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/. Embryol. exp.Morph. Vol. 33, 3, pp. 789-80], 1975 789 Printed in Great Britain A photographic study of development in the living embryo of Drosophila melanogaster By MARY BOWNES 1 From the University of Sussex, Brighton, England SUMMARY The changes which can be seen occurring during the development of a living embryo of Drosophila melanogaster are described in detail, and represented photographically as a series of developmental stages. This provides an easy, but accurate technique for selecting eggs at precise developmental stages for experiments. INTRODUCTION The Drosophila embryo is proving to be an extremely useful organism for studying many aspects of development. The initial rapid synchronous divisions of free nuclei within the yolk provides an excellent situation for studying DNA replication in higher organisms. The recent advances in techniques for injection of Drosophila embryos (Zalokar, 1971; Ulmensee, 1972, 1973; Zalokar, 1973; Okada, Kleinman & Schneiderman, 1974a, b, c) open many paths of study. Maternal-effect embryonic lethals can be cured by injection of normal cytoplasm containing the missing nutrients (Garen & Gehring 1972; Okada et ah 19746). Subsequent biochemical analysis can identify the actual substances involved. The state of determination of nuclei and cells of the embryo can be tested by transplants between eggs. The egg can also be used as an assay system for the determinative properties of nuclei and cytoplasm from somatic cells. The inductive property of polar plasm causes the formation of functional pole cells even when placed at the anterior of the egg (Ulmensee & Mahowald, 1974); the system can therefore be used as a biochemical assay for the determinants of pole cell formation. Injection also provides the possibility for studying basic problems of Drosophila embryology, such as induction of endomitotic chromosome duplication in larval cells. Substances which were previously unable to penetrate the impermeable egg membranes can now be injected at a specific site, time and concentration. For example, colchicine has been used to indicate the sites of endomitotic duplication by injection into embryos at various developmental stages (M. Madhavan, personal communication). 1 Author's address: Center for Pathobiology, University of California, Irvine, California 92664, U.S.A. 790 M. BOWNES Poulson (1937, 1950) and Sonnenblick (1950) have studied Drosophila embryology in great detail. Bull (1952) has studied head involution. Ede and Counce (1956) prepared a cinematographic study of normal development in living embryos and described how each of the developing systems, e.g. germband extension and gut formation, can be seen to change in the living embryo, and Imaizumi (1958) divided normal development into 20 stages and briefly described the changes within each stage. This study is primarily designed to describe, step by step, the changes in external appearance of the developing embryo and to relate these changes briefly to the internal morphological movements known to be occurring in the embryo. It provides a method for selecting eggs at exact developmental stages for experiments, rather than relying on timed collections and subsequent ageing of the eggs. This is necessary since there is so much variation in the development time between different stocks, or even within a single stock under certain experimental conditions, e.g. temperature variation. It also provides a useful way to recognize the stage at which an egg begins to develop abnormally in mutant stocks or after experimental treatment. It should be remembered, however, that development is a continuous, dynamic process, and that stages are merely useful 'labels', helpful in identifying eggs, and in simplifying detailed descriptions. It remains essential for a fuller understanding of the development of the Drosophila embryo to refer to the detailed work of the authors cited above. MATERIALS AND METHODS Eggs from Oregon R females were collected at 25 °C on agar plates coated with yeast paste. They were immediately dechorionated with 3 % sodium hypochlorite for 5 min, then placed on a slide in 0-9 % sodium chloride. The coverslip was supported with pieces of a second coverslip to prevent bursting of the egg. The photographs in this paper (taken on a Zeiss Universal compound microscope) represent the visible changes seen in the living embryo. The stages are related to the development time at 25 °C for comparison with previous accounts of Drosophila embryology. For further observations or experimentation, the particular stages can be selected by submerging a number of eggs in 0-9 % sodium chloride, or in paraffin oil, and then viewing them with transmitted light under a good dissecting microscope. However, mounting the eggs as described above and observing them under a compound microscope produces better photographs than the dissecting microscope. RESULTS Embryonic development Stage 1: 0-JA(Fig. 1) When the Drosophila egg is laid, it is protected by the vitelline membrane and the chorion. A detailed description of these membranes and how they are laid 791 Development o/Drosophila embryo M VM PC Stage 3 Stage 2 Stage 1 Cl.F. of Syn.Bl. Stage 5 a 792 M. BOWNES down in oogenesis can be found in King (1970). The chorion can easily be removed by mechanical or chemical methods and development within the egg can then be readily observed. The egg is approximately 0-5 mm long and 0-2 mm wide. The ventral side is rather convex and at the anterior is a small protuberance of the vitelline membrane called the micropyle, through which sperm enter during maturation divisions of the egg nucleus. At the surface of the egg is a thin layer of periplasm, which surrounds the granular yolk mass. When first laid the living egg appears to be of a uniform density. Staining, however, reveals a posterior region of polar plasm containing RNA located in polar granules. Approximately one-third from the anterior of the egg the nucleus begins its synchronous divisions. This is referred to as the nuclear multiplication stage. Stage 2: i - i A (Fig. 1) The embryo shortens within the vitelline membrane leaving clear gaps at the anterior and posterior poles. Within the egg, nuclei are dividing every 10 min. Stage 3: 1-2 h (Fig. 1) After eight nuclear divisions, the nuclei begin to migrate to the surface of the egg, which appears to become granular. A number of pole cells (3-7) can be seen to be pushed off from the yolk edge at the posterior pole. These pole cells continue to divide as development proceeds. Stage 4: 2-2\ h (Fig. 1) Cleavage furrows begin to be visible at the surface as membranes form around the nuclei. This is the syncytial blastoderm stage and the cell membranes gradually extend inwards giving the egg surface a ruffled appearance. Stage 5: 2±-3 h (Figs. 1,2) The cell membranes continue to extend and a columnar layer of partially formed cells is visible around the surface of the egg. The pole cells can be clearly distinguished by their round shape and lie outside the blastoderm at the posterior pole. Stage 6: 3-3% h (Fig. 2) Cell membrane formation is complete and stage 6 a is referred to as the cellular blastoderm stage. The first visible movements are the infolding of the ventral furrow. This appears as a thickening of the blastoderm layer at the mid-region of the ventral surface and indicates the commencement of gastrulation. Histology has shown that the surface cells form the ectoderm and the inner cells the mesoderm of the germ band. Development o/Drosophila embryo 793 Stage 6 Stage 5 b Stage 7 a AMR Stage 7 b Stage 8 a 794 M. BOWNES Stage 7: 3^4\ h (Fig. 2) Invagination of the posterior midgut rudiment can easily be followed in the living embryo. The blastoderm at the posterior pole changes in shape and the posterior midgut rudiment pushes along the dorsal side carrying with it the pole cells. As this proceeds, several infoldings can be seen between the invagination point and the anterior pole. Simultaneously a lateral cleft begins to form towards the anterior of the ventral furrow and the cephalic furrow is completed. The anterior midgut rudiment invaginates during stages 7 b and 7c at a point along the ventral surface anterior to the cephalic furrow. Stage 8: 4^5\ h (Figs. 2, 3) The posterior midgut pocket continues to move forward dorsally as the germ band extends and can be seen to turn into the embryo. The stomodeal rudiment invaginates at a similar position to the anterior midgut invagination in stage 7 c. Stage 9: 5^-8 h (Fig. 3) There is little visible change in the embryo at this time. As the fore- and hindgut rudiments continue to fold into the embryo, it appears light on the outside with an inner dark region. The beginning of some segment formation is seen towards the end of this period. Stage 10: 8-9 h (Figs. 3,4) The head segment material is invaginated; the opening of the stomodeum makes the head region very distinct. The segmentation becomes more distinct ventrally. A dark central patch of yolk becomes narrower at the posterior of the embryo and spreads to the dorsal edge approximately half-way along the anterior-posterior axis. At this point the embryo has a distinct gap between its edge and the vitelline membrane. Germ-band shortening commences towards the end of this period. Stage 11: 9-11 h (Figs. 4, 5) Segments appear dorsally near to this yolk region. The gap between the head and thorax moves forward as head involution begins. The visible changes in the living embryo are the results of germ-band shortening, dorsal closure, and head involution. The segmentation spreads all along the dorsal edge as the yolk region becomes less distinct. The lighter region located along the ventral line is the ventral nervous system. Stage 12: 11-14 h (Figs. 5,6) The head gradually becomes enclosed as the thoracic segments move forward, leaving a clearly visible single mouth opening to the frontal sac and the pharynx. The sac is broad at the anterior and half-way back it begins to narrow to a point Development o/Drosophila embryo Stage 8 b Stage 9 a 795 Stage 9 b HS Stage 9 c Stage 10 a 796 M. BOWNES Staae 10 b Staue l()c Static lOd VNS -DS Stage 11 a Stage 11 c Development o/Drosophila embryo Stage 11 d Stage 11e 797 Stage 12 a MG I Stage 12 b Stage 12 d 798 M. B O W N E S Stage 13 b Sp. Stage 13 c Stage 13 e Development oj Drosophila embryo Stage 14 a Stage 14 b 799 Stage 14 c MH ThS Abd.S. Lateral view Ventral view Dorsal view Stage 14 d Stage 14 e Stage 14 f Fig. 7 800 M. BOWNES at the posterior. A constriction gradually appears in the centre, dividing the yolk mass into an anterior squarish sac and a posterior cone-shaped sac. Stage 13: 14-17 h (Fig. 6) The sacs continually change in shape and more and more gut coils appear in the abdomen as the yolk is gradually digested. The spiracles are clearly visible at this point. Active movements also begin during this period. Stage 14: 17-22 h (Fig. 7) Paired tracheal tubes form and gradually become air-filled. Many small branches radiate from the tracheae which run latero-dorsally. The mouth parts chitinize and can be seen from dorsal or ventral views. The eight segmental boundaries of the abdomen are marked by rows of chaetae ventrally. The three thoracic segments are less distinctly marked, but quite clearly visible. When all the yolk has been absorbed the larva finally hatches by tearing the vitelline membrane at the point of the micropyle using its mouth hooks. This work was supported by a Medical Research Council studentship and the Science Research Council. My thanks to Professor J. H. Sang who provided valuable advice throughout this work. REFERENCES BULL, A. L. (1952). Embryonic lethality produced by over-lapping deficiencies at the vestigial locus. Ph.D. Thesis, Yale University. EDE, D. A. & COUNCE, S. J. (1956). A cinematographic study of the embryology of Drosophila melanogaster. Wilhelm Roux Arch. EntwMech. Org. 148,402-415. GAREN, A. & GEHRING, W. (1972). Repair of the lethal developmental defect in deep orange embryos of Drosophila by injection of normal egg cytoplasm. Proc. natn. Acad. Sci. U.S.A. 69, 2982-2985. ILLMENSEE, K. (1972). Developmental potencies of nuclei from cleavage, preblastoderm and syncytial blastoderm transplanted into unfertilized eggs of Drosophila melanogaster. Wilhelm Roux Arch. EntwMech. Org. 170, 267-298. ILLMENSEE, K. (1973). The potentialities of transplanted early gastrula nuclei of Drosophila melanogaster. Production of their imago descendants by germ-line transplantation. Wilhelm Roux Arch. EntwMech. Org. \1\, 331-343. ILLMENSEE, K. & MAHOWALD, A. P. (1974). Transplantation of posterior polar plasm in Drosophila. 1. Induction of germ cells at the anterior pole of the egg. Proc. natn. Acad. Sci. 71, 1016-1020. IMAIZUMI, T. (1958). Recherches sur l'expression des facteurs letaux hereditaires chez Vembryon de la Drosophile. V. Sur l'embryogenese et le mode des letalites au cours du developpement embryonnaire. Cytologia 23, 270-285. KING, R. C. (1970). Ovarian development in Drosophila melanogaster, pp. 25-27. New York: Academic Press. OKADA, M., KLEINMAN, I. A. & SCHNEIDERMAN, H. A. (1974a). Restoration of fertility in sterilized Drosophila eggs by transplantation of polar cytoplasm. Devi Biol. 37, 43-54. OKADA, M., KLEINMAN, I. A. & SCHNEIDERMAN, H. A. (19746). Repair of a geneticallycaused defect in oogenesis in Drosophila melanogaster by transplantation of cytoplasm from wild-type eggs and by injection of pyrimidine nucleosides. Devi Biol. 37, 55-62. OKADA, M., KLEINMAN, I. A. & SCHNEIDERMAN, H. A. (1974c). Chimeric Drosophila adults produced by transplantation of nuclei into specific regions of fertilized eggs. Devi Biol. 39, 286-294. Development 0/Drosophila embryo 801 POULSON, D. F. (1937). The embryonic development of Drosophila melanogaster. Exposes de Genetique 498, 1-54. POULSON, D. F. (1950). Histogenesis, organogenesis, and differentiation in the embryo of Drosophila melanogaster, Meigen. In Biology o/Drosophila (ed. M. Demerec), pp. 168-270. New York: Hafner. SONNENBLICK, B. P. (1950). The early embryology of Drosophila melanogaster. In Biology of Drosophila (ed. M. Demerec), pp. 62-163. New York: Hafner. ZALOKAR, M. (1971). Transplantation of nuclei in Drosophila melanogaster. Proc. natn. Acad. Sci. U.S.A. 68, 1539-1541. ZALOKAR, M. (1973). Transplantation of nuclei into the polar plasm of Drosophila eggs. Devi Biol. 32, 189-193. {Received 23 September 1974) ABBREVIATIONS Abd.S. AM AMR Bl. CF CI.F. DS H HS M MG MM JV P Abdominal segment Anterior midgut Anterior midgut rudiment Blastoderm cells Cephalic furrow Cleavage furrow Dorsal segmentation Hindgut Head segment Micropyle Midgut sac Mouth hook Nuclei Pharynx PC PM PMR PR Sp. St. Syn.Bl. T ThS VF VM VNS VS Y Pole cells Posterior midgut Posterior midgut rudiment Proctodeal invagination Spiracles Stomodeum Syncytial blastoderm Tracheae Thoracic segment Ventral furrow Vitelline membrane Ventral nervous system Ventral segmentation Yolk