A three-dimensional study of alveologenesis in mouse lung

Abstract

Alveologenesis is the final step of lung maturation, which subdivides the alveolar region of the lung into smaller units called alveoli. Each of the nascent dividers serves as a new gas-exchange surface, and collectively they drastically increase the surface area for breathing. Disruption of alveologenesis results in simplification of alveoli, as is seen in premature infants diagnosed with bronchopulmonary dysplasia (BPD), a prevalent lung disease that is often associated with lifelong breathing deficiencies. To date, a majority of studies of alveologenesis rely on two-dimensional (2D) analysis of tissue sections. Given that an overarching theme of alveologenesis is thinning and extension of the epithelium and mesenchyme to facilitate gas exchange, often only a small portion of a cell or a cellular structure is represented in a single 2D plane. Here, we use a three-dimensional (3D) approach to examine the structural architecture and cellular composition of myofibroblasts, alveolar type 2 cells, elastin and lipid droplets in normal as well as BPD-like mouse lung. We found that 2D finger-like septal crests, commonly used to depict growing alveolar septae, are often artifacts of sectioning through fully established alveolar walls. Instead, a more accurate representation of growing septae are 3D ridges that are lined by platelet-derived growth factor receptor alpha (PDGFRA) and alpha smooth muscle actin (α-SMA)-expressing myofibroblasts, as well as the elastin fibers that they produce. Accordingly in 3D, both α-SMA and elastin were each found in connected networks underlying the 3D septal ridges rather than as isolated dots at the tip of 2D septal crests. Analysis through representative stages of alveologenesis revealed unappreciated dynamic changes in these patterns. PDGFRA-expressing cells are only α-SMA-positive during the first phase of alveologenesis, but not in the second phase, suggesting that the two phases of septae formation may be driven by distinct mechanisms. Thin elastin fibers are already present in the alveolar region prior to alveologenesis, suggesting that during alveologenesis, there is not only new elastin deposition, but also extensive remodeling to transform thin and uniformly distributed fibers into thick cables that rim the nascent septae. Analysis of several genetic as well as hyperoxia-induced models of BPD revealed that the myofibroblast organization is perturbed in all, regardless of whether the origin of defect is epithelial, mesenchymal, endothelial or environmental. Finally, analysis of relative position of PDGFRA-positive cells and alveolar type 2 cells reveal that during alveologenesis, these two cell types are not always adjacent to one another. This result suggests that the niche and progenitor relationship afforded by their close juxtaposition in the adult lung may be a later acquired property. These insights revealed by 3D reconstruction of the septae set the foundation for future investigations of the mechanisms driving normal alveologenesis, as well as causes of alveolar simplification in BPD.

Keywords: Alveologenesis; Development; Lung; Mouse.

Fig. 1

Septal crests represent sections through existing alveolar walls. (A–C) 2D images of individual optical sections of 75 µm lung slice with α-SMA and T1α staining. Optical section in (A) is 3 um above section in (B) and image (B) is 2 um above optical section in (C). Arrows (i–iv) indicate septal crests that converge into closed alveolar walls in another section plane. (D–E) Front and back views of 3D surface rendering of 50 um image stack which includes optical sections shown in (A–C) [see also Movie S1]. Arrows (i–iv) indicate alveolar walls in 3D that correspond to the 2D septal crests indicated in (A–C). (A–E) 40× magnification. All images in following figures are 3D renderings unless otherwise specified.

Fig. 2

α-SMA is expressed in a restricted time window in alveoli. (A–C) Surface rendering of PDGFRA-GFP expression and α-SMA staining in alveolar region at stages P0, P7 and P15. (A) α-SMA fibers surround saccules openings and arterioles at P0. Arrowheads indicate PDGFRA-GFP⁺ cells not surrounding saccules which do not express α-SMA at P0. (B) All PDGFRA-GFP⁺ myofibroblasts are associated with α-SMA fibers surrounding alveoli at P7. (C) PDGFRA-GFP⁺ cells in the alveolar region do not express α-SMA at P15. α-SMA is expression is still found surrounding terminal bronchioles (tb). (A–C) 40× magnification.

Fig. 3

Mouse models of disrupted alveolar formation exhibit altered α-SMA expression. (A–H) α-SMA staining (red) in alveolar region. (E–H) PDGFRA-GFP labels expressing nuclei. (A–B) Pecam-1 mutants, which exhibit impaired alveologenesis due to endothelial defects, shows increased α-SMA expression around alveoli at P7. (C–D) Shhcre;Etv4;5 mutants, which exhibit impaired alveologenesis due to epithelial defects, shows increased α-SMA expression and disorganized fibers around alveoli at P7. (E–F)Tbx4cre;Pdgfra mutants, which exhibit impaired alveologenesis due to myofibroblast defects, shows increased α-SMA expression in an expanded domain at P12. (G–H) Hyperoxia-exposed mice, which mimic alveolar defects observed in human BPD, exhibit α-SMA expression in alveolar region at P15 when normoxia-exposed alveoli no longer express α-SMA.

Fig. 4

Elastin fiber matrix is present at birth and undergoes remodeling during alveologenesis. (A–C) Surface rendering of PDGFRA-GFP expression and elastin staining in alveolar region at stages P0, P7 and P15. (A) Complex elastin fiber matrix is established by P0. All PDGFRA-GFP⁺ cells appear to be associated with elastin fibers. (B) By P7, elastin matrix has been remodeled to form tight bundles and are preferentially associated with the PDGFRA-GFP cells surrounding the AER. (C) By P15, the elastin fiber matrix is more complex with additional small fibers. (A–C) 40× magnification.

Fig. 5

Mouse models of impaired alveologenesis exhibit disorganized elastin matrices. (A–F) Elastin staining (red) in alveolar region. (E–F) PDGFRA-GFP labels the nuclei of expressing cells. (A–B) Tbx4cre;Pdgfra mutants show mesh of elastin fibers lacking thick bundles at P12. (C–D) Shhcre;Etv4;5 mutants show disorganized thin tortuous elastin fibers at P7. (E–F) Hyperoxia-exposed lungs show patches of dense elastin mesh (see top region and bottom right region of image), while other alveolar regions exhibit apparently normal elastin bundles at P15.

Fig. 6

Myofibroblast relationship to AEC2s and lipid droplets. (A) PDGFRA-GFP⁺ myofibroblasts at P7 in alveoli, outlined by T1α staining of AEC1s [see also Movie S5]. (B) AEC2s at P7 in T1α outlined alveoli [see also Movie S6]. (C) Quantification of mean myofibroblasts/alveolus and AEC2s/alveolus at P7. PDGFRA-GFP⁺ myofibroblasts per alveolus: 6.87 cells ± 2.47. SPC⁺ AEC2s per alveolus: 3.00 cells ± 0.94. n = 10 lungs each, minimum of 50 alveoli quantified. (D–F) PDGFRA-GFP expression and SPC staining in alveolar region at stages P0, P7 and P15 [see also Movies S7–S9]. Arrowheads indicate AEC2s that are not in direct apposition to PDGFRA-GFP+ cell. (G) SPC and α-SMA staining at P7 [see also Movie S10]. (H,I) PDGFRA-GFP expression and anti-PDGFRβ staining in alveolar region at P7. Boxed area in H is magnified in I. Arrows indicate PDGFRA-GFP and PDGFRβ double positive cells, arrowheads indicate PDGFRβ single positive cells, asterisks indicate PDGFRA-GFP single positive cells. (J,K) PDGFRA-GFP expression and LipidTOX staining of lipid droplets at P0 and P15 [see also Movie S11 and S12]. A subset of PDGFRA-GFP⁺ cells are associated with lipid droplets at P0 (J), while all PDGFRA-GFP⁺ cells appear closely associated with lipid droplets by P15 (K). Arrowheads in (J) indicate PDGFRA-GFP⁺ cells that are not closely associated with lipid droplets.

Fig. 7

A schematic of alveologenesis. (A) At birth, prior to alveolar formation, the distal-most lung structures are saccules (shown as circular buds) which form off of terminal bronchioles. Arrows indicate saccule openings. (B) During the peak phase of alveologenesis (P4–P12), α-SMA (in red) and closely associated elastin matrices (not shown) is expressed in an organized network, mimicking a “fishnet” pattern in the alveolar region. Peach-colored hemisphere represents epithelial lining of a saccule. Dotted blue circle outlines an alveolar entrance ring (AER) that is underlined by α-SMA. (C) A slice containing several alveoli. Septal ridges (arrowheads) are lined by α-SMA fibers (in red) and closely associated elastin fibers (not shown). Top dark line outline a possible cut 2D view with septal crests (asterisks) that are a result of a section through fully grown septal walls (arrows). (D) Increase and disorganization in α-SMA (red) and elastin matrices (not shown) transforms “fishnet” into “cheese cloth” and would prevent relative rise of ridges into the lumen, thereby impair alveoli formation. (E) At the height of septae formation (P4–12), PDGFRA-GFP⁺ (green) myofibroblasts form a ring at the AER, where they express α-SMA fibers (red) and deposit elastin fibers (blue). These cells may contract as they drive in new septae, meanwhile remodeling the matrix to stabilize newly formed structures.