/. Embryo/, exp. Morph. Vol. 45,pp. 161-172, 1978
Printed in Great Britain © Company of Biologists Limited 1978
Cell culture of individual Drosophila embryos
I. Development of wild-type cultures
By DAVID P. CROSS 1 AND JAMES H. SANG 2
From the School of Biological Sciences, Sussex University
SUMMARY
A new procedure is described for the preparation of in vitro cell cultures from individual
early gastrulae of Drosophila melanogaster. In these cultures several identifiable cell types
differentiate within 24 h (nerve, muscle, fat-body, haemocyte and chitin-secreting): their
initial appearance and continuing development over a period of weeks is described. It is
proposed that this technique may be used to analyse abnormalities of cellular development
in embryonic lethal mutants. Culture in vitro of cells from lethal embryos is seen to have two
broad roles: (1) to test the developmental capacity of individual cell types in a situation where
they are relatively free from possible deleterious interactions with other cell types and are
liberated from the system of the dying embryo, and (2) through the preparation of mixed
cultures from normal and mutant embryos, to determine the influence of the presence of
wild-type cells on observed abnormalities of a particular cell type.
INTRODUCTION
The primary aim of this paper and the accompanying report (Cross & Sang,
1978) is to establish that culture in vitro of cells from single embryos provides a
powerful methodology to aid in the analysis of embryonic lethal mutants of
Drosophila melanogaster. Reviews are available which discuss both the conclusions from, and prospects for, the study of such mutants (Wright, 1970;
Gehring, 1973). In considering the accumulated data, it is clear that little further
progress can be made by describing the development of observable abnormalities in sections of mutant material. In particular, the small size and fragility of
the Drosophila embryo have meant that an impasse has been 'reached in attempts
to expose by operative procedures the extent and nature of mutant produced
embryological changes' (Wright, 1970).
The procedure of culturing is a simple one and involves dissociating the cells
of a single early gastrula, prior to any overt differentiation, and following their
subsequent development in vitro over a period of up to three weeks: under ideal
conditions a range of identifiable cell types will then differentiate in relative
1
Author's address: Zoology Department, University of British Columbia, Vancouver,
Canada.
2
Author's address for reprints: School of Biological Sciences, University of Sussex, Falmer,
Brighton, U.K.
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D. P. CROSS AND J. H. SANG
isolation one from another. In theory, cell culture in vitro provides an analytical procedure which can address several of the questions which are frequently
asked of embryonic lethals; these questions are set out in turn below.
(1) Which cell types are primarily affected by a mutant lesion?
This question has been asked of almost all embryonic lethals that have been
studied in any detail (Wright, 1970) and, with the exception of those cases in
which lethality is prior to blastoderm formation (where the answer is trivial),
it has never been convincingly answered. For instance, the lethal Notch embryo
is the best-studied in Drosophila, but it is still unclear where the primary
abnormality is in a complex and dramatic syndrome of defects (see Wright
1970; Cross & Sang, 1978). The aim of cell culture is to compare the developmental potential of cell types of normal and mutant embryos in a situation
where they are relatively isolated, and in which, therefore, there is a limited
opportunity for those that are primarily abnormal to exert a deleterious influence on other cell types. This issue is raised for all of the mutants that have
been cultured, and in all cases the new data help to define a clearer answer (Cross
& Sang, 1978). It should be emphasized, however, that it is possible that interactions between cell types are necessary to some normal developments in
cultures in vitro, and that a primary abnormality of one cell type could block
a positive influence on another (see section (4) below).
(2) What long-term developments do mutant cells show when freed from the closed
system of the embryo ?
The significance of this question is especially clear when considering lethal
mutants which either display subtle abnormalities or no detectable defects at
the normal hatching time; subsequent cellular developments in such mutants
occur within the system of the dying embryo and are difficult to interpret. The
cell culture system can allow initially subtle defects to manifest themselves more
clearly in the long-term. This point is again important for all mutants studied,
but it is especially so for the white deficiency lethals which undergo an apparently
normal embryogenesis yet fail to hatch from the egg: several cell types from
this mutant initially develop normally in culture, but eventually show conspicuous abnormalities (Cross & Sang, 1978).
(3) Are abnormalities of a mutant cell autonomous?
If abnormalities are discovered in mutant cells, their autonomy can be tested
by preparing mixed cultures of mutant and wild-type cells. Ideally, a cell marker
system should be incorporated into such an experiment so that the genotype of
individual cells can be unequivocally determined; unfortunately, such a system
is not available at the present time. Mixed culture experiments have been used
to assess the autonomy of abnormalities in cultures of Notch and white deficiency
cells (Cross & Sang, 1978).
Cell cultures from Drosophila
163
(4) What roles do interactions between cell types play in normal development ?
This question would be interesting if, for instance, nerve and muscle cells
from a mutant failed to form neuromuscular junctions in culture (formation of
functional neuromuscular junctions has been demonstrated for Drosophila cells
in culture (Seecof etal. 1972)). With the availability of a reliable cell marker, it
would be possible to analyse this defect by preparing mixed cultures of wildtype and mutant cells thereby giving the opportunity for mutant nerve to form
junctions with wild-type muscle and vice versa. In the absence of a cell marker,
such experiments were not undertaken, but they would be desirable in the case
of the Notch mutants where one may speculate that mutant nerve acts to cause
muscle abnormalities.
]n the last few years there have been great improvements in procedures and
media for culturing embryonic cells. It has been shown that dissociated cells
from early gastrula-stage eggs can differentiate into several typical larval cell
types when cultured in vitro (Shields, Dubendorfer & Sang, 1975; Dubendorfer,
Shields & Sang, 1975); however, the system used for such work involves preparation of cells from a mass of embryos, and is unsuited to the study of
abnormalities likely to be found among the cells from embryos of balanced
lethal stocks, where only a fraction of the segregating population is of the
required genotype. Seecof and co-workers have prepared short-term (1-2 days)
cultures from single embryos (Seecof, Alleaume, Teplitz & Gerson, 1971;
Seecof & Donady, 1972) which have been used to study the early differentiation
of nerve and muscle cells (Seecof, Gerson, Donady & Teplitz, 1973; Seecof
Donady & Teplitz, 1973), no other cell types being identifiable. This paper
describes a method, developed from these earlier reports, of culturing the cells
of a single embryo that allows long-term (up to 3 weeks) observation of the
development of five identifiable larval cell types (nerve, muscle, fat-body, chitinsecreting and haemocyte). Detailed descriptions of the development of these
cell types are given. The column-drop cultures employed here give optical properties which are superior to those of the earlier Petri dish cultures (Seecof et ah
1971).
MATERIALS AND METHODS
A wild-type Oregon-K stock was used as a standard for defining the single
embryo culture method.
Using standard procedures, eggs were collected during a 1-2 h period, aged for
about l | h at 25 °C, dechorionated and surface-sterilized with hypochlorite,
washed thoroughly with sterile doubly-distilled water, and transferred to culture
medium in a sterile plastic Petri dish. Methods of selecting and dissociating
embryos were based on those of Seecof et ah (1971). Eggs showing the first signs
of gastrulation, as signalled by the appearance of the amino-proctodeal imagination (Sonnenblick, 1950), were chosen and each embryo was transferred with
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D. P. CROSS AND J. H. SANG
a fine tungsten loop to the surface of a drop of sterile culture medium in a siliconized glass Petri dish. After further ageing, generally for a period of 45 min
at approximately 22 °C, each egg was removed from its drop by touching it with
the side of a micropipette which had an internal tip diameter of 50-70 /im and
was filled with culture medium. The egg, which adhered to this micropipette
with a minimal transfer of fluid, was placed on a dry glass surface. So long as the
egg surface was dry, an embryo could be cleanly punctured with the micropipette and the contents withdrawn to leave a completely collapsed vitelline
membrane. The pipette contents were expelled into a small drop of culture
medium on a siliconized cavity slide, pipetted up and down once or twice to
give good cell dispersal, and the drop increased to a diameter of about 2 mm.
An untreated coverslip was lowered onto the drop and sealed into position with
petroleum jelly. The optimum culture volume was found to be about 2 /.d. The
cultures were then held inverted for about \ h so that the cells should settle and
attach to the coverslip. The cultures produced using these methods developed
better, and the preparations had superior optical qualities to those produced by
previous techniques.
A few cultures were prepared from embryos at the time of selection, without
any further ageing. With such embryos the first step, dissociation, presented the
greatest difficulties. In some cases good cell survival was achieved, but in others
almost complete cell breakdown was observed on culture; only a few cells had
differentiated by 24 h and further development was completely curtailed.
The culture medium was a modification of that described by Shields & Sang
(1977), and was designated M3(BF). It was found that omission of bicarbonate
from Shields and Sang's medium and raising the level of potassium glutamate
to 888 mg/100 ml gave more stable culture conditions for the small drops
employed here. Penicillin G (sodium salt, 500 units/100 ml) and streptomycin
sulphate (10 mg/100 ml) were included in the medium. Active foetal calf serum
was added to 10 %. Cultures were kept in the dark at 25 °C and were examined
24 h after initiation, and thereafter daily for 10 days. If the cultures were continued beyond this, the medium was replaced in part by lifting the coverslip,
thus breaking the column-drop, and replacing the medium (about one third of
the total) left in the well.
RESULTS
Over 350 cultures were prepared from the Oregon-K strain. Many variations
of procedures and conditions were tested during the course of the work. The
procedure outlined above was found to give the consistent appearance of several
identifiable cell types, and the development of each is described below. In
addition, brief mention is made of a number of 'cell types' which regularly
appeared during culture but could not be identified.
Cell cultures from Drosophila
165
Fig. 1. A phase-contrast micrograph of an area of a culture of Oregon-K cells at
25 h, showing numerous nerve axons (examples arrowed). In some areas axons are
gathered into 'bundles'.
• • • .•
Fig. 2. A myotube in a 24 h culture. This example contains at least seven nuclei
(arrowed).
Fig. 3. An apparently syncitial muscle element in a 24 h culture. Note that there are
several nuclei (two arrowed) and that there are no clear signs of dividing cell walls.
Nerve cells
Nerve cells were the most prominent cells in the wild-type cultures at 24 h.
They conformed to the descriptions given by earlier workers; i.e. small, round,
dark cells in which the nucleus was not normally clearly visible (where it was it
filled most of the cell body), and each producing one or more axonal threads. The
neuron cell bodies normally occurred in small clusters but single cells were
sometimes seen. Most cultures had a small number of larger neuron clusters
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D. P. CROSS AND J. H. SANG
Fig. 4. Group of muscle cells (or possibly one syncitial cell) in a 3-day culture. Note
the well-spread form of the cells. Two nuclei are arrowed.
which could be greater than 100 jam across; some of these appeared to consist
of nothing but nerve cells. Nerve axons had a complex organization, even by
24 h (Fig. 1). Over the following 10 days the fibres increased in number and
gathered together into bundles connecting neuron clusters. During this process
of fibre bunching, neuron clusters tended to come together, and axons which
had previously taken an erratic course over the coverslip were drawn straight;
it seems likely that axon shortening was involved.
Muscle cells
In wild-type cultures, muscle cells were of two main types at 24 h: myotubes
and mononucleate myocytes. Myotubes were the most common: they were striplike elements containing from 2 to 12 nuclei, with a smooth outline and prominent points of attachment to the glass substrate, or to a clump of tissue, at each
end. There was no sign of cell walls between the nuclei, which were usually
arranged in an orderly fashion and in the same plane of focus (Fig. 2). A proportion of the myotubes were rather short and spread out; in these the arrangement of the nuclei was rather disorderly. Mononucleate myocytes were present
either singly or in small clusters. In the case of the clusters, whilst they were
dissimilar to the organized myotubes, it was frequently impossible to say that
no fusion of cytoplasms had taken place. Indeed, in some cases there were clear
signs of a limited fusion (Fig. 3). Myocytes normally had two points of attachment to the substrate and were spindle-shaped; some cells had further points
of attachment and took diverse forms. At 24 h, muscular contraction, present
in both myotubes and myocytes, was usually quite weak and sporadic. Jn the
second and third days of culture, contraction became stronger and muscle
figures tended to spread out over the coverslip (myocyte clusters showed this
spreading most markedly and frequently revealed apparent fusion (Fig. 4)).
Observation beyond 24 h did not reveal any transformation of myocyte clusters
into orderly myotubes. After the third or fourth day the muscle elements showed
little further development, and from that time there seemed to be a progressive
Cell cultures from Drosophila
167
Fig. 5. A group of four fat-body cells at 24 h. Note the bright refractile droplets.
Fig. 6. The fat-body cells of Fig. 5 at 10 days.
Fig. 7. A 'colony' of haemocytes at 24 h.
reduction in the number visible in the cultures, some certainly detaching due to
the vigour of their contractions. Isolated active muscle was observed in the
third week, but rarely beyond that.
Fat-body cells
At 24 h fat-body cells also appeared in a variety of forms in the wild-type
cultures. They ranged from a rather spread epithelial state, through a more
condensed form, to small rounded cells, as described by Shields et ah (1975).
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D. P. CROSS AND J. H. SANG
Fig. 8. A group of dividing haemocytes at 8 days.
Fig. 9. A large area of haemocytes in a 17-day culture. Both dividing and
non-dividing forms are present.
In all, the nucleus and nucleolus were prominent, and the cytoplasm was
granular (Fig. 5). The more condensed forms contained a number of bright
refractile droplets, and by 2 days all fat-body cells had developed these bright
droplets and showed a general tendency towards greater rounding and condensation. By 3 days the most advanced cells had increased in size and had filled up
with enlarged droplets. This process of cell and droplet enlargement continued
until between the eighth and twelfth days of culture (Fig. 6). Almost all fatbody cells went through this maturation, but the size of the final product was
somewhat variable, even within a single culture. Mature fat-body cells seemed
to undergo a change in their adhesion properties; they were often seen to wander
over, or fall away from, the coverslip. Further changes were less regular and
could go in one of two directions: either the cytoplasm around the droplets
would clear and become transparent, or the fat contents would be lost to leave
a large, flat form with an irregular outline and prominent nucleus and nucleolus.
Cell cultures from Drosophila
169
Fig. 10. A chitin-secreting cell at 24 h. Note the central refractile body.
Fig. 11. A cluster of chitin-secreting cells at 35 days. Two (pigmented) inner
layers of chitin are arrowed.
Haemocytes
Haemocytes were first discernible as large, granular cells of rather irregular
shape in the wild-type cultures. The smooth cell outline was initially broken
by a few short processes, which eventually developed into a broad, clear,
membranous border around a granular centre. These cells could occur singly,
but large 'colonies' were frequently seen (Fig. 7). At 24 h a range of types, from
the condensed to the large, circular form, could be identified, and by 48 h the
spreading of the cells appeared to be essentially complete. Haemocyte numbers
were very variable: some cultures contained none or very few, whilst others had
50 or more. Those that had a large number generally showed one or more
'colonies'. Groups of cultures made up on different days frequently had
strikingly different numbers of haemocytes; the cause of this was not clear.
At 6 days, or later, cells of new morphology appeared among them - smaller,
condensed and rather grey in appearance (Fig. 8). Groups of these smaller cells
were seen to increase in number, though usually only for 3 or 4 days, after which
they generally reverted to the original larger form. On rare occasions cell multiplication was much more persistant with virtually all of the clear areas of a
culture filling up (Fig. 9). Haemocyte division was a variable event on the coverslip, but not on the bottom of the culture where it invariably happened. There,
phagocytic activity on the part of the colony could readily be seen. Multiplying
colonies could give rise, in the third week or so, to a further form, even larger
than the original type, which was characterized by a streaked surface and bright
granules around the nucleus. Haemocytes were sometimes seen to melanize and
this generally signalled a deterioration of the culture.
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D. P. CROSS AND J. H. SANG
Chit in-secret ing cells
Chitin-secreting cells made their first appearance within the first 3 days of
culture. They could, with difficulty, be identified after only 24 h as single cells
or clusters, with a bright, refractile body in the middle (Fig. 10). Over the next
week or so, these cells showed a steady enlargement, and, through greater
prominence of the refractile bodies, became more easily identified. At around
2 weeks (or up to 5 days earlier or later) inner capsules, which were presumed to
be chitin, were secreted (Fig. 11). In cultures which were successfully maintained
for a month, or more, this secretion process was repeated to give a further
capsule which surrounded the earlier one. Capsules sometimes became heavily
pigmented, especially where large numbers of chitin-secreting cells were involved.
On very rare occasions hairs were seen inside capsules.
Other cell types
Shields et al. (1975) identified two other cell types in their cultures: imaginal
disc and tracheal. The imaginal disc cells formed monolayer vesicles and the
proof of their identity rested on a metamorphosis test in vitro or in vivo (Dubendorfer et al. 1975). Tracheal cells formed monolayer sheets which were capable
of rolling up into tubes inside which a chitin layer was secreted.
Cellular vesicles were observed in the cultures of single embryos, but they
were usually short-lived, never achieved any great size and did not resemble
those described by Shields et al. (1975). Sheets of cells resembling tracheal cells
were observed in one culture, and they did roll up to form short tubes. This
differentiation, along with a number of other isolated occurrences which were
observed in the course of the work, was not pursued.
Further cell types did appear regularly, but they all remained unidentified.
Chief amongst these were several dividing cell types: a fibroblastic form which
appeared in small numbers from the seventh day, and a number of epithelial
cell types. These various types could become prominent in the cultures through
extensive division.
DISCUSSION
The results of culturing cells from single embryos in small, column drops are
comparable to those from cultures of cells from many embryos (Shields et al.
1975); essentially the same cell types differentiate, although not quite as regularly. Certainly there is some variability in cell density in cultures from single
embryos and this may account for the differences in their development. However, the regularity of appearance of nerve, muscle, fat-body, chitin-secreting
and haemocyte cells, and of their subsequent differentiation, demonstrates that
the technique is adequate for the descriptive study of these processes in embryonic
lethal mutants (Cross & Sang, 1978).
The identification of the various cell types depended on visual evidence alone.
The issue of identification has been investigated elsewhere (Seecof et al. 1972;
Cell cultures from Drosophila
171
Shields et al. 1975) and the only serious doubt concerns the cells which have
been labelled as haemocytes here.
The results show that cells taken from early gastrulae (a little over 4 h old at
22 °C) have the same qualitative capacity for differentiation in culture as do
those from 6-8 h embryos (as used in the cultures of Shields & Sang (1970)).
Counce (1973) reviewed earlier evidence and concluded that cells from the late
gastrulae would give the greater range of differentiated cell types. Seecof &
Donady (1972) have claimed that the competence of cells to differentiate in vitro
varies with donor embryo age, between the time of appearance of the aminoproctodealinvaginationandthe stage achieved 45 min later at 22 °C. They found,
for instance, that cells taken from embryos which were 30 min into gastrulation
produced ten times as much differentiation (measured by counts of axons and
pulsating myocytes) as did those from embryos on the point of gastrulation. It
is our feeling that their observations reflect the fact that the cells of the younger
embryos are more liable to disrupt during the dissociation procedure. This idea
is supported by ultrastructural observations which indicate that cytoplasmic
continuity exists between cells and the primitive yolk sac during early gastrulation in the Drosophila embryo (Rickoll, 1976).
The support of the Science Research Council is gratefully acknowledged. D.P.C. held a
S.R.C. research studentship. It is a pleasure to thank Glen Shields for making his invaluable
experience constantly available during the course of the work described in this and the
accompanying paper. David Shellenbarger, Tom Grigliatti and Ted Homyk generously gave
their time to many fruitful discussions during the preparation of the manuscripts.
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{Received 12 October 1977, revised 25 January 1978)