Pulmonary alveolar type I cell population consists of two distinct subtypes that differ in cell fate

Abstract

Pulmonary alveolar type I (AT1) cells cover more than 95% of alveolar surface and are essential for the air-blood barrier function of lungs. AT1 cells have been shown to retain developmental plasticity during alveolar regeneration. However, the development and heterogeneity of AT1 cells remain largely unknown. Here, we conducted a single-cell RNA-seq analysis to characterize postnatal AT1 cell development and identified insulin-like growth factor-binding protein 2 (Igfbp2) as a genetic marker specifically expressed in postnatal AT1 cells. The portion of AT1 cells expressing Igfbp2 increases during alveologenesis and in post pneumonectomy (PNX) newly formed alveoli. We found that the adult AT1 cell population contains both Hopx⁺Igfbp2⁺ and Hopx⁺Igfbp2^- AT1 cells, which have distinct cell fates during alveolar regeneration. Using an Igfbp2-CreER mouse model, we demonstrate that Hopx⁺Igfbp2⁺ AT1 cells represent terminally differentiated AT1 cells that are not able to transdifferentiate into AT2 cells during post-PNX alveolar regeneration. Our study provides tools and insights that will guide future investigations into the molecular and cellular mechanism or mechanisms underlying AT1 cell fate during lung development and regeneration.

Keywords: Igfbp2; alveolar development and regeneration; lineage tracing; pulmonary alveolar type I cells; single cell RNA-seq.

Fig. 1.

Analyze the development of postnatal AT1 cells by single-cell RNA-seq (scRNA-seq). (A) Schematic illustration of the strategy of lung dissociation, AT1 cell sorting, and single-cell RNA sequencing analysis. (B–D) RFP⁺Pdpn⁺GFP⁻CD45⁻ cells were isolated from TAM-treated Sftpc-CreER; Rosa26-Zsgreen; Hopx-tdTomato mice at P3 (B), P15 (C), and P60 (D). The t-distributed stochastic neighbor embedding plots show cells isolated from P3 (B), P15 (C), and P60 (D) lungs can be clustered into four, four, and two main distinct populations, respectively. AT1 cell population is characterized by expressing Hopx and Pdpn (AT1 markers), but not Sftpc (AT2 marker), Pecam1 (endothelial cell marker), and Foxj1 (ciliated cell marker).

Fig. 2.

scRNA-seq analysis shows that postnatal AT1 cells continue to differentiate from P3 to P60. (A) The clustering of genes from randomly picked 1,000 AT1 cells of P3, P15, and P60 lungs. Differentially expressed genes (Dataset S2) can be clustered into five groups according to their expression patterns from P3 to P60. (B and B′) Selected GO terms of genes in group I genes (B) and genes in groups III, IV and V (B′). (C) Density plots of selected genes in group I. In the density plot, x-axis represents the gene expression level, and y axis represents the density of numbers of cells. (D) Density plots of selected genes in groups III, IV and V.

Fig. 3.

The expression of Igfbp2 is associated with alveologenesis and AT1 cell differentiation during alveolar regeneration. (A and B) The percentages of Igfbp2 expressing AT1 cells were quantified (mean ± SEM; n = 3) by immunostaining with anti-Igfbp2 and anti-Hopx antibodies. Arrowheads indicate AT1 cells that express Igfbp2. (C) The t-distributed stochastic neighbor embedding plot of Igfbp2 expression in P3 AT1 cells. (D) The GO analysis of up-regulated genes in Igfbp2⁺ AT1 cells among P3, P15, and P60 lungs. (E–H) Lungs of TAM-treated Sftpc-CreER; Rosa26-mTmG mice (E) at post-PNX day 14 (F), 21, and 45 (G) were analyzed by antibodies against GFP, Igfbp2, and Hopx. Arrowheads indicate the original AT1 cells. Arrows indicate newly differentiated AT1 cells. The percentages (mean ± SEM, n = 3) of newly differentiated AT1 cells expressing Igfbp2 in all newly differentiated AT1 cells are quantified (H). (I–N) Organoids grown from lineage-labeled AT2 cells (I) were collected at post plating day 9 (J) and day 14 (L) and stained with antibodies against Igfbp2 and Hopx (K and M). The proportion (mean ± SEM, n = 3) of Hopx⁺Igfbp2⁺ cells among the Hopx⁺ cells was quantified. (Scale bars: A, F, G, K, and M, 25 μm; J and L, 1 mm.)

Fig. 4.

Igfbp2⁺ AT1 cells maintain their AT1 cell fate during homeostasis. (A–C) Lungs (n = 5) of TAM-treated Igfbp2-CreER; Rosa26-Zsgreen mice were analyzed with antibodies against GFP and Hopx (B and C) or antibodies against GFP and Prospc (D and E). (F–I) Lungs of Igfbp2-CreER; Rosa26-tdTomato mice (TAM-treatment at P5) (F) were analyzed using antibodies against Pdpn, RFP, and Prospc at P6 (G) and P336 (H). Lungs from P336 old mice (H) were analyzed using antibodies against Igfbp2 and RFP (I). (Scale bars: B, D, and G–I, 25 μm.)

Fig. 5.

Igfbp2⁺ AT1 cells are terminal differentiated AT1 cells and can differentiated from Hopx⁺ AT1 cells. (A–C) Lungs of PNX-treated Igfbp2-CreER; Rosa26-Zsgreen mice (A) were analyzed with antibodies against GFP, Hopx, and Prospc (B). (C) Organoids were grown from either Hopx⁺ AT1 cells or GFP⁺ lineage-labeled AT2 cells. (D and E) At post plating day 14, organoids growth Hopx⁺ AT1 cells (D) from were analyzed with antibodies against Igfbp2 and Hopx (E). (F–H) At post plating day 9, some flatten cells at the luminal side of organoids formed from GFP⁺ AT2 cells (F) expressed both Hopx and Pdpn (G). Hopx⁺ cells were then isolated from these day 9 organoids and cultured in a 3D organoid culture system. At post plating day 14, some Hopx⁺ AT1 cells expressed Igfbp2 (H). (I–K) Lineage-labeled Igfbp2⁺ AT1 cells were plated for the alveolar organoid culture (I). No RFP⁺ organoids formed by day 14 of culture (J and K). (Scale bars: B, E, G, and H, 25 μm; D, F, J, and K, 1 mm.)