2 and 3 (P2-P3), these differences are no longer significant. (D and F) Hematoxylin and eosin (H&E) staining showing organoid structure in total AT2s and AT2s^Axin2. (E and G) Immunohistochemical staining for the AT2 and AT1 markers, Sftpc and Pdpn, respectively, demonstrate that total AT2s and AT2s^Axin2 are composed of both differentiated AT2s and AT1s. (H-L) Wnt3a treatment increases CFE while IWP2 treatment inhibits CFE in AT2 derived organoids. Treatment with both IWP2 and Wnt3a shows that inhibition can be rescued, in part, with exogenous Wnt ligand. Quantification of CFE is represented as average CFE ± SEM. Two-tailed student's t-test: *P < 0.05, n > 3 for each group in 4 independent experiments for A-I, n > 2 for each group in 3 independent experiments for J-N.

Figure 6. Wnt activation increases AT2 proliferation and expansion during alveologenesis in vivo

(A-C) Activation of Wnt signaling increases total alveolar epithelial cell and AT2 cell numbers when lineage tracing from P4 to P30 compared to Sftpc^CreERT2:Ctnnb1^+/+ control mice. Both total Nkx2.1+ and Sftpc+ cells are increased in the alveolar region. (D-F) Proliferation as measured by Ki67 staining is increased in Sftpc^CreERT2:Ctnnb1^fl(Ex3)/+ mutants compared to controls. Arrows indicate Ki67+ Sftpc lineage marked cells. (G-J) Wnt activation in AT2s results in clonal expansion of AT2s as noted by increased cell number per clone and increased clone size. Note the larger size of the CFP clone in H as compared to the YFP and RFP clones in G. (K-M) AT1 cell differentiation is unaffected by activation of Wnt signaling in AT2s as noted by co-staining for the YFP lineage mark and Aqp5. Quantification of cell number counts, %Ki67+, average clone size, and % of YFP+ cells is represented as mean ± SEM. Two-tailed student's t-test: *P < 0.05, n > 4 for each group; Two-tailed student's t-test: **P < 0.05 and Fisher's exact test: ***P <0.05, n=142 clones for Sftpc^CreERT2:Ctnnb1^+/+ and n=258 clones for Sftpc^CreERT2:Ctnnb1^fl(Ex3)/+ mutants.

Figure 7. Loss of Wnt signaling in AT2s inhibits proliferation and promotes differentiation into AT1s

(A-C) Proliferation is reduced in Sftpc^CreERT2:Ctnnb1^fl/fl mutant AT2s compared to controls following deletion of β-catenin from P4 to P30. (D-F) Loss of Wnt signaling in Sftpc^CreERT2:Ctnnb1^fl/fl:R26R^Br2.1 mutants from P4 to P30 leads to increased differentiation into AT1s during alveologenesis as noted by the increase in clones exhibiting the spread and flattened morphology of AT1 cells. (G-I) These data were confirmed using the R26R^mTmG reporter to outline the cell surface of the AT1 cells along with Aqp5 co-immunostaining. (J) Illustration depicting the role of re-emergent Wnt signaling in balancing epithelial growth and differentiation during lung alveologenesis. Quantification of % Ki67+, average % of clone, and % of YFP+ cells is represented by mean ± SEM. Two-tailed student's t-test: *P < 0.05, n > 4 for each group; Two-tailed student's t-test: **P < 0.05, n=133 clones for controls and n=203 clones for Sftpc^CreERT2:Ctnnb1^fl/fl mutants.