1994 RESEARCH ARTICLE
Development 140, 1994-2004 (2013) doi:10.1242/dev.088963
© 2013. Published by The Company of Biologists Ltd
Extramacrochaetae imposes order on the Drosophila eye by
refining the activity of the Hedgehog signaling gradient
Carrie M. Spratford and Justin P. Kumar*
SUMMARY
The compound eye of Drosophila melanogaster is configured by a differentiating wave, the morphogenetic furrow, that sweeps
across the eye imaginal disc and transforms thousands of undifferentiated cells into a precisely ordered repetitive array of 800
ommatidia. The initiation of the furrow at the posterior margin of the epithelium and its subsequent movement across the eye field
is controlled by the activity of the Hedgehog (Hh) signaling pathway. Differentiating photoreceptors that lie behind the furrow
produce and secrete the Hh morphogen, which is captured by cells within the furrow itself. This leads to the stabilization of the fulllength form of the zinc-finger transcription factor Cubitus interruptus (Ci155), the main effector of Hh signaling. Ci155 functions as a
transcriptional activator of a number of downstream targets, including decapentaplegic (dpp), a TGFβ homolog. In this report, we
describe a mechanism that is in place within the fly retina to limit Hh pathway activity within and ahead of the furrow. We
demonstrate that the helix-loop-helix (HLH) protein Extramacrochaetae (Emc) regulates Ci155 levels. Loss of emc leads to an increase
in Ci155 levels, nuclear migration, apical cell constriction and an acceleration of the furrow. We find that these roles are distinct from
the bHLH protein Hairy (H), which we show restricts atonal (ato) expression ahead of the furrow. Secondary furrow initiation along
the dorsal and ventral margins is blocked by the activity of the Wingless (Wg) pathway. We also show that Emc regulates and
cooperates with Wg signaling to inhibit lateral furrow initiation.
INTRODUCTION
Overt patterning of the Drosophila retina begins during the third
and final larval instar when a wave of morphogenesis initiates at
the posterior margin of the eye imaginal disc and proceeds across
the epithelium towards the eye-antenna border. The anterior-most
edge of this differentiating wave can be visualized by a dorsoventral groove in the epithelium known as the morphogenetic
furrow (Ready et al., 1976). As the furrow traverses the retinal
epithelium, the field of non-patterned and undifferentiated cells is
transformed into columns of periodically spaced unit eyes called
ommatidia. Because the retina continues to grow while it is
patterned, the movement of the furrow across the eye disc must be
synchronized with the rate of cell proliferation. The phenotypes of
several furrow-stop mutants such as hhbar3 and Drop1 demonstrate
that if the two are uncoupled the resulting adult will contain too few
ommatidia (Ives, 1950; Heberlein et al., 1993; Ma et al., 1993;
Mozer, 2001). In addition, correct patterning is achieved when only
a single furrow initiates and moves across the eye disc. The
initiation of multiple furrows from the margins results in a small,
disorganized eye (Ma and Moses, 1995; Chanut and Heberlein,
1997b; Pignoni and Zipursky, 1997).
Furrow initiation is restricted to the intersection of the posterior
margin and the midline (called the posterior center) by the
JAK/STAT, Hedgehog (Hh) and Wingless (Wg) signaling pathways.
Both hh and unpaired (upd), the ligand for the JAK/STAT pathway,
are expressed at the posterior center (Domínguez and Hafen, 1997;
Borod and Heberlein, 1998; Ekas et al., 2006; Tsai et al., 2007).
Removal of either pathway from the posterior center prevents
Department of Biology, Indiana University, Bloomington, IN 47405, USA.
*Author for correspondence ([email protected])
Accepted 24 February 2013
furrow initiation, while ectopic expression along the margins or
within the anterior regions of the disc results in ectopic furrow
initiation (Heberlein et al., 1995; Ma and Moses, 1995; Pan and
Rubin, 1995; Strutt et al., 1995; Wehrli and Tomlinson, 1995;
Wiersdorff et al., 1996; Domínguez and Hafen, 1997; Borod and
Heberlein, 1998). An additional input into furrow initiation is the
EGF receptor pathway, as its removal also prevents the furrow from
initiating (Kumar and Moses, 2001). By contrast, wg is absent from
the posterior center and is instead expressed along the lateral
margins (Baker, 1988; Ma and Moses, 1995; Treisman and Rubin,
1995). Inhibition of Wg signaling leads to ectopic furrow initiation
at the margins, whereas overexpression of wg within the eye field
proper is sufficient to block furrow initiation and progression (Ma
and Moses, 1995; Treisman and Rubin, 1995; Ekas et al., 2006; Tsai
et al., 2007).
The progression of the furrow across the eye field is under the
control of Hh signaling. The Hh signal is produced and secreted in
differentiating photoreceptors and received by cells that reside
within the furrow. Reception of the Hh signal prevents the cleavage
of the full-length form of Cubitus Interruptus (Ci155 or CiACT) into
its shorter repressor form (Ci75 or CiR) (Pappu et al., 2003). Hh
signals through CiACT to activate transcription of decapentaplegic
(dpp), which encodes a TGFβ homolog, within the morphogenetic
furrow (Heberlein et al., 1993; Borod and Heberlein, 1998;
Greenwood and Struhl, 1999). Reducing hh expression within
developing photoreceptors leads to the loss of dpp expression and
a furrow-stop phenotype (Ives, 1950; Mohler, 1988; Ma et al., 1993;
Heberlein et al., 1993). It appears, however, that dpp plays only a
minor role in furrow progression as clones lacking downstream
components of the Dpp pathway only slow the furrow (Burke and
Basler, 1996; Wiersdorff et al., 1996; Greenwood and Struhl, 1999;
Curtiss and Mlodzik, 2000). Similarly, although dpp is necessary
for furrow initiation and is sufficient to induce ectopic furrows, it
appears to promote furrow initiation by a feedback loop that
DEVELOPMENT
KEY WORDS: Extramacrochaetae, Hairy, Hedgehog, Suppressor of fused, Wingless, Morphogenetic furrow, Eye
RESEARCH ARTICLE 1995
Hh regulation by Emc
MATERIALS AND METHODS
Fly strains and mosaic clones
The following fly stocks were used: (1) w1118; (2) emc-GFPYB0040; (3) emcGFPYB0067; (4) emc-GFPYB0217; (5) emc-GFPCB03369; (6) P(PZ) emc04322
ry506/TM3; (7) y w hs-flp[22]; FRT80B emcAP6/TM6B; (8) y w hs-flp[22];
FRT80B M(3)i55 Ubi-GFP; (9) y w eyflp; (10) y w hs-flp[22]; (11) Act5C
>y+ >GAL4, UAS-GFP; (12) UAS-emc4M; (13) UAS-emc5M; (14) FRT80B
h22/TM3; (15) FRT80B h22 emcAP6/TM3; (16) UAS-Su(fu); (17) UAS-Su(fu)
RNAi; (18) UAS-ci; (19) UAS-ci RNAi; (20) UAS-h; (21) UAS-arm RNAi;
(22) UAS-pan; (23) dpp-lacZ; (24) slimb-lacZ; (25) ci-lacZ; (26) 5⬘F-atolacZ; (27) wg-lacZ; (28) wg1-12/CyO; (29) wgGla/CyO; (30) 54C-GAL4; (31)
ato-GAL4; (32) ey-GAL4; and (33) dpp-GAL4. emc-null clones were
generated with the following genotypes: y w ey-flp; FRT80B emcAP6/
FRT80B M(3)i55 Ubi-GFP and/or y w hs-flp[22}; FRT80B emcAP6/
FRT80B M(3)i55 Ubi-GFP. Overexpression clones were generated with the
following genotypes: (1) y w hs-flp[22]; Act5C >y+ >GAL4, UASGFP/UAS-Su(fu); (2) y w hs-flp[22]; Act5C >y+ >GAL4, UAS-GFP/UASSu(fu) RNAi; (3) y w hs-flp[22]; Act5C >y+ >GAL4, UAS-GFP/UAS-ci;
(4) y w hs-flp[22]; Act5C >y+ >GAL4, UAS-GFP/UAS-ci RNAi; and (5) y
w hs-flp[22]; Act5C >y+ >GAL4, UAS-GFP/UAS-arm RNAi.
Antibodies and microscopy
The following primary antibodies were used: (1) rat anti-ELAV (1:20,
DSHB); (2) mouse anti-Hairy (1:100, a gift from Nadean Brown, UC Davis,
CA, USA); (3) rabbit anti-PKA RII (1:500, a gift from Dan Kalderon,
Columbia University, New York, USA); (4) mouse anti-Fu (1:200, DSHB);
(5) rabbit anti-CK1 (1:500, a gift from Claudio Sunkel, Institute for
Molecular and Cell Biology, Singapore); (6) mouse anti-Arm (1:20, DSHB);
(7) mouse anti-β-Gal (1:100, Promega); (8) mouse anti-GFP (1:100, Santa
Cruz Biotechnology); (9) rat anti-CiACT (1:50, a gift from Robert Holmgren,
Northwestern University, Evanston, IL, USA); (10) mouse anti-GSK (1:50,
DSHB) ; (11) rabbit anti-Cad86C (1:10,000, a gift from Christian Dahmann,
Max Planck Institute, Dresden, Germany); (12) mouse anti-Costal-2 (1:20,
DSHB); (13) mouse anti-Ptc (1:100, DSHB); (14) mouse anti-Smo (1:100,
DSHB); (15) mouse anti-Su[fu] (1:200, DSHB); (16) mouse anti-HA
(1:1000, Santa Cruz Biotechnology); and (17) mouse anti-Myc (1:1000,
Santa Cruz Biotechnology). Fluorophore-conjugated secondary antibodies
and phalloidin-fluorophore conjugate were obtained from Jackson
Laboratories and Molecular Probes. Imaginal discs and adult heads were
prepared as described by Anderson et al. (Anderson et al., 2012).
Number of ommatidial rows and furrow velocity
w1118 flies were allowed to lay eggs for 2 hours at 25°C. Individual eggs
were placed in tubes containing ~500 μl of media and aged for varying
numbers of hours at 25°C. Eye-antennal discs were dissected and stained
with rat anti-ELAV antibodies and phalloidin. The number of ommatidial
rows was determined by counting the number of rows of ELAV-positive
clusters. At least 19 discs were analyzed for each time point. Standard
deviations were calculated in Excel. Furrow velocity was calculated by
dividing the difference between the average number of ommatidial rows at
two consecutive time points by 180 minutes (the time interval between each
time point).
Calculation of degree of furrow advancement through emc and
hairy clones
Images of imaginal discs containing emc-, h- or emc h-null mutant clones
were imported into ImageJ. Three lines were drawn on each eye-antennal
disc: (1) a vertical line marking the position of the most posteriorly located
ommatidial row; (2) a vertical line marking the position of the normal
furrow; and (3) a vertical line marking the position of the furrow that has
advanced through mutant clone. The distance between 1 and 2 was
measured three times and averaged (NF), as was the distance between 1 and
3 (AF). This process was repeated for 30 clones. The contribution made by
emc and h to the distance travelled by the furrow was calculated by dividing
the values for the AF by those for the NF. We then averaged the set of
AF/NF ratios and multiplied by 100 to obtain a final percentage. Standard
deviations were calculated in Excel. An F-test was used to determine equal
or unequal variance. If the F-value was more than 0.05, then P-values were
calculated using a Student’s t-test of equal variance. If the F-value was less
than 0.05, then a Student’s t-test of unequal variance was used.
emc-GFP expression pattern analysis
More than 400 eye-antennal discs from four emc-GFP enhancer trap lines
were dissected and stained with Phalloidin and anti-ELAV antibodies. The
presence or absence of emc transcription in the anterior compartment was
registered against the number of ommatidial rows.
Immunoprecipitation assays
Kc167 cells were transfected with combinations of the following plasmids:
(1) mt-GAL4, (2) UAS-Emc-HA, (3) UAS-Emc-MYC, (4) UAS-H-HA,
(5) UAS-H-MYC. Immunoprecipitations were carried out as described by
Anderson et al. (Anderson et al., 2012). Each experiment was carried out in
both directions (immunoprecipitation with Emc-MYC, immunoblotting
with H-HA and immunoprecipitation with H-HA, immunoblotting with
Emc-MYC).
Transcriptional activation assay
A transcriptional reporter containing five copies of the consensus Ci binding
site was built (IDT) and cloned into a plasmid containing the hsp70
promoter and the firefly luciferase gene. This plasmid was simultaneously
transfected (Qiagen Effectene Transfection Reagent) into Kc167 cells with
plasmids containing the pMT-GAL4 driver and a UAS-Renilla luciferase
reporter. These cells were also transfected with combinations of the
following plasmids: UAS-GFP, UAS-Su(fu) and UAS-CiU. Deleting amino
acids 612-760 from the full-length Ci protein generated the CiU protein that
was used in this assay. This modified protein cannot be cleaved into the
smaller repressor isoform and functions as a transcriptional activator of
Hedgehog target genes (Wang and Holmgren, 1999). Equal amounts of each
construct were transfected into Kc167 cells, which were then incubated for
12 hours, followed by protein induction and an incubation period. The
Promega Dual-Luciferase Reporter Assay System and a Glomax 20/20
DEVELOPMENT
activates hh expression (Chanut and Heberblein, 1997a; Pignoni
and Zipursky, 1997; Borod and Heberlein, 1998). Thus, it appears
that Hh signaling is the main regulator of furrow initiation and
progression.
Here, we focus on the role that extramacrochaetae (emc) plays in
refining the activity of the Hh signaling gradient during furrow
progression. Emc, a helix-loop-helix (HLH) protein, modulates
transcription by sequestering basic helix-loop-helix (bHLH)
proteins away from target DNA enhancers (Ellis et al., 1990; Garrell
and Modolell, 1990; Van Doren et al., 1991). The furrow accelerates
through emc-null mutant clones suggesting that during normal
development Emc functions to regulate the rate of pattern formation
(Bhattacharya and Baker, 2009). However, a clear mechanism by
which this is achieved has remained elusive. Here, we show that
Emc adjusts the rate at which the furrow moves across the eye field
by regulating the levels of the active form of Ci (CiACT). This is
accomplished in part through the regulation of Suppressor of fused
[Su(fu)], a member of the Hh cascade. Its role in the pathway is
poorly understood but here we show that Su(fu) is required to
stabilize CiACT levels. We go on to demonstrate that increasing the
level of CiACT alone is sufficient to accelerate the pace at which the
furrow progresses. We find that emc is also required for regulating
two cellular features of the furrow: nuclear migration and apical
surface constrictions. These roles are different from that of the
bHLH protein Hairy (H), which has also been implicated in
regulating furrow progression. We demonstrate that H is required
for suppressing precocious neurogenesis ahead of the furrow
through repression of atonal (ato). Finally, we demonstrate that emc
functions upstream of wg to prevent furrow initiation along the
margins. Overall, our results indicate that the role of Emc in eye
development is to impose order on the retina by regulating the
activity of the Hh and Wg signaling pathways.
Luminometer were used to detect luminescence signals from both firefly
and Renilla luciferases, which were then divided to generate luminescence
ratios. For each experiment, three independent biological and technical
replicates were conducted, thus the luminescence ratio for each genotype in
Fig. 4M represents the average of nine experimental ratios.
RESULTS
Emc regulates furrow velocity during eye
development
An accurate measure of furrow velocity across the retinal epithelium
is a prerequisite for fully appreciating the role that emc plays in
regulating the pace of pattern formation. Two prior studies of furrow
speed provide conflicting estimates of its velocity. One
measurement indicates that the furrow moves at a constant rate with
one ommatidial row being laid down every 120 minutes (Basler and
Hafen, 1989). A second study determined that an ommatidial row
was generated every 100 minutes in the posterior half of the eye
field. The pace quickens in the anterior half where a row is produced
every 60 minutes (Campos-Ortega and Hofbauer, 1977). These
conflicting measurements prompted us to measure directly the
velocity of the furrow by determining the number of ommatidial
rows that had been produced in eye discs of individually staged
larvae at defined time points (Fig. 1A,B; supplementary material
Table S1). We calculated the velocity of the furrow by dividing the
difference in the number of rows between time points by the 180-
Development 140 (9)
minute interval that we had incorporated into the experiment
(Fig. 1C). We observe a dynamic range to the pace at which the
furrow progresses across the eye field. The generation time for a
single ommatidial row ranges from 35 to 150 minutes (Fig. 1C).
Thus, in contrast to prior studies, we demonstrate that the furrow
moves dynamically across the eye field with alternating periods of
accelerations and decelerations.
We next set out to analyze whether the expression pattern of emc
correlates with the changing velocity of the furrow. Several GFP
enhancer trap lines, including YB0067, which was used throughout
this study, faithfully recapitulate the normal expression pattern of
emc (Fig. 1D-F). In young eye discs, emc is expressed along the
D/V midline (Fig. 1D,E). Several genes, such as four-jointed, that
are involved in the establishment and maintenance of dorsal-ventral
compartment boundaries are expressed along the midline in a
similar pattern to emc (Brodsky and Steller, 1996). This might hint
at an earlier role for emc in compartment boundary formation. In
older third instar eye discs, emc is transcribed throughout the entire
retinal field with low expression levels within the furrow and
significant enrichment ahead of the advancing furrow and at the
equator (Fig. 1F) (Brown et al., 1995). Eye discs were dissected
from larvae of various ages from these enhancer trap lines and the
expression pattern of emc was compared with the number of
ommatidial rows. We find that although emc is expressed at low
levels throughout the eye disc, enriched expression in the anterior
Fig. 1. emc expression and the progression of the furrow is dynamic during development. (A,B) Graphs depicting the average number of
ommatidial rows that are found at various times after egg deposition. (C) The furrow progresses across the eye field at a dynamic rate. (D,E) In young
discs, emc is elevated at the midline. (F) In more mature discs, emc expression is enriched in the anterior compartment. Anterior is towards the right. All
markers are listed in each panel. (G) Graph demonstrating that onset of emc expression ahead of the furrow coincides with the formation of the tenth
row of ommatidia.
DEVELOPMENT
1996 RESEARCH ARTICLE
compartment does not appear until ~10 rows of ommatidia have
been generated (Fig. 1G). We note that the furrow has already begun
to decelerate prior to the onset of this enrichment and that the furrow
continues to accelerate later in development despite the continued
elevated expression of emc throughout the anterior compartment
(Fig. 1C,G). Coupled with our findings that emc is required to slow
the furrow across the entire disc (see below) we conclude that the
lower basal level of Emc protein is sufficient to serve as a brake on
the furrow and that mechanistically Emc is not the rate-limiting
reagent.
Despite the recognition that emc plays a role in regulating the
speed of the furrow, the mechanisms that underlie this activity
remain poorly understood. A recent report using a null emc allele
concluded that emc could regulate furrow velocity on its own but
only in the ventral half of the retina (Bhattacharya and Baker, 2009).
However, emc was removed throughout the entire disc, resulting in
a small, disorganized eye. This complicates the interpretation of the
emc loss-of-function phenotypes (Bhattacharya and Baker, 2009).
We set out to determine unambiguously whether the loss of emc
alone is sufficient to slow the furrow throughout the whole eye field
during the patterning process. Similar to prior reports, discs that are
lacking emc throughout the entire primordium are small and
extremely disorganized. These animals die as pharate adults and the
eye fields contain very few ommatidia and are instead populated
with head cuticle and extra macrochaetae type bristles (Fig. 2A-D).
We then made patches of emc-null mutant tissue and observed
the morphogenetic furrow accelerating through the clone,
irrespective of compartmental location (Fig. 2E-K). Using physical
distance as a measure of how far the furrow has advanced through
wild-type and emc-null mutant tissue, we calculated the contribution
that emc plays in regulating furrow velocity (Fig. 2I-K). We find
that the furrow moves an average of 30% further through the emcnull clones than wild-type tissue. This is unaffected by the size,
width or location of the clone. We confirmed that the furrow was in
fact accelerating through emc mutant tissue by analyzing the dpplacZ transcriptional reporter, which, during the late third instar, is
expressed solely within the advancing furrow (Blackman et al.,
1991; Heberlein et al., 1993; Chanut and Heberlein, 1997a; Chanut
and Heberlein, 1997b). As the furrow passes through emc-null
clones dpp appears to be expressed in a broader swathe of cells and
at higher levels (Fig. 2L-O, arrows). From these results, we
conclude that emc is able to regulate the furrow throughout the
entire eye disc.
Emc controls the pace of furrow movement
through regulation of Hh signaling
Progression of the furrow and dpp activation are both dependent
upon Hh signaling (Heberlein et al., 1993; Ma et al., 1993;
Heberlein et al., 1995; Domínguez and Hafen, 1997; Borod and
Heberlein, 1998). We attempted to ascertain whether furrow
acceleration and increased dpp transcription within emc-null clones
are due to elevated Hh signaling. We first examined the protein
levels of Cubitus interruptus (Ci), a transcription factor that resides
at the bottom of the Hh pathway (Aza-Blanc and Kornberg, 1999).
Two different versions of Ci are found in developing tissues: a fulllength form that functions as a transcriptional activator (CiACT)
(Alexandre et al., 1996; Hepker et al., 1997) and a smaller form that
serves as a repressor (CiR) (Aza-Blanc et al., 1997). CiACT is present
in cells responding to Hh signaling (Méthot and Basler, 1999; Price
and Kalderon, 1999; Wang and Holmgren, 1999). Within wild-type
eye discs the highest levels of CiACT are found within the furrow
(Fig. 3A) (Eaton and Kornberg, 1990). Removal of emc within the
RESEARCH ARTICLE 1997
Fig. 2. The morphogenetic furrow accelerates through emc-null
clones. (A,B) Scanning electron microscopy and confocal images of wildtype retinas. (C,D) Removal of emc from the entire eye disc (ey-flp; FRT80B
emcAP6/FRT80B M(3)i55 Ubi-GFP) leads to severe patterning and
proliferation defects. (E-O) The furrow (assayed by F-actin, ELAV and dpplacZ) accelerates through emc clones (hs-flp[22]; FRT80B emcAP6/FRT80B
M(3)i55 Ubi-GFP). Anterior is towards the right. All markers and
abbreviated genotypes are listed in each panel. NF, normal furrow; AF,
advanced furrow.
furrow results in a significant increase in the level and number of
cells expressing CiACT, suggesting that emc regulates the
progression of the furrow by controlling the transcriptional output
of Hh signaling (Fig. 3B-D).
Several reports have suggested that the Hh pathway is bifurcated
and that the Hh signal can be transduced independently of Ci
(Krishnan et al., 1997; Lessing and Nusse, 1998; Lewis et al., 1999;
Suzuki and Saigo, 2000; Gallet et al., 2000). The existence of this
distal branch point has been disputed (Méthot and Basler, 2001). We
wanted to determine whether Hh-mediated control of the furrow is
through Ci or not. We observed that the furrow accelerates through
clones in which full-length Ci (Ci155) is overexpressed, thereby
suggesting that increased CiACT is sufficient to advance the furrow
and is responsible for the emc mutant phenotype (Fig. 3E-H, arrows).
We next investigated the molecular mechanism by which emc
regulates CiACT levels by generating emc-null clones and examining
the expression levels of known Hh signaling components. Using a
ci-lacZ transcriptional reporter (Fig. 3I), we demonstrate that the
increased levels of CiACT seen within emc-null clones are not due to
an increase in ci transcription (Fig. 3J-L). From this, we conclude
that CiACT levels are elevated in emc clones via regulation of at least
one other member of the Hh signaling network. A systematic screen
DEVELOPMENT
Hh regulation by Emc
1998 RESEARCH ARTICLE
Development 140 (9)
Fig. 3. Emc regulates the active form of the Ci transcription factor
(Ci155). (A,I) Wild-type expression patterns of CiACT and ci-lacZ in third
instar discs. (B-D,J-L) Elevated levels of CiACT seen in emc clones (hsflp[22]; FRT80B emcAP6/FRT80B M(3)i55 Ubi-GFP) are not due to increased ci
transcription. (E-H) Overexpression of full-length Ci (hs-flp[22]; Act5C >y+
>GAL4, UAS-GFP; UAS-Ci155) results in the acceleration of the furrow.
Arrows in E-H indicate accelerated regions of the furrow. Anterior is
towards the right. All markers and abbreviated genotypes are listed in
each panel.
activity by blocking its entry into the nucleus and/or by repressing
target genes within cells that have received the Hh signal
(Kogerman et al., 1999; Lefers et al., 2001; Méthot and Basler,
2000; Wang and Holmgren, 2000; Wang et al., 2000; Taylor et al.,
2002; Ho et al., 2005; Dussillol-Godar et al., 2006). Consistent
with these findings, we observe that Su(fu) is also capable of
inhibiting the ability of CiACT to activate the transcription of a
luciferase reporter that has been fused to an artificial enhancer
containing five perfect Ci-binding sites (Fig. 4M). These results
suggest that in the developing eye Su(fu) is unlikely to cooperate
with CiACT in the activation of Hh pathway target genes that are
needed for furrow progression. Our results are also consistent with
the fact that Su(fu)-null mutant animals have no obvious eye
defects and movement of the furrow is not retarded as it passes
through clones that lack Su(fu) and/or CiACT (Fig. 4E-H) (Préat,
1992; Pappu et al., 2003). These results suggest that emcdependent regulation of CiACT relies on the activity of a yet to be
identified Hh signaling component.
DEVELOPMENT
of Hh signaling components in emc clones revealed that nearly all
Hh signaling components, including hh, patched (ptc), smoothened
(smo), fused (fu), costal-2 (cos-2; cos – FlyBase), slimb (slmb –
FlyBase), protein kinase A (PKA), glycogen synthase kinase-3
(GSK3) and casein kinase-1 (CK1) are not regulated by emc
(supplementary material Fig. 1A-P) (Østerlund and Kogerman,
2006). The lone exception is Su(fu), which encodes a novel PEST
domain-containing protein. In emc-null clones, Su(fu) protein levels
appear elevated (Fig. 4A-D, arrows). This apparent increase in
Su(fu) does not appear to be a response to elevated CiACT levels as
forced increases or decreases in CiACT levels are insufficient to alter
the amount of Su(fu) protein (supplementary material Fig. 2A-H).
Su(fu) appears to be required for maintaining the levels of CiACT
as reductions in Su(fu) levels are sufficient to drastically reduce
levels of CiACT (Fig. 4E-H; supplementary material Fig. S1Q,R)
(Ohlmeyer and Kalderon, 1998). The increase in CiACT levels is not
a result of increased transcription of ci as the ci-lacZ reporter is
unaffected in emc clones despite elevated Su(fu) protein levels
(Fig. 3J-L; Fig. 4B-D). Instead, the increased CiACT levels may
result from post-translational stabilization by Su(fu), which
physically associates with Ci and its mammalian homologs, the Gli
proteins (Monnier et al., 1998; Pearse et al., 1999; Stegman et al.,
2000; Monnier et al., 2002; Paces-Fessy et al., 2004). Su(fu) does
not, however, appear to be sufficient to prevent the full-length
activator form of Ci from being cleaved into the smaller repressor
form as overexpression clones of Su(fu) do not lead to increased
levels of CiACT (Fig. 4I-L).
The role that Su(fu) plays in regulating Ci activity is not well
understood. Several reports suggest that Su(fu) antagonizes Ci/Gli
Fig. 4. Su(fu) regulates CiACT protein but does not promote
transcriptional activation. (A) Distribution of Su(fu) protein in wild-type
retinas. (B-D) Su(fu) levels appear elevated in emc clones (hs-flp[22];
FRT80B emcAP6/FRT80B M(3)i55 Ubi-GFP). (E-H) Reductions in Su(fu) levels
(hs-flp[22]; Act5C >y+ >GAL4, UAS-GFP; UAS-Su(fu) RNAi) reduce CiACT levels
but do not inhibit furrow progression. (I-L) Elevation of Su(fu) levels (hsflp[22]; Act5C >y+ >GAL4, UAS-GFP; UAS-Su(fu)) is not sufficient to increase
CiACT levels. Anterior is towards the right. All markers and abbreviated
genotypes are listed in each panel. (M) Expression of Su(fu) leads to a
reduction in the activation of a luciferase reporter by CiU that is under the
control of a synthetic Ci-responsive element (compare blue and pink
bars).
Emc functions as an inhibitor of apical surface
constriction and nuclear migration
In addition to changes in gene expression profiles, two cellular
features distinguish cells within the furrow from all others cells
within the eye disc. The first is the constriction of apical cellular
surfaces and the second is the basal migration of nuclei. These two
cellular activities result in a dorsoventral indentation in the disc
epithelium, hence the description of this zone as a furrow (Ready et
al., 1976). The constriction of the apical surfaces of cells within the
furrow is dependent upon Hh signaling. A decrease in F-actin
accumulation and Armadillo (Arm) levels (two apical profile
markers) are observed in clones that are mutant for smoothened
(smo), which encodes a transducer of the Hh signal (Corrigall et al.,
2007). The cell shape changes that are induced by Hh signaling are
crucial for limiting the spread of the Hh signal itself. Cells within
clones mutant for the Drosophila homolog of cyclase-associated
protein homolog act up (acu; capt – FlyBase) fail to constrict their
apical profiles, and both Hh and CiACT proteins accumulate more
anteriorly than in surrounding wild-type cells (Benlali et al., 2000).
As our data indicate that emc regulates Hh signaling, we turned our
attention to the cellular dynamics of cells that lack emc. As
expected, Arm and Cad86C, two proteins that localize to adherens
junctions and are enriched within the furrow (Fig. 5A,E) (McCrea
et al., 1991; Schlichting and Dahmann, 2008), are elevated within
emc-null clones (Fig. 5B-D,F-H). Cad86C is a transcriptional target
of Hh signaling (Schlichting and Dahmann, 2008); therefore, the
increase in Cad86C is consistent with the increases that we see in
CiACT levels (Fig. 3B-D). Arm does not appear to play a role in
stabilizing CiACT as expression of an arm RNAi construct has no
effect on CiACT (supplementary material Fig. S3A-D). Sagittal
sections of discs containing emc mutant clones reveal accumulations
of F-actin and cell shape changes within the mutant tissue
(Fig. 5I,K,L). These cellular changes result in depressions at the
apical surface (Fig. 5I, yellow asterisk) that mimic cells within the
endogenous furrow (Fig. 5I, white asterisk).
Nuclear migrations are a second distinguishing cellular feature
of the morphogenetic furrow (Ready et al., 1976; Fischer-Vize and
Mosley, 1994). The basal migration of nuclei within the furrow and
their subsequent apical rise in post-mitotic cells is dependent upon
microtubule motors such as Dynein and associated proteins (Fan
and Ready, 1997; Mosley-Bishop et al., 1999; Swan et al., 1999;
Houalla et al., 2005). We have used this feature to further analyze
the cellular behavior of cells lacking emc. In emc mutant tissue,
nuclei appear to migrate basally (Fig. 5I, yellow arrowhead) as they
do within the normal furrow (Fig. 5I, white arrowhead). The
strongest effect is seen within and ahead of the furrow (Fig. 5J),
suggesting that cells lacking emc behave as if they are in a ‘furrowlike’ state. From these observations, we conclude that emc is
required to prevent changes in cell shape and basal migration of
nuclei, two distinct characteristics of the cells within the furrow that
receive high levels of Hh signaling.
Emc and H cooperate to regulate pattern
formation through different targets
Although it has been demonstrated that the furrow will accelerate
through a clone that is null for emc (Fig. 2E-H) (Bhattacharya and
Baker, 2009) we wanted to determine what role, if any, does h play
in regulating the furrow velocity. h is normally expressed in a stripe
of cells ahead of the morphogenetic furrow (Fig. 6A) (Brown et al.,
1991). Consistent with prior reports, we find that the furrow does
not accelerate through h-null mutant clones (supplementary material
Fig. S4A-G) (Brown et al., 1995). By contrast, the distance traveled
RESEARCH ARTICLE 1999
Fig. 5. Emc regulates Hedgehog-dependent constriction of apical
cell surfaces. (A,E) Arm and Cad86C proteins are enriched within the
furrow. (B-D,F-H) Both Arm and Cad86C appear elevated in emc clones
(hs-flp[22]; FRT80B emcAP6/FRT80B M(3)i55 Ubi-GFP). (I) F-actin
accumulation (yellow asterisk) and nuclei migration (yellow arrow) within
an emc clone. The normal furrow is marked by a white asterisk, whereas a
white arrow marks nuclear migration. The yellow and orange lines
indicate regions of the disc that are viewed in the side sagittal sections.
(J-L) High-resolution images demonstrating apical surface constriction
and nuclear migration (white asterisk indicates the wild-type furrow).
Anterior is towards the right. All markers and abbreviated genotypes are
listed in each panel.
by the furrow through emc h double-null mutant clones is
significantly greater than the distance traveled through just emc
mutant tissue (Fig. 6B-F). A question that arises from these results
concerns the molecular and genetic relationship between emc and h.
As both genes are co-expressed in a subset of cells ahead of the
furrow, we set out to determine whether emc regulates h expression.
In very large emc-null mutant clones, h expression is reduced but
not eliminated from the entire clone (Fig. 6G-I, arrow). In small- to
medium-sized clones, the level of h expression appears unchanged
(Fig. 6J-L). These results suggest that h expression in emc clones
can be restored non-autonomously from surrounding wild-type
DEVELOPMENT
Hh regulation by Emc
2000 RESEARCH ARTICLE
Development 140 (9)
tissue. A potential candidate is the Dpp pathway, which activates h
expression ahead of the furrow (Greenwood and Struhl, 1999).
We then attempted to ascertain whether Emc and H cooperate to
regulate the furrow by forming a biochemical complex. We
performed immunoprecipitation assays on Drosophila Kc167 cell
extracts that contain epitope-tagged versions of both proteins and
were unable to detect any physical interactions between Emc and H
(Fig. 6M). The immunoprecipitations were performed in both
directions and in both instances we failed to detect an Emc-H
interaction. As Emc and H cooperate to regulate the furrow, the lack
of an Emc-H complex suggests that the two proteins function in
different pathways. Forced expression of emc within the furrow
using an atonal-GAL4 driver has no effect on the structure of the
retina (Fig. 6N), whereas expression of h leads to defects in
photoreceptor specification and a mild roughening of the adult eye
(Fig. 6O,P) (Brown et al., 1991). Simultaneous expression of both
factors has a synergistic effect, with neuronal specification being
nearly completely blocked and an adult eye almost devoid of
ommatidia (Fig. 6Q,R). This synergism, which was first reported
by Brown et al. (Brown et al., 1995), supports a model in which
emc and h are functioning separately rather than being bound
together as a biochemical complex.
As the developmental and adult retinal phenotypes of atonal (ato)
mutants are nearly identical to those observed when emc and h are
overexpressed (Fig. 6Q,R) (Jarman et al., 1994; Jarman et al., 1995),
we examined the effects that loss of either emc or h has on ato. An
enhancer element that lies 5⬘ to the ato-coding sequence can direct
expression to the intermediate groups, the R8 equivalence group
and within the R8 cell itself (Sun et al., 1998). Expression of the
ato reporter is reduced in emc-null clones (Fig. 6S-U, arrows),
whereas the reporter is ectopically activated in h-null clones
(Fig. 6V-X, arrows). Ectopic ato expression in h clones is not
accompanied by neuronal development (marked by ELAV),
indicating that ato activation, by itself, is insufficient to induce
neurogenesis. We conclude that emc and h regulate different aspects
of the furrow: h is primarily tasked with repressing ato expression
ahead of the furrow, whereas emc functions to inhibit Hh signaling
and the cellular dynamics of the furrow.
Emc both regulates and cooperates with Wg
signaling to prevent ectopic furrow formation
As emc appears to slow the progression of the normal furrow
through regulation of Hh signaling, we next asked the following
two questions: (1) would elevated levels of emc at the posterior
DEVELOPMENT
Fig. 6. Extramacrochaetae and Hairy regulate different aspects of pattern formation. (A) During normal retinal development, h is expressed in a
stripe ahead of the furrow. (B-E) The furrow accelerates through emc h double mutant clones (hs-flp22]; FRT80B emcAP6 h22/FRT80B M(3)i55 Ubi-GFP).
(F) Comparison of measured furrow acceleration through emc single and emc h double mutant clones. The y-axis refers the percentage advancement of
the furrow through clones when compared with wild-type tissue. (G-L) h expression in emc clones (hs-flp[22]; FRT80B emcAP6/FRT80B M(3)i55 Ubi-GFP).
Arrow in I indicates the non-autonomous rescue of h expression by the surrounding wild-type tissue. (M) Co-immunoprecipitation from Kc167 cells
demonstrating that Emc and H do not form a biochemical complex. (N-R) Simultaneous expression of h and emc by ato-GAL4 results in a synergistic
reduction of eye development. (S-U) Expression of ato within the furrow is reduced in emc clones (arrows). (V-X) Loss of h (hs-flp[22]; FRT80B h22/FRT80B
M(3)i55 Ubi-GFP) results in ectopic ato expression (arrows). Anterior is towards the right. All markers and abbreviated genotypes are listed in each panel.
margin be sufficient to inhibit the initiation of the furrow and (2)
does emc play a role in preventing ectopic furrows from initiating
at the dorsal and ventral margins of the eye disc? The impetus for
asking the first question lies in the fact that Hh signaling is also
required for furrow initiation (Ma et al., 1993; Heberlein et al.,
1993; Chanut and Heberlein, 1995; Heberlein et al., 1995; Ma and
Moses, 1995; Pan and Rubin, 1995; Strutt et al., 1995; Wehrli and
Tomlinson, 1995). However, expression of emc under the control
of either dpp or ey enhancers, both of which drive expression at the
posterior margin of the disc (Blackman et al., 1991; Hauck et al.,
1999), failed to have any observable effect on furrow initiation
(supplementary material Fig. S5A,B).
An earlier study has shown that emc is required for Wingless
(Wg)-dependent repression of bristle formation in the retina
(Cadigan et al., 2002). Wg signaling also plays an important role in
preventing ectopic furrow initiation at the margins of the eye disc
(Ma and Moses, 1995; Treisman and Rubin, 1995), so it seemed
possible that emc may function with wg to prevent ectopic furrow
initiation. emc-null clones that contact the margins result in ectopic
furrow initiation as assayed by dpp-lacZ (Fig. 7A-D, arrow) and
ato-lacZ (supplementary material Fig. S5C-F) reporters. These
ectopic furrows can be seen originating from both the dorsal and
ventral margins. Our next effort was to determine whether emc lies
genetically upstream or downstream of the Wg signaling cascade.
Removal of Wg activity via a temperature-sensitive allele (wg1-12)
(Baker, 1988) has no visible effect on emc transcription (Fig. 7E-H).
Similarly, activation of the Wg pathway through expression of
pangolin (pan) in clones failed to influence emc expression (Fig. 7IL). And finally, neither removal nor overexpression of emc was
sufficient to significantly modify the adult eye phenotype of wgGla
(supplementary material Fig. S6A-D), a gain-of-function allele of
wg (Brunner et al., 1999). These results suggest that emc does not
function downstream of Wg and are consistent with an earlier
observation that elevating wg expression does not induce either emc
or h transcription in the eye disc (Cadigan et al., 2002). We then
used a wg-lacZ reporter, which recapitulates wg transcription in the
eye (Fig. 7M) (Ma and Moses, 1995; Treisman and Rubin, 1995),
to monitor wg transcription in emc-null mutant clones. Under these
circumstances, the wg reporter is silenced along the ventral margin
(Fig. 7N-S, arrows). This indicates that emc lies upstream of wg at
the ventral margin but cooperates with wg to block inappropriate
pattern formation at the dorsal margin.
DISCUSSION
Here, we describe a molecular mechanism by which the HLH
protein Emc refines the activity of the Hh signaling gradient and
thereby ensures that the morphogenetic furrow patterns the retina
evenly and at an appropriate set of velocities (Fig. 8A,B). As the
furrow progresses across the eye field, Hh is secreted by
differentiating photoreceptors and received by cells within the
furrow (Heberlein et al., 1993; Ma et al., 1993). As a result, the
active version of the transcription factor Ci (CiACT) is stabilized,
which in turn leads to the transcriptional activation of dpp, a longrange morphogen (Heberlein et al., 1993; Borod and Heberlein,
1998; Fu and Baker, 2003; Pappu et al., 2003). Dpp is then used to
prepare more anteriorly positioned cells for entry into the furrow
(Greenwald and Struhl, 1999). In addition to activating dpp
expression, Hh signaling within the furrow is required for apical
surface constriction and the transcriptional repression of h, which
encodes a bHLH transcription factor that, at elevated levels, can
induce numerous structural defects (Brown et al., 1991; Fu and
Baker, 2003; Corrigall et al., 2007; Schlichting and Dahmann,
RESEARCH ARTICLE 2001
Fig. 7. Activation of Wingless signaling by Emc prevents ectopic
furrow initiation. (A-D) Ectopic furrows initiate from the margins (arrow)
in the absence of emc (hs-flp[22]; FRT80B emcAP6/FRT80B M(3)i55 Ubi-GFP).
(E-H) emc expression is not altered when wg activity is removed via a
temperature-sensitive mutant wg1-12. (I-L) Activation of Wg signaling (hsflp[22]; Act5C >y+ >GAL4, UAS-GFP; UAS-pan) fails to alter emc expression.
(M-S) wg expression at the ventral (arrows), but not dorsal, margin is lost
in emc clones. Clones in M-P were generated with hs-flp[22]; FRT80B
emcAP6/FRT80B M(3)i55 Ubi-GFP, whereas clones in Q-S were generated
with ey-flp; FRT80B emcAP6/FRT80B M(3)i55 Ubi-GFP. Anterior is towards the
right. All markers and abbreviated genotypes are listed in each panel.
2008). Together with H, Emc is thought to regulate the rate by which
the furrow traverses the retinal epithelium (Brown et al., 1995;
Bhattacharya and Baker, 2009). However, a molecular link between
H/Emc and the signaling pathways controlling furrow velocity has
remained elusive.
In this report, we have demonstrated that Emc regulates the levels
of CiACT and this in turn has a direct impact on the speed at which
the furrow travels across the eye field. The furrow travels through
emc loss-of-function clones, which show elevated levels of CiACT,
~30% faster on average than it does through wild-type tissue. As a
result, the eye does not have sufficient time to proliferate and the
adult retina is small and disorganized (Fig. 8A,B). The elevated
DEVELOPMENT
Hh regulation by Emc
2002 RESEARCH ARTICLE
Development 140 (9)
Fig. 8. Models for the
involvement of Emc in furrow
initiation and progression.
(A,B) During furrow progression,
Emc counterbalances the activity
of Hh signaling via regulation of
CiACT levels. This puts the rates of
pattern formation and
proliferation into equilibrium.
(C) A summary of the genetic
interactions by which Emc
regulates furrow initiation and
progression.
2000; Singh and Choi, 2003). The genetic separation of emc and
wg in the dorsal half of the retina may stem from the need to activate
Wg signaling independently of emc.
Finally, the original description of emc and furrow regulation
proposed that Emc and H cooperate to regulate furrow velocity
(Brown et al., 1995). A subsequent study has suggested that Emc on
its own is solely responsible for the acceleration of the furrow
(Bhattacharya and Baker, 2009). Our data indicate that although the
furrow can accelerate through tissue that is lacking only emc, h does
in fact contribute to the pace of furrow movement. We measured the
rate of the furrow in emc, h double mutant clones and demonstrate that
the furrow proceeds faster through these clones than it does through
clones that just lack emc. It appears that Emc and H regulate different
aspects of furrow progression with Emc regulating the constriction
of apical surfaces, the basal migration of nuclei and the Hh signaling
cascade whereas H, on the other hand, appears to restrict ato
expression in cells that have not yet entered the furrow.
Acknowledgements
We thank Brandon Weasner for technical support with coimmunoprecipitation and luciferase assays; Abby Anderson, Bonnie Weasner
and Brandon Weasner for comments on the manuscript; Nick Baker, Antonio
Baonza, Konrad Basler, Nadean Brown, Lynn Cooley, Michael Buszczak,
Christian Dahmann, Barry Dickson, Wei Du, Robert Holmgren, Daniel
Kalderon, Monn Myat, Ilaria Rebay, Alan Spradling, Claudio Sunkel, Jessica
Treisman, Janice Fischer, Georg Halder, the DSHB, the DGRC and the BDSC for
antibodies and fly stocks; and the Indiana University Light Microscopy Imaging
Center and Olivier Brun for technical support with Leica SP5 confocal
microscope.
Funding
This work is supported by a stipend from the National Institutes of Health (NIH)
GMCS Training Grant [T32-GM007757] to C.M.S. and a grant from the
National Eye Institute [2R01 EY014863] to J.P.K. Deposited in PMC for release
after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.088963/-/DC1
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2004 RESEARCH ARTICLE