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Localized cell death in Drosophila imaginal wing disc epithelium caused by the mutation apterous-blot
DEVELOPMENTAL
BIOLOGY
104.489-496 (1984)
Localized Cell Death in Drosophila lmaginal Wing Disc Epithelium Caused by the Mutation apterous-blot BONNIEJOYSEDLAK,* *Develqvmmtal
REN$MANZO,-~ANDMARYSTEVENS*
Biology Center, University of California, Irvine, California 92717,and ~CorneU Medical College, New York, New York 100l$ Received February 9, 1984 accepted in revised
form April 30, 1984
Drosophila melanogaster carrying the mutation aptemus-blot have blistered wings. Trypan blue stains a patch of dead cells localized to the wing pouch of imaginal discs and the same area shows acid phosphatase (AcPase) activity suggesting that the cell death is lysosomal. Autophagic vacuoles and other secondary lysosomes show AcPase activity within the disc epithelium and enzyme activity is found in fragments of dead cells which have been extruded basally. The cell death, although extensive and confined to the presumptive wing region, does not result in loss of adult structures.
INTRODUCTION
The phenomenon of cell death in development has been observed in both vertebrate and invertebrate tissues, e.g., the selective destruction of tissue which occurs in chick wing (Saunders, 1969) and fly foot pad (Whitten, 1969) results in the patterning of adult limbs from budlike limb precursors. The sculpturing mechanism occurs at precise times in the developmental program of each animal and in both cases cellular destruction is a normal and necessary consequence of either embryonic or postembryonic maturation. Cell death may also play a role in the formation of mutant patterns in Drosophila In this insect many adult structures are derived from the imaginal discs which remain as discrete sacs of undifferentiated tissue throughout larval life. Fate maps of these larval discs define regions of presumptive adult structures (Bryant, 1978). It has been suggested that in vestigial (Fristrom, 1968, 1969; Bownes and Roberts, 1981; O’Brochta and Bryant, 1988), ultravestigial (O’Brochta and Bryant, 1988), scaUupeducx(James and Bryant, 1981), apterousXa&z, Be&, and Cut (Fristrom, 1969) cell death occurs in regions of the wing discs that correspond to areas where there are abnormalities in adult structures. The mutation apteroue-blot (ap”) causes wings to have a blistered surface but otherwise they are similar to wild-type wings. To test the possibility that localized degeneration of wing discs was associated with the abnormal phenotype we studied cellular degeneration at the levels of histology and ultrastructure. Acid phosphatase (AcPase) has been routinely used to identify and define lysosomes (Holtzman, 1976) and in this study the presence of AcPase confirmed the existence of 2’ lysosomes (i.e., autophagic vacuoles) in ap”. The evidence indicates that the cell death observed in the wing pouch 489
region of imaginal discs does not result in wing blade deficiencies perhaps because instead of entire cells being eliminated only portions of cells are removed. METHODS
Rearing animals. Drosophila melanogaster of both ap terms-blot (mutant) and OreR (wild type; control) stocks were reared on artificial media at 25°C in half pint culture bottles. Larvae were collected 120 hr after egg lay (AEL), i.e., late in the third larval instar, and were lightly anesthetized either by submersion in water or with ether fumes before processing the wing imaginal discs for morphological studies. Individual larvae were initially dissected by grasping the mouth hook parts and thorax with forceps and pulling the animal apart to allow free access of the fixative and/or stain to the internal organs. Trypan blue staining of whole wing imaginal discs. Both mutant and wild-type larvae were dissected in insect Gehring’s Ringer solution and immediately placed in a fresh solution of 0.5% trypan blue dissolved in Ringer’s. After staining for 15 min, larvae were washed with Ringer’s and fixed in 2.5% buffered glutaraldehyde (0.05 M sodium phosphate buffer, pH 7.2, with 5% sucrose added). Following dehydration in 70% and absolute alcohols the wing discs were dissected free of the carcass and mounted in euparol medium for observation as whole mounts. Histology and electron micros-. Apm and wild type larvae were fixed in 2.5% buffered glutaraldehyde (0.05 M sodium phosphate buffer, pH 7.2, with 5% sucrose added), postfixed in 1% buffered Os04, washed and stained in block with 2% aqueous uranyl acetate, dehydrated in alcohols, and embedded in Epon 812 resin. oOli!-X06/64 $3.66 Copyright All righta
0 1994 hy Academic Press. Inc. of reproduction in any form reserved.
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wing pouch) which has been identified as the area which will give rise to wing structures (Bryant, 1975). Of the mutant discs 27/28 had the distinctive pattern of staining whereas none of the 18 wild-type discs showed a concentrated uptake of the dye in any area of the disc. It is not sufficient to use trypan blue as the sole indicator of cell death since this marker only identifies lysed cells and does not distinguish between dead epithelial cells and/or the phagocytes which may engulf the dead cells (Bowen, 1981). Thus a second technique (a) Essentially following the method of Locke and was used to stain for lysosomal enzymes and the maSykes (1975) with modifications, i.e., 2.5 mMlead nitrate terial was studied at both the light and electron miwas made up in 0.1 mM sodium acetate buffer pH 5.5 croscopic levels to see if lysosomal cell death affected containing 5% sucrose, and 0.1 mM sodium /3-glycerothe disc epithelium. phosphate served as a substrate. Tissue was incubated Whole mounts of mutant discs stained for acid phosat both 25 and 37°C for either 90 min (EM) or 180 phatase showed a characteristically granular black demin (LM). posit which defines the enzyme, AcPase, confined to the (b) Lead nitrate (1.5 mM) made up in 0.1 M Trisregion which gives rise to the wing surface. Since the maleate buffer pH 5.0 with 1.5 mM sodium B-glyceroarea is identical to that which stains with trypan blue, phosphate as substrate. Tissue was incubated for 30 min the observation (1) confirms the conclusion that cell at both 25 and 3’7°C (Barka and Anderson, 1962). death occurs only in the wing pouch in ap” and (2) Controls consisted of (1) preincubating tissues in 0.01 suggests that cell death is lysosomal. AcPase staining was never seen in wing discs from control (wild type) M sodium fluoride for 30 min or (2) adding the sodium fluoride directly to the reaction media to inhibit AcPase larvae. activity. Whole imaginal discs were placed in 1% ammonium sulfide for 5 min following incubation with the Cytochemical Localization of CeU Death reaction media to allow visualization for light microsThe ultrastructure of epithelial cells of the mutant copy. Other discs were prepared for electron microscopy. wing discs is similar to that of wild-type discs. In discs from either stock the tissue is composed of columnar RESULTS cells with short microvilli at the apex and typical junctions along the lateral plasma membrane (i.e., zonulae Cell Death in Wing Discs; HtitochewGtrg adherentes, septate junctions, and gap junctions). The Animals homozygous for apterow-blot have wings cells are largely undifferentiated; free ribosomes predominate with only a few small cisternae of rough enwhich are similar to wild type but 40-60% of the mutant doplasmic reticulum found along with a moderate numwings are blistered (Fig. 1); the phenotype shows partial ber of mitochondria. Golgi complexes are seen infrepenetrance and expressivity (Stevens and Bryant, 1984). quently; when present, they are of the vesiculate Golgi Whole mounts of wing discs from both wild-type and mutant larvae were stained with either trypan blue or type. Figure 3 is a section through a wild-type wing disc acid phosphatase to show areas of cell death. Figure 2 shows a trypan blue stained wing disc taken from an which emphasizes two characteristics that define the wing pouch region. First, there is a larger concentration apbu larva; the overall morphology and folding pattern of lipid droplets in the wing pouch than is found in other in the mutant resemble that of the wild-type disc. Trypan blue, a vital dye which is selectively taken up regions of the disc epithelium (see also Fristrom, 1969). by cells that have died (Bowen, 1981), was used to iden- Second, the wing pouch basal lamina is composed of tify regions of tissue degeneration in wing discs. In apbu lamellated layers of extracellular matrix plus a thin, discs the exclusion dye was confined to a region (the electron-dense outermost layer (Fig. 3a, inset). In all Histological sections (0.5 pm) were stained with a 1% methylene blue solution and thin sections were stained with lead and uranium before photographing with a Hitachi 12 electron microscope. Acid phosphutase histo/cytochewGtrg. Larvae from both ap” and OreR stocks were fixed with 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for l-l.5 hr at 5°C and tissues were incubated in either of two reaction media:
FIG. 1. A wing from aput shows blistering (arrow) of the wing surface. X500. FIG. 2. Whole mount of ap” wing imaginal disc 120 hr AEL, trypan blue stain indicates a patch of dead cells in the wing pouch area (arrow). X600. FIG. 3. The wing pouch region of a wild-type wing disc is distinguished by a large concentration of lipid droplets and fairly thick basal lamina, composed of a loosely organized lamellar matrix plus an electron dense outer layer. Inset a, wing pouch basal lamina; inset b, proximal fold basal lamina. bc, Blood cell; bl, basal lamina; 1, lipid. X6000; inset a and b, X26,000.
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other parts of the disc a thin dense layer is the sole component of the basal lamina (Fig. 3b, inset). The only structural differences between wild-type and mutant discs are those associated with cell death. The evidence from whole mounts indicated that dead cells resided in the wing pouch region and in Fig. 4, which shows a histological section taken perpendicular to the long axis of a mutant imaginal wing disc, a large area of darkly staining cellular debris is found at the basal side of the wing epithelium and seems to be extruded from the epithelial tissue. The tissue sections provide evidence that the debris, which has accumulated in the region of the wing pouch and extends to the area of the adjacent (proximal) fold, corresponds to the trypan blue stained area seen in whole mounts. The nature of the cellular debris was further analyzed by electron microscopy. Figure 5 which corresponds to region 1 in the histological section (see Fig. 4) shows that the debris is extracellular. It consists of cytoplasmic fragments which are fairly round, variable in size, and contain recognizable remnants of mitochondria and lipid droplets. All fragments lie between the epithelial cell basal plasma membrane and the basal lamina. Some pieces of cytoplasm are so highly condensed that no organelles can be recognized within the fragments, whereas other fragments appear quite electron lucent and are composed mostly of flocculent material plus a few electron dense inclusions. Tissue was incubated with two Gomori type media at both 25 and 37°C to show AcPase activity. Both incubations produced deposits of black precipitate within the epithelial cells, but the incubations run at 37°C resulted in the heaviest deposition of reaction products. In addition to the mutant discs treated to show AcPase activity, controls consisting of (1) untreated wild-type discs, (2) wild-type discs processed for AcPase activity, and (3) both wild-type and mutant discs treated with sodium fluoride to quench AcPase activity were studied. Sodium fluoride was effective in eliminating any AcPase activity from the regions which otherwise had heavy black deposits. The inset in Fig. 4 shows extracellular debris in a disc treated to show AcPase activity and, as in all other treated tissues, the thin sections were not poststained with a heavy metal (i.e., uranium or lead). The tissue was, however, stained in block with uranyl acetate to enhance organelle resolution. The heavy black deposits represent localization of the lysosomal enzyme, i.e., AcPase activity is specifically associated with membrane-bounded bodies within the debris.
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Figure 6 indicates an epithelial region of a mutant disc corresponding to region 2 in Fig. 4. There are several large inclusions located within the columnar epithelial cells of the wing pouch which lie adjacent to the extracellular debris. A higher magnification shows that one of the dense inclusions appears to be membrane bounded and contains mitochondria in addition to free ribosomes defining it as an autophagic vacuole (Fig. 7). Such structures showed AcPase activity and AcPase reaction products were also found associated with a variety of smaller inclusions within the epithelial cells in up”” wing discs. In some cases, it seems that AcPase activity is associated with the ribosomes and/or rough endoplasmic reticulum encircling a lipid inclusion in the wing pouch (Fig. 8). In Fig. 9 a typical vesiculate Golgi complex abuts upon a small dense body containing patches of AcPase reaction product. A few dense bodies are also found in wild type wing discs and AcPase activity is often associated with such dense bodies. Extruded cellular debris is never found in the control discs. DISCUSSION
The apm mutation causes a relatively mild distortion of the adult wing even though a large patch of cell death is localized within the presumptive wing region in imaginal discs, thus indicating that cell death does not always result in major defects in adult structures. Trypan blue staining identifies dead cells in the wing pouch area of up”” and similarly the wing pouch is the area where cell death was described in larvae carrying the mutation vestigial (Bownes and Roberts, 1981;Fristrom, 1968,1969; O’Brochta and Bryant, 1983), Ultravestigial (O’Brochta and Bryant, 1983), and scallopeduc’ (James and Bryant, 1981). In these mutations the wings are much diminished and lack many structures normally found in the adult. ar, a more severe aptemus allele which causes a notch to develop in adult wings, also has cell death occurring in the wing pouch of imaginal discs (Fristrom, 1969). Unlike all of these mutations, the cell death found in apM does not result in a loss of adult structures and the type of cell death is autophagy, not cell elimination. Wyllie et al. (1980) coined the phrase apoptosis to describe a mechanism for cell death, in which single cells condense, then fragment, and are either eliminated by phagocytosis or undergo a secondary lysis. This mechanism has been confused with autophagy. In apoptosis the elimination of dead cells is nonlysosomal, i.e., acid hydrolyses are not produced by the dying cells but
FIG. 4. A methylene blue-stained section through the longitudinal axis of an up” wing disc 120 hr AEL, darkly stained debris is localized at the basal side of the wing pouch. (High magnification micrographs of regions 1 and 2 are shown in Figs. 5 and 6.) 1, Lipid. X1700. FIG. 5. Cellular debris is found between epithelial cell plasma membranes and the basal lamina in a#” wing disc. The area shown corresponds to region 1 in Fig. 4. Inset, AcPase reaction product within the extracellular debris. 1, Lipid. X9oo0, inset, X40,000.
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are introduced from neighboring phagocytic cells. Autophagy, in contrast, represents lysosomal cell death, in which portions of cells die and cell fragments are digested by enzymes produced within the same cells. There is ultrastructural evidence that apoptosis occurs in VQ(O’Brochta and Bryant, 1983) and ap (Fristrom, 1969) but apoptosis is not the mechanism of cell death in up”. The evidence for autophagy is clear since AcPase reaction products have been found within membranebounded autophagic vacuoles residing in otherwise healthy cells, i.e., cells that have normal nuclei. During autophagy the lytic enzyme may be processed and/or packaged as a 1” lysosome at the Golgi complexes and subsequently introduced to the autophagic vacuole (Locke and Sykes, 1975). In up” the small dense bodies which are located directly adjacent to Golgi vesicles show AcPase activity and may be 1” lysosomes, i.e., their proximity to Golgi elements suggest the Golgi as the source of the enzyme. However, these organelles are rare and none of the Golgi membranes shows any AcPase activity. The possibility that ribosomes (Bowen and Ryder, 1974) or rough endoplasmic reticulum (Borges and Thone, 1976) contribute AcPase can not be discounted. AcPase reaction product encircles lipid droplets in the wing pouch of the mutant. AcPase cytochemistry has been classically used to define the lysosome in cell biology and Clark and Russel (1977) used enzyme localization to correlate lysosomal activity in the eye-antenna1 discs of the mutant &!)ts 726 of Lkosophilu with areas of structural deficiencies in the heads of the adult flies. In this study apm AcPase reaction products were confined to the same area in whole wing discs where trypan blue showed cell death. Thus, dying cells within the wing pouch may be destroyed, at least in part, by lysosomes. Dense bodies with AcPase reaction products are found in wild-type as well as mutant wing discs but this observation is expected since lysosomes are nearly ubiquitous organelles. The most striking morphological difference between up” and wild-type wing discs is the cytoplasmic fragments concentrated at the basal side of the wing pouch epithelium. It is likely that the fragments represent cell debris which has been extruded from the epithelial sheet. The amount of basal debris is extensive. Only a few cell fragments are found within any particular epithelial
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cell late in the larval instar (120 AEL) suggesting that the extruded material represents an accumulation from cells which died at earlier stages. In fact wing discs taken from larvae at mid-third instar (96 hr AEL) also have trypan blue stain localized in the wing pouch area indicating that debris has already formed at this time ‘(M. Stevens, unpublished observations). Basal extrusion of cell debris has been reported for other mutants including vg (O’Brochta and Bryant, 19!33), and ecd’ (D. O’Brochta and T. Sliter, personal communication). In apm the fate of the cellular waste is not known. There was no evidence of phagocytosis by adjoining epithelial cells in up”, i.e., whole cells including their nuclei never appeared encircled by neighboring cell membranes. Blood cell phagocytosis may occur. In vg, for example, in which late stages of development (i.e., after pupariation) were studied, blood cells ingest extruded fragments (O’Brochta and Bryant, 1983). Perhaps phagocytosis is not necessary to eliminate the waste since it is clear that in u$” the extruded debris continues to be digested extracellularly. AcPase activity is found associated with some of the autophagic fragments and other fragments within the debris which are filled with flocculent material lack cellular detail as if the cytoplasm was subject to lysis following extrusion. That cell death occurs in ap”“, is extensive, and is confined to the wing pouch area are obvious conclusions from the data but it is not clear how this cell death relates to the rather minor alterations in adult phenotype. A complication in interpreting how larval cell death affects adult pattern is the fact that in up the phenotype is variably expressed, and in addition to the mild blistering of wings described above, an engrailedlike phenotype is found in a small proportion of the flies in the stock described by Whittle and Sprey (1982). It has been proposed that embryonic cells such as imaginal discs have positional information which enables them to participate in making complex adult patterns. Abnormal patterns, e.g., specific deficiencies in adult structures, may be the result of genetically induced cell death which eliminates essential patterning information (Girton and Bryant, 1980). Understanding the relationship between the larval cell death and adult pattern defects in apHt depends upon interpreting the mechanisms underlying the cellular degeneration. Accumulated debris may have little or no effect on pattern
FIG. 6. In addition to the basal debris, there are large dense bodies located within the epithelial cells in the wing pouch The area shown corresponds to region 2 in Fig. 4. db, Dense body; n, nucleus. X7599. FIG. 7. A higher magnification of the membrane-bounded body seen in Fig. 6 shows that this inclusion is an autophagic mitochondria and free ribosomes. m, Mitochondria. X21,999. FIG. 8. AcPase activity is associated with the lipid droplets of the wing pouch; lipid inclusions are often surrounded by reticulum. 1, Lipid; rer, rough endoplasmic reticulum. X60,999 FIG. 9. A dense body located directly adjacent to the vesiculate Golgi complex shows AcPase activity while the Golgi contain any reaction products. G, Golgi complex. X59,ooO.
of ap”’ wing discs. vacuole containing rough endoplasmic membranes
do not
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formation (Rownes and Roberts, 1981) or it may interfere with wing differentiation by preventing the basal surfaces of the disc epithelium from being normally opposed during wing evagination (Fristrom and Fristrom, 1975). It is likely that, since only a portion of each cell is destroyed in up” wing discs in contrast to up where whole cells are removed via apoptosis (Fristrom, 1969), positional information is not entirely removed in up thus there is no loss of pattern elements in flies carrying the less severe allele. The mild distortion of the wing phenotype typically found in apM may merely be a response to a sluggish system which has accumulated metabolic waste products or a system in which some subvital cells (i.e., those undergoing an increase in autophagy) can not contribute fully to the formation of a complete pattern. Alternatively cell death may eliminate presumptive wing structures but regeneration may lead to the replacement of degenerated parts. Thus the adult wing would not appear greatly distorted. This paper was made possible by the support of National Institutes of Health, Grant AG 01979.We thank Dr. Peter Bryant for his helpful comments on the manuscript. REFERENCES BARKA, T., and ANDERSON,P. J. (1962). Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochen~ Cytochem 10.741-753. BORGES,M., and THONE,F. (1976). Further characterization of phosphatase activities, using non-specific substrates. Histochem J. 6, 201-317. BOWEN,I. D. (1981). Techniques for demonstrating cell death. In “Cell Death in Biology and Pathology” (I. D. Bowen and R. A. Lockshin, eds.), pp. 381-446. Chapman & Hall, London. BOWEN,I. D., and RYDER,T. A. (1974). Cell autolysis and deletion in the planarian Polycelis tenuis Iijima. CeU !&sue Res. 154,265-274. BOWNES,M., and ROBERTS,S. (1981). Regulative properties of wing discs from the vestigial mutant of Drosophila mdanogaster. L?@krentiation 18.39-96. BRYANT, P. J. (1978). Pattern formation in imaginal discs. In “The
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Genetics and Biology of Lkosophild (M. Ashburner and T. R. F. Wright, eds.), Vol. 2c, pp. 230336. Academic Press, London. BRYANT,P. J. (1975). Pattern formation in the imaginal wing disc of Lko.wphdu melanogaster: Fate map, regeneration and duplication. J. Exp. Zool 193.49-77. CLARK,W. C., and RUSSELL,M. A. (1977). The correlation of lysosomal activity and adult phenotype in a cell-lethal mutant of Drosophila Llev. Bid 57.160-173. FRISTROM,D. (1968). Cellular degeneration in wing development of the mutant vest&id of Drosophila mdanogaster. J. Cd Bid 39, 433-491.
FRISTROM,D. (1969). Cellular degeneration in the production of some mutant phenotypes in Drosophila m&noga&x Mokc Ga Gewt 103,369-379.
FRISTROM,D., and FRISTROM,J. W. (1975). The mechanism of evagination of imaginal discs of Lkosophilu melunogaater. Dev. Bid 43, l-23.
GIRTON,J. R., and BRYANT,P. J. (1980). The use of cell lethal mutations in the study of Drosophila development. Dev. Bid 77,233-243. HOLTZMAN,E. (1976). “Lysosomes: A Survey.” Springer-Verlag, New York. JAMES, A., and BRYANT, P. J. (1981). Mutations causing pattern deficiencies and duplications in the imaginal wing disk of Drosophila mhncgaster. Dec. Biol 86,39-54. LOCKE,M., and SYKES,A. K. (1975). The role of the Golgi complex in the isolation and digestion of organelles. !&sue CeU 7, 143-158. G’BROCHTA,D. A.. and BRYANT, P. H. (1983). Cell degeneration and elimination in the imaginal wing disc, caused by the mutations vee~andVUmuest~ofL%osophilamela~. WMmRmx18 Arch. 192,285~294. SAUNDERS,J. W. (1969). Death in embryonic systems. Science 154, 604-612. STEVENS,M. E., and BRYANT,P. J. (1984). Apparent genetic complexity generated by developmental thresholds: The aptemus locus in Dro sophila rxelanogoater. Genetics, in preparation. WHMTEN,J. M. (1969).Cell death during early morphogenesis: Parallels between insect limb and vertebrate limb development. Scarce 163, 1456-1457. WHITTLE, J. R. S. (1979). Replacement of posterior by anterior structures in the L?rosophila wing caused by the mutation apteroua-blot J. Embrgol Exp. Morph& 53,291~303. WHITE, R., and SPREY,T. (1982). Correlation between adult transformation and aldehyde oxidase staining pattern in wing discs of Aptwous genotypes in Lkosqvhila mdanogaskr. Wilhelm Ruux’s Arch 191.2%233.
WYLLIE, A. H., KERR, J. F. R., and CURRIE,A. R. (1980). Cell death: The significance of apoptosis. Int Rev. C&d 68.251-306.
BIOLOGY
104.489-496 (1984)
Localized Cell Death in Drosophila lmaginal Wing Disc Epithelium Caused by the Mutation apterous-blot BONNIEJOYSEDLAK,* *Develqvmmtal
REN$MANZO,-~ANDMARYSTEVENS*
Biology Center, University of California, Irvine, California 92717,and ~CorneU Medical College, New York, New York 100l$ Received February 9, 1984 accepted in revised
form April 30, 1984
Drosophila melanogaster carrying the mutation aptemus-blot have blistered wings. Trypan blue stains a patch of dead cells localized to the wing pouch of imaginal discs and the same area shows acid phosphatase (AcPase) activity suggesting that the cell death is lysosomal. Autophagic vacuoles and other secondary lysosomes show AcPase activity within the disc epithelium and enzyme activity is found in fragments of dead cells which have been extruded basally. The cell death, although extensive and confined to the presumptive wing region, does not result in loss of adult structures.
INTRODUCTION
The phenomenon of cell death in development has been observed in both vertebrate and invertebrate tissues, e.g., the selective destruction of tissue which occurs in chick wing (Saunders, 1969) and fly foot pad (Whitten, 1969) results in the patterning of adult limbs from budlike limb precursors. The sculpturing mechanism occurs at precise times in the developmental program of each animal and in both cases cellular destruction is a normal and necessary consequence of either embryonic or postembryonic maturation. Cell death may also play a role in the formation of mutant patterns in Drosophila In this insect many adult structures are derived from the imaginal discs which remain as discrete sacs of undifferentiated tissue throughout larval life. Fate maps of these larval discs define regions of presumptive adult structures (Bryant, 1978). It has been suggested that in vestigial (Fristrom, 1968, 1969; Bownes and Roberts, 1981; O’Brochta and Bryant, 1988), ultravestigial (O’Brochta and Bryant, 1988), scaUupeducx(James and Bryant, 1981), apterousXa&z, Be&, and Cut (Fristrom, 1969) cell death occurs in regions of the wing discs that correspond to areas where there are abnormalities in adult structures. The mutation apteroue-blot (ap”) causes wings to have a blistered surface but otherwise they are similar to wild-type wings. To test the possibility that localized degeneration of wing discs was associated with the abnormal phenotype we studied cellular degeneration at the levels of histology and ultrastructure. Acid phosphatase (AcPase) has been routinely used to identify and define lysosomes (Holtzman, 1976) and in this study the presence of AcPase confirmed the existence of 2’ lysosomes (i.e., autophagic vacuoles) in ap”. The evidence indicates that the cell death observed in the wing pouch 489
region of imaginal discs does not result in wing blade deficiencies perhaps because instead of entire cells being eliminated only portions of cells are removed. METHODS
Rearing animals. Drosophila melanogaster of both ap terms-blot (mutant) and OreR (wild type; control) stocks were reared on artificial media at 25°C in half pint culture bottles. Larvae were collected 120 hr after egg lay (AEL), i.e., late in the third larval instar, and were lightly anesthetized either by submersion in water or with ether fumes before processing the wing imaginal discs for morphological studies. Individual larvae were initially dissected by grasping the mouth hook parts and thorax with forceps and pulling the animal apart to allow free access of the fixative and/or stain to the internal organs. Trypan blue staining of whole wing imaginal discs. Both mutant and wild-type larvae were dissected in insect Gehring’s Ringer solution and immediately placed in a fresh solution of 0.5% trypan blue dissolved in Ringer’s. After staining for 15 min, larvae were washed with Ringer’s and fixed in 2.5% buffered glutaraldehyde (0.05 M sodium phosphate buffer, pH 7.2, with 5% sucrose added). Following dehydration in 70% and absolute alcohols the wing discs were dissected free of the carcass and mounted in euparol medium for observation as whole mounts. Histology and electron micros-. Apm and wild type larvae were fixed in 2.5% buffered glutaraldehyde (0.05 M sodium phosphate buffer, pH 7.2, with 5% sucrose added), postfixed in 1% buffered Os04, washed and stained in block with 2% aqueous uranyl acetate, dehydrated in alcohols, and embedded in Epon 812 resin. oOli!-X06/64 $3.66 Copyright All righta
0 1994 hy Academic Press. Inc. of reproduction in any form reserved.
490
DEVELOPMENTALBIOLOGY
VOLUME104,1984
SEDLAK, MANZO, AND STEVENS
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wing pouch) which has been identified as the area which will give rise to wing structures (Bryant, 1975). Of the mutant discs 27/28 had the distinctive pattern of staining whereas none of the 18 wild-type discs showed a concentrated uptake of the dye in any area of the disc. It is not sufficient to use trypan blue as the sole indicator of cell death since this marker only identifies lysed cells and does not distinguish between dead epithelial cells and/or the phagocytes which may engulf the dead cells (Bowen, 1981). Thus a second technique (a) Essentially following the method of Locke and was used to stain for lysosomal enzymes and the maSykes (1975) with modifications, i.e., 2.5 mMlead nitrate terial was studied at both the light and electron miwas made up in 0.1 mM sodium acetate buffer pH 5.5 croscopic levels to see if lysosomal cell death affected containing 5% sucrose, and 0.1 mM sodium /3-glycerothe disc epithelium. phosphate served as a substrate. Tissue was incubated Whole mounts of mutant discs stained for acid phosat both 25 and 37°C for either 90 min (EM) or 180 phatase showed a characteristically granular black demin (LM). posit which defines the enzyme, AcPase, confined to the (b) Lead nitrate (1.5 mM) made up in 0.1 M Trisregion which gives rise to the wing surface. Since the maleate buffer pH 5.0 with 1.5 mM sodium B-glyceroarea is identical to that which stains with trypan blue, phosphate as substrate. Tissue was incubated for 30 min the observation (1) confirms the conclusion that cell at both 25 and 3’7°C (Barka and Anderson, 1962). death occurs only in the wing pouch in ap” and (2) Controls consisted of (1) preincubating tissues in 0.01 suggests that cell death is lysosomal. AcPase staining was never seen in wing discs from control (wild type) M sodium fluoride for 30 min or (2) adding the sodium fluoride directly to the reaction media to inhibit AcPase larvae. activity. Whole imaginal discs were placed in 1% ammonium sulfide for 5 min following incubation with the Cytochemical Localization of CeU Death reaction media to allow visualization for light microsThe ultrastructure of epithelial cells of the mutant copy. Other discs were prepared for electron microscopy. wing discs is similar to that of wild-type discs. In discs from either stock the tissue is composed of columnar RESULTS cells with short microvilli at the apex and typical junctions along the lateral plasma membrane (i.e., zonulae Cell Death in Wing Discs; HtitochewGtrg adherentes, septate junctions, and gap junctions). The Animals homozygous for apterow-blot have wings cells are largely undifferentiated; free ribosomes predominate with only a few small cisternae of rough enwhich are similar to wild type but 40-60% of the mutant doplasmic reticulum found along with a moderate numwings are blistered (Fig. 1); the phenotype shows partial ber of mitochondria. Golgi complexes are seen infrepenetrance and expressivity (Stevens and Bryant, 1984). quently; when present, they are of the vesiculate Golgi Whole mounts of wing discs from both wild-type and mutant larvae were stained with either trypan blue or type. Figure 3 is a section through a wild-type wing disc acid phosphatase to show areas of cell death. Figure 2 shows a trypan blue stained wing disc taken from an which emphasizes two characteristics that define the wing pouch region. First, there is a larger concentration apbu larva; the overall morphology and folding pattern of lipid droplets in the wing pouch than is found in other in the mutant resemble that of the wild-type disc. Trypan blue, a vital dye which is selectively taken up regions of the disc epithelium (see also Fristrom, 1969). by cells that have died (Bowen, 1981), was used to iden- Second, the wing pouch basal lamina is composed of tify regions of tissue degeneration in wing discs. In apbu lamellated layers of extracellular matrix plus a thin, discs the exclusion dye was confined to a region (the electron-dense outermost layer (Fig. 3a, inset). In all Histological sections (0.5 pm) were stained with a 1% methylene blue solution and thin sections were stained with lead and uranium before photographing with a Hitachi 12 electron microscope. Acid phosphutase histo/cytochewGtrg. Larvae from both ap” and OreR stocks were fixed with 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for l-l.5 hr at 5°C and tissues were incubated in either of two reaction media:
FIG. 1. A wing from aput shows blistering (arrow) of the wing surface. X500. FIG. 2. Whole mount of ap” wing imaginal disc 120 hr AEL, trypan blue stain indicates a patch of dead cells in the wing pouch area (arrow). X600. FIG. 3. The wing pouch region of a wild-type wing disc is distinguished by a large concentration of lipid droplets and fairly thick basal lamina, composed of a loosely organized lamellar matrix plus an electron dense outer layer. Inset a, wing pouch basal lamina; inset b, proximal fold basal lamina. bc, Blood cell; bl, basal lamina; 1, lipid. X6000; inset a and b, X26,000.
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other parts of the disc a thin dense layer is the sole component of the basal lamina (Fig. 3b, inset). The only structural differences between wild-type and mutant discs are those associated with cell death. The evidence from whole mounts indicated that dead cells resided in the wing pouch region and in Fig. 4, which shows a histological section taken perpendicular to the long axis of a mutant imaginal wing disc, a large area of darkly staining cellular debris is found at the basal side of the wing epithelium and seems to be extruded from the epithelial tissue. The tissue sections provide evidence that the debris, which has accumulated in the region of the wing pouch and extends to the area of the adjacent (proximal) fold, corresponds to the trypan blue stained area seen in whole mounts. The nature of the cellular debris was further analyzed by electron microscopy. Figure 5 which corresponds to region 1 in the histological section (see Fig. 4) shows that the debris is extracellular. It consists of cytoplasmic fragments which are fairly round, variable in size, and contain recognizable remnants of mitochondria and lipid droplets. All fragments lie between the epithelial cell basal plasma membrane and the basal lamina. Some pieces of cytoplasm are so highly condensed that no organelles can be recognized within the fragments, whereas other fragments appear quite electron lucent and are composed mostly of flocculent material plus a few electron dense inclusions. Tissue was incubated with two Gomori type media at both 25 and 37°C to show AcPase activity. Both incubations produced deposits of black precipitate within the epithelial cells, but the incubations run at 37°C resulted in the heaviest deposition of reaction products. In addition to the mutant discs treated to show AcPase activity, controls consisting of (1) untreated wild-type discs, (2) wild-type discs processed for AcPase activity, and (3) both wild-type and mutant discs treated with sodium fluoride to quench AcPase activity were studied. Sodium fluoride was effective in eliminating any AcPase activity from the regions which otherwise had heavy black deposits. The inset in Fig. 4 shows extracellular debris in a disc treated to show AcPase activity and, as in all other treated tissues, the thin sections were not poststained with a heavy metal (i.e., uranium or lead). The tissue was, however, stained in block with uranyl acetate to enhance organelle resolution. The heavy black deposits represent localization of the lysosomal enzyme, i.e., AcPase activity is specifically associated with membrane-bounded bodies within the debris.
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Figure 6 indicates an epithelial region of a mutant disc corresponding to region 2 in Fig. 4. There are several large inclusions located within the columnar epithelial cells of the wing pouch which lie adjacent to the extracellular debris. A higher magnification shows that one of the dense inclusions appears to be membrane bounded and contains mitochondria in addition to free ribosomes defining it as an autophagic vacuole (Fig. 7). Such structures showed AcPase activity and AcPase reaction products were also found associated with a variety of smaller inclusions within the epithelial cells in up”” wing discs. In some cases, it seems that AcPase activity is associated with the ribosomes and/or rough endoplasmic reticulum encircling a lipid inclusion in the wing pouch (Fig. 8). In Fig. 9 a typical vesiculate Golgi complex abuts upon a small dense body containing patches of AcPase reaction product. A few dense bodies are also found in wild type wing discs and AcPase activity is often associated with such dense bodies. Extruded cellular debris is never found in the control discs. DISCUSSION
The apm mutation causes a relatively mild distortion of the adult wing even though a large patch of cell death is localized within the presumptive wing region in imaginal discs, thus indicating that cell death does not always result in major defects in adult structures. Trypan blue staining identifies dead cells in the wing pouch area of up”” and similarly the wing pouch is the area where cell death was described in larvae carrying the mutation vestigial (Bownes and Roberts, 1981;Fristrom, 1968,1969; O’Brochta and Bryant, 1983), Ultravestigial (O’Brochta and Bryant, 1983), and scallopeduc’ (James and Bryant, 1981). In these mutations the wings are much diminished and lack many structures normally found in the adult. ar, a more severe aptemus allele which causes a notch to develop in adult wings, also has cell death occurring in the wing pouch of imaginal discs (Fristrom, 1969). Unlike all of these mutations, the cell death found in apM does not result in a loss of adult structures and the type of cell death is autophagy, not cell elimination. Wyllie et al. (1980) coined the phrase apoptosis to describe a mechanism for cell death, in which single cells condense, then fragment, and are either eliminated by phagocytosis or undergo a secondary lysis. This mechanism has been confused with autophagy. In apoptosis the elimination of dead cells is nonlysosomal, i.e., acid hydrolyses are not produced by the dying cells but
FIG. 4. A methylene blue-stained section through the longitudinal axis of an up” wing disc 120 hr AEL, darkly stained debris is localized at the basal side of the wing pouch. (High magnification micrographs of regions 1 and 2 are shown in Figs. 5 and 6.) 1, Lipid. X1700. FIG. 5. Cellular debris is found between epithelial cell plasma membranes and the basal lamina in a#” wing disc. The area shown corresponds to region 1 in Fig. 4. Inset, AcPase reaction product within the extracellular debris. 1, Lipid. X9oo0, inset, X40,000.
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are introduced from neighboring phagocytic cells. Autophagy, in contrast, represents lysosomal cell death, in which portions of cells die and cell fragments are digested by enzymes produced within the same cells. There is ultrastructural evidence that apoptosis occurs in VQ(O’Brochta and Bryant, 1983) and ap (Fristrom, 1969) but apoptosis is not the mechanism of cell death in up”. The evidence for autophagy is clear since AcPase reaction products have been found within membranebounded autophagic vacuoles residing in otherwise healthy cells, i.e., cells that have normal nuclei. During autophagy the lytic enzyme may be processed and/or packaged as a 1” lysosome at the Golgi complexes and subsequently introduced to the autophagic vacuole (Locke and Sykes, 1975). In up” the small dense bodies which are located directly adjacent to Golgi vesicles show AcPase activity and may be 1” lysosomes, i.e., their proximity to Golgi elements suggest the Golgi as the source of the enzyme. However, these organelles are rare and none of the Golgi membranes shows any AcPase activity. The possibility that ribosomes (Bowen and Ryder, 1974) or rough endoplasmic reticulum (Borges and Thone, 1976) contribute AcPase can not be discounted. AcPase reaction product encircles lipid droplets in the wing pouch of the mutant. AcPase cytochemistry has been classically used to define the lysosome in cell biology and Clark and Russel (1977) used enzyme localization to correlate lysosomal activity in the eye-antenna1 discs of the mutant &!)ts 726 of Lkosophilu with areas of structural deficiencies in the heads of the adult flies. In this study apm AcPase reaction products were confined to the same area in whole wing discs where trypan blue showed cell death. Thus, dying cells within the wing pouch may be destroyed, at least in part, by lysosomes. Dense bodies with AcPase reaction products are found in wild-type as well as mutant wing discs but this observation is expected since lysosomes are nearly ubiquitous organelles. The most striking morphological difference between up” and wild-type wing discs is the cytoplasmic fragments concentrated at the basal side of the wing pouch epithelium. It is likely that the fragments represent cell debris which has been extruded from the epithelial sheet. The amount of basal debris is extensive. Only a few cell fragments are found within any particular epithelial
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cell late in the larval instar (120 AEL) suggesting that the extruded material represents an accumulation from cells which died at earlier stages. In fact wing discs taken from larvae at mid-third instar (96 hr AEL) also have trypan blue stain localized in the wing pouch area indicating that debris has already formed at this time ‘(M. Stevens, unpublished observations). Basal extrusion of cell debris has been reported for other mutants including vg (O’Brochta and Bryant, 19!33), and ecd’ (D. O’Brochta and T. Sliter, personal communication). In apm the fate of the cellular waste is not known. There was no evidence of phagocytosis by adjoining epithelial cells in up”, i.e., whole cells including their nuclei never appeared encircled by neighboring cell membranes. Blood cell phagocytosis may occur. In vg, for example, in which late stages of development (i.e., after pupariation) were studied, blood cells ingest extruded fragments (O’Brochta and Bryant, 1983). Perhaps phagocytosis is not necessary to eliminate the waste since it is clear that in u$” the extruded debris continues to be digested extracellularly. AcPase activity is found associated with some of the autophagic fragments and other fragments within the debris which are filled with flocculent material lack cellular detail as if the cytoplasm was subject to lysis following extrusion. That cell death occurs in ap”“, is extensive, and is confined to the wing pouch area are obvious conclusions from the data but it is not clear how this cell death relates to the rather minor alterations in adult phenotype. A complication in interpreting how larval cell death affects adult pattern is the fact that in up the phenotype is variably expressed, and in addition to the mild blistering of wings described above, an engrailedlike phenotype is found in a small proportion of the flies in the stock described by Whittle and Sprey (1982). It has been proposed that embryonic cells such as imaginal discs have positional information which enables them to participate in making complex adult patterns. Abnormal patterns, e.g., specific deficiencies in adult structures, may be the result of genetically induced cell death which eliminates essential patterning information (Girton and Bryant, 1980). Understanding the relationship between the larval cell death and adult pattern defects in apHt depends upon interpreting the mechanisms underlying the cellular degeneration. Accumulated debris may have little or no effect on pattern
FIG. 6. In addition to the basal debris, there are large dense bodies located within the epithelial cells in the wing pouch The area shown corresponds to region 2 in Fig. 4. db, Dense body; n, nucleus. X7599. FIG. 7. A higher magnification of the membrane-bounded body seen in Fig. 6 shows that this inclusion is an autophagic mitochondria and free ribosomes. m, Mitochondria. X21,999. FIG. 8. AcPase activity is associated with the lipid droplets of the wing pouch; lipid inclusions are often surrounded by reticulum. 1, Lipid; rer, rough endoplasmic reticulum. X60,999 FIG. 9. A dense body located directly adjacent to the vesiculate Golgi complex shows AcPase activity while the Golgi contain any reaction products. G, Golgi complex. X59,ooO.
of ap”’ wing discs. vacuole containing rough endoplasmic membranes
do not
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formation (Rownes and Roberts, 1981) or it may interfere with wing differentiation by preventing the basal surfaces of the disc epithelium from being normally opposed during wing evagination (Fristrom and Fristrom, 1975). It is likely that, since only a portion of each cell is destroyed in up” wing discs in contrast to up where whole cells are removed via apoptosis (Fristrom, 1969), positional information is not entirely removed in up thus there is no loss of pattern elements in flies carrying the less severe allele. The mild distortion of the wing phenotype typically found in apM may merely be a response to a sluggish system which has accumulated metabolic waste products or a system in which some subvital cells (i.e., those undergoing an increase in autophagy) can not contribute fully to the formation of a complete pattern. Alternatively cell death may eliminate presumptive wing structures but regeneration may lead to the replacement of degenerated parts. Thus the adult wing would not appear greatly distorted. This paper was made possible by the support of National Institutes of Health, Grant AG 01979.We thank Dr. Peter Bryant for his helpful comments on the manuscript. REFERENCES BARKA, T., and ANDERSON,P. J. (1962). Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochen~ Cytochem 10.741-753. BORGES,M., and THONE,F. (1976). Further characterization of phosphatase activities, using non-specific substrates. Histochem J. 6, 201-317. BOWEN,I. D. (1981). Techniques for demonstrating cell death. In “Cell Death in Biology and Pathology” (I. D. Bowen and R. A. Lockshin, eds.), pp. 381-446. Chapman & Hall, London. BOWEN,I. D., and RYDER,T. A. (1974). Cell autolysis and deletion in the planarian Polycelis tenuis Iijima. CeU !&sue Res. 154,265-274. BOWNES,M., and ROBERTS,S. (1981). Regulative properties of wing discs from the vestigial mutant of Drosophila mdanogaster. L?@krentiation 18.39-96. BRYANT, P. J. (1978). Pattern formation in imaginal discs. In “The
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Genetics and Biology of Lkosophild (M. Ashburner and T. R. F. Wright, eds.), Vol. 2c, pp. 230336. Academic Press, London. BRYANT,P. J. (1975). Pattern formation in the imaginal wing disc of Lko.wphdu melanogaster: Fate map, regeneration and duplication. J. Exp. Zool 193.49-77. CLARK,W. C., and RUSSELL,M. A. (1977). The correlation of lysosomal activity and adult phenotype in a cell-lethal mutant of Drosophila Llev. Bid 57.160-173. FRISTROM,D. (1968). Cellular degeneration in wing development of the mutant vest&id of Drosophila mdanogaster. J. Cd Bid 39, 433-491.
FRISTROM,D. (1969). Cellular degeneration in the production of some mutant phenotypes in Drosophila m&noga&x Mokc Ga Gewt 103,369-379.
FRISTROM,D., and FRISTROM,J. W. (1975). The mechanism of evagination of imaginal discs of Lkosophilu melunogaater. Dev. Bid 43, l-23.
GIRTON,J. R., and BRYANT,P. J. (1980). The use of cell lethal mutations in the study of Drosophila development. Dev. Bid 77,233-243. HOLTZMAN,E. (1976). “Lysosomes: A Survey.” Springer-Verlag, New York. JAMES, A., and BRYANT, P. J. (1981). Mutations causing pattern deficiencies and duplications in the imaginal wing disk of Drosophila mhncgaster. Dec. Biol 86,39-54. LOCKE,M., and SYKES,A. K. (1975). The role of the Golgi complex in the isolation and digestion of organelles. !&sue CeU 7, 143-158. G’BROCHTA,D. A.. and BRYANT, P. H. (1983). Cell degeneration and elimination in the imaginal wing disc, caused by the mutations vee~andVUmuest~ofL%osophilamela~. WMmRmx18 Arch. 192,285~294. SAUNDERS,J. W. (1969). Death in embryonic systems. Science 154, 604-612. STEVENS,M. E., and BRYANT,P. J. (1984). Apparent genetic complexity generated by developmental thresholds: The aptemus locus in Dro sophila rxelanogoater. Genetics, in preparation. WHMTEN,J. M. (1969).Cell death during early morphogenesis: Parallels between insect limb and vertebrate limb development. Scarce 163, 1456-1457. WHITTLE, J. R. S. (1979). Replacement of posterior by anterior structures in the L?rosophila wing caused by the mutation apteroua-blot J. Embrgol Exp. Morph& 53,291~303. WHITE, R., and SPREY,T. (1982). Correlation between adult transformation and aldehyde oxidase staining pattern in wing discs of Aptwous genotypes in Lkosqvhila mdanogaskr. Wilhelm Ruux’s Arch 191.2%233.
WYLLIE, A. H., KERR, J. F. R., and CURRIE,A. R. (1980). Cell death: The significance of apoptosis. Int Rev. C&d 68.251-306.