Epithelial outgrowth through mesenchymal rings drives lung alveologenesis
Nicholas M. Negretti, Yeongseo Son, Philip Crooke, Erin J. Plosa, John T. Benjamin, Christopher S. Jetter, Claire Bunn, Nicholas Mignemi, John Marini, Alice N. Hackett, Meaghan Ransom, Shriya Garg, David Nichols, Susan H. Guttentag, Heather H. Pua, Timothy S. Blackwell, William Zacharias, David B. Frank, John A. Kozub, Anita Mahadevan-Jansen, Evan Krystofiak, Jonathan A. Kropski, Christopher V.E. Wright, Bryan Millis, Jennifer M.S. Sucre
Nicholas M. Negretti, Yeongseo Son, Philip Crooke, Erin J. Plosa, John T. Benjamin, Christopher S. Jetter, Claire Bunn, Nicholas Mignemi, John Marini, Alice N. Hackett, Meaghan Ransom, Shriya Garg, David Nichols, Susan H. Guttentag, Heather H. Pua, Timothy S. Blackwell, William Zacharias, David B. Frank, John A. Kozub, Anita Mahadevan-Jansen, Evan Krystofiak, Jonathan A. Kropski, Christopher V.E. Wright, Bryan Millis, Jennifer M.S. Sucre
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Research Article Development Pulmonology

Epithelial outgrowth through mesenchymal rings drives lung alveologenesis

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.

Authors

Nicholas M. Negretti, Yeongseo Son, Philip Crooke, Erin J. Plosa, John T. Benjamin, Christopher S. Jetter, Claire Bunn, Nicholas Mignemi, John Marini, Alice N. Hackett, Meaghan Ransom, Shriya Garg, David Nichols, Susan H. Guttentag, Heather H. Pua, Timothy S. Blackwell, William Zacharias, David B. Frank, John A. Kozub, Anita Mahadevan-Jansen, Evan Krystofiak, Jonathan A. Kropski, Christopher V.E. Wright, Bryan Millis, Jennifer M.S. Sucre

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Figure 1

Precision-cut lung slices (PCLS) model of alveologenesis ex vivo.

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Precision-cut lung slices (PCLS) model of alveologenesis ex vivo. (A) Fi...
(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.