In eg ins Coope a e Wi h he EGFR/
Ras Pa hway o P ese e Epi helia
Su i al and A chi ec u e in
De elopmen and Oncogenesis
And ea Valencia-Expósi o
1
†
, M. Jesús Gómez-Lama ca
1
,
2
†
, Thomas J. Widmann
3
and
Ma ía D. Ma ín-Be mudo
1
*
1
Cen o Andaluz de Biología del Desa ollo CSIC-Uni e sidad Pablo de Ola ide, Se illa, Spain,
2
Depa amen o de Biología
Celula , Uni e sidad de Se illa, Se illa, Spain,
3
GENYO, G anada, Spain
Adhesion o he ex acellula ma ix (ECM) is equi ed o no mal epi helial cell su i al.
Dis up ion o his in e ac ion leads o a specific ype o apop osis known as anoikis. Ye ,
he e a e physiological and pa hological si ua ions in which cells no connec ed o he ECM
a e p o ec ed om anoikis, such as du ing cell mig a ion o me as asis. The main ecep o s
ansmi ing signals om he ECM a e membe s o he in eg in amily. Howe e , al hough
in eg in-media ed cell-ECM ancho age has been long ecognized as c ucial o epi helial
cell su i al, he in i o significance o his in e ac ion emains o be weighed. In his wo k,
we ha e used he D osophila wing imaginal disc epi helium o analyze he impo ance o
in eg ins as su i al ac o s du ing epi helia mo phogenesis. We show ha educing
in eg in exp ession in he wing disc induces caspase-dependen cell dea h and basal
ex usion o he dead cells. In his case, anoikis is media ed by he ac i a ion o he JNK
pa hway, which in u n igge s exp ession o he p oapop o ic p o ein Hid. In addi ion, ou
esul s s ongly sugges ha , du ing wing disc mo phogenesis, he EGFR pa hway
p o ec s cells unde going cell shape changes upon ECM de achmen om anoikis.
Fu he mo e, we show ha oncogenic ac i a ion o he EGFR/Ras pa hway in in eg in
mu an cells escues hem om apop osis while p omo ing hei ex usion om he
epi helium. Al oge he , ou esul s suppo he idea ha in eg ins p omo e cell su i al
du ing no mal issue mo phogenesis and p e en he ex usion o ans o med cells.
Keywo ds: in eg ins, su i al, JNK, EGFR, oncogenesis
INTRODUCTION
To su i e o die is a cellula decision con olled by en i onmen al cues and physical s imuli. This li e and
dea h decision is deeply influenced by he ex acellula ma ix (ECM) (Me edi h e al., 1993). When cells
lose hei no mal in e ac ions wi h he ECM, he cell cycle is a es ed and a specific o m o apop osis,
knownasanoikis,isini ia ed(F isch and Ruoslah i, 1997). Du ing de elopmen , abe an anoikis al e s
issue a chi ec u e and unc ion, comp omising emb yonic iabili y (Mole e al., 2021). Con e sely,
p og ammed anoikis con ibu es o physiological de elopmen al p ocesses and issue enewal (Gilmo e,
2005;Chia ugi and Giannoni, 2008). Anoikis is also an impo an su eillance mechanism, ensu ing ha
any cell ha loses i s app op ia e posi ion wi hin a issue is a ge ed o dea h. Fo his eason, impai ed
anoikis con ibu es o he malignancy o many cance cells, allowing hem o su i e wi hou ECM
Edi ed by:
Claudia Tanja Mie ke,
Leipzig Uni e si y, Ge many
Re iewed by:
Susan LaFlamme,
Albany Medical College, Uni ed S a es
Dan Be gs alh,
Uni e si y o Roches e , Uni ed S a es
*Co espondence:
Ma ía D. Ma ín-Be mudo
[email p o ec ed]
†
These au ho s ha e con ibu ed
equally o his wo k
Special y sec ion:
This a icle was submi ed o
Cell Adhesion and Mig a ion,
a sec ion o he jou nal
F on ie s in Cell and De elopmen al
Biology
Recei ed: 09 Ma ch 2022
Accep ed: 09 May 2022
Published: 13 June 2022
Ci a ion:
Valencia-Expósi o A,
Gómez-Lama ca MJ, Widmann TJ and
Ma ín-Be mudo MDD (2022) In eg ins
Coope a e Wi h he EGFR/Ras
Pa hway o P ese e Epi helia Su i al
and A chi ec u e in De elopmen
and Oncogenesis.
F on . Cell De . Biol. 10:892691.
doi: 10.3389/ cell.2022.892691
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g June 2022 | Volume 10 | A icle 8926911
ORIGINAL RESEARCH
published: 13 June 2022
doi: 10.3389/ cell.2022.892691
ancho age and acili a ing hei dispe sion [ e iewed in (Zhong and
Resco la, 2012)]. Un a eling he mechanisms egula ing anoikis is
he e o e c ucial o unde s and bo h no mal mo phogenesis and
cance p og ession.
Al hough he concep o ancho age-dependen cell su i al has
been ecognized o many yea s, i was in he ea ly 1990’swheni was
demons a ed ha cells dep i ed o ECM a achmen unde go
anoikis (Me edi h e al., 1993;F isch and F ancis, 1994). Anoikis
can be conside ed a mul is ep p ocess [ e iewed in (Zhong and
Resco la, 2012)].Fi s o all,losso ancho age esul sininac i a iono
ocal complex signalling molecules, including FAK and S c
(Gianco i, 2000;Pa sons, 2003). As a consequence, se e al p o-
su i al pa hways such as PI3K/Ak and Ra /MEK/ERK a e
dis up ed [ e iewed in (Vachon, 2011)].Theseconds epin
anoikis is he simul aneous disassembly o ocal adhesions and he
des abiliza ion o he cy oskele on, which leads o he elease o p o-
apop o ic Bcl-2 p o eins (Ma in and Vuo i, 2004). The final s ep is
he ac i a ion o apop o ic kinases, including JNK, which esul s in an
inc ease in he exp ession and phospho yla ion o he p o-apop o ic
BH3-only p o eins BIM an BMF (Gi nius and Da is, 2017).
Howe e , he oleo JNKinanoikis emainscon o e sial,as,
depending on he cell ype, kinase iso o m and s imulus, i can
pe o m p o- o an i-apop o ic unc ions (Liu and Lin, 2005).
Analysing JNK unc ion in di e en cellula con ex s shall help
esol ing i s deba ed ole in anoikis.
The majo ansmi e s o su i al signals om he ECM a e
membe s o he in eg in amily (F isch and Ruoslah i, 1997;Me edi h
e al., 1993;Vachon, 2011). In eg ins a e a widely exp essed amily o
he e odime ic ansmemb ane glycop o eins, composed o an aand a
βsubuni , ha link he ECM o he ac in cy oskele on (Hynes, 1992).
The e eb a e in eg in amily is comp ised o 18 aand 8 β
ansmemb ane subuni s enabling abou 24 di e en
he e odime ic ecep o s o a di e se a ay o ECM p o eins.
Howe e , only some in eg ins a e capable o egula ing cell
iabili y and hese include he β1in eg insub amily(S upack and
Che esh, 2002). The egula ion o cell su i al media ed by in eg ins
implica es an inc easing complexi y o playe s depending on he
issue, cell ype, specie and he cell di e en ia ion s a e, emphasizing
he need o s udy anoikis in a gi en issue wi hin i s physiological
con ex [ e iewed in (Igno z and Massague, 1987;F isch and
Sc ea on, 2001;Vachon, 2011)]. None heless, he ea ly emb yonic
le hali y obse ed in some e eb a e models lacking β1 in eg ins has
long p ecluded a di ec e idence o a ole o β1 in eg ins as su i al
ac o s in i o (S ephens e al., 1995). The use o cell lineage-specific
gene dele ion app oaches and ansplan a ion expe imen s o
ci cum en ea ly emb yonic le hali y has e ealed ha he ole o
in eg in unc ion in cell su i al in i o is mul i ace ed and some imes
con adic o y. Thus, while deple ion o β1 in eg ins om ei he he
de eloping mouse lens, he epide mis o he endo helia esul s in
apop osis (DiPe sio e al., 2000;Simi skii e al., 2007;Ca lson e al.,
2008), condi ional dele ion in he in es inal epi helium causes anoikis
esis ance, inc eased cell p oli e a ion and de ec i e di e en ia ion
(Jones e al., 2006). Fu he mo e, in some si ua ions, cells a e
p o ec ed om anoikis, such as du ing cell mig a ion o
me as asis. In hese cases, cells escape dea h using a a ie y o
s a egies, including hype ac i a ion o ecep o y osine kinases
(Guadamillas e al., 2011;Paolie al.,2013).
Since he main componen s o he cell dea h machine y a e
e olu iona y conse ed,D osophila, due o i s unique gene ic and
cell biological ad an ages, has become an ideal model sys em o s udy
apop osis in he con ex o a de eloping o ganism. As in mammals,
he caspase amily o cys eine p o eases is essen ial o he egula ion
o apop osis also in D osophila. These p o eins a e cons i u i ely
exp essed as p ocaspases ha ha e low bu significan p o ease
ac i i y. To p e en unwan ed consequences o basal p ocaspase
ac i i y, li ing cells exp ess high le els o he Inhibi o o
Apop osis (IAP) p o eins, Diap1 and Diap2, which ac as E3-
ubiqui in ligases and a ge ac i a o and e ec o caspases o
p o easome deg ada ion [ e iewed in (Hay, 2000)]. Upon an
apop o ic s imulus, Diap1 an agonis s, including hid, eape ( p ),
g im,sickle (skl), ja ac2,anddOmi/H A2 a e ansc ip ionally
ac i a ed and physically in e ac wi h Diap1 [ e iewed in (Xu
e al., 2009)]. This leads o he blockage o i s caspase inhibi o y
unc ion and he deg ada ion o Diap1 by au o-inhibi o y
ubiqui iniza ion. As a consequence, he caspase cascade is
ac i a ed and he apop osome is o med. Fi s o all, ini ia o p o-
caspases, including DRONC, he casp9 o holog, DREDD and DCP-
2, a e ac i a ed by au o-clea age. Then, ini ia o caspases ca alyse he
ac i a ion o he e ec o p o-caspases d ICE and DCP-1, and his in
u n clea e hei subs a es, including cy oskele on p o eins and DNA
me abolic p o eins (Coope e al., 2009). Howe e , despi e he
conse a ion o he cell dea h esponse be ween flies and
e eb a es, li le is known abou he p ocess o anoikis in D osophila.
Using he p imo dium o he D osophila wing, he wing imaginal
disc, as model sys em, we ha e s udied he implica ion o in eg ins in
he egula ion o epi helial cell su i al in he con ex o a de eloping
issue. The o ma ion o he wing s a s du ing ea ly emb yonic
de elopmen when abou 30 cells a e alloca ed o o m he wing
imaginal disc p imo dium. Du ing la al li e, disc cells di ide and
o m an epi helial sac comp ised o a olded columna epi helium on
one side and a squamous epi helium on he o he (Ga cia-Bellido
and Me ian, 1971).Thema u ediscwillgi e ise o headul wing
and no um. A he onse o me amo phosis, local cell shape changes
p omo e he olding o he single-laye ed columna epi helium in o a
bi-laye ed shee in which epi helial cells ace each o he along hei
basal sides (F is om e al., 1993). In eg ins pe o m wo dis inc
unc ions in he wing disc, o hold he wo laye s oge he and o
egula e he cell shape changes unde lying olding (B aban e al.,
1996;Dominguez-Gimenez e al., 2007). Howe e , despi e he
known ole o in eg ins as su i al ac o s in bo h benign and
malignan epi helia [ e iewed in (Gilc ease, 2007)], in eg ins ha e
no ye been implica ed in cell su i al in wing disc epi helial cells.
He e, we show ha low in eg in le els induces caspase-dependen
wing disc cell dea h. This anoikis p ocess is media ed by he
ac i a ion o he JNK pa hway, which in u n induces exp ession
o he p oapop o ic gene hid.Wealsofind ha in eg ins coope a e
wi h he EGFR pa hway o main ain wing disc epi helial cell su i al.
Finally, ou esul s demons a e ha an oncogenic e sion o Ras,
Ras
V12
, blocks anoikis in he wing disc and ha elimina ion o
in eg ins om ans o med Ras
V12
cells s imula es hei basal
ex usion om he wing disc epi helium. Ou esul s un a el new
oles o in eg ins in epi helial cells in i o, p omo ing cell su i al
du ing issue mo phogenesis and p e en ing he ex usion o
ans o med cells.
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Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s
MATERIALS AND METHODS
Fly S ains
The ollowing s ocks we e used: UAS-Ras
V12
(Lee e al., 1996); nub-
Gal4 (Calleja e al., 1996); hid 5′F-GFP (Tanaka-Ma aka su e al.,
2009); UAS-DER
DN
,UAS-Diap1,UAS-puc,ap-Gal4,puc-LacZ, p -
LacZ,andb k-LacZ (Blooming on D osophila S ock Cen e );
mys
RNAi
,UAS-diβ(Ma in-Be mudo and B own, 1999)and
hid
RNAi
(Vienna D osophila RNA-i Cen e ). To gene a e mu an
clonesin hewingdiscweused heFRT/FLP echnique(Chou and
Pe imon, 1992). Mu an clones we e ma ked by he absence o GFP.
The ollowing mu an alleles and ch omosomes we e used: mys
11
[also known as mys
XG43
(Bunch e al., 1992)] and e22c-Gal4 UAS-
flipase (Du y e al., 1998). The e22c-Gal4 d i e is exp essed in some
pos e io wing disc cells and was he e o e used in combina ion wi h
UAS- flp o gene a e la ge mys clones. Flies we e aised a 25°C.
Immunohis ochemis y and Imaging
Wing imaginal discs we e s ained using s anda d p ocedu es and
moun ed in Vec ashield (Vec o Labo a o ies, Bu lingame, CA,
Uni ed S a es). The ollowing p ima y an ibodies we e used: goa
an i-GFP
FICT
(Abcam, 1:500), abbi an i-caspase Dcp1 (Cell
Signaling; 1:100), abbi an i-pJNK (P omega, 1:200), mouse an i-
βGal (P omega, 1:1000), mouse an i-βPS (De elopmen al S udies
Hyb idoma Bank, DSHB, Uni e si y o Iowa, Uni ed S a es, 1:50)
and mouse an i-myc (P omega, 1:100). The seconda y an ibodies
used we e Alexa fluo 488,(Molecula P obes
TM
) and Cy3 and Cy5
(Jackson ImmunoReseach Labo a o ies, Inc.) a 1: 200. DNA was
labelled using Hoechs (Molecula P obes, 1:1000). Con ocal images
we e ob ained using a Leica SP5-MP-AOBS, equipped wi h a Plan-
Apoch oma 40X oil objec i e (NA 1.4).
Quan ifica ion o Fluo escence In ensi y
To quan i y fluo escen in ensi y o he di e en ma ke s, fluo escen
signaling was measu ed om maximum p ojec ions o
30 Z-con ocal sec ions aken wi h 1 μm o in e al pe wing disc
and geno ype. Quan ifica ions we e made in he wing pouch egion,
which was manually selec ed using he FIJI-Image J line selec ion
ool. Mic oscope se ings we e main ained be ween imaging sessions
in each expe imen . The backg ound alue aken om cell- ee
egion was sub ac ed om all da a se ies. Measu emen s o whole
fluo escence in ensi y we e done di iding he mean o all included
pixels in ensi y by he ou lined cell a ea, using he FIJI-Image J
measu e ool. S uden ’s es s we e used o s a is ical compa isons
o fluo escence in ensi y alues.
RESULTS
In eg ins a e Requi ed o he Su i al o
Wing Disc Epi helial Cells
To deepen ou unde s anding o he ole o in eg ins as su i al ac o s
du ing epi helia mo phogenesis, we es ed whe he emo ing in eg ins
in D osophila wing disc epi helial cells a ec ed hei su i al. The
D osophila genome con ains wo in eg in βsubuni s, βPS and βν
[ e iewed in (B own, 1993;Yee and Hynes, 1993). βPS, encoded by
he gene myosphe oid (mys), is he only βchain p esen in he wing
imaginal disc (B aban e al., 1996); Supplemen a y Figu e S1]. Fi s ,
we gene a ed mosaic wing disc epi helia con aining clones o cells
homozygous o he null allele mys
XG43
( om now on mys cells, whi e
a ow in Figu es 1A–B”). To de ec apop osis, we used an an ibody o
clea ed e ec o caspase Dcp-1 (Yu e al., 2002). In con ol wing
imaginal discs, Dcp-1 ac i i y is e y low, being de ec ed only
occasionally in a ew cells sca e ed h oughou he disc
(Figu e 1A,n= 20, whe e n ep esen s he numbe o wing discs
analyzed). In con as , all wing discs con aining mysclonesshowed
apop o ic cells wi hin he clone a ea (Figu e 1B,n=18).Second,we
educed in eg in le els in la ge a eas o he disc using he nubbin
(nub) Gal4 line o exp ess a mys RNAi (nub>mys RNAi)in hewing
pouch (inse in Figu e 1C) egion in he cen e o he disc ha gi es
ise o he adul wing, indica ed wi h a do ed whi e ci cle in all figu es
(B and and Pe imon, 1993;Calleja e al., 1996). Immunos aining o
con ol (n= 30) and expe imen al nub>mys
RNAi
wing imaginal discs
(n= 36) wi h an ibodies agains he βPS p o ein and clea ed Dcp-1
showed ha he exp ession o he mys specificRNAiin hewing
pouch caused a s ong educ ion in βPS p o ein le els (Figu es 1C,C’)
and a obus inc ease in apop osis (Figu es 1C,C’’,E). Thus, in eg in
exp ession in wing disc epi helial cells p e en hem om unde going
cell dea h. I is known ha emb yonic epi helial cells unde going
apop osis a e emo ed om he epi helium by ex usion a he han
phagocy osis (Rosenbla e al., 2001). Simila ly, in eg in-de ec i e cells
we e ex uded (Figu es 1C–C’’) and accumula ed be ween he basal
su ace o he disc epi helium and he ECM, as seen wi h an an ibody
agains he ECM componen Pe lecan (Supplemen a y Figu e S2).
In eg ins can media e bo h signaling and adhesion [ e iewed
in (Adams and Wa , 1993)]. To add ess which in eg in unc ion
is equi ed o acili a e cell su i al in he wing imaginal disc, we
used a chime ic in eg in (diβ) ha lacks in eg in-adhesi e
unc ion bu main ains i s abili y o signal (Ma in-Be mudo
and B own, 1999;Dominguez-Gimenez e al., 2007). We had
p e iously shown ha exp ession o diβin he wing disc inhibi ed
he basal localiza ion o endogenous in eg in [(Supplemen a y
Figu es S3B,B’;(Dominguez-Gimenez e al., 2007)]. He e, we
ound ha i caused an inc ease in apop osis (Supplemen a y
Figu es S3B,B”), s ongly sugges ing ha in eg in signaling is no
su ficien o p e en cell dea h in de ached wing disc cells.
Al oge he , hese esul s p opose ha in eg in-media ed
adhesion o he ECM p omo es cell su i al in he wing disc
epi helium by egula ing caspase ac i i y. To u he suppo his
hypo hesis, we es ed whe he o e exp ession o one o he
D osophila IAP (inhibi o o apop osis) p o eins, Diap1,
supp essed he cell dea h caused by exp ession o mys RNAi
and ound ha i did (Figu es 1D-D’’,E,n= 35).
In eg ins DownRegula e Caspase Ac i i y
by Inhibi ing Hid Exp ession
P e ious expe imen s ha e shown ha o e exp ession o hid o
p induces down egula ion o Diap1 le els and apop osis in
D osophila wing imaginal discs (Milan e al., 1997;Be gan inos
e al., 2010;Yoo e al., 2002). As Diap1 o e exp ession escues cell
dea h due o in eg in down egula ion, in eg ins could p omo e
cell su i al h ough he egula ion o hid and/o p exp ession.
To es his, we analysed hid and p exp ession in con ol and
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g June 2022 | Volume 10 | A icle 8926913
Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s
nub>mys
RNAi
wing discs, using he hid 5′F-GFP [ om now on
hidGFP,(Tanaka-Ma aka su e al., 2009)] and p LacZ
(No ds om e al., 1996) ansc ip ional epo e s. hidGFP
le els in con ol discs (hidGFP;nubGal4) a e ha dly de ec able,
consis en wi h a low occu ence o cell dea h in his issue
(Tanaka-Ma aka su e al., 2009); Figu es 2A-A’’’,E]. This
exp ession was d ama ically up egula ed in nub >mys
RNAi
discs (n= 25, Figu es 2B-B’’’). In con as , he no mal
exp ession pa e n o p LacZ, confined o a small egion o
he no um and wo s ipes in he D-V and A-P bounda ies
[(No ds om e al., 1996), Supplemen a y Figu es S4A,A’],
was no al e ed in nub >mys
RNAi
discs (n= 28,
Supplemen a y Figu es S4B,B’). These esul s sugges ha
in eg ins block hid, bu no p exp ession in no mal wing
disc epi helial cells o p omo e cell su i al. Fu he mo e,
consis en wi h he idea ha D osophila hid ac s ups eam o
Diap1 (Wang e al., 1999;Goyal e al., 2000), we ound ha
exp ession o Diap1 was no able o escue he inc ease in hidGFP
le els obse ed in mys
RNAi
cells (n= 24, Figu es 2C–C’’’).
To u he confi m hid in ol emen in in eg in loss o unc ion-
dependen cell dea h, we es ed whe he educing hid le els could
escue apop osis in nub >mys
RNAi
discs. We ound ha exp ession
FIGURE 1 | In eg ins p omo e cell su i al in he wing imaginal disc. (A–D99 )Maximal p ojec ion o con ocal iews o wing imaginal discs om hi d-ins a la ae
s ained wi h an i-βPS [g een in (A–D), whi e in (A9–D9)], an i-Dcp1 [ ed in (A–D), whi e in (A99 –D99 )] and he nuclea ma ke Hoechs [DNA, blue in (A–D)]. (A) Con ol
nubGal4 wing disc. (B) Wing disc ca ying mys mu an clones (whi e a ow), ma ked by he absence o βPS (g een). (C) Wing disc exp essing GFP o a mys RNAi unde
he con ol o nubGal4,nub >GFP (inse ) and nub >mys
RNAi
, espec i ely. (yz) Con ocal yz sec ion along he whi e do ed line shown in (C).(D) Wing disc co-
exp essing a mys RNAi and Diap-1 unde he con ol o nubGal4,nub >mys
RNAi
;Diap1.(E) Quan ifica ion o Dcp1 le els in wing discs o he designa ed geno ypes. The
do ed whi e ci cles indica e he wing pouch a ea in all figu es. The s a is ical significance o di e ences was assessed wi h a - es , ***, ** and * p alues <0.001, <0.01,
and <0.05, espec i ely. Scale ba in all panels, 30 μm.
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g June 2022 | Volume 10 | A icle 8926914
Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s
o an RNAi agains hid (hidGFP;nub >mys
RNAi
;hid
RNAi
) esul ed
in a significan educ ion o mys RNAi-induced apop osis (n=20,
Figu es 2A-A’’’,B-B’’’,D-D’’’,E). These findings demons a e
ha in eg ins p omo e cell su i al in wing imaginal discs h ough
he nega i e egula ion o hid exp ession.
Al e ing he le els o Decapen aplegic (Dpp), a D osophila
ans o ming g ow h ac o βhomologue), in wing discs esul s in
up egula ion o b inke (b k), a ansc ip ional ep esso ha p e en s
apop osis (Adachi-Yamada e al., 1999;Mo eno e al., 2002). Howe e ,
educing in eg in unc ion does no seem o a ec Dpp signalling, as
he exp ession pa e n o he b k epo e b kLacZ was no al e ed in
nub >mys
RNAi
wing discs (n=26,Supplemen a y Figu e S5B).
Down egula ion o In eg in Exp ession in
Wing Discs Induces JNK Ac i a ion
As men ioned in he in oduc ion, he ole o JNK in anoikis is
con o e sial, as, depending on he cell ype and cellula con ex , i
FIGURE 2 | In eg ins s imula e cell su i al h ough inhibi ion o hid exp ession. (A–D-)Maximal p ojec ion o con ocal iews o hi d-ins a wing imaginal discs
s ained wi h an i-βPS [blue in (A–D), whi e in (A9–D9)], an i-Dcp1 [ ed in (A–D), whi e in (A99 –D99 )] and an i-GFP [g een in (A–D), whi e in (A-–D-)].(A–A-)Con ol
nubGal4 wing disc ca ying a epo e o hid exp ession, hidGFP,hidGFP; nubGal4.(B–B-)hidGFP wing disc exp essing a mys RNAi unde he con ol o nubGal4,
hidGFP;nub >mys
RNAi
.(C–D-)hidGFP wing disc co-exp essing a mys RNAi unde he con ol o nubGal4 wi h ei he Diap1, hidGFP; nub >mys
RNAi
;Diap1 (C–C-),
o a hid RNAi, hidGFP;nub >mys
RNAi
;hid
RNAi
(D–D-).(E) Quan ifica ion o Dcp1 le els in wing discs o he indica ed geno ypes. The s a is ical significance o di e ences
was assessed wi h a - es , ***, ** and * p alues <0.001, <0.01, and <0.05, espec i ely. NS, non-s a is ically significan . Scale ba in all panels, 30 μm.
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Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s

can be ei he essen ial (F isch e al., 1996)o dispensable(Khwaja
and Downwa d, 1997). In D osophila wing discs, JNK ac i a ion
igge s apop osis in esponse o se e al s imuli, including dis o ion
o posi ional in o ma ion (Adachi-Yamada e al., 1999), TNF
signalling (Igakie al.,2002;Mo eno e al., 2002)o i adia ion
(Luo e al., 2007). To s udy he ole o he JNK pa hway in apop osis
induced by in eg in loss o unc ion, we analyzed JNK ac i a ion
using an an ibody agains phospho yla ed JNK (pJNK, Figu e 3).
Compa ed o con ols (n=16,Figu es 3A,A’,D), pJNK le els we e
up egula ed in nub >mys
RNAi
discs (n=18,Figu es 3B,B’,D),
sugges ing ha in eg ins con ol JNK ac i i y in he wing disc. This
was u he confi med using a epo e ha moni o s he
ansc ip ion o a a ge o he JNK pa hway, he JNK inhibi o
pucke ed (pucLacZ)(Adachi-Yamada e al., 1999). We ound ha
while in wild ype wing discs, pucLacZ exp ession was es ic ed o a
small p oximal egion (Adachi-Yamada e al., 1999),
Supplemen a y Figu es S6A,A’), in nub >mys
RNAi
wing discs,
pucLacZ le els we e s ongly up egula ed in he pouch (n=
28,Supplemen a y Figu es S6B,B’).
In some con ex s, JNK signalling can be ac i a ed as a
consequence o apop osis (Ku anaga e al., 2002). To es whe he
ac i a ion o JNK lies ups eam o downs eam o loss o in eg in
unc ion, JNK ac i a ion was assessed in nub >mys
RNAi
;Diap1 wing
discs (Figu e 3). We ound ha pJNK (n=20,Figu es 3C,D)and
pucLacZ (n= 25, Supplemen a y Figu es S6C,C’) le els we e s ill
up egula ed despi e escue o apop osis (Figu e 1D), sugges ing ha
JNK ac i a ion is ups eam o cell dea h due o in eg in loss o
unc ion in wing disc cells. S ess induced cell dea h in he wing disc
ac i a es JNK ups eam o caspase ac i a o p o eins, which, in u n,
ein o ce JNK ac i a ion in a posi i e eedback loop o ampli y he
apop o ic esponse (Shle ko and Mo a a, 2012). He e, we ound
ha , in ac , he inc ease in JNK ac i i y o nub >mys
RNAi
;Diap1
FIGURE 3 | Elimina ion o in eg ins ac i a es he JNK pa hway. (A–C9)Maximal p ojec ion o con ocal iews o wing imaginal discs om hi d-ins a la ae s ained
wi h an i-pJNK [g een in (A–C), whi e in (A9–C9)] and he nuclea ma ke Hoechs [DNA, blue in (A–C)]. (A,A9)Con ol nubGal4 wing disc. (B,B9)Wing disc exp essing a
mys RNAi unde he con ol o nubGal4,nub >mys
RNAi
.(C,C9)Wing disc co-exp essing RNAi agains mys and Diap1 unde he con ol o nubGal4,nub >mys
RNAi
;
Diap1.(D) Quan ifica ion o pJNK le els in wing discs o he indica ed geno ypes. The s a is ical significance o di e ences was assessed wi h a - es , *** and ** p
alues <0.001 and <0.01, espec i ely. Scale ba in all panels, 30 μm.
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Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s
discs was lowe han he one obse ed in nub >mys
RNAi
discs
(Figu es 3B–D). This esul sugges s ha he eedback loop be ween
JNK ac i i y and apop osis obse ed in s ess-induced apop osis may
also ope a e in in eg in loss o unc ion-dependen apop osis.
JNK Media es Apop osis due o Loss o
In eg in Func ion in Wing Disc Cells
To es whe he he JNK pa hway media es cell dea h due o
in eg in loss o unc ion in wing imaginal discs, we analyzed i
o e exp ession o he JNK inhibi o puc supp essed he cell dea h
caused by mys RNAi (nub >mys
RNAi
;puc) and ound ha i did
(Figu es 4A–D,n= 24).
The ela ionship be ween JNK signalling, p o-apop o ic
p o eins and caspase ac i a ion is con ex -dependen . Thus, he
JNK pa hway can ac ups eam o downs eam o p o-apop o ic
genes such as p o hid. Fo ins ance, expe imen s in D osophila S2
cells sugges ed ha Rp could s imula e JNK ac i a ion h ough
DIAP1 deg ada ion (Ku anaga e al., 2002). In con as , JNK
signalling p omo es hid exp ession (and apop osis) in eye discs,
and i is equi ed o p - epo e induc ion in esponse o
adia ion in wing discs (Ku anaga e al., 2002;Mo eno e al.,
2002). In ou sys em, inhibi ion o JNK signalling ia puc
o e exp ession was able o supp ess hid up egula ion in nub >
mys
RNAi
wing discs o a la ge ex en (n=17,Figu es 4C-C’’’,E).
Al oge he , hese esul s allow us o conclude ha educ ion o
in eg in unc ion in wing discs leads o apop osis by s imula ing hid
ansc ip ion h ough JNK ac i a ion.
In eg ins Coope a e Wi h he EGFR
Pa hway o P omo e Cell Su i al
The EGFR ac i a es a ne wo k o signaling pa hways p omo ing
su i al in many di e en cellula con ex s [ e iewed in (Henson
FIGURE 4 | In eg ins p omo e cell su i al by inhibi ing he JNK pa hway. (A–C-)Maximal p ojec ion o con ocal iews o hi d-ins a wing imaginal discs ca ying
he epo e hidGFP, s ained wi h an i-βPS [blue in (A–C), whi e in (A9–C9)], an i-Dcp1 [ ed in (A–C), whi e in (A99 –C99 )] and an i-GFP [g een in (A–C), whi e in (A-–C-)].
(A–A-)Con ol hidGFP;nubGal4 wing disc. (B–B-)hidGFP wing disc exp essing a mys RNAi unde he con ol o nubGal4,hidGFP;nub >mys
RNAi
.(C–C-)hidGFP wing
disc co-exp essing a mys RNAi and he nega i e egula o o he JNK pa hway puc, unde he con ol o nubGal4 hidGFP;nub >mys
RNAi
;puc.(D,E) Quan ifica ion
o Dcp1 (D) and hidGFP (E) le els in wing discs o he indica ed geno ypes. The s a is ical significance o di e ences was assessed wi h a - es , *** and * p alues <
0.001, <0.01, and <0.05, espec i ely. NS, non-s a is ically significan . Scale ba in all panels, 30 μm.
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Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s
and Gibson, 2006)]. In addi ion, expe imen s mainly om cell
cul u e ha e e ealed syne gis ic coope a ion be ween g ow h
ac o s and in eg ins in cell su i al (Regina o e al., 2003;Mi an i
and B ugge, 2002).Fo ins ance, EGFR p omo es su i al
h ough egula ion o he Ras/MAPK pa hway and consequen
inhibi ion o he p oapop o ic gene hid in he eye disc (Be gmann
e al., 1998;Ku ada and Whi e, 1998;Yu e al., 2002). Mo eo e ,
wing disc mu an clones o EGFR o o some o hei
dowms eam e ec o s, such as Ras, a e smalle han hei win
spo , sugges ing a ole o he EGFR pa hway in cell su i al
(Diaz-Benjumea and Ga cia-Bellido, 1990; Diaz-Benjumea and
Ha en, 1994; Zecca and S uhl, 2002). To di ec ly add ess a ole
o EGFR in cell su i al in he wing disc, we exp essed an EGFR
dominan nega i e cons uc (UAS-DER
DN
) unde he con ol o
he nub-Gal4 line and ound i led o inc ease cell dea h in he
pouch egion (nub >DER
DN
,n= 21, Figu es 5A, A’,C,C’,E).
Howe e , in con as o wha happens in nub >mys
RNAi
wing
discs (Figu es 5B, B’,E), whe e cell dea h was ound dis ibu ed
all o e he pouch, exp ession o DER
DN
es ic ed apop osis o
egions o high EGFR ac i i y (Gabay e al., 1997), such as he
wing ma gin and he p esump i e ein e i o ies (yellow a ow
and as e isks in Figu es 5A, A’,C,C, espec i ely). In addi ion,
and con a y o wha happens in he eye, EGFR did no seem o
exe i s p o-su i al unc ion h ough egula ion o hid o p
ansc ip ion, as hidGFP o p -lacZ exp ession we e no al e ed in
nub >DER
DN
wing discs (Figu es 5A, A’,C,C”;Supplemen a y
Figu e S7). We hus conclude ha he EGFR pa hway has a limi ed
ole in he con ol o cell su i al in he wing disc ha is
independen o he ansc ip ional egula ion o he p o-
apop o ic genes hid and p . Finally, we examined a possible
coope a ion be ween in eg ins and he EGFR pa hway o
media e cell su i al. We ound ha he simul aneous co-
exp ession o mys
RNAi
and DER
DN
in wing discs (nub >
mys
RNAi
;DER
DN
) enhanced he cell dea h pheno ype caused by
he exp ession o any o hem on hei own (Figu es 5B–E),
s ongly sugges ing ha in eg ins and he EGFR pa hway ac in
pa alell o p omo e cell su i al in wing imaginal discs.
Oncogenic Ras Supp esses Anoikis in Wing
Discs
Supp ession o anoikis a e de achmen o cance cells om he
ECM is a key s ep in umo me as asis [ e iewed in (Simpson
e al., 2008)]. In ac , he abili y o oncogenic Ras o supp ess
anoikis has long been conside ed c i ical o Ras ans o ma ion.
Howe e , ecen e idence sugges s ha Ras and o he oncogenes
can induce bo h p o- and an i-apop o ic signals depending on he
cell ype and con ex [ e iewed in (Cox and De , 2003)]. In
D osophila, Ras o e ac i a ion ende s cells e ac o y o s ess-
and i adia ion-induced apop osis (Be gmann e al., 1998;
Ku ada and Whi e, 1998;Pinal e al., 2018). To es whe he
oncogenic Ras could p o ec cells om anoikis in he wing
imaginal disc, we o e exp essed an ac i a ed o m o DRas,
Ras
V12
(Ka im and Rubin, 1998) and ound i subs an ially
supp essed he cell dea h pheno ype caused by in eg in
deple ion (nub >mys
RNAi
, as
V12
;Figu es 6A-A’’,6B–B’’,
C–C’’,D,n= 18). These esul s demons a e ha oncogenic
Ras is able o supp ess anoikis in wing imaginal discs. In epi helial
cell lines, oncogenic Ras con e s esis ance o anoikis pa ly by
down egula ing he exp ession o p oapop o ic Bcl-2 membe s
FIGURE 5 | In eg ins and EGFR coope a e o p omo e cell su i al.
(A–D”)Maximal p ojec ion o con ocal iews o wing imaginal discs om hi d-
ins a la ae ca ying he epo e hidGFP, s ained wi h an i-Dcp1 [ ed in
(A–D), whi e in (A9–D9)], an i-GFP [g een in (A–D), whi e in (A’’–D’’)] and
he nuclea ma ke Hoechs [DNA, blue in (A–D)]. (A–D’’)Con ol hidGFP;
nubGal4 wing disc. (B–B’’)hidGFP wing disc exp essing a mys RNAi unde
he con ol o nubGal4,hidGFP;nub >mys
RNAi
.(C–C’’)hidGFP wing disc
exp essing a dominan nega i e o m o he EGFR, DER
DN
, unde he con ol
o nubGal4,hidGFP;nub >DER
DN
.(C–D’’)hidGFP wing disc co-exp essing a
mys RNAi and DER
DN
unde he con ol o nubGal4 hidGFP;nub >mys
RNAi
;
DER
DN
.(E) Quan ifica ion o Dcp1 le els in wing discs o he indica ed
geno ypes. The s a is ical significance o di e ences was assessed wi h a
- es , *** and ** p alues <0.001 and <0.01, espec i ely. NS, non-s a is ically
significan . Scale ba in all panels, 30 μm.
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Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s
(Rak e al., 1995) and by p e en ing down egula ion o
an iapop o ic p o eins (Rosen e al., 2000). In D osophila,i
appea s ha he Ras pa hway egula es Hid ac i i y a he le el
o bo h, p o ein phospho yla ion and gene exp ession, in hid-
induced apop osis (Be gmann e al., 1998;Ku ada and Whi e,
1998). Howe e , we ound ha , in he D osophila wing disc
epi helium, he abili y o oncogenic Ras o con e esis ance o
anoikis does no in ol e ansc ip ional egula ion o hid,as
hidGFP le els we e no al e ed in nub >mys
RNAi
; Ras
V12
(n=
21, Figu es 6A’’’,C’’’) discs compa ed o nub >mys
RNAi
(n= 20,
Figu e 6B’’’).
Ec opic exp ession o Ras
V12
in he do sal compa men o
wing imaginal discs, by means o he ap e ous Gal4 (ap) Gal4 line,
causes issue o e g ow h and cell shape changes, which esul s in
he o ma ion o ec opic olds [ap >Ras
V12
,n= 20, Figu es
7A,A’,C,C’,(P obe and Edga , 2000;Sole Bea y e al., 2021).
Reducing in eg in unc ion also al e s cell shape causing a mild
olding pheno ype (Dominguez-Gimenez e al., 2007), Figu es
7B, B’E, E’]. He e, we ound ha co-exp ession o mys
RNAi
and
Ras
V12
enhanced he cell shape and olding pheno ype caused by
he sole exp ession o any o hem (ap >mys
RNAi
; Ras
V12
,n= 20,
Figu es 7D,D’).
In his wo k, we show ha educing in eg in exp ession
ensued basal ex usion o he dead cells (Figu es 1C–C’’,
Figu es 7F,G). Howe e , e en hough oncogenic K-Ras
MCDK cells su i e and ex ude basally, a mechanism
p oposed o ini ia e in asion in o su ounding issues (Sla um
e al., 2014), we ound ha exp ession o Ras
V12
on i s own in
wing disc cells (ap >Ras
V12
) did no esul in cell ex usion
(Figu e 7H). In s iking con as , he exp ession o Ras
V12
in
FIGURE 6 | Ec opic ac i a ion o he Ras pa hway escues anoikis. (A–C’’’)Maximal p ojec ion o con ocal iews o hi d-ins a wing imaginal discs ca ying he
epo e hidGFP, s ained wi h an i-βPS [blue in (A–C), whi e in (A9–C9)], an i-Dcp1 [ ed in (A–C), whi e in (A’’–C’’)] and an i-GFP [g een in (A–C), whi e in (A-–C-)].
(A–A-)Con ol hidGFP;nubGal4 wing disc. (B–B-)hidGFP wing disc exp essing a mys RNAi unde he con ol o nubGal4,hidGFP;nub >mys
RNAi
.(C–C-)hidGFP wing
disc co-exp essing a mys RNAi and an ac i a ed o m o Ras, Ras
V12
, unde he con ol o nubGal4 hidGFP;nub >mys
RNAi
; Ras
V12
.(D) Quan ifica ion o Dcp1
le els in wing discs o he indica ed geno ypes. The s a is ical significance o di e ences was assessed wi h a - es , *** and * p alues <0.001 and <0.05, espec i ely.
Scale ba in all panels, 30 μm.
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Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s