The development and plasticity of alveolar type 1 cells

Abstract

Alveolar type 1 (AT1) cells cover >95% of the gas exchange surface and are extremely thin to facilitate passive gas diffusion. The development of these highly specialized cells and its coordination with the formation of the honeycomb-like alveolar structure are poorly understood. Using new marker-based stereology and single-cell imaging methods, we show that AT1 cells in the mouse lung form expansive thin cellular extensions via a non-proliferative two-step process while retaining cellular plasticity. In the flattening step, AT1 cells undergo molecular specification and remodel cell junctions while remaining connected to their epithelial neighbors. In the folding step, AT1 cells increase in size by more than 10-fold and undergo cellular morphogenesis that matches capillary and secondary septa formation, resulting in a single AT1 cell spanning multiple alveoli. Furthermore, AT1 cells are an unexpected source of VEGFA and their normal development is required for alveolar angiogenesis. Notably, a majority of AT1 cells proliferate upon ectopic SOX2 expression and undergo stage-dependent cell fate reprogramming. These results provide evidence that AT1 cells have both structural and signaling roles in alveolar maturation and can exit their terminally differentiated non-proliferative state. Our findings suggest that AT1 cells might be a new target in the pathogenesis and treatment of lung diseases associated with premature birth.

Keywords: Alveolar angiogenesis; Cell plasticity; Lung development.

Fig. 1.

AT1 cell growth fuels postnatal alveolar growth as quantified using marker-based stereology. (A) Confocal projection images of whole-mount immunostained mouse lung strips showing that HOPX (solid arrowhead) and cuboidal E-CAD or LAMP3 (open arrowhead) are mutually exclusive. Any apparent partial overlap is due to the projection view of image stacks, which allows better assignment of the staining to the corresponding nucleus. (B) Confocal projection images of a whole-mount immunostained 60 μm section from a P21 Shh^Cre/+; Rosa^RG/+ lung. Cre recombination and therefore RFP is restricted to airway and alveolar epithelial cells. Only endogenous RFP but not GFP from the Rosa^RG allele is detectable. The boxed region is enlarged in the righthand panels showing that RFP-expressing cells are marked in a mutually exclusive manner by HOPX (AT1, solid arrowhead) and E-CAD (AT2, open arrowhead). Beneath is a transverse section (z-axis) view along the dashed line showing that half AT1 (solid arrowhead) and AT2 (open arrowhead) can be discerned and counted at the image borders (double-sided arrow). Asterisk marks ‘escaping' epithelial cells, presumably due to inefficient recombination or RFP expression. (C) The left lung volume, the ratio between AT1 and AT2 cell numbers, the total number of AT1 and AT2 cells, and alveolar surface area made of AT1 and AT2 cells in the left lung are plotted against animal age. Each symbol represents one mouse. Note that 1 cm² equals 10⁸ μm². Scale bars: 10 μm.

Fig. 2.

AT1 cells flatten in conjunction with molecular specification. (A) Confocal images of immunostained strips from E19 Sox9^CreER/+; Rosa^mTmG/+ lungs with recombination induced at E13 (Tam, tamoxifen). The two leftmost images are maximal projection views of branch tips (dashed outlines). Columnar wedge-shaped epithelial progenitors (P) are found in branch tips. Elongated AT1 cells and cuboidal AT2 cells are found in branch stalks and also shown in section views in subsequent images with the epithelial basement membrane outlined with dashes and the lumen (lu) labeled. Only part of a cell is visible in the section view and therefore may have a different morphology from that in the projection view. Note that AT2 cells, unlike AT1 and progenitor cells, are extruded from the lumen. Elongated AT1 cells have nuclear HOPX expression and have lost progenitor and AT2 cell markers, including SOX9 and SFTPC. (B) Confocal images of E19 lung sections. PODXL-expressing AT1 cells (square bracket) are connected with neighboring AT2 cells through apically restricted tight junctions [ZO1 (TJP1), arrowheads]. E-CAD is also enriched apically at AT1-AT2 and AT1-AT1 junctions, as opposed to accumulating throughout the lateral sides between progenitors (A). Dashed line indicates epithelial basement membrane marked by collagen type IV (COL4). Asterisks mark blood vessels that express PODXL, ZO1 and COL4 but not E-CAD. (C) A model of AT1 cell flattening. As columnar wedge-shaped progenitors flatten to become AT1 cells, apical tight junctions (TJ) are maintained whereas lateral junctions are lost. Cell flattening is accompanied by changes in marker expression including SOX9, SFTPC and HOPX. Scale bars: 10 μm.

Fig. 3.

AT1 cells fold in conjunction with alveolar septation. (A) (Top) Confocal projection images of immunostained strips from a P8 Hopx^CreER; Rosa^nTnG/+ lung showing infrequent mistargeting of AT2 cells (asterisk) by Hopx^CreER when induced with 2 mg tamoxifen at E19. Solid arrowhead, AT1 cells; open arrowhead, AT2 cells. To the right is a schematic of a cranial lobe with airways traced according to a real sample stained for SOX2 to illustrate that a strip in the gas exchange compartment is cut along the dashed line and immunostained and the boxed region imaged. The plot shows quantification of AT1 cell total surface area (both the apical and basal surface). Representative examples are shown in the bottom panels. The plot is distributed along the x-axis to avoid excessive overlapping. (Bottom) Confocal projection images (top row) of immunostained strips from Hopx^CreER; Rosa^mTmG/+ lungs at the indicated stages induced with 0.4 mg tamoxifen at E19, with the corresponding surface rendering (bottom row). Arrowheads, ‘valleys' of AT1 cells; Asterisk, AT2 cell. (B) Confocal projection images of immunostained lung strips and corresponding surface rendering (beneath each confocal image). At P4 (left column), primary saccules are island-like and associated with deeper and wider grooves (dashed line, see also the P0 lung in A) with embedded smooth muscles (open arrowhead). Shallow grooves with smooth muscles begin to form on the surface of primary saccules (solid arrowheads). Vessels are associated with all grooves and only appear double layered (asterisk) between primary saccules. At P19 (right column), SMA-expressing myofibroblasts mostly disappear, whereas vessels persist to mark grooves between mature alveoli. Primary saccules are no longer obvious as secondary septa have deepened. Square brackets indicate pre-capillary arterioles surrounded by smooth muscles. (C) (Left) Projection view (top left), surface rendering (top middle and right) and section views at the indicated depth (bottom row) of confocal images of immunostained strips from a P30 Hopx^CreER; Rosa^mTmG/+ lung induced with 0.4 mg tamoxifen at E19. A single AT1 cell (n, nucleus) spans multiple alveoli (1 through 5, demarcated by vessels) and the underlying alveolar sac (sac). (Right) Projection view (top) and surface rendering (bottom) of confocal images of immunostained strips from a P24 Hopx^CreER; Rosa^tdT/+ lung induced with 0.4 mg tamoxifen at P23. The soluble reporter (RFP, pseudocolored green) highlights the nucleus of a single AT1 cell (dashed outline). (D) A model of AT1 cell folding. Flattened AT1 cells undergo a >10-fold expansion in surface area and fold where myofibroblasts temporally and vessels permanently reside. As a result, a mature AT1 cell spans multiple alveoli (alv). Scale bars: 10 μm.

Fig. 4.

Scnn1a-Cre-driven ectopic SOX2 expression reprograms flattened AT1 cells. (A) Confocal projection images of immunostained P0 Scnn1a-Cre; Rosa^mTmG/+ lung strips. Dashed lines denote a branch-like tube at the lobe edge with distal tips containing progenitors in clusters and proximal stalks containing intermingled flattened AT1 and cuboidal AT2 cells. The boxed region is enlarged to the right. Note that all labeled cells are flattened AT1 cells (demarcated by E-CAD junctions) with nuclear HOPX (arrowhead), rather than cuboidal AT2 cells (asterisk). (B) Confocal projection images of immunostained Scnn1a-Cre; Rosa^Sox2/+ lung strips at the indicated stages showing that isolated flattened mutant AT1 cells (demarcated by E-CAD junctions, dashed lines) form clusters with compact cell arrangement over time. (C-E) Projection (C,D) or section (E) view of confocal images of immunostained strips from littermate control (C) and Scnn1a-Cre; Rosa^Sox2/+ (C,E) or Scnn1a-Cre; Rosa^mTmG/Sox2 (D) mutant lungs. (C) Compared with AT1 cells in the control lung or neighboring unrecombined AT1 cells (open arrowhead), mutant AT1 cells (solid arrowhead) lose RAGE expression and have diffuse HOPX expression. Asterisks denote AT2 cells. (D) The Rosa^mTmG allele is easier to recombine than the Rosa^Sox2 allele, generating juxtaposed GFP-labeled AT1 cells with and without SOX2 expression. Compared with control AT1 cells (green outline with black dashed nuclei), mutant AT1 cells are smaller and express the proliferation marker KI67 (green outline with red dashed nuclei). Note additional KI67 expression in mesenchymal cells. (E) Mutant AT1 cells express the basal cell marker P63 (arrowhead). (F) Left two panels show confocal images of a control lung section showing that NKX2.1 is expressed by both AT1 (solid arrowheads) and AT2 (open arrowheads, cuboidal E-CAD staining) cells. All epithelial cell nuclei are genetically marked by GFP to allow co-staining with the rabbit anti-NKX2.1 antibody. Right two panels are confocal images of sections from littermate control and Scnn1a-Cre; Rosa^Sox2/+ mutant lungs. Mutant AT1 cells marked by GFP have nuclear (solid arrowhead) or diffuse (arrow) NKX2.1. Inset shows NKX2.1 single-channel image. NKX2.1 expression in AT2 cells (open arrowhead, cuboidal E-CAD staining) of the mutant lung is not affected. (G) Scatterplot of log₂ fold change in gene expression between two biological replicates. Compared with littermate control (Scnn1a-Cre; Rosa^mTmG/+) AT1 cells, mutant (Scnn1a-Cre; Rosa^Sox2/+) AT1 cells downregulate AT1 markers (green) and upregulate airway markers (red). The number in parenthesis denotes the rank order in fold change among genes that are significantly different between control and mutant. Scale bars: 10 μm.

Fig. 5.

Aqp5^Cre-driven ectopic SOX2 expression reprograms flattened AT1 cells. (A) Confocal projection images of immunostained Aqp5^Cre/+; Rosa^Sox2/+ lung strips at the indicated stages. Some mutant AT1 cells (arrowhead) have retracted their cellular extensions and accumulate a high level of E-CAD at P3. Mutant cell clusters (arrow) are present at P8. (B) Confocal images of sections from littermate control and mutant lungs showing comparable phenotypes of AT1 cells targeted with Aqp5^Cre to those targeted with Scnn1a-Cre (Fig. 4). Mutant AT1 cells (GFP, arrowhead) form clusters, downregulate RAGE (boxed region shown as single-channel images in insets), express KI67 and DNp63 (deltaNp63), and have diffuse (arrow) or nuclear (solid arrowhead) NKX2.1 (single-channel image shown in inset). NKX2.1 expression in AT2 cells (open arrowhead, cuboidal E-CAD staining) of the mutant lung is not affected. KI67 is expressed in non-AT1 cells in the control lung (Fig. S3A). Scale bars: 10 μm.

Fig. 6.

AT1 cells are the source of Vegfa. (A) Combined fluorescence in situ hybridization and immunostaining of wild-type lung sections. (Top) Vegfa RNA localizes to oval-shaped AT1 cell bodies that are positive for NKX2.1 and HOPX (solid arrowheads), but not to LAMP3-positive cuboidal AT2 cells (open arrowhead). (Bottom) The left panel shows, as a technical control, that Sftpc RNA localizes to cuboidal AT2 cells that are positive for SFTPC and LAMP3. Boxed regions are enlarged in subsequent panels. To the right is a colorimetric in situ hybridization of an E18 lung section near the tissue edge showing Vegfa RNA in the alveolar epithelium (arrow, bronchoalveolar duct junction) and, at a lower level, the distal progenitors (open arrow). The epithelium is traced with a dashed line. (B) Section colorimetric in situ hybridization (left two columns) and confocal projection images of immunostained strips (right two columns) from littermate control and Scnn1a-Cre; Rosa^Sox2/+ lungs at the indicated stages. Vegfa expression (solid arrowheads) is lost in mutant AT1 cells, whereas Sftpc expression (open arrowheads) is not affected. Asterisks indicate regions with fewer vessels in the mutant. (C) Section colorimetric in situ hybridization (left) and confocal projection images of immunostained strips (right two columns) from littermate control and Aqp5^Cre/+; Rosa^Sox2/+ lungs at the indicated stages. The numbers indicate the vessel areas (P=0.02, Student's t-test) and the number of endothelial cells (P=0.1, Student's t-test) for three image fields of 318×318×20 μm. Scale bars: 10 μm.

Fig. 7.

Mature AT1 cells retain cellular plasticity. (A) Stereomicroscopy images of lung strips 21 days after recombination induced at P56 showing a robust response of individual AT1 cells (arrowheads) to ectopic SOX2 expression by retracting their cellular extensions. Different doses of tamoxifen were used to achieve comparable recombination efficiency of the Rosa^mTmG (0.4 mg) and Rosa^Sox2 (4 mg daily for three consecutive days) alleles. (B) Confocal projection images of immunostained lung strips at the indicated days after recombination induced at P35. Boxed regions are enlarged in subsequent panels showing that mutant AT1 cells lose extended morphology (green outline at 4 days; arrowhead indicates another mutant cell) and form clusters, doublets or singlets (21 days). The total numbers of each type examined are shown in the bar chart. All singlets examined at 4 days after recombination have extended morphology, indicating their AT1 cell identity. Mutant AT1 cells including singlets have retracted their cellular extensions (surface rendering shown) and accumulate a high level of E-CAD. (C) Confocal images of immunostained lung sections at 9 or 21 days after recombination induced at P56. Boxed regions are shown as single-channel images. Whereas control AT1 cells do not express CCND1 (Fig. S10C), mutant AT1 cells activate CCND1 when they are still singlets (solid arrowhead), retracting their cellular extensions and starting to accumulate E-CAD (punctate staining in inset). Open arrowhead, CCND1-expressing AT2 cells. Scale bars: 100 μm in A; 10 μm in B,C.