Abstract 170: Hippo Signaling Regulates Epicardial Derived Cell Fate through Controlling Mechanical Property

Abstract

Epicardium is the outmost layer of heart and regulates heart development by contributing to the heart tissue and secreting signaling molecules. Epicardial-derived cells, EPDC, is a group of multi-potent cells mainly derive into cardiac fibroblasts and smooth muscle cells. Hippo signaling pathway controls organ growth through regulating cell proliferation. Yap, the effector of Hippo pathway has been shown to regulate mechanosensing and cell fate. However, the function of Hippo signaling in EPDC development has not been studied yet. To study the role of Hippo signaling in development of epicardium and EPDC, we used epicardial specific line, Wt1 CreERT2 , to conditionally delete the components of the pathway, Lats1/2. Cre activity was induced at embryonic day (E)11.5 and Lats1/2 CKO hearts exhibited disorganized coronary vasculature. We used genetic lineage tracing approach to track EPDC and revealed increased endothelial cells deriving from EPDC, suggesting that EPDC changed cell fate. We observed that shape of the epicardial cells became rounded with decreased F-actin in Lats1/2 CKO, which suggested that cell tension was reduced. Since mechanical properties of cellular microenvironment are known to regulate cell shape and influence cell fate determination, we hypothesize that Hippo pathway regulates mechanical property to control cell fate in EPDC. Transcription analysis showed that signals regulating cytoskeleton organization were disrupted, which suggests Hippo regulates cytoskeleton gene expression and affects mechanical force that cell sensed autonomously. Proliferation assay using EdU incorporation showed increased proliferation in EPDC of Lats1/2 CKO was observed, which suggests the changes of external tension cell sensed. To recapitulate the changes of external tension, we plated EPDC onto different stiffness substrate. EPDC of Lats1/2 CKO differentiated into endothelial cells on the soft substrate. On the other hand, Lats1/2 CKO did not derive endothelial cells on the hard substrate and wild type EPDC did not differentiate into endothelial cells on neither soft nor hard substrate. Taken together, our results suggested that Hippo pathway affected EPDC fate determination through controlling internal and external mechanical cues.