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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.
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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
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Development o/Drosophila embryo
M
VM
PC
Stage 3
Stage 2
Stage 1
Cl.F. of
Syn.Bl.
Stage 5 a
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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
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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
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Stage 9 b
HS
Stage 9 c
Stage 10 a
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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
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Stage 12 a
MG
I
Stage 12 b
Stage 12 d
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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
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Stage 14 c
MH
ThS
Abd.S.
Lateral view
Ventral view
Dorsal view
Stage 14 d
Stage 14 e
Stage 14 f
Fig. 7
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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:
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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.
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Development 0/Drosophila embryo
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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
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SONNENBLICK, B. P. (1950). The early embryology of Drosophila melanogaster. In Biology of
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ZALOKAR, M. (1971). Transplantation of nuclei in Drosophila melanogaster. Proc. natn.
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{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