The Identification of Hippo Pathway Mutants
A striking feature of many of the Hippo pathway components is their distinctive mutant phenotype, which results in dramatic overgrowth of mutant tissue in the imaginal discs and in adult organs (reviewed in Edgar,2006; Pan,2007; Saucedo and Edgar,2007). Thus, hippo (hpo), salvador (Sav), and warts (wts) were isolated based on their overgrown adult head phenotype in chemical mutagenesis screens. Mob-as-tumor-suppressor (mats) was a spontaneous mutation that became a part of the Hippo pathway in part due to the similarity of its mutant phenotype in adult heads and imaginal discs to other Hippo pathway mutants. Previously known genes like fat (ft), expanded (ex), and merlin (mer) were identified as components of the Hippo pathway based on a common/shared phenotype. Yorkie (Yki), on the other hand, was identified in a biochemical screen (yeast two-hybrid) for Warts interacting proteins.
The adult heads of flies of Hippo pathway mutants in which over 90% of cells were homozygous mutant were proportionately larger than the other structures but had a normal overall pattern. The mutant cells proliferated faster to out-compete the normal wild-type cells (Fig. 1). The underlying cause of overgrowth observed in the adult eyes, was studied in both the pupal retinae and the eye–antennal imaginal discs. In the adult eye sections, the pattern of ommatidial development was largely unaffected except for the spacing of the ommatidial clusters. To trace back this phenotype, the mid-pupal retinae were studied in which cellular outlines were marked using antibodies. Hippo pathway mutants exhibited a dramatic increase in the numbers of interommatidial cells when compared with wild-type (Fig. 1).
Figure 1. The overgrowth phenotype of Hippo pathway mutants. A–D: In comparison to normal flies (A), the adult heads of flies mutant for Warts (D), Salvador (C), and Hippo (B) show overgrowth and increase in overall size. The mutant cells are generated by a tissue-specific mosaic technique in which the normal cells (+/+) are killed due to the presence of a cell-lethal mutation, the mutant cells are marked by the presence of the white pigment cells, and the heterozygous cells appear orange/red. The eyes of flies mutant for Hippo, Salvador, and Warts are almost completely (95%) white, indicating that the entire eye is composed of mutant cells, whereas in normal wild-type flies using the same technique approximately 50% of the cells are heterozygous. E–H: Pupal retinae stained with an antibody against Discs large, a protein that marks cell outlines, clearly shows a shared phenotype of excess interommatidial cells in Hippo pathway mutants (f–h) compared with wild-type (e).
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The extra interommatidial cells could be due to excessive cell proliferation, increased spacing of photoreceptor clusters during patterning, lack of apoptosis, or a combination thereof. Systematic analyses revealed that this phenotype results because Hippo pathway mutant cells proliferate faster than surrounding wild-type cells and because they do not terminate proliferation when imaginal tissues have reached their normal size. In fact, hpo mutant cells do not deregulate cell proliferation of terminally arrested cells, as cells differentiate normally. In addition, Hippo affects cell survival. In the wild-type pupal retina, extra interommatidial cells are removed by apoptosis, while extra hpo mutant cells are not. Thus, Hippo has a dual function in promoting cell proliferation arrest and apoptosis (Tapon et al.,2002; Harvey et al.,2003; Jia et al.,2003; Udan et al.,2003; Wu et al.,2003; reviewed in Harvey and Tapon,2007). These effects on proliferation arrest and apoptosis are not limited to the eye–antennal disc, but are observed in all disc-derived structures, including wings, halteres, legs, and thorax, suggesting that Hippo function is ubiquitously required. The ubiquitous expression pattern of several Hippo pathway components in imaginal discs supports this conclusion.
The molecular characterization of Hpo (Ste-20 kinase) and Sav (WW domain adaptor), together with the previously known Lats/Wts (Dbf-related kinase), shed important insights on the interactions between the proteins encoded by these genes, and a signaling pathway began to emerge (Jia et al.,2003; Udan et al.,2003; Wu et al.,2003; reviewed in Harvey and Tapon,2007). Since then, the addition of the Mob super-family protein Mats (Lai et al.,2005), two four point one, ezrin, radixin, moesin (FERM) domain-containing proteins Ex and Mer (Hamaratoglu et al.,2006), a transmembrane Proto-Cadherin Ft (Bennett and Harvey,2006; Cho et al.,2006; Silva et al.,2006; Willecke et al.,2006), the transcriptional coactivator Yki (Huang et al.,2005), and the transcription factor Scalloped (Sd; Goulev et al.,2008; Wu et al.,2008; Zhang et al.,2008b) laid down the frame-work of a signaling network from the membrane to the nucleus for the Hippo signaling pathway. Several targets of Hippo signaling were identified during the course of characterization of mutant phenotypes of pathway components, which include Cyclin E (CycE; Kango-Singh et al.,2002; Tapon et al.,2002), Cyclin B (CycB; Tyler and Baker,2007) and E2F (Nicolay and Frolov,2008; all regulating cell proliferation), Drosophila inhibitor of apoptosis protein 1 (DIAP1; apoptosis; Tapon et al.,2002), the cytoskeletal proteins ex (cell shape; Hamaratoglu et al.,2006), the microRNA bantam (Nolo et al.,2006), and target genes that function in other pathways and Hippo signaling like four-jointed (fj; planar cell polarity; Fanto et al.,2003; Cho and Irvine,2004), division abnormally delayed (dally) and dally-like (dlp; Drosophila HSPGs primarily function in the spread of morphogen signals; Baena-Lopez et al.,2008), and wingless (wg; Cho and Irvine,2004) involved in patterning and growth (reviewed in Edgar,2006; Yin and Pan,2007; Reddy and Irvine,2008).
The Kinase Cascade in the Hippo Pathway
The two protein kinases, Hpo and Wts form a core kinase cascade in the Hippo pathway. Hpo is a member of the Ste-20 superfamily of kinases, and acts upstream of Wts, a member of the nuclear Dbf2 related (NDR) family of protein kinases. Hpo activates Wts by direct phosphorylation without affecting the levels of Wts expression (Wu et al.,2003). Hpo-mediated Wts activation is facilitated by Sav, a WW domain-containing protein that probably functions as a scaffold, and also by Mats, a protein that binds directly to Wts and Hpo (Wu et al.,2003; Wei et al.,2007). When activated, Wts can phosphorylate Yki and reduce its activity, and as a consequence Yki is excluded from the nucleus (Dong et al.,2007). The phosphorylation sites important for activation of Hpo and Wts kinases and inactivation of Yki have been mapped in flies and in mammalian systems (Dong et al.,2007; Zhao et al.,2007; Hao et al.,2008; Oh and Irvine,2008; Zhang et al.,2008a). Yki promotes, either directly or indirectly, the transcription of genes that promote growth and cell-cycle progression as well as genes that inhibit apoptosis (Huang et al.,2005; Dong et al.,2007; Zhao et al.,2008a). Thus, increased activity of Hpo and Wts correlates with a reduction in tissue growth and a reduction of Hpo/Wts activity allows growth to occur.
Upstream Components of the Hippo Pathway
Following the discovery of the core kinase cascade, an important question that remained unanswered was: do extracellular signals regulate Hpo signaling? To understand the regulation of the Hippo pathway, a search for additional components of this pathway was initiated. Several candidate genes were tested for their ability to induce Hpo target genes in mutant clones induced in the imaginal discs, and for the signature “extra interommatidial cell” phenotype in the pupal retinae. These efforts led to the discovery of two proteins with FERM domains, Mer and Ex (Hamaratoglu et al.,2006), and the protocadherin Ft as activators of Hpo (Bennett and Harvey,2006; Cho et al.,2006; Silva et al.,2006; Willecke et al.,2006). Individually ex, mer, or ft had very weak effects on the extra interommatidial cell phenotype typical of the loss of Hippo signaling pathway (Willecke et al.,2006). However, loss-of-function clones of mer, ex double mutants and ft strongly induced CycE and Diap1, two well-known targets of the Hippo pathway.
Of interest, simultaneous loss of mer and ex resulted in (a) impaired endocytic trafficking, leading to up-regulation of multiple cell growth and proliferation pathways (Maitra et al.,2006); and (b) enormous overgrowth of the mutant tissue (more than either single mutant), that is similar to the phenotypes of loss of Hpo signaling (Hamaratoglu et al.,2006). Furthermore, characterization of the double mutant phenotype revealed that cells mutant for mer and ex showed all the defects of reduction/down-regulation of Hpo signaling, for example, increased growth, delayed cell cycle exit, ectopic cell survival, and up-regulation of downstream target genes, including diap1, cycE, and ex (Hamaratoglu et al.,2006). Further genetic and biochemical analysis of ex and mer double mutants showed that these two proteins act upstream of the Hpo signaling cascade to regulate proliferation arrest and apoptosis.
However, recently distinct functional requirements of Drosophila Mer and Ex in the Hippo pathway were reported (Pellock et al.,2007). Merlin and Expanded both contain a FERM domain, but structurally Drosophila Ex is distinct from Mer, and phylogenetically distinct from the ERM proteins (Boedigheimer et al.,1993; Bretscher et al.,2002). Earlier studies showed the additive phenotype of the double mutants, and had proposed a redundant function for Ex and Mer (McCartney et al.,2000). Unlike the mer, ex double mutants, loss of ex or mer has distinct effects on growth and apoptosis. mer and ex both induce transcriptional targets of Hippo signaling (CycE and Diap1), but these targets are not robustly induced in mer mutant cells in the imaginal discs (Pellock et al.,2007). Interestingly, loss of ex in the Drosophila eye discs leads to overgrowth as seen by ectopic proliferation (S-phases) in cells that normally cease proliferations and are postmitotic, whereas mer mutant cells induce very few ectopic S-phases, suggesting that ex plays a significant role in cell cycle exit. Loss of ex in the pupal retina has a weak effect such that few extra interommatidial cells survive, whereas mer clones show several extra interommatidial cells. Thus, compared with ex, the apoptotic mechanism is defective in mer mutant cells (Pellock et al.,2007). These data suggest that ex and mer may act together or separately, by means of signaling interactions that converge on the kinase cascade of the Hippo pathway. Alternatively, these observed differences in ex and mer function may reflect the spatial and temporal differences in the expression and/or function of ex and mer. However, the molecular mechanisms that connect ex and mer to the Hpo kinase cascade remain unclear.
Recently, Ft was shown to be the most upstream activator of the Hippo pathway (Bennett and Harvey,2006; Cho et al.,2006; Silva et al.,2006; Willecke et al.,2006, Taylor and Baker,2007). Ft encodes a transmembrane protein with multiple Cadherin repeats in its extracellular domain (Mahoney et al.,1991). This presented the possibility that Hpo signaling may indeed be regulated by an extracellular signal—probably a ligand expressed on the surface of an adjacent cell. The effects of loss of ft on tissue growth and planar polarity were well documented but the underlying mechanism by which ft regulated growth was not known. Loss of ft in mutant clones in imaginal discs caused the induction of transcriptional targets of the Hippo pathway. Interestingly, Ft was shown to regulate Hippo pathway activity by two distinct signaling interactions—one directly through ex and the Hpo kinase cascade leading to a Fat-Ex-Hippo pathway (Bennett and Harvey,2006; Silva et al.,2006; Willecke et al.,2006). The other mechanism showed that Ft directly binds to Dachs (D), an atypical myosin, which in turn regulates the abundance of Wts, thereby regulating the Hippo pathway independently of Hpo and other upstream components leading to the Fat-Dachs-Warts pathway (Cho et al.,2006; Feng and Irvine,2007; reviewed in Hariharan,2006; Yin and Pan,2007; Reddy and Irvine,2008).
In addition to Dachs, a second protocadherin and known interactor of Ft called Dachsous (Ds) was recently shown to be involved in Hpo signaling (Rogulja et al.,2008; Willecke et al.,2008). Ds is proposed to act as a ligand for the Ft receptor for their role in the regulation of planar polarity (Saburi and McNeill,2005). Although attractive, the molecular mechanism by which this binding can regulate growth is complex. Both loss-of-function and overexpression of Ds in imaginal discs caused transcriptional induction of Hpo target genes, albeit in a graded manner along the boundary of Ds expression (Willecke et al.,2008). Furthermore, this effect on the target genes was not restricted to the cells missing or overexpressing Ds, indicating that discontinuities in the levels of Ds induced Hpo target genes and presumably growth. Ds, therefore, may not simply act as a ligand, which would induce target genes proportional to its concentration. However, how are discontinuities in Ds levels translated into signal(s) that regulate growth? The answer to this question remains unclear, because the evidence so far suggests that Ds acts through Ft and Dachs to regulate the activity of Wts in both Ex-dependent (using the Fat-Ex-Hpo pathway; Fig. 2A) and Ex-independent (using the Fat-Dachs-Warts pathway; Fig. 2B) manner (Rogulja et al.,2008; Willecke et al.,2008). Furthermore, flies that express uniform Ds/Four-jointed expression have significantly reduced appendages like wings, legs, and so on, but growth is not abolished. The Ds boundary effect thus accounts for some but not all growth control (Rogulja et al.,2008; Willecke et al.,2008). These data indicate that other signaling mechanisms may act in addition to the Ds boundary effect to control imaginal disc size (Willecke et al.,2008). Thus, other currently unknown signals may act (a) in parallel to the Hippo pathway, or (b) act parallel to Ds in the Hippo pathway (for example, by means of Merlin) to regulate tissue growth.
Figure 2. Models of Hippo signaling. A: The Ft-Ex-Hpo model of Hippo signaling, where the transmembrane receptor Fat (Ft) interacts by means of Expanded (Ex) but not Merlin (Mer), which comprise the upstream regulators of Hippo signaling, interacts with the core kinase cascade of Hippo signaling (which include the Hippo kinase (Hpo), the adaptor protein Salvador (Sav) and Mats, and the downstream kinase Warts (Wts)). The core kinase cascade functions by inhibiting the phosphorylation of the transcriptional coactivator Yorkie (Yki). Unphosphorylated form of Yki binds with its cognate transcription factor Scalloped (Sd) and translocates to the nucleus to control expression of various Hippo target genes. Currently, all cell biological functions of Hippo signaling cannot be explained by the regulation of target genes controlled by the Yki/Sd complex. Thus, other transcription factors can partner with Yki to mediate Hippo functions. The Ft binding partner Dachsous (Ds) is also involved in Hippo signaling by means of both modes of Hippo signaling. B: The Ft-Dachs-Wts Model of Hippo signaling contends that the transmembrane protein Ft does not interact with Ex (or Hpo, Sav and Mats), but interacts with Wts through the atypical myosin, Dachs. Dachs negatively regulates the levels and activity of the Wts kinase, which in turn can signal to Yki. Ft-Dachs pathway is directly involved in the regulation of planar cell polarity. In addition to the Ft-Dachs-Wts interaction, another noncanonical interaction is that between Hippo and the Drosophila homolog of the RAS association factor 1 protein (RASSF1). Hpo and RASSF1 bind with each other by means of their SARAH domains to repress the expression of Sav, and activate Hippo signaling. C: The current model of the Mammalian Hippo signaling pathway shows the widespread structural and functional conservation between the mammalian and Drosophila proteins. The core kinase cascade between MST1/2 (Hpo homologs), hWW45 (Sav homolog), MOB (Mats homolog), and the Lats 1/2 (Warts homolog) is conserved at the molecular level. As in flies, the mammalian Lats1/2 in turn prevent the phosphorylation of YAP and TAZ, the two Yki homologs, and allow YAP/TAZ to translocate to the nucleus to bind the TEAD (Sd homolog) transcription factors to regulate Hippo signaling. The interactions upstream of the kinase cascade remain unclear (marked by dotted arrows and question marks); however, NF2 (Merlin homolog) has been shown to interact by means of YAP to regulate cell proliferation, survival, and contact inhibition. Database searches have identified Mammalian Ex but its role in vivo remains to be defined. The mammalian isoform of Fat (Ft4) has recently been implicated in breast cancer, but its functional interaction with mammalian Hippo pathway has not been worked out yet.
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The Oncogene Yorkie: The Link Between the Kinase Cascade and Transcription
Yki, the Drosophila ortholog of the mammalian transcriptional coactivator Yes-Associated Protein (YAP), was identified as the missing link between Wts and transcriptional regulation in the Hippo pathway (Huang et al.,2005). Yki was identified in a yeast two-hybrid screen using the N-terminal noncatalytic domain of Wts as bait. Loss-of-function of Yki (named after Yorkshire Terriers, a small dog breed) in mutant clones in imaginal discs results in the development of smaller organs/structures. Yki is required for normal tissue growth and diap1 transcription. 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. Yki is a critical target of the Wts/Lats protein kinase and a potential oncogene (Huang et al.,2005). Yki is the first substrate identified for NDR family kinases and is phosphorylated and inactivated by Wts. Biochemical studies showed that Wts-mediated phosphorylation of Yki was stimulated by upstream components of the Hippo pathway (Huang et al.,2005).
At the time of its characterization, it was speculated that like other coactivators, Yki functions by interacting with DNA binding transcription factors. YAP, the mammalian homolog of Yki, is known to function as coactivator for several transcription factors, such as the p53 family member p73 (Strano et al.,2005), the Runt family member PEBP2α (Yagi et al.,1999; Vassilev et al.,2001), and the four TEAD/TEF transcription factors (Yagi et al.,1999; Vassilev et al.,2001; Strano et al.,2005). This interaction is generally mediated by WW domains of YAP, and the PPxY motifs of the cognate transcription factors or by other binding interactions independent of the WW domain. Indeed, the Drosophila TEAD domain transcription factor, scalloped (sd), was identified as the cognate factor that partnered with the N-terminal (TEAD binding) region of Yki to regulate Hpo target genes by three different approaches: yeast two-hybrid screen (Zhang et al.,2008b), GST pull-down experiments (Goulev et al.,2008), and identification of a Sd/TEAD-binding site in the Hippo Response Element (HRE) of the diap1 gene (Wu et al.,2008; Zhang et al.,2008b).
Scalloped and the Transcriptional Regulation of Hippo Target Genes
In Drosophila, Sd belongs to a family of evolutionarily conserved proteins characterized by the presence of a TEA/ATTS DNA-binding domain (Campbell et al.,1991,1992). Sd has been extensively characterized for its role in wing development and is known to physically interact with the product of the vestigial (vg) gene, where the dimer functions as a master gene controlling wing formation (Simmonds et al.,1998; Paumard-Rigal et al., 1998; Halder et al., 1998; Halder and Carroll,2001). The Vg-Sd dimer activates the transcription of several wing-specific genes, including sd and vg themselves. Sd binds with Yki to regulate the transcription of diap1, a target gene of the Hippo pathway (Wu et al.,2008). The interaction between Yki and Sd increases sd transcriptional activity both ex vivo in Drosophila S2 cells and in vivo in Drosophila wing discs and promotes Yki nuclear localization (Wu et al.,2008; Zhang et al.,2008b). The diap1 locus harbors a minimal Sd-binding Hippo Responsive Element (HRE) that mediates transcriptional regulation by the Hippo pathway. Genetic and biochemical studies showed that sd is required for yki-induced tissue overgrowth and target gene expression, and this sd function is conserved in its mammalian homolog (Goulev et al.,2008; Wu et al.,2008; Zhang et al.,2008b; Zhao et al.,2008b; reviewed in Bandura and Edgar,2008). Contrary to Yki, Sd is not expressed in all imaginal tissues. This indicates that Yki-Sd interaction acts in a tissue-specific manner and that other Yki partners must exist that remain to be identified.
Two Mechanisms of Hippo Regulation: The Fat-Ex-Hpo and the Fat-Dachs-Warts Pathways
Three reports showed that Fat functions through a linear pathway that involves Ft, Ex, Hpo, and Wts (Fig. 2A; Bennett and Harvey,2006; Silva et al.,2006; Willecke et al.,2006). This model is based on the following observations: (1) ft mutant cells affected the stability and localization of Ex to the apical surface of the cells. (2) Overexpression of an activated form of Ft could induce Hpo and Wts phosphorylation. (3) The ability of Ft to inhibit Yki activity was reduced by knockdown of hpo or wts by means of RNA interference using a transcriptional assay of Yki activity in cell culture. (4) The most convincing evidence came from genetic epistasis experiments that showed that, as opposed to the requirement of Hpo components for Ft mediated growth regulation, activation of the Hpo pathway at the level of ex or hpo mostly bypassed the requirement for ft. This placed Ft upstream of Ex and other Hpo components. Interestingly, this interaction does not involve Mer (which is proposed to act together with Ex), suggesting that Mer may receive inputs from other upstream signals. Together, these findings suggested that Ft (possibly acting as a receptor) signaled to Ex, which in turn signaled to the kinase cascade. Warts then negatively regulated the activity of the oncoprotein Yki, which then bound its cognate transcription factor Sd to regulate the expression of diap1.
Cho et al. (2006) demonstrated that Ft promoted Wts-mediated Hippo signaling by a different mechanism that involved an unconventional myosin Dachs (Fig. 2B). In the Fat-Dachs-Wts model, the path from Ft to Wts did not go through Hpo or other upstream components of the pathway like Ex or Mer. Three lines of evidence were presented to support this conclusion. (1) The genetic analysis of double-mutant combinations placed ft upstream of dachs, and dachs upstream of both hpo and wts. (2) The levels of Wts protein, but not the levels of Hpo, Sav, Mer, or Mats were reduced in ft mutant tissue. (3) When overexpressed in tissue culture cells, Dachs bound to Wts. Thus, Ft regulated the abundance of Wts by means of Dachs, while Hpo independently regulated the enzymatic activity of Wts. In this view, Wts was the point of convergence of two distinct upstream signals. This mechanism of Fat action was also reported by Feng and Irvine, where Ft acts in parallel to Ex to signal to Wts to regulate growth by means of Hippo signaling pathway (Feng and Irvine,2007).
Of interest, the experiments described in the two different models do not exclude or contradict each other. Indeed, many of the experimental results are consistent with Ft being able to regulate Wts activity in at least two different ways and also with Wts being regulated by signals that are independent of Ft (reviewed in Hariharan,2006). The observation that the phenotype of ftex double mutants is a little more severe than that of ex mutants suggests that Ft can regulate Wts independently of Ex. Also consistent with this conclusion are the results that show that the suppression of ft mutant phenotypes by the overexpression of Hippo pathway components is typically incomplete. In addition, the observation of reduced growth and decreased transcription of Yki targets in cells doubly mutant for ft and Dachs would seem to suggest that the elevated levels of Wts in these cells can still be activated by a pathway that is independent of Ft such as Mer. In addition, the fact that Sd is expressed in a spatially defined pattern and is not expressed in all imaginal discs, suggests that other transcription factors can bind with Yki to regulate the expression of other target genes in the Hippo pathway.
RASSF1 and the Hippo Pathway
Hippo signaling can also be regulated by other Ft-independent mechanisms, for example, through the interaction of the Ras Association Factor 1 (RASSF1) with Salvador and Hippo. RASSF1 belongs to a family of proteins frequently silenced in a variety of solid tumors, mainly by promoter methylation. Mammalian RASSF proteins were known to interact with Mst1 (the Hippo homolog), and after the discovery of the Hippo pathway in flies a homology domain was discovered between Sav, RASSF, and Hippo called the SARAH (Sav-RASSF-Hippo) domain. RASSF proteins typically contain a RA domain and a C-terminal SARAH domain (Polesello et al.,2006; Guo et al.,2007; Avruch et al.,2009). The dRASSF mutant flies are viable, and under controlled conditions uncover a clear growth defect in comparison to wild-type animals. RASSF expression was reduced in clones mutant for a hpo allele that lacked the SARAH domain. Further investigations reveal that the effect on dRASSF is posttranscriptional. Of interest, dRASSF levels were unaffected in clones mutant for other Hippo-pathway members, such as ex, sav, and wts (Polesello et al.,2006; Guo et al.,2007). These results suggest that direct binding to Hpo through its SARAH domain, rather than signaling through the Hippo pathway, is necessary for dRASSF stability. RASSF1 binds to Hpo but not with Sav, and does not form a ternary complex. Hpo was able to bind Sav and dRASSF, however, in two different Hpo complexes—a highly active Sav-associated pool and an inactive dRASSF-associated pool. Thus, Sav can promote Hpo activation. Salvador and RASSF repress each other's expression, and in cell culture, increasing amounts of Sav can displace RASSF bound to Hpo, suggesting that modulation of the inhibitory effects of dRASSF and the activator effects of Sav determine the level of Hippo activation, and organ size (Polesello et al.,2006; Guo et al.,2007).