Amotl1 mediates sequestration of the Hippo effector Yap1 downstream of Fat4 to restrict heart growth.

Although in flies the atypical cadherin Fat is an upstream regulator of Hippo signalling, the closest mammalian homologue, Fat4, has been shown to regulate tissue polarity rather than growth. Here we show in the mouse heart that Fat4 modulates Hippo signalling to restrict growth. Fat4 mutant myocardium is thicker, with increased cardiomyocyte size and proliferation, and this is mediated by an upregulation of the transcriptional activity of Yap1, an effector of the Hippo pathway. Fat4 is not required for the canonical activation of Hippo kinases but it sequesters a partner of Yap1, Amotl1, out of the nucleus. The nuclear translocation of Amotl1 is accompanied by Yap1 to promote cardiomyocyte proliferation. We, therefore, identify Amotl1, which is not present in flies, as a mammalian intermediate for non-canonical Hippo signalling, downstream of Fat4. This work uncovers a mechanism for the restriction of heart growth at birth, a process which impedes the regenerative potential of the mammalian heart.

Extracting 3D cell parameters from dense tissue environments: application to the development of the mouse heart.

MOTIVATION: In developmental biology, quantitative tools to extract features from fluorescence microscopy images are becoming essential to characterize organ morphogenesis at the cellular level. However, automated image analysis in this context is a challenging task, owing to perturbations induced by the acquisition process, especially in organisms where the tissue is dense and opaque. RESULTS: We propose an automated framework for the segmentation of 3D microscopy images of highly cluttered environments such as developing tissues. The approach is based on a partial differential equation framework that jointly takes advantage of the nuclear and cellular membrane information to enable accurate extraction of nuclei and cells in dense tissues. This framework has been used to study the developing mouse heart, allowing the extraction of quantitative information such as the cell cycle duration; the method also provides qualitative information on cell division and cell polarity through the creation of 3D orientation maps that provide novel insight into tissue organization during organogenesis.

Quantitative analysis of polarity in 3D reveals local cell coordination in the embryonic mouse heart.

Anisotropies that underlie organ morphogenesis have been quantified in 2D, taking advantage of a reference axis. However, morphogenesis is a 3D process and it remains a challenge to analyze cell polarities in 3D. Here, we have designed a novel procedure that integrates multidisciplinary tools, including image segmentation, statistical analyses, axial clustering and correlation analysis. The result is a sensitive and unbiased assessment of the significant alignment of cell orientations in 3D, compared with a random axial distribution. Taking the mouse heart as a model, we validate the procedure at the fetal stage, when cardiomyocytes are known to be aligned. At the embryonic stage, our study reveals that ventricular cells are already coordinated locally. The centrosome-nucleus axes and the cell division axes are biased in a plane parallel to the outer surface of the heart, with a minor transmural component. We show further alignment of these axes locally in the plane of the heart surface. Our method is generally applicable to other sets of vectors or axes in 3D tissues to map the regions where they show significant alignment.