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 4

Mesenchymal ring structure dynamics and contraction are required for alveologenesis.

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Mesenchymal ring structure dynamics and contraction are required for alv...
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.