BIOLOGICAL OVERVIEW

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).

The Hippo pathway coactivator Yorkie can reprogram cell fates and create compartment-boundary-like interactions at clone margins

During development, tissue-specific patterns of gene expression are established by transcription factors and then stably maintained via epigenetic mechanisms. Cancer cells often express genes that are inappropriate for that tissue or developmental stage. This study shows that high activity levels of Yki, the Hippo pathway coactivator that causes overgrowth in Drosophila imaginal discs, can also disrupt cell fates by altering expression of selector genes like engrailed (en) and Ultrabithorax (Ubx). Posterior clones expressing activated Yki can down-regulate en and express an anterior selector gene, cubitus interruptus (ci). The microRNA bantam and the chromatin regulator Taranis both function downstream of Yki in promoting ci expression. The boundary between Yki-expressing posterior clones and surrounding wild-type cells acquires properties reminiscent of the anteroposterior compartment boundary; Hedgehog signaling pathway activation results in production of Dpp. Thus, at least in principle, heterotypic interactions between Yki-expressing cells and their neighbors could activate boundary-specific signaling mechanisms (Bairzin, 2020).

Human cancers are characterized by multiple genetic lesions, a subset of which are driver mutations that are thought to be responsible for their tumorous characteristics. It is estimated that most cancers have two to eight driver mutations. This makes it difficult to evaluate the contribution of each mutation to any particular characteristic of the tumor. This study has taken advantage of the ability of single-gene manipulations to cause overgrowth in Drosophila imaginal discs to assess the ability of three different oncogenes to destabilize established patterns of selector gene expression; yki, the Drosophila ortholog of Yap and Taz, is especially potent in doing so. The patterns of expression of En, Ci, and Ubx are established relatively early in embryogenesis and maintained stably in imaginal discs during the larval stages of development. These patterns of expression can be disrupted in clones expressing an activated form of Yki. Expression of a wild-type form of Yki is capable of disrupting these expression patterns in combination with other genetic manipulations such as overexpression of ban or tara. This latter scenario is more likely to apply to human cancers; increased Yap or Taz activity has been described in multiple human cancers, which often also have other genetic lesions (Bairzin, 2020).

These studies show that sd, ban, and tara make important contributions to the pathway by which Yki^CA destabilizes gene expression; reducing the expression of any of these in clones expressing Yki^CA greatly reduces ectopic Ci expression, and increasing expression of both genes can cause ectopic Ci expression. It is likely that other mechanisms function in parallel to destabilize selector gene expression since combined overexpression of ban and tara increased ectopic Ci expression but did not reduce En expression (Bairzin, 2020).

Changing selector gene expression within an overgrowing clone can create interactions at the clone margin that are reminiscent of compartment boundaries and result in the production of morphogens. A recent study showed that forced expression of En in lgl clones can elicit similar phenomena in anterior clones. In addition, Yki^CA clones are often extruded, consistent with previous observations that heterotypic interactions caused by overexpressing patterning genes also promotes extrusion. Previous work found that ci RNA levels were increased in wts mutant tissue, this study did not see ectopic Ci protein expression in wts mutant clones or wild-type Yki-overexpressing clones. This work shows therefore that sustained expression of very high Yki levels is necessary to destabilize expression of selector genes. However, even under these conditions, the effect on ectopic Ci expression is Sd dependent. Moreover, this study shows that even wild-type Yki can, in combination with increased expression of ban or tara, induce ectopic Ci expression. While these changes in gene expression are most obvious with above-physiological levels of Yki, they nevertheless reflect a previously unknown ability of this pathway to alter patterning gene expression and furthermore to change the growth characteristics of neighboring wild-type cells. Differences in selector gene expression between human cancers or precancerous lesions and their wild-type neighbors have received relatively little attention, and the current results call attention to tumor margins as sites where heterotypic interactions could create signaling centers that affect the behavior of tumor cells (Bairzin, 2020).

The Snakeskin-Mesh complex of smooth septate junction restricts Yorkie to regulate intestinal homeostasis in Drosophila

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, loss of two smooth septate junction components, Snakeskin and Mesh, were found to cause a hyperproliferation phenotype. By examining epitope-tagged endogenous Snakeskin and Mesh, this study 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 and (Chen, 2020).

This study used knockin and knockout alleles of Ssk and Mesh to demonstrate their functions in both EBs and ECs to regulate ISC proliferation. Ssk and Mesh have low but detectable expression in the cytoplasm of EBs, while that in ECs is mainly in septate junctions but also with some cytoplasmic localization. Multiple lines of evidence suggest that the Ssk and Mesh expression in EBs is of functional importance. First, the EB driver Su(H) > has expression in fewer cells when compared with the Myo1A > driver but still can cause comparable Upd3 expression and proliferation phenotypes. Second, mutant Ssk and mesh MARCM clones have detectable upd3-promoter-LacZ reporter expression in late EBs, and in addition to that in mature ECs. Third, the Su(H) > Ssk or mesh RNAi had very minor Smurf and lethality phenotype, suggesting that the EB RNAi effects do not linger into mature ECs, but yet can cause strong proliferation phenotypes (Chen, 2020).

Recent reports have expanded the Hpo/Mst pathway to include Misshapen (Msn) and Happyhour (Hppy), as well as their mammalian homologs MAP4K1-7. Many membrane-associated proteins, such as cadherin-like protein FAT, adherens junction protein α-catenin, and tight junction protein Angiomotin are involved in the Hpo/Mst pathway by regulating upstream components. The current results suggest that smooth septate junctions may act as a signaling platform by directly binding to Yki. That previous protein interaction screens conducted in S2 cells had not identified the Yki complex with Ssk or Mesh may be because Ssk and Mesh are expressed much more highly in the gut than in other tissues (Chen, 2020).

Another smooth septate junction component Tsp2A regulates midgut homeostasis through the aPKC-Hpo pathway, possibly involving endosomal trafficking. This study also observed that Ssk and Mesh had detectable expression in cytoplasmic punctae. However, Tsp2A can act in the whole ISC-EB-EC lineage (Xu, 2019), while this study did not observe a function of Ssk and Mesh in early EBs. Therefore, it is possible that Mesh, Ssk, and Tsp2A can form a complex and are components of the smooth septate junction but each may also have independent functions (Chen, 2020).

Disruption of paracellular junctions in adult midgut leads to epithelial disorganization along the digestive track. Loss of tricellular junction protein Gliotactin leads to activation of the JNK pathway in ECs to stimulate ISC proliferation. Prolonged RNAi of Ssk and mesh, especially in ECs, leads to leaky gut and lethality, consistent with loss of septate junction integrity. The depletion of Yki alone after longer RNAi of Ssk or mesh in ECs, however, is not sufficient to suppress all the phenotypes, suggesting that such stress may stimulate multiple downstream response pathways. Regarding Yki target genes, upd3 is the best-characterized target in the midgut. The other well-known targets from imaginal discs, including DIAP1 and Bantam, are not good targets for Yki in the adult midgut. Meanwhile, one report shows that ImpL2 expression is highly increased in response to over-activated Yki in the midgut, and regulates tissue metabolism. The physiological stimulation of ImpL2 by Yki is still unclear. This study has also assayed for ImpL2 in the midgut after Ssk or mesh RNAi, but an increased expression of ImpL2 was not observed. It is speculated that ImpL2 may be a direct or indirect target related to metabolic phenotype induced by cancer-promoting genes, and therefore may not be regulated by the septate junction proteins Ssk or Mesh. Gut leakage may drive inflammation and trigger systematic immune response contributed by hemocytes, which in turn trigger ISC division indirectly. Further investigation will provide a more complete picture of how different pathways are involved (Chen, 2020).

A novel membrane protein Hoka regulates septate junction organization and stem cell homeostasis in the Drosophila gut

Smooth septate junctions (sSJs) regulate the paracellular transport in the intestinal tract in arthropods. In Drosophila, the organization and physiological function of sSJs are regulated by at least three sSJ-specific membrane proteins: Ssk, Mesh, and Tsp2A. This study reports a novel sSJ membrane protein Hoka, which has a single membrane-spanning segment with a short extracellular region, and a cytoplasmic region with the Tyr-Thr-Pro-Ala motifs. The larval midgut in hoka-mutants shows a defect in sSJ structure. Hoka forms a complex with Ssk, Mesh, and Tsp2A and is required for the correct localization of these proteins to sSJs. Knockdown of hoka in the adult midgut leads to intestinal barrier dysfunction, and stem cell overproliferation. In hoka-knockdown midguts, aPKC is up-regulated in the cytoplasm and the apical membrane of epithelial cells. The depletion of aPKC and yki in hoka-knockdown midguts results in reduced stem cell overproliferation. These findings indicate that Hoka cooperates with the sSJ-proteins Ssk, Mesh, and Tsp2A to organize sSJs, and is required for maintaining intestinal stem cell homeostasis through the regulation of aPKC and Yki activities in the Drosophila midgut (Izumi, 2021).

Epithelia separate distinct fluid compartments within the bodies of metazoans. For this epithelial function, occluding junctions act as barriers that control the free diffusion of solutes through the paracellular pathway. Septate junctions (SJs) are occluding junctions in invertebrates and form circumferential belts along the apicolateral region of epithelial cells. In transmission electron microscopy, SJs are observed between the parallel plasma membranes of adjacent cells, with ladder-like septa spanning the intermembrane space. Arthropods have two types of SJs: pleated SJs (pSJs) and smooth SJs (sSJs). pSJs are found in ectoderm-derived epithelia and surface glia surrounding the nerve cord, whereas sSJs are found mainly in the endoderm-derived epithelia, such as the midgut and gastric caeca. Despite being derived from the ectoderm, the outer epithelial layer of the proventriculus (OELP) and the Malpighian tubules also possess sSJs. Furthermore, pSJs and sSJs are distinguished by the arrangement of septa. For example, the septa of pSJs form regular undulating rows, whereas those in sSJs form regularly spaced parallel lines in the oblique sections in lanthanum-treated preparations. To date, more than 20 pSJ-related proteins have been identified and characterized in Drosophila. In contrast, only three membrane-spanning proteins, Ssk, Mesh and Tsp2A, have been reported as specific molecular constituents of sSJs (sSJ proteins) in Drosophila. Therefore, the mechanisms underlying sSJ organization and the functional properties of sSJs remain poorly understood compared with pSJs. Ssk has four membrane-spanning domains; two short extracellular loops, cytoplasmic N- and C-terminal domains, and a cytoplasmic loop. Mesh is a cell-cell adhesion molecule, which has a single-pass transmembrane domain and a large extracellular region containing a NIDO domain, an Ig-like E set domain, an AMOP domain, a vWD domain and a sushi domain. Tsp2A is a member of the tetraspanin family of integral membrane proteins in metazoans with four transmembrane domains, N- and C-terminal short intracellular domains, two extracellular loops and one short intracellular turn. The loss of ssk, mesh and Tsp2A causes defects in the ultrastructure of sSJs and the barrier function against a 10 kDa fluorescent tracer in the Drosophila larval midgut. Ssk, Mesh and Tsp2A interact physically and are mutually dependent for their sSJ localization. Thus, Ssk, Mesh and Tsp2A act together to regulate the formation and barrier function of sSJs. Furthermore, Ssk, Mesh and Tsp2A are localized in the epithelial cell-cell contact regions in the Drosophila Malpighian tubules in which sSJs are present. Recent studies have shown that the knockdown of mesh and Tsp2A in the epithelium of Malpighian tubules leads to defects in epithelial morphogenesis, tubule transepithelial fluid and ion transport, and paracellular macromolecule permeability in the tubules. Thus, sSJ proteins are involved in the development and maintenance of functional Malpighian tubules in Drosophila (Izumi, 2021).

The Drosophila adult midgut consists of a pseudostratified epithelium, which is composed of absorptive enterocytes (ECs), secretory enteroendocrine cells (EEs), intestinal stem cells (ISCs), EC progenitors (enteroblasts: EBs) and EE progenitors (enteroendocrine mother cells: EMCs). The sSJs are formed between adjacent ECs and between ECs and EEs. To maintain midgut homeostasis, ECs and EEs are continuously renewed by proliferation and differentiation of the ISC lineage through the production of intermediate differentiating cells, EBs and EMCs. Recently, it has been reported that the knockdown of sSJ proteins Ssk, Mesh and Tsp2A in the midgut causes intestinal hypertrophy accompanied by the overproliferation of ECs and ISC. These results indicate that sSJs play a crucial role in maintaining tissue homeostasis through the regulation of stem cell proliferation and enterocyte behavior in the Drosophila adult midgut. Furthermore, it has been reported that the loss of mesh and Tsp2A in adult midgut epithelial cells causes defects in cellular polarization, although no remarkable defects in epithelial polarity were found in the first-instar larval midgut cells of ssk, mesh and Tsp2A mutants. Thus, sSJs may contribute to the establishment of epithelial polarity in the adult midgut (Izumi, 2021).

During the regeneration of the Drosophila adult midgut epithelium, various signaling pathways are involved in the proliferation and differentiation of the ISC lineage. Atypical protein kinase C (aPKC) is an evolutionarily conserved key determinant of apical-basal epithelial polarity . Importantly, it has been reported that aPKC is dispensable for the establishment of epithelial cell polarity in the Drosophila adult midgut. It has been reported that aPKC is required for differentiation of the ISC linage in the midgut. The Hippo signaling pathway is involved in maintaining tissue homeostasis in various organs. In the Drosophila midgut, inhibition of the Hippo signaling pathway activates the transcriptional co-activator Yorkie (Yki), which results in accelerated ISC proliferation via the Unpaired (Upd)-Jak-Stat signaling pathway. Recent studies have shown that Yki is involved in ISC overproliferation caused by the depletion of sSJ proteins in the midgut. Furthermore, it has been shown that aPKC is activated in the Tsp2A-RNAi-treated midgut, leading to activation of its downstream target Yki and causing ISC overproliferation through the activation of the Upd-Jak-Stat signaling pathway. Thus, crosstalk between aPKC and the Hippo signaling pathways appears to be involved in ISC overproliferation caused by Tsp2A depletion (Izumi, 2021).

To further understand the molecular mechanisms underlying sSJ organization, a deficiency screen was performed for Mesh localization, and the integral membrane protein Hoka was identified as a novel component of Drosophila sSJs. Hoka consists of a short extracellular region and the characteristic repeating 4-amino acid motifs in the cytoplasmic region, and is required for the organization of sSJ structure in the midgut. Hoka and Ssk, Mesh, and Tsp2A show interdependent localization at sSJs and form a complex with each other. The knockdown of hoka in the adult midgut results in intestinal barrier dysfunction, aPKC- and Yki-dependent ISC overproliferation, and epithelial tumors. Thus, Hoka plays an important role in sSJ organization and in maintaining ISC homeostasis in the Drosophila midgut (Izumi, 2021).

The identification of Ssk, Mesh and Tsp2A has provided an experimental system to analyze the role of sSJs in the Drosophila midgut. Recent studies have shown that sSJs regulate the epithelial barrier function and also ISC proliferation and EC behavior in the midgut. Furthermore, sSJs are involved in epithelial morphogenesis, fluid transport and macromolecule permeability in the Malpighian tubules. This study reports the identification of a novel sSJ-associated membrane protein Hoka. Hoka is required for the efficient accumulation of other sSJ proteins at sSJs and the correct organization of sSJ structure. The knockdown of hoka in the adult midgut leads to intestinal barrier dysfunction, increased ISC proliferation mediated by aPKC and Yki activities, and epithelial tumors. Thus, Hoka contributes to sSJ organization and the maintenance of ISC homeostasis in the Drosophila midgut (Izumi, 2021).

Arthropod sSJs have been classified together based on their morphological similarity. The identification of sSJ proteins in Drosophila has provided an opportunity to investigate whether sSJs in various arthropod species share similarities at the molecular level. However, Hoka homolog proteins appear to be conserved only in insects upon a database search, suggesting compositional variations in arthropod sSJs (Izumi, 2021).

Interestingly, the cytoplasmic region of Hoka includes three YTPA motifs. The same or similar amino acid motifs are also present in the Hoka homologs of other holometabolous insects, such as other Drosophila species, the mosquito, beetle (YTPA motif), butterfly, ant, bee, sawfly, moth (YQPA motif) and flea (YTAA motif), although the number of these motif(s) vary (1 to 3 in Drosophila species, 1 in other holometabolous insects). In contrast, the motif is not present in hemimetabolous insects. The extensive conservation of the YTPA/YQPA/YTAA motif in holometabolous insects suggests that the motif was evolutionarily acquired and plays a critical role in the molecular function of Hoka. It would be interesting to investigate the role of the YTPA/YQPA/YTAA motif in sSJ organization of holometabolous insects (Izumi, 2021).

The extracellular region of Hoka appears to be composed of 13 amino acids alone after the cleavage of the signal peptide, which is too short to bridge the 15-20 nm intercellular space of sSJs. Thus, Hoka is unlikely to act as a cell adhesion molecule in sSJs. Indeed, the overexpression of Hoka-GFP in Drosophila S2 cells did not induce cell aggregation, which is a criterion for cell adhesion activity (Izumi, 2021).

The loss of an sSJ protein results in the mislocalization of other sSJ proteins, indicating that sSJ proteins are mutually dependent for their sSJ localization. In thessk -deficient midgut, Mesh and Tsp2A were distributed diffusely in the cytoplasm. In the mesh mutant midgut, Ssk was localized at the apical and lateral membranes, whereas Tsp2A was distributed diffusely in the cytoplasm. In the Tsp2A-mutant midgut, Ssk was localized at the apical and lateral membranes, whereas Mesh was distributed diffusely in the cytoplasm. Among these three mutants, the mislocalization of Ssk, Mesh or Tsp2A is consistent; Mesh and Tsp2A were distributed in the cytoplasm, whereas Ssk was localized at the apical and lateral membranes. However, in the hoka-mutant larval midgut, Mesh and Tsp2A were distributed along the lateral membrane, whereas Ssk was mislocalized to the apical and lateral membranes. Interestingly, in some hoka mutant midguts, Ssk, Mesh and Tsp2A were localized to the apicolateral region, as observed in the wild-type midgut. Differences in subcellular misdistribution of sSJ proteins between the hoka mutant and the ssk, mesh and Tsp2A-mutants indicate that the role of Hoka in the process of sSJ formation is different from that of Ssk, Mesh or Tsp2A. Ssk, Mesh and Tsp2A may form the core complex of sSJs, and these proteins are indispensable for the generation of sSJs, whereas Hoka facilitates the arrangement of the primordial sSJs at the correct position, i.e. the apicolateral region. This Hoka function may also be important for rapid paracellular barrier repair during the epithelial cell turnover in the adult midgut. Notably, during the sSJ formation process of the outer epithelial layer of the proventriculus (OELP, the sSJ targeting property of Hoka was similar to that of Mesh, implying that Hoka may have a close relationship with Mesh, rather than Ssk and Tsp2A during sSJ development (Izumi, 2021).

The knockdown of hoka in the adult midgut leads to a shortened lifespan in adult flies, intestinal barrier dysfunction, increased ISC proliferation and the accumulation of ECs. These results are consistent with the recent observation for ssk, mesh and Tsp2A-RNAi in the adult midgut. The intestinal barrier dysfunction caused by RNAi for sSJ proteins may permit the leakage of particular substances from the midgut lumen, which may induce particular cells to secrete cytokines and growth factors for ISC proliferation. Alternatively, sSJs or sSJ-associated proteins may be directly involved in the secretion of cytokines and growth factors through the regulation of intracellular signaling in the ECs. In the latter case, it has been shown that Tsp2A knockdown in ISCs/EBs or ECs hampers the endocytic degradation of aPKC, thereby activating the aPKC and Yki signaling pathways, leading to ISC overproliferation in the midgut. Therefore, it has been proposed that sSJs are directly involved in the regulation of aPKC and the Hippo pathway-mediated intracellular signaling for ISC proliferation. This study has shown that the expression of hoka-RNAi together with aPKC-RNAi or yki-RNAi in ECs significantly reduced ISC overproliferation caused by hoka-RNAi. Thus, aPKC- and Yki-mediated ISC overproliferation appears to commonly occur in sSJ protein-deficient midguts. However, the possibility that the leakage of particular substances through the paracellular route may be involved in ISC overproliferation in the sSJ proteins-deficient midgut cannot be excluded (Izumi, 2021).

It has been reported that apical aPKC staining is observed in ISCs but is barely detectable in ECs. This study found that the expression of hoka-RNAi in ECs increased aPKC staining in the midgut. Additionally, in the hoka-RNAi midgut, apical aPKC staining was observed in ISCs and in differentiated cells, including EC-like cells. Thus, apical and increased cytoplasmic aPKC may contribute to ISC overproliferation. Interestingly, EC-like cells in the hoka-RNAi midgut do not always localize aPKC to the apical regions. Apical aPKC staining was detected in EC-like cells mounted by other cells but was barely detectable in the lumen-facing EC-like cells. These mounted cells are thought to be newly generated cells after the induction of hoka-RNAi, which may not be able to exclude aPKC from the apical region in the crowded cellular environment. A recent study showed that aberrant sSJ formation caused by Tsp2A-depletion impairs aPKC endocytosis and increases aPKC localization in the membrane of cell borders. The sSJ proteins, including Hoka, may also regulate endocytosis to exclude aPKC from the apical membrane of ECs. The identification of molecules involved in aPKC-mediated ISC proliferation may provide a better understanding of the aPKC-mediated signaling pathway, as well as the mechanisms underlying the increased expression and apical targeting of aPKC in the ECs deficient for sSJ proteins (Izumi, 2021).

Control of hormone-driven organ disassembly by ECM remodeling and Yorkie-dependent apoptosis

Epithelia grow and shape into functional structures during organogenesis. Although most of the focus on organogenesis has been drawn to the building of biological structures, the disassembly of pre-existing structures is also an important event to reach a functional adult organ. Examples of disassembly processes include the regression of the Mullerian or Wolffian ducts during gonad development and mammary gland involution during the post-lactational period in adult females. To date, it is unclear how organ disassembly is controlled at the cellular level. This study follows the Drosophila larval trachea through metamorphosis and shows that its disassembly is a hormone-driven and precisely orchestrated process. It occurs in two phases: first, remodeling of the apical extracellular matrix (aECM), mediated by matrix metalloproteases and independent of the actomyosin cytoskeleton, results in a progressive shortening of the entire trachea and a nuclear-to-cytoplasmic relocalization of the Hippo effector Yorkie (Yki). Second, a decreased transcription of the Yki target, Diap1, in the posterior metameres and the activation of caspases result in the apoptotic loss of the posterior half of the trachea while the anterior half escapes cell death. Thus, this work unravels a mechanism by which hormone-driven ECM remodeling controls sequential tissue shortening and apoptotic cell removal through the transcriptional activity of Yki, leading to organ disassembly during animal development (Fraire-Zamora, 2021).

This study report how a functional organ, the larval trachea of Drosophila melanogaster, undergoes a hormone-driven disassembly during metamorphosis. The dorsal trunks of the trachea shorten modularly in two sequential phases: (1) an initial progressive phase of a controlled reduction in the trachea length, involving aECM remodeling and cellular shape changes and, as a consequence, (2) Yki inactivation results in a decreased transcription of its target gene (and apoptosis inhibitor) Diap1 in the posterior metameres, resulting in their loss through cell death (Fraire-Zamora, 2021).

It was found that the activation of apoptosis results in the disassembly and loss of only the posterior metameres (Tr6-Tr8). Why are anterior metameres (Tr3-Tr5) not affected? This is an intriguing observation because most of the signaling inputs occur along the whole dorsal trunk. These results suggest that the anterior metameres are 'protected' or 'desensitized' against the signals that occur along the dorsal trunks. The results on downregulation of AbdB suggest that Hox genes play a role in the differential response between anterior and posterior metameres during dorsal trunk disassembly. However, it cannot be excluded that other elements might also contribute, such as the proximity of pools of adult progenitor cells to the anterior metameres. Whether the anterior metameres are protected against MMP-1 activity and cell death through a compartmentalization of the trachea via Hox genes or through signals from the progenitor cells is a matter of future work (Fraire-Zamora, 2021).

The Hpo signaling pathway and its effector Yki/YAP are conserved both in invertebrates and vertebrates where they regulate organ size through the transcriptional control of genes related to proliferation and cell death. While work in mammalian cell cultures has shown that ECM-related inactivation of YAP can lead to an increase in apoptosis, most of the focus has concentrated on how the Hpo signaling pathway controls proliferation, with very few examples on the activation of apoptosis (Fraire-Zamora, 2021).

The current results unveil a new role of the Hpo pathway in the hormone-driven disassembly of an organ through an ECM-triggered inactivation of the Hpo effector Yki, resulting in the triggering of apoptosis on the posterior end of the trachea. Given the conservation of the Hpo pathway, it is speculated that its role in organ disassembly could be of general use in some of the widespread events leading to organ involution (i.e., the controlled regression or shrinkage of an organ) during embryonic development or in homeostatic processes during adult life or aging (Fraire-Zamora, 2021).

Yorkie drives supercompetition by non-autonomous induction of autophagy via bantam microRNA in Drosophila

Mutations in the tumor-suppressor Hippo pathway lead to activation of the transcriptional coactivator Yorkie (Yki), which enhances cell proliferation autonomously and causes cell death non-autonomously. The mechanism by which Yki causes cell death in nearby wild-type cells, a phenomenon called supercompetition, and its role in tumorigenesis remained unknown. This study shows that Yki-induced supercompetition is essential for tumorigenesis and is driven by non-autonomous induction of autophagy. Clones of cells mutant for a Hippo pathway component fat activate Yki and cause autonomous tumorigenesis and non-autonomous cell death in Drosophila eye-antennal discs. This study found that mutations in autophagy-related genes or NF-κB genes in surrounding wild-type cells block both fat^-induced tumorigenesis and supercompetition. Mechanistically, fat mutant cells upregulate Yki-target microRNA bantam, which elevates protein synthesis levels via activation of TOR signaling. This induces elevation of autophagy in neighboring wild-type cells, which leads to downregulation of IκB Cactus and thus causes NF-κB-mediated induction of the cell death gene hid. Crucially, upregulation of bantam is sufficient to make cells to be supercompetitors and downregulation of endogenous bantam is sufficient for cells to become losers of cell competition. These data indicate that cells with elevated Yki-bantam signaling cause tumorigenesis by non-autonomous induction of autophagy that kills neighboring wild-type cells (Nagata, 2022).

The data reveal that the Hippo pathway mutant fat clones cause supercompetition by inducing autophagy-mediated cell death in surrounding wild-type cells via NF-κB-mediated induction of hid. The autophagy induction in wild-type cells depends on Yki-bantam-mediated activation of TOR signaling in neighboring fat mutant cells. This mechanism is similar to what was observed in the elimination of ribosomal protein or Hel25E mutant loser clones when surrounded by wild-type cells. This is particularly interesting in two ways: first, it suggests that different types of cell competition, namely elimination of unfit cells by wild-type cells and elimination of wild-type cells by supercompetitors, are driven by the common mechanism, and second, it indicates that induction of autophagy in loser cells is non-autonomous, as even wild-type cells elevate autophagy when juxtaposed to supercompetitors. Although the mechanism by which autophagy is induced in loser cells nearby winner cells remains unknown, observations in this study in conjunction with the previous data on the elimination of ribosomal protein or Hel25E mutant clones suggest the possibility that relative difference in protein synthesis levels between cells plays a critical role in autophagy induction (Nagata, 2022).

The mechanism by which elevated autophagy induces hid expression via NF-κB still remains to be elucidated. Elevated autophagy results in downregulation of IκB protein Cactus. IκB is known to be degraded by the ubiquitin-proteasome system. On the other hand, elevated autophagy by starvation or rapamycin treatment was shown to cause degradation of IκB and thus activate NF-κB in mouse fibroblast. Together, the data suggest the possibility that IκB is degraded by selective autophagy in losers of cell competition (Nagata, 2022).

The observations of this studsy intriguingly show that non-autonomous cell death in wild-type cells promotes fat-induced tumorigenesis. This supports the idea that cancer cells expand their territories within the tissue by cell competition during malignant progression of tumors. While the mechanism by which wild-type cell death fuels neighboring tumorigenesis is an important open question, it may involve compensatory proliferation triggered by mitogenic factors secreted from dying cells. Intriguingly, it has been reported in Drosophila eye-antennal discs that clones of malignant tumors caused by Ras activation and cell polarity defects induce autophagy in surrounding wild-type cells, which in this case do not cause cell death but provide nutrient such as amino acids to neighboring tumors to promote their growth. Clones of cells overexpressing activated form of Yki were also shown to induce autophagy in neighboring cells, but in this case non-autonomous autophagy does not have a role in promoting tumorigenesis. Thus, non-autonomous autophagy may have multiple roles and mechanisms in regulating tissue homeostasis and tumorigenesis (Nagata, 2022).

Given that the Hippo pathway is conserved throughout evolution and that YAP-mediated cell competition occurs in mammalian systems as well, autophagy-mediated cell death may play an important role in mammalian cell competition. Notably, in a mouse liver cancer model, hyperactivation of YAP in peritumoral hepatocytes triggers regression of primary liver tumors and melanoma-derived liver metastases. Thus, further studies on the mechanism of Hippo-signaling-mediated supercompetition in Drosophila may provide a novel therapeutic strategy against human cancers (Nagata, 2022).

Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye

The canonical function of the Hippo signaling pathway is the regulation of organ growth. How this pathway controls cell-fate determination is less well understood. This study identified a function of the Hippo pathway in cell-fate decisions in the developing Drosophila eye, exerted through the interaction of Yorkie (Yki) with the transcriptional regulator Bonus (Bon), an ortholog of mammalian transcriptional intermediary factor 1/tripartite motif (TIF1/TRIM) family proteins. Instead of controlling tissue growth, Yki and Bon promote epidermal and antennal fates at the expense of the eye fate. Proteomic, transcriptomic, and genetic analyses reveal that Yki and Bon control these cell-fate decisions by recruiting transcriptional and post-transcriptional co-regulators and by repressing Notch target genes and activating epidermal differentiation genes. This work expands the range of functions and regulatory mechanisms under Hippo pathway control (Zhao, 2023).

Tissue growth and cell-fate determination are critical developmental processes controlled by multiple signaling pathways, including the evolutionarily conserved Hippo pathway, whose dysregulation leads to developmental abnormalities and diseases. The core Hippo (Hpo, MST1/2 in mammals)/Warts (Wts, LATS1/2 in mammals) kinase cascade inhibits the activity of the transcriptional coactivator Yorkie (Yki, YAP/TAZ in mammals) by phosphorylation and cytoplasmic retention, whereas unphosphorylated nuclear Yki associates with DNA-binding proteins such as Scalloped (Sd, TEAD1–4 in mammals) to activate gene expression (Zhao, 2023).

The canonical transcriptional targets of the Yki-Sd complex in Drosophila include Cyclin E (CycE), Death-associated inhibitor of apoptosis 1 (Diap1), bantam microRNA (mir-ban), and expanded (ex), which promote proliferation, inhibit apoptosis, and enable negative feedback regulation. Although increasing evidence supports the essential role of the Hippo pathway in cell-fate determination, the cellular mechanisms remain poorly understood (Zhao, 2023).

The Drosophila eye is an excellent model to study gene regulatory networks controlling cell-fate determination. ost of the Drosophila adult head structures develop from the larval eye-antennal disc, with the compound eye and ocelli originating from the eye disc compartment, the antenna and maxillary palp from the antennal compartment, and the head epidermis from the tissues surrounding the two compartments (Zhao, 2023).

Segregation of the mutually antagonistic eye, antennal, and head epidermal fates, which begins at the second instar larval stage (L2), is regulated by several signaling pathways, including Notch, EGFR, Wingless, and Hedgehog, and retinal determination genes such as eyeless (ey) and homothorax (hth). Alteration of these regulatory inputs can cause a switch from one fate to another, leading to partial or, in some cases, complete homeotic transformations of the affected structures (Zhao, 2023).

Key patterning events in the eye are linked to a wave of differentiation called the morphogenetic furrow (MF) that starts in the early third instar larval stage (L3) and proceeds from the posterior to the anterior of the eye disc, resulting in the differentiation of an array of optical units called ommatidia, each consisting of photoreceptor cells, cone cells, primary pigment cells, interommatidial bristles, and secondary and tertiary pigment cells (also called interommatidial cells) (Zhao, 2023).

Previous studies of the Hippo pathway in Drosophila eye differentiation focused on MF progression, terminal differentiation of photoreceptor cells, and formation of peripodial epithelium. Mutant analyses of the Hippo pathway components ex, Merlin (Mer), and mob as tumor suppressor (mats) have suggested an earlier and broader impact of the Hippo pathway in eye specification (Zhao, 2023).

However, the involvement of the Hippo pathway in controlling major cell-fate decisions among the eye, antenna, and head epidermis remains elusive, and the underlying transcriptional mechanisms are unknown. It was reasoned that the Hippo pathway may function in controlling the eye-antenna-epidermis fate determination through yet unknown interactors that regulate the transcriptional output of the Yki-Sd complex. To identify such interactors, proteomic analyses was performed and a Yki-interacting protein, Bonus (Bon) was identified. Bon is the only Drosophila ortholog of mammalian TIF1 family proteins TIF1α (TRIM24), TIF1β (TRIM28/KAP1), TIF1γ (TRIM33), and TIF1δ (TRIM66) TIF1/Bon proteins are chromatin-associated factors that activate or repress transcription by binding to co-regulators and controlling chromatin state. TIF1 proteins play various roles during vertebrate development and are implicated in cancer. Drosophila Bon is essential for nervous system development, embryo patterning, metamorphosis, and cell survival (Zhao, 2023).

Evidence is presented that Bon and the Hippo pathway co-regulate major cell-fate decisions during the development of the Drosophila eye. Yki and Bon bind via WW domain-PPxY motif interactions and cooperate to produce epidermal cells in the eye at the expense of ommatidial cells, while the loss of bon induces ectopic eye markers, suggesting that the Hippo pathway and Bon control the choice between the eye and epidermal fates. The Hippo pathway and Bon also regulate the eye-antennal specification, with Yki and Bon inhibiting the eye fate and promoting the antennal fate. Through the analysis of Bon and Yki protein interactors, multiple transcriptional and post-transcriptional regulators have been identified that are necessary for their control of cell-fate decisions. Transcriptomic and genetic analyses have revealed that Bon and Yki exert their functions by jointly activating epidermal differentiation genes and, unexpectedly, repressing Notch target genes. Overall, this study has identified a function of the Hippo pathway in the eye/antenna/epidermis cell-fate decisions during Drosophila eye development. This function requires the interaction of Yki with Bon, their recruitment of co-regulators, and the joint transcriptional control of a non-canonical set of target genes (Zhao, 2023).

Instead of mediating the previously described independent functions of Yki (growth control) and Bon (PNS differentiation), the Yki-Bon module regulates proper segregation of the eye, epidermal, and antennal fates in the developing eye. This function involves promotion of the epidermal and antennal fates, and suppression of the eye fate, via transcriptional regulation of a distinct set of target genes. This study thus provides a molecular mechanism for the biological function of the Hippo pathway and Bon in cell-fate determination during eye development (Zhao, 2023).

The results suggest that the Hippo pathway and Bon regulate the developmental cell-fate decisions in the eye at two levels. First, the Yki-Bon complex promotes antennal and epidermal fates and suppresses the eye fate during early eye field specification, before the L3 larval stage. This is supported by the phenotypes observed under various genetic manipulations of Bon, Wts, Yki, and Sd during the L1 and L2 larval stages, including the reciprocal transformations of eye and antenna, epidermal outgrowths in the eye, and ectopic eye fate. The Yki-Bon module is thus an essential component of the extensive gene regulatory network that controls these cell-fate decisions in early eye development (Zhao, 2023).

Previous studies showed that ex, Mer, and mats mutants exhibit eye-to-epidermal transformation and an occasional eye-to-antenna transformation (in an ex mutant combination), suggesting that the upstream Hippo pathway may also regulate the Yki-Bon module in fate determination at this stage. Second, after the segregation of the eye/antenna/epidermis fields and the start of MF in L3, the Yki-Bon complex promotes the epidermal cell fate while suppressing ommatidia, whereas Wts counteracts this activity. This is evidenced by the formation of epidermal trichomes in the retina and the suppression of ommatidial cell types, especially cone cells, upon knockdown of wts or overexpression of Bon or Yki with the late eye driver GMR-GAL4. Furthermore, RNA-seq data also revealed cell-fate regulation at the molecular level: the Yki-Bon complex activates epidermal differentiation genes (sha, f, nyo, and neo) and represses Notch targets (E(spl)-C, ct, and TfAP-2) that are required for eye fate establishment and are expressed in ommatidial cells (e.g., ct in cone cells and E(spl)mdelta-HLH in primary pigment cells). Although activation of the Yki-Bon complex at this stage did not exhibit an eye-to-antenna transformation phenotype, certain antennal genes (Gr64s and Or47b) were upregulated, suggesting transformation at the level of gene expression (Zhao, 2023).

This work shows that these fates are not completely defined during the early stages, as the retina could still transform into epidermal tissue and express epidermal and even antennal genes when the Hippo pathway and Bon were modulated after MF formation. Notably, conditional knockout of eya after the MF results in suppression of ommatidia and formation of trichomes in the eye. This suggests that the retinal determination genes are also involved in eye-epidermal fate decisions during later stages of eye development and that trichome induction may be a general biological outcome of interference with the eye vs. epidermis specification after the start of MF. Thus, it is concluded that the eye is still developmentally plastic at late stages, with a latent epidermal fate that is normally inhibited. Interestingly, this fate is revealed in the insect order Strepsiptera, whose compound eyes are composed of optical units that are separated by epidermal tissue bearing trichome-like extensions (Zhao, 2023).

Given Bon’s role in promoting the epidermal fate in the eye, it was asked whether Bon is involved in epidermal differentiation in other tissues. Knockdown of bon by RNAi in the wing with the C5-GAL4 driver did not affect the number of trichomes, but trichome morphology was abnormal, with bon-RNAi wing cells growing thinner trichomes. bon^21B mutant sensory bristles on the notum showed a similar thinning effect, although the Sb clonal marker precluded genotyping surrounding epidermal cells. These results suggest that Bon may contribute to epidermal differentiation in other contexts in addition to its role in the eye (Zhao, 2023).

This study has identified an unexpected layer of control over eye specification exerted by Yki and Bon at the level of Notch target genes. The Hippo pathway has been reported to control cell-fate determination in other biological contexts through regulation of the Notch receptor or ligands. Although several Notch targets were identified that are repressed by Bon and Yki, Notch and its ligands, Serrate and Delta, were not jointly regulated or found in high-confidence Yki or Bon protein interactomes. Therefore, it is proposed that during cell-fate determination in the eye, Bon and Yki repress Notch targets (such as E(spl)-C genes) independently from upstream Notch signaling. It is noted that not all E(spl)-C genes are under Bon and Yki control, implying context-dependent regulation and functional divergence of E(spl)-C genes, as suggested by previous studies (Zhao, 2023).

Both Hippo and Notch contribute to cell proliferation and growth of the eye. The data suggest that Bon is not required for the growth-controlling function of the Hippo pathway. Instead, the Bon-Yki complex directs the acquisition of appropriate cell fates in the eye through the regulation of Notch targets. It is speculated that Bon may function as a switch that redirects some of Hippo pathway activities from growth regulation to cell-fate determination. So far, Drosophila Yki has only been implicated in transcriptional activation. However, studies in mammalian systems have shown that the YAP/TAZ-TEAD complex can also function as a transcriptional repressor on non-canonical target genes (Zhao, 2023).

The repression of Notch targets reported in this study suggests that Drosophila Yki can also function in transcriptional repression, likely via the recruitment of corepressors mediated by Bon. HDAC1 and its associated corepressor complexes repress gene transcription, including Notch targets (Zhao, 2023).

This study identified HDAC1 and its corepressor, CoRest, in the Bon interactome, raising the possibility that Bon and Yki repress Notch target genes in part via recruiting this repressor complex. The involvement of epigenetic regulators is further exemplified by Su(var)2-10, which has a role in chromatin SUMOylation and piRNA target silencing. Interestingly, Su(var)2-10 can suppress eye fate and even induce antennal fate in a sensitized background. Due to the strong genetic interaction between Su(var)2-10 and the Bon-Yki complex, and the identification of the Drosophila SUMO (smt3) in the Bon interactome, chromatin SUMOylation may be involved in gene repression by Bon and Yki. Future studies of chromatin status and epigenetic marks may reveal the mechanistic details of gene repression by Bon and Yki. The Hippo pathway and TIF1 family proteins are conserved and broadly expressed in higher eukaryotes, raising the possibility that they may also function together in other species and developmental processes, such as retinogenesis and hematopoiesis. Thus, the biological functions controlled by the Hippo pathway and Bon, and the underlying molecular mechanisms reported in this study here, may be evolutionarily conserved (Zhao, 2023).

For this study, RNA-seq was performed using pupal eyes when trichomes initiate; however, the pupal eye patterning defects were detectable before trichome initiation. Thus, there might be additional differentially expressed genes at earlier stages of cell-fate determination that were have missed. Cells mutant for a null allele of bon tend to be eliminated, potentially masking additional cell differentiation defects in bon mutant clones. Although this study has largely focused on eye development, it is possible that Bon and Yki interaction has additional functions in other tissues. Further studies are needed to analyze the precise composition of multiprotein complexes involving Yki and Bon, as well as their effects on the target genes that were identified in this study (Zhao, 2023).

GENE STRUCTURE

cDNA clone length - 1427

Bases in 5' UTR - 170

Exons - 4

Bases in 3' UTR - 112

PROTEIN STRUCTURE

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).

date revised: 10 June 2024

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