A Wingless and Notch double-repression mechanism regulates G1–S transition in the Drosophila wing
2008; Springer Nature; Volume: 27; Issue: 11 Linguagem: Inglês
10.1038/emboj.2008.84
ISSN1460-2075
AutoresHéctor Herranz, Lídia Pérez, Francisco A. Martín, Marco Milán,
Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle1 May 2008free access A Wingless and Notch double-repression mechanism regulates G1–S transition in the Drosophila wing Héctor Herranz Héctor Herranz ICREA and Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Lidia Pérez Lidia Pérez ICREA and Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Francisco A Martín Francisco A Martín Centro de Biología Molecular CSIC-UAM, Madrid, Spain Search for more papers by this author Marco Milán Corresponding Author Marco Milán ICREA and Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Héctor Herranz Héctor Herranz ICREA and Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Lidia Pérez Lidia Pérez ICREA and Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Francisco A Martín Francisco A Martín Centro de Biología Molecular CSIC-UAM, Madrid, Spain Search for more papers by this author Marco Milán Corresponding Author Marco Milán ICREA and Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Author Information Héctor Herranz1, Lidia Pérez1, Francisco A Martín2 and Marco Milán 1 1ICREA and Institute for Research in Biomedicine, Barcelona, Spain 2Centro de Biología Molecular CSIC-UAM, Madrid, Spain *Corresponding author. ICREA and Institute for Research in Biomedicine, Parc Científic de Barcelona, Josep Samitier, 1-5, Barcelona 08028, Spain. Tel.: +34 93 4034902; Fax: +34 93 4037109; E-mail: [email protected] The EMBO Journal (2008)27:1633-1645https://doi.org/10.1038/emboj.2008.84 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info 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. 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 describing the cell interactions that take place between boundary (white) and non-boundary (black) cells that lead to activation of Notch (N) and expression of Wg and Cut along the DV boundary of the Drosophila wing primordium. N, Notch; Dl, Delta; Ser, Serrate. Download figure Download PowerPoint 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 (antibody to GFP in pink or white (B, D, F, H, J, Q)). (A, B) Wild-type wing discs labelled to visualize crumbs–lacZ expression (antibody to β-gal, green). Note failure to incorporate BrdU (A) or low levels of E2F1 activity (B) in those cells expressing high levels of the Notch-regulated gene crumbs, which corresponds to the ZNC. The DV compartment boundary is marked in this and other panels (d, dorsal; v, ventral). (C, D) Notchts2 wing discs reared at the restrictive temperature and labelled to visualize Wg protein expression (green). (E, F) ptc–Gal4; UAS–DeltaDN (E) and sal–Gal4; UAS–mamDN (F) wing discs labelled to visualize Senseless (Sens, green in E) or Wg (green in F) protein expression. Sens expression was used to label the DV boundary. Red lines label the domain of DeltaDN or mamDN expression. (G, H) Wing discs with clones of cells lacking Su(H) activity (Su(H)8), marked by the absence of GFP (G) or β-gal (H) in green. The ZNC is marked by horizontal lines in G. (I) Wing disc with clones of cells lacking arrow activity (arrow2) marked by the absence of GFP. The ZNC is marked by horizontal lines. (J) dpp–Gal4 Gal80ts; UAS–TCFDN wing disc reared at 29°C during 30 h and labelled to visualize Wg (green) protein expression. The dpp–Gal4 domain is marked by arrowheads. (K–R) Wild-type (K, N), C96–Gal4; UAS–TCFDN (L, O), C96–Gal4; UAS –TCFDN UAS–Nintra (M, P, Q) and C96–Gal4; UAS–GFP (R) wing discs labelled to visualize Cut (green, K–M) or GFP (green, R) protein expression, BrdU incorporation (red, N–P) or activity of the E2F1 responsive reporter ORC1–GFP (antibody to GFP in pink, Q). The ZNC is marked by horizontal dashed lines. Download figure Download PowerPoint 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 ORC1–GFP (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 ORC1–GFP (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. (A–H) AbruptexM1 (A, B), dpp–Gal4;UAS–Nintra (C, C′, D, D′), ap–Gal4 Gal80ts, UAS–GFP; UAS–TCFDN wing (E, F), ap–Gal4 Gal80ts, UAS–GFP; UAS–Axin (G) and dpp–Gal4 Gal80ts; UAS–TCFDN (H) late third instar wing discs labelled to visualize cells in S-phase by BrdU incorporation (antibody to BrdU in red or white (A, C, C′, E, G)) or expressing the E2F1-responsive reporter OCT1–GFP (antibody to GFP in pink or white (B, D, D′, F, H)). crb–lacZ (antibody to β-Gal in green (A, B)), Wingless (Wg, green (C, C′, D, D′, H)) and GFP (green (E, G)) protein expression is shown. Panels C′ and D′ show higher magnifications of the discs in panels C and D. The anterior–posterior (ap) boundary is labelled to show the cell-autonomous block in G1 imposed by the activity of N. Nintra activity is labelled by the expression of Wg (red line) and the G1 block by reduced levels of BrdU and dE2F activity. (I, J, K) ap–Gal4, Gal80ts, UAS–GFP; UAS–TCFDN (I), wild-type (J) and ap–Gal4 Gal80ts, UAS–GFP; UAS–Nintra (K) early third instar wing discs labelled to visualize BrdU incorporation (red or white) and GFP (green) expression. The DV compartment boundary is marked in all panels (d, dorsal; v, ventral). Wing discs shown in panels E–I were reared at 29°C during 24 h. Download figure Download PowerPoint Figure 4.A Wg and Notch double-repression mechanism regulates G1 progression. (A–D) dpp–Gal4, UAS–GFP; UAS–NintraUAS–E2F (A), ap–Gal4, Gal80ts, UAS–GFP; UAS–TCFDN, UAS–E2F (B), ptc–Gal4, Gal80ts, UAS–GFP; UAS–Nintra, UAS–CycE (C) and ap–Gal4, Gal80ts, UAS–GFP; UAS–TCFDN, UAS–CycE (D) late third instar wing discs labelled to visualize cells in S-phase by BrdU incorporation (antibody to BrdU in red or white) and GFP (green) protein expression. (E–G) ap–Gal4, Gal80ts, UAS–GFP; UAS–TCFDN (E) and dpp–Gal4, Gal80ts, UAS–GFP; UAS–TCFDN (F, F′, G) late third instar wing discs labelled to visualize Wingless protein (Wg, red or white, E, G), wg mRNA (F), wg–lacZ expression (antibody to β-gal, F′) and GFP (green, E) protein expression. Arrowheads in panels F, F′ and G indicate the dpp–gal4 expression stripe. (H) ap–Gal4, Gal80ts, UAS–GFP; UAS–TCFDN, UAS–mamDN late third instar wing disc labelled to visualize cells in S-phase by BrdU incorporation (antibody to BrdU in red or white) and GFP (green) protein expression. (I) Wing disc with clones of cells lacking arrow activity (arrow2), marked by the absence of GFP in green, and labelled to visualize Wg expression (red or white). Note ectopic expression of Wg in the clones (red arrows). (J) ap–Gal4, Gal80ts, UAS–GFP; UAS–TCFDN,UAS–mamDN late third instar wing discs labelled to visualize cells expressing the E2F1-responsive reporter OCT1–GFP (antibody to GFP in pink or white) and Wg (green) protein expression. Wing discs shown in panels B and D–J were reared at 29°C during 30 h. The DV compartment boundary is marked in all panels (d, dorsal; v, ventral). Download figure Download PowerPoint 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 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. (A–N, P) Wild type (A, B), Notchts2 reared at the restrictive temperature for 48 h (C), ap–Gal4, UAS–GFP; UAS–mamDN (D), C96–Gal4; UAS–mamDN (E, H), C96–Gal4; UAS–TCFDN (F), C96–Gal4; UAS–TCFDNUAS–Nintra (G), C96–Gal4; UAS–mamDN UAS–dMycdsRNA (I), dpp–Gal4; UAS–Nintra (J, K), ap–Gal4, Gal80ts, UAS–GFP; UAS–TCFDN (L, N) dpp–Gal4; UAS–Wg (M), and ap–Gal4, Gal80ts, UAS–GFP; UAS–TCFDN, UAS–mamDN (P) late third instar wing discs labelled to visualize dMyc protein (blue or white (A, D, E–J, M, N, P)) or dMyc mRNA (B, C, K, L) expression, and Wingless (Wg, red (A, E, J, M)), Cut (red (F, G)), Distalless (Dll, red (N, P)) or GFP (green (D, N, P)) protein expression. The DV compartment boundary in panels A and B is indicated by a dashed line. Note dMyc expression in GFP-expressing cells abutting the DV compartment boundary in D (compare with A) as well as in ZNC cells lacking Notch activity (C, E, H). (O, Q, R) ap–Gal4, Gal80ts, UAS–GFP; UAS–TCFDN, UAS–dMyc (O), dpp–Gal4; UAS–Nintra, UAS–dMyc (Q, R) late third instar wing discs labelled to visualize cells in S-phase by BrdU incorporation (antibody to BrdU in red or white (O, Q)), or expressing the E2F1-responsive reporter OCT1–GFP (pink (R)). GFP (O) and dMyc (R) protein expression (green) is also shown. Arrowheads in panels Q and R indicate the dpp–gal4 expression stripe. Wing discs shown in panels L and N–P were reared at 29°C during 30 h. (S) Wild-type wing disc labelled to visualize dMyc (blue) and Distalless (Dll, red) protein expression. Download figure Download PowerPoint 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 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) effec
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