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Developmental Biology 339 (2010) 78–88

Contents lists available at ScienceDirect

Developmental Biology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / d e v e l o p m e n t a l b i o l o g y

hth maintains the pool of eye progenitors and its downregulation by Dpp and Hh
couples retinal fate acquisition with cell cycle exit
Carla S. Lopes a, Fernando Casares a,b,⁎
a
CABD-Centro Andaluz de Biologia del Desarrollo Univ. Pablo de Olavide-CSIC, Ctra. Utrera Km 1, Sevilla 41013, Spain
b
IBMC, Univ. do Porto, Rua do campo Alegre 823, Porto 4150-180, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: During Drosophila eye development, recruitment of retinal precursors from a pool of progenitor cells is
Received for publication 24 November 2009 tightly coupled to proliferation control. However, how this coupling operates is still unclear. Here we show
Revised 11 December 2009 that the transcription factor hth, together with eyeless, is required to stimulate proliferation of progenitor
Accepted 12 December 2009
cells. Accordingly, knocking down hth expression results in severely reduced eyes. Our experiments reveal
Available online 28 December 2009
three additional functions for hth: the cell cycle of progenitors is characterized by a relatively long G2
Keywords:
phase, which makes them prone to enter mitosis; hth represses the burst of string/cdc25 expression that
Drosophila precedes G1 arrest, and also the early expression of the proneural gene atonal. Thereby, hth maintains the
ey proliferative and undifferentiated state of eye progenitors. Furthermore, we show that the G1
hth/meis synchronization that characterizes retinal precursors is the result of the spatially controlled repression of
Organ size hth by Dpp and Hh, and not of an actively induced cell cycle arrest. We integrate these results in a
Proliferation model of the early steps of eye development that links proliferation control and differential gene expression
Retina specification with patterning signals.
© 2009 Elsevier Inc. All rights reserved.

Introduction embryogenesis by the expression of the Pax6 genes twin of eyeless

The development of an organ requires a tight coordination
between cell proliferation and cell fate specification. An unbalance
in either one of these processes will affect the final size and
morphology of the organ, compromising its function. This coordina-
tion is exerted by signaling centers, groups of cells that target
progenitor cells, by means of intercellular signals, to couple their
timely exit from the cell cycle with the induction of differentiation
genes. In the case of the eyes, both of invertebrates and vertebrates, a
hedgehog (hh)-expressing signaling center spreads through the eye
primordium organizing a differentiation wave. The velocity of
spreading determines the rate at which progenitors are recruited as
atonal (ato)/Ath-5-expressing quiescent retinal progenitors (Dyer
and Cepko, 2001; Wallace, 2008). However, questions such as what
mechanisms maintain the progenitor state, which are the genes
controlled by the signaling centers to induce the transition from
progenitors into precursors, or how proliferation is coupled to this
transition are still not fully understood.
During the development of the Drosophila eye not only the
coordination between proliferation and differentiation is particularly
obvious, but it is also especially amenable to experimental analysis. In
the fruit fly, the eye primordium (or eye disc) is specified during late
⁎ Corresponding author. IBMC, Univ. do Porto, Rua do campo Alegre 823, Porto 4150-
180, Portugal. Fax: +34 954349376.
E-mail address: fcasfer@upo.es (F. Casares).
(toy) and eyeless (ey) (Czerny et al., 1999). Its development extends
through the three larval stages (L1–3) and part of the pupal phase
(Dominguez and Casares, 2005). In L1, the eye progenitors express,
in addition to ey, the TALE-type homeodomain transcription factor
homothorax (hth) (Pichaud and Casares, 2000). Progenitor prolifer-
ation starts during L2, coinciding with the onset of the expression of
teashirt (tsh) (Bessa and Casares, 2005), and then the retinal
determination genes eyes absent (eya) and sine oculis (so) lock in
the eye fate of the primordium cells (Kenyon et al., 2003; Kumar and
Moses, 2000). However, retinal differentiation only starts at the
beginning of L3. The moving differentiation wave sweeps the
primordium in a posterior to anterior direction with a straight
wave front leaving on its wake differentiating retinal cells. This wave
front is characterized morphologically by an indentation of the
epithelium, called the morphogenetic furrow (MF), a landmark that
separates two broad regions within the primordium: anterior to the
MF cells are undifferentiated, while in and posterior to the MF, retinal
differentiation takes place (Baker, 2007; Wolff and Ready, 1991)
Thus, in an L3 eye primordium, cells can be found in progressively
more differentiated states as one moves from anterior to posterior
(Fig. 1D) (Bessa et al., 2002; reviewed by Pappu and Mardon, 2004).
Cells in the most anterior domain are characterized by the co-
expression of ey, toy, hth, tsh and tsh's paralogue tiptop (tio) (Bessa
et al., 2002, 2009; Czerny et al., 1999). These cells, that retain the
gene expression profile that characterizes the early L2 cells, are
undifferentiated progenitors that proliferate asynchronously. As the

0012-1606/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2009.12.020
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C.S. Lopes, F. Casares / Developmental Biology 339 (2010) 78–88
Fig. 1. Defining domains during L3 eye development. (A–C) The eye primordium in L3
eye-antenna imaginal disc is encircled in yellow. In all figures anterior is to the left and
dorsal is up. The position of the MF is indicated by the yellow arrowhead. (A) Ey and Hth
are co-expressed in the most anterior cells of the eye field. Hth is shown in red and Ey in
green. Ey expression is maintained until the MF. White arrow indicates movement of
MF. (B) Hth expression coincides with the domain of undifferentiated cells, while onset
of Atonal expression defines acquisition of proneural fate. ELAV is expressed in
differentiating PRs, posterior to the MF. (C) L3 disc stained for Hth and the mitotic
marker PH3. Mitotic cells are sparse within the Hth-expressing cells but concentrate in
the FMW just after Hth expression is lost. In (B) and (C) FMW and SMW are indicated
by arrows. (D) Diagram of the L3 eye disc during MF progression. Patterns of expression
of the transcription factors identifying the different domains are shown. In (B) and (D)
the early and late phases of ato expression are marked in red and blue, respectively.
MF advances, progenitor cells undergo a few rapid cell cycles in
what has been called the first mitotic wave (FMW), before turning
into retinal precursors, which transiently arrest their cell cycle in
the G1-phase. The FMW is associated to a peak of expression of
cdc25/string (stg), which acts as mitotic trigger (Mozer and
Easwarachandran, 1999; Thomas et al., 1994). After the FMW, the
G1-arrested precursor region, that spans a band of 15–20 cells,
coincides with the downregulation of hth expression, up-regulation
of the retinal determination genes eya, so and dachshund (dac), and
the induction of the proneural basic helix-loop-helix transcription
factor atonal (ato) (Fig. 1D; reviewed by Pappu and Mardon, 2004;
Voas and Rebay, 2004). Up-regulation of ato expression endows
cells with neural competence (Dokucu et al., 1996; Escudero and
Freeman, 2007; Jarman et al., 1994, 1995). Therefore, this domain
of retinal precursors is also known as the “pre-proneural” or “non-
proliferative” domain (Escudero and Freeman, 2007; Greenwood
and Struhl, 1999). Once within the MF, progenitors are incorporat-
ed into the forming ommatidium by successive rounds of
recruitment, either directly or after a last terminal mitosis
(known as second mitotic wave (SMW) (Fig. 1D; reviewed by
Baker, 2001, 2007).
Two signals have been shown to drive the forward movement of
the MF: hedgehog (hh) and the BMP2/4 decapentaplegic (dpp) (Fu
and Baker, 2003; Greenwood and Struhl, 1999). hh is expressed first
at the posterior margin of the primordium and then in differentiating
photoreceptors (PRs). Hh signals short range to anterior cells that
respond by expressing dpp, a longer reaching signaling molecule
79
(Greenwood and Struhl, 1999; Heberlein et al., 1993). Recent work
shows that hh and dpp act redundantly anterior to the MF to promote
and maintain G1 arrest (Escudero and Freeman, 2007; Firth and
Baker, 2005; Horsfield et al., 1998; Penton et al., 1997; Vrailas and
Moses, 2006) and to induce the expression of ato (Greenwood and
Struhl, 1999; Niwa et al., 2004; Tanaka-Matakatsu and Du, 2008;
Zhang et al., 2006). Therefore, the current model emphasizes a
positive role of hh and dpp, as the MF advances, in jointly inducing
cell cycle exit and proneural gene expression after the FMW.
However, this model does not directly address whether the FMW,
which likely serves as a transient amplification phase to provide for
enough number of retinal precursors, is also positively controlled by
dpp, hh or any other signal. Even more importantly, it is currently
unknown whether hh and dpp exert their roles in cell cycle control
and differentiation through different or shared targets and, in any
case, which targets are those.
Here we show that many of these complex events – the
proliferation pace of progenitor cells and their maintenance in an
uncommitted state, their expansion at the FMW and the synchroni-
zation in G1 of the resulting retinal precursors – are the result of the
interplay between the function of hth and its regulation by hh and
dpp. hth stimulates proliferation of progenitors cells, and this
stimulation requires ey function. The progenitor population is
characterized by a cell cycle with an extended G2 phase and is thus
poised to enter mitosis. The transcriptional repression of hth by dpp
and hh relieves hth repression on stg, specifically at the FMW, so that
cells undergo mitosis at once and, as a consequence, enter G1 also
simultaneously. In addition, the repression of hth permits the
induction of ato, which couples the specification of retinal progeni-
tors with their proliferative arrest. The recent discovery that hth
homologues, the Meis genes, also participate in the maintenance of
the progenitor state through the control of the cell cycle during
vertebrate eye development suggests that the interplay between hth/
meis and hh-based signaling during organogenesis is evolutionary
conserved.
Results
hth maintains proliferation of retinal progenitor cells aided by ey
In L3 discs, two subdomains can be identified anterior to the MF
(Figs. 1A and D): a most anterior domain in which cells co-express ey/
toy, hth and tsh/tio (domain I) and a domain in which hth expression
is lost but ey/toy and tsh/tio expressions are maintained (domain II).
These two domains correspond to the undifferentiated proliferative
progenitors and the retinal precursor, respectively (Bessa et al., 2002;
reviewed by Pappu and Mardon, 2004).
Previous studies suggested that hth is required in the progenitor
domain to maintain cells proliferative and undifferentiated (Bessa
et al., 2002; Pai et al., 1998; Pichaud and Casares, 2000). The
observation that clones of hth-mutant cells grow poorly in the
progenitor domain pointed towards a role for hth in cell prolifer-
ation (Fig. 2A; Bessa et al., 2002). However, it was not solved
whether this phenotype was due to a role of hth in cell proliferation
or in cell survival. To clarify this point, we blocked apoptosis in
clones of hth-mutant cells by co-expression of the pan-caspase
inhibitor p35 (Hay et al., 1994). These clones were still seldom
recovered in the anterior domain (Figs. 2B and C), revealing a
primary role for hth in cell proliferation. That hth does not play a
major role in cell survival is further supported by the fact that hth-
mutant cells are recovered if given a growth advantage by using the
Minute technique (Fig. 2D). Also, downregulation of hth levels in the
eye primordium by means of an RNAi construct (eyN(RNAi)hth)
leads to a significant reduction in size of the adult eye, which further
reinforces the requirement of hth for proliferation of progenitor cells
(Figs. 2E and F).
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80 C.S. Lopes, F. Casares / Developmental Biology 339 (2010) 78–88
In agreement with hth's role in progenitor cells proliferation,
ectopic expression of hth induces overgrowths (Bessa et al., 2002).
However, these overgrowths behave differently according to their
location within the eye disc. Clones in the margin are much larger
than clones in the middle of the disc. The observation that the margin
is a domain of ey expression, and that clones of ectopic expression of
hth, in the middle of the disc, only partially maintain Ey expression
(Bessa et al., 2002) led us to ask to what extent proliferation induced
by hth depends on ey function. To do so, we compared the size of
clones overexpressing hth in the presence of normal ey levels or when
ey expression had been knocked down by means of an RNAi (Figs. 2G
and H; see Materials and methods). Only clones anterior to the MF
were examined, as this is the normal domain of ey expression. hth-
expressing clones grew in the whole anterior domain, including the
non-proliferating domain II (Fig. 2G; Bessa et al., 2002). However, the
growth of these clones was severely reduced when ey levels were also
reduced (Fig. 2H). The average number of cells per clone is
significantly reduced from 36 to 18 upon ey downregulation
(n = 24). Likewise, a reduction in the number of clones recovered
anterior to the MF is also observed, from 7 to 3 (n = 14). These results
indicate that, in the eye primordium, hth requires ey to maintain
progenitors' proliferation.
Downregulation of hth is associated with transition from progenitor into
retinal precursor cell state
In addition to its contribution to cell proliferation, previous studies
suggested that hth is required in the progenitor domain to maintain
the undifferentiated cell state (Bessa et al., 2002; Pai et al., 1998;
Pichaud and Casares, 2000). Moreover, the transition from a
proliferating progenitor into a quiescent retinal precursor cell state
is associated with the loss of hth, which led us to ask what was the
contribution of hth regulation to this transition. To address this
question, first we precisely mapped the pattern of expression of hth
relative to proliferation markers and read-outs of the dpp and hh
signaling pathways. As proliferation markers we used the G2/M
cyclin, cyclin-B (Knoblich and Lehner, 1993) and the mitotic marker
phosphorylated histone-H3 (PH3) (reviewed by Hans and Dimitrov,
2001). We monitored the range of the dpp signal by detecting the
active phosphorylated form of Mad (P-Mad) (reviewed by Affolter
and Basler, 2007), and that of the hh signal through expression of its
target ato (Borod and Heberlein, 1998; Dominguez and Hafen, 1997)
(Figs. 3A–C).
Moving from anterior to posterior within the eye primordium, the
hth-expressing progenitor cells show uniformly high levels of cyclin B
and sparse mitoses, which together suggest that a large fraction of
these cells is in the G2-phase of the cell cycle (Figs. 3A and C). In order
to obtain a quantitative estimate of the steady-state distribution of
progenitors in the different cell cycle phases, we analyzed the DNA-
content profile of hth-expressing progenitors using FACS (Fig. 3D). To
do so, we compared the average profile of undifferentiated cells
(tioNGFP; Fig. S1A; see also Materials and methods) – which includes
proliferating progenitors, rapidly dividing FMW cells and quiescent
precursors – with hth-expressing cells (tioNhthGFP; Fig. S1C). Forced
Fig. 2. Homothorax is required for proliferation of eye progenitors. (A and A′) Clones of
hthB2, a hypomorphic allele, grow poorly in the proliferative domain of the eye imaginal
disc. Mutant tissue is identified by the absence of GFP. Note the small size of anterior
located clones when compared to the twinspots (bright green). Disc is stained for
cyclin B and PH3. (B and C) Expression of the caspase inhibitor p35 is not sufficient to
recover hth-mutant cells anterior to the MF. (B) L3 imaginal disc with neutral clones
(green), induced in parallel to experiment shown in C. (D and D′) hthP2M+ clones
grow in the anterior domain, but the pattern of accumulation of mitotic cyclin B is
sparser than in wild-type tissue. (E and F) Lateral views of control (E: eyNGFP) and
hth-knocked-down (F: eyNdshth) adult fly heads. Downregulation of hth expression
in the developing eye disc causes a dramatic adult eye size reduction. (G and H) ey is
required for the overproliferation phenotype induced by maintenance of hth
expression. (G) Eye imaginal disc with hth-GFP-expressing clones (marked by GFP)
spanning the non-proliferative domain. hth-expressing cells overproliferate and
maintain cyclin B (blue) and Ey (red) expression. (H) Downregulation of ey expression
partially suppresses the overproliferation phenotype of hth expressing cells. The
average number of cells per clone is significantly reduced from 36 to 18 upon ey
downregulation, in hth expressing cells (n = 24).
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C.S. Lopes, F. Casares / Developmental Biology 339 (2010) 78–88
Fig. 3. Transition into retinal precursor state is associated with hth downregulation and
cell cycle exit. (A–C) High magnification views of L3 discs focused around the G1-
arrested precursor domain, stained for Hth, cell cycle markers and read-outs of the Dpp
and Hh signaling pathways. Dashed lines represent the FMW and SMW. (A) Cyclin B is
expressed homogeneously in anterior cells. Its expression is maintained in 4–6 cells after
Hth expression is turned off. Ato and cyclin B define mutually exclusive domains. (B) The
FMW is a domain of transient amplification of multipotent progenitors. Mitotic cells
(PH3-positive) and cells undergoing S-phase (BrdU-positive) concentrate posterior to
Hth domain. (C) Phospho-Mad, a read-out of the Dpp pathway abuts hth-expressing
cells. (D) hth-expressing cells are characterized by an extended G2 cell cycle phase. tio-
Gal4 was used to drive expression of either GFP or hth-GFP in all cells anterior to the MF.
Cell cycle profiles of GFP cells from tioNGFP (black dashed curve) and tioNhth-GFP (green
curve) L3 discs are shown. The percentage of GFP cells at the different stages of the cell
cycle is indicated. The hth-GFP population shows a significant increase in the percentage
of cells in G2/M phase.
expression of hth-GFP dramatically delayed MF movement and retinal
differentiation and L3 eye discs were characterized by homogeneous
expression of cyclin B and low levels of the RD gene eya (Fig. S1D and
data not shown). According to these markers, the cell cycle profile of
tioNGFP-hth corresponds to that of the progenitor cell population
(domain I). We observed that close to 50% of the hth-expressing cells
from tioNhthGFP L3 eye discs are found in the G2/M phase of the cell
cycle, a proportion significantly larger than that of the whole
undifferentiated cell population. Together with our observations
that the domain of hth expression was not particularly enriched in
mitotic cells (Fig. 3B) and that expression of cyclin B was almost
uniform, these results indicate that the cell cycle phasing of
progenitor (i.e., hth-expressing) cells is characterized by a relative
long G2 phase.
After hth expression has been turned off, a stripe of about 4–6 cells
maintains cyclin B expression (Fig. 3A). This hth-negative, cyclin B-
positive stripe is rich in mitoses (Fig. 3B) and corresponds to the FMW.
The pattern of BrdU incorporation in L3 eye discs (Fig. 3B) shows that
cells at the FMW undergo about two rounds of cell division. After the
synchronic cycles at the FMW, cells enter the G1 phase, characterized
by the lack of BrdU incorporation, cyclin B expression and mitoses. It is
after the FMW, in the G1 domain, where the hh target ato becomes
expressed. In addition, the posterior limit of hth abuts the P-Mad
expressing domain (Fig. 3C), indicating that the domains of dpp
signaling and hth expression are mutually exclusive.
hth expression prevents cells cycle exit and normal ato expression
In order to investigate the role of hth function and regulation in the
coordinated transition into retinal precursor state, we analyzed the
81
effects of hth-expressing clones spanning the FMW and the G1-arrested
domain (Fig. 4). Ectopic expression of hth in this domain was sufficient
to delay exit from the cycle, as we detect mitotic (PH3-positive) cells
and maintenance of cyclin B within the clones (Fig. 4A). Moreover,
these hth-expressing cells do not arrest in G1, as they incorporate the S-
phase marker BrdU (Fig. 4B). These results show that hth expression is
sufficient to maintain cell proliferation anterior to the MF.
hth-expressing clones usually contain a few differentiated PRs,
showing that maintenance of hth delays, but does not completely
block PR differentiation (Fig. 4D; Pichaud and Casares, 2000; data not
shown). In order to address how hth maintenance was affecting
retinal differentiation, we checked the expression of the proneural
gene ato, the earliest indicator of the acquisition of neural compe-
tence. During normal eye development, ato expression is initially
expressed uniformly in G1 arrested cells. Within, and immediately
posterior to the MF, ato expression is progressively restricted to the
regularly spaced R8 PRs (Fig. 1B) (Dokucu et al., 1996; Jarman et al.,
1994; Jarman et al., 1995). Our analysis shows that when hth
expression is maintained, the first phase of uniform ato expression
is severely reduced or absent. However, the ato second phase still
occurs (Fig. 4C). In addition, hth-expressing cells differentiated as PRs
and expressed hh, as monitored by the hh-Z reporter (Fig. 4D). All
together, the results presented so far indicate that hth repression is
necessary to simultaneously drive cells out of the cell cycle and to
allow the normal onset of ato expression.
hth represses stg to define the location of the FMW
At the FMW, cells undergo a few rounds of division prior to cell
cycle exit and acquisition of precursor fate. This domain coincides with
a band of strong expression of the G2/M inducer, the phosphatase
encoded by stg (Fig. 5A; Thomas et al., 1994). In agreement with our
previous mapping of hth relative to the FMW, the posterior limit of the
hth expression abuts the band of stg expression (Fig. 5B). Considering
that hth repression is required to allow cell cycle exit of eye
progenitors, we reasoned that the anterior limit of the stg expression
could also be set by hth repression. To test it, we analyzed stg mRNA
expression in clones of gain of function of hth. In these clones, stg
mRNA was autonomously repressed (Fig. 5C). On the contrary, when
hth function was removed in large Minute + clones (hthP2M+), stg
expression spread more anteriorly (Fig. 5D).
L3 eye discs show a burst of stg at the FMW. The finding the Hth
can act as a repressor of stg transcription specifically at the FMW
provides an explanation for the synchronic cell cycle exit observed
after the FMW. Upon hth downregulation, the burst of stg expression
may drive all progenitors in G2 phase into mitosis, followed by cell
cycle exit. Since about 50% of progenitors are in G2 at the time hth is
repressed and stg peaks, the cell cycle synchrony observed at the
FMW may be the result of the partial pre-synchronization of
progenitors in G2, a “pre-mitotic” state (as dubbed by Escudero
and Freeman, 2007). It follows from this model that the repression of
hth has to be tightly coordinated to ensure the mitotic synchronicity
at the FMW. To test this point, we drove a hth-RNAi transgene in the
eye primordium using the tio-GAL4 driver. To maximize the
efficiency of hth's knock-down, we halved the dose of hth in the
genotype (tio-GaL4/UAS-scrGFP; UAS-dshthP2; Figs. 5F and G). Using
this strategy, hth-RNAi-expressing cells showed decreased Hth signal
by mid L3 and, by late L3, Hth was no longer detected (Fig. 5F′ and
not shown). Indeed, in tio-GAL4/UAS-srcGFP; UAS-dshth, +/+,
hthP2discs, the band of dense mitoses that characterizes the FMW
is lost (Figs. 5F and G), as predicted by the model.
hth represses proneural fate independently of its role in proliferation
Maintenance of hth expression sustains proliferation of eye
progenitors, while delaying differentiation onset. One possibility is
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82 C.S. Lopes, F. Casares / Developmental Biology 339 (2010) 78–88

Fig. 4. hth maintains proliferation and delays differentiation of multipotent progenitors. (A–D) Ectopic expression of hth in clones that straddle the G1-arrested precursor cell
domain. (A) Clones of hth-expressing cells maintain cyclin B expression and undergo mitosis, as detected by PH3. (B) hth-expressing cells incorporate BrdU, indicating a bypass of
the developmentally induced G1 arrest. (C) Maintenance of hth expression results in loss of the first phase of ato expression (red line), which marks the onset of the precursor state.
ato expression only occurs in cells that have exited the cell cycle (blue line). (D) PR differentiation, as monitored by expression of hh-Z, is severely delayed in hth-expressing cells.
Arrows point to the approximate positions of FMW and SMW.

that these two processes are directly coupled, and the delayed
acquisition of neural competence is the outcome of anterior cells
being unable to exit cell cycle due to hth expression. Alternatively,
hth could be repressing cell cycle exit and the acquisition of
proneural fate independently. In order to test this point, we co-
expressed stg and hth in clones and examined their effect in the
domain of G1-arrest. If the sole function of hth were exerted
through its control over the cell cycle, then stg expression should
drive hth-expressing cells into mitosis, allowing for their subsequent
G1 arrest, and this should be sufficient to restore normal atonal
expression (Fig. 5E). stg+hth-expressing clones progressed into G1,
as indicated by sparse cyclin B staining, showing that these cells
 Author's personal copy
C.S. Lopes, F. Casares / Developmental Biology 339 (2010) 78–88
overcome the G2 delay imposed by hth. However, the initial phase
of ato expression was still repressed, which shows that hth
repression on the early phase of ato expression is independent of
its role in proliferation.
83
Dpp and Hh promote G1 arrest through downregulation of hth
Our findings that hth controls cell cycle and cell fate offer an
explanation for how these two events are coordinated at the
transition into the precursor cell state. Accordingly, this change in
fate can be looked at as a forward cell state transition that essentially
requires hth downregulation. Yet current models on eye development
imply that this transition is driven by the positive input of Dpp and Hh
signal from the MF (Escudero and Freeman, 2007; Firth and Baker,
2005; Greenwood and Struhl, 1999; Vrailas and Moses, 2006).
Therefore we addressed to what extent these two pathways
contribute to hth regulation.
We observed that maintenance of hth expression within the
domain of G1 arrest results in a phenotype reminiscent to that
described for clones of cells mutant for smoothened (smo) or thick-
veins (tkv), the receptors of the Hh and Dpp signaling pathways,
respectively. Upon mutation of either signaling pathway, proliferation
is transiently maintained within the domain of G1 arrest (Escudero
and Freeman, 2007; Firth and Baker, 2005; Vrailas and Moses, 2006),
MF progression is delayed and there is a failure to up-regulate the first
phase of ato expression (Dominguez, 1999; Dominguez and Hafen,
1997; Greenwood and Struhl, 1999). Therefore, we reasoned that Dpp
and Hh signaling might function through repressing hth in all cells
anterior to the MF. An indication that this could be the case came from
the observation that in mad- clones, where the transduction of the
Dpp signal is blocked, hth is weakly derepressed (Bessa et al., 2002).
We confirmed this in clones of cells mutant for the Dpp receptor tkv.
In tkv− clones, Hth and cyclin B expression was maintained in the G1-
arrested domain, although only partially: hth as well as cyclin B were
still off in the most posterior cells of the clones (Fig. 6A). To determine
the contribution of hth to the delayed MF progression in tkv− cells, we
compared the degree of G1 arrest between tkv− cells and tkv− cells
where hth expression had been knocked-down by means of RNAi (Fig.
6B). In tkv−, RNAi(hth) cells the G1-arrest was restored, as observed
by loss of cyclin B expression as well as normal MF progression. In
addition, the first phase of ato expression is restored (Fig. 6C). These
findings indicate that, during MF progression, hth downregulation by
dpp is sufficient for cell cycle exit and acquisition of neural fate.
Nevertheless, other signals that contribute to hth downregulation
must exist, since in tkv− clones hth derepression is always limited to
the most anterior region within the clone (Fig. 6A). The obvious
candidate is Hh, a shorter-range signal described to act redundantly
with Dpp to promote both cell cycle exit and retinal differentiation
(Escudero and Freeman, 2007; Firth and Baker, 2005; Fu and Baker,
2003; Vrailas and Moses, 2006). Accordingly, if Hh signaling also
Fig. 5. hth can act as a transcriptional repressor of stg and ato. (A) stg mRNA is
expressed in wild-type discs in a sharp stripe. (B) L3 eye imaginal disc stained for Hth
and stg mRNA. hth and stg are expressed in mutually exclusive domains. (C) Ectopic
expression of hth (green) represses stg expression (red). The inset shows the
expression of stg mRNA in the clone. (D) Disc containing a large hth-mutant clone
(hthP2M+). Mutant cells are detected by the lack of beta-galactosidase (orange). The
white line outlines the clone. Within the hth− clone, the stg stripe is wider. Arrows
indicate the width of the stg stripe outside and inside the clone. (E and E′) hth
repression of proneural fate is independent of hth's role in proliferation. Co-expression
of stg in hth-expressing cells (actNNhthGFP+ stg; green) is sufficient to drive cells into
G1, detected by the sparser cycB expression (E) but early phase of atonal expression is
still repressed (E′). In the inset in (E), the expression of cyclin B in the clone is shown
separately. The arrowheads in (A) and (B) mark the approximate position of the MF. (F
and G) Close up view around the FMW region. (F) Eye disc of a tio-GAL4/UAS-srcGFP;
UAS-dshth/hthP2. The domain of tio-GAL4 expression is marked by GFP (and outlined in
F′). Expression of hth and Elav (in the anterior and posterior parts of the disc,
respectively) is shown in the red channel. In the tio-domain, hth signal is dramatically
reduced, except for a patch of wild-type (non-GFP) cells (arrowhead) (F′), and the band
of dense PH3-positive (mitotic) cells that characterizes the FMW is absent (F″, compare
with wild-type in G). Interestingly, a cluster of mitoses, which seems to be a stretch of
the FMW, can be seen in the wild-type patch (arrowhead in F″) after the normal
downregulation of hth (arrow in F″). The yellow band marks the presumptive location
of the FMW.
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C.S. Lopes, F. Casares / Developmental Biology 339 (2010) 78–88 85

played a role in hth downregulation we would expect maintenance
of hth expression in all cells when the dpp and hh pathways are
simultaneously removed. This is the case (Fig. 6D): in smo− tkv−
clones that straddle the precursor domain, the expression of cyclin
B and hth is maintained now in all clone cells. Next, we checked the
effects of downregulating hth in those same smo− tkv− clones. In
smo− tkv−, RNAi(hth) cells the synchronous exit of the cell cycle
was restored, as indicated by the synchronous cyclin B loss (Fig. 6E),
despite the fact that these cells were unable to respond to Dpp and
Hh. Importantly, although smo− tkv−, RNAi(hth) cells undergo
timed cell cycle exit, they fail to upregulate expression of ato (Fig.
6F). These experiments point towards a role for Hh signaling, in
addition to Dpp, as a transcriptional regulator of hth, and show that
cell cycle exit and onset of retinal differentiation induced by both
pathways is essentially achieved through hth repression.
ey in progenitors to stimulate their proliferation. Hth and Ey have
been proposed to be engaged in a protein complex which included
also Tsh (Bessa et al., 2002). More recently, Peng and co-workers
(2009) have shown that Hth and Tsh directly interact with Yorkie
(Yki), the transcriptional coactivator downstream of the Hippo
pathway, to regulate progenitor cells behavior. Our results point to
the possibility that Ey might also be part of the Hth-Tsh-Yki protein
complex. In addition, Hth is known to be produced as two protein
isoforms: a homeodomain-containing form and a homeodomain-less
one (Noro et al., 2006). Since cells that only express the home-
odomain-less isoform (hth100-1) behave as cells carrying the null
allele (Fig. S2), hth's function in progenitors requires the home-
odomain, which is consistent with a direct regulation of target genes
by Hth as part of this complex, as has been reported for the bantam
locus (Peng et al., 2009).

Discussion
Early development of many organs progresses through a series of
consecutive steps: the proliferation of the organ progenitors, their
expansion at a transient amplification phase and the timed exit from
the cell cycle as cells activate their organ-specific differentiation
program (Edlund and Jessell, 1999). Our work shows that during the
development of the Drosophila eye, hth plays instrumental roles in
the coordination of all these events. hth is required for proliferation
of progenitor cells, together with the pax6 gene ey, and contributes
to their synchronization in a “pre-mitotic” state, immediately
anterior to the FMW. By acting as a transcriptional repressor of
stg, specifically at the FMW, hth delays cell cycle exit of progenitors,
while through transcriptional repression of ato, maintains the
progenitor fate. The regulation of these events by hth provides an
explanation for both the synchronic cell cycle exit and acquisition of
precursor fate fundamental for proper cell fate specification in the
developing Drosophila eye.
hth requirement in proliferating retinal progenitors
The undifferentiated territory of the eye primordium is charac-
terized by the expression of ey/toy, tsh/tio and hth. However, the
proliferation within this territory is not uniform, and the change of
its pace – from asynchronous proliferation, through an amplifying
first mitotic wave, to G1 quiescence – correlates with changes in hth
expression. Our results, as well as those of others, indicate that hth
expression is not required for progenitor proliferation per se, but
rather required to stimulate their proliferation (Bessa et al., 2002;
Peng et al., 2009). Although hth− clones grow poorly in the
undifferentiated region of the eye primordium, they can proliferate
significantly if given a growth advantage (this work and Peng et al.,
2009). This suggests that otherwise, hth− cells are outcompeted by
surrounding wild-type cells. This may underlie the suggestion that
hth plays a role in cell survival (Peng et al., 2009). However, our
experiments indicate that apoptosis has a minor impact on the hth
growth phenotype (this work and Peng et al., 2009). The cause of
the proliferative disadvantage of hth-mutant cells remains
unknown.
Despite the fact that hth is not absolutely required for the
proliferation of progenitors, the eyes of eyNhth(RNAi) flies are
dramatically reduced in size, indicating that hth expression is
essential to attain a normal-sized retina. In any case, hth requires
Synchronic cell cycle exit and the role of Dpp and Hh in the
establishment of G1 arrest
Before entering the non-proliferation zone, progenitor cells go
through a transit amplification at the FMW. In the eye disc stg is
transcribed at low and uniform levels throughout, except for the
strong stripe at the FMW. The regulatory mutation stgHwy, which lacks
specifically this expression, also loses the FMW, and consequently
stgHwy adults have reduced eyes (Mozer and Easwarachandran, 1999).
However, the sole expression of stg is not sufficient to explain the
synchronous mitoses that characterize the FMW, because only cells in
the G2 phase of the cell cycle can be driven into mitosis by stg. Our
results show that a hallmark of progenitors is a cell cycle with a
relatively long G2 phase, and that hth maintains cells in this pre-
mitotic state. At the same time, hth represses the eye disc-specific
expression of stg. Therefore, our results suggest that the FMW is the
result, first, of the progenitor-specific cell cycle phasing provided by
hth, and second, of the spatially regulated repression of hth that
releases stg transcription. It is therefore likely that the establishment
of the G1 that characterizes the acquisition of the retinal precursor
state is the consequence of the previous mitotic synchronization at
the FMW.
dpp and hh are known to be required for the G1 arrest after the
FMW (Escudero and Freeman, 2007; Firth and Baker, 2005; Vrailas
and Moses, 2006). However, it was not clear to what extent the
arrest was the result of a positive action of these signaling pathways
on cell cycle regulation or just the consequence of a presynchroniza-
tion, which we favor. Our results show that dpp and hh achieve the
G1 arrest just by repressing hth, taking advantage of the presyn-
chronization of progenitor cells. However, it is likely that, during
normal development, the action of these two signaling pathways is
staggered. Close inspection of the signaling ranges of dpp and hh
indicates that dpp reaches farther anterior than hh. The domain of
activated-Mad extends over the FMW and abuts hth, while that of
the hh target ato reaches cells as they abandon the FMW (Fig. 7A).
Therefore, the primary repressor of hth is dpp (this work; Bessa et al.,
2002; Firth and Baker, 2008). However, in the absence of dpp signal,
the expression of hth and the proliferative state is maintained only
until hh signaling acts. Our results reveal that Hh signaling is
responsible for hth downregulation at the transcriptional level, a
function that had been overlooked in previous studies (Bessa et al.,
2002; Firth and Baker, 2008). Interestingly, in the chick retina,
downregulation of meis2, a hth homologue, is also mediated by Shh

Fig. 6. Dpp and Hh promote the G1 arrest through repression of hth. MARCM-induced clones of tkv−, smo− or tkv−, smo−tkv− double mutant cells analyzed in the precursor cell
domain. Mutant cells are positively marked with GFP (green). (A and A″) Cells mutant for tkv upregulate hth and maintain cyclin B expression in the anterior region of the clone. (B
and B″) Downregulation of hth, through the use of a hth RNAi (dshth) transgene, is sufficient to restore cell cycle exit defect and MF progression of tkv− cells. (C and C″)
Downregulation of hth, in tkv− cells is sufficient to restore ato expression in the precursor domain. (D and D″) Cells double mutant for tkv−,smo− upregulate Hth expression and
maintain high levels of cyclin B in a cell autonomous manner. (E and E″) When dshth is expressed in tkv−,smo− cells, cyclin B is downregulated and cell cycle arrest in the precursor
cell domain is restored. (F and F″) Downregulation of hth by means of a dshth transgene is not sufficient to restore ato expression in tkv−,smo− mutant cells. Discs from experiments
shown in (A) to (C) and (D) to (F) are derived from the same genetic cross. Clones were induced at the same developmental time and discs processed in parallel.
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86 C.S. Lopes, F. Casares / Developmental Biology 339 (2010) 78–88
Fig. 7. Model of coordinated transition from progenitor into precursor cell state during
eye development. (A) Diagram of a transversal view of the eye disc summarizing the
domains of expression of the different players involved in cell state transitions during
L3 eye development, as well as read-outs of the signaling pathways and their range of
action, analyzed in this study. (B) Schematic model of events and interactions taking
place during MF progression, in the L3 eye imaginal disc. hth expression in the most
anterior domain of the eye field contributes to the maintenance of the progenitor cell
state. It acts as a transcriptional repressor of stg and ato, thus delaying cell cycle exit and
neurogenesis onset. Ey is required for hth-dependent proliferation. It is likely that Tsh
and Yki (Peng et al., 2009), also participate in this process as a multimeric complex.
After the trigger of the MF, Dpp and Hh act redundantly to drive MF progression, but
they do so through different mechanisms. Dpp promotes transcriptional down-
regulation of hth, which leads to transcriptional upregulation of stg. As a result,
progenitors are amplified at the FMW and enter G1 in a coordinated way. Hh directly or
indirectly (Greenwood and Struhl, 1999) reinforces hth repression, and also acts
positively on ato expression. Eya/So act positively on stg and ato transcription, a
function that at least in the case of ato, requires Ey (Zhang et al., 2006). The
combination of the different signals within the G1-arrest domain ensures repression of
hth and contributes for the coordination of the cell cycle with the retinal differentiation
program, and ultimately leads to the growth of the eye up to its specific size.
(Heine et al., 2009), pointing to an evolutionarily conserved
regulatory relationship between hh signaling and hth/meis genes
during retinal development. Greenwood and Struhl (1999) suggested
that control of MF progression by hh involves, in addition to the
induction of Dpp, a short-range signal transduced via Raf kinase. It
was recently shown (Firth and Baker, 2008) that shn−mago− mutant
cells, where both the dpp and ras pathways are blocked, derepress
hth very much like the smo−tkv− clones we have reported. These
findings support a model in which Ras acts downstream of hh to
repress hth. Whether other factors are involved and the mechanisms
through which Ras works as a negative regulator of hth are still
unclear. In addition, our results show that hh not only contributes to
the transcriptional repression of hth, but can block its effects even
when hth expression is forcibly maintained.
hth and the coupling of cell cycle exit with acquisition of neural fate
The upregulation of stg and onset of ato expression occur as hth
expression is turned-off. Work from several groups has found that stg
and ato are positively regulated by RD genes (Jemc and Rebay, 2007;
Tanaka-Matakatsu and Du, 2008; Zhang et al., 2006). Therefore, the
tightly controlled expression of both genes is under both negative (by
hth) and positive (by Hh and Dpp) controls. Accordingly, if stg
upregulation were solely dependent on hth repression, one would
expect a loss of the band of stg expression pattern upon loss of hth. In
contrast, we observed stg in a wider domain of cells, suggesting that
although hth can act as a repressor, the burst of stg expression, and
consequently the synchronic cell cycle exit at FMW, requires an
activator. eya and so have been proposed as positive regulators of stg
expression (Jemc and Rebay, 2007). Therefore, the precise positioning
of the stg stripe is the result of a positive input from eya and so and of
a negative one from hth. Since clones of gain of function of hth in the
G1 domain do not repress eya (and presumably so neither), the
regulation of stg by hth must occur parallel or downstream of eya/so.
None of the previously described stg enhancers (Lehman et al., 1999)
reproduces the expression pattern of stg in the eye disc (our
unpublished data). Also, although it was proposed that so and eya
can induce stg expression, reporter constructs containing the putative
So/Eya binding sites also fail to reproduce stg expression in the eye
(Jemc and Rebay, 2007, our unpublished data). Thus, stg expression at
the FMW depends on a still unidentified eye-specific enhancer.
By acting as a repressor of the initial phase of ato expression hth
delays acquisition of neural competence. This repression is cell cycle
independent, i.e., not linked to hth function in cell cycle control. In the
eye, the dynamic expression of ato is under the control of two
separate enhancers (Sun et al., 1998). Activation of ato expression in
precursors and precluster cells relies on a 3′enhancer, that responds to
Ey, So and Hh signaling (Sun et al., 1998; Tanaka-Matakatsu and Du,
2008 and Supplementary Fig. S3; Zhang et al., 2006). Maintenance of
hth blocks the expression of atonal in progenitor domain and
therefore its effect its likely to occur through this 3′ato enhancer.
Whether this repression is direct or indirect is still unknown.
We identify a requirement for ey in hth induced proliferation which
fits the previously proposed model where a complex of Hth/Ey/Tsh is
predicted to participate in the maintenance of the progenitor fate. We
show that this depends on hth and is accomplished via two parallel
pathways: maintenance of proliferative capacity and repression of neural
fate. The finding that ey also participates in ato induction, together with
so (and presumably eya), suggests that cell state transitions, in the
developing eye, might involve the formation of distinct Ey-dependent
complexes. Therefore, hth downregulation ensures forward cell state
transitions. All the regulatory steps described and discussed in the paper
are integrated in the model depicted in Fig. 7B.
We have shown that hth is critical to maintain the balance
between proliferation and cell cycle arrest of eye progenitors. This
function seems to be conserved during the vertebrate eye (Bessa et al.,
2008; Heine et al., 2008) and perhaps this functional conservation
extends to other tissues. For example, Meis1, a vertebrate hth
homologue, is expressed in the most primitive hematopoietic stem
cell subpopulations and in resemblance to hth, its expression is
downregulated upon differentiation (Pineault et al., 2002). Although
it was shown that Meis1 plays an essential role in regulating
progression of MLL-immortalized cells to leukemia stem cells through
regulation of their self-renewal capacity and differentiation arrest
(Wong et al., 2007), its role in normal hematopoiesis remains unclear.
Our findings in the Drosophila eye may provide cues to understand the
tissue specific normal and oncogenic roles of hth/Meis genes, by
identifying targets and mechanisms underlying the function of this
class of transcription factors.
In the Drosophila eye, hth expression is regulated by Wg signalling
(Pichaud and Casares, 2000). Wg has been shown to be expressed in
the prospective head capsule and required to limit retinal specifica-
tion (Baonza and Freeman, 2002; Treisman and Rubin, 1995). wg
homologues, in other insects, have also been shown to be expressed in
an equivalent pattern and to have a repressive role on retina
differentiation (Dong and Friedrich, 2005). In vertebrates, just like
in Drosophila, canonical Wnt signalling was shown to play a role in the
specification of peripheral eye tissues (Cho and Cepko, 2006). And in
the retina, it was recently shown that Wnt signalling (Wnt-2b)
couples maintenance of proliferation of retinal progenitors with the
suppression of neural fates (Agathocleous et al., 2009). Whether the
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C.S. Lopes, F. Casares / Developmental Biology 339 (2010) 78–88
action of Wg/Wnt in organisms other than Drosophila is mediated by
hth/meis genes therefore deserves further attention.
Materials and methods
Genotypes and genetic manipulations
The following hth alleles were used: w; FRT82BhthP2/TM6B, a
strong hypomorph (Kurant et al., 1998); w; FRT82B hthB2/TM6B, a
hypomorph (Rieckhof et al., 1997); and w; FRT82B hth100-1/TM6B, a
truncated form lacking homeodomain (Kurant et al., 2001). Clones of
the hthP2 allele were recovered using the Minute technique (Morata
and Ripoll, 1975). The fly strain yw,hs-flp; FRT82BhsCD2, y+M/TM2
was used. Mutant tissue was identified by the absence of CD2 staining.
hth-mutant clones were generated through mitotic recombination
(Xu and Rubin, 1993) in yw,hs-flp; FRT82B hth⁎/FRT82BubiGFP larvae
(with hth⁎ being either hthB2 or hth100-1) clones. Clones were induced
between 24 and 48 h or 48 and 72 h after egg laying (AEL) by a 45′
heat-shock at 37 °C. The Flip-Out method (Struhl and Basler, 1993)
was used to induce gain of function clones. yw,hs-flp, actNhsCD2NGal4
females (Basler and Struhl, 1994) were crossed with UAS-hthGFP
males (Casares and Mann, 2000), or UAS-hthGFP;UAS-stg. Clones were
induced at 36–60 h AEL by a 15′ heat-shock at 35.5 °C. The reporter
line hhP30 (hhZ; FlyBase) was introduced in the genotype using
standard genetic techniques. Knock-down of ey expression was
achieved with the UAS-(RNAi)ey line (UAS-eyIR, VDRC l106628). w;
UAS- eyIR/CyO;UAS-hthGFP males were crossed to yw,hs-flp,
actNhsCD2NGal4 females. Clones were induced between 36 and 60 h
AEL by a 15′ heat-shock at 35.5 °C. The efficiency of Ey downregulation
in the clones was estimated as the reduction of anti-Ey staining
intensity within the clones compared to the surrounding wild-type
tissue. Signal in the antennal disc and posterior to the furrow was
used to estimate background signal. Measurements were carried out
using ImageJ software on confocal sections Clonal expression of the
(RNAi)ey transgene resulted in a 50% reduction of anti-Ey staining.
ImajeJ software was used to count number of cells per clone. The
MARCM technique (Lee and Luo, 2001) was used to express a UAS-
(RNAi)hth line (UAS-hthIR; Dietzl et al., 2007), in tkv− and smo−tkv−
double mutant cells. The alleles yw; tkva12 FRT40A/CyO and w;tkva12,
smo3 FRT40A/CyO were used (FlyBase). yw, hsFLP,tub-Gal4,UAS-GFP;
FRT40A,tub-Gal80,CyO females were crossed to tkva12 FRT40A/+;
UAS-hthIR/TM6B, Tb or tkva12, smo3 FRT40A/+; UAS-hthIR/TM6B, Tb
males. Larvae were heat-shocked between 36 and 60 h AEL for 45′ at
37 °C. Tb larvae were dissected as controls. Mutant tissue was
positively marked with GFP. To rescue cell death in hth mutant cells,
ywhsFlp122,tub-Gal4,UAS-GFP;;FRT82B,tub-Gal80,CD2y+ females were
crossed to UAS-p35/Cyo,actGFP; FRThthP2/TM6B, Tb males. Discs from
UAS-p35; FRThthP2 and control (Cyo,actGFP; Tb) larvae were dissected
and processed in parallel. Mutant tissue was positively marked with
GFP. In order to knock-down hth in the tio domain efficiently, we
crossed tio-Gal4/tio-Gal4;hthP2/TM6B males to UAS-ScrGFP;UAS-
hthIR/SM6^TM6B females. Experimental and control larvae were
selected as GFP-positive non-Tb and GFP-negative, Tb.
Flow cytometry analysis
The fly strain tio-GAL4 (A4-Gal4 in Tang and Sun, 2002) was used
to drive expression of UAS-transgenes in all cells anterior to the MF.
This Gal4 line reproduces the pattern of expression of tio, a tsh
paralogue, which shares with tsh an identical expression pattern
(Bessa et al., 2009). tio-GAL4 females were crossed to males carrying
two copies of UAS-GFP (tioNGFP) or males w; UAS-GFP; UAS-hthGFP
(tioNGFP-hth) (Casares and Mann, 2000). Cultures were grown at
25 °C for 24 h and larvae transferred to 29 °C for the rest of
development. Dissociation of eye-antennal disc cells was carried out
as described by Neufeld et al. (1998). Dissociated cells were fixed in
87
3.7% formaldehyde in PBS for 20 min, transferred to 70% EtOH/30%
PBS, and kept at 4 °C. Prior to FACS analysis, cells were washed in PBS
and incubated for 1 h at 37 °C in 10 μg/mL RNase-A and 100 μg/mL of
PI for DNA staining. Samples were analyzed in a BD FACSCalibur with
CellQuest software. FlowJo (Tree Star, Inc) was used for data analysis.
At least five independent experiments per genotype were performed.
Percentages of G1, S or G2/M always refer to GFP positive cells. 10000
GFP–positive cells were analyzed in each experiment per genotype.
Immunohistochemistry
Eye imaginal discs were dissected and fixed according to standard
protocols. Primary antibodies used were guinea-pig anti-Hth (Casares
and Mann, 1998), rabbit anti-PH3 (Sigma), rabbit anti β-galactosidase
(Cappel), mouse anti-CD2 (Serotec), rabbit anti-GFP (Molecular
Probes), rabbit anti-Atonal (Jarman et al., 1994), mouse anti-Ey
(Clements et al., 2008) and rabbit anti-cyclin B (Jacobs et al., 1998).
Mouse anti-Eya, rat anti-ELAV 7E8A10, and mouse anti-cyclin B were
from Developmental Studies Hybridoma Bank (Iowa University).
Flourescently labeled secondary antibodies were from Molecular
Probes. Anti-mouse-HRP (Sigma) was used for immunoperoxidase
staining. Digoxigen labelled stg RNA probe (Roche) was produced
from cDNA clone LD47579 (BDGP). Doubly fluorescent antibody
labeling/in situ hybridization experiments were done according to
Jekely and Arendt (2007). For BrdU incorporation assays, eye-
antennal discs were dissected and incubated in 10 μM BrdU in PBS
for 30 min. BrdU was detected with anti-BrdU (Sigma) after treatment
with DNase.
Acknowledgments
We thank J.L. Gómez-Skarmeta, J. Culí, J.R. Martínez-Morales and
members of the Casares lab for comments on the manuscript; A.
Iannini for technical assistance; the CABD Advanced Microscopy and
Cytometry Facility for technical support; and the VDRC for fly strains
and Y.N. Jan for anti-Ato. This work was funded through grants
BFU2006-00349 to F.C. and Consolider ‘From Genes to Shape,’ of
which F.C. is a participant researcher (both from the Spanish Ministry
of Science and Innovation). C.L. was funded by Fundação para a
Ciência e a Tecnologia (Portugal) and Juan de la Cierva Program
(Spanish Ministry of Science and Innovation).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ydbio.2009.12.020.
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