ABSTRACT
The Hippo signaling pathway is widely considered a master regulator of organ growth because of the prominent overgrowth phenotypes caused by experimental manipulation of its activity. Contrary to this model, we show here that removing Hippo transcriptional output did not impair the ability of the mouse liver and Drosophila eyes to grow to their normal size. Moreover, the transcriptional activity of the Hippo pathway effectors Yap/Taz/Yki did not correlate with cell proliferation, and hyperactivation of these effectors induced gene expression programs that did not recapitulate normal development. Concordantly, a functional screen in Drosophila identified several Hippo pathway target genes that were required for ectopic overgrowth but not normal growth. Thus, Hippo signaling does not instruct normal growth, and the Hippo-induced overgrowth phenotypes are caused by the activation of abnormal genetic programs.
Subject(s)
Drosophila melanogaster , Eye , Gene Expression Regulation, Developmental , Hippo Signaling Pathway , Liver , Transcription, Genetic , Transcriptional Coactivator with PDZ-Binding Motif Proteins , YAP-Signaling Proteins , Animals , Mice , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Eye/embryology , Hippo Signaling Pathway/genetics , Liver/embryology , Organ Size , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Trans-Activators/genetics , Transcriptional Coactivator with PDZ-Binding Motif Proteins/metabolism , YAP-Signaling Proteins/metabolismABSTRACT
Chronic liver disease is the result of long term exposure to viruses or toxins such as alcohol, fat and drugs, and forms the basis for the development of liver fibrosis and primary liver cancer. In vitro and in vivo models are key to study the pathways involved in chronic liver disease and for the development of therapeutics. 3D co-culture systems are becoming the in vitro standard, which requires freshly isolated primary hepatic cells. We developed a novel isolation method to simultaneously isolate liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs) and hepatic stellate cells (HSCs). The method exploits the scavenging activity of LSECs, the phagocytic capacity of KCs and the retinoid content of HSCs in vivo to enable direct processing by fluorescence-activated cell sorting without additional antibody binding and washing steps. UFACS3, for UV-FACS-based isolation of 3 non-parenchymal liver cell types, yields functional and pure LSECs (98 ± 1%), KCs (98 ± 1%) and HSCs (97 ± 3%), with less hands-on time from healthy and diseased rodent livers. This novel approach allows a fast and effective combined isolation of sinusoidal cells for further analysis.
