- Email: [email protected]
Cell proliferation, survival, and death in the Drosophila eye
seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 12, 2001: pp. 499–507 doi:10.1006/scdb.2001.0274, available online at http://www.idealibrary.com on
Cell proliferation, survival, and death in the Drosophila eye Nicholas E. Baker the visual field and transmits information to the optic centers of the brain. 1–3 Precise ommatidial structure is important for multiple aspects of compound eye function. Flies infer motion, obviously a critical ability for an airborne insect, using the temporal procession of images across the field of ommatidia. Superposition of R7 and R8 photoreceptors within each ommatidium is thought to be important for color vision and polarization detection. R8 always lies underneath R7, placing the light sensitive rhabdomere organelle of R8 in the shadow of the R7 rhabdomere. 4–6 Such functional constraints probably explain why both the number and type of cells in each ommatidium are so invariant. In this respect the eye differs from some other organs, such as the wing, whose function is more mechanical and can be achieved by an array of largely similar cells whose precise numbers may be less important. Cellular diversity and reproducible structure have contributed to the Drosophila eye becoming an important system for the study of cell fate specification. Eighteen precursor cell fates are specified by postmitotic cell–cell interactions, often involving receptor tyrosine kinase signaling. 7–9 The nineteenth precursor cell divides twice to generate a four-cell sensory bristle organ. Postmitotic fate specification raises the question of how cell numbers are regulated. Cell number depends on the balance of cell proliferation and cell death, both of which are coordinated with cell differentiation. Recent progress has come from studying the regulation of the terminal cell divisions. Such studies show that both cell cycle progression and cell survival are coordinated by short-range signals that can adjust the number of unspecified progenitor cells according to the number of ommatidia required. 10 This mechanism permits fine-tuning of retinal cell number at the G2/M transition of the cell cycle. Temporal and spatial regulation of G1 and S phases also occur but less is known of the mechanisms. An index of genes implicated in retinal proliferation and survival is shown in Table 1.
The Drosophila retina has a precise repeating structure based on the unit eye, or ommatidium. This review summarizes studies of the cell proliferation and survival episodes that affect the number of cells available to make each ommatidium. Late in larval development, as differentiation and patterning begin, the retinal epithelium exhibits striking regulation of the cell cycle including a transient G1 arrest of all cells, followed by a ‘Second Mitotic Wave’ cell cycle that is regulated at the G2/M transition by local intercellular signals. Reiterated episodes of cell death also contribute to precise regulation of retinal cell number. The EGF receptor homolog has multiple roles in retinal proliferation and survival. Key words: proliferation / survival / apoptosis / Drosophila c 2001 Academic Press
The Drosophila retina requires precise cellular architecture Although different in many respects, both Drosophila and vertebrate retinas contain multiple distinct cell types each specialized for particular roles in the perception of light. The numbers of each cell type are important, as well as the cell diversity. This is especially so for Drosophila, where the compound eye is constructed according to a strikingly regular plan [Figure 1(a)]. Each adult Drosophila eye comprises about 800 ommatidia, or unit eyes. Ommatidia have a precise, reproducible structure generated by 19 precursor cells [Figure 1(a)]. Each ommatidium is a stereotyped assembly that focuses light from a small portion of
From the Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. E-mail: [email protected] c
2001 Academic Press 1084–9521 / 01 / 060499+ 09 / $35.00 / 0
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Figure 1. Structure and development of Drosophila retina. (a) Cartoon of retinal structure after specification of all 19 precursor cells and death of supernumerary cells. Eight photoreceptor neurons are surrounded by accessory cone and pigment cells. Intervening pigment cells isolate neighboring ommatidia. Note that a single layer of 2◦ /3◦ pigment cells is sufficient. Key: R1–R8, photoreceptor neurons; ACC, anterior cone cell; ECC, equatorial cone cell; PCC, posterior cone cell; PoCC, polar cone cell; PPC, primary pigment cell; SPC, secondary pigment cell; TPC, tertiary pigment cell; bcg, bristle cell group. For review see Reference 2. (b) Eye imaginal discs differentiate progressively, beginning at the posterior margin and advancing anteriorly (arrow). In this and subsequent figures, posterior is on the right, so that reading from right to left reveals a temporal sequence of increasingly mature retinal development. A ‘second mitotic wave’ of cell divisions occurs posterior to the morphogenetic furrow (MF). Most of these divisions occur between columns 3 and 5. For review see Reference 2. (c) Enlargement of the region near the morphogenetic furrow, showing the stepwise recruitment and differentiation of ommatidial cells. R8 cells are differentiating in column 1 within the morphogenetic furrow, the other precluster cells R2, R3, R4 and R5 soon afterwards. All the other cells re-enter the cell cycle in the SMW and are recruited to further ommatidial fates after column 5 (shaded cells). Figure adapted from Reference 37. Table 1.
Genes involved in eye disc proliferation and survival
tkv, sax, punt, shn
Required for prompt G1 arrest in the morphogenetic furrow in response to Dpp.
rux
Required for G1 arrest since it prevents ectopic activation of G2 cyclins during the long morphogenetic furrow G1.
rca
Required for cyclin A function in normal G2, or the ectopic S phases of rux mutants.
Su(rux)2B, 2C
Mutations affecting all S phases and that can suppress rux.
ato
Required autonomously for R8 specification. Required indirectly for both G1/S and G2/M of the SMW. The requirement in G2/M is attributable to reduced EGFR activation in the absence of differentiating photoreceptors that secrete EGFR ligands. The requirement in G1/S may be due to absence of other factors.
da
Required for both G1/S and G2/M of the SMW. Possibly similar to ato, which requires da as a heterodimeric partner.
spi
The EGFR ligand required for G2/M progression, neural differentiation, and survival, but apparently not for G1 arrest.
egfr
Required for: G1 arrest and neural differentiation of precluster cells R2, R3, R4, R5; mitosis of SMW cells; differentiation of most other ommatidial cells; survival of retinal cells.
N
Required for apoptosis of extra pupal cells as well as for multiple aspects of fate specification.
hid
Proapoptotic gene implicated in retinal cell survival.
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The Drosophila eye imaginal disc and the ‘second mitotic wave’
column: 1
‘Imaginal discs’ are the larval structures that provide the source of adult cells in holometabolous insects that show full metamorphosis. Imaginal discs invaginate from the body wall during embryogenesis and proliferate without differentiating while inside the body cavity of the larva. Within the pupa, imaginal discs differentiate adult structures that replace larval body parts. 11 Ommatidial specification and retinal differentiation begin in the eye imaginal disk during the last larval instar. A ‘morphogenetic furrow’ sweeps from posterior to anterior across the eye disc as progressively more anterior disc regions begin eye differentiation. Cell division temporarily ceases, and the first ommatidial progenitor cells are specified 2 [Figure 1(b) and 1(c)]. Cell cycle behavior posterior to the morphogenetic furrow is correlated with differentiation status (Figure 2). Specification begins with the photoreceptor cells. Single R8 photoreceptor cells are the first to differentiate under the control of the proneural gene atonal (ato). Each R8 cell rapidly recruits R2, R3, R4 and R5 into preclusters each containing these five photoreceptor precursors. Ommatidium assembly fails completely in ato mutants, owing to the critical founding role of the R8 cells. 12 Cells of the precluster begin neural differentiation and never divide again. The other unspecified cells re-enter the cell cycle in the ‘second mitotic wave’ (SMW), the cell cycle that occurs posterior to the morphogenetic furrow. 1,2 During this period the eye disc contains the specified, differentiating, G1-arrested five-cell preclusters surrounded by SMW cells that undergo S phase and mitosis (Figures 1 and 2). The remaining 14 ommatidial precursor cells are born in the SMW. This is essential to generate enough precursor cells to complete each ommatidium. If it is prevented many ommatidia cannot recruit sufficient cells. All cell fates can still occur, however, indicating that the cell division is not linked tightly to specification of any particular cell type. 13 Specification of the remaining 14 precursor cell fates occurs postmitotically during further episodes of signaling through EGFR and other cell surface receptors. 7–9 Much later, in the pupa after all the ommatidial cells have been specified, apoptosis removes any supernumerary cells. 2 Many genes that are important for growth and development of the retina have other prior developmental functions. Although conditional mutant
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recruitment G1 arrest SMW S-phase SMW G2-phase SMW mitosis cell death
Figure 2. Timing of the SMW and differentiation. An enlargement of Figure 1(c) also showing the spatial and temporal extent of particular processes. Recruitment of ommatidial cells occurs in waves. Specification of the R2–R5 precluster cells in response to signals from the R8 cell occurs by column 1 in the morphogenetic furrow. R1 and R6 join in columns 4–5, R7 by column 6, and all the cone cells are recruited by column 10 (for review see Reference 2). G1 of the SMW is a continuation of the G1 arrest that affects all cells anterior to the morphogenetic furrow, initially in response to Dpp signaling. G1 arrest is maintained posterior to the furrow in the five photoreceptor neurons of each ommatidial precluster. S phase of the SMW begins anterior to column 1. All the cells outside the preclusters are in S phase by column 2. S-phase DNA labeling is undetectable posterior to column 4. Many SMW cells pass rapidly through G2phase to mitosis, other cells remain in G2 indefinitely. The first SMW mitosis occurs around column 3; most mitosis occurs between columns 3 and 5. Occasional mitosis occurs more posteriorly. Small amounts of cell death also occur in the eye disc, typically affecting less than one cell per ommatidium. In normal development cell deaths are first observed around column 7. Figure adapted from Reference 10.
alleles exist for some of these loci, mosaic analysis is usually necessary to recover retinas containing clonal patches of mutant cells in animals otherwise developing normally. Introduction of the FLP/FRT site-specific recombination system into Drosophila has facilitated use of mitotic recombination to generate clones of mutant cells in situ in developing eye tissue. By catalyzing chromosome arm exchange during the mitotic cell cycle, single cells homozygous for 501
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separable processes. Neural differentiation depends on activation of the EGFR by the TGF alpha-like Spitz (Spi) protein. By contrast, G1 arrest can occur in the absence of this ligand. 10 The primary source of Spi within each ommatidium is R8. 24,25 The ligand responsible for G1 arrest has yet to be determined. Except for these preclusters, cells synchronously enter S phase of the SMW. Since S phase occurs in mutants that lack nearly all ommatidia it seems unlikely that local induction is involved. 2,15,26,27 There may be a longer range, global signal that triggers cell cycle re-entry because the proneural gene ato which is required for much of eye development is indirectly and non-autonomously required for SMW S phase. 10,27 A requirement for the heterodimer partner for ato, encoded by daughterless (da), in the SMW might also be indirect. 28 It is also possible that down-regulation of the pathway maintaining G1 arrest in the furrow may be sufficient for cells to re-enter the cell cycle, no positive signal being necessary. Unfortunately this possibility is hard to assess since no genes have yet been described that are specifically and autonomously required for G1/S progression of the SMW. Two genes, Su(rux)2B and Su(rux)2C, that are required for the SMW seem to be required for S phases throughout development. 19
mutants of interest can be generated in heterozygous animals. Clones of homozygous cells descended from such recombinants usually survive even when of genotypes that would be lethal to the whole animal. 14
Cell cycle arrest and the morphogenetic furrow Obvious spatial regulation of the cell cycle is first apparent just anterior to the morphogenetic furrow. All the cells arrest in G1 of their cell cycles before any differentiation begins 1,15,16 (Figure 2). G1 arrest appears to require Decapentaplegic (Dpp) signaling initially but then becomes independent of Dpp; G1 arrest is temporarily delayed in clones of cells unable to respond to Dpp, but not prevented indefinitely. Since dpp is transcribed in the morphogenetic furrow, it is thought that a concentration gradient of Dpp protein anterior to the morphogenetic furrow defines a position where cells pause prior to S phase because of the high Dpp level. Consistent with this view, ubiquitous ectopic expression of Dpp is sufficient to cause cell cycle arrest ahead of the morphogenetic furrow. 17,18 After a few hours an unidentified second pathway supplements or replaces Dpp signaling to maintain G1 arrest. The roughex (rux) gene was originally identified as potentially involved, but is now thought to down-regulate G2 cyclins at mitosis. 19–21 Apparently rux is also important to suppress inappropriate activity of the G2-specific cyclin A during this long G1. Consistent with this view, the ectopic S phases in clones of cells mutant for rux require rca1, a gene necessary for the normal function of cyclin A in G2. 22 Both the nature of the G1 arrest signal and the function of the morphogenetic furrow arrest remain uncertain.
G2/M in the second mitotic wave By contrast to the apparently constitutive, synchronous G1/S transition of cells in the SMW, G2/M progression is regulated by local signals 15 (Figure 3). SMW cells arrest in G2 if ommatidia are not present, showing that signals depend on the presence of ommatidia. The signal provided by ommatidia is again the EGFR agonist Spi. Both Spi and its processing and presenting factors are produced in the precluster cells. 10 EGFR activation is both necessary for SMW mitosis and sufficient to induce mitosis in the absence of ommatidia. Activation of EGFR acts in part to activate string (stg ), which can also induce mitosis in the absence of ommatidia. stg encodes the homolog of yeast cdc25, a phosphatase required for cdc2 activation. Stg is limiting for many cell cycles during Drosophila embryogenesis and is the subject of complex transcriptional regulation thought to couple the cell cycle to developmental signals. 29,30 Unlike EGFR activation, targeted stg transcription does not completely rescue the SMW in eye discs lacking ommatidia, suggesting that EGFR signaling has other cell cycle roles besides activating stg. 10
G1/S in the second mitotic wave Posterior to the morphogenetic furrow many cells progress from G1 into S phase (Figure 2). Most cells express cyclin D and E and re-enter the cell cycle in the SMW. 2,23 The five cells differentiating in each precluster are the exception. Precluster cells remain in G1 and never divide again. Maintaining G1 arrest in preclusters depends on ato function in R8 and on EGFR activity in precluster cells R2, R3, R4, R5. ato and EGFR are also required for neural differentiation of these photoreceptor cells. Notably, G1 arrest and neural differentiation appear to be simultaneous but 502
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Figure 3. Regulation of SMW cell number by precluster signaling to the EGFR. The cartoon illustrates how EGFR regulates SMW processes shown in Figure 2. (a)–(c) represent normal eye development. (d), (e) illustrate the consequences of a defect in ommatidium spacing. Shading reflects EGFR activity. (a) Cartoon of the eye disc epithelium around column 1. R8 cells (<) activate EGFR to arrest and recruit four other precluster cells (shaded). Other cells lack EGFR activity, re-enter the cell cycle and perform S phase (unshaded). (b) Column 3. All cells outside preclusters are in the SMW. Precluster contact activates EGFR in most cells (shaded), allowing progression into mitosis. Some cells are not activated and remain in G2 (unshaded). (c) Column 10. After the SMW, EGFR activity (shaded) protects most G1 progenitor cells from cell death. EGFR activity also protects some remaining G2 cells without causing their mitosis (light shading). A few cells die (*). In wild-type strains less than one cell dies per ommatidium, on average. In combination, mitosis and survival yield an average of 17.1 cells per precluster after the SMW. Another two to three cells die per ommatidium in the pupa several days later. Ultimately, 14 further progenitor cells are required to complete each ommatidium (Figure 1). (d) Cartoon of an eye disc around column 3 where a precluster is missing. Precluster contact activates EGFR in neighboring cells (shaded). Many cells lack precluster contacts and arrest in G2 (unshaded). (e) Cartoon of an eye disc around column 10 where a precluster is missing. Reduced mitosis and survival adjust the number of progenitor cells towards the number necessary to complete the extant ommatidia. Symbols as in panel (c).
Local signaling through the EGFR is both essential and limiting in the SMW, so that not all SMW cells complete this cell cycle (Figure 3). In normal development, an average of 9.7 cells enter the SMW for every five-cell precluster, but on average only 7.7 cells complete the cell cycle and divide. On average two cells never receive the mitotic signal and so remain in G2. 10 Conditional cell cycle progression in the SMW provides flexibility to the generation of unspecified progenitor cells (see later).
survival indirectly. For example, since Spi is required for specification and neural differentiation of ommatidial cells, any other EGFR ligand secreted by ommatidial cells would also be affected in clones of cells mutant for Spi. 10 Regulated cell survival further couples progenitor cell number to the ommatidia differentiating in the eye disc. Several days later, once all the ommatidial cells have been specified and are differentiating in the pupa, further cell death occurs to remove approximately two to three extra cells around each ommatidium. A survival signal coming from the cone cells or primary pigment cells of each ommatidium protects nearby cells so that they can survive to differentiate as secondary and tertiary pigment cells and as sensory bristle organs. 31 If cell death is prevented by expression of baculovirus p35, the rescued ‘undead’ cells differentiate as supranumerary pigment cells. 32 Pupal cell death requires Notch signaling. 2,33 Spi and EGFR expression patterns, and misexpression experiments involving activated Ras or the EGFR antagonist Argos suggest that EGFR is required for survival of pupal cells also, probably through regulated transcription and activity of the proapoptotic gene head involution defective (hid). 31,34–36
Survival signals After SMW cells divide, EGFR signaling is then required from about column 6 or 7 onwards to maintain cell survival (Figures 2 and 3). In normal development only 0.2–0.3 eye disc cells die per ommatidium, but if ommatidia are removed or mis-spaced this number increases dramatically. In Ellipse mutants where very few ommatidia differentiate, almost all the SMW cells die. Cells also die in cell clones mutant for spi. Spi secreted from the differentiating ommatidial cells may be the ligand that activates EGFR during survival signaling, although it is also possible that Spi affects 503
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Coordinating cell numbers with neural patterning
normal development eye discs contain cells that survive despite not having divided, and cells that undergo apoptosis even though they performed the EGFR-dependent mitosis. 10 Further work is required to explore how the EGFR can trigger independent implementation of mitotic and survival responses. The relationship between mitosis and fate specification may be less flexible. G2-arrested cells that escape apoptosis appear to lie in between differentiating ommatidia and are not recruited to early cell fates. They have not been examined during the rest of development to see whether they can differentiate at a later time. It may be that such G2-arrested cells normally find themselves amongst the population that die during pupation. When cell death is blocked by expression of baculovirus p35, however, all cells survive and extra pigment cells differentiate. 32 The G2 cells must therefore be amongst the extra pigment cells. The probability that cells can differentiate as pigment cells in G1 or G2 is supported by the differentiation of secondary and tertiary pigment cells in Elp mutants that lack ommatidia; most of these cells are arrested in G2. 15 Even egfr mutant cells can survive into the adult if protected by p35 expression. Such cells differ morphologically from normal cells. They most resemble unpigmented pigment cells. Some apparently produce small rhabdomeres and might be abnormal R8-like cells. Whether the abnormalities are due to the G2 arrest or to other consequences of EGFR signaling deficiency is not known. Whether cells in G2 could be recruited to the earlier primary pigment cell, cone cell or photoreceptor cell fates is uncertain, but we favor the idea that EGFR activation in such cells would inevitably lead to mitosis before any fate specification response, since this is the order of events when EGFR is ectopically activated in eye discs. 10
Both mitotic signaling and survival signaling contribute to matching the number of unspecified progenitor cells to the number of ommatidia (Figure 3). Mitotic signaling through the EGFR serves to increase the number of progenitor cells as required for normal development. In the event of a surplus, distance from the survival signal can eliminate some extra cells. Whereas complete cell division would result in doubling cell number, the SMW has a lower yield because not all the cells that enter the SMW cell cycle complete it or survive. In normal development, the SMW produces about 89% of the theoretical maximum number of progenitor cells that would be obtained if all cells divided and survived. Because ommatidial preclusters are the source of both mitotic and survival signals, the number of unspecified cells generated and surviving through the SMW depends on the number of ommatidia that have already begun differentiation. In most organs cells cycle asynchronously, making the yield of each cell cycle harder to assess. Perhaps it is often less than 100%, reflecting undescribed limits on cell division and survival analogous to those of the SMW.
Coordination and specificity of multiple signals As is well known, EGFR signaling also plays another role inducing postmitotic cells to adopt particular cell fates 7–9 (Figure 2). The same precluster cells whose mitotic signals generate unspecified precursor cells then induce postmitotic cell fate specification. Fate specification follows very rapidly after mitotic signaling, and precedes and overlaps survival signaling. The first cells specified after the SMW, photoreceptor cells R1 and R6, can be identified joining the ommatidia by column 5, which is 3 h or less after they divide 10 (Figure 2). One question concerns the specificity of EGFR signaling in fate specification, mitosis and survival. Do these responses occur independently through different episodes of EGFR signaling, or does the SMW contain only two types of cells: those that experience EGFR signaling, so dividing, surviving and being specified, and those that lack EGFR signaling, therefore arresting, remaining unspecified and dying? It is already clear that some EGFR responses can occur independently. Thus during
Retinal growth before the second mitotic wave It should be emphasized that the EGFR roles in mitosis and survival apply only to this last cell cycle of retina development, not to earlier growth of the eye or to other tissues, which do not depend on receptor tyrosine kinases in the same way. The analysis of mosaic patches deficient for EGFR and related genes relies on the ability of such mutant cells to grow divide and survive up until the SMW, otherwise data could never have been obtained. Because cells lacking either of the downstream factors Ras or Raf are similarly able to grow and survive during much 504
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of eye development, we think that neither EGFR nor any other RTK acting through the canonical RTK–Ras–Raf pathway can be essential for retinal cell division or survival up until the SMW. 37 The fact that these genes are not essential, however, does not mean they have no role. Indeed cells mutant for EGFR, Ras or Raf grow poorly, and analysis of their developmental functions usually relies on the Minute technique to compensate for poor growth. 37,38 In this technique, mosaic analysis is performed in the genetic background of mutations (‘Minutes’) that reduce ribosome function cell autonomously. 39 In this way the EGFR, Ras, or Raf homozygous cells are simultaneously conferred a growth advantage through restoring normal translation capacity. The resulting growth advantage offsets the intrinsic debility of EGFR, Ras or Raf mutant cells. From such data it appears that EGFR is important for normal growth before the SMW but contributes to growth quantitatively without being essential, unlike the SMW where EGFR signaling is indispensable for mitosis. The role of EGFR, Ras and Raf in eye development prior to the SMW may be related to cellular growth and G1/S progression. 40 In studies of the developing undifferentiated wing, Prober and Edgar 41 found that Ras1 promoted cellular growth, in turn elevating cyclin E levels and promoting G1/S progression. If development of the eye before the morphogenetic furrow is similar, EGFR, Ras and Raf could promote proliferation through an effect of cellular growth on G1/S progression. After the morphogenetic furrow G2/M progression would become completely dependent on EGFR activation in the SMW. It is possible that no extracellular signal is essential for proliferation and growth before the morphogenetic furrow. If cellular mass accumulation is the only requirement then it will be interesting to determine the identity, sources and mechanisms of the putative signals promoting such cellular growth.
tution during evolution. The number of ommatidial cells appears to have increased in distinct taxa. 42 Perhaps additional precursor cells can only be generated once ommatidial development has already been initiated; an earlier increase in the number of retinal cells might lead instead to more ommatidia without changing their constitution. 43 If this is the case then more elaborate retinas require a SMW to acquire increased cellular diversity. The G1 arrest of cells within the morphogenetic furrow is a prominent feature of eye development but its function is uncertain. Some evidence that there must be a function comes from the rux mutations which cause morphological abnormalities that are suppressed by restoration of G1 arrest to rux mutants. Therefore inappropriate cell division is the cause of the abnormal morphology. 22 Unless rux mutations also affect G1 arrest of the precluster cells, this suggests that G1 arrest in the morphogenetic furrow is essential. Mitosis interrupts transcription, 44 so one speculation is that a prolonged mitosis-free period may permit transcription of large genes such as N and Dl, whose products accumulate in the morphogenetic furrow and are required for subsequent cell fate specification. 45 In this regard it is noteworthy that cells in other neurogenic regions also exhibit cell cycle arrest before neural fate specification, including the embryonic neuroectoderm and thoracic proneural territories. 46,47 In these other tissues arrest occurs in G2, however. It is also possible that cell cycle arrest is important in a transition from G1/S cell cycle regulation during the phase of rapid imaginal disc growth, when the goal of growth may be to attain particular tissue dimensions, to G2/M cell cycle regulation during the patterning and specification of adult structures, when the precise number and arrangement of cells may be paramount. It is curious that cell survival should be subject to multiple regulation, and that excess retinal cells die during pupation after being preserved through the SMW. 2,31 We do not know why eye development takes this roundabout course. One possibility is that criteria for survival become increasingly stringent as retinal development approaches completion. Another speculation is that eye development recapitulates reductions in pigment cell number that might have occurred during evolution. In the hemipterous bug Oncopeltus, for example, the ommatidial pigment cell lattice is octagonally arranged instead of hexagonally as in Drosophila. 48 The pigment cell lattice of Drosophila is topologically efficient, each of the secondary pigment cells separating two neighboring
Concluding remarks The regulated cell cycle of cells in the morphogenetic furrow and SMW are striking features of Drosophila eye development. We have suggested that the SMW adjusts the number of precursor cells generated in the retina according to the number of ommatidia being made. 10 Such adjustment must be made once initial steps in ommatidium specification have occurred for the number of ommatidia to be estimated. A second potential benefit is for changing ommatidial consti505
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ommatidia and each tertiary pigment cell separating three (Figure 1). Pupal cell death might simplify an atavistic condition in which each ommatidium was surrounded by its own pigment cells; multiple cells separating neighboring ommatidia could be redundant for optical purposes. Most comparative studies of compound eyes pay less attention to pigment cell numbers, however, so at present there is little evidence against which this idea can be judged.
15. Baker NE, Rubin GM (1992) Ellipse mutations in the Drosophila homologue of the EGF receptor affect pattern formation, cell division, and cell death in eye imaginal discs. Dev Biol 150:381–396 16. Thomas BJ, Gunning DA, Cho J, Zipursky L (1994) Cell cycle progression in the developing Drosophila eye: roughex encodes a novel protein required for the establishment of G1. Cell 77:1003–1014 17. Penton A, Selleck SB, Hoffman FM (1997) Regulation of cell cycle synchronization by decapentaplegic during Drosophila eye development. Science 275:203–206 18. Horsfield J, Penton A, Secombe J, Hoffman FM, Richardson H (1998) Decapentaplegic is required for arrest in G1 phase during Drosophila eye development. Development 125:5069–5078 19. Thomas BJ, Zavitz KH, Dong X, Lane ME, Weigmann K, Finley RLJ, Brent R, Lehner CF, Zipursky SL (1997) Roughex down-regulates G2 cyclins in G1. Genes Dev 11:1289–1298 20. Avedisov SN, Krasnoselskaya I, Mortin M, Thomas BJ (2000) Roughex mediates G(1) arrest through a physical association with cyclin A. Mol Cell Biol 20:8220–8229 21. Foley E, Sprenger F (2001) The cyclin-dependent kinase inhibitor Roughex is involved in mitotic exit in Drosophila. Curr Biol 11:151–160 22. Dong X, Zavitz KH, Thomas BJ, Lin M, Campbell S, Zipursky SL (1997) Control of G1 in the developing Drosophila eye: rca1 regulates cyclin A. Genes Dev 11:94–105 23. Finley RLJ, Thomas BJ, Zipursky SL, Brent R (1996) Isolation of Drosophila cyclin D, a protein expressed in the morphogenetic furrow before entry into S phase. Proc Natl Acad Sci 93:3011–3015 24. Tio M, Moses K (1997) The Drosophila TGFalpha homolog Spitz acts in photoreceptor recruitment in the developing retina. Development 124:343–351 25. Baonza A, Casci T, Freeman M (2001) A primary role for the EGF receptor in ommatidial spacing in the Drosophila eye. Curr Biol 396–404 26. Zak NB, Shilo BZ (1992) Localization of DER and the pattern of cell divisions in wild-type and Ellipse eye imaginal discs. Dev Biol 149:448–456 27. Jarman AP, Sun Y, Jan LY, Jan YN (1995) Role of the proneural gene, ato, in formation of Drosophila chordotonal organs and photoreceptors. Development 121:2019–2030 28. Brown NL, Paddock SW, Sattler CA, Cronmiller C, Thomas BJ, Carroll SB (1996) Daughterless is required for Drosophila photoreceptor cell determination, eye morphogenesis, and cell cycle progression. Dev Biol 179:65–78 29. Edgar BA, O’Farrell PH (1989) Genetic control of cell division patterns in the Drosophila embryo. Cell 57:177–187 30. Lehman DA, Patterson B, Johnstone LA, Balzer T, Britton JS, Saint R, Edgar BA (1999) Cis-regulatory elements of the mitotic regulator, string/Cdc25. Development 126:1793–1803 31. Miller DT, Cagan RL (1998) Local induction of patterning and programmed cell death in the developing Drosophila retina. Development 125:2327–2335 32. Hay BA, Wolff T, Rubin GM (1994) Expression of baculovirus P35 prevents cell death in Drosophila. Development 120:2121–2129 33. Cagan RL, Ready DF (1989) Notch is required for successive cell decisions in the developing Drosophila retina. Genes Dev 3:1099–1112 34. Sawamoto K, Taguchi A, Hirota Y, Yamada C, Jin MH, Okano H (1998) Argos induces programmed cell death in the developing Drosophila eye by inhibition of the Ras pathway. Cell Death Differ 5:262–270
Acknowledgements I am grateful to David Tyler for comments and the NIH, HHMI-RRPMS and US ARMY MRMC for funding.
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35. Bergmann A, Agapite J, McCall K, Steller H (1998) The Drosophila gene hid is a direct molecular target of Rasdependent survival signaling. Cell 95:331–341 36. Kurada P, White K (1998) Ras promotes cell survival in Drosophila by downregulating hid expression. Cell 95:319–329 37. Yang L, Baker NE (2001) Role of the EGFR/Ras/Raf pathway in specification of photoreceptor cells in the Drosophila retina. Development 128:1183–1191 38. Dominguez M, Wassarman JD, Freeman M (1998) Multiple functions of the EGF receptor in Drosophila eye development. Curr Biol 8:1039–1048 39. Morata G, Ripoll P (1975) Minutes: mutants of Drosophila autonomously affecting cell division rate. Dev Biol 42: 211–221 40. Neufeld TP, de la Cruz AF, Johnston LA, Edgar BA (1998) Coordination of growth and cell division in the Drosophila wing. Cell 93:1183–1193 41. Prober DA, Edgar BA (2000) Ras1 promotes cellular growth in the Drosophila wing. Cell 100:435–446
42. Ready DF (1989) A multifaceted approach to neural development. Trends Neurosci 12:102–110 43. Neufeld TP, Hariharan IK (2001) Regulation of growth and cell proliferation in eye development, in Drosophila Eye Development (Moses K, ed.) Springer-Verlag (in press) 44. Shermoen AW, O’Farrell PH (1991) Progression of the cellcycle through mitosis leads to abortion of nascent transcripts. Cell 67:303–310 45. Baker NE, Yu SY (1998) The R8-photoreceptor equivalence group in Drosophila : fate choice precedes regulated Delta transcription and is independent of Notch gene dose. Mech Dev 74:3–14 46. Foe VE (1989) Mitotic domains reveal early commitment of cells in Drosophila embryos. Development 107:1–22 47. Usui K, Kimura K-L (1992) Sensory mother cells are selected from among mitotically quiescent clusters of cells in the wing disc of Drosophila. Development 116:601–610 48. Lawrence PA, Shelton PM (1975) The determination of polarity in the developing insect retina. J Embryol Exp Morphol 33:471–486
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Cell proliferation, survival, and death in the Drosophila eye Nicholas E. Baker the visual field and transmits information to the optic centers of the brain. 1–3 Precise ommatidial structure is important for multiple aspects of compound eye function. Flies infer motion, obviously a critical ability for an airborne insect, using the temporal procession of images across the field of ommatidia. Superposition of R7 and R8 photoreceptors within each ommatidium is thought to be important for color vision and polarization detection. R8 always lies underneath R7, placing the light sensitive rhabdomere organelle of R8 in the shadow of the R7 rhabdomere. 4–6 Such functional constraints probably explain why both the number and type of cells in each ommatidium are so invariant. In this respect the eye differs from some other organs, such as the wing, whose function is more mechanical and can be achieved by an array of largely similar cells whose precise numbers may be less important. Cellular diversity and reproducible structure have contributed to the Drosophila eye becoming an important system for the study of cell fate specification. Eighteen precursor cell fates are specified by postmitotic cell–cell interactions, often involving receptor tyrosine kinase signaling. 7–9 The nineteenth precursor cell divides twice to generate a four-cell sensory bristle organ. Postmitotic fate specification raises the question of how cell numbers are regulated. Cell number depends on the balance of cell proliferation and cell death, both of which are coordinated with cell differentiation. Recent progress has come from studying the regulation of the terminal cell divisions. Such studies show that both cell cycle progression and cell survival are coordinated by short-range signals that can adjust the number of unspecified progenitor cells according to the number of ommatidia required. 10 This mechanism permits fine-tuning of retinal cell number at the G2/M transition of the cell cycle. Temporal and spatial regulation of G1 and S phases also occur but less is known of the mechanisms. An index of genes implicated in retinal proliferation and survival is shown in Table 1.
The Drosophila retina has a precise repeating structure based on the unit eye, or ommatidium. This review summarizes studies of the cell proliferation and survival episodes that affect the number of cells available to make each ommatidium. Late in larval development, as differentiation and patterning begin, the retinal epithelium exhibits striking regulation of the cell cycle including a transient G1 arrest of all cells, followed by a ‘Second Mitotic Wave’ cell cycle that is regulated at the G2/M transition by local intercellular signals. Reiterated episodes of cell death also contribute to precise regulation of retinal cell number. The EGF receptor homolog has multiple roles in retinal proliferation and survival. Key words: proliferation / survival / apoptosis / Drosophila c 2001 Academic Press
The Drosophila retina requires precise cellular architecture Although different in many respects, both Drosophila and vertebrate retinas contain multiple distinct cell types each specialized for particular roles in the perception of light. The numbers of each cell type are important, as well as the cell diversity. This is especially so for Drosophila, where the compound eye is constructed according to a strikingly regular plan [Figure 1(a)]. Each adult Drosophila eye comprises about 800 ommatidia, or unit eyes. Ommatidia have a precise, reproducible structure generated by 19 precursor cells [Figure 1(a)]. Each ommatidium is a stereotyped assembly that focuses light from a small portion of
From the Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. E-mail: [email protected] c
2001 Academic Press 1084–9521 / 01 / 060499+ 09 / $35.00 / 0
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Figure 1. Structure and development of Drosophila retina. (a) Cartoon of retinal structure after specification of all 19 precursor cells and death of supernumerary cells. Eight photoreceptor neurons are surrounded by accessory cone and pigment cells. Intervening pigment cells isolate neighboring ommatidia. Note that a single layer of 2◦ /3◦ pigment cells is sufficient. Key: R1–R8, photoreceptor neurons; ACC, anterior cone cell; ECC, equatorial cone cell; PCC, posterior cone cell; PoCC, polar cone cell; PPC, primary pigment cell; SPC, secondary pigment cell; TPC, tertiary pigment cell; bcg, bristle cell group. For review see Reference 2. (b) Eye imaginal discs differentiate progressively, beginning at the posterior margin and advancing anteriorly (arrow). In this and subsequent figures, posterior is on the right, so that reading from right to left reveals a temporal sequence of increasingly mature retinal development. A ‘second mitotic wave’ of cell divisions occurs posterior to the morphogenetic furrow (MF). Most of these divisions occur between columns 3 and 5. For review see Reference 2. (c) Enlargement of the region near the morphogenetic furrow, showing the stepwise recruitment and differentiation of ommatidial cells. R8 cells are differentiating in column 1 within the morphogenetic furrow, the other precluster cells R2, R3, R4 and R5 soon afterwards. All the other cells re-enter the cell cycle in the SMW and are recruited to further ommatidial fates after column 5 (shaded cells). Figure adapted from Reference 37. Table 1.
Genes involved in eye disc proliferation and survival
tkv, sax, punt, shn
Required for prompt G1 arrest in the morphogenetic furrow in response to Dpp.
rux
Required for G1 arrest since it prevents ectopic activation of G2 cyclins during the long morphogenetic furrow G1.
rca
Required for cyclin A function in normal G2, or the ectopic S phases of rux mutants.
Su(rux)2B, 2C
Mutations affecting all S phases and that can suppress rux.
ato
Required autonomously for R8 specification. Required indirectly for both G1/S and G2/M of the SMW. The requirement in G2/M is attributable to reduced EGFR activation in the absence of differentiating photoreceptors that secrete EGFR ligands. The requirement in G1/S may be due to absence of other factors.
da
Required for both G1/S and G2/M of the SMW. Possibly similar to ato, which requires da as a heterodimeric partner.
spi
The EGFR ligand required for G2/M progression, neural differentiation, and survival, but apparently not for G1 arrest.
egfr
Required for: G1 arrest and neural differentiation of precluster cells R2, R3, R4, R5; mitosis of SMW cells; differentiation of most other ommatidial cells; survival of retinal cells.
N
Required for apoptosis of extra pupal cells as well as for multiple aspects of fate specification.
hid
Proapoptotic gene implicated in retinal cell survival.
500
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The Drosophila eye imaginal disc and the ‘second mitotic wave’
column: 1
‘Imaginal discs’ are the larval structures that provide the source of adult cells in holometabolous insects that show full metamorphosis. Imaginal discs invaginate from the body wall during embryogenesis and proliferate without differentiating while inside the body cavity of the larva. Within the pupa, imaginal discs differentiate adult structures that replace larval body parts. 11 Ommatidial specification and retinal differentiation begin in the eye imaginal disk during the last larval instar. A ‘morphogenetic furrow’ sweeps from posterior to anterior across the eye disc as progressively more anterior disc regions begin eye differentiation. Cell division temporarily ceases, and the first ommatidial progenitor cells are specified 2 [Figure 1(b) and 1(c)]. Cell cycle behavior posterior to the morphogenetic furrow is correlated with differentiation status (Figure 2). Specification begins with the photoreceptor cells. Single R8 photoreceptor cells are the first to differentiate under the control of the proneural gene atonal (ato). Each R8 cell rapidly recruits R2, R3, R4 and R5 into preclusters each containing these five photoreceptor precursors. Ommatidium assembly fails completely in ato mutants, owing to the critical founding role of the R8 cells. 12 Cells of the precluster begin neural differentiation and never divide again. The other unspecified cells re-enter the cell cycle in the ‘second mitotic wave’ (SMW), the cell cycle that occurs posterior to the morphogenetic furrow. 1,2 During this period the eye disc contains the specified, differentiating, G1-arrested five-cell preclusters surrounded by SMW cells that undergo S phase and mitosis (Figures 1 and 2). The remaining 14 ommatidial precursor cells are born in the SMW. This is essential to generate enough precursor cells to complete each ommatidium. If it is prevented many ommatidia cannot recruit sufficient cells. All cell fates can still occur, however, indicating that the cell division is not linked tightly to specification of any particular cell type. 13 Specification of the remaining 14 precursor cell fates occurs postmitotically during further episodes of signaling through EGFR and other cell surface receptors. 7–9 Much later, in the pupa after all the ommatidial cells have been specified, apoptosis removes any supernumerary cells. 2 Many genes that are important for growth and development of the retina have other prior developmental functions. Although conditional mutant
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recruitment G1 arrest SMW S-phase SMW G2-phase SMW mitosis cell death
Figure 2. Timing of the SMW and differentiation. An enlargement of Figure 1(c) also showing the spatial and temporal extent of particular processes. Recruitment of ommatidial cells occurs in waves. Specification of the R2–R5 precluster cells in response to signals from the R8 cell occurs by column 1 in the morphogenetic furrow. R1 and R6 join in columns 4–5, R7 by column 6, and all the cone cells are recruited by column 10 (for review see Reference 2). G1 of the SMW is a continuation of the G1 arrest that affects all cells anterior to the morphogenetic furrow, initially in response to Dpp signaling. G1 arrest is maintained posterior to the furrow in the five photoreceptor neurons of each ommatidial precluster. S phase of the SMW begins anterior to column 1. All the cells outside the preclusters are in S phase by column 2. S-phase DNA labeling is undetectable posterior to column 4. Many SMW cells pass rapidly through G2phase to mitosis, other cells remain in G2 indefinitely. The first SMW mitosis occurs around column 3; most mitosis occurs between columns 3 and 5. Occasional mitosis occurs more posteriorly. Small amounts of cell death also occur in the eye disc, typically affecting less than one cell per ommatidium. In normal development cell deaths are first observed around column 7. Figure adapted from Reference 10.
alleles exist for some of these loci, mosaic analysis is usually necessary to recover retinas containing clonal patches of mutant cells in animals otherwise developing normally. Introduction of the FLP/FRT site-specific recombination system into Drosophila has facilitated use of mitotic recombination to generate clones of mutant cells in situ in developing eye tissue. By catalyzing chromosome arm exchange during the mitotic cell cycle, single cells homozygous for 501
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separable processes. Neural differentiation depends on activation of the EGFR by the TGF alpha-like Spitz (Spi) protein. By contrast, G1 arrest can occur in the absence of this ligand. 10 The primary source of Spi within each ommatidium is R8. 24,25 The ligand responsible for G1 arrest has yet to be determined. Except for these preclusters, cells synchronously enter S phase of the SMW. Since S phase occurs in mutants that lack nearly all ommatidia it seems unlikely that local induction is involved. 2,15,26,27 There may be a longer range, global signal that triggers cell cycle re-entry because the proneural gene ato which is required for much of eye development is indirectly and non-autonomously required for SMW S phase. 10,27 A requirement for the heterodimer partner for ato, encoded by daughterless (da), in the SMW might also be indirect. 28 It is also possible that down-regulation of the pathway maintaining G1 arrest in the furrow may be sufficient for cells to re-enter the cell cycle, no positive signal being necessary. Unfortunately this possibility is hard to assess since no genes have yet been described that are specifically and autonomously required for G1/S progression of the SMW. Two genes, Su(rux)2B and Su(rux)2C, that are required for the SMW seem to be required for S phases throughout development. 19
mutants of interest can be generated in heterozygous animals. Clones of homozygous cells descended from such recombinants usually survive even when of genotypes that would be lethal to the whole animal. 14
Cell cycle arrest and the morphogenetic furrow Obvious spatial regulation of the cell cycle is first apparent just anterior to the morphogenetic furrow. All the cells arrest in G1 of their cell cycles before any differentiation begins 1,15,16 (Figure 2). G1 arrest appears to require Decapentaplegic (Dpp) signaling initially but then becomes independent of Dpp; G1 arrest is temporarily delayed in clones of cells unable to respond to Dpp, but not prevented indefinitely. Since dpp is transcribed in the morphogenetic furrow, it is thought that a concentration gradient of Dpp protein anterior to the morphogenetic furrow defines a position where cells pause prior to S phase because of the high Dpp level. Consistent with this view, ubiquitous ectopic expression of Dpp is sufficient to cause cell cycle arrest ahead of the morphogenetic furrow. 17,18 After a few hours an unidentified second pathway supplements or replaces Dpp signaling to maintain G1 arrest. The roughex (rux) gene was originally identified as potentially involved, but is now thought to down-regulate G2 cyclins at mitosis. 19–21 Apparently rux is also important to suppress inappropriate activity of the G2-specific cyclin A during this long G1. Consistent with this view, the ectopic S phases in clones of cells mutant for rux require rca1, a gene necessary for the normal function of cyclin A in G2. 22 Both the nature of the G1 arrest signal and the function of the morphogenetic furrow arrest remain uncertain.
G2/M in the second mitotic wave By contrast to the apparently constitutive, synchronous G1/S transition of cells in the SMW, G2/M progression is regulated by local signals 15 (Figure 3). SMW cells arrest in G2 if ommatidia are not present, showing that signals depend on the presence of ommatidia. The signal provided by ommatidia is again the EGFR agonist Spi. Both Spi and its processing and presenting factors are produced in the precluster cells. 10 EGFR activation is both necessary for SMW mitosis and sufficient to induce mitosis in the absence of ommatidia. Activation of EGFR acts in part to activate string (stg ), which can also induce mitosis in the absence of ommatidia. stg encodes the homolog of yeast cdc25, a phosphatase required for cdc2 activation. Stg is limiting for many cell cycles during Drosophila embryogenesis and is the subject of complex transcriptional regulation thought to couple the cell cycle to developmental signals. 29,30 Unlike EGFR activation, targeted stg transcription does not completely rescue the SMW in eye discs lacking ommatidia, suggesting that EGFR signaling has other cell cycle roles besides activating stg. 10
G1/S in the second mitotic wave Posterior to the morphogenetic furrow many cells progress from G1 into S phase (Figure 2). Most cells express cyclin D and E and re-enter the cell cycle in the SMW. 2,23 The five cells differentiating in each precluster are the exception. Precluster cells remain in G1 and never divide again. Maintaining G1 arrest in preclusters depends on ato function in R8 and on EGFR activity in precluster cells R2, R3, R4, R5. ato and EGFR are also required for neural differentiation of these photoreceptor cells. Notably, G1 arrest and neural differentiation appear to be simultaneous but 502
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Figure 3. Regulation of SMW cell number by precluster signaling to the EGFR. The cartoon illustrates how EGFR regulates SMW processes shown in Figure 2. (a)–(c) represent normal eye development. (d), (e) illustrate the consequences of a defect in ommatidium spacing. Shading reflects EGFR activity. (a) Cartoon of the eye disc epithelium around column 1. R8 cells (<) activate EGFR to arrest and recruit four other precluster cells (shaded). Other cells lack EGFR activity, re-enter the cell cycle and perform S phase (unshaded). (b) Column 3. All cells outside preclusters are in the SMW. Precluster contact activates EGFR in most cells (shaded), allowing progression into mitosis. Some cells are not activated and remain in G2 (unshaded). (c) Column 10. After the SMW, EGFR activity (shaded) protects most G1 progenitor cells from cell death. EGFR activity also protects some remaining G2 cells without causing their mitosis (light shading). A few cells die (*). In wild-type strains less than one cell dies per ommatidium, on average. In combination, mitosis and survival yield an average of 17.1 cells per precluster after the SMW. Another two to three cells die per ommatidium in the pupa several days later. Ultimately, 14 further progenitor cells are required to complete each ommatidium (Figure 1). (d) Cartoon of an eye disc around column 3 where a precluster is missing. Precluster contact activates EGFR in neighboring cells (shaded). Many cells lack precluster contacts and arrest in G2 (unshaded). (e) Cartoon of an eye disc around column 10 where a precluster is missing. Reduced mitosis and survival adjust the number of progenitor cells towards the number necessary to complete the extant ommatidia. Symbols as in panel (c).
Local signaling through the EGFR is both essential and limiting in the SMW, so that not all SMW cells complete this cell cycle (Figure 3). In normal development, an average of 9.7 cells enter the SMW for every five-cell precluster, but on average only 7.7 cells complete the cell cycle and divide. On average two cells never receive the mitotic signal and so remain in G2. 10 Conditional cell cycle progression in the SMW provides flexibility to the generation of unspecified progenitor cells (see later).
survival indirectly. For example, since Spi is required for specification and neural differentiation of ommatidial cells, any other EGFR ligand secreted by ommatidial cells would also be affected in clones of cells mutant for Spi. 10 Regulated cell survival further couples progenitor cell number to the ommatidia differentiating in the eye disc. Several days later, once all the ommatidial cells have been specified and are differentiating in the pupa, further cell death occurs to remove approximately two to three extra cells around each ommatidium. A survival signal coming from the cone cells or primary pigment cells of each ommatidium protects nearby cells so that they can survive to differentiate as secondary and tertiary pigment cells and as sensory bristle organs. 31 If cell death is prevented by expression of baculovirus p35, the rescued ‘undead’ cells differentiate as supranumerary pigment cells. 32 Pupal cell death requires Notch signaling. 2,33 Spi and EGFR expression patterns, and misexpression experiments involving activated Ras or the EGFR antagonist Argos suggest that EGFR is required for survival of pupal cells also, probably through regulated transcription and activity of the proapoptotic gene head involution defective (hid). 31,34–36
Survival signals After SMW cells divide, EGFR signaling is then required from about column 6 or 7 onwards to maintain cell survival (Figures 2 and 3). In normal development only 0.2–0.3 eye disc cells die per ommatidium, but if ommatidia are removed or mis-spaced this number increases dramatically. In Ellipse mutants where very few ommatidia differentiate, almost all the SMW cells die. Cells also die in cell clones mutant for spi. Spi secreted from the differentiating ommatidial cells may be the ligand that activates EGFR during survival signaling, although it is also possible that Spi affects 503
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normal development eye discs contain cells that survive despite not having divided, and cells that undergo apoptosis even though they performed the EGFR-dependent mitosis. 10 Further work is required to explore how the EGFR can trigger independent implementation of mitotic and survival responses. The relationship between mitosis and fate specification may be less flexible. G2-arrested cells that escape apoptosis appear to lie in between differentiating ommatidia and are not recruited to early cell fates. They have not been examined during the rest of development to see whether they can differentiate at a later time. It may be that such G2-arrested cells normally find themselves amongst the population that die during pupation. When cell death is blocked by expression of baculovirus p35, however, all cells survive and extra pigment cells differentiate. 32 The G2 cells must therefore be amongst the extra pigment cells. The probability that cells can differentiate as pigment cells in G1 or G2 is supported by the differentiation of secondary and tertiary pigment cells in Elp mutants that lack ommatidia; most of these cells are arrested in G2. 15 Even egfr mutant cells can survive into the adult if protected by p35 expression. Such cells differ morphologically from normal cells. They most resemble unpigmented pigment cells. Some apparently produce small rhabdomeres and might be abnormal R8-like cells. Whether the abnormalities are due to the G2 arrest or to other consequences of EGFR signaling deficiency is not known. Whether cells in G2 could be recruited to the earlier primary pigment cell, cone cell or photoreceptor cell fates is uncertain, but we favor the idea that EGFR activation in such cells would inevitably lead to mitosis before any fate specification response, since this is the order of events when EGFR is ectopically activated in eye discs. 10
Both mitotic signaling and survival signaling contribute to matching the number of unspecified progenitor cells to the number of ommatidia (Figure 3). Mitotic signaling through the EGFR serves to increase the number of progenitor cells as required for normal development. In the event of a surplus, distance from the survival signal can eliminate some extra cells. Whereas complete cell division would result in doubling cell number, the SMW has a lower yield because not all the cells that enter the SMW cell cycle complete it or survive. In normal development, the SMW produces about 89% of the theoretical maximum number of progenitor cells that would be obtained if all cells divided and survived. Because ommatidial preclusters are the source of both mitotic and survival signals, the number of unspecified cells generated and surviving through the SMW depends on the number of ommatidia that have already begun differentiation. In most organs cells cycle asynchronously, making the yield of each cell cycle harder to assess. Perhaps it is often less than 100%, reflecting undescribed limits on cell division and survival analogous to those of the SMW.
Coordination and specificity of multiple signals As is well known, EGFR signaling also plays another role inducing postmitotic cells to adopt particular cell fates 7–9 (Figure 2). The same precluster cells whose mitotic signals generate unspecified precursor cells then induce postmitotic cell fate specification. Fate specification follows very rapidly after mitotic signaling, and precedes and overlaps survival signaling. The first cells specified after the SMW, photoreceptor cells R1 and R6, can be identified joining the ommatidia by column 5, which is 3 h or less after they divide 10 (Figure 2). One question concerns the specificity of EGFR signaling in fate specification, mitosis and survival. Do these responses occur independently through different episodes of EGFR signaling, or does the SMW contain only two types of cells: those that experience EGFR signaling, so dividing, surviving and being specified, and those that lack EGFR signaling, therefore arresting, remaining unspecified and dying? It is already clear that some EGFR responses can occur independently. Thus during
Retinal growth before the second mitotic wave It should be emphasized that the EGFR roles in mitosis and survival apply only to this last cell cycle of retina development, not to earlier growth of the eye or to other tissues, which do not depend on receptor tyrosine kinases in the same way. The analysis of mosaic patches deficient for EGFR and related genes relies on the ability of such mutant cells to grow divide and survive up until the SMW, otherwise data could never have been obtained. Because cells lacking either of the downstream factors Ras or Raf are similarly able to grow and survive during much 504
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of eye development, we think that neither EGFR nor any other RTK acting through the canonical RTK–Ras–Raf pathway can be essential for retinal cell division or survival up until the SMW. 37 The fact that these genes are not essential, however, does not mean they have no role. Indeed cells mutant for EGFR, Ras or Raf grow poorly, and analysis of their developmental functions usually relies on the Minute technique to compensate for poor growth. 37,38 In this technique, mosaic analysis is performed in the genetic background of mutations (‘Minutes’) that reduce ribosome function cell autonomously. 39 In this way the EGFR, Ras, or Raf homozygous cells are simultaneously conferred a growth advantage through restoring normal translation capacity. The resulting growth advantage offsets the intrinsic debility of EGFR, Ras or Raf mutant cells. From such data it appears that EGFR is important for normal growth before the SMW but contributes to growth quantitatively without being essential, unlike the SMW where EGFR signaling is indispensable for mitosis. The role of EGFR, Ras and Raf in eye development prior to the SMW may be related to cellular growth and G1/S progression. 40 In studies of the developing undifferentiated wing, Prober and Edgar 41 found that Ras1 promoted cellular growth, in turn elevating cyclin E levels and promoting G1/S progression. If development of the eye before the morphogenetic furrow is similar, EGFR, Ras and Raf could promote proliferation through an effect of cellular growth on G1/S progression. After the morphogenetic furrow G2/M progression would become completely dependent on EGFR activation in the SMW. It is possible that no extracellular signal is essential for proliferation and growth before the morphogenetic furrow. If cellular mass accumulation is the only requirement then it will be interesting to determine the identity, sources and mechanisms of the putative signals promoting such cellular growth.
tution during evolution. The number of ommatidial cells appears to have increased in distinct taxa. 42 Perhaps additional precursor cells can only be generated once ommatidial development has already been initiated; an earlier increase in the number of retinal cells might lead instead to more ommatidia without changing their constitution. 43 If this is the case then more elaborate retinas require a SMW to acquire increased cellular diversity. The G1 arrest of cells within the morphogenetic furrow is a prominent feature of eye development but its function is uncertain. Some evidence that there must be a function comes from the rux mutations which cause morphological abnormalities that are suppressed by restoration of G1 arrest to rux mutants. Therefore inappropriate cell division is the cause of the abnormal morphology. 22 Unless rux mutations also affect G1 arrest of the precluster cells, this suggests that G1 arrest in the morphogenetic furrow is essential. Mitosis interrupts transcription, 44 so one speculation is that a prolonged mitosis-free period may permit transcription of large genes such as N and Dl, whose products accumulate in the morphogenetic furrow and are required for subsequent cell fate specification. 45 In this regard it is noteworthy that cells in other neurogenic regions also exhibit cell cycle arrest before neural fate specification, including the embryonic neuroectoderm and thoracic proneural territories. 46,47 In these other tissues arrest occurs in G2, however. It is also possible that cell cycle arrest is important in a transition from G1/S cell cycle regulation during the phase of rapid imaginal disc growth, when the goal of growth may be to attain particular tissue dimensions, to G2/M cell cycle regulation during the patterning and specification of adult structures, when the precise number and arrangement of cells may be paramount. It is curious that cell survival should be subject to multiple regulation, and that excess retinal cells die during pupation after being preserved through the SMW. 2,31 We do not know why eye development takes this roundabout course. One possibility is that criteria for survival become increasingly stringent as retinal development approaches completion. Another speculation is that eye development recapitulates reductions in pigment cell number that might have occurred during evolution. In the hemipterous bug Oncopeltus, for example, the ommatidial pigment cell lattice is octagonally arranged instead of hexagonally as in Drosophila. 48 The pigment cell lattice of Drosophila is topologically efficient, each of the secondary pigment cells separating two neighboring
Concluding remarks The regulated cell cycle of cells in the morphogenetic furrow and SMW are striking features of Drosophila eye development. We have suggested that the SMW adjusts the number of precursor cells generated in the retina according to the number of ommatidia being made. 10 Such adjustment must be made once initial steps in ommatidium specification have occurred for the number of ommatidia to be estimated. A second potential benefit is for changing ommatidial consti505
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ommatidia and each tertiary pigment cell separating three (Figure 1). Pupal cell death might simplify an atavistic condition in which each ommatidium was surrounded by its own pigment cells; multiple cells separating neighboring ommatidia could be redundant for optical purposes. Most comparative studies of compound eyes pay less attention to pigment cell numbers, however, so at present there is little evidence against which this idea can be judged.
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Acknowledgements I am grateful to David Tyler for comments and the NIH, HHMI-RRPMS and US ARMY MRMC for funding.
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