growth regulation is a critical developmental process whose dysfunction can lead to many diseases , including cancer ( conlon and raff , 1999 ) .
the salvador ( sav)/warts ( wts)/hippo ( hpo ) ( swh ) network , identified in drosophila and conserved in mammals , plays a major role in limiting growth by inhibiting cell proliferation and promoting apoptosis ( harvey and tapon , 2007 ; reddy and irvine , 2008 ) .
activation of the upstream kinase hpo allows it to phosphorylate the downstream kinase wts , which in turn phosphorylates and inhibits the transcription coactivator yorkie ( yki ) .
scaffold proteins , such as salvador ( sav ) and mats , potentiate the activity of the hpo / wts complex ( harvey and tapon , 2007 ) .
one upstream input of the pathway is mediated via merlin ( mer ) and expanded ( ex ) , two ferm ( four point one , ezrin , moesin , radixin ) domain proteins ( hamaratoglu et  al . , 2006 ) .
recently , ex was shown to bind yki , and new experiments hint at the existence of an ex / hpo / wts - containing apical complex anchoring yki at the cortex ( badouel et  al . , 2009 ; oh et  al . , 2009 ) .
however , though some upstream members are known , how the swh network is activated remains unclear .
here , we identify the ww - domain - containing protein kibra as a regulator of the swh network . human kibra ( kremerskothen et  al . , 2003 ) is known to be phosphorylated by protein kinase c  ( pkc ) ( buther et  al . , 2004 ) and has recently been reported to have a role in cell migration ( duning et  al . , 2008 ; rosse et  al . , 2009 ) .
in drosophila , kibra had previously been recovered as  a minor hit in several screens for growth regulators ( boutros et  al .
, 2004 ; muller et  al . , 2005 ; ringrose et  al . , 2003 ; tseng and hariharan , 2002 ) but has not been further studied .
our experiments show that kibra associates with mer , ex , and wts and stabilizes the mer / ex interaction .
this suggests that kibra is a component of an apical scaffold that controls swh pathway activation .
we performed an in  vivo screen in the fly wing in order to identify genes implicated in growth control .
transgenic flies bearing rna  interference ( rnai ) constructs generated by the vienna drosophila rnai centre ( vdrc ) ( dietzl et  al . , 2007 ) were crossed to the hedgehog - gal4 ( hh - gal4 ) driver , leading to target gene silencing in the posterior compartment of the wing .
the results of this screen will be described elsewhere . in this context , expressing an rnai line directed against kibra induced overgrowth of the posterior wing compartment ( figure  1b ) compared to control flies ( figure  1a ) .
this phenotype was also observed upon wts depletion ( figure  1c ) . driving the same kibra rnai line in the eye also led to increased organ size ( figures 1d and 1e ) , similarly to a wts rnai line ( figure  1f ) . to exclude off - target effects
, we generated a transgenic line expressing a nonoverlapping rnai construct and observed identical overgrowth phenotypes ( data not shown ) .
furthermore , adult eye sections revealed that kibra knockdown retinas present an excess of interommatidial cells ( iocs ) ( figures 1 g and 1h ) .
the iocs , the last population of cells to differentiate in the eye primordium , give rise to the secondary and tertiary pigment cells that optically isolate the ommatidia in the compound eye from each other .
extra iocs are produced during normal development but are then eliminated by apoptosis at the pupal stage to give rise to the adult lattice ( wolff and ready , 1993 ) .
the presence of extra iocs is a hallmark of swh network loss of function ( kango - singh et  al . , 2002 ;
2002 ) , which reduces retinal apoptosis , as seen in wts rnai adult eye sections ( figure  1i ) .
thus , depletion of kibra elicits a similar phenotype to swh network mutants , suggesting a potential role for kibra in hpo signaling . to study kibra loss of function
, we generated the kibra allele by imprecise excision of the ep747 transposon ( see figure  1j and experimental procedures ) .
this deletion allele , which removes the translation initiation site , is homozygous lethal and may be a null allele for kibra .
kibra flp / frt mutant clones in 40 hr after - puparium - formation ( apf ) retinas present extra iocs ( figure  1k1 m ) , similarly to what was observed in adult eyes with kibra knockdown ( figures 1g1i ) .
we determined apoptotic indexes ( colombani et  al . , 2006 ) during the retinal apoptosis wave ( 28 hr apf ) in pupal retinas containing kibra mutant clones stained with an anti - active caspase-3 antibody .
kibra mutant tissue presents a reduced apoptotic index compared with wild - type ( wt ) areas in the same retinas ( figures 1n1p ; see figures s1a 
we assessed the proliferation rate of kibra mutant cells in imaginal discs , the larval precursors to the adult appendages . by using the flp / frt system under the control of the heat - shock promoter , kibra mutant cells and their wt sister clones
were generated through single recombination events from heterozygous mother cells ( brumby and richardson , 2005 ) .
after several rounds of divisions , the sizes of mutant clones ( no gfp ) and wt twin spots ( two copies of gfp ) were compared , allowing us to estimate the relative proliferation rates of mutant versus wt cells .
the total kibra clone area is 1.57 ( 0.12)-fold larger than the control twin spot area , compared to a ratio of 0.98 ( 0.09 ) when both clones and twin spots are wt ( figures 1q1s ) , indicating that kibra mutant cells grow 1.6 times faster than wt cells .
in addition to cell cycle rates , the timing of cell cycle exit can readily be measured in the eye disc , where cell divisions follow a spatially determined pattern ( wolff and ready , 1993 ) . during the third larval instar , the morphogenetic furrow
anterior to the furrow cells still proliferate asynchronously , while in the furrow cells synchronize in g1 .
immediately posterior to the furrow , cells enter a final round of synchronous s phases , the second mitotic wave ( smw ) .
thus , in wt discs , no s phases ( marked by edu incorporation ) can be observed posterior to the smw ( figure  1 t ) . as expected , hpo mutant cells fail to exit from the cell cycle in a timely manner and present ectopic edu - positive staining posterior to the swm ( figures s1b and s1b ) .
kibra mutant cells exhibit a less pronounced but similar phenotype ( figures 1u and 1v ) .
thus , kibra mutant tissues have a proliferative advantage and an apoptosis defect , consistent with an involvement in the swh network
. the overgrowth defect appears more subtle than that of core pathway members such as wts and is more akin to upstream regulators ( e.g. , ex and mer ) .
several transcriptional targets of the swh network have been identified , such as the drosophila inhibitor of apoptosis 1 ( diap1 ) gene , the cell cycle regulator cyce , the mirna bantam , as well as ex ( harvey and tapon , 2007 ; saucedo and edgar , 2007 ) . in kibra mutant wing or eye discs
, we could not detect a strong change in diap1 , cyce , or ex - lacz reporter levels ( data not shown ) . since overgrowth of kibra mutant cells in the wing is subtle compared to wts mutants ,
it is possible that kibra  plays a relatively minor role in swh signaling in the wing .
accordingly , using an anti - kibra antibody we generated ( figures s2a  s2c ) , we noted that kibra staining in the wing disc is weak and consists of a punctate apical staining which can clearly be observed when kibra is overexpressed in a stripe of cells .
we and others have previously reported that ovarian posterior follicle cells ( pfcs ) are particularly sensitive to swh loss of function ( meignin et  al .
, 2007 ; polesello and tapon , 2007 ; yu et  al . , 2008 ) , leading us to study the kibra phenotype in the ovary .
first , we noted that kibra protein levels are higher in follicle cells than in the wing discs ( figure  s2d 
kibra staining is mainly apical and is severely reduced in kibra clones . similarly to hpo or wts loss of function , kibra loss of function in the pfcs induces an upregulation of the ex - lacz reporter ( figures 2a2b , compare with figures 2c2c ) .
hpo or wts mutant pfcs also show a misregulation of the notch ( n ) pathway and ectopic cell divisions ( meignin et  al .
the n target hindsight ( hnt ) is normally repressed in all follicle cells up to stage 6 and switched on from stage 7 to stage 10b ( figures 2d2e ) ( poulton and deng , 2007 ) .
cut , which is repressed by hnt , presents an opposite pattern of expression ( figures 2h2i ) . in kibra
mutant pfcs from stage 710b egg chambers , hnt expression is lost ( figures 2d2d and 2f2g ) , while cut is ectopically expressed ( figures 2j2k ) .
loss of kibra also leads to perturbation of epithelial integrity , as mutant pfcs show an accumulation of the apical polarity protein apkc and the n receptor ( figures s2f  s2g ) as well as multilayering of the follicular epithelium ( figures 2i2i and 2k2k ) .
ectopic mitotic divisions are also observed in pfcs clones after stage 6 , as detected by phospho - histone h3 ( ph3 ) staining ( figures s2h  s2h ) .
together , these phenotypes are identical to those observed in hpo or wts loss of function , suggesting that kibra is indeed a member of the swh network . to further explore the role of kibra in the swh network , genetic interaction and epistasis experiments were performed .
overexpressing kibra in the eye under the gmr ( glass multimer reporter ) promoter elicits the formation of a small rough eye with frequent ommatidial fusions ( figures s3a  s3b ) .
this phenotype can be partially rescued by removing one copy of the hpo gene ( figures s3c and s3c ) .
in contrast , overexpressing kibra could not rescue the hpo - like overgrowth phenotype induced by yki overexpression ( figures s3d and s3e ) , suggesting that kibra may be an upstream regulator of the pathway .
to conduct epistasis experiments between kibra and yki , we used the marcm system to generate clones of mutant cells while simultaneously overexpressing or depleting other pathway components ( lee and luo , 1999 ) .
marcm clones expressing yki rnai generated with eyflp lead to the formation of a normal eye , because yki - depleted cells are eliminated by apoptosis ( data not shown ) and replaced by wt cells ( figure  s3f ) .
as expected , eyflp kibra marcm clones cause eye overgrowth ( figure  s3 g ) .
, overexpressing kibra in the eye under the gmr promoter induces apoptosis in third instar eye discs , which is suppressed by loss of hpo ( figures 3a3a ) .
together , these epistasis experiments are consistent with kibra being a member of the swh network acting upstream of yki and hpo .
genetic interactions between kibra , mer , and ex , upstream members of the swh network , were then investigated .
expressing a kibra , an ex , or a mer rnai line in the eye under the gmr promoter induces eye overgrowth ( figures s3i  s3l ) .
combined depletion of either ex / kibra or mer / kibra shows stronger phenotypes than individual depletion of these proteins ( figures s3 m and s3n ) .
hsflp marcm clones of various genotypes were generated and scored according to the severity of the wing overgrowth phenotypes , with type 0 representing normal wings and type 4 the strongest overgrowth ( figures 3b and 3c ) .
overexpressing ex or mer in kibra mutant clones significantly rescues the overgrowth of kibra mutant clones ( p < 0.0001 for both genotypes ) .
reciprocally , kibra overexpression was also able to suppress the ex overgrowth phenotype ( p < 0.0001 ) .
thus , we could not determine a strict epistatic relationship between kibra , ex , and mer , consistent with a model whereby kibra , ex , and mer cooperate to control swh pathway activity .
as well as being an upstream regulator of the swh network , ex is  also one of its transcriptional targets ( hamaratoglu et  al . , 2006 ) , as are other upstream regulators ( e.g. , mer , four - jointed , dachsous ) .
since epistasis experiments place kibra at the level of mer and ex , we wished to test whether this is also the case for kibra .
kibra levels were highly upregulated in mer;ex or hpo clones ( figures 3d3e ) , showing an apical localization ( figures 3f3g ) .
similarly , hpo - depleted cultured drosophila s2r+ cells have increased kibra levels ( figure  3j ) . to determine whether kibra is a transcriptional swh network target ,
quantitative rt - pcr experiments were performed on yki - overexpressing and control wing imaginal discs ( figure  3k ) . as expected ,
ex mrna levels were increased ( 2.97  0.25-fold ) in yki - expressing discs compared to control discs . interestingly , kibra mrna levels were also upregulated in yki - expressing discs ( 6.24  2.12-fold ) , confirming that kibra is a yki transcriptional target and suggesting the existence of a possible negative feedback loop regulating kibra expression . because hpo clones present increased levels of kibra as well as mer and ex , these constitute a good system to evaluate the colocalization of those proteins .
thus , kibra , mer , and ex colocalize apically in imaginal disc cells , but are dispensable for each other 's apical sorting , because kibra is still apical in mer;ex clones and mer / ex are normally localized in kibra clones ( figures 3d , 3d , 3f , and 3f and data not shown ) . because kibra colocalizes with mer / ex , a possible association between those proteins was examined by conducting coimmunoprecipitation ( co - ip ) assays in s2r+ cells .
kibra was found to co - ip with ex and mer , but not with hpo or with the negative regulator of hpo , drassf ( polesello et  al . , 2006 )
interestingly , kibra was reported to interact with mer in a large - scale yeast two - hybrid screening study ( formstecher et  al . , 2005 ) .
kibra possesses two ww domains , which are predicted to mediate protein - protein interactions by binding to ppxy motifs .
furthermore , the first ww domain of human kibra was shown to recognize the consensus motif rxppxy in  vitro ( kremerskothen et  al . , 2003 ) .
in flies , mer does not contain any ppxy sites , while ex has two ppxy sites ( pppy and pppy ) and an rxppxy site ( rdpppy ) .
we therefore further investigated the association between kibra and ex by mutating amino acids that are known to be required for ww domains and ppxy sites to interact ( kremerskothen et  al .
a kibra protein mutant for its first ww domain ( p85a ) could no longer co - ip wt ex .
reciprocally , wt kibra could not co - ip an ex protein deficient for its rxppxy site ( p845a ) ( figure  4b ) .
thus , kibra associates with ex through its first ww domain and the ex rxppxy motif .
in contrast , mutating either one or both kibra ww domains does not affect kibra / mer association ( figure  s4a ) .
further assays reveal that both kibra n- and c - terminal fragments are sufficient for the association with mer , but a central stretch ( aa 484857 ) is not ( figure  s4a ) . because the ww motifs , which are required for the ex / kibra association , are located in the n - terminal fragment , this suggests that mer can complex with kibra both through ex and independently of ex .
to further test this possibility , coimmunoprecipitation assays between mer and kibra were performed in ex - depleted cells ( figure  s4b ) .
the kibra / mer immunoprecipitation is not affected by ex depletion , suggesting that ex is not required for the kibra / mer association . because the hpo pathway is highly conserved from drosophila to humans , we tested for potential interactions between human kibra and the human orthologs of ex ( frmd6 ) , mer ( nf2/mer ) , hpo ( mst2 ) , and drassf ( rassf6 ) .
we used split - tev as readout , which is based on tev protease complementation and represents a sensitive method for detecting interactions between membrane - associated proteins ( wehr et  al .
we found that kibra associates with nf2/mer but did not interact with frmd6 , mst2 , or rassf6 ( figure  s4c ) .
interestingly , frmd6 contains only an n - terminally conserved sequence of ex but lacks the entire c - terminal part , which in ex harbors the ppxy motifs .
thus , the missing ppxy motifs and the generally limited level of sequence conservation in frmd6 likely explain the absence of interaction between kibra and frmd6 .
these results imply that the ability to associate with kibra evolved in an ancestral ex / mer - like ferm domain protein and was later lost in frmd6 but retained in nf2/mer . alternatively ,
as kibra complexes with both ex and mer and ex / mer have been reported to directly interact ( mccartney et  al . , 2000 ) , we tested the possibility that kibra could affect the mer / ex interaction .
we performed co - ip assays between mer and ex in cells expressing different levels of kibra protein ( figure  4c ) .
the mer / ex interaction is reduced in kibra - depleted cells compared to wt cells , whereas the interaction is strengthened in cells that express a myc - kibra construct .
thus , the presence of kibra is required to fine - tune the stability of the mer / ex interaction . because kibra complexes with ex and a yki / ex interaction has recently been described ( badouel et  al . , 2009 ) , we sought to determine whether kibra can affect yki activity .
s2r+ cells were treated with rnai against several swh pathway components , and yki phophorylation on ser168 was monitored by western blotting ( figure  4d ) .
the phosphorylation of yki by wts at ser168 leads to yki inactivation and sequestration in the cytoplasm , where it has been reported to bind ex , wts , hpo , and 14.3.3 ( badouel et  al . , 2009 ;
huang et  al . , 2005 ; oh and irvine , 2008 ; oh et  al . , 2009 ) .
lacz rnai - treated cells show a high basal level of phospho - yki ( p - yki ) .
as expected , yki phosphorylation is abolished when wts is depleted , and mildly reduced when the wts cofactor mats ( lai et  al . , 2005 )
treated rnai cells , a yki downward shift can also be observed using  a pan - yki antibody ( figure  4d , second row ) .
when depleted in conjunction with ex , the p - yki signal becomes even further reduced .
this suggests that kibra and ex are required for wts activity on  yki , which prompted us to investigate whether kibra could associate with wts .
co - ip assays reveal that kibra interacts with wts ( figure  4e ) .
wts does not seem to compete with ex for kibra association , because it could still complex with  a form of kibra mutant for its first ww domain . because kibra associates with wts and ex interacts with yki , we investigated whether wts requires kibra / ex to bind yki .
endogenous ips between yki and wts were performed in s2 cells treated with various dsrnas ( figure  4f ) . in these conditions ,
the effect of kibra and ex depletion on yki phosphorylation can also be observed ( see input ) . in control cells ,
this endogenous interaction is unaffected by the individual or combined depletion of ex  and kibra .
these results suggest that ex and kibra are required to activate the swh pathway by nucleating an active hpo / wts kinase cassette , rather than promoting the wts / yki interaction .
our data identify kibra as a regulator of the swh network that associates with ex and mer , with which it is colocalized apically and transcriptionally coregulated .
given that the apical surface of epithelial cells is instrumental in both cell - cell signaling and tissue morphogenesis , we speculate that kibra may cooperate with ex and mer to transduce an extracellular signal , or relay information about epithelial architecture , via the swh network , to control tissue growth and morphogenesis .
recent data have suggested that an apical scaffold machinery containing hpo , wts , and ex recruits yki to the apical membrane , facilitating its inhibitory phosphorylation by wts ( badouel et  al . , 2009 ; oh et  al . , 2009
since kibra associates with ex and is also apically localized , we can hypothesize that kibra is also part of this scaffold and participates in nucleating an active hpo / wts complex and recruiting yki for inactivation .
this view is supported by our finding that kibra complexes with wts and that combined depletion of kibra and ex leads to a strong decrease in yki phosphorylation , but does not disrupt the wts / yki interaction .
our data also suggest that the importance of kibra may be tissue - specific since we observe robust phenotypes in ovaries and hemocyte - derived s2r+ cells , but weaker effects in imaginal discs .
thus , considering the relative levels of expression of ex , mer , and kibra may be important in determining pathway activation .
finally , since mammalian kibra complexes with the nf2/mer tumor suppressor , our findings raise the possibility that human kibra may contribute to tumor suppression in human neurofibromas and potentially other tumors .
for drosophila genotypes , primer sequences , and further experimental details , see supplemental experimental procedures .
the p element of ep747 ( bloomington stock center ) was mobilized using standard genetic techniques , and excisions were screened by pcr ( see supplemental experimental procedures for details ) .
mouse -galactosidase ( promega ) , rabbit anti - cleaved caspase-3 ( asp175 ) ( cell signaling technology ) , rabbit anti - apkc ( santa cruz ) , and mouse nicd ( c17.9c6 , development studies hybridoma bank ) antibodies were used at 1/500 .
mouse anti - arm , anti - cut , and anti - hnt ( n2 7a1 , 2b10 and 1g9 , dshb ) were used at 1/10 and 1/20 .
the edu staining was performed as described in the click - it edu alexa fluor imaging kit ( invitrogen ) .
guinea pig anti - mer , a gift from r. fehon , was used at 1/7500 , and rabbit anti - ex , a gift from a. laughon , at 1/400 . rabbit anti - kibra antibody ( kib18 , 1/100 ) was generated by eurogentec sa ( seraing , belgium ) against a peptide corresponding to the last 15 amino acids of kibra .
ms1096 > > ( control ) and ms1096 > > yki wing discs were dissected in pbs and snap - frozen in liquid nitrogen .
rna isolation and subsequent qrt - pcr reactions were performed as described in genevet et  al .
all statistical analyses performed ( except epistasis analysis ) were assessed by mann - whitney nonparametric tests using the website http://elegans.swmed.edu/leon/stats/utest.html .
the epistasis analysis was made by pairwise comparison after correction for  the batch effect on 42 to 310 flies of each genotype , divided in 4 to 6 cohorts .
the approach used was a three - way log - linear model , against  a null - hypothesis of no interaction between phenotype and population .
the p value indicates whether the pair of populations differ in their phenotype profiles : p  value(kibra ; kibra + uas ex ) = 2.69  10 , p value(kibra ; kibra + uas mer ) = 6.07  10 , p value(ex ; ex + uas kibra ) = 1.76  10 .
 summarythe salvador ( sav)/warts ( wts)/hippo ( hpo ) ( swh ) network controls tissue growth by inhibiting cell proliferation and promoting apoptosis . 
 the core of the pathway consists of a mst and lats family kinase cascade that ultimately phosphorylates and inactivates the yap / yorkie ( yki ) transcription coactivator . 
 the ferm domain proteins merlin ( mer ) and expanded ( ex ) represent one mode of upstream regulation controlling pathway activity . here , we identify kibra as a member of the swh network . 
 kibra , which colocalizes and associates with mer and ex , also promotes the mer / ex association . 
 furthermore , the kibra / mer association is conserved in human cells . 
 finally , kibra complexes with wts and kibra depletion in tissue culture cells induces a marked reduction in yki phosphorylation without affecting the yki / wts interaction . 
 we suggest that kibra is part of an apical scaffold that promotes swh pathway activity . 
Introduction
Results
Discussion
Experimental Procedures