yorkie: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - yorkie

Synonyms - CG4005

Cytological map position - 60B7--8

Function - transcriptional coactivator

Keywords - Hippo/Warts pathway, negative regulation of cell proliferation, cell death, yes-associate protein homolog, oncogene

Symbol - yki

FlyBase ID: FBgn0034970

Genetic map position - 2R

Classification - WW domain, PDZ binding motif

Cellular location - presumably nuclear and cytoplasmic

NCBI links: EntrezGene
Recent literature
Umegawachi, T., Yoshida, H., Koshida, H., Yamada, M., Ohkawa, Y., Sato, T., Suyama, M., Krause, H. M. and Yamaguchi, M. (2017). Control of tissue size and development by a regulatory element in the yorkie 3'UTR. Am J Cancer Res 7(3): 673-687. PubMed ID: 28401020
Regulation of the Hippo pathway via phosphorylation of Yorkie (Yki), the Drosophila homolog of human Yes-associated protein 1, is conserved from Drosophila to humans. Overexpression of a non-phosphorylatable form of Yki induces severe overgrowth in adult fly eyes. This study shows that yki mRNA associates with microsomal fractions and forms foci that partially colocalize to processing bodies in the vicinity of endoplasmic reticulum. This localization is dependent on a stem-loop (SL) structure in the 3' untranslated region of yki. Surprisingly, expression of SL deleted yki in eye imaginal discs also results in severe overgrowth phenotypes. When the structure of the SL is disrupted, Yki protein levels increase without a significant effect on RNA levels. When the SL is completely removed, protein levels drastically increase, but in this case, due to increased RNA stability. In the latter case, it was shown that the increased RNA accumulation is due to removal of a putative miR-8 seed sequence in the SL. These data demonstrate the function of two novel regulatory mechanisms, both controlled by the yki SL element, that are essential for proper Hippo pathway mediated growth regulation.
Tsai, C. R., Anderson, A. E., Burra, S., Jo, J. and Galko, M. J. (2017). Yorkie regulates epidermal wound healing in Drosophila larvae independently of cell proliferation and apoptosis. Dev Biol 427(1):61-71. PubMed ID: 28514643
Yorkie (Yki), the transcriptional co-activator of the Hippo signaling pathway, has well-characterized roles in balancing apoptosis and cell division during organ growth control. Yki is also required in diverse tissue regenerative contexts. In most cases this requirement reflects its well-characterized roles in balancing apoptosis and cell division. Whether Yki has repair functions outside of the control of cell proliferation, death, and growth is not clear. This study shows that Yki and Scalloped (Sd) are required for epidermal wound closure in the Drosophila larval epidermis. Using a GFP-tagged Yki transgene, Yki was shown to transiently translocate to some epidermal nuclei upon wounding. Genetic analysis strongly suggests that Yki interacts with the known wound healing pathway, Jun N-terminal kinase (JNK), but not with Platelet Derived Growth Factor/Vascular-Endothelial Growth Factor receptor (Pvr). Yki likely acts downstream of or parallel to JNK signaling and does not appear to regulate either proliferation or apoptosis in the larval epidermis during wound repair. Analysis of actin structures after wounding suggests that Yki and Sd promote wound closure through actin regulation. In sum, this study found that Yki regulates an epithelial tissue repair process independently of its previously documented roles in balancing proliferation and apoptosis.
Hsu, T. H., Yang, C. Y., Yeh, T. H., Huang, Y. C., Wang, T. W. and Yu, J. Y. (2017). The Hippo pathway acts downstream of the Hedgehog signaling to regulate follicle stem cell maintenance in the Drosophila ovary. Sci Rep 7(1): 4480. PubMed ID: 28667262
The Hippo pathway is conserved and plays important roles in organ size control. The core components of the Hippo pathway are two kinases Hippo (Hpo), Warts (Wts), and a transcription-co-activator Yorkie (Yki). Yki activity is regulated by phosphorylation, which affects its nuclear localization and stability. To determine the role of the Hippo pathway in stem cells, this study examined follicle stem cells (FSCs) in the Drosophila ovary. Yki is detected in the nucleus of FSCs. Knockdown of yki in the follicle cell lineage leads to a disruption of the follicular epithelium. Mitotic clones of FSCs mutant for hpo or wts are maintained in the niche and tend to replace the other FSCs, and FSCs mutant for yki are rapidly lost, demonstrating that the Hippo pathway is both required and sufficient for FSC maintenance. Using genetic interaction analyses, the Hedgehog pathway was demonstrated to act upstream of the Hippo pathway in regulating FSC maintenance. The nuclear localization of Yki is enhanced when the Hedgehog signaling is activated. Furthermore, a constitutively active but not a wild-type Yki promotes FSC maintenance as activation of the Hedgehog signaling does, suggesting that the Hedgehog pathway regulates Yki through a post-translational mechanism in maintaining FSCs.
Huang, J., Reilein, A. and Kalderon, D. (2017). Yorkie and Hedgehog independently restrict BMP production in Escort cells to permit germline differentiation in the Drosophila ovary. Development. PubMed ID: 28619819
Multiple signaling pathways guide the behavior and differentiation of both germline stem cells (GSCs) and somatic stem cells (FSCs) in the Drosophila germarium, necessitating careful control of signal generation, range and responses. Signal integration involves Escort Cells (ECs), which promote differentiation of the GSC derivatives they envelop, provide niche signals for FSCs and derive directly from FSCs in adults. Hedgehog (Hh) signaling induces the Hippo pathway effector Yorkie (Yki) to promote proliferation and maintenance of FSCs but Hh also signals to ECs, which are quiescent. This study shows that in ECs both Hh and Yki limit production of BMP ligands to allow germline differentiation. Loss of Yki produced a more severe germarial phenotype than loss of Hh signaling and principally induced a different BMP ligand. Moreover, Yki activity reporters and epistasis tests showed that Yki does not mediate the key actions of Hh signaling in ECs. Thus, both the coupling and output of Hh and Yki signaling pathways differ between FSCs and ECs despite their proximity and the fact that FSCs give rise directly to ECs.
Song, S., Herranz, H. and Cohen, S. M. (2017). The chromatin remodeling BAP complex limits tumor promoting activity of the Hippo pathway effector Yki to prevent neoplastic transformation in Drosophila epithelia. Dis Model Mech [Epub ahead of print]. PubMed ID: 28754838
SWI/SNF chromatin remodeling complexes are mutated in many human cancers. This report makes use of a Drosophila genetic model for epithelial tumor formation to explore the tumor suppressive role of SWI/SNF complex proteins. Members of the BAP complex (a core complex containing Brahma, Snr1, Bap111, Moira (Mor) core associated with Osa) exhibit tumor suppressor activity in tissue overexpressing the Yorkie (Yki) proto-oncogene, but not in tissue overexpressing EGFR. The BAP complex has been reported to serve as a Yki-binding cofactor to support Yki target expression. However, this study observed that depletion of BAP leads to ectopic expression of Yki targets both autonomously and non-autonomously, suggesting additional indirect effects. Evidence is provided that BAP complex depletion causes upregulation of the Wingless and Dpp morphogens to promote tumor formation in cooperation with Yki.
Fletcher, G. C., Diaz-de-la-Loza, M. D., Borreguero-Munoz, N., Holder, M., Aguilar-Aragon, M. and Thompson, B. J. (2018). Mechanical strain regulates the Hippo pathway in Drosophila. Development 145(5). PubMed ID: 29440303
Animal cells are thought to sense mechanical forces via the transcriptional co-activators YAP (or YAP1) and TAZ (or WWTR1), the sole Drosophila homolog of which is named Yorkie (Yki). In mammalian cells in culture, artificial mechanical forces induce nuclear translocation of YAP and TAZ. This study shows that physiological mechanical strain can also drive nuclear localisation of Yki and activation of Yki target genes in the Drosophila follicular epithelium. Mechanical strain activates Yki by stretching the apical domain, reducing the concentration of apical Crumbs, Expanded, Kibra and Merlin, and reducing apical Hippo kinase dimerisation. Overexpressing Hippo kinase to induce ectopic activation in the cytoplasm is sufficient to prevent Yki nuclear localisation even in flattened follicle cells. Conversely, blocking Hippo signalling in warts clones causes Yki nuclear localisation even in columnar follicle cells. No evidence was found for involvement of other pathways, such as Src42A kinase, in regulation of Yki. Finally, the results in follicle cells appear generally applicable to other tissues, as nuclear translocation of Yki is also readily detectable in other flattened epithelial cells such as the peripodial epithelium of the wing imaginal disc, where it promotes cell flattening.
Yu, J. and Pan, D. (2018). Validating upstream regulators of Yorkie activity in Hippo signaling through scalloped-based genetic epistasis. Development 145(4). PubMed ID: 29467233
Genetic studies in Drosophila have been instrumental in characterizing the Hippo pathway, which converges on the co-activator Yorkie to regulate target gene transcription. A routinely used strategy to interrogate upstream regulators of Yorkie involves the examination of selected Hippo target genes upon loss or gain of function of a suspected pathway regulator. A caveat with this strategy is that aberrant expression of a given Hippo target per se does not distinguish whether it is caused by changes in Yorkie or Yorkie-independent inputs converging on the same target gene. Building on previous findings that the DNA-binding transcription factor Scalloped mediates both Yorkie overexpression and loss-of-function phenotypes yet is itself dispensable for normal eye development, this study describes a simple strategy to distinguish these possibilities by analyzing double-mutant clones of scalloped and a suspected Yorkie regulator. Proof of principle is provided that this strategy can be used effectively to validate canonical Yorkie regulators and to exclude proteins that impact target expression independent of Yorkie. The described methodology and reagents should facilitate efforts to assess the expanding repertoire of proteins implicated in regulation of Yorkie activity.
Gerlach, S. U., Eichenlaub, T. and Herranz, H. (2018). Yorkie and JNK control tumorigenesis in Drosophila cells with cytokinesis failure. Cell Rep 23(5): 1491-1503. PubMed ID: 29719260
Cytokinesis failure may result in the formation of polyploid cells, and subsequent mitosis can lead to aneuploidy and tumor formation. Tumor suppressor mechanisms limiting the oncogenic potential of these cells have been described. However, the universal applicability of these tumor-suppressive barriers remains controversial. This study used Drosophila epithelial cells to investigate the consequences of cytokinesis failure in vivo. Cleavage defects trigger the activation of the JNK pathway, leading to downregulation of the inhibitor of apoptosis DIAP1 and programmed cell death. Yorkie overcomes the tumor-suppressive role of JNK and induces neoplasia. Yorkie regulates the cell cycle phosphatase Cdc25/string, which drives tumorigenesis in a context of cytokinesis failure. These results highlight the functional significance of the JNK pathway in epithelial cells with defective cytokinesis and elucidate a mechanism used by emerging tumor cells to bypass this tumor-suppressive barrier and develop into tumors.
Manning, S. A., Dent, L. G., Kondo, S., Zhao, Z. W., Plachta, N. and Harvey, K. F. (2018). Dynamic fluctuations in subcellular localization of the Hippo pathway effector Yorkie in vivo. Curr Biol 28(10): 1651-1660.e1654. PubMed ID: 29754899
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. 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. 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, there is 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, live multiphoton microscopy imaging was used to investigate the dynamics of an endogenously tagged Yorkie-Venus protein in growing epithelial organs. The majority of Yorkie was found to rapidly traffic 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, this study 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.
Sander, M., Eichenlaub, T. and Herranz, H. (2018). Oncogenic cooperation between Yorkie and the conserved microRNA miR-8 in the wing disc of Drosophila. Development. PubMed ID: 29945869
Tissue growth has to be carefully controlled to generate well-functioning organs. microRNAs are small noncoding RNAs that modulate the activity of target genes and play a pivotal role in animal development. Understanding the functions of microRNAs in development requires the identification of their target genes. This study finds that the conserved microRNA miR-8/miR-200 controls tissue growth and homeostasis in the Drosophila wing imaginal disc. Upregulation of miR-8 causes the repression of Yorkie, the effector of the Hippo pathway in Drosophila, and reduces tissue size. Remarkably, coexpression of Yorkie and miR-8 causes the formation of neoplastic tumors. Upregulation of miR-8 represses the growth inhibitor brinker, and depletion of brinker cooperates with Yorkie in the formation of neoplastic tumors. Hence, miR-8 modulates a positive growth regulator, Yorkie, and a negative growth regulator, brinker. Deregulation of this network can result in the loss of tissue homeostasis and the formation of tumors.
Mach, J., Atkins, M., Gajewski, K. M., Mottier-Pavie, V., Sansores-Garcia, L., Xie, J., Mills, R. A., Kowalczyk, W., Van Huffel, L., Mills, G. B. and Halder, G. (2018). Modulation of the Hippo pathway and organ growth by RNA processing proteins. Proc Natl Acad Sci U S A. PubMed ID: 30257938
The Hippo tumor-suppressor pathway regulates organ growth, cell proliferation, and stem cell biology. Defects in Hippo signaling and hyperactivation of its downstream effectors-Yorkie (Yki) in Drosophila and YAP/TAZ in mammals-result in progenitor cell expansion and overgrowth of multiple organs and contribute to cancer development. Deciphering the mechanisms that regulate the activity of the Hippo pathway is key to understanding its function and for therapeutic targeting. However, although the Hippo kinase cascade and several other upstream inputs have been identified, the mechanisms that regulate Yki/YAP/TAZ activity are still incompletely understood. To identify new regulators of Yki activity, Drosophila was screened for suppressors of tissue overgrowth and Yki activation caused by overexpression of atypical protein kinase C (aPKC), a member of the apical cell polarity complex. This screen identified mutations in the heterogeneous nuclear ribonucleoprotein Hrb27C that strongly suppressed the tissue defects induced by ectopic expression of aPKC. Hrb27C was required for aPKC-induced tissue growth and Yki target gene expression but did not affect general gene expression. Genetic and biochemical experiments showed that Hrb27C affects Yki phosphorylation. Other RNA-binding proteins known to interact with Hrb27C for mRNA transport in oocytes were also required for normal Yki activity, although they suppressed Yki output. Based on the known functions of Hrb27C, it is concluded that Hrb27C-mediated control of mRNA splicing, localization, or translation is essential for coordinated activity of the Hippo pathway.
Pan, Y., Alegot, H., Rauskolb, C. and Irvine, K. D. (2018). The dynamics of hippo signaling during Drosophila wing development. Development. PubMed ID: 30254143
Tissue growth needs to be properly controlled for organs to reach their correct size and shape, but the mechanisms that control growth during normal development are not fully understood. This study reports that the activity of the Hippo signaling transcriptional activator Yorkie gradually decreases in the central region of the developing Drosophila wing disc. Spatial and temporal changes in Yorkie activity can be explained by changes in cytoskeletal tension and biomechanical regulators of Hippo signaling. These changes in cellular biomechanics correlate with changes in cell density, and experimental manipulations of cell density are sufficient to alter biomechanical Hippo signaling and Yorkie activity. The pattern of Yorkie activity in older discs was also related to patterns of cell proliferation. These results establish that spatial and temporal patterns of Hippo signaling occur during wing development, that these patterns depend upon cell-density modulated tissue mechanics, and that they contribute to the regulation of wing cell proliferation.
Francis, D., Chanana, B., Fernandez, B., Gordon, B., Mak, T. and Palacios, I. M. (2019). YAP/Yorkie in the germline modulates the age-related decline of germline stem cells and niche cells. PLoS One 14(4): e0213327. PubMed ID: 30943201
The properties and behaviour of stem cells rely heavily on signaling from the local microenvironment. At the apical end of Drosophila testis, self-renewal and differentiation of germline stem cells (GSCs) are tightly controlled by distinct somatic cells that comprise a specialised stem cell niche known as the hub. The hub maintains GSC homeostasis through adhesion and cell signaling. The Salvador/Warts/Hippo (SWH) pathway, which suppresses the transcriptional co-activator YAP/Yki via a kinase cascade, is a known regulator of stem cell proliferation and differentiation. This study shows that increasing YAP/Yki expression in the germline, as well as reducing Warts levels, blocks the decrease of GSC numbers observed in aging flies, with only a small increase on their proliferation. An increased expression of YAP/Yki in the germline or a reduction in Warts levels also stymies an age-related reduction in hub cell number, suggesting a bilateral relationship between GSCs and the hub. Conversely, RNAi-based knockdown of YAP/Yki in the germline leads to a significant drop in hub cell number, further suggesting the existence of such a SC-to-niche relationship. All together, these data implicate the SWH pathway in Drosophila GSC maintenance and raise questions about its role in stem cell homeostasis in aging organisms.
Borreguero-Munoz, N., Fletcher, G. C., Aguilar-Aragon, M., Elbediwy, A., Vincent-Mistiaen, Z. I. and Thompson, B. J. (2019). The Hippo pathway integrates PI3K-Akt signals with mechanical and polarity cues to control tissue growth. PLoS Biol 17(10): e3000509. PubMed ID: 31613895
The Hippo signalling pathway restricts cell proliferation in animal tissues by inhibiting Yes-associated protein (YAP or YAP1) and Transcriptional Activator with a PDZ domain (TAZ), coactivators of the Scalloped (Sd or TEAD) DNA-binding transcription factor. Drosophila has a single YAP/TAZ homolog named Yorkie (Yki) that is regulated by Hippo pathway signalling in response to epithelial polarity and tissue mechanics during development. This study shows that Yki translocates to the nucleus to drive Sd-mediated cell proliferation in the ovarian follicle cell epithelium in response to mechanical stretching caused by the growth of the germline. Importantly, mechanically induced Yki nuclear localisation also requires nutritionally induced insulin/insulin-like growth factor 1 (IGF-1) signalling (IIS) via phosphatidyl inositol-3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1 or PDPK1), and protein kinase B (Akt or PKB) in the follicular epithelium. Similar results were obtained in the developing Drosophila wing, where Yki becomes nuclear in the mechanically stretched cells of the wing pouch during larval feeding, which induces IIS, but translocates to the cytoplasm upon cessation of feeding in the third instar stage. Inactivating Akt prevents nuclear Yki localisation in the wing disc, while ectopic activation of the insulin receptor, PI3K, or Akt/PKB is sufficient to maintain nuclear Yki in mechanically stimulated cells of the wing pouch even after feeding ceases. Finally, IIS also promotes YAP nuclear localisation in response to mechanical cues in mammalian skin epithelia. Thus, the Hippo pathway has a physiological function as an integrator of epithelial cell polarity, tissue mechanics, and nutritional cues to control cell proliferation and tissue growth in both Drosophila and mammals.
Skouloudaki, K., Christodoulou, I., Khalili, D., Tsarouhas, V., Samakovlis, C., Tomancak, P., Knust, E. and Papadopoulos, D. K. (2019). Yorkie controls tube length and apical barrier integrity during airway development. J Cell Biol. PubMed ID: 31315941
Epithelial organ size and shape depend on cell shape changes, cell-matrix communication, and apical membrane growth. The Drosophila melanogaster embryonic tracheal network is an excellent model to study these processes. This study shows that the transcriptional coactivator of the Hippo pathway, Yorkie (YAP/TAZ in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water tightness of tracheal tubes by transcriptional regulation of the delta-aminolevulinate synthase gene (Alas). It is concluded that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality.
Sidor, C., Borreguero-Munoz, N., Fletcher, G. C., Elbediwy, A., Guillermin, O. and Thompson, B. J. (2019). Mask family proteins ANKHD1 and ANKRD17 regulate YAP nuclear import and stability. Elife 8. PubMed ID: 31661072
Mask family proteins were discovered in Drosophila to promote the activity of the transcriptional coactivator Yorkie (Yki), the sole fly homolog of mammalian YAP (YAP1) and TAZ (WWTR1). The molecular function of Mask, or its mammalian homologs Mask1 (ANKHD1) and Mask2 (ANKRD17), remains unclear. Mask family proteins contain two ankyrin repeat domains that bind Yki/YAP as well as a conserved nuclear localisation sequence (NLS) and nuclear export sequence (NES), suggesting a role in nucleo-cytoplasmic transport. This study shows that Mask acts to promote nuclear import of Yki, and that addition of an ectopic NLS to Yki is sufficient to bypass the requirement for Mask in Yki-driven tissue growth. Mammalian Mask1/2 proteins also promote nuclear import of YAP, as well as stabilising YAP and driving formation of liquid droplets. Mask1/2 and YAP normally colocalise in a granular fashion in both nucleus and cytoplasm, and are co-regulated during mechanotransduction.
Schaub, C., Rose, M. and Frasch, M. (2019). Yorkie and JNK revert syncytial muscles into myoblasts during Org-1-dependent lineage reprogramming. J Cell Biol. PubMed ID: 31591186
Lineage reprogramming has received increased research attention since it was demonstrated that lineage-restricted transcription factors can be used in vitro for direct reprogramming. The ventral longitudinal musculature of the adult Drosophila heart has been reported to arise in vivo by direct lineage reprogramming from larval alary muscles, a process that starts with the dedifferentiation and fragmentation of syncytial muscle cells into mononucleate myoblasts and depends on Org-1 (Drosophila Tbx1). This study sheds light on the events occurring downstream of Org-1 in this first step of transdifferentiation and shows that alary muscle lineage-specific activation of Yorkie plays a key role in initiating the dedifferentiation and fragmentation of these muscles. An additional necessary input comes from active dJNK signaling, which contributes to the activation of Yorkie and furthermore activates dJun. The synergistic activities of the Yorkie/Scalloped and dJun/dFos transcriptional activators subsequently initiate alary muscle fragmentation as well as up-regulation of Myc and piwi, both crucial for lineage reprogramming.
Gao, Y., Zhang, X., Xiao, L., Zhai, C., Yi, T., Wang, G., Wang, E., Ji, X., Hu, L., Shen, G. and Wu, S. (2019). Usp10 modulates the Hippo pathway by deubiquitinating and stabilizing the transcriptional coactivator Yorkie. Int J Mol Sci 20(23). PubMed ID: 31795326
The Hippo signaling pathway is an evolutionarily conserved regulator that plays important roles in organ size control, homeostasis, and tumorigenesis. As the key effector of the Hippo pathway, Yorkie (Yki) binds to transcription factor Scalloped (Sd) and promotes the expression of target genes, leading to cell proliferation and inhibition of apoptosis. Thus, it is of great significance to understand the regulatory mechanism for Yki protein turnover. This study provides evidence that the deubiquitinating enzyme ubiquitin-specific protease 10 (Usp10) binds Yki to counteract Yki ubiquitination and stabilize Yki protein in Drosophila S2 cells. The results in Drosophila wing discs indicate that silence of Usp10 decreases the transcription of target genes of the Hippo pathway by reducing Yki protein. In vivo functional analysis ulteriorly showed that Usp10 upregulates the Yki activity in Drosophila eyes. These findings uncover Usp10 as a novel Hippo pathway modulator and provide a new insight into the regulation of Yki protein stability and activity.
Chen, H. J., Li, Q., Nirala, N. K. and Ip, Y. T. (2020). The Snakeskin-Mesh Complex of Smooth Septate Junction Restricts Yorkie to Regulate Intestinal Homeostasis in Drosophila. Stem Cell Reports. PubMed ID: 32330445
Tight junctions in mammals and septate junctions in insects are essential for epithelial integrity. This study shows that, in the Drosophila intestine, smooth septate junction proteins provide barrier and signaling functions. During an RNAi screen for genes that regulate adult midgut tissue growth, it was found that loss of two smooth septate junction components, Snakeskin and Mesh, caused a hyperproliferation phenotype. By examining epitope-tagged endogenous Snakeskin and Mesh, it was demonstrated that the two proteins are present in the cytoplasm of differentiating enteroblasts and in cytoplasm and septate junctions of mature enterocytes. In both enteroblasts and enterocytes, loss of Snakeskin and Mesh causes Yorkie-dependent expression of the JAK-STAT pathway ligand Upd3, which in turn promotes proliferation of intestinal stem cells. Snakeskin and Mesh form a complex with each other, with other septate junction proteins and with Yorkie. Therefore, the Snakeskin-Mesh complex has both barrier and signaling function to maintain stem cell-mediated tissue homeostasis.

Coordination between cell proliferation and cell death is essential to maintain homeostasis in multicellular organisms. In Drosophila, these two processes are regulated by a pathway involving the Ste20-like kinase Hippo (Hpo) and the NDR family kinase Warts (Wts; also called Lats). Hpo phosphorylates and activates Wts, which in turn, through unknown mechanisms, negatively regulates the transcription of cell-cycle and cell-death regulators such as cycE and diap1. Yorkie (Yki), the Drosophila ortholog of the mammalian transcriptional coactivator yes-associated protein (YAP), has been identified as a missing link between Wts and transcriptional regulation. Yki (named for its loss-of-function phenotype after a very small breed of dog, the Yorkshire Terrier) is required for normal tissue growth and diap1 transcription and is phosphorylated and inactivated by Wts. Overexpression of yki phenocopies loss-of-function mutations of hpo or wts, including elevated transcription of cycE and diap1, increased proliferation, defective apoptosis, and tissue overgrowth. Thus, Yki is a critical target of the Wts/Lats protein kinase and a potential oncogene (Huang, 2005).

The increase in cell number that accompanies the growth of an organ or organism results from the balanced coordination of three simultaneous processes, including cell growth, cell proliferation, and cell death. Cell growth is a prerequisite for cell proliferation during normal organ growth, and sustained cell proliferation must be coupled to appropriate cell growth. With appropriate cell growth, a net increase in cell number in a growing organ depends on the rate at which they are generated via cell proliferation, as well as the rate at which they are eliminated by cell death (apoptosis). How cell proliferation and cell death are coordinated during tissue growth and homeostasis is yet to be completely understood, and this mechanism must be intact throughout life to prevent diseases such as cancer (Huang, 2005).

Recent studies in mice and fruit flies have revealed two distinct modes in which cell proliferation and cell death could be coupled. In the first mode, increased proliferation, such as that resulting from activation of the Myc oncogene, is coupled in an obligatory fashion to increased cell death. Such coupling between proliferation and apoptosis provides an important failsafe mechanism to prevent inappropriate proliferation of somatic cells. In the second mode, increased proliferation, such as that resulting from activation of the microRNA bantam, or inactivation of the tumor suppressors hippo (hpo), salvador (sav), and warts (wts), is accompanied by an inhibition of cell death. Here, suppression of cell death might allow the overproliferating cells to overcome proliferation-induced apoptosis, thus resulting in a robust increase in organ size. In many aspects, these circumstances resemble certain cancer cells, which display both increased cell proliferation and suppressed cell death (Huang, 2005 and references therein).

hpo, sav, and wts (also called lats) were identified from genetic screens in Drosophila for negative regulators of tissue growth. Inactivation of any of these genes results in increased cell proliferation and reduced apoptosis. hpo encodes a Ste20 family protein kinase, sav encodes a protein containing WW and coiled-coil domains, and wts encodes an NDR (nuclear Dbf-2-related) family protein kinase. Studies have suggested that these genes function in a common pathway that coordinately regulates cell proliferation and apoptosis by targeting the cell-cycle regulator CycE and the cell-death inhibitor DIAP1. Using a combination of genetic and biochemical assays, it has been shown that Hpo, Sav, and Wts define a novel protein kinase cascade wherein Hpo, facilitated by Sav, phosphorylates Wts (Wu, 2003). It was further demonstrated that this pathway, hereafter referred to as the Hpo pathway, negatively regulates the transcription of diap1 (Wu, 2003). It is worth noting that this model differs significantly from an alternative model by others that suggests that this pathway regulates DIAP1 posttranscriptionally through phosphorylation of DIAP1 by Hpo. Another unresolved issue in Hpo signaling concerns the molecular mechanism of the Wts/Lats kinase. While previous studies have identified a number of putative targets for this tumor suppressor, including the G2/M regulator cdc2 and the actin regulators zyxin and LIMK1, none of them could account for the excessive overgrowth associated with wts mutant clones. Thus, the most critical target of the Wts/Lats kinase has remained elusive (Huang, 2005 and references therein).

yorkie (yki) has now been identified as the elusive target of the Wts/Lats tumor suppressor. yki encodes the Drosophila ortholog of yes-associated protein (YAP), a transcriptional coactivator in mammalian cells (Yagi, 1999; Strano, 2001; Vassilev, 2001). Yki is required for normal tissue growth and diap1 transcription and is phosphorylated and inactivated by Wts. Overexpression of yki phenocopies loss-of-function mutations of hpo, sav, or wts. Taken together, these studies identify a missing link between Hpo signaling and transcriptional control and provide further support for the model implicating the Hpo signaling pathway in transcriptional regulation of diap1. These studies further reveal a functional conservation between YAP and Yki and implicate YAP as a potential oncogene in mammals (Huang, 2005).

Activation of yki leads to massive tissue overgrowth that resembles the loss-of-function phenotype of hpo, sav, or wts. To probe the physiological function of yki, the “flip-out” technique was used to generate clones of cells in which yki is overexpressed during development. yki-overexpressing clones lead to marked overgrowth in adult epithelial structures. Wing imaginal discs containing multiple yki-overexpressing clones reach up to eight times the area of control wing discs raised under identical conditions. Besides the overgrowth phenotype, adult cuticles secreted by yki-overexpressing cells display an unusual texture. In yki-overexpressing clones on the notum, the apical surface of the epidermal cells is domed such that cell-cell boundaries are visible between adjacent cells, whereas cell boundaries are not visible in the neighboring wild-type tissues. Both the overgrowth and the abnormal cell morphology caused by yki overexpression closely resemble those shown previously for hpo and wts mutant cells, suggesting that these genes might function in a common pathway (Huang, 2005).

Cell-doubling time for control and yki-overexpressing cells in the wing imaginal disc was determined by analyzing well-separated flip-out clones 48 hr post clone induction. The cell-doubling time for wild-type and yki-overexpressing clones (30 pairs of clones analyzed) was 16.1 hr and 12.0 hr, respectively. Thus, like mutant clones of hpo or wts, yki-overexpressing cells multiply faster. Notably, while cells in the control clones intermingle with their neighbors and form wiggly borders, yki-overexpressing cells minimize their contacts with their neighbors and form round smooth borders. This phenotype indicates distinct adhesive properties of the yki-overexpressing cells and resembles that seen with loss-of-function wts clones. FACS analysis shows that yki-overexpressing cells have a similar cell-cycle profile and cell size distribution as compared to wild-type cells. Thus, like loss-of-function of hpo (Wu, 2003), activation of yki does not accelerate a particular phase of the cell cycle. Rather, each phase of the cell cycle is proportionally accelerated (Huang, 2005).

Activation of yki in the eye imaginal disc leads to increased number of interommatidial cells without affecting photoreceptor differentiation. Focus was placed on the eye imaginal disc, a pseudostratified epithelium in which cell differentiation, proliferation, and apoptosis occur in a highly stereotyped manner. In the third instar, the morphogenetic furrow (MF) traverses the eye disc from posterior to anterior. Cells anterior to the MF are undifferentiated and divide asynchronously, whereas cells in the MF are synchronized in the G1 phase of the cell cycle. Posterior to the MF, cells either exit the cell cycle and differentiate or undergo one round of synchronous division (second mitotic wave, SMW) before differentiation. These cells assemble into approximately 750 ommatidia, leaving behind approximately 2000 superfluous cells that are eliminated by a wave of apoptosis ~36 hr after puparium formation (APF) (Huang, 2005).

To investigate whether activation of yki perturbs photoreceptor differentiation, the neuronal marker Elav was examined. yki-overexpressing ommatidial clusters have the normal complement of photoreceptor cells. The spacing between adjacent ommatidial clusters is increased due to the presence of extra interommatidial cells. The formation of extra interommatidial cells is most evident in pupal eye discs, when yki-overexpressing clones contain many additional cells between photoreceptor clusters. Thus, like loss-of-function of hpo, sav, or wts, yki overexpression results in an increased number of uncommitted, interommatidial cells without affecting early retina patterning (Huang, 2005).

Activation of yki leads to increased cell proliferation and decreased apoptosis. To pinpoint the developmental cause of yki-induced overgrowth, cell proliferation and apoptosis were monitored in eye imaginal discs. In wild-type eye discs, cells posterior to the MF undergo a synchronous second mitotic wave (SMW) that can be revealed as a band of BrdU-positive cells. Few BrdU-positive cells are found posterior to the SMW. In yki-overexpressing clones, cells fail to undergo cell-cycle arrest posterior to the SMW and continue cell cycles as shown by BrdU incorporation as well as M phase marker phospho-histone H3 (PH3). Thus, yki overexpression results in increased cell proliferation (Huang, 2005).

Using the TUNEL assay, cell death was monitored in the pupal retina at a point when a wave of apoptosis normally removes excess interommatidial cells around 36 hr APF. Strikingly, cell death was significantly suppressed in yki-overexpressing clones, even though abundant apoptosis was detected in the neighboring wild-type cells. Thus, normal developmental cell death is largely inhibited by yki overexpression (Huang, 2005).

The mechanisms of how body and organ size are regulated are just beginning to be understood. Recent studies in Drosophila have implicated a number of pathways in the coordinate control of cell growth, proliferation, and apoptosis, which ultimately regulate body and organ size. The insulin/Tsc/TOR signaling network, for example, plays a major role in coordinating organ growth with environmental cues such as nutrients. The Hpo signaling pathway, in contrast, might contribute to an intrinsic size 'checkpoint' that normally stops growth when a given organ reaches its characteristic size. Thus, molecular elucidation of the Hpo signaling pathway should provide important insights into size-control mechanisms in development (Huang, 2005).

Previous studies of the Wts/Lats tumor suppressor have failed to identify any target of this kinase that could account for its potent growth-regulatory activity. This study has provided genetic and biochemical evidence implicating Yki, the Drosophila ortholog of the mammalian coactivator protein YAP, as a direct, critical target of Wts/Lats in the Hpo pathway. Yki associates with and is phosphorylated by Wts. Moreover, Wts-mediated phosphorylation of Yki is stimulated by upstream components of the Hpo pathway, and the extent of Yki phosphorylation induced by Hpo pathway components in vitro correlates with the severity of the overexpression phenotype caused by the respective transgenes in vivo. Most importantly, overexpression of yki phenocopies loss of hpo, sav, or wts, while loss of yki results in the opposite phenotype, and epistasis analyses unambiguously places yki downstream of hpo, sav, and wts. Taken together, these results provide compelling evidence that Yki is a critical target of Wts/Lats in the Hpo pathway. It is further speculated that the relationship between Yki and Hpo signaling is likely conserved during evolution since overexpression of mammalian YAP is able to rescue the lethality associated with hyperactivation of the Hpo pathway in Drosophila. The functional conservation between Yki and YAP further suggests that YAP might function as an oncogene in mammals (Huang, 2005).

Yki is the first substrate identified for NDR family kinases, which include, besides Wts/Lats, Cbk1, Dbf2, and Dbf20 in budding yeast, Sid2 and Orb6 in fission yeast, Cot-1 in Neurospora, Sax-1 in C. elegans, Trc in Drosophila, and NDR1 and NDR2 in mammals (reviewed by Tamaskovic, 2003). The NDR family kinases are involved in diverse events in cell-cycle and cell morphogenesis, such as maintaining cell polarity (Cbk1 and Orb6), coordinating CDK inactivation and cytokinesis (Dbf2, Dbf20, and Sid2), and neuronal morphogenesis (Sax-1). Despite their diverse cellular functions, all NDR family kinases share similar structural features, such as the insertion of 30-60 amino acids between kinase subdomains VII and VIII, the presence of conserved activation loop and hydrophobic motif, and the presence of N-terminal noncatalytic domain (Tamaskovic, 2003). These common features suggest that NDR family kinases may employ similar mechanisms to interact with their substrates and regulators. Along this line, it is suggested that the approach described in this study, which uses the N-terminal noncatalytic domain of Wts as yeast two-hybrid bait, might provide a general method to discover substrates for other NDR family kinases (Huang, 2005).

A model has been proposed whereby Hpo, somehow facilitated by Sav, phosphorylates Wts (Wu, 2003). While this model implied that phosphorylation of Wts leads to activation of its kinase activity, it was not possible to directly test this due to the lack of an appropriate assay that measures pathway activity downstream of Wts. The identification of Yki as a Wts substrate provides a new tool to evaluate the earlier model. Consistent with the previous model implicating Hpo as an activating kinase of Wts, it has been shown that in S2 cells, the phosphorylation of Yki induced by transfected Wts is dependent on the endogenous Hpo protein. Furthermore, the in vitro kinase activity of Wts toward Yki is strongly stimulated when Wts is coexpressed with Hpo-Sav. It is suggested that such a relationship between Hpo and Wts is likely conserved during evolution. Indeed, a recent study (Chan, 2005) has demonstrated the activation of the mammalian Lats1 kinase by the mammalian Hpo homologs Mst1/Mst2 (Huang, 2005).

It is worth noting that several Ste20-like kinases have been implicated in the activation of NDR kinases. Such examples include the activation of Wts by Hpo (Wu, 2003; Chan, 2005), the activation of Dbf2 by Cdc15, the regulation of Orb6 by Pak1, and the regulation of Sid2 by Sid1. Thus, activation by Ste20-like kinases might represent a general mechanism for regulating NDR kinases. In retrospect, the difficulties in identifying substrates for NDR kinases might be due to their substrate specificity in conjunction with a requirement for activation by upstream kinases. Another emerging feature of the NDR kinases concerns their regulation by the Mob family of small regulatory proteins, which have been found to associate with multiple NDR family kinases, such as Dbf2, Orb6, Sid2, Cbk1, NDR1, and NDR2 (Tamaskovic, 2003). In Drosophila, Mats, a Mob family protein, has recently been identified as a tumor suppressor gene that likely regulates Wts in the Hpo signaling pathway (Lai, 2005). Thus, regulation by Mob family proteins likely represents an important and shared feature of modulating NDR family kinases (Huang, 2005 and references therein).

Previous studies of Hpo signaling have suggested two contrasting models on how this pathway regulates the cell-death regulator DIAP1. Using a diap1-lacZ reporter to follow diap1 transcription, elevated diap1 transcription was observed in mutant clones of hpo, sav, or wts that closely matches the increase in DIAP1 protein levels. Based on these results, it was proposed that the Hpo pathway negatively regulates diap1 at the level of transcription (Wu, 2003). However, an alternative model suggested that Hpo regulates DIAP1 posttranscriptionally by directly phosphorylating DIAP1, thus promoting its degradation. This model was largely based on two lines of evidence, including in situ hybridization showing unchanged diap1 mRNA level in mutant clones and the ability of Hpo to phosphorylate DIAP1 in vitro. It is noted, however, that in situ hybridization used in the latter studies did not involve the marking of mutant clones and thus may be less definitive than the diap1-lacZ reporter. A major drawback of the posttranscriptional model is that it cannot easily account for the involvement of Wts in the Hpo pathway. A direct link between Hpo and DIAP1 inevitably implies Wts as acting upstream or in parallel with Hpo, which is contradictory to other studies of the NDR kinases that generally place them downstream of the Ste20-like kinases (Huang, 2005 and references therein).

If the Hpo signaling pathway regulates diap1 via a transcriptional mechanism, then there should exist transcriptional regulator(s) that control diap1 transcription and whose activity may be regulated by the Hpo signaling pathway. Furthermore, such transcriptional regulator(s) must account for the mutant phenotypes resulting from deregulation of the Hpo pathway, such as changes in diap1 transcription and overgrowth. This current study demonstrates that Yki represents such a regulator, thus further supporting the previous model implicating the Hpo pathway in regulating diap1 transcription (Huang, 2005).

Understanding the molecular mechanisms by which the Hpo pathway regulates diap1 transcription will provide important insights into the developmental coordination of tissue growth and apoptosis. Like other coactivators, Yki presumably functions by interacting with DNA binding transcription factors. YAP, the mammalian homolog of Yki, is known to function as coactivator for a number of transcription factors, such as the p53 family member p73 (Strano, 2001), the Runt family member PEBP2α (Yagi, 1999), and the four TEAD/TEF transcription factors (Vassilev, 2001). This interaction is generally mediated by WW domains of YAP and PPxY motifs of the cognate transcription factors. At present, it is unclear whether any of the reported mammalian proteins represents the physiological partner for YAP. Along this line, it is worth noting that while the reported ability of YAP to transactivate p73 in cultured mammalian cells is more suggestive of a tumor suppressor function for YAP (Basu, 2003), these studies clearly implicate Yki and YAP as potential oncogenes. One interesting possibility (Lowe, 2004) is that the reported coupling of mammalian YAP to p73 might represent a fail-safe mechanism to limit the oncogenic potential of YAP in much the same way as cell death is obligatorily linked to oncogene activation (Huang, 2005).

An important direction in the future is to identify the DNA binding transcription factor that partners with Yki to regulate gene transcription; identifying the factor should provide critical insights into how Yki (and likely YAP as well) could function as a potent oncogene. This effort should be facilitated by the dissection of the diap1 promoter and the identification of a minimal Hpo-responsive element that confers transcriptional regulation of diap1 by the Hpo pathway. With such a DNA element, one should be able to identify the DNA binding transcription factor that partners with Yki to regulate the transcription of diap1 and other Hpo-pathway-responsive genes (Huang, 2005).

Many components of the Hpo pathway are conserved throughout evolution, suggesting that this emerging pathway might play a similar role in mammals. Indeed, previous studies have shown that human homologs of wts, hpo, and mats could rescue the respective Drosophila mutants. Moreover, mice lacking a wts homolog are prone to tumor formation, and the human orthologs of sav and mats are mutated in several cancer cell lines. Such conservation is further extended in the current study, showing that Yki and YAP have similar biological activity when assayed in Drosophila. These results suggest that the Hpo signaling pathway might play a conserved role in mammalian growth control. Furthermore, inactivation of growth suppressors of the Hpo pathway, such as Hpo, Sav, Wts, and Mats, and hyperactivation of growth promoters of the pathway, such as YAP, are likely to contribute to mammalian tumorigenesis (Huang, 2005).

Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules

Adherens junctions (AJs) and cell polarity complexes are key players in the establishment and maintenance of apical-basal cell polarity. Loss of AJs or basolateral polarity components promotes tumor formation and metastasis. Recent studies in vertebrate models show that loss of AJs or loss of the basolateral component Scribble (Scrib) cause deregulation of the Hippo tumor suppressor pathway and hyperactivation of its downstream effectors Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ), homologs of Drosophila Yorkie. However, whether AJs and Scrib act through the same or independent mechanisms to regulate Hippo pathway activity is not known. This study dissects how disruption of AJs or loss of basolateral components affect the activity of the Drosophila YAP homolog Yorkie (Yki) during imaginal disc development. Surprisingly, disruption of AJs and loss of basolateral proteins produced very different effects on Yki activity. Yki activity was cell-autonomously decreased but non-cell-autonomously elevated in tissues where the AJ components E-cadherin (E-cad) or α catenin (α-cat) were knocked down. In contrast, scrib knockdown caused a predominantly cell-autonomous activation of Yki. Moreover, disruption of AJs or basolateral proteins had different effects on cell polarity and tissue size. Simultaneous knockdown of α-cat and scrib induced both cell-autonomous and non-cell-autonomous Yki activity. In mammalian cells, knockdown of E-cad or α-cat caused nuclear accumulation and activation of YAP without overt effects on Scrib localization and vice versa. Therefore, these results indicate the existence of multiple, genetically separable inputs from AJs and cell polarity complexes into Yki/YAP regulation. (Yang, 2014).

This report addresses the effects of AJs and basolateral cell polarity determinants on the activity of the Hippo pathway in Drosophila imaginal discs. Knockdown of AJs and basolateral components both induced ectopic activation of Yki. However, knockdown of AJs and basolateral proteins had strikingly different effects on Yki. Disruption of the basolateral module induced mainly a cell-autonomous increase in Yki activity, whereas knockdown of AJs caused non-autonomous induction of Yki reporters. Therefore, these data identify and genetically uncouple multiple different molecular pathways from AJs and the basolateral module that regulate Yki activity (Yang, 2014).

These studies further show that knockdown of AJs induces cell-autonomous reduction of Yki activity and causes cell death and decreased size of Drosophila imaginal discs. Likewise, E-cad and :alpha;-cat mutant clones do not survive in imaginal discs. This effect may be mediated by LIM domain proteins of the Zyxin and Ajuba subfamilies, which regulate Hippo signaling by directly inhibiting Wts/Lats kinases and by interacting with Salvador (Sav), an adaptor protein that binds to the Hpo/MST kinases. A recent report shows that α-Cat recruits Ajuba and indirectly Wts to AJs and loss of Ajuba leads to activation of Wts and hence phosphorylation and inhibition of Yki and diminished tissue size. Thus, α-cat mutant cells may inactivate Yki because they lose Ajuba function (Yang, 2014).

In contrast, in mammalian systems, several in vivo and in vitro studies have shown the opposite effect on Hippo signaling upon AJ disruption; knockdown of E-cad or α-cat caused an increase in cell proliferation and nuclear accumulation of YAP, and conditional knockout of α-cat in mouse skin cells caused tumor formation and elevated nuclear YAP staining. This suggests that AJ components have a tumor suppressor function in mammals. The observation that Scrib is mislocalized upon disruption of AJs in several different mammalian cell lines suggested that YAP activation could be due to the concomitant disruption of the basolateral module. However, the finding that acute disruption of AJs can cause YAP activation without disrupting Scrib localization and vice versa indicates that AJs and the basolateral module also act independently on the Hippo pathway in mammalian cells. In mammalian cells, α-Cat forms a complex with YAP and 14-3-3 proteins, thereby sequestering phosphorylated YAP at the plasma membrane. However, α-Cat may function as a tumor suppressor only in epidermal stem cells, as conditional deletion of α-cat in differentiated cells only caused a mild phenotype with no overgrowth and tumor formation. Therefore, it is possible that the negative regulation of YAP by α-Cat is cell type-specific, although further testing is required to fully address this issue (Yang, 2014).

The non-cell-autonomous effect of AJ knockdown on the Hippo pathway is an intriguing phenomenon. Several groups reported non-autonomous effects on the Hippo pathway in Drosophila in other mutant conditions. Disrupting the expression gradients of the atypical Cadherin Dachsous or that of its regulator Four-jointed, clones of cells mutant for the tumor suppressor genes vps25 or hyperplastic discs (hyd) , clones of cells overexpressing Src64, or overexpression of the proapoptotic gene reaper or the JNK signaling ligand eiger all cause non-autonomous activation of Yki. This non-autonomous activation of Yki may be part of a regenerative response that stimulates cell proliferation in cells neighboring tissue defects. The signals that activate Yki in these situations are not known, nor is it known whether these mutant conditions activate the same or different signaling mechanisms. The non-autonomous activation of Yki around cells with AJ knockdown may be mediated by changes in mechanical forces. AJs are important for maintaining tension between cells across epithelia, and disruption of AJs leads to an imbalance of apical tension. Mechanical forces are known to regulate the Hippo pathway, and YAP/TAZ act as mediators of mechanical cues from the cellular microenvironment such as matrix stiffness. In particular, the Zyxin and Ajuba family LIM domain proteins can act as sensors of mechanical forces and may be involved in the non-autonomous activation of Yki. The effects on Hippo signaling of solely changing Zyxin and Ajuba may not be as strong as those described here, and these proteins may thus cooperate with other molecular conduits to regulate the activity of the Hippo pathway in response to changes in AJ strength. Unraveling these mechanisms will provide important new insights into understanding how cells interact with neighboring cells to regulate proliferation, apoptosis, and the Hippo pathway (Yang, 2014).

It is currently unknown whether AJs also exert non-autonomous effects on the Hippo pathway in mammalian tissues. Amphiregulin, an EGF ligand, is a downstream target of YAP and can induce non-cell-autonomous cell proliferation through EGFR signaling. However, it is not known whether YAP itself is activated non-cell-autonomously to contribute to the hyper-proliferation phenotypes observed upon disruption of AJs in vivo and in vitro. It will be interesting to determine whether AJs and other cell-cell signaling mechanisms also have non-cell-autonomous effects on the activity of YAP in mammalian tissues, for example during regeneration (Yang, 2014).

Finally, the apical proteins aPKC and Crb modulate the activity of the Hippo pathway, and many Hippo pathway components are apically localized, which is important for their activity. The data presented in this study add to these findings. Disruption of AJs causes reduced Yki activity, despite the fact that Crb and Mer are mislocalized. Thus, AJs and cell polarity components regulate Yki activity through multiple, genetically separable inputs. It will be interesting to decipher all of the different underlying molecular mechanisms of how AJs and basolateral proteins regulate the Hippo pathway and how these mechanisms evolved in Drosophila and in mammals (Yang, 2014).

Yorkie promotes transcription by recruiting a histone methyltransferase complex

Hippo signaling limits organ growth by inhibiting the transcriptional coactivator Yorkie. Despite the key role of Yorkie in both normal and oncogenic growth, the mechanism by which it activates transcription has not been defined. This paper reports that Yorkie binding to chromatin correlates with histone H3K4 methylation and is sufficient to locally increase it. Yorkie can recruit a histone methyltransferase complex through binding between WW domains of Yorkie and PPxY sequence motifs of NcoA6, a subunit of the Trithorax-related (Trr) methyltransferase complex. Cell culture and in vivo assays establish that this recruitment of NcoA6 contributes to Yorkie's ability to activate transcription. Mammalian NcoA6, a subunit of Trr-homologous methyltransferase complexes, can similarly interact with Yorkie's mammalian homolog YAP. The results implicate direct recruitment of a histone methyltransferase complex as central to transcriptional activation by Yorkie, linking the control of cell proliferation by Hippo signaling to chromatin modification (Oh, 2014).

Transcriptional activators increase transcription through recruitment of transcriptional proteins or through chromatin modification. Each of these encompasses a wide range of specific mechanisms, including interaction with core subunits of RNA polymerase, interaction with Mediator proteins, interaction with chromatin remodeling complexes, or interaction with complexes that influence posttranslational modifications of histones, such as acetylation or methylation. Previous studies have observed that Yki and YAP could interact with Mediator subunits, ATP-dependent chromatin remodeling complexes, and other transcription factors such as GAGA. Nonetheless, based on the results described in this study, it is argued that a key mechanism by which Yki activates transcription is increasing H3K4 methylation through recruitment of the Trr HMT complex. Most notably, point mutations in Yki that specifically impair its ability to interact with NcoA6 abolish its transcriptional activity, and this transcriptional activity is restored by fusion with NcoA6. Moreover, the essential role of Yki as a transcriptional coactivator for its DNA binding partner Sd can be bypassed by fusing NcoA6 directly with Sd (Oh, 2014).

These observations tie Yki's transcriptional activity most directly to NcoA6, and the argument that this reflects a necessary and sufficient role for H3K4 methylation in transcriptional activation by Yki rests in part on the identity of NcoA6 as a component of the Trr HMT complex. This argument receives further support from several additional observations: the strong, genome-wide correlation between Yki's association with chromatin and H3K4 methylation; the increased H3K4 methylation when Yki competent to interact with NcoA6 is targeted to a novel chromosomal location; the similar decreases in expression of Yki target genes when either NcoA6 or Trr are reduced by RNAi in cultured cells or in vivo; and the recent biochemical demonstration that H3K4 methylation of chromatin by MLL2, a Trr-homologous complex in mammals, could increase transcription in in vitro assays (Oh, 2014).

NcoA6 and Trr have previously been linked to transcriptional activation by nuclear hormone receptors. NcoA6 is believed to play an analogous role in transcriptional activation by nuclear receptors, i.e., its direct binding to these transcription factors recruits the Trr HMT complex or its mammalian homologs. However, a distinct structural motif (LxxLL) within NcoA6 mediates interactions with nuclear receptors. Thus, NcoA6 appears to act as a multifunctional adaptor protein that can link different classes of transcriptional activators to Trr/MLL2/3 HMT complexes, which as is established in this study are involved not only in transcriptional activation induced by nuclear receptors but also by Yki and its mammalian homologs (Oh, 2014).

Crosstalk between Hippo signaling and other pathways has been observed at the level of transcription factors, including physical interactions between Yki, YAP and TAZ, and β-catenin and SMADs, which are transcriptional effectors of Wnt and BMP signaling, respectively. Thus, the current observations raise the possibility that Trr-dependent H3K4 methylation could also contribute to transcriptional activation by these pathways (Oh, 2014).

In humans, NCOA6 has been identified as a gene commonly amplified and overexpressed in breast, colon, and lung cancers (it is also known as Amplified in breast cancer). In mice, gene-targeted mutations have implicated NcoA6 in promoting growth during development and wound healing. These roles in promoting growth are reminiscent of YAP, which is similarly required for growth during embryonic development and wound repair and linked to these cancers when amplified or activated. Thus, while functional studies linking mammalian NCOA6 to cell survival, growth, and cancer have previously been interpreted as a reflection of its role as a coactivator of transcription mediated by nuclear hormone receptors, the current results, together with analysis of MLL2 binding by ChIP-seq, argue that at least part of its effects reflect its role as a cofactor of YAP (Oh, 2014).

A notable feature of Hippo signaling is the recurrence of WW domains or PPxY motifs in multiple pathway components. Within Yki, YAP, and TAZ, the WW domains serve a dual role. They facilitate inhibition, as major negative regulators, including Warts/Lats, Expanded (in Drosophila), and Angiomotin (in mammals), utilize PPxY motifs to bind Yki, YAP, and TAZ and promote their cytoplasmic localization. Conversely, they also facilitate activation, through binding to Wbp2 and, as is shown in this study, NcoA6. It seems unlikely to be coincidental that key positive and negative partners of Yki/YAP/TAZ bind the same structural motifs. Rather, this shared recognition of the same motifs may have evolved to ensure tight on/off regulation of Yki/YAP/TAZ-dependent transcription (Oh, 2014).

JNK and Yorkie drive tumor progression by generating polyploid giant cells in Drosophila

Epithelial cancer tissues often possess polyploid giant cells, which are thought to be highly oncogenic. However, the mechanisms by which polyploid giant cells are generated in tumor tissues and how such cells contribute to tumor progression remain elusive. Cells mutant for the endocytic gene rab5 in Drosophila imaginal epithelium exhibit enlarged nuclei. This study finds that mutations in endocytic 'neoplastic tumor-suppressor' genes, such as rab5, vps25, erupted, or avalanche result in generation of polyploid giant cells. Genetic analyses on rab5-defective cells reveal that cooperative activation of JNK and Yorkie generates polyploid giant cells via endoreplication. Mechanistically, Yorkie-mediated upregulation of Diap1 cooperates with JNK to downregulate the G2/M cyclin CycB, thereby inducing endoreplication. Interestingly, malignant tumors induced by Ras activation and cell polarity defect also consist of polyploid giant cells, which are generated by JNK and Yorkie-mediated downregulation of CycB. Strikingly, elimination of polyploid giant cells from such malignant tumors by blocking endoreplication strongly suppressed tumor growth and metastatic behavior. These observations suggest that JNK and Yorkie, two oncogenic proteins activated in many types of human cancers, cooperatively drive tumor progression by generating oncogenic polyploid giant cells (Cong, 2018).

Polyploid giant cells, which contain multiples of the diploid genome equivalents, are often observed in human cancer tissues. Such polyploidy can be generated by endoreplication, a cell cycle variation that gives rise to genomic contents by replicating DNA in the absence of cell division. Polyploid giant cancer cells were shown to be more tumorigenic than normal diploid cancer cells. However, the mechanisms by which polyploid giant cells are generated in tumors and how they contribute to tumor progression remain elusive (Cong, 2018).

In Drosophila imaginal epithelia, loss-of-function mutations in the endocytic genes, such as rab5, vps25, erupted (ept), or avalanche (avl) cause neoplastic tissue overgrowth and therefore these genes are called 'neoplastic tumor-suppressors'. Previously found that cells deficient for Rab5, a small GTPase essential for generating early endosomes, induce non-autonomous overgrowth of surrounding tissue when induced as mosaic clones in the imaginal disc (Takino, 2014). Mechanistically, loss-of-Rab5 causes activation of Eiger (a tumor necrosis factor (TNF) homolog)-JNK signaling and EGFR-Ras signaling, which cooperate together to activate the Hippo pathway effector Yorkie (Yki, a YAP homolog), leading to upregulation of a secreted growth factor Unpaired (Upd, an IL-6 homolog) (Takino, 2014). Intriguingly, this study also noticed that such Rab5-deficient cells exhibited enlarged nuclei, which was suppressed by inhibition of JNK signaling, Ras signaling, or Yki activity, although the underlying mechanisms and its function have been unknown (Cong, 2018).

This study also analyzed Rab5-defective cells in detail and found that rab5 mutation generates polyploid giant cells through endoreplication. Genetic analyses reveal that JNK and a Yki-target Diap1 (Drosophila inhibitor-of-apoptosis protein 1) cooperate to induce endoreplication in Rab5-defective cells via downregulation of the G2/M cyclin CyclinB (CycB). Furthermore, this study also showed that generation of such polyploid giant cells is essential for tumor growth and metastasis in a Drosophila model of malignant tumors bearing Ras activation and cell polarity defect (Cong, 2018).

Serpin facilitates tumor-suppressive cell competition by blocking Toll-mediated Yki activation in Drosophila

Normal epithelial tissue exerts an intrinsic tumor-suppressive effect against oncogenically transformed cells. In Drosophila imaginal epithelium, clones of oncogenic polarity-deficient cells mutant for scribble (scrib) or discs large (dlg) are eliminated by cell competition when surrounded by wild-type cells. In this study, a genetic screen in Drosophila identified Serpin5 (Spn5), a secreted negative regulator of Toll signaling, as a crucial factor for epithelial cells to eliminate scrib mutant clones from epithelium. Downregulation of Spn5 in wild-type cells leads to elevation of Toll signaling in neighboring scrib cells. Strikingly, forced activation of Toll signaling or Toll-related receptor (TRR) signaling in scrib clones transforms scrib cells from losers to supercompetitors, resulting in tumorous overgrowth of mutant clones. Mechanistically, Toll activation in scrib clones leads to c-Jun N-terminal kinase (JNK) activation and F-actin accumulation, which cause strong activation of the Hippo pathway effector Yorkie that blocks cell death and promotes cell proliferation. These data suggest that Spn5 secreted from normal epithelial cells acts as a component of the extracellular surveillance system that facilitates elimination of pre-malignant cells from epithelium (Katsukawa, 2018).

Clones of oncogenic polarity-deficient cells are actively eliminated from Drosophila imaginal epithelium when surrounded by normal tissue, indicating the existence of intrinsic tumor-suppression mechanism by cell competition. The present study shows that normal epithelial cells secrete Spn5 to facilitate the tumor-suppressive cell competition by antagonizing Toll signaling activation in polarity-deficient cells. Elevation of Toll signaling in polarity-deficient cells transforms them from losers to supercompetitors, which leads to tumorous overgrowth of mutant tissue. Thus, Spn5 acts as a component of the extracellular surveillance system that eliminates oncogenic cells by cell competition. It is not known at this stage why scrib cells are more sensitive to loss of spn5 to upregulate Toll signaling compared to surrounding wild-type cells. One possible mechanism that drives Toll activation in scrib cells would be JNK activation, which was shown to be sufficient to activate Toll signaling (Katsukawa, 2018).

Interestingly, it has been shown that activation of TRR signaling in losers of Myc- or Minute-induced cell competition causes losers' death through nuclear factor κB (NF-κB)-mediated induction of cell death gene hid or rpr, respectively. Consistent with this report, it has been shown in Drosophila larval fat bodies that activation of Toll signaling leads to inactivation of Yki, which may cause hid- or rpr-mediated cell death because one of the important Yki targets is a caspase inhibitor diap1. These observations intriguingly indicate that Toll signaling has opposite roles in different types of cell competition; while Toll activation promotes elimination of losers in Myc- or Minute-induced cell competition, it suppresses elimination of polarity-deficient losers in tumor-suppressive cell competition. Importantly, however, in both cases, Toll or TRR signaling seems to act as an oncogenic signaling that promotes expansion of pre-malignant winner clones within the tissue. Consistent with these findings in Drosophila, it has been reported that upregulation of Toll-like receptors (TLRs) is associated with tumor growth and progression in some human cancers. In addition, one of the human orthologs of Drosophila Spn5, SpnA5, has been shown to inhibit breast cancer growth and metastasis, and its expression level is decreased in renal cell carcinoma and sarcoma. These observations, together with the data from Drosophila genetics, suggest that Toll signaling drives tumorigenesis by promoting supercompetition of oncogenic cell clones (Katsukawa, 2018).

The mechanism by which Toll activation in polarity-deficient cells leads to Yki activation is an important open question for future studies. One possible mechanism is co-activation of JNK and Ras signaling in Toll-activated scrib cells, as these two pathways have shown to cooperate to induce Yki activation through F-actin accumulation and Wts inactivation. Interestingly, it has been shown in mammalian systems that the TLR signaling activates JNK signaling and that several TLRs activate EGFR-Ras signaling upon immune response. Given that signaling molecules identified in Drosophila are all conserved, similar Toll-mediated regulation of tumorigenesis could be involved in human cancer (Katsukawa, 2018).

Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis

To compensate for accumulating damages and cell death, adult homeostasis (e.g., body fluids and secretion) requires organ regeneration, operated by long-lived stem cells. How stem cells can survive throughout the animal life remains poorly understood. This study shows that the transcription factor Shavenbaby (Svb, OvoL in vertebrates) is expressed in renal/nephric stem cells (RNSCs) of Drosophila and required for their maintenance during adulthood. As recently shown in embryos, Svb function in adult RNSCs further needs a post-translational processing mediated by the Polished rice (Pri) smORF peptides and impairing Svb function leads to RNSC apoptosis. Svb interacts both genetically and physically with Yorkie (YAP/TAZ in vertebrates), a nuclear effector of the Hippo pathway, to activate the expression of the inhibitor of apoptosis DIAP1. These data therefore identify Svb as a nuclear effector in the Hippo pathway, critical for the survival of adult somatic stem cells (Bohere, 2018).

The results show that Shavenbaby is expressed and required for the maintenance of adult renal stem cells in flies, supporting the conclusion that the OvoL/Svb family of transcription factors plays a key and evolutionarily-conserved role in the behavior of progenitors/stem cells (Bohere, 2018).

The role of Svb in adult stem cell maintenance in flies requires both a fine control of its expression and of its transcriptional activity. Svb expression in RNSCs involves at least two separable enhancers, driving similar expression patterns. Svb was one of the first cases to reveal the functional importance of apparently redundant (or shadow) enhancers in the phenotypic robustness of regulatory networks across tissues and development stages. The data suggest that a similar cis-regulatory architecture is also underlying the control of adult stem cells (Bohere, 2018).

RNSCs maintenance further requires a proper post-translational maturation of the Svb protein, in response to Pri smORF peptides. During both embryonic and post-embryonic development, the main role of Pri peptides is to provide a temporal control of Svb activity, conveying systemic steroid signaling. It is therefore possible that Pri smORF peptides also connect genetic networks to hormonal control for the regulation of adult stem cells. Recent work has shown that various smORF peptides contribute to the regulation of developmental pathways, muscle formation and physiology, etc., and the current findings extend their influence to the control of adult stem cells. It has been proposed that the emerging field of smORF peptides may open innovative therapeutic strategies, for example peptidomimetic drugs, which might also be of interest for regenerative medicine (Bohere, 2018).

The current results establish that a main function of Svb in adult stem cells is mediated by a functional interplay with the Hippo pathway, well established for its roles in the control of adult stem cells. The results indicate that Svb behaves as a nuclear effector of Hippo, relying on a direct interaction with Yorkie in order to protect stem cells from apoptosis, at least in part through the regulation of DIAP1 expression. Analysis of genome-wide binding events further suggests that the Svb/Yki interaction is involved in the control of a broader set of Hippo-regulated genes, including during development. Since both Pri and Ubr3 are also essential for the survival of adult stem cells, it is interesting to note that Ubr3 protects the DIAP1 protein from degradation, and direct binding of Ubr3 on the activated form of DIAP1 is elicited in the presence of Pri peptides. Therefore, in addition to the control of DIAP1 expression (via Svb), Ubr3 and Pri could also stabilize the DIAP1 protein to protect stem cells from apoptosis. Although initially restricted to TEAD transcription factors, the number of Yorkie (YAP/TAZ) nuclear partners is rapidly growing. Interestingly, recent work has shown a direct interaction of YAP/TAZ with the pro-EMT factors Snail/Slug, in the control of stem cell renewal and differentiation. As previously reported for intestinal stem cells, this study shows that pro-EMT regulators are also required for preventing premature differentiation of renal stem cells. While pro-EMT and OvoL factors are often viewed as antagonistic factors, in vivo studies in Drosophila stem cells show that they both contribute to their maintenance, Svb/Yki preventing their apoptosis and EMT factors their differentiation. Since many studies have implicated the Hippo pathway, pro-EMT and OvoL/Svb factors in various tumors, new insights into their functional interactions in adult stem cells may provide additional knowledge directly relevant to understand their connections in human cancers (Bohere, 2018).

Opposing transcriptional and post-transcriptional roles for Scalloped in binary Hippo-dependent neural fate decisions

The Hippo tumor suppressor pathway plays many fundamental cell biological roles during animal development. Two central players in controlling Hippo-dependent gene expression are the TEAD transcription factor Scalloped (Sd) and its transcriptional co-activator Yorkie (Yki). Hippo signaling phosphorylates Yki, thereby blocking Yki-dependent transcriptional control. In post-mitotic Drosophila photoreceptors, a bistable negative feedback loop forms between the Hippo-dependent kinase Warts/Lats and Yki to lock in green vs blue-sensitive neuronal subtype choices, respectively. Previous experiments indicate that sd and yki mutants phenocopy each other's functions, both being required for promoting the expression of the blue photoreceptor fate determinant melted and the blue-sensitive opsin Rh5. This study demonstrates that Sd ensures the robustness of this neuronal fate decision via multiple antagonistic gene regulatory roles. In Hippo-positive (green) photoreceptors, Sd directly represses both melt and Rh5 gene expression through defined TEAD binding sites, a mechanism that is antagonized by Yki in Hippo-negative (blue) cells. Additionally, in blue photoreceptors, Sd is required to promote the translation of the Rh5 protein through a 3'UTR-dependent and microRNA-mediated process. Together, these studies reveal that Sd can drive context-dependent cell fate decisions through opposing transcriptional and post-transcriptional mechanisms (Xie, 2019).

Ensuring that the correct complement of genes remains on or off in any given cell type is an essential feature of multicellular organisms. This is particularly critical in the peripheral nervous system, where exclusive sensory receptor expression is necessary for selective and specific activation of a given sensory neuron. Such exclusion is well-established in the visual system of most animals, where individual photoreceptors (PRs) express a single opsin photopigment and repress the expression of others to prevent sensory overlap. The gene regulatory mechanisms underlying this mutual exclusion, however, are still under investigation (Xie, 2019).

The Drosophila eye has long served as a powerful model to understand the functions and architecture of gene regulatory networks underlying PR subtype cell fate specification. Each of the approximately 750 individual eye units (ommatidia) in the Drosophila compound eye contains 8 PRs. Based on the specific opsin that is expressed in the R8 photoreceptor, two major ommatidial subtypes, pale (p) and yellow (y), are present in the adult eye. Pale ommatidia are primarily defined based on the expression of the blue-sensitive opsin, Rhodopsin 5 (Rh5), while yellow ommatidia express the green-sensitive opsin, Rh6. These ommatidial subtypes are randomly distributed through the eye in a 30:70 blue:green ratio, and are established and maintained through a bistable negative feedback loop between two signaling molecules: the pleckstrin homology-containing protein Melted (Melt) and the Hippo signaling kinase Warts (Wts, aka Lats) (Xie, 2019).

Wts is a core component of the Hippo kinase complex that phosphorylates and inactivates the transcriptional co-activator Yorkie (Yki). Hippo signaling is best understood in the context of growth regulation, where Wts and Yki function in a homeostatic feedback loop: Wts blocks Yki function and Yki initiates its own inactivation by promoting Hippo pathway gene expression. In contrast, in post-mitotic PR fate decisions, Yki promotes the expression of the wts repressor, melt, generating a double-negative 'on/off' feedback loop between wts and Yki that ensures two stably maintained fate choices. In green PRs, Hippo signaling promotes the expression of green fate determinants (wts and Rh6), and prevents the expression of Yki-dependent blue fate determinants (melt and Rh5). In blue PRs, Yki promotes melt, thereby repressing wts and inhibiting Hippo signaling, further promoting Yki-dependent activation of blue fate effectors and suppression of green fate effectors. Thus, Wts-positive (Yki-inactive) cells adopt the default green/wts/Rh6 fate, while Wts-negative (Yki-active) cells acquire the blue/melt/Rh5 fate (Xie, 2019).

Yki, a YES-associated protein (YAP), is a transcriptional co-activator that does not bind DNA itself, but instead requires a DNA-binding partner. The primary binding partners for Yki/Yap factors are members of the TEAD family of transcription factors. In Drosophila, the single TEAD family member is encoded by Scalloped (Sd). Sd/TEAD and Yki/YAP can physically interact and together activate TEAD-site-containing reporter expression in vitro. Furthermore, in ectopic yki conditions, sd/TEAD is essential for yki/YAP to induce tissue overgrowth and activate target gene expression. However, in vivo, sd mutants do not phenocopy yki growth phenotypes and sd mutants do not show changes in yki target gene expression. These data suggest that Sd and Yki use distinct mechanisms to control tissue size. Studies aimed at addressing this conundrum have shown that in developing wing, eye, and follicle cells, Sd functions as a transcriptional repressor under 'Hippo-on' conditions to inhibit cell growth, and that in 'Hippo-off' cells, Yki antagonizes Sd repression to promote growth regulatory genes. This suggests that Sd and Yki can play opposite roles during growth (Xie, 2019).

In post-mitotic PRs, it has been previously shown that sd mutants phenocopy yki's knockdown phenotype in PR subtype fate specification: both sd and yki are necessary to promote blue PR fate and inhibit green PR fate. Combined, these findings suggest that sd and yki function together in this cell fate specification event. This study investigated the molecular basis underlying this interaction. Sd was found to play roles at both the transcriptional and post-transcriptional level to ensure blue vs green PR subtype fate decisions. At the transcriptional level, Sd directly represses blue fate effector gene expression in Hippo (Wts)-positive green PRs, and Yki antagonizes this repression in Hippo (Wts)-negative blue PRs. This is consistent with previously reported antagonism between Sd and Yki. In addition to this function, it was found that Sd promotes blue fate through a post-transcriptional, microRNA (miRNA)-dependent process in Wts-negative blue PRs, revealing a cooperative interaction with Yki in promoting blue PR fate. Together, these new findings elucidate a multi-tiered regulatory network involving the Drosophila TEAD transcription factor that functions at both the transcriptional and post-transcriptional level to precisely specify neuronal subtype fate (Xie, 2019).

The mutually exclusive expression of sensory receptor genes in sense organs is essential to prevent sensory input overlap in the mature organism. This study shows that, in the fly retina, the TEAD factor Sd achieves this in blue and green PRs using two different mechanisms: direct transcriptional repression of the blue fate determinant melt and blue Rh5 opsin genes in green photoreceptors, and relief of post-transcriptional control of the Rh5 mRNA in blue photoreceptors. In addition, Yki, a major Sd cofactor, antagonizes Hippo-specific and Sd-dependent repression of melt and Rh5 to promote blue PR fate. Thus, Sd and Yki play multiple roles to ensure a robust bistable cell fate decision in post-mitotic sensory neurons (Xie, 2019).

The antagonistic relationship between Sd repression and Yki de-repression is similar to the model previously proposed in cell cycle control. Nevertheless, the mechanisms by which Sd represses gene expression in green PRs remains unknown. In cell growth, for instance, repression is mediated in part through Tgi, a Tondu domain containing protein, which Yki competes with to alleviate repression. However, no significant change was detected in Rh5 protein or reporter expression with knockdown of Tgi in PRs, suggesting the existence of another Sd co-repressor in this system. Indeed, a zinc finger protein Nerfin-1 was recently identified as a Tgi-independent Sd co-repressor that participates in Hippo-dependent cell growth and competition during Drosophila eye development (Guo, 2019). Preliminary studies showed that knockdown of nerfin-1 led to an expansion of Rh5-expressing blue PRs at the expense of green PRs, comparable to the expanded expression of Sd site mutants in the melt and Rh5 reporters. Therefore, Nerfin-1 is very likely to be at least one Sd co-repressor during blue- and green PR fate specification in the Drosophila eye. Combined, these findings suggest Sd repression activity is a general mechanism in controlling the output of the Hippo pathway (Xie, 2019).

If the role of Sd in green PRs were solely to repress Rh5 transcription, then Rh5 mRNA levels might be expected to be elevated in sd mutants relative to controls. Instead, a ~50% reduction was observed. This observation could reflect two possibilities, which are not mutually exclusive. First, based on previous and unpublished findings that Otd cooperates with Yki to activate Rh5 in Hippo-negative blue PRs, it is expected that in sd mutants, where all R8s switch to Hippo-positive (and hence Yki-inactive) green PRs, Rh5 activation in green PRs would be reduced. Second, since the current studies suggest a new role for miRNAs in the post-transcriptional control of Rh5, it is possible that Rh5 mRNA stability is affected in sdmutants (Xie, 2019).

In terms of the post-transcriptional control of Rh5, it was demonstrated that the Rh5 3' UTR was required to prevent its co-expression with Rh6 in sd knockdown green PRs. In addition, the simultaneous knockdown of sd and miRNA processing machinery genes led to Rh5 protein de-repression (and co-expression with Rh6) in a substantial subset of green R8 cells. Together, these data suggest miRNA-dependent regulation of Rh5 depends on Sd, either directly or indirectly. It is posited that, as a transcription factor, Sd prevents the transcription of Rh5-directed miRNA genes. However, follow-up studies will be important for defining the complete repertoire of miRNA-dependent events involved in this Hippo-directed cell fate decision. For example, possible differences in an pRh5 reporter and endogenous Rh5 protein were reported in retinas mutant for the transcription factor PvuII-PstI homology 13 (pph13). While this disparity could be due to the rhabdomere defects observed in pph13 mutants, there is potential for a role for Pph13 in Rh5 post-transcriptional regulation. Finally, it is possible that the Rh5 3'UTR recruits other non-coding RNAs or proteins to regulate its expression (Xie, 2019).

Combined, the bimodal functions of Sd in Yki-vs Wts-positive cells form a feedforward regulatory module in post-mitotic PR fate decisions, robustly preventing sensory receptor overlap. Feedforward modules between transcription factors and miRNAs have been previously reported in neuronal differentiation and other biological processes. For example, the proto-transcription factor c-Myc can directly activate E2F1 transcription, but also limit E2F1 translation by activating miR-175p and miR-20a. In contrast to the c-Myc-miRNAs-E2F1 activation module, which fine-tunes a proliferative signal in dividing cells, however, the Sd-miRNA-Rh5 repression module ensures a robust ON-OFF switch in the terminal PR differentiation process. If similar mechanisms take place during Hippo-dependent cell growth remains to be determined (Xie, 2019).

Whether yki is also involved in Sd's post-transcriptional control in blue PRs remains unresolved, as yki itself is essential for blue PR fate, and hence, Rh5-expressing cells. Previous studies have demonstrated that Yki is important for the activation of at least one miRNA to promote cell growth (i.e. bantam). However, in the case of Rh5 regulation, the miRNA must be repressed in Yki-expressing cells, rather than activated. In this context, it is worth noting that the Yki ortholog YAP has been shown to mediate widespread miRNA suppression in tumor cells (Hippo-negative) by sequestering an RNA helicase p72/DDX-17, a regulatory component of microRNA-processing machinery. Comparably, the results suggest that the miRNA(s) is/are inactive in Yki-positive blue PRs in order to allow Rh5 protein expression. These findings raise the possibility that YAP/Yki- and TEAD/Sd-dependent regulation of miRNA biogenesis is a universal mechanism in control of the Hippo signaling pathway in tissue growth and neuronal cell fate decisions (Xie, 2019).


cDNA clone length - 1427

Bases in 5' UTR - 170

Exons - 4

Bases in 3' UTR - 112


Amino Acids - 418
(AAZ42161 )

Structural Domains

Yki is most closely related to the human yes-associated protein (YAP, also called YAP65) (Sudol, 1994), with 31% identity between the two proteins. Both proteins contain two WW domains, protein-protein interaction modules of 35-40 amino acids that are known to interact with PPXY-containing polypeptides. The similarity between Yki and YAP extends beyond the WW domains and includes a stretch of sequence similarity at the N-terminal part of the proteins. The WW domains of Yki are required for its interaction with Wts. While initially isolated as a protein that interacts with the SH3 domain of the Yes proto-oncogene, the involvement of YAP in Yes signaling has not been validated (Sudol, 1994). Notably, the corresponding SH3 binding region (Sudol, 1994) is absent in the Drosophila Yki protein. In contrast, YAP has been implicated as a transcriptional coactivator, a class of transcriptional regulators that do not bind to DNA themselves but associate with DNA binding transcription factors and supply or stimulate transcriptional activation of the cognate transcription factors. Specifically, YAP has been shown to function as a coactivator for a number of transcription factors, such as the p53 family transcription factor p73 (Strano, 2001), the Runt family protein PEBP2α (Yagi, 1999), and the TEAD/TEF family transcription factors (Vassilev, 2001). However, these studies have been performed exclusively in cultured mammalian cells and little is known about the physiological function of YAP (Huang, 2005).

yorkie: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 2 December 2018

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