• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of embojLink to Publisher's site
EMBO J. Jun 4, 2008; 27(11): 1633–1645.
Published online May 1, 2008. doi:  10.1038/emboj.2008.84
PMCID: PMC2426722

A Wingless and Notch double-repression mechanism regulates G1–S transition in the Drosophila wing

Abstract

The control of tissue growth and patterning is orchestrated in various multicellular tissues by the coordinated activity of the signalling molecules Wnt/Wingless (Wg) and Notch, and mutations in these pathways can cause cancer. The role of these molecules in the control of cell proliferation and the crosstalk between their corresponding pathways remain poorly understood. Crosstalk between Notch and Wg has been proposed to organize pattern and growth in the Drosophila wing primordium. Here we report that Wg and Notch act in a surprisingly linear pathway to control G1–S progression. We present evidence that these molecules exert their function by regulating the expression of the dmyc proto-oncogene and the bantam micro-RNA, which positively modulated the activity of the E2F transcription factor. Our results demonstrate that Notch acts in this cellular context as a repressor of cell-cycle progression and Wg has a permissive role in alleviating Notch-mediated repression of G1–S progression in wing cells.

Keywords: bantam, dMyc, E2F, micro-RNA

Introduction

During development of multicellular organisms, Wnt/Wingless (Wg)- and Notch-signalling pathways are involved in the determination of a variety of cell fates and in the control of tissue growth (Bray, 2006; Clevers, 2006). In many cellular contexts, genetic manipulations that change the activities of these two proteins result in cancer (Bienz and Clevers, 2000; Radtke and Raj, 2003). Thus, tight regulation of Notch and Wnt/Wg is crucial for proper development and survival of multicellular organisms.

The wing primordium of Drosophila is a very suitable model system to define, at a genetic and cellular level, the role of these pathways in the development of highly proliferative tissues. Cell-fate specification by Notch and Wg is a well-known and defined process; by contrast, little is known about their role in the control of cell proliferation. The wing primordium arises as a group of 30–40 cells in the embryonic ectoderm that proliferates during the three larval stages to reach a final size of around 50 000 cells (García-Bellido and Merriam, 1971; Madhavan and Schneiderman, 1977). Early in development, the wing becomes subdivided into a dorsal (D) and a ventral (V) cell population, or compartment, by the activity of the LIM-Homedomain transcription factor Apterous in D cells (Diaz-Benjumea and Cohen, 1993; Blair et al, 1994), and cell interactions between D and V cells lead to activation of Notch at the compartment boundary (Diaz-Benjumea and Cohen, 1995; de Celis et al, 1996). During the third instar larval stage, Notch activity induces Wg expression at the dorsal–ventral (DV) boundary (Diaz-Benjumea and Cohen, 1995; de Celis et al, 1996), and another set of cell interactions between boundary and nearby non-boundary cells takes place to maintain Notch activity and Wg expression at the DV boundary (Figure 1). The DV boundary has a role in organizing growth of the whole-wing primordium (Diaz-Benjumea and Cohen, 1993). Notch and Wg have been postulated as being responsible for this organizing activity, although data are controversial (Giraldez and Cohen, 2003; Johnston and Sanders, 2003). Late in development, cells at the DV boundary are characterized by cell-cycle arrest and define the so-called Zone of Non-proliferating Cells (ZNC; O'Brochta and Bryant, 1985; Phillips and Whittle, 1993). Wg has been reported to induce this cell-cycle arrest (Johnston and Edgar, 1998; Duman-Scheel et al, 2004).

Figure 1
Wg- and Notch-mediated cell interactions involved in DV boundary formation. (A) Late third instar wing imaginal disc labelled to visualize Wg (green) and Senseless (red) protein expression in boundary and non-boundary cells, respectively. (B) Illustration ...

Here we have revised the role of Wg and Notch in the control of cell proliferation and present evidence that a Wg and Notch double-repression mechanism controls G1–S transition in the wing primordium. Our results indicate that these signalling molecules exert their function through regulation of the bantam micro-RNA (Brennecke et al, 2003) and the proto-oncogene dmyc (Johnston et al, 1999). Our work clarifies and simplifies the role of Notch and Wg in cell-cycle control in the Drosophila wing and provides a very suitable model by which to analyse the function of Notch- and Wg-signalling pathways in the regulation of the cell-cycle machinery.

Results and discussion

The ZNC is defined by Notch activity

Stable activation of Notch and expression of Wg along the DV boundary relies on a positive feedback loop between boundary and non-boundary cells (Figure 1). Boundary cells activate the receptor Notch and express Wg, whereas non-boundary cells respond to Wg and signal back by expressing the Notch ligands Serrate and Delta. Boundary and non-boundary cells are characterized late in development by their cell-cycle arrest and constitute the ZNC (O'Brochta and Bryant, 1985). Anterior non-boundary cells are arrested in G2 by the activity of Wg (Johnston and Edgar, 1998; Figure 8A). Wg exerts its function through its target genes achaete and scute, which inhibit the expression of cdc25/string, the universal eukaryotic regulator of the G2/M transition. Posterior cells and anterior boundary cells are arrested in G1 (Johnston and Edgar, 1998). This arrest has also been postulated to be defined by the activity of Wg (Johnston and Edgar, 1998; Duman-Scheel et al, 2004). Two observations suggest that Notch, and not Wg, is responsible for the cell-cycle arrest in G1. First, the Wg-signalling pathway is blocked in boundary cells by activity of Notch, as recently reported (Buceta et al, 2007; Figure 1B). Second, some low-threshold target genes of Notch, like crumbs, are expressed in a broader domain that includes both boundary and non-boundary cells and corresponds to the ZNC (Figure 2A and B; Herranz et al, 2006). Here we have readdressed the role of Wg and Notch in this process.

Figure 2
The ZNC is defined by the activity of Notch. Late third instar wing discs labelled to visualize cells in S-phase by BrdU incorporation (antibody to BrdU in red or white (A, C, E, G, I, N, O, P)) or expressing the E2F1-responsive reporter ORC1–GFP ...

The ZNC is characterized by its failure to incorporate bromodeoxyuridine (BrdU) (O'Brochta and Bryant, 1985) or by the reduced activity levels of the E2F1 transcription factor (Johnston and Edgar, 1998; Figure 2A and B). E2F proteins, such as Drosophila E2F1, regulate the expression of a number of genes required for S-phase, and their activity is inhibited by the Retinoblastoma (Rb) proteins like Drosophila Rbf (Rb-familiy protein, reviewed by Dyson, 1998). The Rb pathway is a key regulator of the G1–S transition and Drosophila Rbf is required for G1 arrest in the ZNC (Duman-Scheel et al, 2004). Activity of E2F1, labelled as dE2F in the Figures, can be visualized by observing the expression of the E2F1-responsive reporter ORC1GFP (Asano and Wharton, 1999). It consists of the ORC1 promoter bearing functional E2F-binding sites driving the expression of an unstable Ftz–GFP–Myc fusion protein. As the half-life of this protein is short, this reporter monitors newly synthesized protein and permits examination of cell-cycle-regulated transcription in vivo.

We first analysed the role of Notch in defining the ZNC. In a temperature-sensitive Notch loss-of-function background (Nts2) reared for 48 h at restrictive (29°C) temperature, cells at the ZNC incorporated BrdU and expressed ORC1GFP (Figure 2C and D). Blocking Notch activation by expression of a dominant-negative form of the Notch ligand Delta or the nuclear Notch effector Mastermind (Giraldez et al, 2002) caused similar effects (Figure 2E and F). Clones of cells located at the ZNC and mutant for the Suppressor of Hairless (Su(H)) transcription factor, the nuclear mediator of Notch signalling (Bray, 2006), incorporated BrdU and expressed the E2F1-responsive reporter (Figure 2G and H; see also Supplementary Figure S1). We used a fluorescence-associated cell sorter (FACS) to collect data on the cellular DNA content of dissociated, GFP-sorted, ZNC wing disc cells, and to confirm the role of Notch in defining the G1 block. Blocking Notch signalling in these cells led to a decrease in the fraction of cells in G1 (Supplementary Figure S1). Altogether, these results imply that the activity of the Notch-signalling pathway is required for the cell-cycle arrest in G1 that takes place at the ZNC.

Wg has been reported to be required for the G1 arrest at the ZNC, as blocking Wg signalling in all wing margin cells causes cells to enter into S-phase (in C96; UAS–TCFDN larvae; Figure 2N and O; and Johnston and Edgar, 1998; Duman-Scheel et al, 2004; see also Figure 2R for the pattern of expression of the C96–Gal4 driver). We further analysed the requirement of the Wg pathway in this process. Unexpectedly, clones of cells mutant for arrow, the Wg co-receptor (Wehrli et al, 2000), and located at the ZNC, did not incorporate BrdU (Figure 2I; Supplementary Figure S1; 74 out of 84 clones located at the ZNC did not incorporate BrdU; n (scored discs)=33). Similarly, expression in a subset of ZNC cells of a dominant-negative form of the LEF-1/TCF transcription factor (TCFDN), the nuclear mediator of canonical Wg signalling (Logan and Nusse, 2004), did not increase BrdU nor E2F1 activity levels either (Figure 2J; Supplementary Figure S1). Altogether, these results indicate that Wg signalling is not required for the cell-cycle arrest in G1 that occurs at the ZNC. The effects reported on G1 progression after blocking Wg signalling in all wing-margin cells (Johnston and Edgar, 1998; Duman-Scheel et al, 2004) can be explained by the Notch- and Wg-dependent positive feedback loop that operates at the DV boundary (Figure 1B), which may also compromise the activity of Notch in these conditions. Indeed, expression of the Notch-regulated genes cut and wg was reduced (Figure 2K and L; and data not shown), and increasing Notch activity levels, by means of expression of an activated form of the Notch receptor (NINTRA), counteracted the effects of TCFDN expression and restored cut expression (Figure 2M), reduced E2F activity (Figure 2Q) and BrdU incorporation (Figure 2P). Blocking Wg signalling only in a subset of wing-margin cells did not compromise Notch activity, as reported by expression of cut and wingless (Figures 3H, 4F and F′). This might be caused by the fact that the Notch- and Wg-dependent positive feedback loop that operates at the DV boundary is not affected in these conditions. Note also that TCFDN expression in these experiments was temporally controlled by the Gal4/Gal80ts system (see Materials and methods for details) and induced during the third instar stage (30 h before dissection) to circumvent the requirement of Wg signalling in this feedback loop.

Figure 3
Regulation of E2F activity by Notch and Wg. (AH) AbruptexM1 (A, B), dppGal4;UASNintra (C, C′, D, D′), apGal4 Gal80ts, UASGFP; UASTCFDN wing (E, F), apGal4 Gal80ts, UAS ...
Figure 4
A Wg and Notch double-repression mechanism regulates G1 progression. (AD) dpp–Gal4, UAS–GFP; UASNintraUASE2F (A), ap–Gal4, Gal80ts, UAS–GFP; UAS–TCFDN, UAS–E2F (B), ptc–Gal4, ...

A G1–S checkpoint controlled by a Wg and Notch double-repression mechanism

Notch activity is required in ZNC cells to induce cell-cycle arrest in G1, most probably by a reduction in E2F1 activity levels (Duman-Scheel et al, 2004). We then examined whether ectopic activation of Notch could reduce E2F1 activity levels and cause cell-cycle arrest in the rest of the wing cells, the so-called wing-blade cells (Figure 1A). For this purpose, we monitored these parameters in wing discs mutant for the Notch gain-of-function allele AbruptexM1 or expressing an activated form of the Notch receptor (NINTRA). In both cases, the ZNC was expanded (Figure 3A–D), as revealed by the reduced activity levels of E2F1 and the failure to incorporate BrdU. Notch induced this cell-cycle arrest in all cells located in the presumptive wing blade and at any time during the third instar larval stage (Figure 3C and K). Reduction in the activity of E2F1 mediated this cell-cycle arrest, as overexpression of E2F (known to bypass the negative effects of Rbf protein) restored cell-cycle progression, as monitored by BrdU incorporation (Figure 4A). To confirm that these cells were arrested in G1, we coexpressed NINTRA together with cyclin E, the key regulator and rate-limiting factor controlling G1–S transition in wing cells (Knoblich et al, 1994; Neufeld et al, 1998). Interestingly, these cells incorporated BrdU (Figure 4C, compare with Supplementary Figure S2). Coexpression of cdc25/string, the rate-limiting factor of G2–M transition, was not able to rescue the failure to incorporate BrdU (Supplementary Figure S2). Altogether, these results indicate that high levels of Notch induce a G1 block by reducing the activity of E2F1. We noted that high levels of Notch activity were able to induce an increase in BrdU incorporation and E2F activity in the proximal part of the wing (wing hinge, data not shown), and this increase might be the cause of the overgrowth phenotype observed in AbruptexM1 wing discs.

Notch activation along the DV boundary induces Wg expression. Wg protein is secreted and forms a long-range protein gradient that reaches every wing-blade cell (Zecca et al, 1996; Neumann and Cohen, 1997; Strigini and Cohen, 2000). We then analysed the role of Wg in G1–S progression in these cells. For this purpose, we blocked the Wg pathway by expressing the dominant-negative form of TCF (TCFDN) or axin, and monitored BrdU incorporation and E2F1 activity levels. TCFDN or axin expression was temporally controlled by the Gal4/Gal80ts system (see Materials and methods for details) and induced during the third instar stage (30 h before dissection) to circumvent the requirement of Wg signalling in cell survival and wing-fate specification (Giraldez and Cohen, 2003; Johnston and Sanders, 2003). We chose to use strong Gal4 drivers to completely block the Wg pathway. Interestingly, high levels of expression of TCFDN or axin caused cell-cycle arrest in wing-blade cells. Those cells unable to transduce the Wg signal did not incorporate BrdU (Figure 3E and G; Supplementary Figure S1), and in these cells the expression levels of the E2F1-responsive reporter ORC1–GFP were reduced (Figure 3F and H). All cells located in the presumptive wing blade and at any time during the third instar larval stage required activation of the Wg pathway to pass through S-phase (Figure 3E, G and I; compare with Figures 2A and and3J).3J). This cell-cycle arrest was due to a reduction in E2F1 activity levels, as coexpression of E2F rescued the cell proliferation defects (Figure 4B). Expression of cyclin E, and not cdc25/stg, was able to restore cell-cycle progression in the presence of TCFDN (Figure 4D; Supplementary Figure S2). Taken together, these results indicate that Wg signalling is required in wing blade cells to reach the appropriate levels of E2F1 activity to trigger G1 progression.

We noticed that reduced Wg signalling was also able to compromise the mitotic activity of wing blade cells (Supplementary Figure S2). We then analysed the possible regulation of the G2–M transition in these conditions. In wing cells expressing TCFDN, coexpression of cdc25/string, the rate-limiting factor of G2–M transition, was not able to rescue mitotic activity and BrdU incorporation (Supplementary Figure S2). In contrast, expression of cyclin E was able to rescue both of them (Supplementary Figure S2; Figure 4D). Thus, the cell-cycle arrest imposed by lack of Wg signalling takes place only in G1.

Notch- and Wg-signalling pathways exert opposite effects on G1–S transition. Notch imposes G1 arrest whereas Wg is required for G1–S progression. Interestingly, Notch is known to be repressed by the activity of Wg at different levels of its pathway during wing development (Axelrod et al, 1996; Rulifson et al, 1996; Micchelli et al, 1997; de Celis and Bray, 1997; Neumann and Cohen, 1998). Consistent with this, blocking the Wg pathway in the dorsal compartment or in a stripe along the anterior–posterior compartment boundary induced ectopic Notch activation, as monitored by the expression of Wg protein, wg mRNA and wg–lacZ (Figure 4E–G), and clones of cells mutant for arrow, the Wg co-receptor, and located far away from the DV boundary express Wg (Figure 4I). These observations suggest that the opposite effects of Notch and Wg in the G1–S transition could be explained by a Wg-mediated repression of the Notch pathway and a consequent alleviation of the G1 block. To test this, we blocked the Notch pathway in cells unable to transduce the Wg signal and analysed their capacity to progress through G1. Interestingly, wing cells expressing the dominant-negative form of the nuclear Notch effector Mastermind (MamDN), together with TCFDN in the dorsal compartment, incorporated BrdU and increased E2F1 activity levels (Figure 4H and J, compare with Figure 3E and F). Note that the expression pattern of Wg is restored (compare Figure 4E and J). These results imply that a Wg and Notch double-repression mechanism controls G1–S transition in wing cells.

dMyc and bantam micro-RNA mediate regulation of G1–S by Wg and Notch

The proto-oncogene dMyc promotes G1–S progression in Drosophila cells (Johnston et al, 1999) and the cell-cycle arrest in G1 that occurs at the ZNC is defined by the absence of dMyc expression (Johnston et al, 1999; Duman-Scheel et al, 2004; Figure 5A and B). We then analysed the regulation of dMyc expression by the activities of Notch and Wg. Interestingly, and consistent with the above results, downregulation of dMyc expression at the ZNC is defined by the activity of Notch, and not Wg (Johnston et al, 1999; Duman-Scheel et al, 2004). Blocking Notch activation by a dominant-negative form of Mastermind (Giraldez et al, 2002), or by using a Notchts2 loss-of-function background raised at the restrictive temperature during 48 h, led to expression of dMyc at the ZNC (Figure 5C–E). The effects reported on dMyc expression after blocking Wg signalling in all ZNC cells (Johnston et al, 1999; Duman-Scheel et al, 2004; see also Figure 5F) can be explained again by compromised Notch activity, due to the Wg and Notch positive feedback loop that operates at the DV boundary. Increasing Notch activity levels was able to counteract the effects of TCFDN expression (Figure 5G).

Figure 5
Regulation of dMyc expression by Notch and Wg. (AN, P) Wild type (A, B), Notchts2 reared at the restrictive temperature for 48 h (C), apGal4, UASGFP; UASmamDN (D), C96Gal4; UASmamDN (E, H), C96 ...

Expression of activated forms of the receptor Notch (NINTRA), or the transcription factor Suppressor of Hairless (Su(H)-VP16), led to a reduction in dMyc expression levels in wing-blade cells (Figure 5J and K; and Supplementary Figure S3). Ectopic expression of Wg in these cells did not cause such a reduction (Figure 5M). The cell-cycle arrest in G1 that takes place in cells expressing NINTRA was at least in part due to a reduction in dMyc expression levels, as coexpression of dMyc induced cells to enter into the S-phase, as shown by BrdU incorporation (Figure 5Q). This observation correlates with the upregulation of E2F1 activity levels in these cells (Figure 5R). We then examined the contribution of reduced levels of dMyc in the G1 block that occurs at the ZNC. To address this, we analysed the ability of reduced dMyc expression to impose a G1 block at the ZNC in a situation of reduced Notch activity. In wing discs expressing a dominant-negative form of Mastermind along the DV boundary, dMyc expression and BrdU incorporation were increased in ZNC cells (Figure 5H). Decreasing dMyc protein levels in these cells, by means of a dMyc RNA interference construct, restored the ZNC, as revealed by the absence of BrdU incorporation (Figure 5I). These results indicate that reduced dMyc levels caused by the activity of Notch contribute to define the G1 block at the ZNC.

As reported above, a Wg and Notch double-repression mechanism induces G1–S transition in wing cells not located at the ZNC. This mechanism correlates with the presence of dMyc expression, as wing cells unable to transduce the Wg signal (expressing TCFDN) showed reduced levels of dMyc (Figure 5L and N) and coexpression of MamDN, together with TCFDN-rescued dMyc expression levels (Figure 5P). More interestingly, regulation of G1–S transition by this double-repression mechanism was mediated by the presence of dMyc, as coexpression of dMyc together with TCFDN rescued G1–S progression, as visualized by BrdU incorporation (Figure 5O). We noticed that the Wg and Notch double-repression mechanism did not regulate the expression of other Wg-regulated genes like Distalless, as wing cells unable to transduce the Wg signal lost expression of Distalless independently of MamDN coexpression (Figure 5N and P, compare with Figure 5S).

Altogether, these results indicate that dMyc mediates the activities of Notch and Wg in the regulation of G1–S transition in the wing disc, defining the G1 cell-cycle arrest at the ZNC and facilitating G1 progression in the rest of cells. Interestingly, there was a striking correlation between dMyc expression and the activity of the bantam micro-RNA, which controls cell-cycle progression in the Drosophila wing (Figures 6A and and7A;7A; Brennecke et al, 2003). For this reason, we then analysed the role of bantam in this process. To monitor bantam activity, we used a green fluorescent protein (GFP) bantam sensor that expresses GFP under the control of a ubiquitously active tubulin promoter and has two perfect bantam target sites in its 3′ untranslated region (Brennecke et al, 2003). When present, the bantam micro-RNA reduces GFP expression through its RNA interference (RNAi) effect. The expression pattern of GFP is thus a negative image of the activity pattern of bantam micro-RNA. Brennecke et al (2003) showed that the ZNC is characterized by higher levels of the bantam sensor (Figure 6A), and ectopic expression of bantam induces ZNC cells to enter into S-phase, as shown by BrdU incorporation. We also monitored E2F activity levels in these cells and verified that levels of E2F activity rose upon bantam expression in ZNC cells (Figure 6M), thereby indicating that absence of bantam contributes to inducing G1 cell-cycle arrest through regulation of E2F1 activity.

Figure 6
Regulation of bantam activity by Notch and Wg. (AG; I, J) Wild type (A, A′, E), apGal4; UASmamDN (B), dppGal4; UASNintra (C, C′), dpp–Gal4; UASWg (D), C96Gal4; UAS ...
Figure 7
Crosstalk between dMyc and bantam. (A) Wild-type wing disc labelled to visualize the bantam sensor (blue) and dMyc protein expression (red and white). (B) ptcGal4; UASbantamGFP wing disc showing dMyc expression (red and white) and GFP ...

We then analysed the regulation of bantam activity by Notch and Wg. Blocking Notch activation by means of a dominant-negative form of Mastermind (Giraldez et al, 2002) reduced the expression of the bantam sensor at the ZNC (Figure 6B), and expression of an activated form of the Notch receptor (NINTRA) led to an increase in its expression levels in wing-blade cells (Figure 6C and C′). The activity of Notch is not mediated by Wg, as Wg ectopic expression did not cause reduction of bantam activity levels (Figure 6D), and Wg signalling was not required for repression of bantam activity in ZNC cells, either. Again, the reported effects of Wg signalling on bantam activity (Brennecke et al, 2003) can be explained by compromised Notch activity, as increased Notch activity levels were able to counteract the effects of TCFDN expression on bantam activity levels (Figure 6E–G).The cell-cycle arrest in G1 that occurs in cells expressing NINTRA was at least, in part, due to a reduction in bantam expression levels, as coexpression of bantam induced cells to enter into the S-phase, as shown by BrdU incorporation (Figure 6L). We then examined the contribution of reduced bantam activity in defining the G1 block that takes place at the ZNC. In wing discs with reduced Notch signalling along the DV boundary, BrdU incorporation was increased in ZNC cells (Figure 5H). We noted that Notch activity levels were not completely reduced in these conditions, as Wg and Cut were still expressed in some cells at the DV boundary (Figures 5E and and6H).6H). Thus, this background might be able to respond to changes in the levels of bantam activity. Indeed, halving the dose of bantam restored the ZNC, as revealed by the absence of BrdU incorporation (Figure 6H). These results indicate that reduced bantam activity contributes to define the G1 block at the ZNC.

In the rest of the wing cells, a Wg and Notch double-repression mechanism controls G1–S transition. There is again a striking correlation between the activity levels of bantam and this double-repression mechanism. Indeed, wing cells unable to transduce the Wg signal (expressing TCFDN) showed increased expression of the bantam sensor (Figure 6I), and coexpression of MamDN, together with TCFDN, restored the expression levels of the bantam sensor back to the endogenous ones (Figure 6J). More interestingly, regulation of the G1–S transition by this double-repression mechanism was mediated by the presence of bantam, as coexpression of bantam, together with TCFDN, rescued G1 cell-cycle progression, as visualized by BrdU incorporation (Figure 6K).

dMyc and bantam micro-RNA are two independent effectors of Notch and Wg

Notch imposed a G1 arrest at the ZNC by reducing the activity of bantam and the expression of dMyc. Wg repressed Notch and facilitated G1 progression in the rest of wing cells through dMyc and bantam. Expression of either of these genes was sufficient to induce G1 progression and bypass the effects of Notch or absence of Wg. We then questioned whether these effectors are independently regulated by Notch and Wg, or, alternatively, whether their expression depends on each other.

Overexpression of bantam induced an increase in dMyc protein expression in wing-blade cells and at the ZNC (Figure 7B, compare with Figure 7A), and overexpression of dMyc caused an increase in the activity levels of bantam (Figure 7E). By contrast, clones of cells mutant for bantam did not show reduction in dMyc protein levels (Figure 7C and D), and reduced levels of dmyc did not considerably affect bantam activity levels (Figure 7G). Altogether, these results indicate that dMyc and bantam are able, when overexpressed, to modulate each other's expression or activity levels, but are independently regulated by Notch and Wg.

Are dMyc and bantam two independent effectors regulating G1 progression? The evidence for dMyc is clear, as in the presence of Notch or absence of Wg signalling, dMyc expression had the capacity to rescue G1 progression (Figure 5M, O and P), and this happened in the presence of reduced levels of bantam activity. Note that dMyc overexpression was able to increase bantam activity levels only very mildly at the ZNC where Notch activity is high (Figure 7E), and was completely unable to restore the low activity levels of bantam imposed by Notch activation (compare Figures 6C and and7F).7F). By contrast, bantam might rescue G1 progression through regulation of dMyc (Figure 7B). Note that in this case bantam is able to bypass the effect of Notch at the ZNC and induce an increase in dMyc expression (Figure 7B). We then analysed the capacity of bantam to induce G1 progression after reducing dMyc expression levels. Expression of bantam, together with a dmyc double-stranded RNA (dsRNA) construct, induced G1–S progression in wing cells (Figure 7H), and this happened in the presence of very low levels of dMyc (Figure 7H). Altogether, these results imply that bantam and dMyc are two independent effectors of Wg and Notch that regulate G1 progression.

These effectors are able to substitute each other when overexpressed in situations of reduced Wg signalling or high levels of Notch activity. However, their endogenous levels do not appear to do so, as loss of dMyc or bantam activity in clones of cells compromises cell-cycle progression (Johnston et al, 1999; Brennecke et al, 2003), and reduced levels of either dMyc or bantam, were able to define the ZNC (Figures 5 and and6).6). This suggests that the endogenous levels of both effectors are rate-limiting. Interestingly, coexpression of both effectors at endogenous, wing-blade levels was able to drive G1–S progression in ZNC cells. As shown in Figure 7I, ZNC cells with wing-blade levels of bantam activity and dMyc protein were able to enter S-phase, as monitored by BrdU incorporation. Thus, wing cells require the activity of both bantam and dMyc to drive G1–S transition.

Concluding remarks

Here we have analysed the role of Notch and Wg in the regulation of G1–S progression in the Drosophila wing and have provided evidence that a surprisingly linear pathway, based on a Wg and Notch double-repression mechanism, controls this transition (Figure 8B). Notch activity autonomously blocks cells in G1 and defines the ZNC at the DV boundary of the Drosophila wing. Wg activity is required in wing-blade cells to repress Notch signalling and allow cells to enter the S-phase. These signalling molecules exert their function in a linear pathway through the bantam micro-RNA and the dmyc proto-oncogene, two independent effectors controlling the activity of the E2F1 transcription factor and regulate G1-to-S progression. Notch is known to be repressed by the activity of Wg at different levels of its pathway (Axelrod et al, 1996; Rulifson et al, 1996; Micchelli et al, 1997; de Celis and Bray, 1997; Neumann and Cohen, 1998), but the level at which Wg represses Notch activity to facilitate dMyc expression, bantam activity and G1–S progression remains to be elucidated.

Figure 8
Different effects of Notch and Wg in cell-cycle control in the Drosophila wing. (A) Illustration describing the control of the G2-to-M transition in the anterior (a) compartment by the activity of Wg through its target genes achaete and scute (ac ...

There is evidence in mammals that Notch can act as a tumour-suppressor gene or as an oncogene depending on the cellular context (reviewed by Radtke and Raj, 2003). The cellular context should then modulate the differential response of the cells to changes in Notch activity, in some cells leading to hyper-proliferation, and in others to quiescence. How this context and the differential response are defined at the molecular level remains unclear. Drosophila, again, provides a very suitable model system to address this issue. Although our results highlight that Notch is involved in the inhibition of G1–S progression in wing discs, in other developmental contexts Notch exerts a proliferative function (Baonza and Freeman, 2005; Firth and Baker, 2005). Interestingly, in both cases, Notch exerts an effect through Rbf and E2F to positively or negatively control the G1–S transition. We, therefore, speculate that the molecular context is then defined by the effectors available, like dMyc and bantam, or by the presence of nuclear factors that act as a switch in the ultimate activation/inactivation of E2F through Rbf.

It is interesting to note that there are three consensus sequences for binding of Su(H) to DNA in the dMyc gene (data not shown), and in the particular case of human T-cell acute lymphoblastic leukaemia/lymphoma, c-myc proto-oncogene acts as a direct downstream target of Notch1 (Sharma et al, 2006; Weng et al, 2006). These results raise the possibility that dMyc might be a direct target of Notch in the Drosophila wing. However, the fact that dMyc is negatively regulated by Notch and an activated form of Su(H) (Su(H)-VP16) is able to repress dMyc expression, suggests that Notch regulates dMyc through a Notch target gene that may act as a repressor.

The DV boundary behaves as an organizer of growth in the developing wing, as ectopic DV boundaries induce non-autonomous proliferation of the surrounding tissue, and loss of the DV boundary leads to absence of wing tissue (Diaz-Benjumea and Cohen, 1993). Notch has been shown to mediate this organizing activity (Irvine and Wieschaus, 1994; de Celis et al, 1996), and previous reports showed that Wg is not sufficient to promote growth in the absence of an endogenous DV boundary (Klein and Arias, 1998; Baena-Lopez and Garcia-Bellido, 2003; Zecca and Struhl, 2007). These results are consistent with the data obtained in this work and suggest that the organizing activity of the DV boundary should not be attributed to the activity of Wg. Our results indicate that the organizing activity of Notch is strictly non-autonomous, as Notch induces cell-cycle arrest in a cell-autonomous manner. In this context, Wg signalling alleviates Notch-mediated repression of G1–S progression in wing cells. The role of Wg can be defined as permissive rather than instructive, as wing cells unable to respond to Notch and Wg do proliferate. Altogether, these results indicate that unknown signalling molecules regulated by Notch might exert the organizing activity of the wing DV boundary.

Materials and methods

Drosophila strains

crbM11. M2 (crb–lacZ in the text), UAS–SerDN and UAS–DlDN (Herranz et al, 2006); E2F1-responsive reporter (Asano and Wharton, 1999), UAS–dE2F1 UAS–DP (UAS–E2F in the text; Neufeld et al, 1998, a gift from B Edgar), UAS–dMyc (Johnston et al, 1999, a gift from B Edgar), banΔ1, ban–sensor–GFP and UAS–bantam–GFP (Brennecke et al, 2003, a gift from S Cohen), and UAS–dMycRNAi (Vienna Drosophila RNAi Center), UAS–mamDN (Helms et al, 1999). Other stocks are described in Flybase.

Antibodies

Guinea pig anti-Senseless (Nolo et al, 2000, a gift from H Bellen), mouse anti-Wg and mouse anti-BrdU are described in the Developmental Studies Hybridoma Bank; rabbit anti-β-gal (Cappel) was also used. Anti-dMyc antibody was raised in guinea pig against recombinant full-length dMyc protein. The specificity of the anti-dMyc antibody was tested in wing discs overexpressing dMyc and in wing discs in which dMyc expression is reduced by means of a dsRNA construct of dmyc (see Supplementary Figure S4). BrdU staining, antisense digoxigenin-labelled RNA probes of dMyc and wg mRNAs and in situ hybridization was performed as described by Milan et al (1996).

Spatiotemporal gene expression targeting in Drosophila

We made use of the TARGET system developed by McGuire et al (2004). Adult flies carrying the Gal4 drivers ptc–Gal4, dpp–Gal4 or ap–Gal4 and the thermosensitive version of the Gal4 repressor Gal80 (Gal80ts) under the control of the tubulin promoter (tubulin–Gal80ts) were crossed with transgenic flies bearing one or more UAS transgenes. Flies were allowed to lay eggs over a period of 24 h at 18°C. The progeny was then grown at 18°C to maintain the Gal4/UAS system in a switched-off condition and transferred to 29°C for a range of periods during larval development to induce Gal4/UAS-dependent expression.

Larval genotypes

The TARGET system: An external file that holds a picture, illustration, etc.
Object name is emboj200884i1.jpg

Supplementary Material

Supplementary Figures S1

Supplementary Figures S2

Supplementary Figures S3

Supplementary Figures S4

Supplementary Figures Legends

Acknowledgments

We thank H Bellen, S Campuzano, S Cohen, B Edgar, C Estella, M Llimargas, E Martí, G Morata, J-P Vincent, R Wharton, the Bloomington Stock Center and the Developmental Studies Hybridoma Bank for flies and reagents; L Johnston, Laura Buttitta, BA Edgar and three anonymous reviewers for comments on the manuscript; and T Yates for help with manuscript preparation. HH was funded by a Juan de la Cierva postdoctoral contract (Ministerio de Educación y Ciencia), and MM laboratory was funded by a grant from the Dirección General de Investigación Científica y Técnica (BFU2004-00167/BMC), a Grant from the Generalitat de Catalunya (2005 SGR 00118) and by intramural funds.

References

  • Asano M, Wharton RP (1999) E2F mediates developmental and cell cycle regulation of ORC1 in Drosophila. EMBO J 18: 2435–2448 [PMC free article] [PubMed]
  • Axelrod JD, Matsuno K, Artavanis-Tsakonas S, Perrimon N (1996) Interaction between wingless and Notch signaling pathways mediated by Dishevelled. Science 271: 1826–1832 [PubMed]
  • Baena-Lopez LA, Garcia-Bellido A (2003) Genetic requirements of vestigial in the regulation of Drosophila wing development. Development 130: 197–208 [PubMed]
  • Baonza A, Freeman M (2005) Control of cell proliferation in the Drosophila eye by Notch signaling. Dev Cell 8: 529–539 [PubMed]
  • Bienz M, Clevers H (2000) Linking colorectal cancer to Wnt signaling. Cell 103: 311–320 [PubMed]
  • Blair SS, Brower DL, Thomas JB, Zavortink M (1994) The role of apterous in the control of dorsoventral compartmentalization and PS integrin gene expression in the developing wing of Drosophila. Development 120: 1805–1815 [PubMed]
  • Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7: 678–689 [PubMed]
  • Brennecke J, Hipfner DR, Stark A, Russell RB, Cohen SM (2003) bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113: 25–36 [PubMed]
  • Buceta J, Herranz H, Canela-Xandri O, Reigada R, Sagues F, Milan M (2007) Robustness and stability of the gene regulatory network involved in DV boundary formation in the Drosophila wing. PLoS ONE 2: e602. [PMC free article] [PubMed]
  • Clevers H (2006) Wnt/beta-catenin signaling in development and disease. Cell 127: 469–480 [PubMed]
  • de Celis JF, Bray S (1997) Feedback mechanisms affecting Notch activation at the dorsoventral boundary in the Drosophila wing. Development 124: 3241–3251 [PubMed]
  • de Celis JF, Garcia-Bellido A, Bray SJ (1996) Activation and function of Notch at the dorsal–ventral boundary of the wing imaginal disc. Development 122: 359–369 [PubMed]
  • Diaz-Benjumea FJ, Cohen SM (1993) Interaction between dorsal and ventral cells in the imaginal disc directs wing development in Drosophila. Cell 75: 741–752 [PubMed]
  • Diaz-Benjumea FJ, Cohen SM (1995) Serrate signals through Notch to establish a Wingless-dependent organizer at the dorsal/ventral compartment boundary of the Drosophila wing. Development 121: 4215–4225 [PubMed]
  • Duman-Scheel M, Johnston LA, Du W (2004) Repression of dMyc expression by Wingless promotes Rbf-induced G1 arrest in the presumptive Drosophila wing margin. Proc Natl Acad Sci USA 101: 3857–3862 [PMC free article] [PubMed]
  • Dyson N (1998) The regulation of E2F by pRB-family proteins. Genes Dev 12: 2245–2262 [PubMed]
  • Firth LC, Baker NE (2005) Extracellular signals responsible for spatially regulated proliferation in the differentiating Drosophila eye. Dev Cell 8: 541–551 [PubMed]
  • García-Bellido A, Merriam JR (1971) Parameters of the wing imaginal disc development of Drosophila melanogaster. Dev Biol 24: 61–87 [PubMed]
  • Giraldez AJ, Cohen SM (2003) Wingless and Notch signaling provide cell survival cues and control cell proliferation during wing development. Development 130: 6533–6543 [PubMed]
  • Giraldez AJ, Perez L, Cohen SM (2002) A naturally occurring alternative product of the mastermind locus that represses notch signalling. Mech Dev 115: 101–105 [PubMed]
  • Helms W, Lee H, Ammerman M, Parks AL, Muskavitch MA, Yedvobnick B (1999) Engineered truncations in the Drosophila mastermind protein disrupt Notch pathway function. Dev Biol 215: 358–374 [PubMed]
  • Herranz H, Stamataki E, Feiguin F, Milan M (2006) Self-refinement of Notch activity through the transmembrane protein Crumbs: modulation of gamma-secretase activity. EMBO Rep 7: 297–302 [PMC free article] [PubMed]
  • Irvine K, Wieschaus E (1994) fringe, a boundary specific signalling molecule, mediates interactions between dorsal and ventral cells during Drosophila wing development. Cell 79: 595–606 [PubMed]
  • Johnston LA, Edgar BA (1998) Wingless and Notch regulate cell-cycle arrest in the developing Drosophila wing. Nature 394: 82–84 [PubMed]
  • Johnston LA, Prober DA, Edgar BA, Eisenman RN, Gallant P (1999) Drosophila myc regulates cellular growth during development. Cell 98: 779–790 [PubMed]
  • Johnston LA, Sanders AL (2003) Wingless promotes cell survival but constrains growth during Drosophila wing development. Nat Cell Biol 5: 827–833 [PubMed]
  • Klein T, Arias AM (1998) Different spatial and temporal interactions between Notch, wingless, and vestigial specify proximal and distal pattern elements of the wing in Drosophila. Dev Biol 194: 196–212 [PubMed]
  • Knoblich JA, Sauer K, Jones L, Richardson H, Saint R, Lehner CF (1994) Cyclin E controls S phase progression and its downregulation during Drosophila embryogenesis is required for the arrest of cell proliferation. Cell 77: 107–120 [PubMed]
  • Logan CY, Nusse R (2004) The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 20: 781–810 [PubMed]
  • Madhavan MM, Schneiderman HA (1977) Histological analysis of the dynamics of growth of imaginal discs and histoblast nests during the larval development of Drosophila melanogaster. Rouxs Arch Dev Biol 183: 269–305
  • McGuire SE, Mao Z, Davis RL (2004) Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila. Sci STKE 2004: pl6. [PubMed]
  • Micchelli CA, Rulifson EJ, Blair SS (1997) The function and regulation of cut expression on the wing margin of Drosophila: Notch, Wingless and a dominant negative role for Delta and Serrate. Development 124: 1485–1495 [PubMed]
  • Milan M, Campuzano S, Garcia-Bellído A (1996) Cell-cycling and patterned cell proliferation in the wing primordium of Drosophila. Proc Natl Acad Sci USA 93: 640–645 [PMC free article] [PubMed]
  • Neufeld TP, de la Cruz AF, Johnston LA, Edgar BA (1998) Coordination of growth and cell division in the Drosophila wing. Cell 93: 1183–1193 [PubMed]
  • Neumann CJ, Cohen SM (1997) Long-range action of Wingless organizes the dorsal–ventral axis of the Drosophila wing. Development 124: 871–880 [PubMed]
  • Neumann CJ, Cohen SM (1998) Boundary formation in the Drosophila wing: the POU-domain protein Nubbin attenuates Notch activity. Science 281: 409–413 [PubMed]
  • Nolo R, Abbott LA, Bellen HJ (2000) Senseless, a Zn finger transcription factor, is necessary and sufficient for sensory organ development in Drosophila. Cell 102: 349–362 [PubMed]
  • O'Brochta DA, Bryant PJ (1985) A zone of non-proliferating cells at a lineage restriction boundary in Drosophila. Nature 313: 138–141 [PubMed]
  • Phillips R, Whittle JRS (1993) wingless expression mediates determination of peripheral nervous system elements in late stages of Drosophila wing disc development. Development 118: 427–438 [PubMed]
  • Radtke F, Raj K (2003) The role of Notch in tumorigenesis: oncogene or tumour suppressor? Nat Rev Cancer 3: 756–767 [PubMed]
  • Rulifson EJ, Micchelli CA, Axelrod JD, Perrimon N, Blair SS (1996) wingless refines its own expression domain on the Drosophila wing margin. Nature 384: 72–74 [PubMed]
  • Sharma VM, Calvo JA, Draheim KM, Cunningham LA, Hermance N, Beverly L, Krishnamoorthy V, Bhasin M, Capobianco AJ, Kelliher MA (2006) Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc. Mol Cell Biol 26: 8022–8031 [PMC free article] [PubMed]
  • Strigini M, Cohen SM (2000) Wingless gradient formation in the Drosophila wing. Curr Biol 10: 293–300 [PubMed]
  • Wehrli M, Dougan ST, Caldwell K, O'Keefe L, Schwartz S, Vaizel-Ohayon D, Schejter E, Tomlinson A, DiNardo S (2000) arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature 407: 527–530 [PubMed]
  • Weng AP, Millholland JM, Yashiro-Ohtani Y, Arcangeli ML, Lau A, Wai C, Del Bianco C, Rodriguez CG, Sai H, Tobias J, Li Y, Wolfe MS, Shachaf C, Felsher D, Blacklow SC, Pear WS, Aster JC (2006) c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma. Genes Dev 20: 2096–2109 [PMC free article] [PubMed]
  • Zecca M, Basler K, Struhl G (1996) Direct and long-range action of a Wingless morphogen gradient. Cell 87: 833–844 [PubMed]
  • Zecca M, Struhl G (2007) Recruitment of cells into the Drosophila wing primordium by a feed-forward circuit of vestigial autoregulation. Development 134: 3001–3010 [PubMed]

Articles from The EMBO Journal are provided here courtesy of The European Molecular Biology Organization
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...