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Ras Signaling Pathway
Current Biology 17, 728–733, April 17, 2007 ª2007 Elsevier Ltd All rights reserved
DOI 10.1016/j.cub.2007.03.023
Report Capicua Regulates Cell Proliferation Downstream of the Receptor Tyrosine Kinase/Ras Signaling Pathway Ai-Sun Kelly Tseng,1 Nicolas Tapon,1,4 Hiroshi Kanda,3 Seden Cigizoglu,1 Lambert Edelmann,1 Brett Pellock,1,2 Kristin White,2 and Iswar K. Hariharan1,3,* 1 Massachusetts General Hospital Cancer Center 2 Cutaneous Biology Research Center Charlestown, Massachusetts 02129 3 Department of Molecular and Cell Biology University of California, Berkeley Berkeley, California 94720
Summary Signaling via the receptor tyrosine kinase (RTK)/Ras pathway promotes tissue growth during organismal development and is increased in many cancers [1]. It is still not understood precisely how this pathway promotes cell growth (mass accumulation). In addition, the RTK/Ras pathway also functions in cell survival, cell-fate specification, terminal differentiation, and progression through mitosis [2–7]. An important question is how the same canonical pathway can elicit strikingly different responses in different cell types. Here, we show that the HMG-box protein Capicua (Cic) restricts cell growth in Drosophila imaginal discs, and its levels are, in turn, downregulated by Ras signaling. Moreover, unlike normal cells, the growth of cic mutant cells is undiminished in the complete absence of a Ras signal. In addition to a general role in growth regulation, the importance of cic in regulating cell-fate determination downstream of Ras appears to vary from tissue to tissue. In the developing eye, the analysis of cic mutants shows that the functions of Ras in regulating growth and cell-fate determination are separable. Thus, the DNA-binding protein Cic is a key downstream component in the pathway by which Ras regulates growth in imaginal discs. Results and Discussion Inactivation of capicua Results in Increased Growth but Does Not Affect Cell-Fate Determination in the Developing Eye We performed a genetic screen, by using mitotic recombination in the developing eye, for mutations that allow homozygous mutant cells to outgrow their wild-type neighbors [8]. In addition to mutations in genes, such as Tsc1, Tsc2, Pten, salvador, warts and hippo, that encode negative regulators of growth (reviewed in [9]) and result in grossly enlarged eyes, we identified mutations where the only observable abnormality was an overrepresentation of mutant over wild-type tissue. Four such
*Correspondence: [email protected] 4 Present address: Cancer Research UK, London Research Institute, London WC2 3PX, United Kingdom.
mutations belonged to a single lethal complementation group. Eyes containing mutant clones showed an increased relative representation of mutant tissue over wild-type tissue (Figures 1C–1E) when compared to the parent chromosome used in the screen (Figure 1B). Eyes containing mutant clones also consistently contained more ommatidia (mean = 763 ommatidia; n = 6) and were thus slightly larger than eyes containing clones that were homozygous for the parent chromosome (mean = 703 ommatidia; n = 6, p = 0.0037). Otherwise, the eyes were normal in appearance. All four alleles failed to complement the lethality of cicfetU6 and cicfetE11, which are alleles of capicua (cic) [10]. Mutations in the cic locus (also known as fettucine and bullwinkle) have previously been isolated in screens for mutations that disrupt either embryonic patterning or patterning of the eggshell [10–12], but the role of cic as a negative regulator of growth has not been described previously. cic encodes a protein with a single highmobility group (HMG)-box that localizes to the nucleus and that is likely to bind DNA via its HMG-box motif. Each of the four mutant chromosomes isolated in our screen has a mutation in the coding region of the cic gene (Figure 1A). An antibody that recognizes the C-terminal portion of Cic stains nuclei throughout the eye imaginal disc. There is a stripe of increased expression immediately anterior to the morphogenetic furrow and reduced expression in the morphogenetic furrow itself (Figure 1I). Staining is not detected in clones of cicQ474X cells (Figure S1 in the Supplemental Data online), thus confirming that the antibody recognizes the C-terminal portion of the Cic protein. In the eye imaginal disc, loss-of-function mutations in cic appear to increase tissue growth but do not seem to perturb cell-fate specification or differentiation. cic mutant ommatidia were indistinguishable from wild-type ommatidia in terms of the size, number, and arrangement of photoreceptor cells in the adult retina (Figure 1F) and appear to develop normally at earlier stages (Figure 1G). Discs containing cic clones also showed normal patterns of BrdU incorporation throughout the eye imaginal disc (Figure 1H). However, cic clones anterior to the morphogenetic furrow contained a 2- to 3-fold higher density of cyclin-E-positive cells per unit of pixel area than wild-type clones (n = 15, p < 0.0001) (Figures 2A and 2B), consistent with the increased rate of cell proliferation in mutant clones (see below). As in wild-type discs, no BrdU incorporation was observed in cic mutant discs posterior to the second mitotic wave (Figure 1H), and ectopic cyclin E protein was not observed in cic clones posterior to the second mitotic wave (data not shown). The patterns of mitosis as assessed by staining with anti-phospho-histone H3 [13] were also unchanged (data not shown). Thus, cic cells maintain a relatively normal pattern of S phases and mitoses in the eye disc and are still able to exit from the cell cycle in a timely manner. In mature pupal eye discs, occasional
Capicua Negatively Regulates Growth Downstream of Ras 729
Figure 1. cic Mutant Cells Have a Proliferative Advantage over Wild-Type Cells but Undergo Normal Cell-Fate Determination (A) Cic protein showing the location of the HMG box (shown in black) and the positions of the mutations. In three instances (cicQ219X, cicQ474X, and cicQ677X) the mutations change a CAG (Gln) codon to a TAG (stop), resulting in the generation of a truncated protein. The fourth allele, cicR505W, changes an arginine residue within the HMG box to a tryptophan. (B–E) Adult mosaic eyes containing tissue homozygous for the parent chromosome bearing the FRT82B element (B), the cicQ474X (C), the cicR505W (D), and the cicQ677X (E) alleles. Mutant tissue is shown in white, and wild-type twin-spot tissue is shown in red. The cicQ677X mutation, which is predicted to make a truncated protein that still has an intact HMGbox, has a slightly weaker phenotype than the other alleles (E), indicating that the CicQ677X protein may retain some of its function. (F) Retinal section of an adult eye containing cicQ474X mutant clones. Mutant ommatidia do not have the white pigment but are otherwise indistinguishable from wild-type ommatidia. (G) Larval eye imaginal disc showing normal photoreceptor differentiation marked by antiElav staining (shown in green) of cic mutant tissue as compared to wild-type tissue. Mutant clones fail to stain with anti-b-galactosidase (b-gal) (shown in blue). The approximate position of the morphogenetic furrow is indicated by the arrowhead. (H) BrdU incorporation (shown in red) showing S phases in the larval third instar eye disc. Mutant clones fail to express GFP (shown in green). (I) Expression of the Cic protein in the third instar eye disc as shown by anti-Cic staining (green). The anterior is to the right in all panels. Scale bars represent the following for individual panels: (F), 10 mm; and (G)–(I), 50 mm. (For details of methods, see Supplemental Experimental Procedures.)
extra interommatidial cells are observed in mutant clones, suggesting that cic cells may have a subtle defect in developmental apoptosis (data not shown). To examine the growth characteristics of cic cells at greater resolution, we dissociated and analyzed cells from the eye and wing discs of early third instar larvae (120 hr AED) by flow cytometry. The distribution of mutant cells in the different phases of the cell cycle as assessed by their DNA content was very similar to that of wild-type cells, as was cell size as assessed by forward scatter in cells of the eye disc (data not shown) or the wing disc (Figures 2C and 2D). As in the adult eye and the eye imaginal disc, the area occupied by mutant clones in the wing disc was larger than the corresponding wild-type twin spots, suggesting that the mutant cells collectively grow (accumulate mass) more quickly than their wild-type neighbors (Figure 2E). Also, mutant clones typically contained more cells than their wild-type twin spots (Figure 2F). The inferred population doubling time calculated from the median clone size was 10.3 hr in mutant clones compared to 12.3 hr in the wild-type twin spots. The simplest interpretation of all of these observations is that cic cells have an increased rate of growth (mass accumulation) compared to wild-type cells but maintain a normal size because of a commensurate acceleration of the cell cycle. These findings indicate that a normal function of cic is to restrict cell growth in both the eye and wing imaginal discs.
Cic Levels Are Regulated by Ras Signaling in the Eye Disc Previous work has shown that the levels of Cic protein are responsive to the level of signaling via RTKs and Ras. In the embryo, the level of Cic protein in the terminal regions is decreased upon signaling via the Tor RTK [12]. Activation of Ras in the cells of the wing imaginal disc also reduces Cic levels in those cells [14]. In eye discs, loss-of-function clones of Egfr (Figures 3A–3C) or Ras (Figures 3D–3I), although small, had clearly elevated levels of Cic protein. Conversely, clones of cells expressing the activated form of Ras, Ras (Val12), had reduced levels of Cic (Figure 3J–3L). Thus, as in other tissues, increased signaling via the Egfr/Ras pathway reduces Cic protein levels in the eye disc. Furthermore, studies with mutations in the effector domain of Ras suggest that Ras regulates Cic primarily via the Raf/ MAPK pathway (Figure S2). This is consistent with a recent study that has shown a direct interaction between Cic and MAPK [15]. Inactivation of cic Enables Cells to Grow without Ras Function In the eye imaginal disc, clones of RasDC40b [16], a null allele of Ras, were much smaller than their wild-type twin spots. Strikingly, clones of cells that were mutant for both cic and RasDC40b were indistinguishable from cic clones in that they were typically larger than their twin spots (Figures 4A–4D and Figure S3). Thus, the loss of
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Figure 2. Changes in Cic Levels Do Not Alter Cell-Cycle Phasing or Cell Size but Affect Growth and Cell Proliferation In all panels, profiles of control populations are shown in green; cic mutant populations are shown in black. (A) Larval eye discs containing either clones of the FRT82B parent chromosome (GFP-negative) and wild-type sister clones (GFP-positive) (shown in top panels) or cicQ474X (GFP-negative) and wild-type sister clones (GFP-positive) (shown in bottom panels) stained with anti-cyclin E (shown in red). Arrowheads indicate the approximate position of the morphogenetic furrow. The anterior is to the right. (B) Quantification of number of cyclin-E-positive cells. Clone area was measured with the histogram function of Adobe Photoshop. cic mutant clones contain 2.3-fold more cyclin-E-positive cells per 1000 pixel area when compared to wild-type clones (n = 15, p < 0.0001). In contrast, control FRT82B clones contain similar numbers of CycE-positive cells as compared to wild-type clones (n = 10, p = 0.69). Error bars indicate SDs from the mean value. (C and D) Analysis of DNA content (C) and cell size (D) by flow cytometry of dissociated cells from wing imaginal discs. The forward scatter (FSC) ratio of cic mutant population to control population is shown (0.97). (E) Areas of individual cic mutant clones and their corresponding wild-type twin spots calculated with the histogram function of Adobe Photoshop. (F) Number of cells in individual cic clones and their corresponding wild-type sister clones arranged in order of increasing number. Both the observed increase in clonal area (E) and cell numbers (F) in mutant clones are significant as measured by a paired student’s t test (p < 0.0001). (For details of methods, see Supplemental Experimental Procedures).
cic function completely bypasses the requirement for Ras in promoting cell growth. In contrast to the result obtained with cic, clones that were doubly mutant for Ras as well as a different negative regulator of growth, Tsc1 [9], were no larger than Ras clones (Figures 4Q–4S).
Hence, the ability of cic to suppress the growth defect of Ras clones is specific and not a general property of negative regulators of growth. Also, cic mutations did not suppress the growth defect resulting from mutations in the Insulin Receptor (InR) [17], Akt [18],
Capicua Negatively Regulates Growth Downstream of Ras 731
Figure 3. Cic Expression Can Be Modulated by Changes in Ras or Egfr Signaling (A–L) Third instar eye imaginal discs. The anterior is to the right. The morphogenetic furrow is indicated by an arrowhead. (A), (D), (G), and (J) show anti-Cic staining (shown in red). Clones of Egfr [k05115] (A–C) or RasDC40b (D–I) grow poorly and thus are very small. Levels of Cic are increased in Egfr or Ras mutant clones (A, D, and G) but are lowered in cells overexpressing RasV12 (J). (B), (E), and (H) show b-galactosidase staining to mark wild-type cells in the disc (shown in green). Mutant cells fail to stain for b-gal. (K) shows that cells overexpressing RasV12 express GFP (shown in green). (C), (F), (I), and (L) show merged images of Cic and b-gal (or GFP) stainings. Scale bars represent the following for individual panels: (C), (I), and (L), 10 mm; and (F), 50 mm. (For details of methods, see Supplemental Experimental Procedures.)
and Rheb [19, 20] (Figure S4). Thus, cic mutations appear capable of rendering cell growth independent of Rasmediated signaling but not independent of InR/PI3K- or Tor-mediated signaling. Taken together, these findings support the notion that the ability of Cic to restrict cell growth is specific to its function as a downstream component of the Ras pathway. Cic Mutations Do Not Affect Cell-Fate Determination in the Developing Eye In addition to promoting tissue growth, the recruitment of photoreceptor cell precursors to the developing ommatidia occurs via reiterated use of the EGFR/Ras pathway [4]. Clones of cells that are mutant for RasDC40b do not contain clusters of cells expressing the neural marker Elav [4, 6], and instead they contain only the regularly spaced single Elav-positive nuclei that belong to the R8 photoreceptor cells. Although clones doubly mutant for cic and RasDC40b are of normal size, they, like Ras clones, contain single nuclei that stain with antiElav (Figures 4E–4L) and express the R8-specific marker Senseless (data not shown). Thus, loss of cic function does not bypass the requirement for Ras function in the specification of photoreceptor cells R1–R7. Mutations in Tsc1 suppress the requirement for Ras neither
in growth nor in photoreceptor differentiation (Figures 4Q–4T). Thus, adult eyes containing clones doubly mutant for cic and RasDC40b have large patches of tissue lacking any recognizable ommatidia (Figures 4M–4P). In retinal sections, there are no photoreceptor cells in the cic Ras double-mutant clones, and all the photoreceptor cells at the borders of the clone are wild-type for Ras (data not shown). Thus, although they exhibit impaired photoreceptor differentiation, cic Ras doublemutant clones are not impaired in their growth and, unlike Ras clones, are not outcompeted by neighboring cells. Indeed, the phenotype of cells doubly mutant for Ras and cic is extremely similar to that of large Ras clones that are generated in a Minute background [5], suggesting that cic mutations primarily rescue the growth disadvantage of Ras clones. Thus, in the eye disc, there may be a branching of the Egfr/Ras pathway. One branch, functioning via Cic, appears important for growth regulation, whereas the other branch, acting via Pnt, appears important for photoreceptor cell-fate specification. In contrast to Ras clones, clones of pnt in the eye imaginal disc do not show a marked growth defect (Figure S5), suggesting that pnt has a minor role in regulating tissue growth in the eye disc.
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Figure 4. Loss of cic Suppresses the PoorGrowth Phenotype of Ras Mutant Clones but Does Not Rescue the Differentiation Defect (A–L) Third instar eye imaginal discs containing clones of the FRT82B parent chromosome (A, E, and I), cicQ474X (B, F, and J), RasDC40b (C, G, and K), and RasDC40b, cicQ474X (D, H, and L) were generated by eyFLP-induced mitotic recombination. Wild-type cells are marked by b-galactosidase expression (shown in red) in the discs (A–D). (E)–(H) show mutant clones that were examined for photoreceptor differentiation with anti-Elav staining (shown in green). (I)–(L) show merged images of anti-Elav and anti-b-gal stainings. The inset in (L) shows regularly spaced single Elav-positive nuclei in the mutant clone. (M–P) Adult fly eyes containing clones of FRT82B parent chromosome (M), cicQ474X (N), RasDC40b (O), and RasDC40b, cicQ474X (P). Mutant tissue appears white, and wild-type tissue appears red. A greater relative representation of mutant tissue is seen in discs and adult eyes containing cic mutant clones (B and N) than those containing FRT82B control clones (A and M). Ras mutant clones are poorly represented in the disc (C) and are not visible in the adult eye (O). Discs with clones doubly mutant for Ras and cic (D) resemble discs with cic clones alone (B) with respect to clone size. In the adult eye, although tissue deficient in both Ras and cic is observed (P), the mutant tissue lacks the normal ommatidial architecture. The scale bar in (I) represents 50 mm. (Q–T) Discs containing Ras Tsc1 (double-mutant) clones (Q–S) where mutant clones fail to express GFP (shown in green). Mutant clones are small in the imaginal disc, lack normal ommatidia and are absent in the adult eye (T). (For details of methods, see Supplemental Experimental Procedures.)
In mammalian cells, several extracellular growth factors that act via RTKs increase the activity of cyclin D/ Cdk4 or cyclin D/Cdk6 complexes [21] that can phosphorylate and inactivate the retinoblastoma protein (pRb) and thus promote S phase entry. However, it is still unclear how inactivation of pRb can cause cell growth (mass accumulation). At least in Drosophila, the role of Cic appears distinct from cyclin D because neither are cyclin D protein levels elevated in cic clones (data not shown) nor is the growth advantage of cic cells over wild-type cells compromised in flies that completely lack Cdk4/6 function (Figure S6). Other studies suggest that Ras can promote cell growth by stabilizing Myc protein via MAPK-mediated phosphorylation [22, 23]. This mode
of Ras function also appears to be dispensable under conditions where cic function is inactivated but may still be relevant at physiological levels of Ras signaling. Notably, our data also show that Cic also functions as a negative regulator of tissue growth in the wing disc. However, in this tissue, Cic has a role in specifying cell fates as well because others have shown that cic mutations result in the formation of ectopic vein tissue [14]. Thus, although the role of Cic as a regulator of growth in imaginal discs appears to be general, the importance of Cic in pathways that regulate cell-fate determination may vary from one tissue to another. The human and mouse genome each appear to have a single cic ortholog [12, 24] whose function in the
Capicua Negatively Regulates Growth Downstream of Ras 733
regulation of growth has not been addressed to date. However, a recent study that determined the DNA sequence of 13,023 genes from 11 breast and 11 colorectal cancers found missense mutations in the human cic ortholog in three of the breast cancers [25]. Although the functional consequences of these mutations have not been evaluated, these data suggest that Cic may indeed function in restricting cell growth in human cells. Supplemental Data Supplemental Data include Experimental Procedures and six figures and are available with this article online at http://www. current-biology.com/cgi/content/full/17/8/728/DC1/. Acknowledgments We thank Donald Morisato for providing the cicfetU6 and cicfet-E11 alleles prior to publication and for helpful discussions, Terry OrrWeaver for the cyclin E antibody, JoAnn Yetz-Aldape for help with flow cytometry, Nick Baker, Bruce Edgar, Jordi Casanova, Ernst Hafen, and Sean Oldham for fly stocks, and Nick Dyson for discussions. This work was funded by National Institutes of Health grants RO1 GM61672 to I.K.H. and GM 55568 to K.W. H.K. was supported in part by a fellowship from the Japan Society for the Promotion of Science, and B.P. was supported by a Massachusetts Biomedical Research Council Tosteson Postdoctoral Fellowship.
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20. Received: December 19, 2006 Revised: February 20, 2007 Accepted: March 5, 2007 Published online: March 29, 2007
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DOI 10.1016/j.cub.2007.03.023
Report Capicua Regulates Cell Proliferation Downstream of the Receptor Tyrosine Kinase/Ras Signaling Pathway Ai-Sun Kelly Tseng,1 Nicolas Tapon,1,4 Hiroshi Kanda,3 Seden Cigizoglu,1 Lambert Edelmann,1 Brett Pellock,1,2 Kristin White,2 and Iswar K. Hariharan1,3,* 1 Massachusetts General Hospital Cancer Center 2 Cutaneous Biology Research Center Charlestown, Massachusetts 02129 3 Department of Molecular and Cell Biology University of California, Berkeley Berkeley, California 94720
Summary Signaling via the receptor tyrosine kinase (RTK)/Ras pathway promotes tissue growth during organismal development and is increased in many cancers [1]. It is still not understood precisely how this pathway promotes cell growth (mass accumulation). In addition, the RTK/Ras pathway also functions in cell survival, cell-fate specification, terminal differentiation, and progression through mitosis [2–7]. An important question is how the same canonical pathway can elicit strikingly different responses in different cell types. Here, we show that the HMG-box protein Capicua (Cic) restricts cell growth in Drosophila imaginal discs, and its levels are, in turn, downregulated by Ras signaling. Moreover, unlike normal cells, the growth of cic mutant cells is undiminished in the complete absence of a Ras signal. In addition to a general role in growth regulation, the importance of cic in regulating cell-fate determination downstream of Ras appears to vary from tissue to tissue. In the developing eye, the analysis of cic mutants shows that the functions of Ras in regulating growth and cell-fate determination are separable. Thus, the DNA-binding protein Cic is a key downstream component in the pathway by which Ras regulates growth in imaginal discs. Results and Discussion Inactivation of capicua Results in Increased Growth but Does Not Affect Cell-Fate Determination in the Developing Eye We performed a genetic screen, by using mitotic recombination in the developing eye, for mutations that allow homozygous mutant cells to outgrow their wild-type neighbors [8]. In addition to mutations in genes, such as Tsc1, Tsc2, Pten, salvador, warts and hippo, that encode negative regulators of growth (reviewed in [9]) and result in grossly enlarged eyes, we identified mutations where the only observable abnormality was an overrepresentation of mutant over wild-type tissue. Four such
*Correspondence: [email protected] 4 Present address: Cancer Research UK, London Research Institute, London WC2 3PX, United Kingdom.
mutations belonged to a single lethal complementation group. Eyes containing mutant clones showed an increased relative representation of mutant tissue over wild-type tissue (Figures 1C–1E) when compared to the parent chromosome used in the screen (Figure 1B). Eyes containing mutant clones also consistently contained more ommatidia (mean = 763 ommatidia; n = 6) and were thus slightly larger than eyes containing clones that were homozygous for the parent chromosome (mean = 703 ommatidia; n = 6, p = 0.0037). Otherwise, the eyes were normal in appearance. All four alleles failed to complement the lethality of cicfetU6 and cicfetE11, which are alleles of capicua (cic) [10]. Mutations in the cic locus (also known as fettucine and bullwinkle) have previously been isolated in screens for mutations that disrupt either embryonic patterning or patterning of the eggshell [10–12], but the role of cic as a negative regulator of growth has not been described previously. cic encodes a protein with a single highmobility group (HMG)-box that localizes to the nucleus and that is likely to bind DNA via its HMG-box motif. Each of the four mutant chromosomes isolated in our screen has a mutation in the coding region of the cic gene (Figure 1A). An antibody that recognizes the C-terminal portion of Cic stains nuclei throughout the eye imaginal disc. There is a stripe of increased expression immediately anterior to the morphogenetic furrow and reduced expression in the morphogenetic furrow itself (Figure 1I). Staining is not detected in clones of cicQ474X cells (Figure S1 in the Supplemental Data online), thus confirming that the antibody recognizes the C-terminal portion of the Cic protein. In the eye imaginal disc, loss-of-function mutations in cic appear to increase tissue growth but do not seem to perturb cell-fate specification or differentiation. cic mutant ommatidia were indistinguishable from wild-type ommatidia in terms of the size, number, and arrangement of photoreceptor cells in the adult retina (Figure 1F) and appear to develop normally at earlier stages (Figure 1G). Discs containing cic clones also showed normal patterns of BrdU incorporation throughout the eye imaginal disc (Figure 1H). However, cic clones anterior to the morphogenetic furrow contained a 2- to 3-fold higher density of cyclin-E-positive cells per unit of pixel area than wild-type clones (n = 15, p < 0.0001) (Figures 2A and 2B), consistent with the increased rate of cell proliferation in mutant clones (see below). As in wild-type discs, no BrdU incorporation was observed in cic mutant discs posterior to the second mitotic wave (Figure 1H), and ectopic cyclin E protein was not observed in cic clones posterior to the second mitotic wave (data not shown). The patterns of mitosis as assessed by staining with anti-phospho-histone H3 [13] were also unchanged (data not shown). Thus, cic cells maintain a relatively normal pattern of S phases and mitoses in the eye disc and are still able to exit from the cell cycle in a timely manner. In mature pupal eye discs, occasional
Capicua Negatively Regulates Growth Downstream of Ras 729
Figure 1. cic Mutant Cells Have a Proliferative Advantage over Wild-Type Cells but Undergo Normal Cell-Fate Determination (A) Cic protein showing the location of the HMG box (shown in black) and the positions of the mutations. In three instances (cicQ219X, cicQ474X, and cicQ677X) the mutations change a CAG (Gln) codon to a TAG (stop), resulting in the generation of a truncated protein. The fourth allele, cicR505W, changes an arginine residue within the HMG box to a tryptophan. (B–E) Adult mosaic eyes containing tissue homozygous for the parent chromosome bearing the FRT82B element (B), the cicQ474X (C), the cicR505W (D), and the cicQ677X (E) alleles. Mutant tissue is shown in white, and wild-type twin-spot tissue is shown in red. The cicQ677X mutation, which is predicted to make a truncated protein that still has an intact HMGbox, has a slightly weaker phenotype than the other alleles (E), indicating that the CicQ677X protein may retain some of its function. (F) Retinal section of an adult eye containing cicQ474X mutant clones. Mutant ommatidia do not have the white pigment but are otherwise indistinguishable from wild-type ommatidia. (G) Larval eye imaginal disc showing normal photoreceptor differentiation marked by antiElav staining (shown in green) of cic mutant tissue as compared to wild-type tissue. Mutant clones fail to stain with anti-b-galactosidase (b-gal) (shown in blue). The approximate position of the morphogenetic furrow is indicated by the arrowhead. (H) BrdU incorporation (shown in red) showing S phases in the larval third instar eye disc. Mutant clones fail to express GFP (shown in green). (I) Expression of the Cic protein in the third instar eye disc as shown by anti-Cic staining (green). The anterior is to the right in all panels. Scale bars represent the following for individual panels: (F), 10 mm; and (G)–(I), 50 mm. (For details of methods, see Supplemental Experimental Procedures.)
extra interommatidial cells are observed in mutant clones, suggesting that cic cells may have a subtle defect in developmental apoptosis (data not shown). To examine the growth characteristics of cic cells at greater resolution, we dissociated and analyzed cells from the eye and wing discs of early third instar larvae (120 hr AED) by flow cytometry. The distribution of mutant cells in the different phases of the cell cycle as assessed by their DNA content was very similar to that of wild-type cells, as was cell size as assessed by forward scatter in cells of the eye disc (data not shown) or the wing disc (Figures 2C and 2D). As in the adult eye and the eye imaginal disc, the area occupied by mutant clones in the wing disc was larger than the corresponding wild-type twin spots, suggesting that the mutant cells collectively grow (accumulate mass) more quickly than their wild-type neighbors (Figure 2E). Also, mutant clones typically contained more cells than their wild-type twin spots (Figure 2F). The inferred population doubling time calculated from the median clone size was 10.3 hr in mutant clones compared to 12.3 hr in the wild-type twin spots. The simplest interpretation of all of these observations is that cic cells have an increased rate of growth (mass accumulation) compared to wild-type cells but maintain a normal size because of a commensurate acceleration of the cell cycle. These findings indicate that a normal function of cic is to restrict cell growth in both the eye and wing imaginal discs.
Cic Levels Are Regulated by Ras Signaling in the Eye Disc Previous work has shown that the levels of Cic protein are responsive to the level of signaling via RTKs and Ras. In the embryo, the level of Cic protein in the terminal regions is decreased upon signaling via the Tor RTK [12]. Activation of Ras in the cells of the wing imaginal disc also reduces Cic levels in those cells [14]. In eye discs, loss-of-function clones of Egfr (Figures 3A–3C) or Ras (Figures 3D–3I), although small, had clearly elevated levels of Cic protein. Conversely, clones of cells expressing the activated form of Ras, Ras (Val12), had reduced levels of Cic (Figure 3J–3L). Thus, as in other tissues, increased signaling via the Egfr/Ras pathway reduces Cic protein levels in the eye disc. Furthermore, studies with mutations in the effector domain of Ras suggest that Ras regulates Cic primarily via the Raf/ MAPK pathway (Figure S2). This is consistent with a recent study that has shown a direct interaction between Cic and MAPK [15]. Inactivation of cic Enables Cells to Grow without Ras Function In the eye imaginal disc, clones of RasDC40b [16], a null allele of Ras, were much smaller than their wild-type twin spots. Strikingly, clones of cells that were mutant for both cic and RasDC40b were indistinguishable from cic clones in that they were typically larger than their twin spots (Figures 4A–4D and Figure S3). Thus, the loss of
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Figure 2. Changes in Cic Levels Do Not Alter Cell-Cycle Phasing or Cell Size but Affect Growth and Cell Proliferation In all panels, profiles of control populations are shown in green; cic mutant populations are shown in black. (A) Larval eye discs containing either clones of the FRT82B parent chromosome (GFP-negative) and wild-type sister clones (GFP-positive) (shown in top panels) or cicQ474X (GFP-negative) and wild-type sister clones (GFP-positive) (shown in bottom panels) stained with anti-cyclin E (shown in red). Arrowheads indicate the approximate position of the morphogenetic furrow. The anterior is to the right. (B) Quantification of number of cyclin-E-positive cells. Clone area was measured with the histogram function of Adobe Photoshop. cic mutant clones contain 2.3-fold more cyclin-E-positive cells per 1000 pixel area when compared to wild-type clones (n = 15, p < 0.0001). In contrast, control FRT82B clones contain similar numbers of CycE-positive cells as compared to wild-type clones (n = 10, p = 0.69). Error bars indicate SDs from the mean value. (C and D) Analysis of DNA content (C) and cell size (D) by flow cytometry of dissociated cells from wing imaginal discs. The forward scatter (FSC) ratio of cic mutant population to control population is shown (0.97). (E) Areas of individual cic mutant clones and their corresponding wild-type twin spots calculated with the histogram function of Adobe Photoshop. (F) Number of cells in individual cic clones and their corresponding wild-type sister clones arranged in order of increasing number. Both the observed increase in clonal area (E) and cell numbers (F) in mutant clones are significant as measured by a paired student’s t test (p < 0.0001). (For details of methods, see Supplemental Experimental Procedures).
cic function completely bypasses the requirement for Ras in promoting cell growth. In contrast to the result obtained with cic, clones that were doubly mutant for Ras as well as a different negative regulator of growth, Tsc1 [9], were no larger than Ras clones (Figures 4Q–4S).
Hence, the ability of cic to suppress the growth defect of Ras clones is specific and not a general property of negative regulators of growth. Also, cic mutations did not suppress the growth defect resulting from mutations in the Insulin Receptor (InR) [17], Akt [18],
Capicua Negatively Regulates Growth Downstream of Ras 731
Figure 3. Cic Expression Can Be Modulated by Changes in Ras or Egfr Signaling (A–L) Third instar eye imaginal discs. The anterior is to the right. The morphogenetic furrow is indicated by an arrowhead. (A), (D), (G), and (J) show anti-Cic staining (shown in red). Clones of Egfr [k05115] (A–C) or RasDC40b (D–I) grow poorly and thus are very small. Levels of Cic are increased in Egfr or Ras mutant clones (A, D, and G) but are lowered in cells overexpressing RasV12 (J). (B), (E), and (H) show b-galactosidase staining to mark wild-type cells in the disc (shown in green). Mutant cells fail to stain for b-gal. (K) shows that cells overexpressing RasV12 express GFP (shown in green). (C), (F), (I), and (L) show merged images of Cic and b-gal (or GFP) stainings. Scale bars represent the following for individual panels: (C), (I), and (L), 10 mm; and (F), 50 mm. (For details of methods, see Supplemental Experimental Procedures.)
and Rheb [19, 20] (Figure S4). Thus, cic mutations appear capable of rendering cell growth independent of Rasmediated signaling but not independent of InR/PI3K- or Tor-mediated signaling. Taken together, these findings support the notion that the ability of Cic to restrict cell growth is specific to its function as a downstream component of the Ras pathway. Cic Mutations Do Not Affect Cell-Fate Determination in the Developing Eye In addition to promoting tissue growth, the recruitment of photoreceptor cell precursors to the developing ommatidia occurs via reiterated use of the EGFR/Ras pathway [4]. Clones of cells that are mutant for RasDC40b do not contain clusters of cells expressing the neural marker Elav [4, 6], and instead they contain only the regularly spaced single Elav-positive nuclei that belong to the R8 photoreceptor cells. Although clones doubly mutant for cic and RasDC40b are of normal size, they, like Ras clones, contain single nuclei that stain with antiElav (Figures 4E–4L) and express the R8-specific marker Senseless (data not shown). Thus, loss of cic function does not bypass the requirement for Ras function in the specification of photoreceptor cells R1–R7. Mutations in Tsc1 suppress the requirement for Ras neither
in growth nor in photoreceptor differentiation (Figures 4Q–4T). Thus, adult eyes containing clones doubly mutant for cic and RasDC40b have large patches of tissue lacking any recognizable ommatidia (Figures 4M–4P). In retinal sections, there are no photoreceptor cells in the cic Ras double-mutant clones, and all the photoreceptor cells at the borders of the clone are wild-type for Ras (data not shown). Thus, although they exhibit impaired photoreceptor differentiation, cic Ras doublemutant clones are not impaired in their growth and, unlike Ras clones, are not outcompeted by neighboring cells. Indeed, the phenotype of cells doubly mutant for Ras and cic is extremely similar to that of large Ras clones that are generated in a Minute background [5], suggesting that cic mutations primarily rescue the growth disadvantage of Ras clones. Thus, in the eye disc, there may be a branching of the Egfr/Ras pathway. One branch, functioning via Cic, appears important for growth regulation, whereas the other branch, acting via Pnt, appears important for photoreceptor cell-fate specification. In contrast to Ras clones, clones of pnt in the eye imaginal disc do not show a marked growth defect (Figure S5), suggesting that pnt has a minor role in regulating tissue growth in the eye disc.
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Figure 4. Loss of cic Suppresses the PoorGrowth Phenotype of Ras Mutant Clones but Does Not Rescue the Differentiation Defect (A–L) Third instar eye imaginal discs containing clones of the FRT82B parent chromosome (A, E, and I), cicQ474X (B, F, and J), RasDC40b (C, G, and K), and RasDC40b, cicQ474X (D, H, and L) were generated by eyFLP-induced mitotic recombination. Wild-type cells are marked by b-galactosidase expression (shown in red) in the discs (A–D). (E)–(H) show mutant clones that were examined for photoreceptor differentiation with anti-Elav staining (shown in green). (I)–(L) show merged images of anti-Elav and anti-b-gal stainings. The inset in (L) shows regularly spaced single Elav-positive nuclei in the mutant clone. (M–P) Adult fly eyes containing clones of FRT82B parent chromosome (M), cicQ474X (N), RasDC40b (O), and RasDC40b, cicQ474X (P). Mutant tissue appears white, and wild-type tissue appears red. A greater relative representation of mutant tissue is seen in discs and adult eyes containing cic mutant clones (B and N) than those containing FRT82B control clones (A and M). Ras mutant clones are poorly represented in the disc (C) and are not visible in the adult eye (O). Discs with clones doubly mutant for Ras and cic (D) resemble discs with cic clones alone (B) with respect to clone size. In the adult eye, although tissue deficient in both Ras and cic is observed (P), the mutant tissue lacks the normal ommatidial architecture. The scale bar in (I) represents 50 mm. (Q–T) Discs containing Ras Tsc1 (double-mutant) clones (Q–S) where mutant clones fail to express GFP (shown in green). Mutant clones are small in the imaginal disc, lack normal ommatidia and are absent in the adult eye (T). (For details of methods, see Supplemental Experimental Procedures.)
In mammalian cells, several extracellular growth factors that act via RTKs increase the activity of cyclin D/ Cdk4 or cyclin D/Cdk6 complexes [21] that can phosphorylate and inactivate the retinoblastoma protein (pRb) and thus promote S phase entry. However, it is still unclear how inactivation of pRb can cause cell growth (mass accumulation). At least in Drosophila, the role of Cic appears distinct from cyclin D because neither are cyclin D protein levels elevated in cic clones (data not shown) nor is the growth advantage of cic cells over wild-type cells compromised in flies that completely lack Cdk4/6 function (Figure S6). Other studies suggest that Ras can promote cell growth by stabilizing Myc protein via MAPK-mediated phosphorylation [22, 23]. This mode
of Ras function also appears to be dispensable under conditions where cic function is inactivated but may still be relevant at physiological levels of Ras signaling. Notably, our data also show that Cic also functions as a negative regulator of tissue growth in the wing disc. However, in this tissue, Cic has a role in specifying cell fates as well because others have shown that cic mutations result in the formation of ectopic vein tissue [14]. Thus, although the role of Cic as a regulator of growth in imaginal discs appears to be general, the importance of Cic in pathways that regulate cell-fate determination may vary from one tissue to another. The human and mouse genome each appear to have a single cic ortholog [12, 24] whose function in the
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regulation of growth has not been addressed to date. However, a recent study that determined the DNA sequence of 13,023 genes from 11 breast and 11 colorectal cancers found missense mutations in the human cic ortholog in three of the breast cancers [25]. Although the functional consequences of these mutations have not been evaluated, these data suggest that Cic may indeed function in restricting cell growth in human cells. Supplemental Data Supplemental Data include Experimental Procedures and six figures and are available with this article online at http://www. current-biology.com/cgi/content/full/17/8/728/DC1/. Acknowledgments We thank Donald Morisato for providing the cicfetU6 and cicfet-E11 alleles prior to publication and for helpful discussions, Terry OrrWeaver for the cyclin E antibody, JoAnn Yetz-Aldape for help with flow cytometry, Nick Baker, Bruce Edgar, Jordi Casanova, Ernst Hafen, and Sean Oldham for fly stocks, and Nick Dyson for discussions. This work was funded by National Institutes of Health grants RO1 GM61672 to I.K.H. and GM 55568 to K.W. H.K. was supported in part by a fellowship from the Japan Society for the Promotion of Science, and B.P. was supported by a Massachusetts Biomedical Research Council Tosteson Postdoctoral Fellowship.
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20. Received: December 19, 2006 Revised: February 20, 2007 Accepted: March 5, 2007 Published online: March 29, 2007
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