Studies of the Hippo signaling pathway have revealed a highly complex network of interacting regulatory mechanisms that together control pathway output and tissue growth. An overriding theme of this regulation is that it functions to link basic cellular architecture and processes to pathway function, thereby allowing tissue growth to be coordinated with cell shape and tissue morphogenesis. One such mechanism involves the transmembrane protein Crumbs and its partner Expanded, which recruit and activate the core kinases, Tao-1, Hippo, and Warts, to the junctional cortex. In parallel, Kibra and its partner Merlin recruit and activate pathway components at the apicomedial cell cortex. Both mechanisms physically and functionally interact with apical and basolateral polarity components, leading to significant cross talk between these pathways. Importantly, mechanical tension, generated within cells by actomyosin contractile networks and transmitted between cells through intercellular junctions, controls pathway output via multiple mechanisms in distinct cellular domains. In this review, we discuss these regulatory mechanisms, with particular attention to those that function upstream of the core kinases, their organization within epithelial cells, and how this organization allows cells to sense mechanical tension to shape growth in developing tissues.

The Hippo signaling pathway plays a key role in organ size control in normal development and tumorigenesis. While many components of this pathway are well understood, its upstream regulation remains unclear. Among the most enigmatic upstream regulators are the protocadherins Dachsous and Fat. These transmembrane proteins regulate a growth-promoting complex composed of the atypical myosin Dachs, the adaptor protein Dlish, and the palmitoyltransferase Approximated (together termed the core complex). We propose that by default the core complex promotes growth and that Dachsous and Fat, which previously have been thought to act antagonistically, can also function synergistically to repress core complex function and therefore restrict growth. Understanding the molecular mechanisms underlying Dachsous-Fat signaling offers insight into how multicellular organisms precisely control organ size.

The Hippo signaling pathway is conventionally known to restrict tissue growth in animals. Genetic studies have also shown that loss of Hippo pathway components leads to defects in asymmetric cell division in Drosophila neural stem cells, known as neuroblasts. The hallmark of neuroblast division is the asymmetric localization of aPKC/Bazooka (Par-3)/Par-6 complex, termed the Par complex, to the apical cell cortex. However, the localization of the Hippo pathway components in neuroblasts remains unknown. Here, we report that two key activators of the Hippo pathway, Kibra and Salvador, polarize to the apical cortex of mitotic neuroblasts. We show that apical polarity, via the activity of aPKC, and F-actin dynamics synergize to drive Kibra polarization. Together, these results provide further insights into the relationship between apical polarity and Hippo pathway organization and suggest a possible mechanism by which pathway activity is regulated during neuroblast asymmetric division.

The Hippo signaling pathway plays a key role in organ size control in normal development and tumorigenesis. While many components of this pathway are well understood, its upstream regulation remains unclear. Among the most enigmatic upstream regulators are the protocadherins Dachsous and Fat. These transmembrane proteins regulate a growth-promoting complex composed of the atypical myosin Dachs, the adaptor protein Dlish, and the palmitoyltransferase Approximated (together termed the core complex). We propose that by default the core complex promotes growth and that Dachsous and Fat, which previously have been thought to act antagonistically, can also function synergistically to repress core complex function and therefore restrict growth. Understanding the molecular mechanisms underlying Dachsous-Fat signaling offers insight into how multicellular organisms precisely control organ size.

Fluorescent proteins (FPs) are essential tools in biology. The utility of FPs depends on their brightness, photostability, efficient folding, monomeric state, and compatibility with different cellular environments. Despite the proliferation of available FPs, derivatives of the originally identified Aequorea victoria green fluorescent protein often show superior behavior as fusion tags. We recently generated msGFP2, an optimized monomeric superfolder variant of A. victoria GFP. Here, we describe two derivatives of msGFP2. The monomeric variant msYFP2 is a yellow superfolder FP with high photostability. The monomeric variant moxGFP2 lacks cysteines but retains significant folding stability, so it works well in the lumen of the secretory pathway. These new FPs are useful for common imaging applications.

Two protocadherins, Dachsous and Fat, regulate organ growth in Drosophila via the Hippo pathway. Dachsous and Fat bind heterotypically to regulate the abundance and subcellular localization of a “core complex” consisting of Dachs, Dlish, and Approximated. This complex localizes to the junctional cortex where it represses Warts. Dachsous is believed to promote growth by recruiting and stabilizing this complex, while Fat represses growth by promoting its degradation. Here, we examine the functional relationships between the intracellular domains of Dachsous and Fat and the core complex. While Dachsous promotes the accumulation of core complex proteins in puncta, it is not required for their assembly. Indeed, the core complex accumulates maximally in the absence of both Dachsous and Fat. Furthermore, Dachsous represses growth in the absence of Fat by removing the core complex from the junctional cortex. Fat similarly recruits core complex components but promotes their degradation. Our findings reveal that Dachsous and Fat coordinately constrain tissue growth by repressing the core complex.

The Hippo pathway is an evolutionarily conserved regulator of tissue growth that integrates inputs from both polarity and actomyosin networks. An upstream activator of the Hippo pathway, Kibra, localizes at the junctional and medial regions of the apical cortex in epithelial cells, and medial accumulation promotes Kibra activity. Here, we demonstrate that cortical Kibra distribution is controlled by a tug-of-war between apical polarity and actomyosin dynamics. We show that while the apical polarity network, in part via atypical protein kinase C (aPKC), tethers Kibra at the junctional cortex to silence its activity, medial actomyosin flows promote Kibra-mediated Hippo complex formation at the medial cortex, thereby activating the Hippo pathway. This study provides a mechanistic understanding of the relationship between the Hippo pathway, polarity, and actomyosin cytoskeleton, and it offers novel insights into how fundamental features of epithelial tissue architecture can serve as inputs into signaling cascades that control tissue growth, patterning, and morphogenesis.

The Hippo pathway is an evolutionarily conserved regulator of tissue growth. Multiple Hippo signaling components are regulated via proteolytic degradation. However, how these degradation mechanisms are themselves modulated remains unexplored. Kibra is a key upstream pathway activator that promotes its own ubiquitin-mediated degradation upon assembling a Hippo signaling complex. Here, we demonstrate that Hippo complex-dependent Kibra degradation is modulated by cortical tension. Using classical genetic, osmotic, and pharmacological manipulations of myosin activity and cortical tension, we show that increasing cortical tension leads to Kibra degradation, whereas decreasing cortical tension increases Kibra abundance. Our study also implicates Par-1 in regulating Kib abundance downstream of cortical tension. We demonstrate that Par-1 promotes ubiquitin-mediated Kib degradation in a Hippo complex-dependent manner and is required for tension-induced Kib degradation. Collectively, our results reveal a previously unknown molecular mechanism by which cortical tension affects Hippo signaling and provide novel insights into the role of mechanical forces in growth control.

The Hippo pathway is an evolutionarily conserved regulator of tissue growth. Multiple Hippo signaling components are regulated via proteolytic degradation. However, how these degradation mechanisms are themselves modulated remains unexplored. Kibra is a key upstream pathway activator that promotes its own ubiquitin-mediated degradation upon assembling a Hippo signaling complex. Here, we demonstrate that Hippo complex-dependent Kibra degradation is modulated by cortical tension. Using classical genetic, osmotic, and pharmacological manipulations of myosin activity and cortical tension, we show that increasing cortical tension leads to Kibra degradation, whereas decreasing cortical tension increases Kibra abundance. Our study also implicates Par-1 in regulating Kib abundance downstream of cortical tension. We demonstrate that Par-1 promotes ubiquitin-mediated Kib degradation in a Hippo complex-dependent manner and is required for tension-induced Kib degradation. Collectively, our results reveal a previously unknown molecular mechanism by which cortical tension affects Hippo signaling and provide novel insights into the role of mechanical forces in growth control.

The Hippo (Hpo) pathway regulates tissue growth in many animals. Multiple upstream components promote Hpo pathway activity, but the organization of these different inputs, the degree of crosstalk between them, and whether they are regulated in a distinct manner is not well understood. Kibra (Kib) activates the Hpo pathway by recruiting the core Hpo kinase cassette to the apical cortex. Here, we show that the Hpo pathway downregulates Drosophila Kib levels independently of Yorkie-mediated transcription. We find that Hpo signaling complex formation promotes Kib degradation via SCF Slimb -mediated ubiquitination, that this effect requires Merlin, Salvador, Hpo, and Warts, and that this mechanism functions independently of other upstream Hpo pathway activators. Moreover, Kib degradation appears patterned by differences in mechanical tension across the wing. We propose that Kib degradation mediated by Hpo pathway components and regulated by cytoskeletal tension serves to control Kib-driven Hpo pathway activation and ensure optimally scaled and patterned tissue growth.

Ventral furrow formation, the first step in Drosophila gastrulation, is a well-studied example of tissue morphogenesis. Rho1 is highly active in a subset of ventral cells and is required for this morphogenetic event. However, it is unclear whether spatially patterned Rho1 activity alone is sufficient to recapitulate all aspects of this morphogenetic event, including anisotropic apical constriction and coordinated cell movements. Here, using an optogenetic probe that rapidly and robustly activates Rho1 in Drosophila tissues, we show that Rho1 activity induces ectopic deformations in the dorsal and ventral epithelia of Drosophila embryos. These perturbations reveal substantial differences in how ventral and dorsal cells, both within and outside the zone of Rho1 activation, respond to spatially and temporally identical patterns of Rho1 activation. Our results demonstrate that an asymmetric zone of Rho1 activity is not sufficient to recapitulate ventral furrow formation and reveal that additional, ventral-specific factors contribute to the cell- and tissue-level behaviors that emerge during ventral furrow formation.

Examining the subcellular localization of Hippo pathway components has helped elucidate the molecular mechanisms that regulate the pathway. Here we describe methods for performing live imaging of fluorescently tagged Hippo pathway components in Drosophila wing imaginal discs.

The Hippo signaling pathway regulates tissue growth and organ development in many animals, including humans. Pathway activity leads to inactivation of Yorkie (Yki), a transcriptional coactivator that drives expression of growth-promoting genes. In addition, Yki has been shown to recruit chromatin modifiers that enhance chromatin accessibility and thereby enhance Yki function. Here, we asked whether changes in chromatin accessibility that occur during DNA replication could also affect Yki function. We found that depletion of the chromatin assembly complex-1 (CAF-1) complex, a histone chaperone that is required for nucleosome assembly after DNA replication, in the wing imaginal epithelium leads to increased Hippo pathway target gene expression but does not affect expression of other genes. Yki shows greater association with target sites when CAF-1 is depleted and misregulation of target gene expression is Yki-dependent, suggesting that nucleosome assembly competes with Yki for pathway targets post-DNA replication. Consistent with this idea, increased target gene expression is DNA replication dependent and newly replicated chromatin at target sites shows marked nucleosome depletion when CAF-1 function is reduced. These observations suggest a connection between cell cycle progression and Hippo pathway target expression, providing insights into functions of the Hippo pathway in normal and abnormal tissue growth.

The Hippo signaling pathway is an evolutionarily conserved mechanism that controls organ size in animals. Yorkie is well known as a transcriptional coactivator that functions downstream of the Hippo pathway to positively regulate transcription of genes that promote tissue growth. Recent studies have shown that increased myosin activity activates both Yorkie and its vertebrate orthologue YAP, resulting in increased nuclear localization and tissue growth. Here we show that Yorkie also can accumulate at the cell cortex in the apical junctional region. Moreover, Yorkie functions at the cortex to promote activation of myosin through a myosin regulatory light chain kinase, Stretchin-Mlck. This Yorkie function is not dependent on its transcriptional activity and is required for larval and adult tissues to achieve appropriate size. Based on these results, we suggest that Yorkie functions in a feedforward "amplifier" loop that promotes myosin activation, and thereby greater Yorkie activity, in response to tension.

The Hippo pathway is emerging as a key evolutionarily conserved signaling mechanism that controls organ size. Three membrane-associated proteins, Kibra, Merlin, and Expanded, regulate pathway activity, but the precise molecular mechanism by which they function is still poorly understood. Here we provide evidence that Merlin and Kibra activate Hippo signaling in parallel to Expanded at a spatially distinct cellular domain, the medial apical cortex. Merlin and Kibra together recruit the adapter protein Salvador, which in turn recruits the core kinase Hippo. In addition, we show that Crumbs has a dual effect on Hippo signaling. Crumbs promotes the ability of Expanded to activate the pathway but also sequesters Kibra to downregulate Hippo signaling. Together, our findings elucidate the mechanism of Hippo pathway activation by Merlin and Kibra, identify a subcellular domain for Hippo pathway regulation, and demonstrate differential activity of upstream regulators in different subcellular domains.

The large protocadherin Fat functions to promote Hippo pathway activity in restricting tissue growth. Loss of Fat leads to accumulation of the atypical myosin Dachs at the apical junctional region, which in turn promotes growth by inhibiting Warts. We previously identified Approximated (App), a DHHC domain palmitoyltransferase, as a negative regulator of Fat signaling in growth control. We show here that App promotes growth by palmitoylating the intracellular domain of Fat, and that palmitoylation negatively regulates Fat function. Independently, App also recruits Dachs to the apical junctional region through protein protein association, thereby stimulating Dachs's activity in promoting growth. Further, we show that palmitoylation by App functions antagonistically to phosphorylation by Discs-overgrown, which activates Fat. Together, these findings suggest a model in which App promotes Dachs activity by simultaneously repressing Fat via posttranslational modification and recruiting Dachs to the apical junctional region, thereby promoting tissue growth.

Much of the Hippo and planar cell polarity (PCP) signaling mediated by the Drosophila protocadherin Fat depends on its ability to change the subcellular localization, levels and activity of the unconventional myosin Dachs. To better understand this process, we have performed a structure-function analysis of Dachs, and used this to identify a novel and important mediator of Fat and Dachs activities, a Dachs-binding SH3 protein we have named Dlish. We found that Dlish is regulated by Fat and Dachs, that Dlish also binds Fat and the Dachs regulator Approximated, and that Dlish is required for Dachs localization, levels and activity in both wild type and fat mutant tissue. Our evidence supports dual roles for Dlish. Dlish tethers Dachs to the subapical cell cortex, an effect partly mediated by the palmitoyltransferase Approximated under the control of Fat. Conversely, Dlish promotes the Fat-mediated degradation of Dachs.