The imaginal discs of Drosophila provide a favorable system for the analysis of the mechanisms controlling developmental cell proliferation, because of the separation in time between cell proliferation and differentiation, and the facility with which controlling genes can be identified and characterized. Imaginal discs are established in the embryo, and grow by cell proliferation throughout the larval period. Proliferation terminates in a regular spatial pattern during the final stages of larval development and the first day of pupal development. Cell proliferation can be locally reactivated in growth-terminated imaginal discs by removing part of the disc and culturing the remaining fragment in an adult host. The pattern of proliferation in these fragments suggests that cell proliferation in imaginal discs is controlled by direct interactions between cells and their neighbors. Proliferation appears to be stimulated by positional information differences, and these differences are reduced by the addition of new cells during tissue growth. Genes involved in cell proliferation control have been identified by collecting and analyzing recessive lethal mutations which cause overgrowth of imaginal discs. In some of these mutants (fat, Igd, c43, dco) the overgrowing tissue is hyperplastic; it retains its single-layered epithelial structure and is capable of differentiating. In two of the hyperplastic mutants (dco and c43), the imaginal discs show a failure of gap-junctional cell communication, suggesting that this form of cell communication may be involved in termination of proliferation. In other mutants the overgrowing disc tissue is neoplastic: it loses its structure and ability to differentiate, becoming a tumorous growth. The two genes that give a neoplastic phenotype (dig and lgl) have been cloned and cDNAs of one of them (1gl) sequenced. The lgl gene encodes a cell surface molecule with significant homology to calcium-dependent cell adhesion molecules (cadherins). The expression of lgl at the time of termination of cell proliferation suggests that there are changes in the way that cells interact with one another at these times, and that these changes may be implemented by cell adhesion molecules. Direct cell contact within the epithelium, as well as signalling through gap junctions, appears to be involved in the cell interactions needed for the termination of cell proliferation. Mutations in genes encoding the Drosophila homologs of growth factors, growth factor receptors and oncogenes usually show an effect on cell-fate decisions rather than cell proliferation control, but this may be because oncogenic mutations in these genes would be dominant lethals and would therefore not be identified by conventional genetic analysis.

The remarkably uniform size and shape of organs is the outcome of a highly controlled program of cell proliferation that occurs during embryonic and postembryonic organ development, but we know little of how these growth patterns are controlled. One of the least understood aspects of the problem is the question of what causes organs to terminate growth at the appropriate final size and cell number. Our approach to this problem is to find mutations that interfere with growth termination during development of imaginal discs in larvae of the fruit fly Drosophila, and use them to identify the corresponding genetic functions at the molecular level. It is analogous to the strategy of identifying human growth-control genes by cloning and analyzing tumor suppressor genes (Sager, 1989).

Imaginal discs are sacs of undifferentiated epithelium in the Drosophila larva. They have no function in the larval insect, but during metamorphosis they undergo substantial morphogenesis and differentiation to give rise to various parts of the adult body surface (see Bryant, 1978). The precursors of the adult appendages (1egs, wings, antennae, mouthparts) as well as the eyes and genitalia develop from imaginal discs, whereas the abdomen develops from small nests of cells in the larval integument called histoblasts. Many internal organs develop from precursors known as imaginal nests or rings, and the central nervous system develops from precursor cell populations, known as proliferation centers, within the larval central nervous system (see Bryant and Levinson, 1985).

Imaginal discs provide an ideal experimental system in which to analyze mechanisms of cell proliferation control. They have a simple histological structure, consisting mainly of a single-layered columnar epithelium along with some muscle precursor cells basal to the epithelium. One of their advantages is that cell proliferation occurs during the larval period and is completed before differentiation begins in the pupal period. Thus the control of cell proliferation can be studied in the absence of complications due to simultaneous overt differentiation, as occurs in most tissues of vertebrates.

After the discs are established in the embryo, their cell numbers increase exponentially during the larval period, then the growth rate slows down towards the end of larval development (Bryant and Levinson, 1985). Cell proliferation in the wing disc stops in a predictable spatial pattern during the late third larval instar and early pupal period, as shown by Schubiger and Palka (1987) (Fig. 1). The first cells to stop proliferating at the end of the larval period are located in a zone about ten cells wide at the position of the presumptive dorsal/ventral margin of the wing (O’Brochta and Bryant, 1985); the presence of this zone accounts for the cell lineage restriction at this position which has been observed in mitotic recombination experiments (Bryant, 1970; Garcia-Bellido et al. 1976). A second, narrower band of non-proliferating cells intersects the first one at right angles and coincides in position with the presumptive third longitudinal wing vein; surprisingly, it does not coincide with the anterior/posterior lineage restriction and compartment boundary (Garcia-Bellido et al. 1976) which lies nearby. By 3-4 h after puparium formation, cell proliferation has terminated over the entire disc epithelium except for a row of cells along the anterior wing margin where bristles and nerve cell bodies will later develop. Measurements of DNA content indicate that the arrested cells are in the G2 stage of the cell cycle (Fain and Stevens, 1982; Graves and Schubiger, 1982). After the epithelium has secreted the pupal cuticle there is a further cycle of mitosis between 12 and 24 h after puparium formation. This round of division occurs in a spatially ordered way, beginning in the cells along the presumptive wing veins and spreading into the inter-vein regions. After the final round of mitosis, the disc epithelium enters the stage of adult differentiation and produces the adult wing cuticle.

Fig. 1.

Summary diagram of the spatial patterns of DNA replication in wing development. The time line at the bottom indicates the developmental time in hours after egg deposition (AEL) and after pupariation (AP). Asterisks: periods of frequent mitoses. From 15 to 21 h AP mitotic figures are especially abundant. Continuous lines: periods with abundant S-phase nuclei in the margin (M) in vein 3 (L3), and in the intervein regions (I-V). Broken lines: periods with few nuclei replicating. The figures at the top represent the spatial patterns of four selected periods (wandering larva [W.L.], 6h, 12h, and 18h AP). (From Schubiger and Palka, 1987).

Fig. 1.

Summary diagram of the spatial patterns of DNA replication in wing development. The time line at the bottom indicates the developmental time in hours after egg deposition (AEL) and after pupariation (AP). Asterisks: periods of frequent mitoses. From 15 to 21 h AP mitotic figures are especially abundant. Continuous lines: periods with abundant S-phase nuclei in the margin (M) in vein 3 (L3), and in the intervein regions (I-V). Broken lines: periods with few nuclei replicating. The figures at the top represent the spatial patterns of four selected periods (wandering larva [W.L.], 6h, 12h, and 18h AP). (From Schubiger and Palka, 1987).

Since imaginal discs terminate cell proliferation at a reproducible cell number, and since the adult derivatives of imaginal discs are made up of pattern elements (e.g. bristles, sensilla, hairs) that are derived from individual cells or fixed small numbers of cells, it might be assumed that cell proliferation in imaginal discs terminates after a certain number of cell divisions. However, several studies show that growth termination in imaginal discs is not a function of a cell-division counting mechanism. The amount of proliferation within individual cell clones marked by mitotic recombination during normal development is highly variable, and clones in different individual flies occupy partly overlapping territories on the adult structure (Bryant and Schneiderman, 1969; Bryant, 1970), showing that the lineage pattern is variable. High doses of X-irradiation during larval development can kill or mitotically inhibit up to 75% of the cells in imaginal discs, without causing noticeable abnormalities of the adult structure. The surviving cells compensate and form the adult structure by undergoing additional proliferation, as shown by enlargement of mitotic recombination clones compared to controls (Haynie and Bryant, 1977). When individual cell clones are genetically altered by mitotic recombination so that they have a higher or lower proliferation rate compared to the remaining cells, they contribute more or less to the adult structure respectively, but the structure is normal (Simpson, 1976,1979; Simpson and Morata, 1980,1981). Thus the cell proliferation pattern is naturally variable and can be experimentally altered without noticeably altering the size or pattern of the structure produced. It therefore seems likely that proliferation is controlled by interactions between cells, independently of their lineage history. Our working hypothesis (Bryant and Simpson, 1984) is that cell proliferation is controlled by the developing map of positional information (Wolpert, 1971) in the disc and that cell proliferation stops when the map is complete.

Although the imaginal discs are undifferentiated epithelia, experimental analysis has revealed that different parts of a full-grown disc are already different from one another, so that if isolated they will construct specific parts of the adult structure (see Bryant, 1978). This covert pattern of differentiation indicates that a detailed positional information map must have become established during the growth of the disc.

Growth termination, under normal conditions, coincides with hormonal changes occurring at the onset of metamorphosis, but it also occurs in the absence of these hormonal changes. When whole discs from early larvae are implanted into the growth-permissive environment of the female adult abdomen, they grow until they reach approximately their normal final size and shape, then they stop growing (Bryant and Levinson, 1985). Thus under permissive growth conditions, the attainment of a correct size and shape is due to disc-intrinsic properties, consistent with the idea that it is controlled by the positional information map.

Studies of transplanted disc fragments show that growth termination in regenerating imaginal disc fragments is also controlled by interactions between cells and their neighbors within the disc epithelium. When fragments of imaginal discs are cultured in adult female flies for several days before transfer to larval hosts for metamorphosis, the cuticular patterns produced after metamorphosis reveal that the tissue fragment has either regenerated or duplicated during the culture period (see Bryant, 1978). Bryant and Fraser (1988) investigated the pattern of cell proliferation in a 3/4 fragment of the wing disc, which regenerates the missing sector by cell proliferation over a period of several days. Individual cells in the starting fragment were marked by iontophoretic injection of high molecular weight, lysinated rhodamine dextran, and the positions of the marked cells were determined by fluorescence microscopy before and after various culture times. The results show that cells along the two wound edges are brought together by wound healing during the first day of the culture period. The marked cells then move apart over the next three days as new cells are added between them. The results indicate that intercalary tissue growth is restricted to the immediate region of the wound, and does not spread significantly into adjacent tissue. During the regenerative growth, the new cells become specified to make pattern elements that would normally intervene between the confronted cells, a process termed intercalation (French et al. 1976).

The pattern of DNA synthesis during intercalation has been monitored by analyzing the incorporation of tritiated thymidine, which is detected by autoradiography of serially sectioned disc fragments (O’Brochta and Bryant, 1987) or of the thymidine analog bromodeoxyuridine, which is detected by immunolocalization on whole mounts (Bryant and Fraser, 1988). Intense and localized DNA synthesis occurs adjacent to the healed wound, starting at 16 to 18 h after the beginning of the culture period (Fig. 2). DNA synthesis is stimulated over a distance of a few cell diameters from the wound, and localized cell proliferation continues for three to more than five days. The results suggest that DNA synthesis is stimulated by the positional information discontinuity across the healed wound, and that the stimulus is attenuated as the positional information discontinuity is reduced by the addition of cells carrying intermediate positional values.

Fig. 2.

Pattern of incorporation of bromodeoxyuridine into 3/4 wing disc fragments in vitro after various periods of culture in vivo, visualized by indirect immunofluorescence using a monoclonal antibody against BrdU. (A) Oh; (B) 4h; (C) 12h; (D) 18h; (E) 24h; (F) 48 h. The position of the wound vertex (A,B) or healed wound (C-F) is indicated by an arrow. In (A) and (B), the wound has not yet healed, and there is no localized BrdU incorporation. In (C), the wound has begun to heal, but there is still no localized incorporation. In (D-F), a cluster of labeled cells can be seen adjacent to the healed wound. Trails of weakly labeled cells along the disc margin can be seen in (D) and (E). (From Bryant and Fraser, 1988).

Fig. 2.

Pattern of incorporation of bromodeoxyuridine into 3/4 wing disc fragments in vitro after various periods of culture in vivo, visualized by indirect immunofluorescence using a monoclonal antibody against BrdU. (A) Oh; (B) 4h; (C) 12h; (D) 18h; (E) 24h; (F) 48 h. The position of the wound vertex (A,B) or healed wound (C-F) is indicated by an arrow. In (A) and (B), the wound has not yet healed, and there is no localized BrdU incorporation. In (C), the wound has begun to heal, but there is still no localized incorporation. In (D-F), a cluster of labeled cells can be seen adjacent to the healed wound. Trails of weakly labeled cells along the disc margin can be seen in (D) and (E). (From Bryant and Fraser, 1988).

The possible role of gap-junctional cell communication across the healed wound has been investigated by injecting small fluorescent dye molecules into cells close to the wound region, and determining the degree to which the dye passes into or across the wound area (Bryant and Fraser, 1988). In undamaged imaginal wing discs, there is a directional bias but, in contrast to a report in the literature (Weir and Lo, 1982), there are no fixed boundaries to this form of communication (Fraser and Bryant, 1985). In disc fragments, dye movement across the wound was not detectable after one day of culture. Since DNA synthesis is already underway in the growth zone at this time, we conclude that gap-junctional communication across the wound is not necessary for initiating the local cell proliferation involved in intercalary regeneration. After two days there was some dye transfer into the growth zone from cells outside it, but not as much as in other directions. After three days there was significant dye transfer into the growth zone. The time of re-establishment of gap-junctional communication therefore indicates that this form of cell communication is not involved in stimulating regenerative cell proliferation, but suggests that it could be involved in terminating it.

Genes involved in cell proliferation control in imaginal discs have been identified by selecting recessive mutations (imaginal disc overgrowth mutations) which cause the imaginal discs to continue growing by cell proliferation beyond the normal limits during an extended larval period. In some cases, the mutant animals remain in the larval stage for several weeks rather than the normal four days. The mutations are all recessive lethals, causing death of the animal either as a late larva or as a pupa which forms after a prolonged larval period. They fall into two categories depending on the effect of the mutant lesion on the histological structure of the imaginal discs.

1. Hyperplastic overgrowth mutants

Five genetic loci are known in which mutations give the hyperplastic overgrowth phenotype (Table 1). In these mutants, the imaginal discs show abnormal folding patterns early in their development, then they grow to several times their normal size during the extended larval period. They maintain their single-layered epithelial structure and their ability to differentiate, but the additional tissue causes the imaginal disc to attain an abnormal morphology with additional lobes and folds as compared to normal (Fig. 3). In Igd and possibly in other mutants, not only the imaginal discs but also other groups of proliferating cells whose function is to produce internal adult derivatives (the imaginal rings for the foregut, hindgut, and salivary glands) show overgrowth during the extended larval period (Bryant and Levinson, 1985).

Table 1.

Imaginal disc overgrowth mutants of Drosophila melanogaster

Imaginal disc overgrowth mutants of Drosophila melanogaster
Imaginal disc overgrowth mutants of Drosophila melanogaster
Fig. 3.

Wing imaginal discs from wild type and fat homozygotes. (A, B) Wild type at 4 and 5 days. (C-G) fatfd at 4, 5, 7, 8, and 9 days. DF, distal folds; N, presumptive notum; PF, proximal folds; T, trachea; WP, wing pouch; arrow, some of the extra folds mentioned in text. Bar, 0.5 mm. (From Bryant et al. 1988).

Fig. 3.

Wing imaginal discs from wild type and fat homozygotes. (A, B) Wild type at 4 and 5 days. (C-G) fatfd at 4, 5, 7, 8, and 9 days. DF, distal folds; N, presumptive notum; PF, proximal folds; T, trachea; WP, wing pouch; arrow, some of the extra folds mentioned in text. Bar, 0.5 mm. (From Bryant et al. 1988).

In most of the hyperplastic overgrowth mutants, homozygotes are sometimes able to pupariate and to differentiate adult cuticular structures, although the resulting abnormal adult fly (pharate adult) is unable to emerge from the pupal case. The structures produced show many abnormalities specific to the genetic locus.

Pharate adults which are homozygous for lethal fat mutations show a characteristic syndrome of abnormalities, including invaginations and évaginations of cuticle from the body surface, completely separated cuticle vesicles (both internal and external to the body surface) and bristle polarity rosettes (Fig. 4; Bryant et al. 1988). These defects, especially the separated vesicles, suggest that the mutations interfere with cell adhesion within the epithelium. They show a striking resemblance to the set of abnormalities in the wing of the moth Manduca that are produced by transplanting pieces of pupal integument to inappropriate positions along the proximal/distal axis (Nardi and Kafatos, 1976a,6), and that have been interpreted as resulting from disruption of an adhesion gradient. It has therefore been suggested (Bryant et al. 1988) that the basic defect in fat is in cell adhesion. Defective cell adhesion could not only lead to the observed abnormalities of morphogenesis, but it could also interfere with the cell communication needed for proliferation control.

Fig. 4.

Cuticular abnormalities in legs from fat,d pharate adults. (A) Male first leg with enlarged coxa, laterally expanded and truncated tarsus (t), and vesicles with internal bristles in coxa (c) and femur (f). (B) Male first leg coxa, showing vesicles (v) apparently budding from ingrowth (i). Bars, 50 μ m. (From Bryant et al. 1988).

Fig. 4.

Cuticular abnormalities in legs from fat,d pharate adults. (A) Male first leg with enlarged coxa, laterally expanded and truncated tarsus (t), and vesicles with internal bristles in coxa (c) and femur (f). (B) Male first leg coxa, showing vesicles (v) apparently budding from ingrowth (i). Bars, 50 μ m. (From Bryant et al. 1988).

Pharate adults of dco show some similarities to fat pharate adults including separated cuticular vesicles, albeit at a much lower frequency than in fat (Jursnich et al. 1990). Both mutants, and c43, show substantial widening and disruption of segmentation in the distal part of the leg, the tarsus (Bryant, 1987). Igd shows a different set of abnormalities, notably a drastic reduction (to about 20% of normal) in the number of bristles and other cuticular sense organs produced, subdivision of the tarsus into only two segments rather than the usual five, and large-scale mirror symmetrical pattern duplications in the leg discs when the larvae are reared under crowded conditions (Bryant and Schubiger, 1971).

Since growth termination in normal and regenerating wild-type discs appears to depend on cell interactions within the imaginal disc, there may be abnormalities in the way that cells interact in the mutant discs. Gap-junctional communication has been tested by injecting fluorescent marker dye into individual disc cells and observing passage into adjacent cells (dye coupling). Discs from wild type, fat and Igd show extensive dye coupling at all tested stages, but discs from four-day dco and c43 larvae show complete lack of dye coupling (Jursnich et al. 1990). At later stages, when these discs are already overgrown, they start to show some dye coupling. Some dco genotypes, and c43, also show a substantial reduction in the surface density of gap junctions between imaginal disc cells when compared to wild type (Ryerse and Nagel, 1984; Jursnich et al. 1990). Although the defects in gap-junctional communication and in cell proliferation control may be unrelated pleiotropic effects of these mutations, another possibility is that the gap-junctional defect is responsible for the failure of dco and c43 mutant discs to terminate cell proliferation. The latter possibility would be consistent with the common finding of decreased gap-junctional communication in transformed cells (Sheridan, 1989).

2. Neoplastic overgrowth mutants

In addition to causing overgrowth, these mutations cause breakdown of the single-layered epithelial structure of the imaginal discs, converting them into spongy masses of tissue as they continue to grow beyond the normal final size. The disc cells become cuboidal in shape, show irregular apical-basal polarity, and lose the ability to differentiate even after transplantation into wild-type hosts. The abnormal discs are therefore tumorous or neoplastic growths, in contrast to the hyperplastic overgrowth mutants in which the imaginal discs maintain a much more normal histological structure. According to current usage, the wild-type alleles of these genes are tumor suppressor genes.

Two genes are known in which mutations give the neoplastic overgrowth phenotype: discs large (dig) (Stewart et al. 1972; Murphy, 1974) and giant larvae (1gl) (Gateff, 1978; Table 1). Mutations in both of these genes cause the imaginal discs to grow by cell proliferation beyond their normal final size, transform into solid tumors, fuse with one another and the brain, and lose their ability to differentiate (Fig. 5; Woods and Bryant, 1989). lgl mutations also cause overproliferation of neuroblasts in the larval brain, giving rise to ganglion mother cells which fail to differentiate into neurons (Gateff, 1978). This converts the tissue into a transplantable and invasive malignant neuroblastoma (Gateff and Schneiderman, 1974).

Fig. 5.

Wing imaginal discs from wild type (A-C) and dlgAX-2 hemizygotes (D—I); (A,B) wild type discs at 3 and 4 days; (C) wing, haltere and third leg disc at 5 days, with their connections intact; (D,E) mutant wing discs at 3 and 4 days; (F-I) mutant wing, haltere, and third leg disc complex at 5,6,8, and 11 days. Note the progressive fusion between these three discs. W, Wing disc; H, haltere disc; 3L, third leg disc. Bar, 0.5 mm. (From Woods and Bryant, 1989).

Fig. 5.

Wing imaginal discs from wild type (A-C) and dlgAX-2 hemizygotes (D—I); (A,B) wild type discs at 3 and 4 days; (C) wing, haltere and third leg disc at 5 days, with their connections intact; (D,E) mutant wing discs at 3 and 4 days; (F-I) mutant wing, haltere, and third leg disc complex at 5,6,8, and 11 days. Note the progressive fusion between these three discs. W, Wing disc; H, haltere disc; 3L, third leg disc. Bar, 0.5 mm. (From Woods and Bryant, 1989).

The Igl gene produces two transcripts of 5.7 and 4.3 kb in size which are detected mainly during embryogenesis and at the larval-pupal transition (Mechler et al. 1985). Sequencing of cDNA fragments from Igl revealed a conceptual reading frame with a coding capacity for a 130 × 103Mr protein. Fragments of the cDNA were incorporated into bacterial expression vectors to make fusion proteins, which were then used to raise antibodies. These antibodies detect a protein of the appropriate size both during embryogenesis and at the end of larval development (Klarnbt and Schmidt, 1986). The protein is found at the outer surface of dissociated cells and is attached to the cell membrane in an unknown way (Klâmbt et al. 1989). Sequence similarity of the deduced protein to those of cadherins (Fig. 6; Klambt et al. 1989), a family of calcium-dependent cell adhesion molecules in vertebrates (Takeichi, 1988), and the presence of an RGDV motif (Lützelschwab et al. 1987) which could function as a binding site for cell-cell or cell-matrix interaction (Ruoslahti and Pierschbacher, 1986), suggest that the protein is involved in cell adhesion.

Fig. 6.

Igl and L-CAM protein structure. Schematic drawing of the structural organization of similar protein regions in L-CAM as an example of a cadherin molecule and the Igl protein. The L-CAM protein (Gallin et al. 1987) spans the membrane (TM domain) and has a 16 × 103Mr intracellular domain (i). The Igl protein exhibits no transmembrane domain (Lützelschwab et al. 1987) and is attached to the membrane and/or the extracellular matrix by an unknown mechanism. Four regions conserved between L-CAM and Igl amino acid sequences are indicated. A and B are repeats containing putative N-glycosylation sites. B is a modified A-type region. C and D represent smaller regions containing negatively and positively charged amino acids, respectively, o, outside, i, inside the cell; TM, transmembrane domain (from Klambt et al. 1989). The bar represents 100 amino acids.

Fig. 6.

Igl and L-CAM protein structure. Schematic drawing of the structural organization of similar protein regions in L-CAM as an example of a cadherin molecule and the Igl protein. The L-CAM protein (Gallin et al. 1987) spans the membrane (TM domain) and has a 16 × 103Mr intracellular domain (i). The Igl protein exhibits no transmembrane domain (Lützelschwab et al. 1987) and is attached to the membrane and/or the extracellular matrix by an unknown mechanism. Four regions conserved between L-CAM and Igl amino acid sequences are indicated. A and B are repeats containing putative N-glycosylation sites. B is a modified A-type region. C and D represent smaller regions containing negatively and positively charged amino acids, respectively, o, outside, i, inside the cell; TM, transmembrane domain (from Klambt et al. 1989). The bar represents 100 amino acids.

In sections of embryos which have been stained with antibodies against the Igl protein, the antigen is detected predominantly in developing larval structures (Fig. 7). The nervous system contains much less of the protein, except in areas where nerve cells are in the process of axonogenesis (Klambt et al. 1989). The protein is also transiently expressed in pole cells and in the neuroblasts of the presumptive optic lobes of the brain (Klambt and Schmidt, 1986). The protein is expressed again mainly in imaginal discs and in the central nervous system at the end of larval development.

Fig. 7.

Localization of lgl protein in a parasagittal section of a wild type Drosophila embryo. Binding of antibody to the lgl protein was visualized by indirect immunofluorescence, vc, ventral nerve cord. (O. Schmidt, unpublished).

Fig. 7.

Localization of lgl protein in a parasagittal section of a wild type Drosophila embryo. Binding of antibody to the lgl protein was visualized by indirect immunofluorescence, vc, ventral nerve cord. (O. Schmidt, unpublished).

The Igl protein is present in those tissues, both imaginal and larval, which are about to stop cell proliferation and begin differentiation (Klambt and Schmidt, 1986; Klambt et al. 1989). In homozygous mutant tissues, the cells show reduced contact with one another, cuboidal rather than columnar shape, and excessive proliferation. When transplanted into wild type hosts, mutant brain cells dissociate from the implant, invade host tissues and eventually kill the host (Gateff, 1978). These results indicate that the Igl protein functions in cell adhesion, and support the idea that cell proliferation is controlled by interaction between cells and their neighbors, with the signalling process dependent on intimate and direct cell contact.

Although both larval and imaginal primordia express the lgl protein in wild type embryos (Fig. 7), only imaginal primordia are phenotypically affected by lgl mutations (Gateff, 1978). The absence of an effect on larval tissues in homozygous embryos could be due to provision of sufficient wild type gene product by the heterozygous mother (Klambt et al. 1989), or due to compensation of embryonic lgl functions by other gene products. The amount of rescue by either means must be insufficient to supply the imaginal disc cells, either because they have a higher requirement or because the rescuing gene products are diluted out to subthreshold levels during the extensive cell proliferation occurring in these tissues.

Different imaginal disc overgrowth mutants show different patterns of overgrowth, the exact pattern of extra folds and lobes being characteristic for each gene (Bryant, 1987). This suggests that these mutations exert their effects directly on the imaginal discs, rather than by affecting disc growth indirectly through an effect on some other system such as the endocrine system. This point has also been demonstrated by transplanting mutant imaginal discs into wild-type adult hosts and observing their growth properties. In all of the mutants discussed above, the growth abnormality is disc-autonomous; that is, the transplanted tissue shows overgrowth whereas wild type discs cease growth at a characteristic size and cell number (1gl, Gateff and Schneiderman, 1974; wild type andZgd, Bryant and Levinson, 1985; c43, Martin et al. 1977; fat, Bryant et al. 1988; dco, Jursnich et al. 1990; dig, D. Sponaugle and D. Woods, unpublished observations). Thus, the growth-control defects are intrinsic to the imaginal discs, rather than being a result of the extended larval period or a systemic defect. The results are therefore consistent with the regeneration experiments that show the importance of disc-intrinsic growth-control mechanisms.

In all of the imaginal disc overgrowth mutants so far described, the overgrowth occurs during a greatly prolonged larval life, which is followed by death of the mutant animal either as a larva or as a pupa. In dig, fat, Igd, dco, and c43, the prolonged larval life is associated with very low levels of the molting hormone 20-hydroxyecdysone during and after the time when this hormone peaks in the wild type (F. Sehnal and P. Bryant, unpublished results). Although in principle this endocrine defect may be an independent, pleiotropic effect of the mutations, we feel that it is more likely that the endocrine defect is a secondary result of the imaginal disc overgrowth. The most direct evidence for this idea is from recent work by Poodry and Woods (1990) who have shown that in dig the delay in development can be prevented by eliminating imaginal discs with ionizing radiation. Not only is the delay prevented, but the irradiated animals, unlike the controls, are able to pupariate.

It has been known for many years that the presence of a regenerating imaginal disc in a larval insect can delay the onset of metamorphosis of the host, suggesting that growing imaginal disc tissue sends an inhibitory signal to the endocrine system. In the moth Ephestia kühniella, extirpation of a single wing disc from a mature larva (just prior to cocoon-spinning) resulted in a pupation delay of about eight days in those animals which regenerated the wing, but no delay in the animals which failed to regenerate (Pohley, 1965). If two wing discs were regenerating, the delay was about 1.5 days longer. Madhavan and Schneiderman (1969) conducted similar experiments on another moth, Galleria mellonella, and showed that extirpation of one wing disc delayed pupation by five days, extirpation of two discs caused an eight-day delay, and extirpation of all four wing discs caused a 14-day delay. Both Pohley (1965) and Madhavan and Schneider-man (1969) suggested that the regenerating imaginal discs somehow reduced the concentration of ecdysone in the animal.

In principle, the delay of pupation which is caused by imaginal disc extirpation could be mediated by a hormone, or it could be mediated by a signal transmitted from the imaginal discs to the endocrine glands via the nervous system. However, it is almost certain that the signal is an endocrine one, since a similar delay can be produced by the implantation of regenerating tissue into the hemocoel of an otherwise normal host. For example, when imaginal wing discs were implanted into last-instar Ephestia larvae, they underwent duplication and delayed pupation of the host by 8.6 days (forewing) or 7.7 days (hindwing) (Rahn, 1972). When wing discs which had already been cultured in one host for various periods were implanted into a second host, they delayed its pupation by an amount which decreased with the age of the regenerate. This meant that the total culture time available to the implant before pupation was practically constant. The prepupal phase was prolonged until regeneration (more properly in this case, duplication) was complete (Rahn, 1972). Similar results were obtained by Dewes (1975, 1979) using the regenerating half-genital disc of Ephestia. Apparently, a very important control system links developing tissues and the endocrine system in insects, and this system ensures that different parts of the insect develop in harmony. However, the nature of the signal from regenerating tissues is not known, and the question of how the endocrine glands and/or their target tissues are affected by the regenerating tissue has received little attention.

Simpson et al. (1980) have reported an ingenious demonstration of regeneration-induced developmental delay in Drosophila using several genetic techniques. Extensive regeneration in many imaginal discs of the same larva can be induced by applying a sublethal heat pulse to a larva carrying a temperaturesensitive, cell-lethal mutation (Girton and Bryant, 1980). These treated animals show delayed pupariation (Russell, 1974; Simpson and Schneiderman, 1975), and the amount of delay is correlated with the amount of extra growth that occurs during regeneration (Simpson et al. 1980). Furthermore, by heat-treating gynandromorphs carrying different proportions of wild type and mutant tissue, Simpson et al. (1980) showed that the pupariation delay was greater with larger amounts of mutant tissue. Elimination of entire imaginal discs (which, in Drosophila, is not followed by regeneration) did not delay pupariation, indicating that it is regeneration per se, rather than any other heat-induced damage, that delays pupariation. Furthermore, Simpson et al. (1980) showed that, as in the studies on other insects, the wild type (non-regenerating) tissues in the delayed animals were not enlarged. This is consistent with other results indicating that each imaginal disc has an intrinsic growth limit.

If regenerating imaginal discs cause developmental delay by an endocrine mechanism, this would suggest that imaginal tissues secrete a factor which inhibits release of 20-hydroxyecdysone from the endocrine glands. In the overgrowth mutants, the extended larval life could be a result of continuous production of this inhibitor by the imaginal discs.

Each zygotic lethal mutation causes death of the animal at a characteristic developmental stage, so phenotypic analysis of mutants can reveal functions of the affected gene only up to that stage. The imaginal disc overgrowth mutants die as late larvae or pupae. However, using special methods, it has been shown that the normal alleles of some of these genes also function in the adult diming gametogenesis. In some cases this has been shown by testing the function of mutant germ cells transplanted into wild type hosts in the embryonic stage; in other cases homozygous mutant clones have been induced in the germ line of an otherwise heterozygous adult by X-ray induced mitotic recombination. In the case of dig, homozygous germ-line clones in the ovary produce eggs in which embryonic development is disrupted because of the lack of normal gene product supplied during oogenesis. In the maternally affected embryos, an additional layer of neuroblasts is formed in the gastrula and subsequent overgrowth of the nervous system prevents completion of dorsal closure (Perrimon, 1988). The normal gene is therefore required for both cell proliferation control and epithelial integrity in the embryos, just as it is required in imaginal discs. One of the dig transcripts is limited to the adult ovaries and is missing in several of the viable heteroallelic combinations (Woods and Bryant, 1989), so this may be responsible for the maternal-effect lethality shown in these genotypes.

Most of the other imaginal disc overgrowth mutations have recently been tested by germ-line transplantation and/or mitotic recombination, and all of those tested (1gl, dco, igd, fat, c43) interfere with functions of the female germ line (Szabad et al. 1990), preventing the production of viable progeny. In one case (Igd) the number of cells in the egg chamber was over 40 compared to the 16 (15 nurse cells and one oocyte) found normally, suggesting an effect on cell proliferation control. In the other cases the mutant germline either failed to produce eggs or produced eggs which failed to complete their development without showing any obvious abnormalities in cell proliferation control. In imaginal discs the mutations appear to affect cell proliferation control by interfering with cell interactions controlling proliferation. We suggest that in the female germ line the mutations might also interfere with cell interactions (for example, between the oocyte and nurse cells, or between the oocyte and the surrounding follicle cells) but that these interactions control aspects of egg development other than cell proliferation.

Another approach to the problem of cell proliferation control in Drosophila is to use mutational analysis to investigate the functions of genes which show homology to various molecules thought to be involved in the control of growth and cell proliferation in vertebrates. Table 2 lists the genes showing growth factor, growth factor receptor, or oncogene homology for which a mutant phenotype has been identified in Drosophila. Most of these genes have recently been reviewed in detail by Hoffmann (1989). Surprisingly, for twelve of the thirteen genes listed the mutations cause a change in cell fate, whereas in only two cases is there an indication of a direct effect on growth or cell proliferation.

Table 2.

Drosophila genes showing homology to growth factors, growth factor receptors, and oncogenes, for which mutant phenotypes have been described

Drosophila genes showing homology to growth factors, growth factor receptors, and oncogenes, for which mutant phenotypes have been described
Drosophila genes showing homology to growth factors, growth factor receptors, and oncogenes, for which mutant phenotypes have been described

Mutations in four of the genes listed in Table 2 lead to cell-fate changes in the neurogenic ectoderm of the embryo. This region normally undergoes patterning to produce intermingled neuroblasts and epidermal precursors, in which neuroblasts make up about 25% of the total cells (Hartenstein and Campos-Ortega, 1984). Mutations of the Notch, Delta and crumbs genes (which encode molecules containing multiple EGF-like repeats) interfere with this process, leading to an abundance of neuroblasts at the expense of epidermal cells (the neurogenic phenotype; Lehmann et al. 1983). Mutations at other loci including Enhancer of split, which interacts with Notch and shows homology to myc (Knust and Campos-Ortega, 1989), also produce a neurogenic phenotype (Knust et al. 19875). Notch mutations also interfere with other cell-fate decisions during neurogenesis in the larval peripheral nervous system (Hartenstein and Campos-Ortega, 1986), bristle cell patterning (L. Held, personal communication) and development of the compound eye of the adult (Cagan and Ready, 1989). The neurogenic mutants do show an effect on cell proliferation in that the embryonic central nervous system is overgrown, but this is an indirect effect due to conversion of epidermal cells into neuroblasts, which are characterized by a greater proliferative potential than epidermis (Technau and Campos-Ortega, 1986).

The only genes on the list in which mutations might directly cause cell proliferation abnormalities are raf, in which lethal mutations cause absence or underdevelopment of imaginal discs and rings, brain lobes, lymph glands and gonads (Nishida et al. 1988) and torpedo, in which one of the zygotic effects is underdevelopment of the wing, haltere and eye imaginal discs (Clifford and Schubach, 1989). Even in these cases, it is not known whether the underdevelopment is due to inhibition of cell proliferation rather than degeneration.

The general conclusion from the Drosophila results is that these gene products in vivo usually have a role in pattern formation rather than in cell proliferation control. Although it is generally accepted that growth factors are often involved in controlling cell differentiation as well as proliferation, the scarcity of effects on proliferation is perhaps surprising. However, the phenotypes listed in Table 2 are those resulting from genetic loss of function. If mutations in these genes led to activation similar to the oncogene activation that is seen in the vertebrate oncogenes, they would show a dominant gain of function. Dominant or semidominant gain-of-function mutations have, in fact, been reported for Enhancer of split (Knust et al. 19876), torso (Strecker et al. 1989) and achaete-scute (Garcia-Alonso and Garcia-Bellido, 1986), but in all cases the resultant phenotype is a cell-fate change in the opposite direction from that caused by genetic loss of function, rather than an effect on cell proliferation. A dominant gain-of-function mutation leading to excess cell proliferation would probably behave as a dominant lethal, and this is one class of mutations which Drosophila geneticists cannot easily study. Such alterations would not have been identified as spontaneous mutations and would not have been recovered in conventional mutagenesis screens. Possibly they could be identified if they were temperature-sensitive, but such mutants have not been reported. Therefore, the lack of clear effects on cell proliferation among the mutants listed in Table 2 could be, at least in part, due to our inability to identify and work with dominant lethal mutations.

Dr Bryant’s research is supported by grants from the National Science Foundation and the Monsanto Company.

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