Poster U17 - Substratum stiffness and Yap dynamics regulate differentiation of bipotent alveolar progenitors

Introduction: The fundamental building block of the lung, the alveolus, is composed of type I (AT1) and type II (AT2) alveolar epithelial cells, which arise from a common bipotent progenitor. AT1 cells are large and flat to facilitate the exchange of gases with the bloodstream, while AT2 cells are cuboidal and produce surfactant to maintain lung patency. Although AT1 cells account for 95% of the surface area of alveoli, there are at least twice as many AT2 cells in the lung1. The molecular signals that generate this precise ratio during the differentiation of bipotent progenitors are unknown. Yap is a component of the Hippo signaling pathway that is essential for AT1-cell fate2 but is also necessary for AT2-cell proliferation3. In response to mechanical forces, Yap translocates from the cytoplasm to the nucleus, where it regulates transcription. Cell differentiation is affected both by Yap nuclear localization as well as the relative rates of Yap nuclear import and export4. Here we use synthetic substrata of different stiffnesses to quantify Yap dynamics as bipotent progenitors differentiate into AT1 cells.

Materials and

Methods: Mice were maintained following protocols approved by the Princeton University Institutional Animal Care and Use Committee (IACUC). Embryos were isolated from timed pregnant dams and lung lobes were obtained by microdissection. Embryonic lungs were digested with dispase to generate a single cell-suspension, which was subjected to magnetic-activated cell sorting to isolate Epcam+/CD45-/CD31- bipotent progenitor cells. Synthetic substrata were prepared using polyacrylamide as previously described5. Isolated cells were cultured for up to 72 hours on synthetic substrata of different stiffness, and assessed every 24 hours by immunofluorescence analysis and in-situ hybridization for markers of differentiation and targets of Yap signaling. Bipotent progenitors isolated from Yap-emiRFP670 embryonic lungs were cultured on synthetic substrata and time-lapse microscopy was performed to track the dynamics of Yap nuclear localization during differentiation.

Results, Conclusions, and Discussions: Bipotent progenitor cells cultured on stiff substrata exhibited a larger projected area and expressed higher levels of the AT1 marker Pdpn. Immunofluorescence analysis revealed that Yap was localized to the nucleus as bipotent progenitor cells spread and differentiated on stiff substrata. Consistently, in-situ hybridization analysis confirmed that Yap-target genes were upregulated in bipotent progenitors cultured on stiff substrata. Time-lapse imaging of Yap-emiRFP670-expressing progenitors revealed that cells undergo cycles of high and low levels of nuclear Yap as they differentiate towards an AT1-cell fate. Our data thus suggest that the nuclear localization of Yap correlates with expression of AT1-cell markers, implying that Yap localization can be used to tune the differentiation of bipotent alveolar progenitors. We are currently using optogenetics to mimic the dynamics of Yap nuclear cycling independently of substratum stiffness to determine if we can control the ratio of AT1 and AT2 cells that differentiate from bipotent progenitors.

References:
1. Yang J, et al, Development, 2015, 143, 54.
2. Shiraishi K, et al, Cell, 2023, 186, 1.
3. Liu, et al, Cell Reports, 2016, 16, 1810.
4. Meyer K, et al, Nat Commun, 2023, 14, 6929.

Acknowledgements (Optional): RECODE NSF 2134935