- THIS ARTICLE
-
Abstract
- Full Text (PDF)
- Alert me when this article is cited
- Alert me if a correction is posted
- SERVICES
- Similar articles in this journal
- Similar articles in PubMed
- Alert me to new issues of the journal
- Download to citation manager
- Reprints & Permissions
- CITING ARTICLES
- Citing Articles via HighWire
- Citing Articles via Google Scholar
- GOOGLE SCHOLAR
- Articles by Tseng, A.-S. K.
- Articles by Hariharan, I. K.
- Search for Related Content
- PUBMED
- PubMed Citation
- Articles by Tseng, A.-S. K.
- Articles by Hariharan, I. K.
An Overexpression Screen in Drosophila for Genes That Restrict Growth or Cell-Cycle Progression in the Developing Eye
Ai-Sun Kelly Tsenga and Iswar K. Hariharanaa Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, Massachusetts 02129
Corresponding author: Iswar K. Hariharan, Bldg. 149, 13th St., Charlestown, MA 02129., hariharan{at}helix.mgh.harvard.edu (E-mail)
Communicating editor: K. V. ANDERSON
 | ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
We screened for genes that, when overexpressed in the proliferating cells of the eye imaginal disc, result in a reduction in the size of the adult eye. After crossing the collection of 2296 EP lines to the ey-GAL4 driver, we identified 46 lines, corresponding to insertions in 32 different loci, that elicited a small eye phenotype. These lines were classified further by testing for an effect in postmitotic cells using the sev-GAL4 driver, by testing for an effect in the wing using en-GAL4, and by testing for the ability of overexpression of cycE to rescue the small eye phenotype. EP lines identified in the screen encompass known regulators of eye development including hh and dpp, known genes that have not been studied previously with respect to eye development, as well as 19 novel ORFs. Lines with insertions near INCENP, elB, and CG11518 were characterized in more detail with respect to changes in growth, cell-cycle phasing, and doubling times that were elicited by overexpression. RNAi-induced phenotypes were also analyzed in SL2 cells. Thus overexpression screens can be combined with RNAi experiments to identify and characterize new regulators of growth and cell proliferation.
THE progression of a cell through the cell cycle is subject to a number of controls. Both extracellular and intracellular signals combine to regulate the activity of proteins that directly control cell-cycle progression. Moreover, passage from one cell-cycle state to another is controlled by checkpoints that act to ensure that a cell has completed the requirements of each stage prior to proceeding to the next stage. For example, in response to mitogenic signals, active complexes of cyclins and cyclin-dependent kinases (cdks) are formed and act positively to drive the cell though S phase. If the cell fails to repair DNA damage, negative controls such as cdk inhibitors (CKIs) halt cell-cycle progression. Negative regulators of the cell cycle have been the focus of many studies partly because many of them function as tumor suppressor genes.
In vertebrates, many negative cell-cycle regulators have been identified. These include proteins that directly regulate the cell cycle, such as the retinoblastoma (Rb) protein and its relatives, the CIP/KIP class of cdk inhibitors, as well as proteins that function in signaling pathways that eventually regulate growth or cell-cycle progression such as p53, PTEN, and APC. The Drosophila genome encodes at least two Rb-related genes (
We have chosen to use a screen that utilizes gain-of-function mutations to identify genes that restrict growth or cell-cycle progression. Classical genetic screens induce mutations using either X-irradiation or chemical mutagens such as ethyl methanesulfonate. These mutagens mostly generate loss-of-function mutations. Such an approach has led to the discovery of many important genes and pathways that regulate cell proliferation but may also fail to identify genes with subtle loss-of-function phenotypes and genes with redundant functions. In mice, a deletion of the gene encoding the CKI p21 does not result in obvious phenotypic abnormalities; these mice are viable and fertile (
To facilitate systematic misexpression screens in Drosophila, Rørth has established a collection of 2300 Drosophila stocks referred to as EP lines (
| MATERIALS AND METHODS |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Drosophila strains and cultures:
Flies were grown on a standard cornmeal medium at 25° unless otherwise specified. The UAS-p21 and UAS-dacapo stocks were generated in our laboratory (J. DE NOOIJ, M.-G. WANG and I. K. HARIHARAN, unpublished data). The UAS-Rbf transgenic line was a gift from N. Dyson. The ey-GAL4 driver line was a gift from J. Treisman. The EP collection was obtained from the Berkeley Drosophila Genome Project (BDGP;
In situ hybridizations and immunohistochemistry:
cDNA clones of the inner centromeric protein (INCENP; LD34828) and CG11518 (GH25362) were obtained from Research Genetics (Huntsville, AL). A 482-bp fragment of the predicted third exon of the elbowB open reading frame (ORF; CG4220) was amplified by PCR and cloned into pBluescript SK+ vector (Promega, Madison, WI). Each linearized cDNA was used as a template for in vitro transcription using the digoxigenin RNA labeling kit (Boehringer Mannheim, Indianapolis). RNA in situs were performed as described previously (
Flow cytometry and cell-cycle analysis:
The genotypes used for fluorescence-activated cell sorter (FACS) analysis were as follows: (1) y w hsFLP/+; EP2039/+; Act5c > CD2 > GAL4 UAS-GFPNLSS65T/+; (2) y w hsFLP/+; +; EP1076/Act5c > CD2 > GAL4 UAS-GFPNLSS65T; and (3) y w hsFLP/+; EP2340/+; Act5c > CD2 > GAL4 UAS-GFPNLSS65T/+. For flow cytometry, larvae were heat-shocked at 72 hr after egg deposition (AED) to induce EP expression. At 96 hr AED, third instar wing imaginal discs were dissected and processed as described (
RNA interference and Northern analysis:
dsRNA was generated using the Ribomax Large Scale RNA Production-T7 kit (Promega). Drosophila SL2 cells were seeded in a six-well culture dish at a concentration of 1.0 x 106 cells/ml in Schneider's medium supplemented with 10% fetal bovine serum. Cells were washed once with Schneider's medium and then incubated with 50 µg (elB, noc) or 25 µg of dsRNA (INCENP) in 1 ml of Schneider's medium. After 30 min, 2 ml of Schneider's medium (with fetal bovine serum) was added. Cell counts were performed each day using a hemocytometer. After 4 days, cells were harvested, fixed, and stained with propidium iodide. FACS analysis was performed using a Cytomation MoFlo instrument. Data were analyzed using FlowJo (Tree Star). To assess the effects of RNA-mediated interference (RNAi) on target mRNA levels, Northern analysis was performed. Total RNA was isolated using the Trizol reagent (Sigma, St. Louis) and processed as described (
| RESULTS |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Overexpression of known negative cell-cycle regulators causes reduced eye phenotypes:
To identify genes that function in restricting tissue growth, we used the ey-GAL4 driver line (
To examine the effects of expressing known negative regulators of the cell cycle in the developing eye imaginal disc, we first crossed flies carrying UAS-dacapo (
|
Identification of novel negative cell-cycle regulators in Drosophila through a gain-of-function screen:
To identify novel regulators of tissue growth, each EP line was crossed to flies expressing ey-GAL4 and the subsequent progeny were screened for reduced eye size. We tested 2296 individual EP lines in this manner. Fifty-three (2.3%) of the lines displayed phenotypic abnormalities. Of the 53 EP lines, 3 lines displayed ey-GAL4-dependent lethality; 4 lines gave slightly rough eyes; and 46 EP lines showed reduced eye size of varying severity (Table 1). The lethality is likely due to low levels of ey-GAL4-driven EP expression in other tissues that perturbs the development of essential organs and affects organism viability.
|
A major advantage of performing a screen utilizing the EP lines is that the P-element insertion in most of the lines has been identified with respect to the completed genomic sequence. Thus the genes adjacent to the site of insertion of the P element can be identified rapidly. The 46 EP lines identified from the screen as having ey-GAL4-dependent reduced eye phenotypes represent insertions at 32 different genetic loci (Table 1). Thirteen lines contain insertions in known genes. As expected, some of these, including hedgehog (hh) and decapentaplegic (dpp), have well-characterized functions in eye development. Other genes identified include regulators of the cytoskeleton (Rac2 and pebble) and fringe (fng), a modulator of extracellular signals. Also identified were Kruppel-homolog 1 (Kr-h1) and elbowB (elB), two putative transcriptional regulators. The remaining 19 loci have conceptual ORFs that have been identified by the sequencing project. Not all insertions were located in the 5' region of the ORFs. In one instance, the EP2340 line, the EP element inserted within the coding region of the gene and is likely to generate a protein with an N-terminal truncation when compared to its wild-type counterpart. In two instances, EP1595 and EP2419, the EP elements had inserted downstream of the coding region so as to direct antisense transcription. Thus, for those two lines, the ey-GAL4-dependent reduced eye phenotypes may represent a partial or complete loss-of-function phenotype.
From the primary screen, we identified 32 genes that, when overexpressed in the developing eye imaginal disc, result in a reduced eye phenotype. In addition to identifying genes with known functions in eye development, we also identified previously characterized genes that hitherto had not been studied in the context of eye development. Finally, we showed that increased expression of 19 novel ORFs also results in a small eye phenotype.
Further classification of genes identified in the screen:
Overexpression of genes can perturb eye development in a variety of ways. We are most interested in identifying genes that restrict cell growth and cell proliferation. Another category of genes likely to be identified in this screen includes those that, when overexpressed, are toxic to cells and result in cell death. To help identify this class of genes and to help define some of the mechanisms by which overexpression results in a reduced eye phenotype, we tested the identified lines in three other ways.
We reasoned that a subset of the genes identified may reduce eye size because overexpression of these genes is toxic and kills cells. If so, then overexpression of these genes is likely to affect both cycling cells and postmitotic cells. In contrast, genes that perturb cell proliferation are more likely to have an effect only in cycling cells. To help distinguish between these two classes of genes, we crossed the selected EP lines to a sevenless-GAL4 (sev-GAL4) driver line. The sev-GAL4 driver directs expression of GAL4 specifically in the postmitotic cells of the eye imaginal discs. Expression of p21, Rbf, and dacapo UAS transgenes under sev-GAL4 control resulted in wild-type adult eyes, demonstrating that overexpression of known negative regulators of the cell cycle has no discernible effect on postmitotic cells (data not shown). Of the 32 genes tested with the sev-GAL4 driver, ectopic expression of 9 genes generated phenotypically abnormal eyes while 16 genes gave wild-type eyes (Table 1). Thus insertions in 9 of 32 loci have an effect in postmitotic cells, indicating that the overexpressed gene may be toxic to all cells or, alternatively, may perturb the growth or differentiation of postmitotic cells. For 16 of 32 loci, overexpression in postmitotic cells shows no effect, suggesting that overexpression of these genes may be able to interfere only with the function of actively cycling cells. Surprisingly, a significant number of loci (7 of 32 loci) exhibited lethality in combination with the sev-GAL4 driver. This could be due to leaky expression in essential organs.
A second way to subdivide the genes identified in the screen was to determine whether the effect generated by overexpression was tissue specific. It is possible that some of the genes identified in the screen may disrupt processes that are specific to eye development. Thus overexpression of these genes in other tissues may not give a detectable phenotype. In contrast, overexpression of known negative regulators of the cell cycle has been shown to inhibit the proliferation of a variety of cell types. The engrailed-GAL4 driver (en-GAL4), which directs expression in the posterior compartment of the wing (
A third method used to classify the lines was aimed at establishing a link with cell-cycle regulation by testing for the ability of cyclinE (cycE) overexpression to rescue the small eye phenotype. It has previously been shown that the effect of dacapo overexpression in the embryo can be overcome by coexpression of a positive regulator, cycE (
In summary, we identified 32 genes that, when overexpressed in the developing eye imaginal disc, result in a reduced eye phenotype. Further classification of these genes showed that 23 of the 32 genes have a sev-GAL4-dependent phenotype, suggesting that these genes may cause toxicity in postmitotic cells or have roles in differentiation. In addition, 19 of the genes appear to have functions in wing development, as demonstrated by their en-GAL4-dependent phenotypes. Finally, by their ability to show suppression of the reduced eye phenotype by coexpression of cyclin E, 14 genes appear to genetically interact with a regulator of G1-S progression.
Identification of loci encoding potential negative regulators of cell proliferation:
We conducted a more detailed analysis of four lines that together represent three different loci. From the secondary analysis of the selected EP lines, four lines (EP965, EP2039, EP2340, and EP1076) that seemed more likely to overexpress negative regulators of growth or proliferation emerged. Expression of these lines under ey-GAL4 control results in reduced eye size (Fig 2) but gives no detectable phenotype when expressed under sev-GAL4 control, suggesting that overexpression does not result in toxicity to all cells. Second, en-GAL4-directed expression of these lines results in a reduced number of cells in the posterior compartment of the wing, indicating a general effect of these genes on cell growth and cell proliferation. Third, coexpression of cycE, a positive regulator of the cell cycle, can suppress the reduced eye size generated by overexpression of these genes alone. We therefore reasoned that these genes may function to regulate cell-cycle progression during Drosophila development. The four lines chosen represent insertions at three loci: INCENP, elB, and the gene designated CG11518.
|
To establish that the predicted ORFs downstream of the respective EP insertions were indeed being expressed ectopically in the presence of GAL4 drivers, digoxigenin-labeled antisense RNA probes were made using the cDNA corresponding to INCENP and CG11518. In the case of elB, a cDNA clone was not available and therefore a fragment of the predicted third exon was amplified by PCR and used as the template to generate antisense RNA probes. In the presence of GMR-GAL4 (directing GAL4 expression behind the morphogenetic furrow in the eye imaginal disc), the antisense probes specifically detected expression of the INCENP and CG11518 in the expected pattern in the EP2340 and EP1076 lines, respectively, confirming that these genes were indeed overexpressed in those EP lines (Fig 3A and Fig B). Because of the apparent high and ubiquitous level of elB expression in the eye imaginal disc (data not shown), we looked at specific GAL4-directed elB expression during embryogenesis in the EP965 and EP2039 lines. In both cases, the elB antisense probe demonstrated expression of the elB RNA in the striped paired pattern in embryos (Fig 3C and Fig D).
|
Characterization of growth and cell proliferation in the EP2340 line:
The P element in the EP2340 line is inserted within the coding region of the gene encoding INCENP. Originally identified in a screen that utilized monoclonal antibodies to identify proteins tightly associated with the chicken mitotic chromosome scaffold (
|
Because the EP element is inserted within the INCENP coding region (Fig 4A), transcripts generated from the P element would result in the translation of a protein that lacks the first 119 amino acids. In vertebrates, it has been shown that the N-terminal region is required for proper targeting of INCENP during mitosis. Despite the divergence in sequence, it is likely that the N-terminal domain of Drosophila INCENP has a similar function. Thus it is conceivable that the inhibition of tissue growth in the EP2340 line may have resulted from the overexpression of an INCENP protein with an N-terminal truncation. To test whether overexpression of wild-type INCENP also affected tissue growth, we generated transgenic flies carrying a gene encoding full-length INCENP under the control of GAL4-responsive UAS elements. In the presence of ey-GAL4, overexpression of full-length INCENP resulted in a small eye phenotype (Fig 4C) indistinguishable from that observed in the EP2340 line, as compared to control eyes (Fig 4B). Thus, it is likely that the N-terminally truncated INCENP produced by EP2340 overexpression functions in this situation in a manner similar to its full-length counterpart.
To characterize the proliferative properties of cells expressing EP2340, we examined cell size and cell-cycle phasing in green fluorescent protein (GFP)-marked clones of cells in the wing imaginal disc and compared their properties to control cells expressing GFP. Expression of EP2340 did not have any observable effect on cell size as assessed by the forward scatter parameter. However, EP2340 overexpression resulted in a small increase in the proportion of cells with a 4N DNA content (cells presumed to be in G2; Fig 4D). We also determined the population doubling time of cells overexpressing EP2340. A potential pitfall in doubling time measurements is the effect of cell competition, a phenomenon in which growth-disadvantaged cells are eliminated by apoptosis. Hence, to eliminate cell death, all doubling times were measured while coexpressing the caspase inhibitor, p35 (
To assess the consequences of reducing INCENP function we used RNAi in SL2 cells in culture (
|
Characterization of insertions at the elbowB locus:
The lines EP965 and EP2039 have insertions that are 171 bp apart and in the same orientation, 
16.7 kb upstream of the gene, elB (CG4220). Even though no cDNA has been identified for this gene so far, the EP2039 insertion was recently shown to be a weak elB allele, and a putative open reading frame (BG:DS06238.3) was identified by the Berkeley Drosophila Genome Project (
|
We examined the properties of proliferating cells in the wing imaginal disc in the EP2039 line. Compared to the wild-type control, overexpression of the EP2039 line did not have any observable effects on cell size as determined by forward scatter (Fig 6B). However, EP2039 overexpression did result in a slight change in cell-cycle phasing. There is a small but reproducible increase in the G2/M population (Fig 6B), suggesting that EP2039 overexpression may be able to restrict either entry into or passage through mitosis. We determined the population doubling time of cells overexpressing EP2039. Overexpression of EP2039 and p35 resulted in a doubling time of 18.6 hr, which was 26% longer than the doubling time of 14.8 hr calculated for the control cells expressing p35 alone (Fig 6C). An increased doubling time with no change in cell size is consistent with a decreased rate of growth (mass accumulation) with a concomitant slowing of the cell cycle.
RNAi was used to reduce the levels of elB in SL2 cells. elB is expressed at extremely low levels in SL2 cells. Expression could not be detected by Northern blotting (data not shown), but could be detected at low levels using Affymetrix oligonucleotide microarrays (O. STEVAUX, D. DIMOVA and N. DYSON, personal communication). There was no change in the growth properties of the treated SL2 cells, although it is important to point out that we could not monitor the change in the level of elB expression (data not shown). We also considered the possibility that elB and noc might have shared functions. However, RNAi experiments using dsRNA for both elB and noc showed no effect on cell division (data not shown).
Characterization of the EP1076 insertion:
The line EP1076 contains an EP insertion in the 5'-UTR of the transcription unit designated CG11518. It encodes an 815-amino-acid protein containing a plant homology domain (PHD) zinc-finger motif (Fig 7A) in its C-terminal region (amino acids 750–802). This motif is found in the polycomb and trithorax group genes in Drosophila (
|
In cells of the wing imaginal disc, overexpression of EP1076 did not have any observable effects on cell size as assessed by forward scatter (Fig 7B). However, EP1076 overexpression also resulted in a small increase in the population of cells with a 4N DNA content (Fig 7B). Cells overexpressing EP1076 and p35 have a doubling time of 17.5 hr, which is 10% longer than the doubling time of 15.9 hr calculated for control cells expressing p35 alone (Fig 7C). Thus, overexpression appears to result in a small decrease in the growth rate of the population. Since overexpression of cyclin E was able to restore normal eye size in this EP line, we examined the properties of the "rescued" cells. Overexpression of cyclin E does not restore normal cell-cycle phasing. Cells overexpressing both cyclin E and EP1076 behave much like cells overexpressing cyclin E alone. More than 80% of cells have a 4N DNA content. However, coexpression of cyclin E reduces doubling time by 12% when compared to cells expressing EP1076 alone and the cell size is slightly reduced (data not shown). Thus cyclin E appears to restore eye size by restoring growth and not by correcting the cell-cycle abnormalities induced by EP1076 overexpression.
Using RNAi, we reduced the levels of CG11518 RNA in SL2 cells (Fig 5E) and examined their proliferative properties. There was no discernible effect on cell division or growth (data not shown).
| DISCUSSION |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
To identify genes that restrict tissue growth in vivo, we screened for genes that, when overexpressed, reduce the size of the Drosophila eye. A screen based on overexpression may identify genes that are missed in screens that depend on phenotypes elicited by loss-of-function mutations, especially in cases in which those phenotypes are subtle or in which the gene has a redundant function. After screening 2296 EP lines using the ey-GAL4 driver to direct gene expression to proliferating cells of the eye imaginal disc, we identified 53 lines (2.3%) that resulted in a reduction in eye size. Screens using other GAL4 driver lines have elicited phenotypes in 2–7% of the lines screened (50% of the EP lines were oriented with the promoter toward the immediately adjacent ORF, this would imply that 4% of appropriately oriented insertions (46 of 1148) would generate a phenotype. This is a rough estimate of the percentage of genes that can generate this phenotype since there are many instances where more than one EP line has an insertion in the same locus. The genome sequencing effort identified 13,600 ORFs in the entire genome. On the basis of the number of lines identified in our screen, we would predict that a screen of the entire genome would identify on the order of 500 genes that could generate this phenotype.
Screens that have been conducted by others in some instances have sought phenotypes that are elicited by disrupting cellular processes that are very different from those that regulate disc growth. In one screen, a pan-neural GAL4 driver was used to identify genes that disrupted synapse formation (
Categories of genes identified:
Of the 32 loci identified in the screen, 13 represent known genes. Several of these genes have functions related to cell division. INCENP homologs are important for chromosome alignment and segregation (
Among the cell-cycle regulators, it was surprising that line EP2584 was not identified in our screen. This line has an insertion upstream of the dacapo gene and was detected in the screen for genes that affected the development of the es organ. We have shown that overexpression of dap using the ey-GAL4 driver and the UAS-dap transgene was able to reduce eye size dramatically (Fig 1). Thus it seems likely that the levels of dap expressed in the EP(2)2584 line are lower and that this lower level of expression is sufficient to disrupt the formation of the es organ (
Another group of genes identified includes genes such as fng, dpp, and hh, which function in patterning proliferation and cell fate determination in the eye imaginal disc. For some of these genes, previous studies suggest a likely mechanism of action. For example, fng has an important role in patterning cell proliferation in the eye imaginal disc. Expression of fng in the ventral half of the eye imaginal disc creates a border of fng+ and fng- cells at the equatorial border. This leads to activation of Notch along the border and via an unknown mechanism promotes cell proliferation in the entire disc (
Our identification of dpp in this screen is consistent with the previous observation that overexpression of dpp in the eye imaginal disc reduces the number of cells in S phase (
Other known genes identified in the screen include signaling molecules (Kr-H1, Rac2, and Traf1) and a receptor (Hr39). The Kr-H1 gene was identified by its sequence homology to Kruppel, a Drosophila segmentation gene (
Rac2 is a member of the Rho family of GTPases (
Traf1 is the Drosophila ortholog of the tumor necrosis factor-receptor-associated factor and appears to be an activator of Jun kinase (
Hormone receptor-like in 39 (Hr39) encodes a nuclear hormone receptor that acts to repress alcohol dehydrogenase transcription (
Properties of INCENP, elB, and CG11518:
We conducted a more detailed characterization of three of the genes that were identified in the screen. The properties of INCENP have been examined in some detail in vertebrate cells (reviewed by
Our studies using RNAi in SL2 cells are consistent with the functions described for INCENP; we observe large polyploid cells. However, we have also shown that overexpression of either full-length or an N-terminally truncated version of INCENP in the eye imaginal disc reduces disc growth and the size of the adult eye. Overexpression of INCENP in cells of the wing disc appears to result in a slowing of cell proliferation as assessed by an increase in the population doubling time. If this were the result of a slowing down of progression through mitosis, one would expect that the rate of growth (mass accumulation) would continue as before and would result in an increase in cell size. However, INCENP overexpression does not appear to change cell size. This indicates that the rate of growth has slowed and there is an equivalent slowing of the cell cycle such that normal cell size is maintained. It is difficult to explain this observation on the basis of the roles described for INCENP in cell division. INCENP binds to microtubules and the overexpression of INCENP in vertebrate cells in culture has been shown to disrupt the microtubule network. This might lead to an impairment of growth in interphase cells as we have observed in cells from the wing disc.
The elB gene encodes protein with a Zn-finger motif. It is most similar to the Drosophila gene noc (
The CG11518 gene encodes a protein with a PHD zinc finger that may function in chromatin remodeling. Overexpression of CG11518 elicits the same phenotype as elB, albeit a weaker one. The extension of division time is less pronounced but the change in cell-cycle phasing is of a similar magnitude. As with elB, a reduction in its function in SL2 cells using RNAi resulted in no measurable change in doubling time (data not shown). One possible explanation of the phenotypes elicited by overexpression is that both elB and CG11518 may function in repressing the expression of genes that promote growth (mass accumulation).
Given that at least two of these genes appear to reduce growth when overexpressed, it is somewhat surprising that normal growth is restored by cyclin E overexpression. In clones of cyclin E overexpressing cells in the wing disc, cell-cycle phasing is altered but growth does not change (
Concluding remarks:
We have identified insertions in 32 loci that, when overexpressed, reduce the growth of the eye imaginal disc with a resulting decrease in the size of the adult eye. Flow cytometry and division time estimates of cells that overexpress the EP insertions allowed us to examine the consequences of overexpressing these genes at the cellular level. RNAi now provides a way of assessing the effects of a reduction in gene function on growth and cell-cycle progression in tissue culture cells. Thus the combination of a gain-of-function screen and an examination of the isolated genes using phenotypes induced by RNAi in cell culture is likely to facilitate the identification of more novel regulators of growth and cell proliferation.
| ACKNOWLEDGMENTS |
|---|
We thank T. Laverty for the EP lines; N. Dyson, B. Edgar, B. Hay, C. Lehner, and J. Treisman for fly stocks; S. Schelble for technical assistance; S. Jiang and J. Yetz-Aldape for help with flow cytometry; O. Stevaux for help with RNAi; J. Monterecy for injections; and members of the Hariharan, Settleman, and Dyson labs for generous help and advice. K.T. was funded by predoctoral fellowships from the National Science Foundation and the MGH Fund for Medical Discovery. I.K.H. is a Faculty Scholar of the Richard Saltonstall Charitable Foundation. This work was funded in part by a grant from the National Eye Institute (EY11632) to I.K.H.
Manuscript received December 15, 2001; Accepted for publication June 17, 2002.
| LITERATURE CITED |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
AASLAND, R., T. J. GIBSON, and A. F. STEWART, 1995 The PHD finger: implications for chromatin-mediated transcriptional regulation. Trends Biochem. Sci. 20:56-59.[Medline]
ABDELILAH-SEYFRIED, S., Y. M. CHAN, C. ZENG, N. J. JUSTICE, and S. YOUNGER-SHEPHERD et al., 2000 A gain-of-function screen for genes that affect the development of the Drosophila adult external sensory organ. Genetics 155:733-752.
ADAMS, M. D., S. E. CELNIKER, R. A. HOLT, C. A. EVANS, and J. D. GOCAYNE et al., 2000 The genome sequence of Drosophila melanogaster. Science 287:2185-2195.
ADAMS, R. R., S. P. WHEATLEYA, A. M. GOULDSWORTHY, S. E. KANDELS-LEWIS, and M. CARMENA et al., 2000 INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow. Curr. Biol. 10:1075-1078.[Medline]
ADAMS, R. R., M. CARMENA, and W. C. EARNSHAW, 2001a Chromosomal passengers and the (aurora) ABCs of mitosis. Trends Cell Biol. 11:49-54.[Medline]
ADAMS, R. R., H. MAIATO, W. C. EARNSHAW, and M. CARMENA, 2001b Essential roles of Drosophila inner centromere protein (INCENP) and Aurora B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation. J. Cell Biol. 153:865-880.
AINSZTEIN, A. M., S. E. KANDELS-LEWIS, A. M. MACKAY, and W. C. EARNSHAW, 1998 INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1. J. Cell Biol. 143:1763-1774.
ASHBURNER, M., S. MISRA, J. ROOTE, S. E. LEWIS, and R. BLAZEJ et al., 1999 An exploration of the sequence of a 2.9-Mb region of the genome of Drosophila melanogaster: the Adh region. Genetics 153:179-219.
AYER, S., N. WALKER, M. MOSAMMAPARAST, J. P. NELSON, and B. Z. SHILO et al., 1993 Activation and repression of Drosophila alcohol dehydrogenase distal transcription by two steroid hormone receptor superfamily members binding to a common response element. Nucleic Acids Res. 21:1619-1627.
BASLER, K., P. SIEGRIST, and E. HAFEN, 1989 The spatial and temporal expression pattern of sevenless is exclusively controlled by gene-internal elements. EMBO J. 8:2381-2386.[Medline]
BOWTELL, D. D., M. A. SIMON, and G. M. RUBIN, 1989 Ommatidia in the developing Drosophila eye require and can respond to sevenless for only a restricted period. Cell 56:931-936.[Medline]
CAPLEN, N. J., J. FLEENOR, A. FIRE, and R. A. MORGAN, 2000 dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference. Gene 252:95-105.[Medline]
CHEAH, P. Y., Y. B. MENG, X. YANG, D. KIMBRELL, and M. ASHBURNER et al., 1994 The Drosophila l(2)35Ba/nocA gene encodes a putative Zn finger protein involved in the development of the embryonic brain and the adult ocellar structures. Mol. Cell. Biol. 14:1487-1499.
CHO, K. O. and K. W. CHOI, 1998 Fringe is essential for mirror symmetry and morphogenesis in the Drosophila eye. Nature 396:272-276.[Medline]
CLEMENS, J. C., C. A. WORBY, N. SIMONSON-LEFF, M. MUDA, and T. MAEHAMA et al., 2000 Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl. Acad. Sci. USA 97:6499-6503.
COOKE, C. A., M. M. HECK, and W. C. EARNSHAW, 1987 The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J. Cell Biol. 105:2053-2067.
DATAR, S. A., H. W. JACOBS, A. F. DE LA CRUZ, C. F. LEHNER, and B. A. EDGAR, 2000 The Drosophila cyclin D-Cdk4 complex promotes cellular growth. EMBO J. 19:4543-4554.[Medline]
DENG, C., P. ZHANG, J. W. HARPER, S. J. ELLEDGE, and P. LEDER, 1995 Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 82:675-684.[Medline]
DE NOOIJ, J. C. and I. K. HARIHARAN, 1995 Uncoupling cell fate determination from patterned cell division in the Drosophila eye. Science 270:983-985.
DE NOOIJ, J. C., M. A. LETENDRE, and I. K. HARIHARAN, 1996 A cyclin-dependent kinase inhibitor, Dacapo, is necessary for timely exit from the cell cycle during Drosophila embryogenesis. Cell 87:1237-1247.[Medline]
DOBIE, K. W., C. D. KENNEDY, V. M. VELASCO, T. L. MCGRATH, and J. WEKO et al., 2001 Identification of chromosome inheritance modifiers in Drosophila melanogaster. Genetics 157:1623-1637.
DOMINGUEZ, M. and J. F. DE CELIS, 1998 A dorsal/ventral boundary established by Notch controls growth and polarity in the Drosophila eye. Nature 396:276-278.[Medline]
DU, W. and N. DYSON, 1999 The role of RBF in the introduction of G1 regulation during Drosophila embryogenesis. EMBO J. 18:916-925.[Medline]
DU, W., M. VIDAL, J. E. XIE, and N. DYSON, 1996 RBF, a novel RB-related gene that regulates E2F activity and interacts with cyclin E in Drosophila. Genes Dev. 10:1206-1218.
HAMMOND, S. M., E. BERNSTEIN, D. BEACH, and G. J. HANNON, 2000 An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293-296.[Medline]
HARDEN, N., H. Y. LOH, W. CHIA, and L. LIM, 1995 A dominant inhibitory version of the small GTP-binding protein Rac disrupts cytoskeletal structures and inhibits developmental cell shape changes in Drosophila. Development 121:903-914.[Abstract]
HARIHARAN, I. K., K. Q. HU, H. ASHA, A. QUINTANILLA, and R. M. EZZELL et al., 1995 Characterization of rho GTPase family homologues in Drosophila melanogaster: overexpressing Rho1 in retinal cells causes a late developmental defect. EMBO J. 14:292-302.[Medline]
HAUCK, B., W. J. GEHRING, and U. WALLDORF, 1999 Functional analysis of an eye specific enhancer of the eyeless gene in Drosophila. Proc. Natl. Acad. Sci. USA 96:564-569.
HAY, B. A., T. WOLFF, and G. M. RUBIN, 1994 Expression of baculovirus P35 prevents cell death in Drosophila. Development 120:2121-2129.[Abstract]
HAYASHI, S., 1996a A Cdc2 dependent checkpoint maintains diploidy in Drosophila. Development 122:1051-1058.[Abstract]
HAYASHI, S., 1996 Drosophila imaginal development and the escargot gene. Tanpakushitsu Kakusan Koso 41:1095-1103, b (in Japanese).[Medline]
HAZELETT, D. J., M. BOUROUIS, U. WALLDORF, and J. E. TREISMAN, 1998 decapentaplegic and wingless are regulated by eyes absent and eyegone and interact to direct the pattern of retinal differentiation in the eye disc. Development 125:3741-3751.[Abstract]
HORSFIELD, J., A. PENTON, J. SECOMBE, F. M. HOFFMAN, and H. RICHARDSON, 1998 decapentaplegic is required for arrest in G1 phase during Drosophila eye development. Development 125:5069-5078.[Abstract]
HUANG, A. M. and G. M. RUBIN, 2000 A misexpression screen identifies genes that can modulate RAS1 pathway signaling in Drosophila melanogaster. Genetics 156:1219-1230.
JIAO, R., M. DAUBE, H. DUAN, Y. ZOU, and E. FREI et al., 2001 Headless flies generated by developmental pathway interference. Development 128:3307-3319.
KAITNA, S., M. MENDOZA, V. JANTSCH-PLUNGER, and M. GLOTZER, 2000 Incenp and an aurora-like kinase form a complex essential for chromosome segregation and efficient completion of cytokinesis. Curr. Biol. 10:1172-1181.[Medline]
KRAUT, R., K. MENON, and K. ZINN, 2001 A gain-of-function screen for genes controlling motor axon guidance and synaptogenesis in Drosophila. Curr. Biol. 11:417-430.[Medline]
LANE, M. E., K. SAUER, K. WALLACE, Y. N. JAN, and C. F. LEHNER et al., 1996 Dacapo, a cyclin-dependent kinase inhibitor, stops cell proliferation during Drosophila development. Cell 87:1225-1235.[Medline]