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First published online November 7, 2006
doi: 10.1242/10.1242/dev.02686
1 Department of Molecular and Cellular Biology, One Baylor Plaza, Houston, TX
77030, USA.
2 Program in Developmental Biology, Baylor College of Medicine, One Baylor
Plaza, Houston, TX 77030, USA.
3 Department of Ophthalmology, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA.
* Authors for correspondence (e-mail: asingh{at}bcm.edu; kchoi{at}bcm.tmc.edu)
Accepted 2 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila eye, Cell survival, Cell death, JNK Signaling, Wg Signaling, Lobe, Serrate
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Wolff and Ready, 1993). Prior
to furrow initiation, it is important to establish the dorsal and ventral
compartments for the growth of early eye discs (for a review, see
Singh et al., 2005b). Recent
studies suggest that the ground or default state of the entire early eye
imaginal primordium is ventral, and that the onset of expression of the dorsal
selector gene pannier (pnr), establishes the dorso-ventral
(DV) lineage in the eye (Maurel-Zaffran
and Treisman, 2000; Singh and
Choi, 2003).
L and Ser regulate ventral eye growth
(Singh et al., 2005b).
L acts upstream of Ser, a Notch (N) ligand in the ventral
eye (Chern and Choi, 2002;
Singh et al., 2005b). Loss of
L or Ser (hereafter L/Ser) function exhibits
distinct mutant phenotypes depending on the stage of DV eye patterning. Early
loss-of-function (LOF) of L/Ser, when the entire eye field is in the
default ventral state, results in the loss of the entire eye. However, LOF of
L/Ser after the onset of pnr expression, during second
larval instar of development, results in the loss of only the ventral half of
the eye (Singh and Choi, 2003;
Singh et al., 2005b). This
suggests that ventral-specific cells are selectively lost from the developing
eye of L/Ser mutants by a currently unknown mechanism. One possible
mechanism for the loss of eye pattern in L/Ser mutants may be due to
the induction of cell death in the ventral cells.
In the Drosophila eye, active caspases execute apoptosis
(Song et al., 1997;
Fraser et al., 1997;
Meier et al., 2000;
Hays et al., 2002;
Yu et al., 2002). Inhibitor of
apoptosis proteins (IAPs), a highly conserved class of proteins, negatively
regulate caspase activity (Miller,
1999; Wang et al.,
1999; Goyal et al.,
2000; Lisi et al.,
2000; Wilson et al.,
2002). The Drosophila pro-apoptotic genes
wrinkled [also known as head involution defect
(hid), and hereafter referred to as hid], reaper
(rpr) and grim (White et
al., 1994; Grether et al.,
1995; Chen et al.,
1996) in turn negatively regulate IAPs. These genes encode the
members of Hid-Reaper-Grim (HRG) complex, which binds to and inactivates IAPs
(Ryoo et al., 2002;
Yoo et al., 2002;
Holley et al., 2002). The
overexpression of the baculovirus protein P35 can block caspase-dependent cell
death (Hay et al., 1994).
However, there are also some caspase-independent cell-death pathways. For
example, cell death induced by extrinsic signals, such as UV-irradiation,
causes DNA damage and consequently triggers P53-dependent cell death
(Brodsky et al., 2000;
Ollmann et al., 2000).
In the growing discs, apoptosis can be induced by a variety of stimuli,
such as inappropriate levels of morphogens or extracellular signaling
(Mehlen et al., 2005).
Interestingly, the morphogens, such as Decapentaplegic (Dpp; a homolog of
transforming growth factor-ß) and Wingless (Wg; a Wnt homolog protein),
that are required for early developmental steps, cause cell death when
ectopically induced in a developing wing imaginal disc
(Adachi-Yamada et al., 1999;
Ryoo et al., 2004). During eye
development, one of the many functions of Wg signaling is to induce apoptosis
by activating the expression of hid, rpr and grim in
ommatidia at the periphery of the eye during the pupal stage
(Lin et al., 2004;
Cordero et al., 2004).
When secreted morphogens attain inappropriate levels in the wing disc,
activation of the c-Jun N-terminal kinases (JNKs) of the mitogen-activated
protein-kinase super family occurs
(Adachi-Yamada et al., 1999).
This JNK-signal activation leads to caspase-3 activation, which induces cell
death to eliminate cells with aberrant morphogen signal and to correct the
morphogen gradient (Adachi-Yamada et al.,
1999; Adachi-Yamada and
O'Connor, 2002; Moreno et al.,
2002). Caspase-3 plays a central role in many types of apoptosis,
whereas JNK activation elicits a limited group of apoptotic events
(Davis, 2000). In some cases,
JNK signaling can induce caspase-independent cell death. Thus, the
JNK-signaling pathway regulates apoptosis as well as other fundamental cell
behaviors such as differentiation and morphogenesis
(Adachi-Yamada and O'Connor,
2004; Stronach,
2005). In Drosophila, JNK signaling has a core signaling
module consisting of Hemipterous (Hep, a JNK kinase), Basket (Bsk, a JNK) and
Jun (Stronach, 2005). JNK
signaling is manifested by the expression levels of puckered
(puc), a gene encoding a dual-specificity phosphatase that forms a
negative feedback loop by downregulating the activity of JNK
(Martin-Blanco et al., 1998;
Adachi-Yamada et al., 1999).
Despite the known function of JNK signaling in morphogenetic cell death, its
role in apoptosis during early eye imaginal disc development is not known.
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We propose that L and Ser, members of the N-signaling pathway, are required for cell survival during early eye development. We also present evidence that L and Ser promote cell survival through a mechanism that involves inactivation of the Wg signaling pathway. In the early eye, LOF of L/Ser results in the upregulation of Wg and the induction of caspase-dependent cell death. Our results demonstrate that, during the second larval instar stage, a time window prior to the initiation of retinal differentiation, the ventral cells are preferentially sensitive to Wg-dependent cell death. Wg is known to induce JNK signaling in the wing. In the eye, blocking the JNK signaling pathway can significantly rescue the L/Ser-mutant phenotype. Lastly, we found that the L/Ser-mutant phenotype observed in the eye is a result of the cumulative effect of the induction of the JNK-signaling pathway and of caspase-dependent cell death. Thus, we have identified a cell-survival mechanism required for early eye development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), y w; Lrev6-3FRT42D/CyO,
L2/CyO, Lsi,
(Chern and Choi, 2002;
Singh and Choi, 2003),
UAS-SerDN (Hukriede et
al., 1997), wg1
(Couso and Martinez-Arias,
1994), wgCX3, wgCX4, UAS-wg
(Azpiazu and Morata, 1998),
wgIL114
(Nusslein-Volhard et al.,
1984), wg-LacZ/CyO
(Kassis et al., 1992),
UAS-sggS9A, ey-GAL4
(Hazelett et al., 1998),
UAS-arm (Zecca et al.,
1996), UAS-dTCFDN
(van de Wetering et al.,
1997), pucE69, UAS-puc
(Martin-Blanco et al., 1998),
hepr75/FM7 (Glise et
al., 1995), bsk2 FRT40/CyO, UAS-bsk
(Boutros et al., 1998),
UAS-bskDN
(Adachi-Yamada et al., 1999),
UAS-DJunaspv7 (Treier
et al., 1995), UAS-p53 and UAS-p53DN
(Brodsky et al., 2000;
Ollmann et al., 2000),
UAS-P35 (Hay et al.,
1994), UAS-diap1
(Lisi et al., 2000), and
Df(3L)H99/TM6B (Abbott and
Lengyel, 1991). We used a GAL4/UAS system for targeted
misexpression studies (Brand and Perrimon,
1993). All GAL4/UAS crosses were performed at 18, 25 and 29°C,
unless specified, to sample different induction levels.
Genetic mosaic analysis
LOF clones were generated using the FLP/FRT system of mitotic recombination
(Xu and Rubin, 1993). To
generate LOF clones of L in eye, eyFLP; FRT42D ubi-GFP
females were crossed to Lrev6-3 FRT42D males. MARCM clones
were generated (Lee and Luo,
1999) by crossing yw, hs-FLP, tub-GAL4, UAS-GFP-6xMYC-NLS;
tubP-GAL80, FRT42/CyO virgins to Lrev6-3 FRT42D;
UAS-sggS9A or Lrev FRT42D;
UAS-dTCFDN males.
Immunohistochemistry
Eye-antenna discs were dissected from wandering third instar larvae and
stained following the standard protocol
(Singh et al., 2002).
Antibodies used were mouse rabbit anti-ß-galactosidase (1:200) (Cappel);
chicken anti-GFP (1:200; Upstate Biotechnology); rat anti-Elav (1:100); mouse
22C10 (1:20); mouse anti-Wg (1:20) (Developmental Studies Hybridoma Bank);
rabbit anti-Drosophila Ice (Drice) (a gift from B. Hay, California
Institute of Technology, CA, USA); and rabbit anti-Dlg (a gift from K. Cho,
Baylor College of Medicine, Houston, TX, USA). Secondary antibodies (Jackson
Laboratories) used were goat anti-rat IgG conjugated with Cy5 (1:200); donkey
anti-rabbit IgG conjugated to Cy3 (1:250) or donkey anti-mouse IgG conjugated
to FITC (1:200); and donkey anti-chicken IgG conjugated to FITC.
Detection of cell death
Apoptosis was detected by TUNEL assays. Eye-antennal discs, after
secondary-antibody staining (Singh et al.,
2002), were blocked in 10% normal goat serum in phosphate buffered
saline with 0.2% Triton X-100 (PBT) and labeled for TUNEL assays using a
cell-death detection kit from Roche Diagnostics.
Temperature shift regimen
Eggs were collected from a synchronous culture for a period of 2 hours.
Each egg collection was divided into several batches in different vials. These
independent batches of cultures were reared at 16.5°C, except for a single
shift to 29°C in a 24-hour time window during different periods of
development spanning from first instar to the late third instar of larval
development. After the incubation at 29°C, these cultures were returned to
16.5°C for the latter part of development.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and the expression of the cell-proliferation
marker phospho-Histone H3, was not significantly affected in LOF clones of
Lrev6-3 (Lrev), a null allele of
L (Chern and Choi,
2002), and L2/+-mutant eye discs (data not
shown). L2, a dominant-negative allele of L,
shows loss of ventral eye in the L2/+ background in 100%
of flies (Fig. 4A,B)
(Singh and Choi, 2003;
Singh et al., 2005a). We
overexpressed cyclin A, cyclin B and cyclin E to accelerate cell-cycle
progression in the L2/+-mutant background and did not find
any significant rescues (data not shown). These results suggested that the
cell-proliferation arrest may not play a significant role in the
loss-of-ventral-eye phenotype in L mutation.
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; Ollmann et al.,
2000). Overexpression of a Drosophila p53 homolog
(hereafter p53) in the eye, by eyeless (ey)-GAL4
(ey>p53) results in reduced eyes
(Fig. 2A). In the
L2/+-mutant eye, overexpression of p53
(L2/+; ey>p53) did not enhance the
L2/+-mutant phenotype of ventral eye loss
(Fig. 2B,B'). We verified
our results by overexpressing dominant-negative p53,
UAS-p53DN, in the eye (L2/+;
ey>p53DN) and found that the
L2/+-mutant phenotype was not rescued (data not shown).
Our results suggest that cell death from the loss of L gene function
is p53-independent.
We then tested whether the cell death observed because of the loss of
L function was caspase-dependent by determining the effects of
altering the level of caspase-pathway gene functions in the
L2/+ background. Overexpression of the baculovirus P35 in
the Drosophila eye (ey>P35) selectively blocks
caspase-dependent cell death (Hay et al.,
1994), and resulted in eye discs that resemble those of wild-type
flies in size (Fig. 2C).
Overexpression of P35 in the eye of L-mutants
(L2/+; ey>P35) restored the
loss-of-ventral-eye phenotype to more than three-quarters of the wild-type eye
size in 40% of the flies (Fig.
2D,D', Table
1). In addition, a range of weaker rescue phenotypes (partial
rescue of L2/+ ventral eye loss) was observed in the
remaining 60% of L2/+; ey>P35 flies.
Overexpression of diap1 (also known as thread - Flybase), an
inhibitor of apoptosis, in the L-mutant background
(L2/+; ey>diap1) rescued the
L2/+-mutant phenotype
(Fig. 2F,F'). The
frequency of strong rescues (i.e. more than three-quarters of the wild-type
eye size) was 27% (Table 1),
and a range of intermediate and weaker rescues were observed in the remaining
progeny. The ey>diap1 control flies showed wild-type eye size
(Fig. 2E).
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; Bergmann et al.,
2003). We tested the role of the HRG complex in the cell death
caused by loss of L gene function. When the gene dosage for HRG was
reduced to half of that observed in wild type in the L2/+
background (L2/+; H99/+) using the
Df(3L)H99 deficiency, which uncovers all three genes
(White et al., 1994), the
L2/+ phenotype of loss-of-ventral-eye was rescued to near
wild type in the eye imaginal disc and in the eyes of 38% of adult flies
(Fig. 2H,H',
Table 1). The control
H99/+ eye discs were normal in size
(Fig. 2G). We verified our
results using other L mutations [Lrev and
Lsi, a hypomorph allele of L
(Chern and Choi, 2002)] to
rule out any allele-specific interactions (data not shown). These results
suggest that members of the HRG complex are required for the L-mutant
phenotype of loss-of-ventral-eye.
During DV patterning of the eye, Ser acts downstream of L
(Chern and Choi, 2002).
Dominant-negative Ser (SerDN) is commonly used to sample the
Ser LOF phenotypes in the eye (Kumar and Moses, 2000;
Singh and Choi, 2003).
Overexpression of SerDN in the eye by an ey-GAL4
driver (ey>SerDN) resulted in the loss of the
ventral-half of the eye (Fig.
2I). We tested whether the Ser LOF phenotype of
loss-of-ventral-eye is due to the induction of caspase-dependent cell death.
Overexpression of P35 in the SerDN background
(ey>SerDN+P35) significantly rescued the loss
of the ventral-half of the eye imaginal disc and the adult eye
(Fig. 2J,J') in 32%
(22/69) of flies, whereas remaining flies showed weaker rescues. As the
frequency of strong rescues by blocking caspase-dependent cell death was less
than 40%, it suggested that caspase-dependent cell death is one of the major,
but not the sole, reason for the loss-of-ventral-eye in
L/Ser mutants.
wg acts antagonistically to L gene function
Ectopic upregulation of Wg in the wing is known to induce cell death
(Adachi-Yamada et al., 1999).
In a genetic screen, we identified Shaggy (Sgg), an antagonist of Wg Signaling
(Hazelett et al., 1998;
Heslip et al., 1997), as the
modifier of the L-mutant phenotype
(Singh et al., 2005a). To test
if the loss of L results in the abnormal induction of Wg signal and
thereby causes cell death, we overexpressed the members of the Wg-signaling
pathway to increase or decrease the levels of Wg signaling and sampled its
effect on the L-mutant phenotype. Overexpression of wg in
the eye of L2/+ mutants (L2/+;
ey>wg) completely eliminated the eye field in the eye imaginal disc
and in adult the eye (Fig.
3B,B', Table
1). Overexpression of wg in the eye (ey>wg)
suppressed eye development (Fig.
3A) (Lee and Treisman,
2001), but the phenotypes are much weaker than
L2/+; ey>wg. Increasing the levels of
Armadillo (Arm) in the wild-type eye (ey>arm) resulted in small
eyes (Fig. 3C), whereas, in the
L2/+ background, L2/+;
ey>arm eliminated the entire eye field
(Fig. 3D,D'). Similar
phenotypes were observed upon overexpression of Dishevelled (Dsh)
(Table 1). Thus, increasing
levels of canonical Wg signaling can enhance the L-mutant
phenotype.
We blocked Wg signaling by overexpressing antagonists of the Wg signaling
pathway. Overexpression of Sgg in the eye (ey>Sgg) does not affect
the eye size (Fig. 3E)
(Singh et al., 2002), whereas,
in the eye of L2/+ mutants (L2/+;
ey>Sgg), overexpression significantly rescued the ventral eye loss in
26% of flies (Fig. 3F,F',
Table 1). In the remaining
progeny, weaker rescue phenotypes were seen. The transcription factor TCF is
the downstream target of Wg signaling, and is inhibited by overexpression of
the N-terminal deleted dominant-negative form of TCF (dTCFDN)
(van de Wetering et al.,
1997). Overexpression of dTCFDN in the eye
(ey>dTCFDN) does not affect eye size
(Fig. 3G)
(Singh et al., 2002), whereas
in the eye of L2/+ mutants (L2/+;
ey>dTCFDN), it significantly rescued the phenotype of the
loss of ventral eye in 23% of flies (Fig.
3H,H', Table
1). Thus, reducing levels of Wg signaling can rescue the
L-mutant phenotype. Similar results were seen in eye discs in which
Ser function was abolished along with increased or decreased levels
of Wg signaling (see Fig. S1 in the supplementary material).
L is required for the repression of Wg expression in the ventral eye
Our results demonstrate that Wg signaling acts antagonistically to
L function in the ventral eye and raise the possibility that
L may act upstream to Wg signal transduction. Therefore, we tested
genetic interaction between L and wg. Loss of L
results in preferential loss-of-ventral-eye pattern in the eye disc
(Fig. 4A) and adult eye
(Fig. 4B)
(Singh and Choi, 2003). The
sections of the L2/+-mutant adult eye showed selective
loss of the ventral ommatidial clusters
(Fig. 4C,C'). When we
generated recombinant chromosome harboring both L and wg
mutations (L2, wg1/CyO), the
loss-of-ventral-eye was strongly rescued to near wild-type eye size in 35% of
flies (Fig. 4D,E,
Table 1). In adult eye sections
of the double mutant fly (L2, wg1/CyO), we
confirmed that ommatidia in the rescued ventral eye
(Fig. 4E) are indeed of the
ventral polarity (Fig.
4F,F'). Similar results were found using other alleles of
L (Lsi and Lrev) and
wg (wgCX3 and wgCX4). This
suggests that the L-mutant phenotype in the ventral eye is probably
caused by ectopic induction of Wg expression.
|
;
Heberlein et al., 1998).
During eye development, the reporter gene reliably represents Wg expression
(Royet and Finkelstein, 1997).
In the third instar wild-type eye disc, wg-lacZ is expressed in the
dorsal, and weakly in ventral, polar margins, anterior to the MF (i.e. on the
DV margins of the undifferentiated eye disc;
Fig. 4G). In the
L2/+-mutant eye disc, the expression of the wg
reporter is ectopically induced on the ventral eye margin posterior to the MF
(Fig. 4H).
To test the possibility that L represses Wg expression only in the
ventral eye, we generated LOF clones of Lrev by the
genetic-mosaics approach (Xu and Rubin,
1993). The LOF clones of L do not show any phenotype in
the dorsal eye, whereas clones in the ventral eye result in the loss of eye
(Singh and Choi, 2003).
Consistent with this observation, LOF clones of L in the ventral eye
showed ectopic induction and upregulation of Wg expression on the posterior
and lateral margin of the ventral eye, whereas dorsal eye clones did not show
ectopic Wg induction (Fig.
4I,I').
Loss of L/Ser induces ectopic Wg and the downstream caspase Drice
Wg induces cell death to eliminate excess ommatidia at the periphery of the
pupal eye (Lin et al., 2004;
Cordero et al., 2004). To test
whether Wg is responsible for the induction of cell death in the eye mutant
for L/Ser, we looked at the expression of the downstream effector
caspase Drice and Wg in LOF clones of L in the eye. The LOF clone of
L at the ventral margin of the eye disc, posterior to the
morphogenetic furrow, showed ectopic induction of activated Drice caspase
accompanied by the induction of Wg (Fig.
5A-A'''). Similar results were observed with Ser
(data not shown). Thus, during eye development, L/Ser function to
prevent cell death, which is most probably induced by aberrant Wg
signaling.
It can also be argued that changes in the eye size because of activation of Wg signaling could be due to changes in the pattern of differentiation or of proliferation rather than cell death. To rule out this possibility and to support that Wg signaling is directly responsible for the cell death of ventral eye cells in L-mutant eye discs, we blocked Wg signaling only in the cells lacking L gene function. We overexpressed Sgg (Fig. 5B) or dTCFDN (Fig. 5C) in the Lrev clones by MARCM analysis and found that these LOF clones of L marked by GFP reporter expression in the ventral eye can no longer eliminate eye and cannot induce activated Drice in these clones. We counted 11 MARCM clones for Sgg overexpression in the ventral eye, and seven for dTCFDN overexpression, and none showed the loss-of-ventral-eye phenotype.
L inactivates Wg during the second larval instar
To understand the physiological relevance of the genetic interaction
between L and wg, we looked for the developmental time
window for Wg to inhibit ventral eye growth. We employed a conditional mutant
wgIL114 that encodes a temperature-sensitive Wg protein
(Baker, 1988;
Treisman and Rubin, 1995). The
cultures harboring L and temperature-sensitive
wgIL114 mutations were maintained at 16.5°C, the
temperature at which mutant Wg protein is functional for most Wg activities
(Baker, 1988). The cultures
were shifted to the restrictive temperature of 29°C in a 24-hour time
window during different stages of development
(Fig. 6A). The rationale was to
block Wg function in the L2/+ background and to look for
the time window in which reducing wg function can rescue the
loss-of-ventral-eye phenotype. Based on sampling of the phenotypes from
different batches, we found that reducing wg function during the
second instar of larval development can significantly rescue the
L-mutant phenotype of ventral eye loss
(Fig. 6A,C). However, reducing
wg function very early during the first instar or later during third
instar of larval eye development had no significant effect on the
L-mutant phenotype (Fig.
6A,B). These results suggest that L and wg act
antagonistically during the second instar of larval eye development before the
onset of retinal differentiation.
|
L blocks ectopic induction of the JNK signaling pathway
Ectopic induction of Wg can induce JNK signaling in developing wing
imaginal discs (Adachi-Yamada et al.,
1999; Moreno et al.,
2002). Therefore, we tested whether elimination of the ventral eye
pattern in the eye of L mutants is affected by the induction of the
JNK signaling pathway. The extent of activation of the Drosophila JNK
pathway can be monitored by puc-lacZ expression
(Adachi-Yamada et al., 1999).
In the eye disc, puc-lacZ is expressed in the peripodial
cells (Adachi-Yamada, 2002). In
the L2/+ mutant eye disc, there was a strong induction of
puc-lacZ on margins of the ventral eye
(Fig. 7A). We also checked
puc-lacZ expression in the LOF clones of Lrev and
found that puc-lacZ was ectopically induced in the
L-mutant cells near the ventral eye margin
(Fig. 7B,B'; arrow). In
the dorsal eye clones, puc-lacZ was not induced
(Fig. 7B,B'; arrowhead).
Puc downregulates JNK activity by a negative-feedback loop
(Martin-Blanco et al., 1998;
McEwen and Peifer, 2005).
Thus, overexpression of Puc can be used to repress JNK activity.
Overexpression of Puc alone (ey>puc) does not affect eye size
(Fig. 7C). However, in the
L2/+- mutant background, puc overexpression
(L2/+; ey>puc) resulted in the rescue of the
ventral eye loss in 36% of flies (Fig.
7D,D', Table
1).
Inhibition of JNK signaling by the use of a dominant-negative form of Drosophila JNK (ey>bskDN) resulted in wild-type eye (Fig. 7E), whereas in L2/+; ey>bskDN, it rescued the loss-of-ventral-eye phenotype in 34% of flies (Fig. 7F, Table 1). Reducing the levels of Drosophila JNK kinase (DJNKK) encoded by hep, strongly rescued the L2/+-mutant phenotype in 32% of hepr75/+; L2/+ flies (Fig. 7G). Overexpression of the activated form of Bsk (ey>bskAct) alone resulted in eyes smaller than those of wild type (Fig. 7H). Consistently, when we increased the levels of Bsk by overexpressing the activated form of Bsk in the L2/+-mutant background (L2/+; ey>bskAct), the loss-of-ventral-eye phenotype was enhanced to near complete loss of eye in 39% of flies (Fig. 7I,I', Table 1). Finally, increasing JNK signaling by the overexpression of the activated form of Drosophila Jun (L2/+; ey>junaspv7) resulted in the strong enhancement of the L2/+- mutant phenotype to a very small eye or no eye in 36% of flies (Fig. 7K,K', Table 1), whereas overexpression of Drosophila Jun alone (ey>junaspv7) resulted in weaker eye reduction (Fig. 7J). Our results demonstrate that the ventral eye cells lacking L gene function ectopically induce the JNK signaling pathway.
L-mutant phenotype depends on both JNK signaling and caspase-dependent cell death
We found that blocking JNK signaling or caspase-dependent cell death
individually did not completely rescue the L-mutant eye and strong
rescues were seen in less than 40% of total progeny. JNK signaling is known to
induce caspase-independent cell death. To test the possibility that the cell
death observed in the L-mutant background results from the cumulative
outcome of both caspase-independent/JNK-signaling mediated cell death and
caspase-dependent cell death, we blocked both JNK signaling and
caspase-dependent cell death together in the L-mutant background.
Overexpression of P35 and Puc (ey>P35+puc), which blocks
caspase-dependent cell death and JNK-signaling, respectively, results in
wild-type eye (Fig. 8A).
Overexpression of P35 and Puc in the L2/+-mutant eye
(L2/+; ey>P35+puc) rescued the ventral eye
loss to a near wild-type eye (Fig.
8B,B') in 69% of flies and pharates
(Table 1). There was a greater
than 1.5-fold increase in the frequency of strong rescues seen in these
mutants than by blocking caspase-dependent cell death or JNK signaling alone.
Our results suggest that L blocks cell death by preventing caspase
induction, as well as by activating JNK signaling.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and
that L/Ser are required for development of the ventral eye
(Chern and Choi, 2002;
Singh and Choi, 2003;
Singh et al., 2005a;
Singh et al., 2005b). During
early eye development, little or no cell death is observed. At the mid-pupal
stage, programmed cell death plays an important role in the selective removal
of a large number of excess undifferentiated cells in the interommatidial
space that fail to be recruited to the ommatidia
(Miller and Cagan, 1998;
Rusconi et al., 2000). During
this time window of pupal development, EGFR and Ras act as survival cues
(Silver and Rebay, 2005;
Bergmann et al., 2002; Yang and Baker,
2003). Surprisingly, there is little information concerning genes
responsible for cell survival during early larval eye imaginal disc
development. Our studies show that, during early eye development, L
and Ser are required for the survival of ventral eye cells. We found
that one of the major reasons for the elimination of the ventral eye cells in
L/Ser mutants is due to the induction of caspase-dependent cell
death. We found that L- and Ser-mutant phenotypes can be
rescued by blocking inappropriate induction of (i) Wg, (ii)
JNK-signaling-mediated cell death and (iii) caspase-dependent cell death.
Role of Wg in L/Ser function in the eye
In animal tissues, Wg is required to drive developmental patterning. Wg is
produced in a restricted area and is distributed either by diffusion or by
transport to generate a concentration gradient throughout the tissue to induce
proper differentiation (Tabata,
2001; Adachi-Yamada and
O'Connor, 2004). In the developing wing imaginal disc, Wg has also
been shown to promote growth. By contrast, abnormal expression of Wg or Dpp
triggers aberrant differentiation signals that result in the induction of
apoptotic cell death in the wing disc
(Adachi-Yamada et al., 1999).
However, it is difficult to directly extrapolate results from the wing disc to
the eye disc because of organ-specific functions of Wg.
In the eye, Wg has complex functions at different stages of development:
(i) prior to eye differentiation, Wg is involved in growth and in the
establishment of the dorsal eye fate (McNeill et al., 1998; Maurel-Zaffaran
and Treisman, 2000); (ii) during eye differentiation, initiation of the
morphogenetic furrow by hedgehog (hh) is restricted to the
posterior margin by the presence of Wg, which represses hh and
dpp at the lateral eye margins
(Ma and Moses, 1995;
Treisman and Rubin, 1995;
Dominguez and Hafen, 1997);
and (iii) in the pupal stage, Wg is responsible for inducing apoptosis by
activating the expression of hid, rpr and grim in ommatidia
at the periphery of the eye (Lin et al.,
2004; Cordero et al.,
2004).
We found that, during early eye development, L and Ser are required to
repress Wg signaling in the ventral eye disc. Our genetic-interaction studies
demonstrate that Wg expression is ectopically induced in the L-mutant
background (Figs 3,
4,
5 and
6). Here, we propose a model in
which L and Ser downregulate the level of Wg activity and
expression in the eye. Loss-of-function of L/Ser, induce higher
levels of Wg, which is coincident with the elimination of the ventral eye
pattern by ectopic induction of caspase-dependent cell death
(Fig. 9). Because blocking
caspase-dependent cell death in L/Ser-mutant backgrounds results in
striking but incomplete rescues of the loss-of-ventral-eye phenotypes, it
suggests that L/Ser-mutant eye phenotypes are not solely due to the
induction of caspase-dependent cell death. It is possible that ectopic
upregulation of Wg signaling in the LOF of L/Ser causes abnormal
induction of JNK signaling or that L/Ser LOF can induce JNK signaling
independently (Fig. 9).
Upregulation of JNK signaling can also induce caspase-independent cell death.
It is possible that the loss of L/Ser can result in cell death caused
by both caspase-dependent and caspase-independent mechanism. This may be one
of the underlying developmental mechanisms for the early cell-survival
function of L and Ser
(Fig. 9). However, there can be
several other interesting possibilities. Other studies have shown that JNK can
be activated downstream of rpr, and that it affects the extent of
rpr-induced cell death (Kuranaga
et al., 2002). Also, wg can be induced downstream of
hid and diap1 (Ryoo et
al., 2004; Huh et al.,
2004). Therefore, one can suggest an alternative model where a low
level of apoptosis induced by hid, rpr and grim is augmented
by a secondary activation of JNK and Wg, which ultimately results in eye
ablation.
|
;
Ryoo et al., 2004;
Perez-Garizo et al., 2005). In cellular injury, Diap1 is inhibited, whereas
the JNK pathway and Wg/Dpp are induced in the apoptotic cells. Secretion of
these factors stimulates the growth or proliferation in competent neighboring
cells and leads to compensatory proliferation
(Ryoo et al., 2004). In other
scenarios in which morphogenetic cell death occurs, altered levels of
morphogenetic signals give rise to abnormal cell types, which are frequently
removed by activating apoptotic signals. Morphogenetic cell death is one of
the strategies employed by the developing field to correct its morphogen
gradient by eliminating cells with abnormal levels of morphogen by inducing
the JNK-signaling pathway (Adachi-Yamada et
al., 1999; Adachi-Yamada and
O'Connor, 2004). Cell death caused by loss of L/Ser function results in the induction of both JNK- and Wg-signaling pathways. However, the outcome is different from that seen in compensatory proliferation or morphogen-gradient correction. Instead of compensatory growth in neighboring cells, LOF of L/Ser triggers ectopic signaling, which can be neither corrected nor compensated for. As a consequence, the affected tissue, in this case the ventral half of the eye discs, cannot be rescued. It results in the loss of the ventral or entire eye field, depending upon the domain of function of these survival factors. Our results demonstrate that one of the essential roles of L and Ser is their requirement for the survival of early proliferating cells in the eye.
Role of Notch signaling in cell survival in the early eye
During development, N signaling is involved in many processes, including
cell-fate commitment, cell-fate specification and cell adhesion
(Schweisguth, 2004). In the
Drosophila eye, N signaling plays important roles in compound eye
morphogenesis, such as DV patterning, cell proliferation and differentiation
in the eye. However, N signaling has not been shown to promote cell survival
during early eye development. Our studies raise the possibility of the role of
the Ser-N pathway in cell survival during early eye development. Earlier, the
extremely reduced or complete loss of eye field in N mutant eye disc was
interpreted as being caused by a loss of proliferation. Our data raises
another possibility: that N signaling may be playing an important role in cell
survival.
The compound eye of Drosophila shares similarities with the
vertebrate eye. There is conservation at the level of genetic machinery, as
well as in the processes of differentiation
(Hartenstein and Reh, 2002;
Peters, 2002). Thus, the
information generated in Drosophila can be extrapolated to higher
organisms (Bier, 2005). As Wnt
signaling induces programmed cell death in patterning the vasculature of the
vertebrate eye (Lobov et al.,
2005), it will be important to study what molecules prevent Wg
signaling from inducing cell death during early eye development. Mutations in
Jagged1, the human homolog of Ser, is known to cause
autosomal-dominant developmental disorder, called Alagille's syndrome, which
also affects eye development (Alagille et
al., 1987; Li et al.,
1997). Hence, it would be interesting to study what roles N
pathway genes play in cell survival during early eye development and in the
early onset of retinal diseases.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/23/4771/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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