Lgl, aPKC, and Crumbs Regulate the Salvador/Warts/Hippo Pathway through Two Distinct Mechanisms

Background: The Drosophila neoplastic tumor suppressor Lethal (2) giant larvae (Lgl) controls apicobasal cell polarity and proliferation. We have previously shown that lgl(-) clones in the developing eye exhibit ectopic proliferation and suppress apoptosis without affecting apicobasal cell polarity. Ectopic expression of the apical polarity regulators atypical protein kinase C (aPKC) and Crumbs also leads to increased cell proliferation and/or survival. Here we investigate how these cell polarity regulators control proliferation and survival. Results: We report that depletion of lgl in eye epithelial tissue, where polarity is maintained, results in upregulation of targets of the Salvador/Warts/Hippo (SWH) tumor suppressor pathway. Consistent with this, the SWH pathway transcriptional coactivator Yorkie is hyperactivated in Lgl-deficient tissue and is rate limiting for lgl(-) phenotypes. Overexpression of the apical polarity regulators Crumbs or aPKC also leads to ectopic expression of SWH pathway targets without affecting polarity. We show that Lgl depletion or aPKC overexpression results in comislocalization of Hippo and Ras-associated domain family protein (RASSF), consistent with RASSF's ability to block Hippo activation by Salvador. In contrast, Crumbs overexpression leads to mislocalization of Expanded away from the apical cortex, which is predicted to deregulate the pathway. Conclusions: Collectively, our data reveal that the cell polarity regulators Lgl, aPKC, and Crumbs regulate the SWH pathway by two distinct pathways: Lgl acts antagonistically to aPKC to regulate Hippo and RASSF localization, whereas Crumbs regulates Expanded localization. Thus, our study implicates Lgl, aPKC, and Crumbs as regulators of tissue growth via the SWH pathway.