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. 2025 Jan 7;10(4):e187876.
doi: 10.1172/jci.insight.187876.

Epithelial outgrowth through mesenchymal rings drives lung alveologenesis

Nicholas M Negretti  1 , Yeongseo Son  1 , Philip Crooke  2 , Erin J Plosa  1 , John T Benjamin  1 , Christopher S Jetter  1 , Claire Bunn  1 , Nicholas Mignemi  3 , John Marini  4 , Alice N Hackett  1 , Meaghan Ransom  1 , Shriya Garg  1 , David Nichols  5 , Susan H Guttentag  1 , Heather H Pua  6 , Timothy S Blackwell  3   5   7 , William Zacharias  8 , David B Frank  9 , John A Kozub  10   11   12 , Anita Mahadevan-Jansen  10   11   13 , Evan Krystofiak  3 , Jonathan A Kropski  3   5   7   14 , Christopher Ve Wright  3   14   15 , Bryan Millis  11   14 , Jennifer Ms Sucre  1   3   14   15
Affiliations

Epithelial outgrowth through mesenchymal rings drives lung alveologenesis

Nicholas M Negretti et al. JCI Insight. .

Abstract

Determining how alveoli are formed and maintained is critical to understanding lung organogenesis and regeneration after injury. To study the cellular dynamics of this critical stage of lung development, we have used scanned oblique-plane illumination microscopy of living lung slices to observe alveologenesis in real time at high resolution over several days. Contrary to the prevailing notion that alveologenesis occurs by airspace subdivision via ingrowing septa, we found that alveoli form by ballooning epithelial outgrowth supported by contracting mesenchymal ring structures. Systematic analysis has produced a computational model of finely timed cellular structural changes that drive normal alveologenesis. With this model, we can now quantify how perturbing known regulatory intercellular signaling pathways and cell migration processes affects alveologenesis. In the future, this paradigm and platform can be leveraged for mechanistic studies and screening for therapies to promote lung regeneration.

Keywords: Development; Organogenesis; Pulmonology.

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Figures

Figure 1
Figure 1. Precision-cut lung slices (PCLS) model of alveologenesis ex vivo.
(A) Five-day-old mT/mG transgenic mice fluorescently reporting for alveolar epithelial, mesenchymal, or endothelial cells were imaged by scanned oblique-plane illumination (SOPi) microscopy. (B) PCLS were fixed immediately after preparation on P5, or after 48 hours in culture and stained with H&E. Alveolar septal tip length was calculated and compared between PCLS taken from the same lung, comparing P5 PCLS slices to P5 PCLS from the same lung after 48-hour culture (n = 6 mice from 2 separate litters, total of 11 PCLS per condition, with each point representing the average tip length values calculated from 9–10 images of an individual PCLS replicate), showing histological changes equivalent in many respects to H&E-stained sections from P5 or P7 mice, as reflected by alveolar septal tip length. Scale bars: 40 μm. **P < 0.001 by Student’s t test. (C) PCLS were imaged by scanning electron microscopy. Scale bars: 10 μm. Images are representative of SEM from n = 3 mice per condition/time point.
Figure 2
Figure 2. Alveolar type 2 cells undergo changes in cell shape that are associated with differentiation into alveolar type 1 cells.
PCLS from mT/mG;Sftpc-CreERT2 mice (with tamoxifen given on P3 and P4) were volumetrically imaged and displayed as a 3D projection (GFP green, tdTomato magenta), representative of n = 9 imaging movies from 3 mice. (A) 3D projection of a still image from the start of the 72-hour imaging period. (B) 3D projection of a still image from the end of the same imaging period. Scale bars: 30 μm. (C) The same Sftpc-GFP–labeled cell viewed from 3 different orientations showing the dramatic cell-shape change from round to flat over 42 hours. Scale bars: 10 μm. (D) Quantification of sphericity of individual cells in C making this transition (representative of n = 83 cells counted). (E) Lungs from mT/mG;Sftpc-CreERT2 mice given tamoxifen on P3 and P4 and harvested on P5 (left) and P7 (right), immunostained with antibodies against GFP (green) and PDPN (white), analyzed by RNA in situ hybridization for Sftpc (red), with DAPI counterstaining (blue) to mark DNA. Scale bars: 10 μm. (F) Quantification of percentage Sftpc+ and PDPN+ cells among total GFP+ cells on P5 and P7, with each data point indicating the average value from 8–10 images of an individual mouse. **P < 0.01; ***P < 0.001 by 1-tailed Student’s t test (n = 5–6 mice on P5, 8 mice on P7). (G) Sequential still frames from 4D imaging of PCLS from mT/mG;Ager-CreERT2 mice (with tamoxifen given on P3 and P4). Scale bars: 30 μm. Representative of n = 9 imaging movies from 3 mice.
Figure 3
Figure 3. Alveologenesis is characterized by epithelial ballooning outgrowth.
(A) PCLS from mT/mG;Sftpc-CreERT2 mice (with tamoxifen given on P3 and P4) were volumetrically imaged and displayed as a 3D projection (GFP green, tdTomato magenta). Left: 3D projection of a still image from the start of the 72-hour imaging period. Scale bars: 20 μm. (B) 3D projection of a still image from the end of the same imaging period. (C–E) Insets of 3 different areas of epithelial cells clustering with ballooning outgrowth and elongation, as representative areas of this process, which occurs asynchronously across the PCLS. Scale bars: 10 μm. (F) Imaris surface rendering of Sftpc-GFP+ epithelial cells (green) moving through ring-like structures (magenta) and expanding in the formation of neo-alveoli shown in 2 perspectives. Scale bars: 10 μm. (G) PCLS from mT/mG;Sftpc-CreERT2 mice given tamoxifen on P3 and P4 and harvested on P5, live imaged with a probe for F-actin (blue, segmented to only show GFP+ cells), demonstrating polymerized actin (white arrows) at the leading edge of the epithelial cell just before movement. Scale bars: 10 μm. All images are representative of n = 9 movies from 3 mice, each with 3 regions of interest imaged per experiment.
Figure 4
Figure 4. Mesenchymal ring structure dynamics and contraction are required for alveologenesis.
PCLS from mT/mG;Pdgfra-Cre mice were volumetrically imaged via SOPi for 72 hours. Representative of n = 5 mice, each with 3 regions of interest imaged per experiment Comparison of (A) a single plane with (B) a volumetric rendering of the Z-stack from the same area demonstrates that putative “septal tips” (white arrows) are in fact part of a 3D ring structure. (C) Imaris-based surface rendering of thick, tissue-cleared sections from the lungs of mT/mG;Pdgfra-Cre mice on P5 and P14 demonstrates gradual loss of the ring structure over the course of alveologenesis, with Pdgfra+ cells in blue (left) showing the complex ring network, with individual rings highlighted in red (middle) and shown in the context of adjacent growing airspaces (right). (D) Whole-mount tissue-cleared RNA in situ hybridization of thick sections with probes for hallmark genes of alveolar myofibroblasts Wnt5a (top) and Fgf18 (bottom) demonstrate colocalization with GFP (green). (E) Still frames from excerpt of larger 72-hour 4D imaging of lungs from mT/mG;Pdgfra-Cre mice demonstrate the dynamic movement of individual cells to form ring structures and gradual contraction of individual rings over time. (F) PCLS from these mice administered the ML-7 inhibitor after 20 hours of imaging dramatically decreased the displacement of Pdgfra+ cells (inset) quantified by a change in rate of movement of individual cells over time (G). All scale bars: 10 μm.
Figure 5
Figure 5. Endothelial cells form a dynamic complex vascular network during alveologenesis.
PCLS from mT/mG;Vecad-Cre mice were volumetrically imaged by SOPi for 48 hours. All images are representative of n = 9 movies from 3 mice. (A) Still frames from this imaging period (inset) show movement and elongation of some individual cells (white arrows) and changes in network complexity as quantified by (B) number of branch points and length of individual filaments. (C) Filament length at 2 different points in time during the imaging period represented graphically with a colorimetric scale (shorter filaments in pink, longer filaments in blue). All scale bars: 10 μm.
Figure 6
Figure 6. Modulation of the Wnt pathway disrupts alveologenesis, with decreased epithelial ballooning movements and changes in shape.
(A) H&E staining of PCLS made on P5 and cultured for 72 hours under control conditions and with the addition of Wnt activator CHIR-99021 or Wnt inhibitor XAV-939 demonstrate abnormal alveologenesis with both modulators. Scale bars: 100 μm. Still projections from (B) control, (C) CHIR-99021–, or (D) XAV-939–treated PCLS imaged over time. Scale bars: 10 μm. (E) Control, CHIR-, or XAV-treated PCLS from mT/mG;Sftpc-CreERT2 mice were immunostained for GFP (green) and PDPN (white) and analyzed by RNA in situ hybridization for Sftpc (magenta), with DAPI counterstaining (blue) to mark DNA. Scale bars: 10 μm. (F) Individual alveologenesis events by epithelial cells were scored in a blinded manner from PCLS from CHIR (Wnt activator), XAV (Wnt inhibitor), or vehicle control. ****P < 0.001 by 1-way ANOVA followed by Dunnett’s multiple-comparison test with Bonferroni’s correction. n = 5 for CHIR exposure. n = 7–9 movies from 3 mice per condition, with a minimum of 5 PCLS immunostained per group.
Figure 7
Figure 7. Computational modeling of alveologenesis based on membrane/cell shape–tracking data parameterizes epithelial outgrowth.
(A–C) Computational modeling of representative single alveolar buds from (A) control, (B) CHIR-99021–treated (Wnt activator), and (C) XAV-939–treated (Wnt inhibitor) PCLS imaged over time. Area, perimeter, and the area/perimeter ratio (a proxy of complexity) were calculated. Data shown are representative of individual alveoli from n = 4 movies that were analyzed per condition. (D) Proposed schematic of alveologenesis characterized by epithelial outgrowth from a foundational mesenchymal ring.
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