The Drosophila Hippo pathway transcription factor Scalloped and its co-factors alter each other's chromatin binding dynamics and transcription in vivo.

The Hippo pathway is an important regulator of organ growth and cell fate. The major mechanism by which Hippo is known to control transcription is by dictating the nucleo-cytoplasmic shuttling rate of Yorkie, a transcription co-activator, which promotes transcription with the DNA binding protein Scalloped. The nuclear biophysical behavior of Yorkie and Scalloped, and whether this is regulated by the Hippo pathway, remains unexplored. Using multiple live-imaging modalities on Drosophila tissues, we found that Scalloped interacts with DNA on a broad range of timescales, and enrichment of Scalloped at sites of active transcription is mediated by longer DNA dwell times. Further, Yorkie increased Scalloped's DNA dwell time, whereas the repressors Nervous fingers 1 (Nerfin-1) and Tondu-domain-containing growth inhibitor (Tgi) decreased it. Therefore, the Hippo pathway influences transcription not only by controlling nuclear abundance of Yorkie but also by modifying the DNA binding kinetics of the transcription factor Scalloped.

Basal spot junctions of Drosophila epithelial tissues respond to morphogenetic forces and regulate Hippo signaling.

Organ size is controlled by numerous factors including mechanical forces, which are mediated in part by the Hippo pathway. In growing Drosophila epithelial tissues, cytoskeletal tension influences Hippo signaling by modulating the localization of key pathway proteins to different apical domains. Here, we discovered a Hippo signaling hub at basal spot junctions, which form at the basal-most point of the lateral membranes and resemble adherens junctions in protein composition. Basal spot junctions recruit the central kinase Warts via Ajuba and E-cadherin, which prevent Warts activation by segregating it from upstream Hippo pathway proteins. Basal spot junctions are prominent when tissues undergo morphogenesis and are highly sensitive to fluctuations in cytoskeletal tension. They are distinct from focal adhesions, but the latter profoundly influences basal spot junction abundance by modulating the basal-medial actomyosin network and tension experienced by spot junctions. Thus, basal spot junctions couple morphogenetic forces to Hippo pathway activity and organ growth.

The Hippo pathway uses different machinery to control cell fate and organ size.

The Hippo pathway is a conserved signaling network that regulates organ growth and cell fate. One such cell fate decision is that of R8 photoreceptor cells in the Drosophila eye, where Hippo specifies whether cells sense blue or green light. We show that only a subset of proteins that control organ growth via the Hippo pathway also regulate R8 cell fate choice, including the STRIPAK complex, Tao, Pez, and 14-3-3 proteins. Furthermore, key Hippo pathway proteins were primarily cytoplasmic in R8 cells rather than localized to specific membrane domains, as in cells of growing epithelial organs. Additionally, Warts was the only Hippo pathway protein to be differentially expressed between R8 subtypes, while central Hippo pathway proteins were expressed at dramatically lower levels in adult and pupal eyes than in growing larval eyes. Therefore, we reveal several important differences in Hippo signaling in the contexts of organ growth and cell fate.

The regulation of Yorkie, YAP and TAZ: new insights into the Hippo pathway.

The Hippo pathway is a highly conserved signalling pathway that regulates multiple biological processes, including organ size control and cell fate. Since its discovery, genetic and biochemical studies have elucidated several key signalling steps important for pathway activation and deactivation. In recent years, technical advances in microscopy and genome modification have allowed new insights into Hippo signalling to be revealed. These studies have highlighted that the nuclear-cytoplasmic shuttling behaviour of the Hippo pathway transcriptional co-activators Yorkie, YAP and TAZ is far more dynamic than previously appreciated, and YAP and TAZ are also regulated by liquid-liquid phase separation. Here, we review our current understanding of Yorkie, YAP and TAZ regulation, with a focus on recent microscopy-based studies.

The dPix-Git complex is essential to coordinate epithelial morphogenesis and regulate myosin during Drosophila egg chamber development.

How biochemical and mechanical information are integrated during tissue development is a central question in morphogenesis. In many biological systems, the PIX-GIT complex localises to focal adhesions and integrates both physical and chemical information. We used Drosophila melanogaster egg chamber formation to study the function of PIX and GIT orthologues (dPix and Git, respectively), and discovered a central role for this complex in controlling myosin activity and epithelial monolayering. We found that Git's focal adhesion targeting domain mediates basal localisation of this complex to filament structures and the leading edge of migrating cells. In the absence of dpix and git, tissue disruption is driven by contractile forces, as reduction of myosin activators restores egg production and morphology. Further, dpix and git mutant eggs closely phenocopy defects previously reported in pak mutant epithelia. Together, these results indicate that the dPix-Git complex controls egg chamber morphogenesis by controlling myosin contractility and Pak kinase downstream of focal adhesions.

Dynamic Fluctuations in Subcellular Localization of the Hippo Pathway Effector Yorkie In Vivo.

The Hippo pathway is an evolutionarily conserved signaling network that integrates diverse cues to control organ size and cell fate. The central downstream pathway protein in Drosophila is the transcriptional co-activator Yorkie (YAP and TAZ in humans), which regulates gene expression with the Scalloped/TEA domain family member (TEAD) transcription factors [1-8]. A central regulatory step in the Hippo pathway is phosphorylation of Yorkie by the NDR family kinase Warts, which promotes Yorkie cytoplasmic localization by stimulating association with 14-3-3 proteins [9-12]. Numerous reports have purported a static model of Hippo signaling whereby, upon Hippo activation, Yorkie/YAP/TAZ become cytoplasmic and therefore inactive, and upon Hippo repression, Yorkie/YAP/TAZ transit to the nucleus and are active. However, we have little appreciation for the dynamics of Yorkie/YAP/TAZ subcellular localization because most studies have been performed in fixed cells and tissues. To address this, we used live multiphoton microscopy to investigate the dynamics of an endogenously tagged Yorkie-Venus protein in growing epithelial organs. We found that the majority of Yorkie rapidly traffics between the cytoplasm and nucleus, rather than being statically localized in either compartment. In addition, discrete cell populations within the same organ display different rates of Yorkie nucleo-cytoplasmic shuttling. By assessing Yorkie dynamics in warts mutant tissue, we found that the Hippo pathway regulates Yorkie subcellular distribution by regulating its rate of nuclear import. Furthermore, Yorkie's localization fluctuates dramatically throughout the cell cycle, being predominantly cytoplasmic during interphase and, unexpectedly, chromatin enriched during mitosis. Yorkie's association with mitotic chromatin is Scalloped dependent, suggesting a potential role in mitotic bookmarking.

A Drosophila RNAi library modulates Hippo pathway-dependent tissue growth.

Libraries of transgenic Drosophila melanogaster carrying RNA interference (RNAi) constructs have been used extensively to perform large-scale functional genetic screens in vivo. For example, RNAi screens have facilitated the discovery of multiple components of the Hippo pathway, an evolutionarily conserved growth-regulatory network. Here we investigate an important technical limitation with the widely used VDRC KK RNAi collection. We find that approximately 25% of VDRC KK RNAi lines cause false-positive enhancement of the Hippo pathway, owing to ectopic expression of the Tiptop transcription factor. Of relevance to the broader Drosophila community, ectopic tiptop (tio) expression can also cause organ malformations and mask phenotypes such as organ overgrowth. To enhance the use of the VDRC KK RNAi library, we have generated a D. melanogaster strain that will allow researchers to test, in a single cross, whether their genetic screen of interest will be affected by ectopic tio expression.

Warts Opens Up for Activation.

Warts is the central effector kinase of the Hippo growth-control pathway. In this issue of Developmental Cell, by assessing Warts conformation in vivo, Vrabioiu and Struhl (2015) report that the Mob family protein Mats regulates Warts activity allosterically, independent of phosphorylation by Hippo.

An in vivo large-scale chemical screening platform using Drosophila for anti-cancer drug discovery.

Anti-cancer drug development involves enormous expenditure and risk. For rapid and economical identification of novel, bioavailable anti-tumour chemicals, the use of appropriate in vivo tumour models suitable for large-scale screening is key. Using a Drosophila Ras-driven tumour model, we demonstrate that tumour overgrowth can be curtailed by feeding larvae with chemicals that have the in vivo pharmacokinetics essential for drug development and known efficacy against human tumour cells. We then develop an in vivo 96-well plate chemical screening platform to carry out large-scale chemical screening with the tumour model. In a proof-of-principle pilot screen of 2000 compounds, we identify the glutamine analogue, acivicin, a chemical with known activity against human tumour cells, as a potent and specific inhibitor of Drosophila tumour formation. RNAi-mediated knockdown of candidate acivicin target genes implicates an enzyme involved in pyrimidine biosynthesis, CTP synthase, as a possible crucial target of acivicin-mediated inhibition. Thus, the pilot screen has revealed that Drosophila tumours are glutamine-dependent, which is an emerging feature of many human cancers, and has validated the platform as a powerful and economical tool for in vivo chemical screening. The platform can also be adapted for use with other disease models, thus offering widespread applications in drug development.

Homeodomain-interacting protein kinase regulates Hippo pathway-dependent tissue growth.

The Salvador-Warts-Hippo (SWH) pathway is an evolutionarily conserved regulator of tissue growth that is deregulated in human cancer. Upstream SWH pathway components convey signals from neighboring cells via a core kinase cassette to the transcription coactivator Yorkie (Yki). Yki controls tissue growth by modulating activity of transcription factors including Scalloped (Sd). To date, five SWH pathway kinases have been identified, but large-scale phosphoproteome studies suggest that unidentified SWH pathway kinases exist. To identify such kinases, we performed an RNA interference screen and isolated homeodomain-interacting protein kinase (Hipk). Unlike previously identified SWH pathway kinases, Hipk is unique in its ability to promote, rather than repress, Yki activity and does so in parallel to the Yki-repressive kinase, Warts (Wts). Hipk is required for basal Yki activity and is likely to regulate Yki function by promoting its accumulation in the nucleus. Like many SWH pathway proteins, Hipk's function is evolutionarily conserved as its closest human homolog, HIPK2, promotes activity of the Yki ortholog YAP in a kinase-dependent fashion. Further, HIPK2 promotes YAP abundance, suggesting that the mechanism by which HIPK2 regulates YAP has diverged in mammals.