Merlin, the protein product of the Neurofibromatosis type-2 gene, acts as a tumour suppressor in mice and humans. Merlin is an adaptor protein with a FERM domain and it is thought to transduce a growth-regulatory signal. However, the pathway through which Merlin acts as a tumour suppressor is poorly understood. Merlin, and its function as a negative regulator of growth, is conserved in Drosophila, where it functions with Expanded, a related FERM domain protein. Here, we show that Drosophila Merlin and Expanded are components of the Hippo signalling pathway, an emerging tumour-suppressor pathway. We find that Merlin and Expanded, similar to other components of the Hippo pathway, are required for proliferation arrest and apoptosis in developing imaginal discs. Our genetic and biochemical data place Merlin and Expanded upstream of Hippo and identify a pathway through which they act as tumour-suppressor genes.

Proliferation and apoptosis must be precisely regulated to form organs with appropriate cell numbers and to avoid tumour growth. Here we show that Hippo (Hpo), the Drosophila homologue of the mammalian Ste20-like kinases, MST1/2, promotes proper termination of cell proliferation and stimulates apoptosis during development. hpo mutant tissues are larger than normal because mutant cells continue to proliferate beyond normal tissue size and are resistant to apoptotic stimuli that usually eliminate extra cells. Hpo negatively regulates expression of Cyclin E to restrict cell proliferation, downregulates the Drosophila inhibitor of apoptosis protein DIAP1, and induces the proapoptotic gene head involution defective (hid) to promote apoptosis. The mutant phenotypes of hpo are similar to those of warts (wts), which encodes a serine/threonine kinase of the myotonic dystrophy protein kinase family, and salvador (sav), which encodes a WW domain protein that binds to Wts. We find that Sav binds to a regulatory domain of Hpo that is essential for its function, indicating that Hpo acts together with Sav and Wts in a signalling module that coordinately regulates cell proliferation and apoptosis.

The Hippo tumour suppressor pathway is a conserved signalling pathway that controls organ size. The core of the Hpo pathway is a kinase cascade, which in Drosophila involves the Hpo and Warts kinases that negatively regulate the activity of the transcriptional coactivator Yorkie. Although several additional components of the Hippo pathway have been discovered, the inputs that regulate Hippo signalling are not fully understood. Here, we report that induction of extra F‐actin formation, by loss of Capping proteins A or B, or caused by overexpression of an activated version of the formin Diaphanous, induced strong overgrowth in Drosophila imaginal discs through modulating the activity of the Hippo pathway. Importantly, loss of Capping proteins and Diaphanous overexpression did not significantly affect cell polarity and other signalling pathways, including Hedgehog and Decapentaplegic signalling. The interaction between F‐actin and Hpo signalling is evolutionarily conserved, as the activity of the mammalian Yorkie‐orthologue Yap is modulated by changes in F‐actin. Thus, regulators of F‐actin, and in particular Capping proteins, are essential for proper growth control by affecting Hippo signalling.

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.

Taken together, our data identify a cell-surface molecule that may act as a receptor of the Hippo signaling pathway.