1 Institute of Genetics, National Yang-Ming University, Taipei 111, Taiwan,
Republic of China
2 Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan,
Republic of China
* Author for correspondence (e-mail: mbyhsun{at}ccvax.sinica.edu.tw)
Accepted 29 April 2004
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SUMMARY |
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Key words: Drosophila, Eye, Cell proliferation, Growth, Notch, upd, eyg
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Introduction |
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In Drosophila, the compound eye of adult fly is composed of about
750 ommatidia that each contains eight photoreceptors and 12 accessory cells
(Ready et al., 1976). All
these cells are derived from the eye-antennal disc that invaginates from the
ectoderm of the embryo and grows inside the larva. In embryo stage, there are
6-23 cells determined as eye-antennal disc primordium. These cells rapidly
proliferate without differentiation in the first and second instar. In early
third instar, cells at the posterior margin of the eye discs start to
progressively differentiate into ommatidial clusters in a posterior to
anterior direction. The front of the differentiation wave is mark by an indent
called the morphogenetic furrow (MF). The MF is a moving boundary that
separates undifferentiated from differentiated tissue. The differentiation of
eye is complete during metamorphosis. Loss-of-function and gain-of-function
mutations in a number of genes can cause an alteration in the eye size. The
study of these genes has provided some knowledge on the genetic control of eye
size.
The activation of Notch-mediated signaling along dorsoventral (DV)
midline can form an organizer of eye growth and patterning
(Cho and Choi, 1998;
Dominguez and de Celis, 1998
;
Kenyon et al., 2003
;
Papayannopoulos et al., 1998
).
The organizer is established by restricted expression of the Notch
ligand, Delta (Dl) and Serrate (Ser).
Dl is expressed in the dorsal side and Ser is detected
preferentially in the ventral side along midline before the initiation of
differentiation (Cho and Choi,
1998
). This expression pattern creates the DV axis and specifies
the expression domain of Notch (N) in the DV boundary
(Cho and Choi, 1998
;
Dominguez and de Celis, 1998
;
Papayannopoulos et al., 1998
).
N activation in DV boundary is known to play an essential role both
in promoting the growth and in regulating patterning during development of
Drosophila wing and tetrapod limb
(Irvine, 1999
). In eye
development, reducing N, as in Nts mutant grown
at the non-permissive temperature during the second instar stage, caused an
eyeless to headless phenotype
(Shellenbarger and Mohler,
1978
). Blocking N signaling by misexpressing the antagonists
Hairless (H) or a dominant-negative form of N
(NDN) can abolish the eye formation
(Kurata et al., 2000
).
Conversely, targeted activation of N induced strong mitotic activity
in eye discs and caused hyperplasia in adult eyes
(Go et al., 1998
;
Kurata et al., 2000
). These
observations indicated that N is required for the growth of eye
discs. But it is not clear which gene(s) is the downstream effector in this
process. It is also not clear how a localized activation of Notch signaling at
the DV border can affect the growth of the entire eye disc.
eye gone (eyg) is another gene that regulates eye size.
It encodes a Pax transcription factor (Jun
et al., 1998; Jones et al.,
1998
; Jang et al.,
2003
). Loss-of-function eyg mutants show a phenotypic
series ranging from mild reduction of eye size to a headless phenotype that
lacks structures derived from the eye-antennal disc
(Jang et al., 2003
;
Dominguez et al., 2004
). In
eyg mutants, the eye disc is reduced even before photoreceptor
differentiation. Blocking apoptosis by expressing the anti-apoptotic P35
(Hay et al., 1994
) in eye disc
does not rescue the reduced size (Jang et
al., 2003
). These results suggest that eyg plays a role
in the early growth of the eye disc. Consistent with this interpretation,
targeted expression of eyg can cause overgrowth in the eye disc
(Jang et al., 2003
). Like
N, eyg is expressed in the central region of eye discs in second
instar (Cho and Choi, 1998
;
Jang et al., 2003
). The
similarity in expression and in mutant phenotype suggests that eyg
may be a target of Notch and may be an effector of N signaling for eye growth.
However, eyg encodes a transcription factor, so is expected to affect
target gene expression autonomously. For the locally expressed eyg to
affect global growth in the eye disc, it must induce some signaling molecule,
which then promotes long-range cell proliferation.
The Unpaired (Upd) protein, a ligand for the Jak/STAT signaling pathway
(Harrison et al., 1998), was
recently shown to promote cell proliferation in eye disc
(Bach et al., 2003
;
Tsai and Sun, 2004
). Upd is
expressed in the center of the posterior margin of eye disc
(Tsai and Sun, 2004
). This is
the site where the DV border (N activation) intersects the posterior margin,
making upd a candidate target for N. Loss-of-function upd
mutations caused a reduction of eye size, while overexpression of upd
caused enlargement of the eye (Bach et al.,
2003
; Tsai and Sun,
2004
). Upd promotes cell cycle only in the undifferentiated cells
anterior to the MF (Tsai and Sun,
2004
), which mimics the early eye disc before MF initiated. In
addition, Upd can induce cyclin D expression
(Tsai and Sun, 2004
). Thus,
the proliferative function of Upd may be directly linked to the cell cycle
genes. Mostly importantly, Upd protein can distribute over a long distance and
can exert the proliferative effect over a long distance
(Tsai and Sun, 2004
). So,
upd is an ideal candidate to turn the localized N activation into a
global signal for proliferation.
In this study, we provide evidences to show that the N signal at the DV border induces eyg expression, which then induces upd expression. This functional link explains how the localized N signal from the DV organizer can non-autonomously affect the growth of an entire organ.
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Materials and methods |
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Clonal induction
Positively labeled flp-out expression clones were generated by crossing
UAS-lines to hs-FLP22;
Act5C>y+>GAL4 UAS-GFPS65T
(Ito et al., 1997). Heat-shock
induction of hs-FLP22 was at 37°C for 1 hour
at 24-48 hours after egg laying for UAS-eyg, and at 48-72 hours after
egg laying for UAS-Nact, UAS-NDN,
UAS-Ser and UAS-Dl. Mutant clones were induced
by the FLP-FRT method (Xu and Rubin,
1993
). For Su(H)SF8 mutant clones,
hs-FLP22; 2XP[ubi-nls-GFP]FRT40A females
were crossed to Su(H)SF8 FRT40A males. Heat shock
induction of hs-FLP22 was at 37°C for 1 hour
at 24-48 hours after egg laying.
Immunohistochemistry
Late third instar larval imaginal discs were dissected and stained. Primary
antibodies were rat anti-Elav (1:500), mouse anti-WG, mouse anti-DAC (1:200,
Developmental Studies Hybridoma Bank, University of Iowa) and rabbit
anti-ß-galactosidase (1:2000, Cappel). Secondary antibodies (Jackson
ImmunoResearch) were Cy3 anti-rabbit, Cy5 anti-rabbit, Cy3 anti-rat, Cy5
anti-rat FITC anti-mouse and Cy5 anti-mouse.
In situ hybridization
upd antisense probe and hybridization procedure are as described
previously (Tsai and Sun,
2004).
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Results |
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eyg mediates N signal to induce cell proliferation
As eyg expression is activated by N signaling, we asked whether
eyg acts downstream of N signal to promote cell proliferation. The
eyg1/eygM3-12 mutants have no eye
(Fig. 1D). Removing one copy of
Hairless (H), an antagonist of N signaling, can partially
restore the eye size (Fig. 4A).
Targeted expression of Nact
(Fig. 4B) or Su(H)
(Fig. 4C) can also partially
restore the eye size of eyg1/eygM3-12
mutants. One interpretation is that N acted through an
eyg-independent mechanism to affect cell proliferation, which can
compensate for the loss of eyg. Another interpretation is that N
enhanced the eyg expression in the hypomorphic
eyg1 allele. So we tested with the
eygM3-12 null mutant. eygM3-12 is a
deletion that deleted the eyg transcription unit but does not extend
to the adjacent toe gene (Jang et
al., 2003). Removing one copy of H resulted in flies with
a complete head except the eye (Fig.
4D; compare with Fig.
1F). In the third instar larva, expression of
eyg-lacZ is detected in the antennal disc but not in the eye
disc, which is still highly reduced (Fig.
4E). Therefore, when eyg is null, increasing N signaling
cannot promote cell proliferation in the eye disc. The rescue of
eyg1/eygM3-12 mutant by N signaling
must be through the hyper-activation of the eyg1
allele.
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N and eyg activates upd expression
Another gene known to promote cell proliferation is the unpaired
(upd) gene, which encodes a ligand for the Domeless (Dome) receptor
and signals through the Jak/STAT pathway
(Harrison et al., 1998). In
second and early third instar eye disc, upd mRNA is expressed at the
junction of eye disc and the optic stalk
(Tsai and Sun, 2004
).
Loss-of-function upd mutants have small eyes, whereas misexpression
of upd can induce non-autonomous proliferation of the
undifferentiated cells of the eye disc
(Bach et al., 2003
;
Chen et al., 2002
;
Chen et al., 2003
;
Tsai and Sun, 2004
). The site
of upd expression is where the posterior margin intersects with the
DV border, which corresponds to N activation. So we tested the relationship
between upd and the N/eyg pathway.
An upd-lacZ enhancer trap line was used to monitor the
upd expression (Faucheux et al.,
2001; Tsai and Sun,
2004
). Although upd mRNA was not detectable in late third
instar eye disc, the upd-lacZ can be detected in the pattern
reflecting the expression in the early eye disc
(Fig. 6A), probably owing to
perdurance of the reporter protein. Clonal induction of
Nact can induce upd-lacZ expression, but
only in cells that are near the posterior margin
(Fig. 6B,C). Conversely, clonal
induction of NDN can suppress the
upd-lacZ expression (Fig.
6D,E). However, not all cells expressing NDN have lost
the upd-lacZ expression (Fig.
6D,E), perhaps because of the perdurance of the reporter protein.
Alternatively, the upd-lacZ in some cells is induced by short-range
signal from neighboring cells. To avoid these problem, we examined
upd expression by in situ hybridization in Nts
mutant eye disc. In Nts shifted to the restrictive
temperature during eye development, the eye disc is reduced in size and has no
detectable upd mRNA (Fig.
6G,H). Both the gain-of-function and loss-of-function experiments
showed that N acts upstream to induce upd expression.
Targeted expression of NDN, driven by the
ey-GAL4, completely blocked eye development
(Fig. 4F). Co-expression of
upd and NDN rescued eye development and caused a
slightly enlarged eye (Fig.
6F). Thus, upd is epistatic to N.
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Discussion |
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Eyg is a transcription factor, so must activate the transcription of some
genes that promote cell proliferation. Upd is reported to act through the
Jak/STAT signaling pathway to promote cell proliferation
(Tsai and Sun, 2004). We
demonstrate that upd expression is dependent on eyg and N
signaling (Fig. 6B-E,
Fig. 7D-F). Furthermore, when
the upstream N signaling or eyg is reduced, overexpression of
upd can rescue the growth defect
(Fig. 6F, Fig. 7G). The overgrowth effect
due to overexpression of the upstream N or eyg is blocked
when the downstream upd is defective. Our results suggest that
upd is a major effector for the growth promotion by N and
eyg.
Our results have demonstrated the functional link from Notch to
eyg to upd in the promotion of eye growth. The link to
upd solved a long-standing problem. N signaling is activated locally
at the border between the dorsal Dl-expressing cells and the ventral
Ser-expressing cells. How does a localized activation of N signal promote cell
proliferation throughout the entire eye disc? The finding of eyg as
the major mediator of N function did not solve the problem, as Eyg is a
transcriptional factor and is expected to affect target gene expression
autonomously. The link from eyg to upd provided a solution,
as Upd is a diffusible signaling molecule. Upd protein can distribute over a
long distance and exert long-range non-autonomous effect to promote cell
proliferation (Tsai and Sun,
2004). So the localized N activation can locally activate
eyg, which then turns on upd expression, probably through a
short-range signal. The Upd signal then acts over a long range to promote cell
proliferation in the early eye disc.
Mechanisms of induction
Although we demonstrated that N activates eyg, and
eyg activates upd, these transcriptional activation may be
direct or indirect. When novel DV borders were created by ectopic expressing
Dl or Ser, eyg was induced non-autonomously at the border of
these clones (Fig. 3D-I). We
also noted that in Su(H) mutant clones, mutant cells at the border of
the clone can still express eyg-lacZ
(Fig. 2G-I). These observations
suggest that N may induce a short-range signal, which then activates
eyg expression. Alternatively, the apparent non-autonomous induction
may be due to perdurance of the reporter protein in cells that were once close
to the clone border. The induction of upd by eyg also may be
indirect. Clonal expression of eyg also induced upd
expression non-autonomously (Fig.
6E,F). In addition, based on RNA in situ hybridization,
eyg expression in the eye disc did not extend to the posterior margin
(Jang et al., 2003), so does
not overlap with the expression domain of upd
(Tsai and Sun, 2004
). These
suggested that the effect of eyg on upd expression may be
indirect. However, an eyg enhancer trap line showed reporter
expression extending to the posterior margin
(Dominguez et al., 2004
). Thus,
we do not exclude the possibility that Eyg can directly activate the
expression of upd.
The activation of eyg and upd are context dependent. Nact does not induce eyg expression in antenna and wing discs. In the eye disc, Nact induced eyg expression only in the region anterior to the MF, and not within the wg expression domain in the lateral margin (Fig. 2C-F). Similarly, Nact and eyg can only induce upd expression at the margin (Fig. 5B,C; Fig. 6E,F), but not in the center of the eye disc. Nact induce upd at the posterior margin but not lateral margins, while eyg can induce upd in the lateral margins but not in the posterior margin. The context dependence indicates that additional factors are involved to determine the specificity of induction.
In a late third instar eye disc, eyg is expressed in an equatorial
domain that does not overlap with the disc margin, so cannot induce
upd. In early eye disc, eyg expression domain comes closer
to the posterior margin (Jang et al.,
2003). Thus, the induction of upd by eyg is
likely at second instar, which is consistent with the timing of upd
expression (Tsai and Sun,
2004
).
More than a linear pathway
Although eyg plays an important role in mediating the
growth-promoting N signal, it is probably not the only effector. In the
eygM3-12 null mutant, ey>Nact does
not rescue the endogenous eye field, but can still induce proliferation to
provide the antennal disc and an extra eye field
(Fig. 5). Thus, N can induce
proliferation by an eyg-independent mechanism. The effect on antenna
and on eye seems to be separate, because ey>Nact can
induce a large antenna disc with duplicate or triplicate antennal field
without rescue of the eye disc (Fig.
5E). As N can induce upd, but not eyg, in the
posterior margin, the induction of upd can also be through an
eyg-independent mechanism.
Nact can induce overgrowth in the central domain of the eye disc (Fig. 2C,D). In these, eyg, but not upd, is induced. In addition, the overgrowth does not extend much beyond the clone. Ectopic eyg in the central domain also induced proliferation without inducing upd. In eyg1 mutant, there is no upd-lacZ expression in eye disc (Fig. 6D), but the eye is only slightly reduced. These results suggest that the N signaling and eyg can induce local proliferation independent of upd.
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ACKNOWLEDGMENTS |
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