Notch (N) signal is activated at the dorsoventral (DV) border of the Drosophila eye disc and is important for growth of the eye disc. In this study, we showed that the Pax protein Eyg is a major effector mediating the growth promotion function of N. eyg transcription is induced by N signaling occurring at the DV border. Like N, eyg controls growth of the eye disc. Loss of N signaling can be compensated by overexpressing eyg, whereas loss of the downstream eygblocked the function of N signaling. In addition, we showed that Nand eyg could induce expression of upd, which encodes the ligand for the Jak/STAT pathway and acts over long distance to promote cell proliferation. Loss of eyg or N can be compensated by overexpressing upd. These results suggest that upd is a major effector mediating the function of eyg and N. The functional link from N to eyg to upd explains how the localized Notch activation can achieve global growth control.

Introduction

The size of an organ is controlled by multiple processes that have to be coordinately regulated. The Drosophila compound eye has been used extensively as an excellent model system to study the growth control of an organ. In this study, we provide a link from a localized signal to a long-range signal to coordinate the growth of the entire organ.

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 Notchligand, 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 eygmay 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 updmutations caused a reduction of eye size, while overexpression of updcaused 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 updexpression. This functional link explains how the localized N signal from the DV organizer can non-autonomously affect the growth of an entire organ.

Materials and methods

Fly stocks

Fly culture and crosses were performed according to standard procedure at 25°C unless otherwise noted. In these experiments, transgenic lines used were UAS-eyg (Jang et al., 2003), UAS-Nact(Go et al., 1998), UAS-NDN(Rebay et al., 1993), UAS-H (Go et al.,1998), UAS-Su(H), UAS-Ser, UAS-Dl,y w hsFLP12; Sco/CyO, y w hsFLP22; CxD/TM3, Sb and Act5C>y+>GAL4, UAS-GFPS65T were kindly provided by G. Doughty,A. Nagel and J. E. Treisman, respectively. UAS-upd(Harrison et al., 1998; Zeidler et al., 1999) was from N. Perrimon. The GAL4 driver strains used were dpp-GAL4(Staehling-Hampton et al.,1994) and ey-GAL4(Quiring et al., 1994). Alleles used were eyg1 (weak mutant), eygM3-12 (null mutant)(Jang et al., 2003) and HE31 (Bailey and Posakony, 1995). upd-lacZ (also called PD)(Sun et al., 1995) was identified by a P{lacW}-mediated enhancer/silencer screen(Sun et al., 1995).

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-β-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).

Results

eyg controls cell proliferation in the early eye disc

eyg is expressed in the embryonic eye primordium and in the larval eye disc (Jang et al., 2003; Dominguez et al., 2004). In loss-of-function eyg mutants, the eye disc is reduced. The size reduction is apparent in early third instar eye disc and cannot be rescued by blocking apoptosis (Jang et al.,2003), suggesting that eyg controls cell proliferation in early eye disc. We have identified a genomic fragment (E2-EX) from the eyg-toe locus that specifies eyg expression in the embryonic eye primordium to the second instar eye-antennal disc, but not in the third instar eye-antennal disc (not shown). When this fragment was used to drive the expression of eyg by the GAL4-UAS system(Brand and Perrimon, 1993), the eyg mutant phenotypes can be partially rescued. The eyg1/eygM3-12 has no eye(Fig. 1A), but can be rescued to have one small eye (37%; Fig. 1B) or two small eyes (42%). The eygM3-12homozygotes are headless (Fig. 1C), but can be rescued to have small head with antennae(Fig. 1D, 64%). These results indicate that eyg has a role in regulating cell proliferation in the early eye disc. When eyg is ectopically expressed by dpp-GAL4 (Jang et al.,2003) or clonally induced by the flp-out method(Fig. 1E,F), it caused overgrowth in the eye disc and in proximal regions of the antennal disc. Clones posterior to the MF caused only one or two extra ommatidia, perhaps because the time before the passage of the MF is limited. Thus, eygis a potent regulator of cell proliferation.

Fig. 1.

eyg promotes growth of the early eye disc. The eyg1/eygM3-12 mutants have no eye (A), but the phenotype can be rescued by expressing eyg driven by the E2-XS enhancer fragment (B). The eygM3-12 homozygotes are headless (C), but the phenotype can be rescued by expressing eygdriven by the E2-XS enhancer fragment (D). (E,F) Clonal expression of eyg (marked by GFP, green) can induce overgrowth (arrows) in the eye disc and antenna disc. Clones posterior to the MF caused only a few extra ommatidia (arrowhead). (E) DIC image.

Fig. 1.

eyg promotes growth of the early eye disc. The eyg1/eygM3-12 mutants have no eye (A), but the phenotype can be rescued by expressing eyg driven by the E2-XS enhancer fragment (B). The eygM3-12 homozygotes are headless (C), but the phenotype can be rescued by expressing eygdriven by the E2-XS enhancer fragment (D). (E,F) Clonal expression of eyg (marked by GFP, green) can induce overgrowth (arrows) in the eye disc and antenna disc. Clones posterior to the MF caused only a few extra ommatidia (arrowhead). (E) DIC image.

eyg expression is induced by Notch signaling

eyg is expressed at the equatorial region in the eye disc from late second instar to late third instar(Jang et al., 2003; Dominguez et al., 2004). The equatorial region is the dorsoventral boundary, which is also the region of N activation (Cho and Choi,1998). N regulates cell proliferation in the eye disc(Cho and Choi, 1998; Dominguez and de Celis, 1998). In Nts mutant that had grown at the non-permissive temperature during the second instar stage, the eye size is reduced(Shellenbarger and Mohler,1978). In such flies, the expression of the mini-whitereporter gene in the eygM3-12 enhancer trap line(Fig. 2A) is also reduced in the eye (Fig. 2B). This result suggests that N signaling is required for eyg expression in the eye. Clonal expression of a constitutively activated N(Nact) in the eye disc can induce overgrowth when located anterior to the MF (Fig. 2C,D). A clone near the lateral margin caused non-autonomous overgrowth, while a central clone caused only local overgrowth. Anterior to the MF, the eyg-lacZ (using the eygM3-12 enhancer trap line) was induced in the overgrown tissue(Fig. 2D,F, arrow) except when the clones were located within the wg expression domains in the dorsal and ventral margins (Fig. 2D,F, arrowhead). By contrast, WG level was enhanced by Nact only when the clone lies within the wgdomain (Fig. 2D,E, arrowhead). Posterior to the MF, the Nact clones did not induce eyg-lacZ expression but inhibited neural differentiation(not shown), owing to the inhibition of ato expression by N(Baker et al., 1996). In the antennal disc (Fig. 2C-F) and wing disc (not shown), the Nact clones caused tissue overgrowth but did not induce eyg-lacZ expression. These results suggested that eyg transcription is specifically induced by N in the undifferentiated cells in the central equatorial region of the eye disc.

Fig. 2.

Notch signaling induces eyg transcription in eye disc. (A) eygM3-12/TM3 fly is an enhancer trap line with a P[lacW] inserted in the eyg locus. The mini-whitereporter in P[lacW] reflected the eyg expression pattern and in eygM3-12/TM3 shows the eye pigmentation pattern in an anterior-central region of adult eye. (B) The reporter expression in eygM3-12/TM3 is reduced in the Ntsmutant background. The eggs were laid at 18°C, raised at 18°C for 5 days, then shifted to 29°C until adults eclosed. The eye size is reduced.(C-F) Clones of ectopic Nact (marked by GFP, green) caused tissue overgrowth in eye-antennal disc. (C) DIC image. (D,F) eyg-lacZ (eygM3-12, stained by anti-β-galactosidase, red) expression is activated (arrow) within the Nact clones in eye discs except in the wg domain(arrowhead). (E) WG (stained by anti-WG, blue) is enhanced (arrowhead) within the wg endogenous expressing domain. (G-L) In most Su(H)SF8 clones (marked by the absence of GFP, green) eyg-lacZ (eygM3-12, stained by anti-β-galactosidase, red) expression were not detected. But in a few clones, the mutant cells at the border of the clone still expressed eyg-lacZ.

Fig. 2.

Notch signaling induces eyg transcription in eye disc. (A) eygM3-12/TM3 fly is an enhancer trap line with a P[lacW] inserted in the eyg locus. The mini-whitereporter in P[lacW] reflected the eyg expression pattern and in eygM3-12/TM3 shows the eye pigmentation pattern in an anterior-central region of adult eye. (B) The reporter expression in eygM3-12/TM3 is reduced in the Ntsmutant background. The eggs were laid at 18°C, raised at 18°C for 5 days, then shifted to 29°C until adults eclosed. The eye size is reduced.(C-F) Clones of ectopic Nact (marked by GFP, green) caused tissue overgrowth in eye-antennal disc. (C) DIC image. (D,F) eyg-lacZ (eygM3-12, stained by anti-β-galactosidase, red) expression is activated (arrow) within the Nact clones in eye discs except in the wg domain(arrowhead). (E) WG (stained by anti-WG, blue) is enhanced (arrowhead) within the wg endogenous expressing domain. (G-L) In most Su(H)SF8 clones (marked by the absence of GFP, green) eyg-lacZ (eygM3-12, stained by anti-β-galactosidase, red) expression were not detected. But in a few clones, the mutant cells at the border of the clone still expressed eyg-lacZ.

Delta (Dl) and Serrate (Ser) are two ligands for the N receptor. Dlrev10 and SerRX82 transheterozyous mutant has normal eye size. In Dlrev10, SerRX82and eygM3-12 triple heterozygous mutant, there is a reduction of eye size and mini-white reporter expression(Fig. 3A-C). Based on this genetic interaction, we tested which ligand contributed to this growth ability. When Dl or Ser are clonally expressed in the eye disc, eyg-lacZ was generally not induced within the clones(data not shown). However, when the Dl-expressing clone is located in the ventral part of the eye disc, eyg was non-autonomously induced at the border of the clone (Fig. 3G-I). Similarly, in dorsal Ser-expressing clones, eyg is induced non-autonomously at the clone border(Fig. 3D-F). The eyginduction only occurs when the Dl- and Ser-expressing clones are located near the central region of the eye disc. In eye discs, Delta is normally expressed in the dorsal side and Serrateis detected in the ventral side (Cho and Choi, 1998). Thus, the ventral Dl-expressing clones and dorsal Ser-expressing clones created novel DV borders, which should cause N activation. eyg induction is therefore consistent with N activation.

Fig. 3.

eyg is induced at new DV border created by Ser- or Dl-expressing clones. (A-C) In Dlrev10,SerRX82 and eygM3-12 triple heterozygous mutant, the eye size and eyg expression (eye pigmentation based on the mini-white reporter in eygM3-12) were both reduced (1% of flies are like A; 98% are like B; 1% are like C). (D-I) Ectopic expressing clones were marked by GFP (green). Discs were stained using anti-Elav (blue) and anti-β-galactosidase (eygM3-12,red) antibodies. (D-F) When the Ser-expressing clone is located in the dorsal side near the DV midline of eye disc, eyg-lacZ is induced non-autonomously at the border of the clone (arrow). E,F are higher magnifications of D. (G-I) When the Dl-expressing clone is located in the ventral side near the DV midline of eye disc, eyg-lacZ is induced non-autonomously at the border of the clone (arrow). H,I are higher magnifications of G.

Fig. 3.

eyg is induced at new DV border created by Ser- or Dl-expressing clones. (A-C) In Dlrev10,SerRX82 and eygM3-12 triple heterozygous mutant, the eye size and eyg expression (eye pigmentation based on the mini-white reporter in eygM3-12) were both reduced (1% of flies are like A; 98% are like B; 1% are like C). (D-I) Ectopic expressing clones were marked by GFP (green). Discs were stained using anti-Elav (blue) and anti-β-galactosidase (eygM3-12,red) antibodies. (D-F) When the Ser-expressing clone is located in the dorsal side near the DV midline of eye disc, eyg-lacZ is induced non-autonomously at the border of the clone (arrow). E,F are higher magnifications of D. (G-I) When the Dl-expressing clone is located in the ventral side near the DV midline of eye disc, eyg-lacZ is induced non-autonomously at the border of the clone (arrow). H,I are higher magnifications of G.

Su(H) is a downstream component of N signaling and is required for the growth control of eye disc by N (Li and Baker, 2001). In Su(H)SF8 mutant clones(marked by the absence of the GFP marker), eyg-lacZ is absent or reduced (Fig. 2G-L). This demonstrates that the activation of eyg is through the canonical N signaling pathway. In a few clones, eyg-lacZ is still expressed at the cells adjacent to wild-type tissue(Fig. 2G-I).

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/eygM3-12mutants. 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 eyg1allele.

Fig. 4.

eyg mediates the growth promoting function of Notch signaling. eyg1/eygM3-12 mutants are eyeless with complete penetrance (see Fig. 1D). (A)In eyg1, HE31/eygM3-12 the eye size is partially restored. The eye size is also partially restored in (B) dpp-GAL4 UAS-Nact (abbreviated as dpP>Nact) and (C) dpp>Su(H) in the eyg1/eygM3-12 mutant background. (D,E) When one copy of H was removed in eygM3-12,HE31/eygM3-12, the flies have complete head structures but no eye (D) and eyg-lacZ (anti-β-galactosidase,red) is detected in antennal discs, but not in eye discs (E). The disc is also stained with anti-DAC (green) to mark the antennal disc. (F) In ey>NDN flies, the eye formation is completely inhibited. (G) When eyg and NDN were co-expressed(ey>NDN+eyg), the eye size was partially restored. (H)In ey>H flies, the eye formation is completely abolished. (I) In ey>H+eyg flies, the eye size is partially restored.

Fig. 4.

eyg mediates the growth promoting function of Notch signaling. eyg1/eygM3-12 mutants are eyeless with complete penetrance (see Fig. 1D). (A)In eyg1, HE31/eygM3-12 the eye size is partially restored. The eye size is also partially restored in (B) dpp-GAL4 UAS-Nact (abbreviated as dpP>Nact) and (C) dpp>Su(H) in the eyg1/eygM3-12 mutant background. (D,E) When one copy of H was removed in eygM3-12,HE31/eygM3-12, the flies have complete head structures but no eye (D) and eyg-lacZ (anti-β-galactosidase,red) is detected in antennal discs, but not in eye discs (E). The disc is also stained with anti-DAC (green) to mark the antennal disc. (F) In ey>NDN flies, the eye formation is completely inhibited. (G) When eyg and NDN were co-expressed(ey>NDN+eyg), the eye size was partially restored. (H)In ey>H flies, the eye formation is completely abolished. (I) In ey>H+eyg flies, the eye size is partially restored.

We further tested whether expression of the activated N in eye disc(ey>Nact) can rescue eygM3-12homozygous mutant. Most animals of such genotype die at the larval stage, only 6% survived to the pharate stage. In these pharates, the head development can be rescued, but the eye is usually absent(Fig. 5A). In about 20% of these pharates, the eye is partially rescued(Fig. 5B). Very rarely, the antenna is duplicated (Fig. 5C). When the discs were examined, the antenna disc is always present and has eyg-lacZ expression(Fig. 5D-F). A few of the antennal disc has duplicated or triplicated antennal field, as indicated by the eyg-lacZ expression domain(Fig. 5F). The endogenous eye field (based on the location of the optic stalk and Bolwig nerve; Fig. 5D-F, arrow) is highly reduced, lacks eyg-lacZ expression and has no photoreceptor differentiation (Fig. 5D-F),but an extra eye field is induced dorsal to the endogenous eye field in about 36% of these discs (Fig. 5D,E). The extra eye field can have eyg-lacZ expression and photoreceptor differentiation (Fig. 5E),which accounts for the presence of small adults eyes. These results suggest that eyg is required for the N-mediated proliferation in the endogenous eye field. Outside of the eyg domain in the eye disc, N signaling can induce cell proliferation by an eyg-independent mechanism. As these cells have ey, they are competent for eye development.

Fig. 5.

Effect of N induction in eyg null mutant. Nact was induced by ey-GAL4 (abbreviated as ey> Nact)in eygM3-12 homozygous mutant background. (A) Eighty percent of the pharates have head with no eyes. (B) Twenty percent have partially restored eye. (C) A few flies have a duplicated antennae. (D-F) The antennal discs are restored in size and have eyg-lacZ expression(green). (F) A disc has triplicated antennal fields (indicated by the eyg-lacZ expression). Occasionally one of the antennal fields can be recognized morphologically but lacks eyg-lacZ expression (not shown).(D-F) The endogenous eye field (based on the location of the optic stalk and Bolwig nerve; arrow) is highly reduced, lacks eyg-lacZ expression and has no photoreceptor differentiation (ELAV-staining, red). (D,E) In about 36%of the clones, an extra eye field (arrowhead) is induced dorsal to the endogenous eye field. The extra eye field can have eyg-lacZ (green)expression and photoreceptor differentiation (ELAV, red). The ocellus is marked by ELAV (*). (G) The eyg-lacZ expression pattern(green) in wild type is used for comparison.

Fig. 5.

Effect of N induction in eyg null mutant. Nact was induced by ey-GAL4 (abbreviated as ey> Nact)in eygM3-12 homozygous mutant background. (A) Eighty percent of the pharates have head with no eyes. (B) Twenty percent have partially restored eye. (C) A few flies have a duplicated antennae. (D-F) The antennal discs are restored in size and have eyg-lacZ expression(green). (F) A disc has triplicated antennal fields (indicated by the eyg-lacZ expression). Occasionally one of the antennal fields can be recognized morphologically but lacks eyg-lacZ expression (not shown).(D-F) The endogenous eye field (based on the location of the optic stalk and Bolwig nerve; arrow) is highly reduced, lacks eyg-lacZ expression and has no photoreceptor differentiation (ELAV-staining, red). (D,E) In about 36%of the clones, an extra eye field (arrowhead) is induced dorsal to the endogenous eye field. The extra eye field can have eyg-lacZ (green)expression and photoreceptor differentiation (ELAV, red). The ocellus is marked by ELAV (*). (G) The eyg-lacZ expression pattern(green) in wild type is used for comparison.

If eyg acts downstream of N signaling to control growth, then when N signaling is blocked, overexpression of eyg should be able to rescue the phenotype. Targeted expression of a dominant-negative N(NDN) by the ey-GAL4 caused an `eyeless'phenotype (Fig. 4F)(Kurata et al., 2000). Co-expression of eyg and NDN restored the eye size to nearly normal (Fig. 4G). Similarly, expression of H driven by the ey-GAL4 completely blocked eye development(Fig. 4H), while co-expression of eyg with H can restore the eye size(Fig. 4I). These results indicate that eyg is a major effector mediating N signal to promote growth in the eye disc.

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 Ntsmutant 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.

Fig. 6.

Notch induces upd expression. (A) upd-lacZ(anti-β-galactosidase, red) is expressed at the junction of optic stalk and the posterior margin of eye disc. (B,C) upd-lacZ (red) is induced(arrowhead) in the Nact-expressing clones (marked by GFP,green), but only when the clone is located at the posterior margin of the eye disc. Even large clones in the central region of the eye disc and in antenna disc caused no induction on upd-lacZ. (D,E) NDN-expressing clone (marked by GFP, green) inhibited upd-lacZ (red) expression (arrow). Whereas ey>NDN caused the complete absence of eye development(see Fig. 4F), ey>NDN+upd (F) completely restored the eye size. (G,H) upd RNA in situ hybridization. (G) upd is expressed at the center of the posterior margin in wild-type late second instar eye-antenna disc. (H) Nts eggs were laid at 18°C for 24 hours,cultured at 18°C for 6 days, then shifted to 29°C for 24 hours and dissected for hybridization. Only males were selected for disc dissection. The Nts discs can be further identified by the reduction of eye disc size.

Fig. 6.

Notch induces upd expression. (A) upd-lacZ(anti-β-galactosidase, red) is expressed at the junction of optic stalk and the posterior margin of eye disc. (B,C) upd-lacZ (red) is induced(arrowhead) in the Nact-expressing clones (marked by GFP,green), but only when the clone is located at the posterior margin of the eye disc. Even large clones in the central region of the eye disc and in antenna disc caused no induction on upd-lacZ. (D,E) NDN-expressing clone (marked by GFP, green) inhibited upd-lacZ (red) expression (arrow). Whereas ey>NDN caused the complete absence of eye development(see Fig. 4F), ey>NDN+upd (F) completely restored the eye size. (G,H) upd RNA in situ hybridization. (G) upd is expressed at the center of the posterior margin in wild-type late second instar eye-antenna disc. (H) Nts eggs were laid at 18°C for 24 hours,cultured at 18°C for 6 days, then shifted to 29°C for 24 hours and dissected for hybridization. Only males were selected for disc dissection. The Nts discs can be further identified by the reduction of eye disc size.

upd also genetically interacts with eyg. os1is a hypomorphic allele of upd and exhibits small eyes(Fig. 7A). eyg1 is a hypomorphic eyg allele and also exhibits small eyes. os1; eyg1 double mutants have no eye (Fig. 7B). Removing one copy of eyg (in eygM3-12/+heterozygotes) also enhanced the small eye phenotype of os1 (Fig. 7C). In eyg1 mutant eye disc, upd-lacZ expression is absent(Fig. 7D). Clonal expression of eyg can induce upd-lacZ expression when the clone is at the eye disc margin (Fig. 7E,F). So eyg acts upstream to regulate upd expression. In eyg1/eygM3-12 mutant, which has no eye(Fig. 1D), targeted expression of upd (dpp>upd) can completely restore the eye size(Fig. 7G). Conversely, in the os1 mutant, targeted expression of eyg(ey>eyg) did not rescue the small eye phenotype(Fig. 7H). This is in contrast to the enlarged eye because of ey>eyg in wild-type background(Fig. 7I). These results suggest that eyg acts upstream to induce upd transcription and its effect on cell proliferation is largely mediated through upd.

Fig. 7.

upd expression is induced by eyg and mediates eyg function. (A) The hypomorphic upd allele os1 has small eyes. Similarly, the hypomorphic eyg1 mutants also have small eyes. (B) The os1; eyg1 double mutants have total absence of eyes. (C) The os1 eye phenotype was enhanced by removing one copy of eyg in eygM3-12/+ heterozygote. (D) upd-lacZ (anti-β-galactosidase, red) expression is not detected in eyg1 mutant eye disc. Arrow indicates its normal expression site. (E,F) Clonal expression of eyg (marked by GFP,green) at the eye disc margin non-autonomously induced upd-lacZ (red)expression (arrowhead in F, magnified view of fromed area in E. (G) dpp>upd fully rescued the eye development which is absent in eyg1/eygM3-12. The eyes are slightly larger than wild-type (compare with Fig. 2A). (H) In os1 mutant, the overexpression of eyg (ey>eyg) did not cause eye enlargement, which ey>eyg did in wild-type background (I).

Fig. 7.

upd expression is induced by eyg and mediates eyg function. (A) The hypomorphic upd allele os1 has small eyes. Similarly, the hypomorphic eyg1 mutants also have small eyes. (B) The os1; eyg1 double mutants have total absence of eyes. (C) The os1 eye phenotype was enhanced by removing one copy of eyg in eygM3-12/+ heterozygote. (D) upd-lacZ (anti-β-galactosidase, red) expression is not detected in eyg1 mutant eye disc. Arrow indicates its normal expression site. (E,F) Clonal expression of eyg (marked by GFP,green) at the eye disc margin non-autonomously induced upd-lacZ (red)expression (arrowhead in F, magnified view of fromed area in E. (G) dpp>upd fully rescued the eye development which is absent in eyg1/eygM3-12. The eyes are slightly larger than wild-type (compare with Fig. 2A). (H) In os1 mutant, the overexpression of eyg (ey>eyg) did not cause eye enlargement, which ey>eyg did in wild-type background (I).

Discussion

Turning a localized signal to a global growth signal

Notch signaling controls the growth of the eye disc. In this study, we showed that the Eyg Pax protein is a major effector mediating the growth promoting function of N. N is activated at the DV boundary of the early eye disc. This equatorial N signal then activates eyg expression at the transcriptional level. When N signal is reduced, eyg expression is reduced (Fig. 2B,G-J). When N signal is elevated, eyg expression is induced(Fig. 2C-F). Induction of eyg expression occurs at the DV border between the dorsal Dl-expressing and the ventral Ser-expressing cells(Fig. 3). Furthermore, when the upstream N signal is blocked, overexpression of eyg can rescue the growth defect in the eye (Fig. 4F-I), whereas increasing N signaling cannot rescue the eye-growth defect caused by the downstream eyg gene(Fig. 4D,E). Our analysis showed that the induction of eyg by N is dependent on the ligands Dl and Ser, and involves the effector Su(H)and the antagonist H. Thus, the localized activation of N signal is transmitted to the induction of a transcription factor, Eyg, which then promotes cell proliferation. A recent paper(Dominguez et al., 2004) came to the same conclusion.

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 updexpression 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 eygexpression 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 updexpression 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 updexpression (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.

Acknowledgements

We thank Drs G. Doughty, A. Nagel, J. E. Treisman, N. Perrimon and the Bloomington Stock Center for providing the fly stocks. We are grateful to Drs Cheng-Ting Chien and Gwo-Jen Liaw for valuable suggestions and Maria Dominguez for communicating unpublished results. We thank Chen-Shu Hsieh, Chun-lan Hsu and Yu-Chi Yang for preparing fly food; Mei-Li Chen for maintaining fly stocks; and Chiou-Yang Tang for transgenic flies. This study was supported by grants (NSC 91-2312-B-001-006 and NSC 92-2312-B-001-006) to Y.H.S. from the National Science Council of the Republic of China.

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