Polar granules and pole cells in the embryo of Calliphora erythrocephala: ultrastructure and [ 3 H]leucine labelling

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1 /. Embryol exp. Morph. Vol. 57, pp. 79-9, Printed in Great Britain Company of Biologists Limited 198 Polar granules and pole cells in the embryo of Calliphora erythrocephala: ultrastructure and [ H]leucine labelling By ANDERS LUNDQUIST 1 AND HADAR EMANUELSSON 1 From the Zoophysiological Institute, University of Lund, Sweden SUMMARY The polar granules in Calliphora undergo a gradual fragmentation during early cleavage, but reaggregate after pole-cell formation. Autoradiographic analysis showed that the pole cells in Calliphora acquire a higher [ H]leucine label than the rest of the embryo during the blastoderm stage. Such an increased label was not seen in the pole plasm before pole-cell formation or in the pole cells during gastrulation. Electron microscopic autoradiography revealed that the polar granules are substantially labelled during the blastoderm stage. At the same time, characteristic nuclear blebs appear in the pole cells. The observations are consistent with the hypothesis that polar granules contain maternal messenger RNA, which is released and translated into proteins. INTRODUCTION The idea that the egg contains cytoplasmic determinants has long been held in developmental biology. It was most successfully applied to the determination of the germ cells experimentally studied in chaetognath.es, amphibians, nematodes, and insects (reviewed by Beams & Kessel, 1974; Eddy, 1975). The pole plasm is the hindmost cytoplasmic region in the fly egg. It contains determinants required for germ-cell differentiation (reviewed by Mahowald, 1977; Agrell & Lundquist, 197; Counce, 197). The polar granules are specific organelles found in the pole plasm, but absent from other parts of the egg. The pole cells bud off from the posterior pole during cleavage after the nuclei have reached the surface of the egg syncytium. They enclose the pole plasm with the polar granules. The pole cells are the sole progenitors of the adult germ cells. If the pole plasm of the Drosophila egg is damaged or destroyed, no pole cells are formed (e.g. Geigy, 191; Poulson & Waterhouse, 196; Hathaway & Selman, 1961; Graziosi & Micali, 1974) and if it is transplanted to an incapacitated posterior pole or to ectopic loci, potentially functional pole cells are formed there (Illmensee & Mahowald, 1974, 1976; Okada, Kleinman& Schneiderman, 1974; Warn, 1975; Illmensee, Mahowald & Loomis, 1976). Thus, it is well 1 Authors' address: Zoophysiological Institute, University of Lund, Helgonavagen B, S Lund, Sweden. 6-2

2 "8 A. LUNDQUIST AND H. EMANUELSSON established that some factor necessary for pole cell formation resides in the pole plasm. The polar granules in Drosophila contain RNA (Mahowald, 1962, 1971; Counce, 196). They begin to lose their RNA after fertilization, and the RNA is no longer cytochemically detectable by the blastoderm stage when the pole cells have stopped dividing (Mahowald, 1971). During pole-cell formation, the polar granules in Drosophila are fragmented and surrounded by polysomes (Counce, 196; Mahowald, 1968). Therefore, the suggestion that the polar granule RNA is a long-lived maternal messenger RNA, which is translated into germ-cell-determining proteins (Mahowald, 1968). Light microscopy has shown that the polar granules are fragmented during pole-cell formation also in Calliphora (Noack, 191; Alleaume, 1971) and they undergo conspicuous ultrastructural changes in Coelopa (Schwalm, Simpson & Bender, 1971). As the polar granules in higher dipterans seem to be active when the pole cells appear, it was suggested that they are the factor causing pole-cell formation, but the direct evidence is uncertain. The available general studies on the protein synthesis in the fly egg disagree about the pole plasm. In Musca, an increased label was found in the pole plasm before pole-cell formation (Pietruschka & Bier, 1972). In Drosophila, labelling increased only in the early pole cells (Zalokar, 1976). In this study, the amino acid labelling of Calliphora pole cells and polar granules was examined with ~both light and electron microscopic autoradiography. Moreover, some ultrastructural features of polar granules and pole cells in Calliphora are described for the first time. MATERIALS AND METHODS The culture of the flies {Calliphora erythrocephala Meig.), the collection of the eggs, the development of the embryos at 2 C, and the treatment of eggs for electron microscopy were earlier described in detail (Lundquist & Emanuelsson, 1979). The eggs were fixed with 2-5 % glutaraldehyde and 4 % formaldehyde in cacodylate buffer, ph 7-2 (Karnovsky, 1965), and postfixed with 1 % OsO 4. Eggs for autoradiography were permeabilized according to a method modified after Limbourg & Zalokar (197). Dechorionized eggs (Lundquist & Emanuelsson, 1979) were placed on a piece of moist tissue paper (Kleenex) inside a small cup of stainless-steel screen. The cup was immersed in n-octane for 15 sec. The octane was thoroughly blotted off, and any remaining octane was evaporated for 15 sec in a humid air stream. The cup was immersed in incubation medium,.and the eggs were covered with another piece of tissue paper. The cup was then taken up, partly drained, and incubated suspended in a moist chamber at 2 C for or 6 min. In this way, the eggs and a thin layer of medium were sandwiched between two layers of tissue paper, thus ensuring adequate oxygen.supply and least mechanical damage to the eggs. Shaw's medium (Shaw, 1956), -which is suitable for Calliphora embryo culture (Davis, Krause & Krause, 1968),

3 Polar granules in Calliphora 81 Table 1. The condition of permeabilized Calliphora eggs immediately after incubation in Shaw's medium Number of eggs Incubation time (min) Age of the eggs at fixation (h) Normal Possibly* abnormal Clearly! abnormal Total % 6 H H V Immediately after incubation, the eggs were fixed with Bradley-Carnoy solution ( parts absolute ethanol, 1 part glacial acetic acid, 4 parts chloroform). Whole-mounts were prepared, essentially according to Agrell (1962). * Delayed development of the nuclear sphere or an unusually large number of vitellophages. t Generally abnormalities of the nuclear sphere or the blastoderm. % Some eggs either did not develop or aborted after a few cleavage divisions. These eggs are probably unfertilized and were not included was used. The medium contained 7-4 MBq/ml (= 2/*Ci/ml) L-[4,5- H]- leucine (2-1 TBq/mmol (=57 Ci/mmol); Radiochemical Centre, Amersham). The leucine content of the medium was about times lower than the natural leucine content in fly eggs (Chen, Hanimann & Briegel, 1967). This is probably an important reason why a comparatively high radioactivity in the medium and a long incubation are necessary to ensure adequate labelling. On the other hand, the retention of free labelled leucine (Peters & Ashley, 1967) would be reduced because of the resulting low specific activity in the endogenous leucine pool and the prolonged incubation. But under the present conditions, acid-insoluble label of amino acids in the Drosophila egg is nearly half the total label (Limbourg & Zalokar, 197; Zalokar, 1976). Therefore, such retention should present no problem. Eggs of Calliphora incubated for 6 min with [ H]leucine (1-85 MBq/ ml) during oxygen shortage did not show autoradiographically-detectable label. Tables 1 and 2 record the development of the permeabilized eggs. Eggs of all stages develop normally in the medium for 1 h, but eggs permeabilized and incubated during cleavage do not hatch. Possibly, cleavage eggs receive mechanical injuries, which manifest only at a later stage. The eggs lose some turgor during permeabilization, and this makes them more sensitive to mechanical

4 82 A. LUNDQUIST AND H. EMANUELSSON Treatment Permeabilized, min in medium, paraffin oil Dechorionized, paraffin oil Dechorionized Table 2. The development of permeabilized Calliphora eggs after incubation in Shaw's medium Age of the eggs after incubation in medium (h) 2± ± i ± 4 Hatched larvae Unhatched larvae* Number of eggs Segmented embryosf A Pigmented embryosj Eggs without pigmentation Total Permeabilized eggs were incubated in Shaw's medium for min and transferred to paraffin oil in a water-saturated oxygen atmosphere. The results were scored a few hours after the end of the normal hatching period. The values were similar when the eggs were incubated in medium for 6 min except for a decrease in the number of 'hatched larvae' and a corresponding increase in the number of 'segmented embryos'. The unpermeabilized control eggs were only decborionized. They were transferred to paraffin oil during the blastoderm stage and treated as above or allowed to develop on moist filter paper in air. The hatching percentage was always higher after the latter treatment. * Otherwise apparently normal. t Usually with minor segmental defects or abnormal mouth hooks. % Usually with grossly abnormal segmentation or no visible segments. The cuticular pigmentation appears shortly before hatching. Unfertilized eggs included. FIGURE 1 Light-microscopic autoradiographs of pole plasm and pole cells in Calliphora eggs labelled with [ H]leucine for min. (A) Posterior pole with pole plasm during intravitelline cleavage (6 min). (B) Pole-cell buds (12 min). (C) Pole cells during the cellularization of the blastoderm (18 min). (D) Pole cell with polar granules (arrows) in the yolk region near the posterior midgut invagination. This is phase II of pole-cell migration. Early germ-band-elongation stage (27 min). PC, Pole cell; PMI, posterior midgut invagination; Y, yolk. Notice the heavy label of the pole cells in the blastoderm. See also Table. Section thickness: 1 //,m. Exposure time: 27 days.

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6 84 A. LUNDQUIST AND H. EMANUELSSON damage before they have acquired a firmer structure by the cellularization of the blastoderm. The Calliphora egg should be more susceptible to such damage than the smaller Drosophila egg. After incubation, eggs for autoradiography were fixed for 2 h at room temperature with Karnovsky fixative (six changes). The vitelline membrane was removed in the fixative after h. The embryos were washed with cacodylate buffer at + 4 C overnight (at least five changes), postfixed with 1 % OsO 4 in the same buffer, bulk-stained with -5 % uranyl acetate and 1 % phosphotungstic acid, and embedded in Vestopal W. Thin sections for light microscopic autoradiography (1 pm) were covered with liquid nuclear emulsion (Ilford K2) according to the dipping method and exposed for 6-27 days. The auto radiograms were developed in Kodak D 19 (5 min, 2 C), rinsed in distilled water (1 sec), fixed in Kodak F24 (6 min, 2 C), and stained with Richardson's azure II. Ultrathin sections for electron microscopic autoradiography were vacuumcoated with carbon, covered with a monolayer of nuclear emulsion (Ilford L4) according to the loop method, and exposed for 4-24 weeks. The preparations were developed in Kodak D 19 (2 min, 2 C), rinsed in distilled water ( sec), fixed in 15% Na 2 S 2 O ( min, 2 C), and finally washed in distilled water (2 min). Examination was made with a Philips EM electron microscope at the Zoological Institute, University of Lund. RESULTS At 2 C the Calliphora egg has completed nine intravitelline synchronous nuclear divisions 9 min after egg-laying, when the dividing nuclei reach the periphery of the egg. The remaining four nuclear divisions are partially synchronous and occur in the syncytial blastoderm (Lundquist & Emanuelsson, 1979). The pole cells bud off during the second or third blastodermal division (Fig. IB). They proliferate at the posterior end during the ensuing cellularization of the blastoderm (from 15 min; Fig. 1C) and most of them are brought into the egg interior with the posterior midgut invagination during gastrulation (between 24 and 27 min; Fig. ID). Pole cells migrate into the yolk region in FIGURES 2 AND Electron microscopic autoradiographs of polar granules in Calliphora eggs labelled with [ H]leucine for min. The stages are: (2A) Intravitelline cleavage (6 min). (2B) Pole-cell formation (12 min). ( A) Cellularization of the blastoderm (18 min). (B) Early germ-band elongation (27 min). dmvb, Dark multivesicular body; ER, endoplasmic reticulum; N, nucleus; M, mitochondrion; PG, polar granule. Notice that the polar granules are fragmented during pole-cell formation and heavily labelled during the cellularization of the blastoderm. Exposure time: 2 weeks (2A), 9 weeks (2B, A), and 24 weeks (B).

7 Polar granules in Calliphora 85 FIGURE 2

8 86 A. LUNDQUIST AND H. EMANUELSSON FlGURE

9 Polar granules in Calliphora 87 Table. Autoradiographic [ s H]leucine labelling of the pole plasm and the pole cells in Calliphora eggs Age of the eggs (min)* Stage 1 Egg no. 6 Pole plasm during early cleavage 12 Pole-cell buds - 15 Pole cells in the + - syncytial blastoderm 18 Pole cells during the cellular ization of the blastoderm 21 Pole cells during the cellular ization of the blastoderm 24 Pole cells at the onset of gastrulationf 27 Pole cells during the early - - germ-band-elongation stage The grain density over the pole cells was compared with that of the adjacent blastoderm cells:, similar grain density; +, higher grain density; ++, much higher grain density;, some pole cells (*S 5%) with no increased grain density. * The eggs were labelled for min in medium containing [ H]leucine (74 MBq/ml = 2/tCi/ml) and fixed at (he indicated age. t The eggs were often weakly labelled at this stage. two phases, before and after gastrulation (reviewed by Counce, 197; for Calliphora, see Alleaume, 1971). The polar granules in Calliphora consist of a network of dense material. During early cleavage, most of them are irregular in shape and relatively large (up to 1 /<m), sometimes with annular profiles (Fig. 2A). Sometimes, they are chained together. Subsequently, they become smaller. During pole-cell formation, small profiles (less than c. -5 /im) are gathered in a perinuclear zone (Fig. 2B). Towards the end of the nuclear cleavage period, large granules reappear. Later, very large circular or annular profiles become predominant (up to 2/MTO, although small profiles are still seen at gastrulation, especially near the nucleus (Fig. ). Pieces of endoplasmic reticulum are often very close to the polar granules of the pole cells (Figs 2, ). Extranuclear annulate lamellae (Lundquist & Emanuelsson, 1979) are present in the pole-cells (Fig. 4B), but almost absent from the blastoderm. Dark multivesicular bodies were regularly found in the pole plasm and the pole cells (Fig. 2B). Characteristic blebs are seen on the nuclear envelope in the pole cells of the blastoderm (Fig. 4). Such a bleb consists of an out-pocketing of the outer nuclear envelope membrane. It contains a circular membrane-enclosed profile filled with electron-dense material and is often associated with a nuclear pore. The blebs are first seen in the early pole cells, where they are very frequent

10 88 A. LUNDQUIST AND H. EMANUELSSON during the cellularization of the blastoderm. They are only occasionally observed at the late blastoderm stage and in the early gastrula. They are never seen outside the pole cells. The light-microscopic autoradiographs display an even distribution of silver grains over the pole plasm and the pole cells after [ H]leucine labelling. This applies to all stages. Both the nucleus and the cytoplasm are labelled. The grain density over the pole plasm or the pole cells compared with the adjacent part of the egg was judged semiquantitatively (Table ; Fig. 1). No difference was observed before pole-cell formation, but thereafter, the pole cells acquire a more intense label. This difference disappears at the late blastoderm stage and in the early gastrula. The highest grain density recorded in the egg was that over the pole cells during the early cellularization of the blastoderm. For electron-microscopic autoradiography, a min pulse with [ H]leucine was generally used (Figs 2, ). Both min and 6 min pulses were made during the cellularization of the blastoderm, but the grain distributions were not detectably different. Silver grains over the polar granules are present at all stages, but the polar-granule label follows the general pole-cell label. There is an approximately even distribution of label between the polar granules and other parts of the cytoplasm and between cytoplasm and nuclei. The polar granules are thus heavily labelled during the cellularization of the blastoderm. The silver grains are often seen over the periphery of the granules. The label of the pole cell nuclei is often found close to the nuclear envelope, and silver grains are sometimes seen near nuclear blebs. DISCUSSION In the CalHphora egg the time course of fragmentation and reaggregation of the polar granules parallels the rise and fall in amino-acid labelling of the pole cells. The peak in polar-granule fragmentation during pole-cell formation precedes the peak in pole-cell labelling by about one hour. That applies also to Drosophila, except that no clear-cut decrease in pole-cell label compared to other cells was detected in ovo (Mahowald, 1968; Zalokar, 1976). The results of Allis, Underwood, Caulton & Mahowald (1979) do not contradict the interpretation that at least a slight decrease occurs in cultured Drosophila pole cells. Because RNA synthesis in the pole cells is very low or absent in both species (Zalokar, 1976; Lamb & Laird, 1976; Lundquist & Emanuelsson, preliminary results), FIGURE 4 Nuclear blebs in the pole cells of CalHphora eggs during the cellularization of the blastoderm (18 min). Each bleb consists of an evagination of the outer nuclear membrane and contains a membranous vesicle. AL, Annulate lamella; B, bleb; N, nucleus; NE, nuclear envelope; M, mitochondrion; PG, polar granule. (A)Blebbing in an egg labelled with [ H]leucine. Exposure time: 9 weeks. (B) Bleb in an unlabelled egg. The black dot is an artifact.

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12 9 A. LUNDQUIST AND H. EMANUELSSON the amino-acid label should be attributed to translation of stored messenger RNA. In Calliphora, the polar granules are substantially labelled with [ H]- leucine, especially peripherally, when the label of the pole cells is high. In this respect, by showing a label comparable to the ground plasm, polar granules differ from the somatic inclusions: the yolk region shows less label than the periplasm in Calliphora (unpublished) and Drosophila (Zalokar, 1976). The correlation between polar-granule fragmentation and amino-acid labelling is compatible with the idea (Mahowald, 1968, 1977) that stored mrna is released from the fragmented polar granules and later translated into proteins. The changes in amino-acid label are interpreted as local changes in protein synthetic activity. Alternatively, there is an exceedingly large, transient change in the leucine pool size or the permeability of the pole cells only. This is not likely. Moreover, pole cells acquire increased label only some time after their formation. As pointed out by Zalokar (1976), this indicates that the newly formed pole cells continue to share their amino-acid pools with the blastoderm. Some translocation of label to the polar granules may occur. But since the total amount of polar-granule material apparently does not increase before gastrulation (Rabinowitz, 1941; Counce, 196), such translocation is unlikely to be of major importance. The vesicle-containing nuclear blebs of the pole cells have not previously been recorded. Interestingly, the blebbing coincides with the increased amino-acid labelling in the pole cells and silver grains were sometimes seen near the blebs. But more evidence is difficult to obtain, as it is not yet possible to shorten the labelling pulse. The polar granules are thought to be responsible for pole-cell formation. When the polar granules in the Drosophila egg are removed from the pole plasm by centrifugation, no pole cells are formed at the posterior pole. However, the dislodged polar granules do not give rise to pole cells in other parts of the egg (Imaizumi, 1958; Jazdowska-Zagrodziriska, 1966). Obviously, direct evidence is, as yet, lacking. In some lower dipterans, certain chromosomes are eliminated from all somatic nuclei during cleavage, whereas the full chromosome set is retained in the pole cells, i.e. in the germ line. In gall midges, the polar granule material seems to protect the pole cells from chromosome elimination, but it is not needed for pole cells to form (Geyer-Duszyiiska, 1959; Nicklas, 1959; Bantock, 197). Chromosome elimination is not known in higher dipterans, but somatic loss of chromatin does occur. In Calliphora and Drosophila, a specific terminal chromosome fragment is lost in the early embryo, presumably from all somatic nuclei. In Calliphora, such fragments appear during the last three nuclear divisions of the syncytial blastoderm and in the gastrula (Melander, 196). Pseudochiasmata such as those believed to precede this chromosome diminution were previously observed in Drosophila. They probably affect the X chromosome (Rabinowitz, 1941). As a second working hypothesis, we tentatively suggest that the polar

13 Polar granules in Calliphora 91 granules prevent chromosome diminution in the pole cells of Calliphora and Drosophila. Chromosome diminution occurs after polar-granule fragmentation and coincides with the increased [ H]leucine label and the nuclear blebbing in the pole cells. Moreover, a unified hypothesis explaining the function of the polar granules in all dipterans should be appealing. The hypothesis is testable, because it offers cytological markers. The tools needed to determine the function (or functions) of the polar granules might soon become available. As yet, cell fractions enriched in polar granules have been isolated (Allis, Waring & Mahowald, 1977; Waring, Allis & Mahowald 1978) and culture methods for pole cells have recently been designed (Allis, Underwood, Caulton & Mahowald, 1979). Grants from the Magnus Bergvall Foundation and the Swedish Natural Science Research Council supported this work. We express our gratitude to Mrs Annagreta Petersen for invaluable technical aid, to Miss Inger Norling for printing the electron micrographs, and to Mrs Marianne Andersson for typing the manuscript. REFERENCES AGRELL, I. (1962). Mitotic gradients in the early insect embryo. Ark. Zool. 15, AGRELL, I. P. S. & LUNDQUIST, A. (197). Physiological and biochemical changes during insect development. In The Physiology of Insect a, vol. 1 (ed. M. Rockstein), pp New York: Academic Press. ALLEAUME, N. (1971). Contribution a 1'analyse experimentale des facteurs de la determination et de la differenciation des ebauches dans le germe des Dipteres superieurs (Calliphora erythrocephala Meig. et Drosophila melanogaster Meig.). Thesis, University of Bordeaux I, France. ALLIS, C. D., WARING, G. L. & MAHOWALD, A. P. (1977). Mass isolation of pole cells from Drosophila melanogaster. Devi Biol. 56, ALLIS, C. D., UNDERWOOD, E. M., CAULTON, J. H. & MAHOWALD, A. P. (1979). Pole cells of Drosophila melanogaster in culture. Normal metabolism, ultrastructure, and functional capabilities. Devi Biol. 69, BANTOCK, C. R. (197). Experiments on chromosome elimination in the gall midge, Mayetiola destructor. J. Embryol. exp. Morph. 24, BEAMS, H. W. & KESSEL, R. G. (1974). The problem of germ cell determinants. Int. Rev. Cytol. 9, CHEN, P. S., HANIMANN, F. & BRIEGEL, H. (1967). Freie Aminosauren und Derivate in Eiern von Drosophila, Culex und Phormia. Rev. Suisse Zool. 74, COUNCE, S. J. (196). Developmental morphology of polar granules in Drosophila. J. Morph. 112, COUNCE, S. J. (197). The causal analysis of insect embryogenesis. In Developmental Systems: Insects, vol. 2 (ed. S. J. Counce and C. H. Waddington), pp New York: Academic Press. DAVIS, C. W. C, KRAUSE, J. & KRAUSE, G. (1968). Morphogenetic movements and segmentation of posterior egg fragments in vitro {Calliphora erythrocephala Meig., Diptera). Wilhelm Roux Arch. EntwickMech. Org. 161, EDDY, E. M. (1975). Germ plasm and the differentiation of the germ cell line. Int. Rev. Cytol. 4, GEIGY, R. (191). Action de l'ultra-violet sur le pole germinal dans Pceuf de Drosophila melanogaster (castration et mutabilite). Rev. Suisse Zool. 8, GEYER-DUSZYNSKA, I. (1959). Experimental research on chromosome elimination in Cecidomyidae (Diptera). /. exp. Zool. 141, GRAZIOSI, G. & MICALI, F. (1974). Differential response to ultraviolet irradiation of the polar cytoplasm of Drosophila eggs. Wilhelm Roux Arch. EntwickMech. Org. 175,

14 92 A.LUNDQUIST AND H. EMANUELSSON HATHAWAY, D. S. & SELMAN, G. G. (1961). Certain aspects of cell lineage and morphogenesis studied in embryos of Drosophila melanogaster with an ultraviolet micro-beam. /. Embryo/. exp. Morph. 9, ILLMENSEE, K. & MAHOWALD, A. P. (1974). Transplantation of posterior polar plasm in Drosophila. Induction of germ cells at the anterior pole of the egg. Proc. natn. Acad. Sci., U.S.A. 71, ILLMENSEE, K. & MAHOWALD, A. P. (1976). The autonomous function of germ plasm in a somatic region of the Drosophila egg. Expl Cell Res. 97, ILLMENSEE, K., MAHOWALD, A. P. &LOOMIS, M. R. (1976). The ontogeny of germ plasm during oogenesis in Drosophila. Devi Biol. 49, IMAIZUMI, T. (1958). Recherches sur l'expression des facteurs letaux hereditaires chez l'embryon de la Drosophile. VI. Une experience de centrifugation sur l'oeuf de la mouche sauvage. Cytologia 2, JAZDOWSKA-ZAGRODZINSKA, B. (1966). Experimental studies on the role of 'polar granules' in the segregation of pole cells in Drosophila melanogaster. J. Embryol. exp. Morph. 16, KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 17A-18A. LAMB, M. M. & LAIRD, C. D. (1976). Increase in nuclear poly (A)-containing RNA at syncytial blastoderm in Drosophila melanogaster embryos. Devi Biol. 52, LIMBOURG, B. & ZALOKAR, M. (197). Permeabilization of Drosophila eggs. Devi Biol. 5, LUNDQUIST, A. & EMANUELSSON, H. (1979). Membrane production and yolk degradation in the early fly embryo (Calliphora erythrocephala Meig.): an ultrastructural analysis. J. Morph. 161, MAHOWALD, A. P. (1962). Fine structure of pole cells and polar granules in Drosophila melanogaster. J. exp. Zool. 151, MAHOWALD, A. P. (1968). Polar granules of Drosophila. II. Ultrastructural changes during early embryogenesis. J. exp. Zool. 167, MAHOWALD, A. P. (1971). Polar granules of Drosophila. IV. Cytochemical studies showing loss of RNA from polar granules during early stages of embryogenesis. /. exp. Zool. 176, MAHOWALD, A. P. (1977). The germ plasm of Drosophila: an experimental system for the analysis of determination. Am. Zool. 17, MELANDER, Y. (196). Chromatid tension and fragmentation during the development of Calliphora erythrocephala Meig. (Diptera). Hereditas 49, NICKLAS, R. B. (1959). An experimental and descriptive study of chromosome elimination in Miastor spec. (Cecidomyidae, Diptera). Chromosoma 1, 1-6. NOACK, W. (191). Beitrage zur Entwicklungsgeschichte der Musciden. Z. wiss. Zool. 7, OKADA, M., KLEINMAN, I. A. & SCHNEIDERMAN, H. A. (1974). Restoration of fertility in sterilized Drosophila eggs by transplantation of polar cytoplasm. Devi Biol. 7, PETERS, T., Jr. & ASHLEY, C. A. (1967). An artefact in radioautography due to binding of free amino acids to tissues byfixatives. /. Cell Biol., 5-6. PIETRUSCHKA, F. & BIER, K. (1972). Autoradiographische Untersuchungen zur RNS- und Proteinsynthese in der friihen Embryogenese von Musca domestica. Wilhelm Roux Arch. EntwickMech. Org. 169, POULSON, D. F. & WATERHOUSE, D. F. (196). Experimental studies on pole cells and midgut differentiation in Diptera. Aust. J. biol. Sci. 1, RABINOWITZ, M. (1941). Studies on the cytology and early embryology of the egg of Drosophila melanogaster. J. Morph. 69, 1-5. SCHWALM, F. E., SIMPSON, R. & BENDER, H. A. (1971). Early development of the kelp fly, Coelopa frigida (Diptera). Ultrastructural changes within the polar granules during pole cell formation. Wilhelm Roux Arch. EntwickMech. Org. 166, SHAW, E. I. (1956). A glutamic acid-glycine medium for prolonged maintenance of high mitotic activity in grasshopper neuroblasts. Expl Cell Res. 11,

15 Polar granules in Calliphora 9 WARING, G. L., ALLIS, C. D. & MAHOWALD, A. P. (1978). Isolation of polar granules and the identification of polar granule-specific protein. Devi Biol. 66, WARN, R. (1975). Restoration of the capacity to form pole cells in UV-irradiated Drosophila embryos. J. Embryol. exp. Morph., ZALOKAR, M. (1976). Autoradiographic study of protein and RNA formation during early development of Drosophila eggs. Devi Biol. 49, (Received 6 November 1979, revised 16 January 198) EMB 57

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