C29-04 Sustained Yap/Taz Activity Is Required to Maintain AT1 Cell Identity During Alveolar Repair

Abstract Rationale Alveolar type 1 (AT1) cells are thin cells that lay near alveolar capillaries to conduct gas exchange between the lung and blood. During injury, AT1 cells are lost, and during repair the alveolar type 2 (AT2) cell acts as a facultative progenitor cell to restore the AT1 cell populations. Hippo pathway components YAP and TAZ are essential in guiding AT2 to AT1 differentiation as deletion of Yap/Taz in AT2 cells results in loss of AT2 to AT1 differentiation. Recent work has demonstrated that loss of Yap/Taz in Hopx+ cells leads to these cells losing AT1 features and adopting a cuboidal AT2 like shape. Hopx is expressed in both mature and transitional AT1 cells and airway cell populations leading to some questions around the role of Yap/Taz in AT1 cell maturation and maintenance. Herein we tested the hypothesis that sustained Yap/Taz activity is required to maintain mature AT1 cell identity. Methods Tamoxifen was administered to AT1-specific Yap/Taz deleted lineage-tracing mice (RtknCreert2tdTomato Yapfl/fl/Tazfl/fl, referred to as YTAT1del) or wild-type (RtknCreert2tdTomato) 3-weeks prior to challenging the mice with single-dose bleomycin (0.08iU or saline control delivered via intratracheal intubation). Lungs were collected 21-days post-injury and lung injury and fibrosis were assessed by Ashcroft scoring. Cd326+ Epithelial cells were collected and grown in alveolar organoids to assess organoid generation efficiency from the lineage traced AT1 cells. Immunofluorescence analysis of cell-type markers was used to define cell-type specific differentiation from lineage traced AT1 cells. Results AT1-specific deletion of Yap/Taz resulted loss of Hopx+ lineage traced cells in YTAT1del mice (44% Hopx+/Lin ++/-8.4stdev) compared to control mice (89%+/-4.5) in saline controls. Bleomycin injured YTAT1del lungs had lineage traced cells that no longer expressed Hopx (7.1% Hopx+/Lin ++/-3.9) and now expressed Sp-C (30.9% Sp-C+/Lin ++/-11.4) compared to 84% Hopx+/Lin ++/-5.8 and 0.09% Sp-C+/Lin ++/-0.16 expressing cells in control mice. Analysis of fibrotic remodeling showed that YTAT1del mice had worsened repair by Ashcroft score. Alveolar organoids generated from lineage traced WT or YTAT1del mice showed that no organoids formed from lineage traced wild-type cells, whereas alveolar organoids were readily generated (53.4 +/-18.8 per well) from YTAT1del lineage traced cells indicating that these cells lose AT1-like features and can proliferate to generate alveolar organoids. Conclusion These studies demonstrate that persistent Yap/Taz activity is required to maintain AT1 cell identity, and YTAT1del mice have worsened alveolar repair following bleomycin injury suggesting these abnormal AT1 cells do not contribute to repair. This abstract is funded by: Parker B. Francis Foundation