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Developmental studies in Drosophila
Copyright © 1972 by Academic Press, Inc. All rights of reproduction in any form reserved
292
J. ULTRASTRUCTURERESEARCH40, 292--312 (1972)
Developmental Studies in
Drosophila
VI. Ultrastructural Analysis of the Salivary Glands of Drosophila pseudoobscuraDuring the Late Larval Period M. J. E. HARROD and C. D. KASTRITSIS1
Department of Cell Biology, The University of Texas, Southwestern Medical School, Dallas, Texas 75235 Received October 8, 1971, and in revised form February 18, 1972 Salivary gland cells of Drosophilapseudoobscura were examined electron microscopically and cytochemically during the late larval stage of development. The data indicate that several synthetic processes are occurring in the glands during this period. The first, a presumably digestive secretion, is produced throughout the gland in the earliest stages studied, and only in the most proximal cells with increasing time. Mucoprotein granules, which are secreted and utilized as a "glue" at the time of puparium formation, are produced in the most distal cells of the gland beginning at about 100-106 hours after hatching, and are eventually found throughout the medial region as well, but never in the proximal portion of the gland. Such granules are produced in the Golgi regions and have nonmucoprotein "caps" at their periphery. Several types of material are also found within the cisternae of the rough endoplasmic reticulum of the cells; the function and significance of these crystals, granules, and aggregations of granules is unknown. Lysosomal activity is present in the cells from the earliest stages, and increases with time. In a previous report (15) we have described the ultrastructural changes which occur in the salivary glands of Drosophila pseudoobscura during the period of transition from the larval to the pupal stages. Briefly, the process involves the following events: Large numbers of mucoprotein granules are found within the gland cells at the end of the larval period, and are subsequently secreted just prior to puparium formation; these form the "glue" that attaches the pupal case to its substrate. After puparium formation the residual granules are attacked by lysosomes and degraded by a process of crinophagy (6). A further secretion of vesicles packaged by the Golgi regions occurs during the prepupal period, concomitant with an increase in the number of secondary lysosomes. Histolysis of the gland cells occurs, both by the process of z Present address: Department of General Biology, Aristotelian University of Thessaloniki, Thessaloniki, Greece.
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autolysis, presumably due to lysosomal enzymes, and by phagocytosis, by cells from the hemolymph. The present report deals with the events from the time when mucoprotein synthesis begins, through the production of the granules, and their accumulation and stoiage at the end of larval life. The data obtained in concurrent studies also undertaken in this laboratory on the cytogenetic aspects of development, as determined by the chromosomal puffing changes (37) and the biochemical changes which occur, as determined by microelectrophoretic studies on the proteins of the glands (26) will, it is hoped, correlate with the ultrastructural findings, thereby providing a useful contribution to the study of development.
MATERIALS AND METHODS The experimental animals used in this study were third-instar larvae of a standard strain of Drosophila pseudoobscura, from Mather, California. Salivary glands from larvae aged 96, 100, 106, 114, 122, 130, and 144 hours after hatching were utilized in this series of experiments. The fixation, embedding, and observation of the sections were carried out as previously described (15). Sections were cut from the proximal, medial, and distal regions of the gland at each stage to provide a basis of comparison of the activities in these different areas.
RESULTS
96-Hour stage At 96 hours after hatching the larvae are near the midpoint of the third-instar period. The salivary glands are small and relatively uniform in diameter throughout their length. Sections through proximal, medial, and distal regions of the gland reveal that, although the distal cells are somewhat larger, overall there is less variation in the ultrastructural appearance of the cells of the three regions than at any other stage studied. The individual cells of a given region of the gland display a noticeable degree of polarity, as can be seen in Fig. 1. The apical border of the cell is composed of microvilli that extend into the lumen of the gland. The large nucleus with its prominent nucleolus and polytene chromosomes occupies the central region of the cell. The basal border of the cell, covered by a fine basement membrane, forms infoldings which extend for some distance into the cell. In those portions of the gland where the basal surface is openly exposed to the hemolymph, the infoldings tend to appear as open spaces, sometimes containing small vesicles of unknown origin. Occasionally they are seen to end almost in contact with cisternae of the rough endoplasmic reticulum (Fig. 3). In areas of the gland that are in contact with the fat body, only the 2 0 -- 721834 J . Ultrastructure Research
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basement membrane separates the two organs, and the infoldings are tightly apposed along their length, appearing in the sections as dense lines. Cells of other' types are occasionally found in contact with the basal surface. Muscle cells are oriented lengthwise along the proximal third of the gland, although they apparently do not form a continuous layer; their appearance in the sections is rather rare. Blood cells, presumably phagocytes, are also found in some sections. Adjacent cell boundaries display complex interdigitations. Near the apical cell surface tight junctions are often visible, along with various types of desmosomes; toward the basal side of the gland the lateral boundaries usually terminate in one of the infoldings of the basal membrane. Along the length of the cell boundary are found septate desmosomes. A striking contrast in the density of two adjacent cells is frequently seen, thus allowing the easy tracing of the cell boundary between them, even under low magnifications. The cytoplasm of the cells is rich in free ribosomes as well as in rough endoplasmic reticulum, which is often arranged in parallel arrays of cisternae. Mitochondria with long filamentous, oval, and rounded profiles are abundant and display no particular orientation throughout the cell. The cells are rich in Golgi Iegions, which present a rounded, vesicular appearance, rather than the flattened saccules of classic Golgi morphology. Small, smooth tubular elements are also seen between the rough endoplasmic reticulum and the Golgi vesicles. Closely associated with the Golgi regions are secretory vesicles containing a flocculent material identical in appearance to material found in the terminal cisternae of some of the Golgi complexes (Fig. 2). These secretory vesicles are often seen near the apical border of the cell. In several instances they are fused with the cell membrane so that their contents are free to enter the lumen, which, at this stage, contains material identical in appearance to that of the vesicles. The cytoplasm contains numerous microtubules, whose position can generally be determined by zones of almost " e m p t y " cytoplasm immediately surrounding them. Lipid inclusions are seen frequently, largely confined to the basal region of the cells. They are usually oval in section and slightly more electron dense than the Golgi cisternae.
FIG. 1. 96 Hours. A portion of one cell, containing the large nucleus with its prominent nucleolus (Nu) and polytene chromosomes (Chr), which have not ieached their ultimate degree of polyteny in this cell. The apical membrane of the cell forms microvilli (My) bordering the lumen (Lu) of the gland. The cytoplasm contains numerous mitochondria (Mi), many arrays of rough endoplasmic reticulum (ER) as well as free ribosomes, and a number of Golgi zones (Go). The basal cell membrane (Ba) forms infoldings and is covered by a basement membrane. × 6 400.
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Near the basal surface of the gland, small multivesicular bodies are frequently encountered. In addition, occasional autophagic vacuoles are found, surrounded by several layers of membrane, and containing cytoplasmic elements such as ribosomes, vesicles of rough endoplasmic reticulum, and mitochondria (Fig. 11). Two types of intracisternal inclusions are found at this stage. More frequently seen are electron-dense granules, 0.2 # to 0.3/z in diameter, almost round in section, and situated within rounded cisternae of rough endoplasmic reticulum (Fig. 4). Their occurrence is confined to the basal portion of the cells. Occasionally, similar granules are found within irregularly shaped cisternae which also contain other amorphous, slightly electron-dense material. Less frequently seen, but usually abundant in the cells where they occur, are crystalline inclusions within cisternae of the rough endoplasmic reticulum (14). At low magnifications they are difficult to distinguish from sections of mitochondria, but at high magnification the crystalline structure becomes clearly evident (Fig. 4). In longitudinal section they have a length of as much as 8 #; the individual subunits have a diameter of about 200 A. Figures 5 and 6 indicate that a connection may exist between a cisterna containing a crystalline inclusion and one containing a dense intracisternal granule. The crystalline inclusions, like the intracisternal granules, are confined to the basal portion of the cells. As previously stated, the proximal, medial, and distal portions of the gland at 96 hours are quite similar in ultrastructural appearance. Cell size differences exist, as do differences in the degree of polytenization of the chromosomes, which is detectable as a lack of prominent banding when seen in section (Fig. 1). Within a given region of the gland, cell to cell differences are frequently noticeable. One distal cell may appear to be active in secretion; the adjacent cell may show no sign of such activity.
lO0-Hour stage At 100 hours the salivary gland cells are very similar in general appearance to those of the 96-hour stage. The same cytoplasmic organelles are present, and secretion of the flocculent material packaged by the Golgi regions is continuing in most of the cells examined. A striking difference is seen in a minority of the glands. In cells from the distal
]FIG. 2. 96 Hours. Golgi regions (Go) with their associated smooth tubular elements are seen near the apical border of a cell (upper right). Secretory vesicles (SV) containing a flocculent material are found near the Golgi regions and also near the microvilli (My) at the lumen, x 30 000. FIG. 3. 96 Hours. Basal cell surface exposed to hemolymph. The basement membrane (BM) covers numerous infoldings (In) of the basal cell membrane, one of which is seen to terminate (arrow) almost in contact with a cisterna of the rough endoplasmic reticulum (ER). × 30 000.
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region and, in a few instances, the medial region, the production and accumulation of membrane-bound, relatively electron dense granules occurs. Such granules are never seen in the proximal region of the gland where the previously described secretion of the flocculent material continues to occur. These granules are derived from Golgi regions. In the earliest stages of synthesis observed, small irregularly shaped accumulations of slightly less electron dense material are seen within the terminal cisternae of the Golgi (Fig. 7). Fusion of such smaller elements apparently occurs to form larger granules which range, at this stage, from 0.25 # to 1.1 # in diameter. Some relatively large granules are occasionally seen in association with the Golgi and were apparently synthesized as such (Fig. 8). An interesting feature of these granules is the presence of somewhat more electron dense "caps" at their periphery (Figs. 9 and 10). The "caps" are also produced in the Golgi, and fusion of " c a p " and granule may take place within that area, or may occur at some distance from the site of synthesis (Fig. 14). Some "caps" are found to contain regions of even greater density in the form of small, round spots, sometimes two or more per "cap." The material within the terminal Golgi cisternae and the body of the granules gives a positive reaction for the presence of periodic acid-reactive carbohydrate when the silver methenamine technique of R a m b o u r g (31) is utilized (15). The "caps" show no reaction, indicating that they are of a different chemical composition from the remainder of the granule. These granules represent the substance secreted by the salivary glands just prior to puparium formation, a mucoprotein "glue," which attaches the pupal case to its substrate. They will be referred to as mucoprotein granules throughout the remainder of the text. Dense intracisternal granules and crystalline inclusions (both negative to the R a m bourg technique), are seen in 100-hour glands, in cells of the proximal region as well as those of the medial and distal regions. Degenerative areas, presumably of lysosomal origin, are found within the cytoplasm in the basal portion of some of the cells. These areas, always membranebound, contain amorphous material of low electron density and are usually seen to be in contact with cytoplasmic constituents such as cisternae of endoplasmic retic-
Fio. 4.96 Hours. Basal portion of a cell showing crystalline inclusions (Cry) and dense intracisternal granules (arrows). Mitochondria (Mi) can be readily distinguished from crystalline inclusions at this magnification, x 6 400. FIG. 5. 100 Hours. Two crystalline inclusions and a dense intracisternal granule in close proximity. Arrow indicates extension of granule's cisterna toward that of crystalline inclusions, x 81 000. Fie. 6. 100 Hours. Serial section showing apparent connection between the cisternae and the presence of material similar in density to the crystalline inclusions within the connecting cisternae. ×81 000.
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ulum and mitochondria. They are frequently seen with mucoprotein granules at their periphery, or in some cases, within the membrane-bound area (Fig. 14). Other smaller, membrane-bound structures containing recognizable cytoplasmic elements aie also found. These autophagic vacuoles are seen throughout the cell and contain membranous residues (Fig. 12) as well as other identifiable structures such as mitochondria.
106 to 122-Hour stages Throughout this portion of the third instar a gradient of mucoprotein production by the cells is evident. By 106 hours the distal cells of all glands contain some mucoprotein granules. Some medial cells are also seen to be active in mucoprotein synthesis, with great variations in distribution and size of granules from cell to cell. No mucoprotein is found in the proximal region, but the secretmy activity previously described is still occurring in this region. At 114 hours the granules in the distal regions are larger, up to 2/~ in diameter, and more numerous (Fig. 14), as are those of the medial region, although the latter are still of an average size smaller than those of the distal region. Similar variations exist at 122 hours. At this stage secretory activity in the proximal region has diminished, but no mucoprotein granules are found in that portion of the gland. The dense intracisternal granules are still common, and crystalline inclusions are found sporadically. In addition to the autophagic vacuoles and the degenerative areas of the earlier stages, an additional type of lysosome is found in some cells. Morphologically identical to the secondary lysosomes of later instar stages, these bodies contain myelin-figure-like inclusions of dense, membranous lamellae within an amorphous, moderately electron dense, membrane-bound matrix. Cytoplasmic organelles and mucoprotein granules are often found in contact with such bodies (Fig. 13).
130 and 144 Hour stages Anterior spiracle eversion, the first indication of the onset of puparium formation, occurs in D. pseudoobscura at 144_+ 6 hours. Thus the 130- and 144-hour stages studied represent the latter portion of the mucoprotein synthesis period, and the stages at which granule size and numbers reach a maximum. The same gradient of distribuFIG. 7. 100 Hours. Early stage of mucoprotein production showing small mucoprotein granules (MG) within the cisternae of the Golgi (Go). x 28 000. Fro. 8. 100 Hours. Formation of mucoprotein granule (MG) within a Golgi zone (Go) having a double layer of flattened saccules. × 28 000. Fro. 9. Late third instar. Interaction between granules. Fine, membranous elements are seen between the granules in a region of almost "empty" cytoplasm (arrows). x 27 000. FIG. 10. Late third instar. Apparent fusion of two mucoprotein granules. Arrow indicates membrane still separating the two granules, x 25 000.
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tion is still evident. The proximal region of the gland is never seen to be involved in mucoprotein synthesis. The medial portion of the gland is still an area of transition. Although the production of mucoprotein granules reaches its peak during this period there are still quite obvious variations in the size and density of granules among the cells of this region. In the distal region of the gland the cells are packed with mucoprotein granules which have at this point in development reached their maximum diameter of as much as 3.5 #, although many smaller granules are still present. Interactions between individual granules are seen. Obvious fusions occur (Fig. 10), and some granules are seen to be connected by areas of peculiar appearance, almost devoid of cytoplasmic elements, which contain fine, membranous or vesicular extensions between the granules (Fig. 9). Whole groups of granules are seen to be connected in this manner in some sections. The other cytoplasmic components are relatively unchanged. The endoplasmic reticulum forms well ordered arrays among the granules. Although the cisternae may be somewhat dilated, no contents are visible within most of them. Occasional cisternae which appear to be partly rough and partly smooth are found in the vicinity of the Golgi regions, between the arrays of rough cisternae and the smooth tubular elements which are adjacent to the Golgi vesicles. No material is visible in the Golgi regions, with the exception of the most terminal cisternae, some of which contain the mucoprotein product. Secondary lysosomes are present in most cells and are slightly larger than in previous stages. In addition to the previously described dense intracisternal granules and crystals an additional type of intracisternal inclusion not seen in previous stages, appears with the 130-hour stage. Consisting of aggregations of small, round bodies of varying electron-densities, ranging in size from 0.5 # to 0.15 #, they are found within greatly dilated cisternae of the rough endoplasmic reticulum (16). Small granules of similar size and shape are frequently found in separate cisternae near the larger aggrega-
FIG. 11. 100 Hours. A membrane-bound autophagic vacuole (AV) contains particles identical in size to the ribosomes of the surrounding cytoplasm. A secretory vesicle (SV) is seen near the lumen (Lu). x 35 000. FIG. 12. 100 Hours. An autophagic vacuole containing membranous structures (arrow) as well as membrane bound inclusions containing material resembling ribosomes. × 35 000. FIG. 13. 114 Hours. A secondary lysosome (Ly) consisting of a membrane bound accumulation of moderately electron-dense material containing myelin-figure-like inclusions. Mitochondria (Mi) and mucoprotein granules (MG) are seen to be in contact with the body at its periphery. A Golgi region (Go) is seen still active in the production of mucoprotein, x 15 500.
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tions. Den s e intracisternal granules a p p e a r near the aggregations in some sections, b u t no c o n n e c t i o n between the cisternae of the t w o types has been observed. The nuclear inclusions previously described
(13, 15) as
occurring in late third instar
an d thereafter,have also been f o u n d in all stages described here.
DISCUSSION
The data presented in this report indicate that several synthetic processes occur in the salivary gland cells of D. pseudoobscura during the late larval period. The finding that synthesis and secretion of a material derived from the Golgi regions of the cell, presumably protein in nature, occurs at least during part of the feeding period in larval D. pseudoobscura, and that such synthesis ceases at the stage when the larvae leave the food in preparation for pupation, strongly suggests that the secretory product may be active in digestion. Such an accessory digestive function has been described for the salivary glands of Drosophila (4), although definitive evidence for this interpretation is lacking. Hsu (16) described the production of material by the Golgi regions and its subsequent secretion in salivary gland cells of "young" D. melanogaster larvae (exact stage unspecified). He interpreted this secretion to be digestive in nature, since it occurred only during the feeding period of larval life. Patterson et al (27) described the presence of peptidase in extracts of the salivary glands of D. melanogaster, a finding which is suggestive of a possible digestive function. In more recent studies, Berendes (3) described the synthesis and secretion of substances presumed to be digestive enzymes, throughout the gland of D. hydei in early larval stages and confined to the most proximal cells at late third instar. This secretory product is composed of small granules, 0.35 # to 0.85 # in diameter, membranebound, composed of a number of particles, and often appearing partially dissolved. The occurrence of the secretion in D. pseudoobscura follows the pattern described by Berendes (3) for D. hydei; such activity is seen throughout the gland prior to the production of mucoprotein by the salivary gland, and is confined to the proximal portion of the gland during the late third instar. The appearance of the secretory granules in the different species of Drosophila which have been described is quite different. Instead of the dense, partially dissolved granules described by Berendes, the secretory vesicles seen in D. pseudoobscura are electron lucent and contain small FIG. 14. 114 Hours. A cell from the distal region of the gland containing many mucoprotein granules. Unattached "caps" can be seen in some areas, as well as granules which appear to be in the process of fusion of "cap" and granule. A degenerative area is seen at lower right with several granules within its boundaries. The nucleus (N) contains a small nuclear inclusion adjacent to the membrane (arrow). The banding pattern of the polytene chromosomes (Chr) is more readily apparent at this stage than in Fig. 1. x 6 400.
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quantities of flocculent material. Such a morphological variation in what are presumably functionally similar products is of interest. Similar findings have been reported in some other genera. Phillips and Swift (30) described the production and secretion of a specific granule during the feeding period of larval development in Sciara coprophila which is not found after the larvae cease feeding. They interpreted this as evidence of a digestive function. Oschman and Betridge (24) demonstrated that the secretory droplets produced by the salivary glands of Calliphora erythrocephala, which are presumed to contain amylase, have an electron-lucent appearance and contain flocculent material. These granules are more similar in appearance to those of D. pseudoobscura than are the granules described in the other species of Drosophila. Mucoprotein production by the salivary gland cells of D. pseudoobscura is initiated in the most distal cells of the gland at 100-106 hours after hatching. The sequence of events apparently involves: the synthesis of protein within the rough endoplasmic reticulum, which is abundant in the cells engaged in mucoprotein production; the packaging of this protein and the addition of a polysaccharide component within the confines of the Golgi regions to produce the mucoprotein granules (7); the formation of nonpolysaccharide "caps" within the same or different Golgi regions; the fusion of granules and "caps" to produce the typical mucoprotein granule observed in the gland; and the enlargement of the granules through fusions among themselves. The region of the gland in which mucoprotein can be found extends proximally with time, as can be confirmed with PAS staining of the whole glands. Just prior to secretion there is a gradient of distribution, with the distal cells containing more and larger granules; however, all but the most proximal cells appear to contain some. The synthesis and secretion of mucoprotein by larval dipteran salivary glands was grossly misinterpreted by early investigators, who observed the process but were unaware of its significance. The mucoprotein granules were described as secretion vesicles associated with increased salivary function during the last hours of feeding (33), and as food storage granules derived from transformed mitochondria (16). The first satisfactory explanation of the purpose of the secretion was published by Fraenkel (8) and Fraenkel and Brookes (9). They described the secretory process in D. melanogaster and in Phormia regina, and concluded that it was this material, secreted just prior to puparium formation, whicb formed the "glue" that attaches the pupal case to its substrate. This clarification of the function of the secretion was followed by a number of studies which touched upon the method of its production within the cells, utilizing the newly available technique of electron microscopy to provide information at the ultrastructural level. Gay (10) observed that secretion granules were present in salivary glands of mid-
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third instar larvae of D. melanogaster, that they increased in number and size during late larval life, and that they disappeared from the cytoplasm when the PAS-positive secretion was discharged into the lumen. She theorized that there was a correlation between the appearance of nuclear blebs during this stage and the production of mucoprotein secretory granules; such blebs were suggested as a possible transport mechanism for genetic information by transformation of the portions of nuclear membrane into endoplasmic reticulum, which she considered to be the site of formation of the mucoprotein. Similar blebs of the nuclear membrane are found in D. pseudoobscura in association with nuclear inclusions (13, 15), but their occurrence is not confined to the mucoprotein production stage; they are found in similar numbers after the secretion of mucoprotein, during the onset of histolysis of the gland. This evidence suggests that it is unlikely that the blebs are involved in mucoprotein synthesis. Although the endoplasmic reticulum is undoubtedly involved in mucoprotein synthesis, the Golgi regions are seen to be the site of granule formation and the site of the polysaccharide component's synthesis, as indicated by the results obtained by the silver methenamine technique. The cytoplasm of cells engaged in mucoprotein production--indeed, the cytoplasm of all cells studied--is rich in organelles, contains a great quantity of rough endoplasmic reticulum and few smooth-membraned elements, and has large numbers of free ribosomes. In contrast to the D. pseudoobscura data, Berendes (3) characterized the cytoplasmic organization in the mucoprotein producing cells of D. hydei as poor in cytoplasmic organelles, with the endoplasmic reticulum consisting of smooth membranes, and noted that free ribosomes were seldom found. The ultrastructural composition of the mucoprotein granules of D. pseudoobscura differs from the description of Berendes and deBruyn (2) and Berendes (3) for D. hydei. In contrast to the fine granular appearance of the mucoprotein in D. hydei, the mucoprotein of D. pseudoobscura has a very definite crystalline substructure. This finding agrees with that of Rizki (32) for D. melanogaster. The swellings and bulbs at the margin of the granules in D. hydei could conceivably represent a phenomenon similar to the "caps" of D. pseudoobscura, but the description of their substructure as being identical to that of the body of the granule, and the observation that they appear to be due to the fusion of bodies of differing sizes makes this appear unlikely. Fusion of "caps" and granules does occur in D. pseudoobscura, but the "caps" are composed of a material of differing ultrastructural appearance, are of a greater density when stained with nonspecific electron stains, and are different in chemical composition from the body of the granule, as indicated by the results obtained with the silver methenamine technique.
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Berendes and deBruyn (2) described two types of granules in the mucoprotein producing cells, but unfortunately did not include a figure of the second type; no cytochemical characterization was made. It would be of interest to know whether both types contain the polysaccharide component of the mucoprotein, or if a difference exists in the chemical composition of the two types of granules. Since the body and the "cap" portion of the mucoprotein granules of D. pseudoobscura are produced separately by the cell and undergo fusion to produce the composite secretory granule, it is at least conceivable that the two entities are produced as separate bodies in D. hydei and do not fuse, but are secreted concurrently. In addition, Vidal et al. (38) have described a heterogeneous ultrastructure for the mucoprotein granules of D. melanogaster when they are stained with ammoniacal silver carbonate, which is believed to stain basic proteins. Regions of differing affinity for the stain are irregularly distributed over the body. It would be interesting to know if a similar pattern of distribution exists for the polysaccharide component. Berendes (3) also commented on the pattern of distribution of mucoprotein in D. hydei; he noted that in the region between the most proximal cells, which were still producing the presumably digestive secretion, and the distal cells, which contained the large "glue" granules, were cells which contained either small granules or none, which he termed a transitional zone. He correlated the production of "glue" with the degree of polyteny of the cell's nucleus, and characterized the transitional zone as one in which increases in polytenization were occurring. These findings are in agreement with the D. pseudoobscura data. Berendes' transitional zone is the same morphologically as the medial region of the gland, described previously as a zone of transition with respect to mucoprotein production. Although no attempt was made to measure the degree of polytenization, itis evident that in cells such as those represented by Fig. 1, the chromosomes are not as large and well banded as those seen in later stages. No mucoprotein is seen in these less polytenized cells, and cells which are active in granule formation are seen to have chromosomes which appear more polytene (Fig. 14). Similar ultrastructural investigations of salivary gland mucoprotein secretion have been carried out in some other dipteran genera. Such analyses by Phillips and Swift (30) of Sciara coprophila, Macgregor and Mackie (22) of Simulium niditifrons, and Kloetzel and Laufer (20) of Chironomus thumrni revealed that the process of synthesis of the mucoprotein secretory products shares certain similar features, in spite of the different functions served by the product in different genera. The process of mucoprotein synthesis, like all synthetic processes within the cells, is presumably under genetic control. Concomitant work on D. pseudoobscura has revealed a specific puff, which appears at approximately 106 hours and disappears at spiracle eversion (37), and the appearance of a specific protein component of the
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salivary glands between 96 and 120 hours, which also disappears at spiracle eversion
(26). Although the evidence is incomplete, these findings may coirelate with the observation that mucoprotein synthesis is observed ultrastructurally to begin around 100 to 106 hours after hatching, and that the mucoprotein disappears from the gland, either by secretion or by the crinophagic process, during the spiracle eversion period. Although the protein described by Pasteur (26) is not a mucoprotein, it may well represent a nonmucoprotein component of the secretion, which is known to contain smaller polypeptide molecules (29). Alternatively, it is possible that it represents the "cap" portion of the mucoprotein granules, which is presumably protein in nature. Correlation of puffing patterns with specific protein production has been made in only one instance in the literature thus far. Beermann (1) found that a particular secretory granule in the cells of the special lobe of Chironomus paIlidivittatus appeared only when a specific Balbiani ring was present on the chromosomes. Absence of this Balbiani ring, as was noted in C. tentans, correlated with absence of the granules. Biochemical analyses by Grossbach (11, 12) indicated that one protein component which was present in C. pallidivittatus was not present in C. tentans. He also found that the Balbiani ring described by Beermann was present in all glands which produced the protein. Other work suggestive of such correlations, but lacking the complete evidence required, has been published. Berendes (3) found a specific puff in D. hydei which was active only in the cells of the part of the gland which contained mucoprotein. It appeared before granule production and receded shortly before secretion. This puff was not found in the proximal part of the gland. Such findings are of importance to the study of development, since they provide an opportunity to observe '"cause and effect" relationships which few other systems can offer. A particularly interesting finding is the occurrence of various types of material within the cisternae of the rough endoplasmic reticulum at different stages of development. The crystalline inclusions (Fig. 4), found rather commonly in the early stages studied and sporadically thereafter, appear to be involved in some manner with the dense intracisternal granules which are also present. The observation of rough cisternae connecting the cisternae containing the two types of inclusions suggests that one form may be producing the other (Figs. 5 and 6); probably the dense intracisternal granules are derived from the crystalline material, since their occurrence is more common, and they are found in cells without crystalline inclusions, while the opposite is never true. The aggregations of small dense granules were found infrequently; it is difficult to speculate upon their possible function, or significance. 2 I -- 721834 J . Ultrastructure Research
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The presence of granules within the cisternae of the rough endoplasmic reticulum has been reported in other secretory tissues (17, 25, 36). It has been suggested that such granules are further processed or packaged in the Golgi region before secretion. No bodies resembling the cisternal contents have been found within the Golgi regions of the salivary gland cells, but it is possible that an alteration in morphology might occur if such a process were taking place. It has also been shown that, in some cells, secretion of cisternal contents may occur by direct fusion of the membrane of the endoplasmic reticulum with the plasma membrane of the cell (34). Although intracisternal granules have been found near the apical cell surface, they have not been observed in the process of being secreted, nor have similar structures been found within the lumen. Other aspects of the ultrastructural findings may be significant in providing information about the synthetic and metabolic processes of the salivary gland cells of
D. pseudoobscura. The deep infoldings of the basal cell membrane appear to be involved in transport, and may play a role in the uptake of water soluble substances from the hemolymph, since similar infoldings are commonly found in epithelia engaged in water transport (29). The process does not involve pinocytosis of the classic "coated vesicle" variety (35). The proximity of the extracellular space, bounded by the infoldings, to the intracellular space, within the cisternae of the rough endoplasmic reticulum (Fig. 3), suggests a possible route of entry into the cell for metabolites. The concentration of multivesicular bodies near the basal cell surface in D. pseudoobscura is similar to that described by Locke and Collins (21) in tissues of Calpodes ethlius, in which protein uptake was occurring. Sequestration of blood proteins and their hydrolysis by the enzymes of the multivesicular bodies were suggested as a possible source of amino acids in metabolically active tissues. The multivesicular bodies in D. pseudoobscura contain acid phosphatase, as indicated by the results obtained with the Gomori technique (15). Such multivesicular bodies are formed from small Golgi vesicles and appear to constitute primary lysosomes (6) in the salivary gland at this stage; they are thus capable of participating in such proteolytic activities. It is thus seen that the ultrastructural findings in D. pseudoobscura suggest that uptake of proteins from the hemolymph probably occurs; a similar uptake of protein from the fat body may occur, particularly in the later third instar stages when vesicles have been seen just within the basement membrane of the salivary gland which resemble vesicles within the adjacent fat body. These conclusions ate supported by Pasteur and Kastritsis' (26) data which indicate that throughout most of development all the nonspecific proteins found in the salivary gland are also found in the hemolymph and fat body, or only in the fat body. Such proteins may be produced
DROSOPHILA SALIVARY GLANDS
311
in the fat body and either stored there or released into the hemolymph, from which they are taken up by the salivary glands; uptake of hemolymph proteins by the fat body may occur with subsequent storage in the fat body and eventual transfer directly to the salivary gland. Both processes would account for the ultrastructural observations. The occurrence of varying types of lysosomes within the salivary gland cells at different stages of development is of interest. The purpose of autophagic vacuoles such as those of the earliest stages described in tissues which are seemingly "healthy" is not apparent. Napolitano (23) noted the presence of similar cytolysomes in metabolically active cells, and suggested that they might constitute a prelude to cytolysis, or that they might result from an acute reorientation of normal metabolic processes within the cell. The somewhat larger lysosomal bodies seen in the earlier stages, which contain membranous residues (Fig. 13), apparently the "indigestible" remnants of organelles which have been degraded, are considered to represent slightly more advanced stages of the autolytic process. The lysosomal areas encountered in cells engaged in the early stages of mucoprotein synthesis appear to degrade some of the granules soon after they are produced. The presence of periodic acid-reactive carbohydrate throughout such an area has been observed with the silver methenamine technique. Such degradation may be accidental; the properties of their respective membranes may be such that contact between the two bodies is sufficient to cause fusion and the ultimate attack of the granule's contents. Other cytoplasmic elements, particularly vesicles of endoplasmic reticulum and mitochondria are seen in contact with the lysosomal membrane. Their membranes probably serve as the source of the myelin-figure-like inclusions seen in secondary lysosomes of the later stages (Fig. 13). The authors wish to acknowledge the technical help of Mrs Helen M. McNeil and the secretarial help of Mrs Madelon Smith. This work was supported in part by Grants GM-16736-03 and FR-05426-09 from the U.S. Department of Health, Education, and Welfare, and a grant from the Ruth Jackson foundation of Dallas, Texas. M. J. E. Harrod was a holder of a University of Texas predoctoral fellowship during the course of this investigation. The work presented is a portion of a dissertation submitted in partial fulfillment of the requirements for the degree of Docter of Philosophy. REFERENCES 1. BEERMANN,W. Chromosoma 12, 1 (1961). 2. BERENDES H. D. and DEBRUYN, W. C., Z. Zellforsch. Mikrosk. Anat. 59, 142 (1963). 3. - Chromosoma 17, 35 (1965). 4. BODENSTEIN,D., in DEMEREC, M. (Ed.), Biology of Drosophila. Hafner, New York, 1965.
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5. CARO,L. G. and PALADE,G. E., J. Cell Biol. 20, 473 (1964). 6. DE DUVE, C., it/DINGLE, J. T. and FELL, H. B., (Eds.), Lysosomes in Biology and Pathology. Wiley, New York, 1969. 7. FAVARD, P., in LIMA-DE-FARIA,A., (Ed.), Handbook of Molecular Cytology. American Elsevier, New York, 1969. 8. FRAENKEL,G., Biol. Bull. 103, 285 (1952). 9. FRAENKEL,G. and BROOKES,V. J., Biol. Bull. 105, 442 (1953). 10. GAY, H., Cold Spring Harbor Symp. Quant. Biol. 21, 257 (1956). 1 ]. GROSSBACH,U., ~Intl. Zool. Fenn. 5, 37 (1968). 12. - Chromosoma 28, 136 (1969). 13. HARROD, M. J. E. and KASTR1TSlS,C. D., Drosophila Inform. Serv. 46, 142 (1971). 14. - - - - J. Invert. Pathol. 18, 297 (1971). 15. - J. Ultrastruct. Res. 38, 482 (1972). 16. Hsu, W. S., Quart. J. Microsc. Sci. 89, 410 (1948). 17. ICH1KAWA,A., J. Cell Biol. 24, 369 (1965). 18. JACOB, J. and JURAND, A., J. Insect Physiol. 9, 849 (1963). 19. - Ibid. 11, 1337 (1965). 20. KLOETZEL,J. A. and LAUFER, H., J. UItrastruct. Res. 29, 15 (1969). 21. LOCKE, M. and COLLINS,J. V., J. Cell Biol. 26, 857 (1965). 22. MACGREGOR,H. C. and MACKIE, J. B., J. Cell Sci. 2, 137 (1967). 23. NAPOLITANO,L., J. Cell Biol. 18, 478 (1963). 24. OSCHMAN,J. L. and BERRIDGE,M. J., Tissue Cell2, 281 (1970). 25. PALADE, G. E., in BOYD, J. D., JOHNSON,F. R. and LEVER,J. D., (Eds.), Electron Microscopy in Anatomy. Williams and Wilkins, Baltimore, Maryland, 1961. 26. PASTEUR,N. and KASTR~TSlS,C. D., Develop. Biol. 26, 525 (1971). 27. PATTERSON,E. K., DACKERMAN,M. E. and SCHULTZ, J., J. Gen. Physiol. 32 607 (1949). 28. PEASE, D. C., J. Biophys. Biochem. Cytol., Suppl. 2, 203 (1956). 29. PERKOWSKA,E., Exp. Cell Res. 32, 259 (1963). 30. PmLLIPS, D. M. and SWIFT, H., J. Cell Biol. 27, 395 (1965). 31. RAMBOUR~,A., J. Histochem. Cytochem. 15, 409 (19567). 32. RIZKI, T. M., J. Cell Biol. 32, 531 (1967). 33. Ross, E. B., J. Morphol. 64, 471 (1939). 34. Ross, R. and BEND~TT, E. P., J. Cell Biol. 27, 83 (1965). 35. ROTH, T. F. and PORTER, K. R., in BREESE, S. S., JR. (Ed.), Proc. lnt. Cong. Electron Microsc. 5th, 1962. 36. SIEKEVITZ,P. and PALADE, G. E., J. Biophys. Biochem. Cytol. 4, 309 (1958). 37. STOCKER,A. J. and KASTR1TSlS, C. D., Chromosoma, in press (1972). 38. VIDAL, O. R., SPmITO, S. and RIVA, R., Experientia 27, 178 (1971).
292
J. ULTRASTRUCTURERESEARCH40, 292--312 (1972)
Developmental Studies in
Drosophila
VI. Ultrastructural Analysis of the Salivary Glands of Drosophila pseudoobscuraDuring the Late Larval Period M. J. E. HARROD and C. D. KASTRITSIS1
Department of Cell Biology, The University of Texas, Southwestern Medical School, Dallas, Texas 75235 Received October 8, 1971, and in revised form February 18, 1972 Salivary gland cells of Drosophilapseudoobscura were examined electron microscopically and cytochemically during the late larval stage of development. The data indicate that several synthetic processes are occurring in the glands during this period. The first, a presumably digestive secretion, is produced throughout the gland in the earliest stages studied, and only in the most proximal cells with increasing time. Mucoprotein granules, which are secreted and utilized as a "glue" at the time of puparium formation, are produced in the most distal cells of the gland beginning at about 100-106 hours after hatching, and are eventually found throughout the medial region as well, but never in the proximal portion of the gland. Such granules are produced in the Golgi regions and have nonmucoprotein "caps" at their periphery. Several types of material are also found within the cisternae of the rough endoplasmic reticulum of the cells; the function and significance of these crystals, granules, and aggregations of granules is unknown. Lysosomal activity is present in the cells from the earliest stages, and increases with time. In a previous report (15) we have described the ultrastructural changes which occur in the salivary glands of Drosophila pseudoobscura during the period of transition from the larval to the pupal stages. Briefly, the process involves the following events: Large numbers of mucoprotein granules are found within the gland cells at the end of the larval period, and are subsequently secreted just prior to puparium formation; these form the "glue" that attaches the pupal case to its substrate. After puparium formation the residual granules are attacked by lysosomes and degraded by a process of crinophagy (6). A further secretion of vesicles packaged by the Golgi regions occurs during the prepupal period, concomitant with an increase in the number of secondary lysosomes. Histolysis of the gland cells occurs, both by the process of z Present address: Department of General Biology, Aristotelian University of Thessaloniki, Thessaloniki, Greece.
DROSOPHILA SALIVARY GLANDS
293
autolysis, presumably due to lysosomal enzymes, and by phagocytosis, by cells from the hemolymph. The present report deals with the events from the time when mucoprotein synthesis begins, through the production of the granules, and their accumulation and stoiage at the end of larval life. The data obtained in concurrent studies also undertaken in this laboratory on the cytogenetic aspects of development, as determined by the chromosomal puffing changes (37) and the biochemical changes which occur, as determined by microelectrophoretic studies on the proteins of the glands (26) will, it is hoped, correlate with the ultrastructural findings, thereby providing a useful contribution to the study of development.
MATERIALS AND METHODS The experimental animals used in this study were third-instar larvae of a standard strain of Drosophila pseudoobscura, from Mather, California. Salivary glands from larvae aged 96, 100, 106, 114, 122, 130, and 144 hours after hatching were utilized in this series of experiments. The fixation, embedding, and observation of the sections were carried out as previously described (15). Sections were cut from the proximal, medial, and distal regions of the gland at each stage to provide a basis of comparison of the activities in these different areas.
RESULTS
96-Hour stage At 96 hours after hatching the larvae are near the midpoint of the third-instar period. The salivary glands are small and relatively uniform in diameter throughout their length. Sections through proximal, medial, and distal regions of the gland reveal that, although the distal cells are somewhat larger, overall there is less variation in the ultrastructural appearance of the cells of the three regions than at any other stage studied. The individual cells of a given region of the gland display a noticeable degree of polarity, as can be seen in Fig. 1. The apical border of the cell is composed of microvilli that extend into the lumen of the gland. The large nucleus with its prominent nucleolus and polytene chromosomes occupies the central region of the cell. The basal border of the cell, covered by a fine basement membrane, forms infoldings which extend for some distance into the cell. In those portions of the gland where the basal surface is openly exposed to the hemolymph, the infoldings tend to appear as open spaces, sometimes containing small vesicles of unknown origin. Occasionally they are seen to end almost in contact with cisternae of the rough endoplasmic reticulum (Fig. 3). In areas of the gland that are in contact with the fat body, only the 2 0 -- 721834 J . Ultrastructure Research
294
HARROD AND KASTRITSIS
basement membrane separates the two organs, and the infoldings are tightly apposed along their length, appearing in the sections as dense lines. Cells of other' types are occasionally found in contact with the basal surface. Muscle cells are oriented lengthwise along the proximal third of the gland, although they apparently do not form a continuous layer; their appearance in the sections is rather rare. Blood cells, presumably phagocytes, are also found in some sections. Adjacent cell boundaries display complex interdigitations. Near the apical cell surface tight junctions are often visible, along with various types of desmosomes; toward the basal side of the gland the lateral boundaries usually terminate in one of the infoldings of the basal membrane. Along the length of the cell boundary are found septate desmosomes. A striking contrast in the density of two adjacent cells is frequently seen, thus allowing the easy tracing of the cell boundary between them, even under low magnifications. The cytoplasm of the cells is rich in free ribosomes as well as in rough endoplasmic reticulum, which is often arranged in parallel arrays of cisternae. Mitochondria with long filamentous, oval, and rounded profiles are abundant and display no particular orientation throughout the cell. The cells are rich in Golgi Iegions, which present a rounded, vesicular appearance, rather than the flattened saccules of classic Golgi morphology. Small, smooth tubular elements are also seen between the rough endoplasmic reticulum and the Golgi vesicles. Closely associated with the Golgi regions are secretory vesicles containing a flocculent material identical in appearance to material found in the terminal cisternae of some of the Golgi complexes (Fig. 2). These secretory vesicles are often seen near the apical border of the cell. In several instances they are fused with the cell membrane so that their contents are free to enter the lumen, which, at this stage, contains material identical in appearance to that of the vesicles. The cytoplasm contains numerous microtubules, whose position can generally be determined by zones of almost " e m p t y " cytoplasm immediately surrounding them. Lipid inclusions are seen frequently, largely confined to the basal region of the cells. They are usually oval in section and slightly more electron dense than the Golgi cisternae.
FIG. 1. 96 Hours. A portion of one cell, containing the large nucleus with its prominent nucleolus (Nu) and polytene chromosomes (Chr), which have not ieached their ultimate degree of polyteny in this cell. The apical membrane of the cell forms microvilli (My) bordering the lumen (Lu) of the gland. The cytoplasm contains numerous mitochondria (Mi), many arrays of rough endoplasmic reticulum (ER) as well as free ribosomes, and a number of Golgi zones (Go). The basal cell membrane (Ba) forms infoldings and is covered by a basement membrane. × 6 400.
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Near the basal surface of the gland, small multivesicular bodies are frequently encountered. In addition, occasional autophagic vacuoles are found, surrounded by several layers of membrane, and containing cytoplasmic elements such as ribosomes, vesicles of rough endoplasmic reticulum, and mitochondria (Fig. 11). Two types of intracisternal inclusions are found at this stage. More frequently seen are electron-dense granules, 0.2 # to 0.3/z in diameter, almost round in section, and situated within rounded cisternae of rough endoplasmic reticulum (Fig. 4). Their occurrence is confined to the basal portion of the cells. Occasionally, similar granules are found within irregularly shaped cisternae which also contain other amorphous, slightly electron-dense material. Less frequently seen, but usually abundant in the cells where they occur, are crystalline inclusions within cisternae of the rough endoplasmic reticulum (14). At low magnifications they are difficult to distinguish from sections of mitochondria, but at high magnification the crystalline structure becomes clearly evident (Fig. 4). In longitudinal section they have a length of as much as 8 #; the individual subunits have a diameter of about 200 A. Figures 5 and 6 indicate that a connection may exist between a cisterna containing a crystalline inclusion and one containing a dense intracisternal granule. The crystalline inclusions, like the intracisternal granules, are confined to the basal portion of the cells. As previously stated, the proximal, medial, and distal portions of the gland at 96 hours are quite similar in ultrastructural appearance. Cell size differences exist, as do differences in the degree of polytenization of the chromosomes, which is detectable as a lack of prominent banding when seen in section (Fig. 1). Within a given region of the gland, cell to cell differences are frequently noticeable. One distal cell may appear to be active in secretion; the adjacent cell may show no sign of such activity.
lO0-Hour stage At 100 hours the salivary gland cells are very similar in general appearance to those of the 96-hour stage. The same cytoplasmic organelles are present, and secretion of the flocculent material packaged by the Golgi regions is continuing in most of the cells examined. A striking difference is seen in a minority of the glands. In cells from the distal
]FIG. 2. 96 Hours. Golgi regions (Go) with their associated smooth tubular elements are seen near the apical border of a cell (upper right). Secretory vesicles (SV) containing a flocculent material are found near the Golgi regions and also near the microvilli (My) at the lumen, x 30 000. FIG. 3. 96 Hours. Basal cell surface exposed to hemolymph. The basement membrane (BM) covers numerous infoldings (In) of the basal cell membrane, one of which is seen to terminate (arrow) almost in contact with a cisterna of the rough endoplasmic reticulum (ER). × 30 000.
298
HARROD AND KASTRITSIS
region and, in a few instances, the medial region, the production and accumulation of membrane-bound, relatively electron dense granules occurs. Such granules are never seen in the proximal region of the gland where the previously described secretion of the flocculent material continues to occur. These granules are derived from Golgi regions. In the earliest stages of synthesis observed, small irregularly shaped accumulations of slightly less electron dense material are seen within the terminal cisternae of the Golgi (Fig. 7). Fusion of such smaller elements apparently occurs to form larger granules which range, at this stage, from 0.25 # to 1.1 # in diameter. Some relatively large granules are occasionally seen in association with the Golgi and were apparently synthesized as such (Fig. 8). An interesting feature of these granules is the presence of somewhat more electron dense "caps" at their periphery (Figs. 9 and 10). The "caps" are also produced in the Golgi, and fusion of " c a p " and granule may take place within that area, or may occur at some distance from the site of synthesis (Fig. 14). Some "caps" are found to contain regions of even greater density in the form of small, round spots, sometimes two or more per "cap." The material within the terminal Golgi cisternae and the body of the granules gives a positive reaction for the presence of periodic acid-reactive carbohydrate when the silver methenamine technique of R a m b o u r g (31) is utilized (15). The "caps" show no reaction, indicating that they are of a different chemical composition from the remainder of the granule. These granules represent the substance secreted by the salivary glands just prior to puparium formation, a mucoprotein "glue," which attaches the pupal case to its substrate. They will be referred to as mucoprotein granules throughout the remainder of the text. Dense intracisternal granules and crystalline inclusions (both negative to the R a m bourg technique), are seen in 100-hour glands, in cells of the proximal region as well as those of the medial and distal regions. Degenerative areas, presumably of lysosomal origin, are found within the cytoplasm in the basal portion of some of the cells. These areas, always membranebound, contain amorphous material of low electron density and are usually seen to be in contact with cytoplasmic constituents such as cisternae of endoplasmic retic-
Fio. 4.96 Hours. Basal portion of a cell showing crystalline inclusions (Cry) and dense intracisternal granules (arrows). Mitochondria (Mi) can be readily distinguished from crystalline inclusions at this magnification, x 6 400. FIG. 5. 100 Hours. Two crystalline inclusions and a dense intracisternal granule in close proximity. Arrow indicates extension of granule's cisterna toward that of crystalline inclusions, x 81 000. Fie. 6. 100 Hours. Serial section showing apparent connection between the cisternae and the presence of material similar in density to the crystalline inclusions within the connecting cisternae. ×81 000.
?
•
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ulum and mitochondria. They are frequently seen with mucoprotein granules at their periphery, or in some cases, within the membrane-bound area (Fig. 14). Other smaller, membrane-bound structures containing recognizable cytoplasmic elements aie also found. These autophagic vacuoles are seen throughout the cell and contain membranous residues (Fig. 12) as well as other identifiable structures such as mitochondria.
106 to 122-Hour stages Throughout this portion of the third instar a gradient of mucoprotein production by the cells is evident. By 106 hours the distal cells of all glands contain some mucoprotein granules. Some medial cells are also seen to be active in mucoprotein synthesis, with great variations in distribution and size of granules from cell to cell. No mucoprotein is found in the proximal region, but the secretmy activity previously described is still occurring in this region. At 114 hours the granules in the distal regions are larger, up to 2/~ in diameter, and more numerous (Fig. 14), as are those of the medial region, although the latter are still of an average size smaller than those of the distal region. Similar variations exist at 122 hours. At this stage secretory activity in the proximal region has diminished, but no mucoprotein granules are found in that portion of the gland. The dense intracisternal granules are still common, and crystalline inclusions are found sporadically. In addition to the autophagic vacuoles and the degenerative areas of the earlier stages, an additional type of lysosome is found in some cells. Morphologically identical to the secondary lysosomes of later instar stages, these bodies contain myelin-figure-like inclusions of dense, membranous lamellae within an amorphous, moderately electron dense, membrane-bound matrix. Cytoplasmic organelles and mucoprotein granules are often found in contact with such bodies (Fig. 13).
130 and 144 Hour stages Anterior spiracle eversion, the first indication of the onset of puparium formation, occurs in D. pseudoobscura at 144_+ 6 hours. Thus the 130- and 144-hour stages studied represent the latter portion of the mucoprotein synthesis period, and the stages at which granule size and numbers reach a maximum. The same gradient of distribuFIG. 7. 100 Hours. Early stage of mucoprotein production showing small mucoprotein granules (MG) within the cisternae of the Golgi (Go). x 28 000. Fro. 8. 100 Hours. Formation of mucoprotein granule (MG) within a Golgi zone (Go) having a double layer of flattened saccules. × 28 000. Fro. 9. Late third instar. Interaction between granules. Fine, membranous elements are seen between the granules in a region of almost "empty" cytoplasm (arrows). x 27 000. FIG. 10. Late third instar. Apparent fusion of two mucoprotein granules. Arrow indicates membrane still separating the two granules, x 25 000.
i
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tion is still evident. The proximal region of the gland is never seen to be involved in mucoprotein synthesis. The medial portion of the gland is still an area of transition. Although the production of mucoprotein granules reaches its peak during this period there are still quite obvious variations in the size and density of granules among the cells of this region. In the distal region of the gland the cells are packed with mucoprotein granules which have at this point in development reached their maximum diameter of as much as 3.5 #, although many smaller granules are still present. Interactions between individual granules are seen. Obvious fusions occur (Fig. 10), and some granules are seen to be connected by areas of peculiar appearance, almost devoid of cytoplasmic elements, which contain fine, membranous or vesicular extensions between the granules (Fig. 9). Whole groups of granules are seen to be connected in this manner in some sections. The other cytoplasmic components are relatively unchanged. The endoplasmic reticulum forms well ordered arrays among the granules. Although the cisternae may be somewhat dilated, no contents are visible within most of them. Occasional cisternae which appear to be partly rough and partly smooth are found in the vicinity of the Golgi regions, between the arrays of rough cisternae and the smooth tubular elements which are adjacent to the Golgi vesicles. No material is visible in the Golgi regions, with the exception of the most terminal cisternae, some of which contain the mucoprotein product. Secondary lysosomes are present in most cells and are slightly larger than in previous stages. In addition to the previously described dense intracisternal granules and crystals an additional type of intracisternal inclusion not seen in previous stages, appears with the 130-hour stage. Consisting of aggregations of small, round bodies of varying electron-densities, ranging in size from 0.5 # to 0.15 #, they are found within greatly dilated cisternae of the rough endoplasmic reticulum (16). Small granules of similar size and shape are frequently found in separate cisternae near the larger aggrega-
FIG. 11. 100 Hours. A membrane-bound autophagic vacuole (AV) contains particles identical in size to the ribosomes of the surrounding cytoplasm. A secretory vesicle (SV) is seen near the lumen (Lu). x 35 000. FIG. 12. 100 Hours. An autophagic vacuole containing membranous structures (arrow) as well as membrane bound inclusions containing material resembling ribosomes. × 35 000. FIG. 13. 114 Hours. A secondary lysosome (Ly) consisting of a membrane bound accumulation of moderately electron-dense material containing myelin-figure-like inclusions. Mitochondria (Mi) and mucoprotein granules (MG) are seen to be in contact with the body at its periphery. A Golgi region (Go) is seen still active in the production of mucoprotein, x 15 500.
I
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tions. Den s e intracisternal granules a p p e a r near the aggregations in some sections, b u t no c o n n e c t i o n between the cisternae of the t w o types has been observed. The nuclear inclusions previously described
(13, 15) as
occurring in late third instar
an d thereafter,have also been f o u n d in all stages described here.
DISCUSSION
The data presented in this report indicate that several synthetic processes occur in the salivary gland cells of D. pseudoobscura during the late larval period. The finding that synthesis and secretion of a material derived from the Golgi regions of the cell, presumably protein in nature, occurs at least during part of the feeding period in larval D. pseudoobscura, and that such synthesis ceases at the stage when the larvae leave the food in preparation for pupation, strongly suggests that the secretory product may be active in digestion. Such an accessory digestive function has been described for the salivary glands of Drosophila (4), although definitive evidence for this interpretation is lacking. Hsu (16) described the production of material by the Golgi regions and its subsequent secretion in salivary gland cells of "young" D. melanogaster larvae (exact stage unspecified). He interpreted this secretion to be digestive in nature, since it occurred only during the feeding period of larval life. Patterson et al (27) described the presence of peptidase in extracts of the salivary glands of D. melanogaster, a finding which is suggestive of a possible digestive function. In more recent studies, Berendes (3) described the synthesis and secretion of substances presumed to be digestive enzymes, throughout the gland of D. hydei in early larval stages and confined to the most proximal cells at late third instar. This secretory product is composed of small granules, 0.35 # to 0.85 # in diameter, membranebound, composed of a number of particles, and often appearing partially dissolved. The occurrence of the secretion in D. pseudoobscura follows the pattern described by Berendes (3) for D. hydei; such activity is seen throughout the gland prior to the production of mucoprotein by the salivary gland, and is confined to the proximal portion of the gland during the late third instar. The appearance of the secretory granules in the different species of Drosophila which have been described is quite different. Instead of the dense, partially dissolved granules described by Berendes, the secretory vesicles seen in D. pseudoobscura are electron lucent and contain small FIG. 14. 114 Hours. A cell from the distal region of the gland containing many mucoprotein granules. Unattached "caps" can be seen in some areas, as well as granules which appear to be in the process of fusion of "cap" and granule. A degenerative area is seen at lower right with several granules within its boundaries. The nucleus (N) contains a small nuclear inclusion adjacent to the membrane (arrow). The banding pattern of the polytene chromosomes (Chr) is more readily apparent at this stage than in Fig. 1. x 6 400.
306
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quantities of flocculent material. Such a morphological variation in what are presumably functionally similar products is of interest. Similar findings have been reported in some other genera. Phillips and Swift (30) described the production and secretion of a specific granule during the feeding period of larval development in Sciara coprophila which is not found after the larvae cease feeding. They interpreted this as evidence of a digestive function. Oschman and Betridge (24) demonstrated that the secretory droplets produced by the salivary glands of Calliphora erythrocephala, which are presumed to contain amylase, have an electron-lucent appearance and contain flocculent material. These granules are more similar in appearance to those of D. pseudoobscura than are the granules described in the other species of Drosophila. Mucoprotein production by the salivary gland cells of D. pseudoobscura is initiated in the most distal cells of the gland at 100-106 hours after hatching. The sequence of events apparently involves: the synthesis of protein within the rough endoplasmic reticulum, which is abundant in the cells engaged in mucoprotein production; the packaging of this protein and the addition of a polysaccharide component within the confines of the Golgi regions to produce the mucoprotein granules (7); the formation of nonpolysaccharide "caps" within the same or different Golgi regions; the fusion of granules and "caps" to produce the typical mucoprotein granule observed in the gland; and the enlargement of the granules through fusions among themselves. The region of the gland in which mucoprotein can be found extends proximally with time, as can be confirmed with PAS staining of the whole glands. Just prior to secretion there is a gradient of distribution, with the distal cells containing more and larger granules; however, all but the most proximal cells appear to contain some. The synthesis and secretion of mucoprotein by larval dipteran salivary glands was grossly misinterpreted by early investigators, who observed the process but were unaware of its significance. The mucoprotein granules were described as secretion vesicles associated with increased salivary function during the last hours of feeding (33), and as food storage granules derived from transformed mitochondria (16). The first satisfactory explanation of the purpose of the secretion was published by Fraenkel (8) and Fraenkel and Brookes (9). They described the secretory process in D. melanogaster and in Phormia regina, and concluded that it was this material, secreted just prior to puparium formation, whicb formed the "glue" that attaches the pupal case to its substrate. This clarification of the function of the secretion was followed by a number of studies which touched upon the method of its production within the cells, utilizing the newly available technique of electron microscopy to provide information at the ultrastructural level. Gay (10) observed that secretion granules were present in salivary glands of mid-
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third instar larvae of D. melanogaster, that they increased in number and size during late larval life, and that they disappeared from the cytoplasm when the PAS-positive secretion was discharged into the lumen. She theorized that there was a correlation between the appearance of nuclear blebs during this stage and the production of mucoprotein secretory granules; such blebs were suggested as a possible transport mechanism for genetic information by transformation of the portions of nuclear membrane into endoplasmic reticulum, which she considered to be the site of formation of the mucoprotein. Similar blebs of the nuclear membrane are found in D. pseudoobscura in association with nuclear inclusions (13, 15), but their occurrence is not confined to the mucoprotein production stage; they are found in similar numbers after the secretion of mucoprotein, during the onset of histolysis of the gland. This evidence suggests that it is unlikely that the blebs are involved in mucoprotein synthesis. Although the endoplasmic reticulum is undoubtedly involved in mucoprotein synthesis, the Golgi regions are seen to be the site of granule formation and the site of the polysaccharide component's synthesis, as indicated by the results obtained by the silver methenamine technique. The cytoplasm of cells engaged in mucoprotein production--indeed, the cytoplasm of all cells studied--is rich in organelles, contains a great quantity of rough endoplasmic reticulum and few smooth-membraned elements, and has large numbers of free ribosomes. In contrast to the D. pseudoobscura data, Berendes (3) characterized the cytoplasmic organization in the mucoprotein producing cells of D. hydei as poor in cytoplasmic organelles, with the endoplasmic reticulum consisting of smooth membranes, and noted that free ribosomes were seldom found. The ultrastructural composition of the mucoprotein granules of D. pseudoobscura differs from the description of Berendes and deBruyn (2) and Berendes (3) for D. hydei. In contrast to the fine granular appearance of the mucoprotein in D. hydei, the mucoprotein of D. pseudoobscura has a very definite crystalline substructure. This finding agrees with that of Rizki (32) for D. melanogaster. The swellings and bulbs at the margin of the granules in D. hydei could conceivably represent a phenomenon similar to the "caps" of D. pseudoobscura, but the description of their substructure as being identical to that of the body of the granule, and the observation that they appear to be due to the fusion of bodies of differing sizes makes this appear unlikely. Fusion of "caps" and granules does occur in D. pseudoobscura, but the "caps" are composed of a material of differing ultrastructural appearance, are of a greater density when stained with nonspecific electron stains, and are different in chemical composition from the body of the granule, as indicated by the results obtained with the silver methenamine technique.
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Berendes and deBruyn (2) described two types of granules in the mucoprotein producing cells, but unfortunately did not include a figure of the second type; no cytochemical characterization was made. It would be of interest to know whether both types contain the polysaccharide component of the mucoprotein, or if a difference exists in the chemical composition of the two types of granules. Since the body and the "cap" portion of the mucoprotein granules of D. pseudoobscura are produced separately by the cell and undergo fusion to produce the composite secretory granule, it is at least conceivable that the two entities are produced as separate bodies in D. hydei and do not fuse, but are secreted concurrently. In addition, Vidal et al. (38) have described a heterogeneous ultrastructure for the mucoprotein granules of D. melanogaster when they are stained with ammoniacal silver carbonate, which is believed to stain basic proteins. Regions of differing affinity for the stain are irregularly distributed over the body. It would be interesting to know if a similar pattern of distribution exists for the polysaccharide component. Berendes (3) also commented on the pattern of distribution of mucoprotein in D. hydei; he noted that in the region between the most proximal cells, which were still producing the presumably digestive secretion, and the distal cells, which contained the large "glue" granules, were cells which contained either small granules or none, which he termed a transitional zone. He correlated the production of "glue" with the degree of polyteny of the cell's nucleus, and characterized the transitional zone as one in which increases in polytenization were occurring. These findings are in agreement with the D. pseudoobscura data. Berendes' transitional zone is the same morphologically as the medial region of the gland, described previously as a zone of transition with respect to mucoprotein production. Although no attempt was made to measure the degree of polytenization, itis evident that in cells such as those represented by Fig. 1, the chromosomes are not as large and well banded as those seen in later stages. No mucoprotein is seen in these less polytenized cells, and cells which are active in granule formation are seen to have chromosomes which appear more polytene (Fig. 14). Similar ultrastructural investigations of salivary gland mucoprotein secretion have been carried out in some other dipteran genera. Such analyses by Phillips and Swift (30) of Sciara coprophila, Macgregor and Mackie (22) of Simulium niditifrons, and Kloetzel and Laufer (20) of Chironomus thumrni revealed that the process of synthesis of the mucoprotein secretory products shares certain similar features, in spite of the different functions served by the product in different genera. The process of mucoprotein synthesis, like all synthetic processes within the cells, is presumably under genetic control. Concomitant work on D. pseudoobscura has revealed a specific puff, which appears at approximately 106 hours and disappears at spiracle eversion (37), and the appearance of a specific protein component of the
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salivary glands between 96 and 120 hours, which also disappears at spiracle eversion
(26). Although the evidence is incomplete, these findings may coirelate with the observation that mucoprotein synthesis is observed ultrastructurally to begin around 100 to 106 hours after hatching, and that the mucoprotein disappears from the gland, either by secretion or by the crinophagic process, during the spiracle eversion period. Although the protein described by Pasteur (26) is not a mucoprotein, it may well represent a nonmucoprotein component of the secretion, which is known to contain smaller polypeptide molecules (29). Alternatively, it is possible that it represents the "cap" portion of the mucoprotein granules, which is presumably protein in nature. Correlation of puffing patterns with specific protein production has been made in only one instance in the literature thus far. Beermann (1) found that a particular secretory granule in the cells of the special lobe of Chironomus paIlidivittatus appeared only when a specific Balbiani ring was present on the chromosomes. Absence of this Balbiani ring, as was noted in C. tentans, correlated with absence of the granules. Biochemical analyses by Grossbach (11, 12) indicated that one protein component which was present in C. pallidivittatus was not present in C. tentans. He also found that the Balbiani ring described by Beermann was present in all glands which produced the protein. Other work suggestive of such correlations, but lacking the complete evidence required, has been published. Berendes (3) found a specific puff in D. hydei which was active only in the cells of the part of the gland which contained mucoprotein. It appeared before granule production and receded shortly before secretion. This puff was not found in the proximal part of the gland. Such findings are of importance to the study of development, since they provide an opportunity to observe '"cause and effect" relationships which few other systems can offer. A particularly interesting finding is the occurrence of various types of material within the cisternae of the rough endoplasmic reticulum at different stages of development. The crystalline inclusions (Fig. 4), found rather commonly in the early stages studied and sporadically thereafter, appear to be involved in some manner with the dense intracisternal granules which are also present. The observation of rough cisternae connecting the cisternae containing the two types of inclusions suggests that one form may be producing the other (Figs. 5 and 6); probably the dense intracisternal granules are derived from the crystalline material, since their occurrence is more common, and they are found in cells without crystalline inclusions, while the opposite is never true. The aggregations of small dense granules were found infrequently; it is difficult to speculate upon their possible function, or significance. 2 I -- 721834 J . Ultrastructure Research
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The presence of granules within the cisternae of the rough endoplasmic reticulum has been reported in other secretory tissues (17, 25, 36). It has been suggested that such granules are further processed or packaged in the Golgi region before secretion. No bodies resembling the cisternal contents have been found within the Golgi regions of the salivary gland cells, but it is possible that an alteration in morphology might occur if such a process were taking place. It has also been shown that, in some cells, secretion of cisternal contents may occur by direct fusion of the membrane of the endoplasmic reticulum with the plasma membrane of the cell (34). Although intracisternal granules have been found near the apical cell surface, they have not been observed in the process of being secreted, nor have similar structures been found within the lumen. Other aspects of the ultrastructural findings may be significant in providing information about the synthetic and metabolic processes of the salivary gland cells of
D. pseudoobscura. The deep infoldings of the basal cell membrane appear to be involved in transport, and may play a role in the uptake of water soluble substances from the hemolymph, since similar infoldings are commonly found in epithelia engaged in water transport (29). The process does not involve pinocytosis of the classic "coated vesicle" variety (35). The proximity of the extracellular space, bounded by the infoldings, to the intracellular space, within the cisternae of the rough endoplasmic reticulum (Fig. 3), suggests a possible route of entry into the cell for metabolites. The concentration of multivesicular bodies near the basal cell surface in D. pseudoobscura is similar to that described by Locke and Collins (21) in tissues of Calpodes ethlius, in which protein uptake was occurring. Sequestration of blood proteins and their hydrolysis by the enzymes of the multivesicular bodies were suggested as a possible source of amino acids in metabolically active tissues. The multivesicular bodies in D. pseudoobscura contain acid phosphatase, as indicated by the results obtained with the Gomori technique (15). Such multivesicular bodies are formed from small Golgi vesicles and appear to constitute primary lysosomes (6) in the salivary gland at this stage; they are thus capable of participating in such proteolytic activities. It is thus seen that the ultrastructural findings in D. pseudoobscura suggest that uptake of proteins from the hemolymph probably occurs; a similar uptake of protein from the fat body may occur, particularly in the later third instar stages when vesicles have been seen just within the basement membrane of the salivary gland which resemble vesicles within the adjacent fat body. These conclusions ate supported by Pasteur and Kastritsis' (26) data which indicate that throughout most of development all the nonspecific proteins found in the salivary gland are also found in the hemolymph and fat body, or only in the fat body. Such proteins may be produced
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in the fat body and either stored there or released into the hemolymph, from which they are taken up by the salivary glands; uptake of hemolymph proteins by the fat body may occur with subsequent storage in the fat body and eventual transfer directly to the salivary gland. Both processes would account for the ultrastructural observations. The occurrence of varying types of lysosomes within the salivary gland cells at different stages of development is of interest. The purpose of autophagic vacuoles such as those of the earliest stages described in tissues which are seemingly "healthy" is not apparent. Napolitano (23) noted the presence of similar cytolysomes in metabolically active cells, and suggested that they might constitute a prelude to cytolysis, or that they might result from an acute reorientation of normal metabolic processes within the cell. The somewhat larger lysosomal bodies seen in the earlier stages, which contain membranous residues (Fig. 13), apparently the "indigestible" remnants of organelles which have been degraded, are considered to represent slightly more advanced stages of the autolytic process. The lysosomal areas encountered in cells engaged in the early stages of mucoprotein synthesis appear to degrade some of the granules soon after they are produced. The presence of periodic acid-reactive carbohydrate throughout such an area has been observed with the silver methenamine technique. Such degradation may be accidental; the properties of their respective membranes may be such that contact between the two bodies is sufficient to cause fusion and the ultimate attack of the granule's contents. Other cytoplasmic elements, particularly vesicles of endoplasmic reticulum and mitochondria are seen in contact with the lysosomal membrane. Their membranes probably serve as the source of the myelin-figure-like inclusions seen in secondary lysosomes of the later stages (Fig. 13). The authors wish to acknowledge the technical help of Mrs Helen M. McNeil and the secretarial help of Mrs Madelon Smith. This work was supported in part by Grants GM-16736-03 and FR-05426-09 from the U.S. Department of Health, Education, and Welfare, and a grant from the Ruth Jackson foundation of Dallas, Texas. M. J. E. Harrod was a holder of a University of Texas predoctoral fellowship during the course of this investigation. The work presented is a portion of a dissertation submitted in partial fulfillment of the requirements for the degree of Docter of Philosophy. REFERENCES 1. BEERMANN,W. Chromosoma 12, 1 (1961). 2. BERENDES H. D. and DEBRUYN, W. C., Z. Zellforsch. Mikrosk. Anat. 59, 142 (1963). 3. - Chromosoma 17, 35 (1965). 4. BODENSTEIN,D., in DEMEREC, M. (Ed.), Biology of Drosophila. Hafner, New York, 1965.
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5. CARO,L. G. and PALADE,G. E., J. Cell Biol. 20, 473 (1964). 6. DE DUVE, C., it/DINGLE, J. T. and FELL, H. B., (Eds.), Lysosomes in Biology and Pathology. Wiley, New York, 1969. 7. FAVARD, P., in LIMA-DE-FARIA,A., (Ed.), Handbook of Molecular Cytology. American Elsevier, New York, 1969. 8. FRAENKEL,G., Biol. Bull. 103, 285 (1952). 9. FRAENKEL,G. and BROOKES,V. J., Biol. Bull. 105, 442 (1953). 10. GAY, H., Cold Spring Harbor Symp. Quant. Biol. 21, 257 (1956). 1 ]. GROSSBACH,U., ~Intl. Zool. Fenn. 5, 37 (1968). 12. - Chromosoma 28, 136 (1969). 13. HARROD, M. J. E. and KASTR1TSlS,C. D., Drosophila Inform. Serv. 46, 142 (1971). 14. - - - - J. Invert. Pathol. 18, 297 (1971). 15. - J. Ultrastruct. Res. 38, 482 (1972). 16. Hsu, W. S., Quart. J. Microsc. Sci. 89, 410 (1948). 17. ICH1KAWA,A., J. Cell Biol. 24, 369 (1965). 18. JACOB, J. and JURAND, A., J. Insect Physiol. 9, 849 (1963). 19. - Ibid. 11, 1337 (1965). 20. KLOETZEL,J. A. and LAUFER, H., J. UItrastruct. Res. 29, 15 (1969). 21. LOCKE, M. and COLLINS,J. V., J. Cell Biol. 26, 857 (1965). 22. MACGREGOR,H. C. and MACKIE, J. B., J. Cell Sci. 2, 137 (1967). 23. NAPOLITANO,L., J. Cell Biol. 18, 478 (1963). 24. OSCHMAN,J. L. and BERRIDGE,M. J., Tissue Cell2, 281 (1970). 25. PALADE, G. E., in BOYD, J. D., JOHNSON,F. R. and LEVER,J. D., (Eds.), Electron Microscopy in Anatomy. Williams and Wilkins, Baltimore, Maryland, 1961. 26. PASTEUR,N. and KASTR~TSlS,C. D., Develop. Biol. 26, 525 (1971). 27. PATTERSON,E. K., DACKERMAN,M. E. and SCHULTZ, J., J. Gen. Physiol. 32 607 (1949). 28. PEASE, D. C., J. Biophys. Biochem. Cytol., Suppl. 2, 203 (1956). 29. PERKOWSKA,E., Exp. Cell Res. 32, 259 (1963). 30. PmLLIPS, D. M. and SWIFT, H., J. Cell Biol. 27, 395 (1965). 31. RAMBOUR~,A., J. Histochem. Cytochem. 15, 409 (19567). 32. RIZKI, T. M., J. Cell Biol. 32, 531 (1967). 33. Ross, E. B., J. Morphol. 64, 471 (1939). 34. Ross, R. and BEND~TT, E. P., J. Cell Biol. 27, 83 (1965). 35. ROTH, T. F. and PORTER, K. R., in BREESE, S. S., JR. (Ed.), Proc. lnt. Cong. Electron Microsc. 5th, 1962. 36. SIEKEVITZ,P. and PALADE, G. E., J. Biophys. Biochem. Cytol. 4, 309 (1958). 37. STOCKER,A. J. and KASTR1TSlS, C. D., Chromosoma, in press (1972). 38. VIDAL, O. R., SPmITO, S. and RIVA, R., Experientia 27, 178 (1971).