BioMed Cen al
Page 1 o 13
(page numbe no o ci a ion pu poses)
BMC De elopmen al Biology
Open Access
Resea ch a icle
Mechanism o G1 a es in he D osophila eye imaginal disc
Luis M Escude o and Ma hew F eeman*
Add ess: MRC Labo a o y o Molecula Biology, Hills Road, Camb idge CB2 0QH, UK
Email: Luis M Escude o - lme@m c-lmb.cam.ac.uk; Ma hew F eeman* - m 1@m c-lmb.cam.ac.uk
* Co esponding au ho
Abs ac
Backg ound: Mos di e en ia ing cells a e a es ed in G1-phase o he cell cycle and his
p oli e a i e quiescence appea s impo an o allow di e en ia ion p og ammes o be execu ed. An
example occu s in he D osophila eye imaginal disc, whe e all cells a e synch onized and a es ed in
G1 phase p io o making a a e choice ei he o ini ia e he i s ound o pho o ecep o
di e en ia ion o o e-en e one e minal mi osis.
Resul s: We ha e analysed he mechanism o his empo ally egula ed G1-phase in o de o
de elop an in eg a ed model o his p oli e a i e egula ion. We ind ha an o e lapping se o cell
cycle inhibi o s combine o o m an e icien ba ie o cell cycle p og ession. This ba ie depends
on bo h he p ima y sec e ed signals ha d i e e inal de elopmen , Dpp and Hh. Each o hese has
dis inc , as well as pa ially o e lapping unc ions, in ensu ing ha Cyclin E and dE2F1 a e kep in
check. Addi ionally, inhibi ion o Cyclin A by Roughex is essen ial, and his egula ion is independen
o Dpp and Hh.
Conclusion: One implica ion o hese esul s is o u he suppo he idea ha Cyclin A has
impo an unc ions in S-phase en y as well as in mi osis. The unexpec edly complex ne wo k o
egula ion may e lec he impo ance o cells being uni o mly eady o espond o he induc i e
signals ha coo dina e e inal di e en ia ion.
Backg ound
All euka yo es use he same undamen al machine y o
d i e cell cycle p og ession bu mul icellula o ganisms
ace he addi ional challenge o egula ing he ime and
place o p oli e a ion h oughou de elopmen . A well
conse ed aspec o his de elopmen al egula ion is ha
cells no mally a es in G1-phase o he cell cycle p io o
di e en ia ion. This p o ides a quiescen s age o di e -
en ia ion o begin and, once cells s a o adop hei e -
minal a e, mos will ne e e-en e a p oli e a i e s a e. In
umo igenesis, howe e , e-en y o a es ed cells in o
abno mal p oli e a ion can occu [1].
The D osophila eye p o ides an expe imen ally amenable
example o de elopmen ally egula ed p oli e a ion and
has he e o e been ex ensi ely s udied as a model. Di e -
en ia ion o he indi idual ace s (omma idia) o he com-
pound eye occu s in a mo ing wa e om he pos e io o
he an e io o he eye imaginal disc, implying ha he e
is a g adien o inc easing cell ma u i y om an e io o
pos e io in he disc. The on o he wa e o de elop-
men is ma ked by an inden a ion known as he mo pho-
gene ic u ow (MF) [2]. Se e al hou s p io o he passage
o he MF (and he e o e an e io o i ) all cells in he eye
disc a es in G1-phase [3,4]. A e he MF passes, hose
cells ha ha e no ye s a ed o di e en ia e as pho o e-
Published: 2 Ma ch 2007
BMC De elopmen al Biology 2007, 7:13 doi:10.1186/1471-213X-7-13
Recei ed: 13 Sep embe 2006
Accep ed: 2 Ma ch 2007
This a icle is a ailable om: h p://www.biomedcen al.com/1471-213X/7/13
© 2007 Escude o and F eeman; licensee BioMed Cen al L d.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0),
which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
BMC De elopmen al Biology 2007, 7:13 h p://www.biomedcen al.com/1471-213X/7/13
Page 2 o 13
(page numbe no o ci a ion pu poses)
cep o s e-en e S-phase o one e minal ound o di i-
sion, he "second mi o ic wa e" [5].
He e we ocus on he mechanisms esponsible o es ab-
lishing and main aining G1-a es o cells p io o he MF
(in wha we call non-p oli e a i e egion, NPR). Speci i-
cally, we aim o unde s and how cells become synch o-
nous and a es in G1-phase and o lea n how he signals
ha d i e he wa e o eye de elopmen di ec his p ocess.
This aspec o de elopmen al con ol o p oli e a ion in
D osophila is no well unde s ood, al hough i has been
in es iga ed in a numbe o di e en con ex s including
he eye, he wing and he emb yo [4,6-12]. Mos signi i-
can ly in he eye, Decapen aplegic (Dpp, he D osophila
homologue o BMP ligands) signalling is equi ed o
main ain G1 a es in he an e io egion o he a es ed
zone. Dpp appea s o ep ess Cyclin E, since he emo al
o se e al CycE inhibi o s [7,13,14] causes a simila phe-
no ype o he lack o he Dpp ecep o , hick eins ( k ) o
o he o e exp ession o a CycE ansgene [12]. Mo e
ecen ly, Fi h and Bake ha e concluded ha Dpp and
Hedgehog (Hh) ac edundan ly, and be ween hem a e
su icien o a es cells in G1 [7]. This in ol emen o
Dpp and Hh is consis en wi h hei oles as he p ima y
sec e ed signals ha d i e MF p og ession. Finally, he
o e exp ession o Cyclin A o he absence o he cyclin
kinase inhibi o Roughex (Rux) can also induce S-phase
en y in he NPR [4,15]. Toge he , hese ea lie s udies
ha e iden i ied a numbe o di e en mechanisms o con-
ol o G1 a es bu i is no able ha no common check-
poin has been iden i ied and no uni ied iew o he
p ocess has ye been p oposed.
P o iding an explana ion o his lack o a clea model,
ou esul s indica e ha in he eye disc no single compo-
nen is ully esponsible o he de elopmen al cell cycle
a es p io o he mo phogene ic u ow. We show ha
i s , Hh and Dpp oge he p omo e en y in o mi osis o
he cells p io o G1 a es , he eby d i ing cells in o G1-
phase and ini ia ing he NPR. Then, an o e lapping se o
cell cycle inhibi o s combine o o m an e icien and
obus ba ie o cell cycle p og ession. This ba ie
depends pa ially on he inhibi ion o Cyclin E and dE2F1
ac i i y, also unde he con ol o Dpp and Hh. Howe e ,
in con as o p e ious wo k [7], ou esul s show ha he
ole o Hh and Dpp in main aining G1 a es , is la gely
con ined o he an e io pa o he NPR. The inhibi ion o
Cyclin A by Rux becomes he majo ba ie o S-phase
en y in he pos e io egion and, signi ican ly, his is
independen o bo h Dpp and Hh. This analysis o he el-
a i e impo ance o he di e en playe s in ol ed in G1
a es allows us o in eg a e hem in o an o e all model o
signal- egula ed synch onisa ion and p oli e a i e a es .
Resul s
De ining he egion o G1 a es in he eye imaginal disc
Be ween he p oli e a ing cells in he an e io o he eye
imaginal disc and he SMW is a b oad band o cells ha
a e a es ed in G1-phase [3,16]; hey span abou 11–14
ows o cells. Gi en he accep ed iew ha he mo phoge-
ne ic u ow mo es o wa d, on a e age, by one ow o
omma idia (3–4 cell ows) e e y 70 min [17], we es ima e
ha on a e age cells emain in his G1 a es o i e o six
hou s. In e es ingly, his is only sligh ly longe han he
es ima e o he no mal G1 phase o cells in he p oli e a -
ing egion o he disc. This is based on he obse a ion
ha he doubling ime o cells in he eye disc is app oxi-
ma ely 12 hou s [18], and ha he p opo ion o cells in
G1 phase in p oli e a ing disc cells is one hi d [19]. These
es ima es imply ha he o ma ion o his non-p oli e a-
i e egion (NPR) depends signi ican ly on cell cycle syn-
ch oniza ion as hey app oach he MF, as well as on
speci ic a es mechanisms.
In his pape we de ine he NPR as co esponding o he
absence o B dU inco po a ion an e io o he SMW (Fig.
1A–C). So, om an e io o pos e io in he eye disc a e 1)
he egion whe e undi e en ia ed cells p oli e a e an-
domly; 2) he NPR (which includes he mo phogene ic
u ow); and 3) he second mi o ic wa e (Fig. 1C). No e
ha in he an e io pa o he NPR, cells do no en e G1
a es un il a e mi osis, so he las cells o be de ec ed by
B dU s aining ac ually en e G1 one o wo ows la e ,
a e comple ion o G2 and M-phases. The ac i i y o
dE2F1 (a homologue o mammalian E2F, a ansc ip ion
ac o esponsible o he exp ession o many S-phase
componen s [20]) is la gely absen om he NPR, as
measu ed by he exp ession o a PCNA-GFP epo e [21],
which con ains he dE2F1 binding sequence om he p o-
mo e o he PCNA gene (Figu e 1B, E). We obse ed ha
he down egula ion o B dU s aining coincides p ecisely
wi h he onse o A onal (A o) exp ession [22,23] an e io
o he u ow (Fig. 1A, B, D), and wi h he loss o Homo -
ho ax (H h) exp ession (no shown). Howe e , in clones
o a o o h h mu an cells en e G1 a es no mally, imply-
ing ha hese genes a e no essen ial o es ablishing he
NPR.
To cha ac e ise he ana omy o he NPR u he , we ha e
used 3-dimensional econs uc ion so wa e o image he
disc (Fig. 1D, E). This clea ly showed ha he mo phoge-
ne ic u ow o ms he pos e io egion o he NPR. I also
allowed a clea iew o he 'pe ipodial memb ane', he
laye o squamous cells ha o e lie he disc-p ope and
which can in luence aspec s o disc de elopmen [24].
B dU posi i e cells a e p esen in he pe ipodial mem-
b ane, bu hese we e andomly dis ibu ed, indica ing he
absence o coo dina ion o G1 a es be ween he wo
adjacen epi helia. To p e en he B dU posi i e cells in
BMC De elopmen al Biology 2007, 7:13 h p://www.biomedcen al.com/1471-213X/7/13
Page 3 o 13
(page numbe no o ci a ion pu poses)
he pe ipodial memb ane being misin e p e ed as disc
p ope cells, in all subsequen con ocal p ojec ions we
used A o exp ession as a e e ence o disc p ope .
The NPR equi es ep ession o Cyclin E, dE2F1 and Cyclin
A
In D osophila, he main ac i a o s o S-phase en y a e Cyc-
lin E [25], dE2F1 [20,26] and Cyclin A [7,9,15,27]. All a e
inac i e in he NPR and he p o ein le els o Cyclins E and
A a e e y low in his egion [4,12]. To examine he ela-
i e impo ance o he absence o hese S-phase igge s in
he main enance o G1 a es , we o e exp essed hem
using he lip-ou Gal4 echnique [28]. This echnique
pe mi s he di ec compa ison be ween o e exp essing
cells and neighbou ing wild- ype cells, he eby p o iding
a mo e de ailed iew o he pheno ype han is possible
wi h simple Gal4 misexp ession. The ec opic exp ession
o Cyclin E induced B dU inco po a ion e y e icien ly in
he an e io egion o he NPR (Fig. 2A and [12]). Mo e
a ely, we obse ed a ew cells in S-phase in mo e pos e-
Pa e n o p oli e a ion and G1 a es in he eyeFigu e 1
Pa e n o p oli e a ion and G1 a es in he eye. (A) P ojec ion o se e al con ocal sec ions o a WT eye disc showing
A madillo (g een), Ela ( ed) and A o (blue) localiza ion. The mo phogene ic u ow (MF, a owhead in all igu es) is high-
ligh ed by accumula ion o A madillo (β-ca enin), which ou lines cell memb anes. A o exp ession appea s be ween 6–8 cells
an e io o he MF and becomes limi ed o he R8 pho o ecep o jus pos e io o he u ow. Ela is exp essed in all he pho-
o ecep o cells pos e io o he A o exp essing R8. In his and all subsequen igu es, an e io is o he le . (B) WT eye disc
showing he inco po a ion o B dU ( ed) and he exp ession o PCNA-GFP epo e (indica ing dE2F1 ac i a ion, g een) and A o
p o ein (blue). An e io o he u ow he e is a non-p oli e a i e egion, wi hou B dU inco po a ion (whi e ba ). The pa e n
o PCNA-GFP exp ession coincides wi h he egions o p oli e a ion. (C) Scheme showing he di e en p oli e a ing egions o
he eye disc. The panel shows a d awing o he disc in 1B. The inse shows a pic u e o he whole disc. F om an e io o pos e-
io : o ange ma ks he egion o undi e en ia ed cells ha p oli e a e andomly; he NPR is ma ked in blue (i s ex en is
ma ked by he whi e ba ), wi h he da ke zone showing he mo phogene ic u ow (also ma ked by an a owhead); inally, he
ed band ma ks he egion o he second mi o ic wa e. (D) Z-axis econs uc ion o a WT eye disc. The ou lines o he cells
a e shown by A madillo (A m) exp ession (g een). The pe ipodial memb ane appea s a he op wi h some B dU posi i e cells
( ed). In he disc p ope he e is a high accumula ion o A m p o ein in he apical pa o he MF (a owhead). S-phase nuclei in
he SMW a e basally loca ed (a ow), whe eas an e io o he NPR, B dU posi i e nuclei a e mo e apical. The NPR includes
he u ow cells ( ha accumula e high apical le els o A m) and be ween 4–6 ows o mo e an e io cells. A o exp essing cells
(blue) a e es ic ed o he disc p ope . (E) The cy oplasmic exp ession o PCNA-GFP epo e (blue) is seen in cells ha a e
comple ing he cell cycle in he mos an e io pa o he NPR, bu is no exp essed in he cells o he MF ( ha accumula e E-
cadhe in in he apical egion, in g een). In he SMW he cells wi h B dU posi i e nuclei also exp ess he PCNA-GFP.
Ela
A o
A m
B dU
A o
PCNA
B dU
E-cadh
PCNA
B dU
A m
A o
AB
DE
NPR
SECOND
MITOTIC
WAVE
AP
C
BMC De elopmen al Biology 2007, 7:13 h p://www.biomedcen al.com/1471-213X/7/13
Page 4 o 13
(page numbe no o ci a ion pu poses)
io egions o he NPR. Cyclin E egula es dE2F1 ac i i y
by inhibi ing Rb [29], he D osophila homologue o he
e inoblas oma ac o . Consis en wi h his, cells o e ex-
p essing Cyclin E exp essed he dE2F1 epo e cons uc ,
PCNA-GFP (Fig. 2B).
O e exp ession o UAS-dE2F1 (combined wi h i s obli-
ga e pa ne UAS-Dp) [30] caused a weake pheno ype
han Cyclin E, al hough he pa e n was simila : in his
case, occasional B dU posi i e cells we e seen bu only in
he an e io egion o he NPR (Fig. 2C). This esul is con-
sis en wi h he loss o unc ion pheno ype o b , he
main inhibi o dE2F1 ac i i y, which causes ec opic S-
phase cells only in he mos an e io egions o he NPR
[13]. Taken oge he , he o e exp ession o Cyclin E and
dE2F1/Dp in he NPR implies ha a leas one addi ional
mechanism mus p omo e G1 a es in he pos e io cells
o he NPR; unlike he an e io cells, hey a e e ac o y o
o e exp ession o Cyclin E o dE2F1/Dp. No e ha
because o he p og essi e de elopmen o he e ina, he
an e io cells ma u e in o he pos e io cells, so his di e -
en esponsi eness o Cyclin E and dE2F1/Dp ac ually ep-
esen s a de elopmen al di e ence a he han a spa ial
one.
The co-o e exp ession o UAS-cycE and UAS-dE2F1/Dp
p oduced a sligh ly s onge pheno ype han UAS-cycE
o e exp ession alone (in which endogenous dE2F1 is also
ac i a ed): S-phase is e icien ly ac i a ed in he an e io
bu less so in he pos e io pa o he NPR (Fig. 2D). This
esul is consis en wi h he ea lie epo ha in clones
mu an o bo h b and dap, inhibi o s o dE2F1 and Cyc-
lin E, espec i ely, he cells in he whole o he NPR do no
a es [7]; in ou hands hese double mu an b dap clones
can only some imes induce ec opic B dU s aining in he
an e io egion o he NPR and ha e e en weake e ec s in
he pos e io egion (no shown). We ake he di e ence
be ween he mo e powe ul e ec o ec opic Cyclin E and
dE2F1, and he weake e ec o b dap o indica e ha
o e exp ession o ac i a o s is a mo e powe ul igge
han emo al o na u al ep esso s.
Cyclin A has a less well de ined ole in S-phase ac i a ion
bu he e idence o i s pa icipa ion is ne e heless s ong
[7,15,27]. In e es ingly, i s o e exp ession in he NPR
ga e a di e en esul o Cyclin E o dE2F1. UAS-cycA
induced ec opic B dU s aining in he whole wid h o he
NPR, bu was mos e icien in he mos pos e io domain,
whe e Cyclin E and dE2F1 a e less e ec i e (Fig. 2E and
[15]). The combina ion o bo h ansgenes, UAS-cycA and
UAS-cycE s ongly induced ec opic S-phase en y in cells
h oughou he NPR: almos all o e exp essing cells ailed
o a es in G1 (Fig. 2F).
O e all, ou da a p o ide s ong e idence ha G1 a es in
he NPR is con olled by mul iple mechanisms wi h dis-
inc egional e ec s. Speci ically, in he an e io egion o
he NPR, which co esponds o he ea ly s ages o G1
a es , he ep ession o Cyclin E and dE2F1 ac i i y is c i -
ically impo an , while in he pos e io hal , whe e he
cells a e mo e ma u e, he ep ession o Cyclin A becomes
necessa y o he main enance o hese cells in he G1
phase.
Con ol o G1 a es by Hh and Dpp
The NPR is he ea lies isible change in cells as hey en e
he wa e o de elopmen ha will e en ually p oduce he
adul e ina. Ul ima ely, he whole wa e o eye de elop-
men is p opaga ed by sec e ed Dpp and Hh signals. Con-
sis en wi h his, i has p e iously been epo ed ha Dpp
is esponsible o he G1-a es in he an e io pa o he
NPR [8,11]; mos ecen ly, Fi h and Bake ha e epo ed
ha Dpp and Hh ac edundan ly o induce he whole
NPR [7]. To unde s and he mechanism in de ail by which
Dpp and Hh cause cells o exi om p oli e a ion and
a es /synch onise in G1, we gene ically blocked he wo
pa hways, ei he alone o oge he . We used mu a ions in
Thick Veins (Tk ), he Dpp ecep o ; Smoo hened (Smo),
he memb ane associa ed ansduce o Hh signalling;
Mad, an essen ial in acellula ansduce o Dpp signal-
ling; and Ci, a nuclea e ec o o Hh signalling. No e ha
Ci has cons i u i e ep esso ac i i y as well as being a Hh-
dependen ac i a o [31], implying ha loss o Ci is no
always equi alen o loss o Smo. Fo his eason, simul a-
neous loss o Smo and Tk ep esen s he bes way o ana-
lysing he loss-o - unc ion pheno ypes o he Hh and Dpp
pa hways in he eye disc [32,33].
Ini ial synch onisa ion o cells as hey en e he NPR
One o he ea lies signs o he NPR is an inc ease in he
numbe o mi o ic cells immedia ely p io o he G1 a es
egion. This is seen in wild ype discs as a well de ined line
o phosphohis one H3 (pH3) posi i e cells immedia ely
an e io o he NPR (Fig. 3A), and con as s wi h he much
mo e spa sely sca e ed pH3-posi i e cells u he an e-
io . We obse ed ha in he smo k double mu an clones
(bu no in he k o smo clones alone, no shown) his
clea alignmen o mi o ic cells was los (Fig. 3B), imply-
ing ha cells we e no longe being e icien ly d i en in o
mi osis. No e ha pH3 only s ains cells in o a sho
pe iod o he cell cycle, so i s absence canno be aken as
a sign o p oli e a i e a es . Ins ead we in e p e his
esul o indica e a unc ion o Hh and Dpp in accele a -
ing h ough mi osis hose cells ha a e al eady 'p e-
mi o ic' o in la e s ages o he cell cycle.
BMC De elopmen al Biology 2007, 7:13 h p://www.biomedcen al.com/1471-213X/7/13
Page 5 o 13
(page numbe no o ci a ion pu poses)
A di e en ial equi emen o Dpp and Hh signalling in he pos e io
and an e io o he NPR
Loss o smo alone causes a sligh delay in en y o he NPR,
by one o wo ows o cells, bu no ec opic B dU s aining
occu s in he es o he egion [7]. We examined clones o
k as well as double mu an clones o Mad ci, o k ci, o
smo k (Fig. 3C–H). All ou mu an condi ions caused
ec opic B dU inco po a ion and Cyclin B exp ession in he
an e io egion o he NPR bu none caused signi ican
ec opic S-phase induc ion in he pos e io egion ( his is
especially clea in he case o Cyclin B s aining (Fig. 3F,
3H). O hese ou condi ions, smo k was he only one in
which occasional B dU posi i e cells we e de ec ed in he
pos e io egion (6 ou o 32 clones; no mo e han 2–3
posi i e cells in any clone). Th ee poin s eme ge om
hese da a. Fi s , hey imply a clea di e ence be ween he
main enance o G1 in he an e io and pos e io o he
NPR. Second, Fi h and Bake epo ed ec opic B dU
inco po a ion and Cyclin B accumula ion in Mad ci clones
in he whole NPR, no jus he an e io egion. We used
he same alleles as hey did bu did no obse e any pos-
e io B dU s aining. Thi d, he e is no e idence o a
E ec o o e exp ession o cyclins in he NPRFigu e 2
E ec o o e exp ession o cyclins in he NPR. All panels excep B show B dU s aining ( ed) o eye disc ha bou ing di -
e en o e exp ession clones ma ked wi h GFP (g een). (A) The o e exp ession o CycE is able o induce ec opic B dU inco -
po a ion wi h a high e iciency in he an e io pa o he NPR, bu less so he pos e io , whe e only a ew cells en e S phase.
(B) The PCNA-GFP epo e (g een) is ac i e (a ows) in he CycE o e exp ession clones (ma ked by p esence o β-gal in ed)
wi hin he NPR. (C) In dE2F1 o e exp ession clones he e is B dU inco po a ion only in he an e io pa o he NPR. (D) The
o e exp ession o CycE and dE2F1 can induce inco po a ion o B dU in an e io and pos e io cells o he NPR. (E) CycA
o e exp essing cells inco po a e B dU in any pa o he NPR, bu wi h a highe e iciency in he pos e io pa , whe e mos
cells a e B dU posi i e. (F) Simul aneous o e exp ession o CycE and CycA induces a high p opo ion o B dU inco po a ion in
any pa o he NPR.
U-cycE
U-dE2F
U-cycE U-cycE
U-cycE
U-cycA
U-cycA
U-dE2F1
A A’ B B’
C C’ D D’
E E’ F F’

BMC De elopmen al Biology 2007, 7:13 h p://www.biomedcen al.com/1471-213X/7/13
Page 6 o 13
(page numbe no o ci a ion pu poses)
Dpp and Hh con ol G1 a es p ima ily in he an e io o he NPRFigu e 3
Dpp and Hh con ol G1 a es p ima ily in he an e io o he NPR. (A) WT eye disc showing he cells in mi osis as
ma ked by phosphohis one H3 an ibody (PH3, in ed) and he A o pa e n o exp ession (blue). The whi e a ow ma ks he
line o synch onized mi osis an e io o he NPR in A and B. In he egion an e io o his line he equency o phosphohis one
H3 posi i e cells is e y low (as e isk) (B) Eye disc wi h a la ge smo3 k a12 M+ clone (absence o g een). The yellow a ow
ma ks he egion whe e he dis inc line o mi osis (pH3, ed; he so-called i s mi o ic wa e) is los . (C) k a12 clones (absence
o g een) showing some B dU posi i e cells ( ed) in he an e io pa bu no in he pos e io (a ows). (D) Simila ly, in k a12
ci94 clones (ma ked by he absence o Ci an ibody in g een) he e a e no B dU posi i e cells in he pos e io egion o he NPR
( ed). The a ows ma k some B dU cells in he an e io pa o he NPR. (E-F) Mad12 ci94 clones (ma ked by absence o Ci,
g een) s ained o B dU (E) and CycB (F) in ed. The a ows ma k clones in he pos e io pa o he NPR whe e he e is no
ec opic B dU inco po a ion o CycB accumula ion. (G) smo3 k a12 clones (a ows) ma ked by absence o β-gal, in g een
(a ows). The pic u e shows a ep esen a i e clone in he lowe pa o he panel wi h no ec opic B dU posi i e cells ( ed) (H)
Ec opic CycB ( ed) accumula es only in he an e io egion o he NPR in smo3 k a12 clones (absence o g een). This is high-
ligh ed in clones ha span he whole NPR, whe e he e is clea CycB accumula ion ha does no each he mos pos e io
cells (a ows).
Ci
B dU B dU
Ci
CycB CycB
B dU
GFP
GFP
CycB
B dU
CycB
Ci
B dU B dU
k
-
ci
-
k
-
Mad
-
ci
-
Mad
-
ci
-
smo
-
k
-
smo
-
k
-
smo
-
k
-
B dU
PH3
A o PH3
PH3
BGal PH3
WT
A A’ B B’
C C’ D D’
E E’ F F’
G G’ H H’
*
BMC De elopmen al Biology 2007, 7:13 h p://www.biomedcen al.com/1471-213X/7/13
Page 7 o 13
(page numbe no o ci a ion pu poses)
equi emen o Ci in his p ocess: k ci clones a e indis-
inguishable om clones o k alone.
Redundancy o Smo and Tk in he egula ion o dE2F1 ac i i y
Loss o k and smo oge he caused ec opic dE2F1 ac i i y
(as assayed by PCNA-GFP exp ession) in he whole NPR
(Fig. 4B), while loss o k alone ac i a ed dE2F1 in he
an e io egion alone (Fig. 4A); loss o smo alone had no
e ec on dE2F1 ac i i y (no shown). The e o e bo h sig-
nals con ibu e, in a pa ially edundan manne , o he
no mal down egula ion o dE2F1 ac i i y in he NPR.
No e ha his ac i a ion o dE2F1 is no su icien o
induce ec opic S-phase en y in he pos e io egion o he
NPR ( ecall ha he e was almos no ec opic B dU o Cyc-
lin B accumula ion in pos e io smo k clones), which is
consis en wi h ou obse a ion abo e ha ec opic dE2F1
is also no su icien o igge S-phase in his same egion.
A non- edundan equi emen o Hh signalling in he exp ession o
Dacapo
Loss o smo alone abolished he exp ession o Dacapo
(Dap), an inhibi o o CycE ac i i y [34,35]. Ca e ul
examina ion showed ha Dap exp ession begins in he
pos e io domain o he NPR and emains de ec able
h ough o he pos e io o he SMW (Fig. 4C, 4D). We
obse ed ha Dap exp ession disappea s in he smo3
clones, bu no in he k a12 clones o ci94 clones (Fig. 4C,
4D and no shown). This implies ha Dap exp ession is
no dependen on Dpp signalling. The esul wi h ci clones
implies ha , consis en wi h ea lie epo s [36,37], he e
is no posi i e ole o Ci in Hh signalling in his con ex .
We also obse ed Dap down egula ion when he cons i-
u i e Ci ep esso o m, UAS-ciCELL was o e exp essed in
clones (no shown). We he e o e in e ha Hh signalling
is needed o emo e he Ci ep esso o m in he NPR,
a he han di ec ly ac i a ing Dap exp ession.
These gene ic expe imen s wi h he Hh and Dpp pa hways
poin o se e al subs an ial conclusions. 1) Bo h pa hways
a e equi ed o p omo e he las coo dina ed mi osis p e-
ious o he G1 a es . 2) G1 a es in he an e io egion
o he NPR depends p ima ily on Dpp signalling. 3) Mo e
gene ally, G1 a es is egula ed di e en ially in he an e-
io and pos e io o he NPR; in nei he egion is Ci-
dependen ac i a ion equi ed. 4) The e is a edundancy
be ween Hh and Dpp in he con ol o dE2F1 ac i i y in
he pos e io o he NPR. 5) The e is a non- edundan
equi emen o Hh o ac i a e Dap exp ession (and
he eby inhibi Cyclin E) in he pos e io o he NPR; his
ac i a ion equi es Smo bu , again, no Ci. Toge he , hese
da a imply ha while he e is some edundancy be ween
Hh and Dpp signalling in he con ol o he NPR, non-
o e lapping unc ions can also be iden i ied. They also
imply ha , con a y o an ea lie epo [7], Hh and Dpp
signalling do no comp ise he whole mechanism o G1
a es : in he pos e io egion o he NPR, some o he ac-
o mus p e en cells om S-phase en y, e en when Hh
and Dpp signalling a e comple ely blocked.
A seconda y ole o A onal in main aining G1 a es ?
A o exp ession in he NPR is induced by bo h Hh and Dpp
pa hways [32]. Toge he wi h he in ol emen o o he
p oneu al genes in G1 a es in he wing ma gin [10], his
sugges s a possible ole o his gene in he es ablishmen
o he NPR. Howe e , we did no obse e ec opic B dU
posi i e cells in A o loss-o - unc ion clones encompassing
he NPR (Fig. 4E). On he o he hand, abou 55% o
A onal o e exp ession clones showed a clea p ecocious
en y in o he NPR (Fig. 4F), sugges ing ha A onal may
in luence mi o ic p og ession p io in he NPR.
Rux is necessa y o he G1 a es in he NPR
The obse a ion ha Cyclin A o e exp ession induced
B dU inco po a ion in he NPR sugges ed ha Cyclin A
inhibi ion is impo an o he a es o hese cells. We
he e o e examined he pheno ype o emo ing Roughex
(Rux), a cy oplasmic inhibi o o Cyclin A ac i i y
[4,15,38,39]. ux mu an eye discs ha e inc eased p oli e -
a ion an e io and pos e io o he mo phogene ic u ow
[4]. To dissec his pheno ype p ecisely, we induced loss o
unc ion clones o he null allele ux8. In dis inc ion o he
loss o smo and k , cells in he mos pos e io pa o he
NPR a e no longe a es ed in G1, demons a ed by he
high numbe o cells ha inco po a e B dU and he accu-
mula ion o Cyclin B (Fig. 5A–B); an e io cells a e
a ec ed only sligh ly and many emain a es ed in G1
(Fig. 5A). This pheno ype is simila o ha ob ained by
o e exp essing Cyclin A and suppo s he idea ha Rux
ep esses Cyclin A ac i i y and plays a signi ican ole in
main aining G1 a es , pa icula ly in he pos e io o he
NPR.
ux8 clones also displayed sligh ec opic ac i a ion o he
PCNA-GFP ansgene in he NPR (Fig. 5C) sugges ing ha
he powe ul ac i a ion o S-phase in hese clones migh
ely on mo e han jus Cyclin A ac i a ion. Howe e , since
Rux is epo ed o be a speci ic egula o o Cyclin A, and
is he e o e no expec ed o a ec dE2F1 ac i i y di ec ly,
we es ed whe he his up egula ion o dE2F1 was ig-
ge ed by Cyclin A ac i i y. Indeed, Cyclin A o e exp es-
sion was also able o induce weakly he PCNA epo e in
he NPR (Fig. 5D), sugges ing ha he sligh gain o dE2F1
ac i i y in ux clones was indi ec .
Rux an ibodies a e no sensi i e enough o de ec he
endogenous p o ein by immuno luo escence, so he
exp ession pa e n o ux in he eye disc was analysed
using a ux-lacZ ansgene ha is able o escue he ux
pheno ype and is he e o e hough o ep esen ai h ully
he exp ession o he gene [15]. ux-lacZ is exp essed gen-
BMC De elopmen al Biology 2007, 7:13 h p://www.biomedcen al.com/1471-213X/7/13
Page 8 o 13
(page numbe no o ci a ion pu poses)
e ally in he eye disc, including in he NPR, wi h a
dec ease jus pos e io o he mo phogene ic u ow as he
second mi o ic wa e ini ia es (Fig. 5F). Exp ession o ux
was no dependen on Dpp o Hh signalling: ux-lacZ was
no down- egula ed in smo k double mu an clones. On
he con a y, ux-lacZ was ele a ed, indica ing ha Hh and
Dpp no mally ep ess ux exp ession in he second mi o ic
wa e (Fig. 5G). This esul is consis en wi h ou o he
da a ha imply ha Hh and Dpp a e impo an de e mi-
nan s o G1 a es only in he an e io domain o he NPR.
In he pos e io domain, when he cells ha e de eloped
u he , Cyclin A and Rux become he key egula o s o G1
a es .
Discussion
Mi o ic synch ony s. G1 A es
In his pape we ha e ocused on he mechanism o G1
a es in he eye imaginal disc, he i s o e sign o e inal
di e en ia ion. Al hough he NPR has some imes been
conside ed o be me ely he beginning o he mo phoge-
ne ic u ow, ou 3D image analysis clea ly shows ha i
ini ia es well be o e he cells al e hei shape. Jus an e io
o he cells a es ed in G1, he e is a inc eased numbe o
mi o ic cells sugges ing a coo dina ed ac i a ion o hei
en y in o mi osis. This could ep esen he subse o cells
ha a e in S o G2 phases and ha a e accele a ed h ough
he cell cycle in o de o become a es ed in G1. We ha e
Fac o s con ibu ing o G1 a es Figu e 4
Fac o s con ibu ing o G1 a es . (A-B) PCNA-GFP exp ession (g een) in discs wi h k a12 (A) and smo3 k a12 (B) clones.
Up egula ion in he pos e io pa o he NPR only occu s in he smo3 k a12 clones. (C-D) Eye disc wi h smo3 (C) and k a12 (D)
clones (absence o GFP in ed), s ained o Dap an ibody in g een. The loss o smo, bu no k , causes he loss o Dap in he
u ow (a ows). (E) Eye disc wi h a o1 M+ clones (absence o g een) showing no ec opic B dU inco po a ion ( ed) in he NPR.
(F) UAS-a o o e exp ession clones (ma ked by GFP in g een) can inhibi CycB accumula ion ( ed) close o he an e io pa o
he NPR (a ow in F').
BMC De elopmen al Biology 2007, 7:13 h p://www.biomedcen al.com/1471-213X/7/13
Page 9 o 13
(page numbe no o ci a ion pu poses)
shown ha Hh and Dpp bo h ha e a no el ole in his i s
mi o ic synch onisa ion. This could be media ed by S ing
( he homologue o Cdc25 phospha ase in D osophila)
since i s RNA is up egula ed in hese cells [4]. Ob iously,
no all cells will be poised o en e mi osis immedia ely
upon ecei ing hese signals bu we imagine ha hei
unc ion is o d i e cells h ough mi osis wi h as li le
delay as possible.
Based on se e al lines o published e idence and ou da a
[17-19], we es ima e ha cells in he NPR spend only a li -
le mo e ime in G1 phase han he p oli e a ing an e io
cells o he eye disc (see abo e). Coupled wi h he sha p
onse o G1-a es , his implies ha he NPR is a leas
pa ly a consequence o cells being igge ed o en e G1
synch onously. Howe e , he obse a ion ha he loss o
k causes ec opic S-phase en y in he an e io pa o he
NPR, and he ac ha G1-phase is ex ended, albei no
g ea ly, bo h indica e he exis ence o a mechanism o
p e en ing cells p ecociously en e ing S-phase in he NPR.
In o he wo ds, we conclude ha he NPR is a conse-
Rux is necessa y o main ain G1 a es in he pos e io o he NPRFigu e 5
Rux is necessa y o main ain G1 a es in he pos e io o he NPR. (A-B) ux8 clones (absence o β-galac osidase in
g een) s ained o B dU (A) and CycB (B) in ed. Bo h ec opic B dU inco po a ion and CycB accumula ion lie p edominan ly in
he pos e io pa o he NPR, al hough occasional B dU posi i e cells a e also seen mo e an e io ly. (C-D) PCNA-GFP epo e
exp ession (g een in C and D, and whi e in C' and D') in discs wi h ux8 clones (absence o ed in C) and CycA o e exp ession
clones (ma ked by p esence o β-galac osidase ( ed in D). The PCNA-GFP epo e is sligh ly ac i a ed in he ux clone in he
NPR (a ow in C') and in cells o e exp essing CycA in he NPR (a ow in D'). (F-G) ux-lacZ ansgene exp ession ( ed) in a
WT disc (F) and in a disc ha bou ing smo3 k a12 double mu an clones. The ansgene is exp essed in he NPR, he posi ion o
which is localised by he exp ession o A o (blue in F), and i s le els dec eases jus pos e io o he MF. The le els o ux-lacZ
inc ease in he double mu an cells o smo and k (ma ked by absence o g een in G). The ba indica es he wid h o he NPR.