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The Hippo pathway plays a key role in controlling organ growth in many animal species and its deregulation is associated with different types of cancer. Understanding the regulation of the Hippo pathway and discovering upstream regulators is thus a major quest. Interestingly, while the core of the Hippo pathway contains a highly conserved kinase cascade, different components have been identified as upstream regulators in Drosophila and vertebrates. However, whether the regulation of the Hippo pathway is indeed different between Drosophila and vertebrates or whether these differences are due to our limited analysis of these components in different organisms is not known. Here we show that the mouse Fat4 cadherin, the ortholog of the Hippo pathway regulator Fat in Drosophila, does not apparently regulate the Hippo pathway in the murine liver. In fact, we uncovered an evolutionary shift in many of the known upstream regulators at the base of the arthropod lineage. In this evolutionary transition, Fat and the adaptor protein Expanded gained novel domains that connected them to the Hippo pathway, whereas the cell-adhesion receptor Echinoid evolved as a new protein. Subsequently, the junctional adaptor protein Angiomotin (Amot) was lost and the downstream effector Yap lost its PDZ-binding motif that interacts with cell junction proteins. We conclude that fundamental differences exist in the upstream regulatory mechanisms of Hippo signaling between Drosophila and vertebrates.

How organ size is controlled in mammals is not currently understood. In Drosophila the Hippo signaling pathway functions to suppress growth in imaginal discs and has been suggested to control organ size. To investigate the role of hippo signaling in regulation of mammalian organ size we have generated conditional alleles of Sav1 , mst1 , and mst2 , orthologs of Drosophila Salvador and hippo , respectively. Specific deletion of both mst1 and mst2 in hepatocytes results in significantly enlarged livers due to excessive proliferation. By the age of 5–6 months, mst1/2 conditional mutant livers have multiple foci of liver tumors, indicating that the combined activities of mst1 and mst2 act as redundant tumor suppressors in hepatocytes. Similar findings were obtained with liver-specific deletion of Sav1 , a second core Hippo signaling component that facilitates activation of mst1 and mst2 . Tumors from sav1 mutants exhibited varied morphology, suggesting a mixed-lineage origin of tumor-initiating cells. Transcriptional profiling of liver tissues from both mst1/2 and sav1 conditional mutants revealed a network of Hippo signaling regulated genes with specific enrichment for genes involved in immune and inflammatory responses. Histological and immunological characterization of mst1/2 double mutant liver tissues revealed abundant accumulation of adult facultative stem cells termed oval cells in periductal regions. Because oval cells induction is commonly associated with liver injury and tumor formation, it is likely that these cells contribute to the enlarged livers and hepatomas that we observe in sav1 and mst1/2 mutants. Taken together, our results demonstrate that the Hippo signaling pathway is a critical regulator of mammalian liver growth and a potent suppressor of liver tumor formation.

Defects in apical-basal cell polarity and abnormal expression of cell polarity determinants are often associated with cancer in vertebrates. In Drosophila , abnormal expression of apical-basal determinants can cause neoplastic phenotypes, including loss of cell polarity and overproliferation. However, the pathways through which apical-basal polarity determinants affect growth are poorly understood. Here, we investigated the mechanism by which the apical determinant Crumbs (Crb) affects growth in Drosophila imaginal discs. Overexpression of Crb causes severe overproliferation, and we found that loss of Crb similarly results in overgrowth of imaginal discs. Crb gain and loss of function caused defects in Hippo signaling, a key signaling pathway that controls tissue growth in Drosophila and mammals. Manipulation of Crb levels caused the up-regulation of Hippo target genes, genetically interacted with known Hippo pathway components, and required Yorkie, a transcriptional coactivator that acts downstream in the Hippo pathway, for target gene induction and overgrowth. Interestingly, Crb regulates growth and cell polarity through different motifs in its intracellular domain. A juxtamembrane FERM domain-binding motif is responsible for growth regulation and induction of Hippo target gene expression, whereas Crb uses a PDZ-binding motif to form a complex with other polarity factors. The Hippo pathway component Expanded, an apically localized adaptor protein, is mislocalized in both crb mutant cells and Crb overexpressing tissues, whereas the other Hippo pathway components, Fat and Merlin, are unaffected. Taken together, our data show that Crb regulates growth through Hippo signaling, and thus identify Crb as a previously undescribed upstream input into the Hippo pathway.