J. Embryo!, exp. Morph. Vol. 23, J, pp. 53-69, 1970
Printed in Great Britain
53
Characteristics of
five cell types appearing during in vitro culture
of embryonic material from Drosophila
melanogaster
By G L E N S H I E L D S 1 AND J A M E S H. SANG 1
From the School of Biology, University of Sussex, England
Although attempts have been made over a number of decades to achieve
successful tissue culture of Drosophila material (see Schneider, 1964), progress
has been held back until recently by a lack of basic information on the chemical
and physiological characteristics of the haemolymph of the organism necessary
for a reasoned formulation of a culture medium. In 1963, Begg & Cruickshank
published details of the mineral composition, osmotic pressure and pH of the
haemolymph of third instar larvae, and this has provided the basis for a more
satisfactory approach, as reflected in an increasing amount of fruitful work
since reported. Much of this work has been done with embryonic material.
Horikawa & Fox (1964) claimed continuous multiplication of a small type of
early embryonic cell; Lesseps (1965) described re-aggregation of dissociated
embryonic cells in culture, with possible development of muscle cells, nerve
cells and oenocytes in the aggregates; in the same year, Echalier, Ohanessian &
Brun (1965) noted the emergence of free contractile elements and a polygonal type
of cell in embryonic cultures, with the later development of a fibroblastic
type of cell ; Gvozdev & Kakpakov (1968) reported the appearance of a small type
of cell growing in multilayer clumps as well as of fibroblast- and epithelialtype cells, and Seecof & Unanue (1968) reported the development of free
neuron-like cells and muscle cells. Results are presented below of a further
investigation into the behaviour of Drosophila melanogaster embryonic cells in
culture, and the characteristics of five distinct cell types are described. These
comprehend the types mentioned above (and to some extent there is necessarily
repetition of the earlier work) but, as well as bringing the previously isolated
observations into a single study, greater detail is given of the form and mode of
development of the elements, and some account of the dynamic activities of
some of them is included.
1
Authors'1 address: School of Biology, University of Sussex, Brighton, England.
4-2
54
G. SHIELDS AND J. H. SANG
MATERIALS AND METHODS
Eggs from a cross between the wild-type Oregon S and tu bw: st su-tu strains
of Drosophila melanogaster were used as the source of embryonic material.
Several hundred pairs of flies were allowed to lay for 2 h at 25 °C on killed
yeast paste medium to eliminate old eggs, and then for a further 2 h on fresh
medium for the true collection of 500-1000 eggs. These were incubated at 25 °C
for 6 h to give 6-8 h embryos, thoroughly washed into distilled water to get
rid of most of the yeast, and suspended in 30 % sucrose solution and allowed
to float to the surface to separate from the remaining yeast. They were dechorionated and surface-sterilized by holding for 10 min in a freshly prepared
and filtered saturated solution of calcium hypochlorite. On occasion 1-3 h,
10—12 h or 14-16 h embryos were used, and they were handled in the same
manner.
Cell suspensions were prepared by homogenizing and dissociating the
eggs in a salts/trypsin solution containing 0-30 % NaCl, 0-29 % KCl, 0-09 %
NaH 2 P0 4 .2H 2 0, 6-00 % sucrose and 010 % Difco trypsin, made up in doubly
distilled water and brought to pH 6-9 with 1 % NaOH. Working under sterile
conditions, the eggs, after thorough washing with doubly distilled water to
remove the hypochlorite, were homogenized lightly in 1 ml of this solution
(using a 5 ml glass-teflon homogenizer with a 0-15-0-25 mm clearance), centrifuged down at 800 rev/min for 3 min and washed twice with fresh lots to remove
subcellular debris, and resuspended in a further 1 ml of solution at 25 °C for
10 min, with occasional shaking, to dissociate. The dissociated cells were then
spun down and suspended in about 1 ml of the culture medium. Preparation of
the eggs for dissociation took about I h. The trypsinization inhibited further
development of the embryonic cells for 3-4 h, appearances of differentiated
elements in the cultures being delayed about that much compared with nontrypsinized cultures.
The dissociation achieved was generally fairly good, but clumps of cells of
various sizes always remained. For this reason accurate cell counts on the
suspension could not be made. A range of three dilutions was usually used, the
first being made sufficiently dense to give a nearly confluent monolayer when
the cells settled out, and the second and third being at i-x and £x this
concentration.
The culture procedure used was a modification of the hanging-drop method
(employing depression slides with cavity 15 mm diameter and 0-8 mm depth),
with the drop made sufficiently large and compact to make contact with
the floor of the depression chamber when the latter was lowered on to it. The
column of liquid so formed was generally quite stable as long as the drop was
not made too large, and provided greatly improved conditions for phasecontrast microscopy. Slides were kept inverted for 1 h after mounting to allow
the cells to settle out and adhere to the coverslip; but not longer, to avoid
Drosophila embryonic cells
55
settling out of subcellular debris. They were then incubated, in the dark, at
25 °C.
Medium changes, generally performed every 3 days, were rendered slightly
more difficult by the column-drop method of culture, but could be accomplished
without loss of too many cultures by carefully swivelling the coverslip to free
one of its corners, lifting by this to break the column and free the coverslip
completely, removing the half of the drop left in the depression slide and placing
an equal volume of fresh medium on the half left on the coverslip, and reforming the column by replacing the slide in the original way.
Table 1. Composition of the culture medium (mg/100 ml)
Salts
MgS0 4 .7H 2 0
CaCI 2 .6H 2 0
KCl
NaCl
NaH 2 P0 4 .2H 2 0
KHC03
Organic acid salts
Monosodium malate 2H 2 0
Monosodium a-ketoglutarate
Disodium fumarate
Disodium succinate 6H 2 0
Amino acids
Monosodium L-glutamate
L-Aspartic acid
L-Threonine
L-Serine
L-Asparagine
L-Glutamine
L-Proline
513
174
313
86
88
18
95
42
8
14
246
15
50
35
30
60
40
L-Glycine
L-a-Alanine
L-Valine
L-Methionine
L-lsoleucine
L-Leucine
L-Tyrosine
L-Phenylalanine
L-/?-Alanine
L-Histidine
L-Trytophan
L-Arginine
L-Lysine
L-Cystine
L-Cysteine
Glutathione
Sugar
Glucose
Vitamins, etc.
TC Yeastolate (Difco)
50
165
42
12
27
40
26
24
10
55
10
50
68
20
80
0-5
460
200
Made up in doubly glass-distilled H20, and pH brought to 6-9 with 1 % NaOH. Sterilized
by milliporefiltrationand foetal bovine serum (Flow Laboratories) added to 10%.
The culture medium had the composition given in Table 1. This gives the
metal ions at close to their levels in third instar haemolymph, with correction
made in the case of Na + and K+ for the introduction of the serum. The pH is
slightly above the haemolymph level, and osmotic pressure (equivalent to about
1-00% NaCl) slightly below. The amino acid composition is based on the
analyses of Chen & Hanimann (1965) for the free amino acid content of wholelarva extracts of first instar larvae.
RESULTS
The cells settle out and attach by 1 h, and when first viewed are beginning to
flatten down. They continue to do this over the first few hours, and by 3 h the
56
G. SHIELDS AND J. H. SANG
cell form is fairly distinctive. At this time (3 h) most of the cells are fairly
similar, varying somewhat in size, but nearly all rather angular, with quite
densely granular cytoplasm, and a large, clear nucleus often showing a nucleolus. The angularity is due to a number of slender, tapering, sometimes simply
branched, processes of up to 1 cell-length long, which are often further extended
into fine filaments of clear cytoplasm which may be up to 3 cell-lengths long.
The latter are usually unbranched and straight, and show probing and exploratory movement over the coverslip surface. The processes are constantly being
withdrawn and put out in new directions, or completely retracted with new ones
extended, and the cells as a whole show continuous change of shape, often
exhibiting a tendency to assume an elongated or spindle form, with a single
process from one end.
A proportion of the cells appear to be somewhat larger than average, with
more heavily charged cytoplasm, a more rounded outline and more rhizoidallooking processes (i.e. shorter, stouter and more branched), and these may
represent an earlier type of embryonic cell, since they are more numerous in
cultures from 3-5 h embryos.
A third type of cell occasionally seen, emerging from undissociated cell
masses at 1-3 h after mounting, has a large bulbous body, with dense, rather
heavily granulated cytoplasm, a large nucleus with a quite distinct nucleolus,
and long, slender, tentacle-like processes that are put out only in the direction
in which the cell is travelling. These cells emerge and creep rapidly away into
the surrounding medium—moving up to 3 cell lengths in I h—but they soon
become stationary, and seem generally to degenerate within a few hours.
The cells as a whole remain active up to about 6 h after mounting, but then
increasing numbers begin to withdraw their processes, round up and fall away
(as a fair number do from the beginning), so that by 12 h the cultures have
thinned out considerably. A high proportion of cells remain viable, however,
merely retracting their fine filaments, perhaps partly rounding up and becoming
more quiescent, and amongst these the cell types to be described begin to appear.
Five main types are eventually recognizable: a neuron-like type, a muscle
type, an epithelial type, a macrophage-like type and a fibroblast type. They are
dealt with separately below.
Neuron-like cells
The most easily recognized of the cell types are the neuron-like cells. The
chief distinguishing feature of these is one or more long (up to 20 cell-lengths)
nerve-like cytoplasmic threads. In their mature form (Fig. 1B) the cells are uniformly small, round and grey-looking, consisting essentially of a nucleus (with
a not very clear nucleolus) surrounded by only a very thin layer of cytoplasm,
most of the latter being extended into the threads. They are usually monopolar
(giving off one main thread), but some are bipolar, and a few multipolar.
The threads adhere closely to the coverslip, follow an irregular path, and at
their simplest are unbranched and of uniform narrow width. More usually,
Drosophila embryonic cells
In Figs. 1-4, unless otherwise stated, all cells derived from 6-8 h embryos.
FIGURE 1
(A) Neuron-like cells at 18 h of culture. Cells still rather flattened and angular.
(B) Neuron-like cells at 3 days of culture. Cell body rounded and thread system
well developed.
(C) Clumps of neuron-like cells at 3 days of culture, showing connecting thread
tracts.
(D, E) Small neuron-like cells from 14-16 h embryos at 4 and 6 h of culture, showing pulling together by connecting threads.
57
58
G. SHIELDS AND J. H. SANG
however, they show a simple form of branching, with a thicker main axis giving
off a few primary branches which have a number of finer offshoots, and some
are very complexly branched. Often there are rows of fine laterals on either side
of part or the whole of the main axes, and there may be varicosities along the
length of the thread. Cross-connexions often exist between the branches of a
single cell, and between the branches of different cells, and sometimes direct
main strand linkage between cells is seen. Very complex networks may be built
up. In addition, where cells are closely clustered together, their threads are very
often gathered into thick tracts.
The neuron-like cells, which form a high proportion of the surviving cells,
mostly begin to appear between 6 and 12 h after mounting, and continue to
mature over the next few days. The ones which appear earlier arise from cells
that are initially somewhat smaller and rounder than the normal embryonic
cells (i.e. closer to the final body form), and develop merely by putting out and
extending their threads over a period of hours. The majority, however, seem to
derive from more typical-looking embryonic cells that pass through an angular
phase and assume the final form by the cell body becoming smaller and rounder
as the thread system extends. There appears to be division of some of the larger
embryonic cells to give more of these cells during the first 12 h after mounting.
At 12 h most of the cells are of a rather intermediate appearance, with a
well-developed though rather fine thread system, but with the cell body still
somewhat flattened, sometimes angular and with a fair amount of cytoplasm
around the nucleus (Fig. 1 A). During the next few days the thread networks
increase in extent and complexity, and, although most isolated cells observed
do not appear to alter the main position of their threads once these are laid
down, merely thickening and extending the latter, there seems to be some
gathering in of free cells into clumps. Actual pulling together of free cells has
been observed in some cases, with a cell putting out a thread (sometimes a
number radially) which shows slow random movement over its surroundings
until it makes contact with another thread or cell, when it thickens and seems
to fuse with the latter, and is pulled in by shortening and tautening of the bond
so formed. Movement together of cells as far apart as 3-4 cell-lengths over a
period of 3-4 h has been seen.
This pulling together mostly occurs in the early stages of development, and
perhaps partly accounts for the fact that many of the neuron-like cells occur in
small monolayer groups or multilayer clusters. Most multilayer clusters derive
from the division of the larger embryonic cells already described, and from the
clumps of cells present in the initial suspension. These aggregates readily form
thread tracts, which develop strongly over the first 3 days of incubation, thickening, lengthening and eventually joining up the clusters (Fig. 1 C). The tracts
often seem to grow straight out towards nearby clusters, and sometimes appear
to change direction to grow more directly towards one, indicating a possible
tropic attraction. On reaching a cluster they link with it and straighten still
Drosophila embryonic cells
59
further by shortening and tautening as in the case of the individual cells described above. The tension set up often pulls cell clusters together, or into a
position of balance between two or more opposing tracts.
With medium change the thread networks continue to develop over the
4th and 5th days, and in thick cultures reach extreme degrees of complexity.
Both threads and cells then survive well, but show inevitable slow deteriorative
changes and losses, and are gradually reduced over the next few weeks. Few
persist beyond 7 weeks.
The yield of neuron-like cells is greatly increased when 1-3 h embryo cultures
are used, the majority of surviving cells seeming to give rise to them. Then, because
of the lower proportion of other cell types developing, and the tendency of these
to degenerate soon and fall away, they may eventually come to survive in practically pure culture.
With 10-12 h embryos, there are initially many more of the near mature—
small and round—cells that develop merely by extending the threads, and thread
network formation begins sooner. With yet older (14-16 h) embryos (where the
cell yield is considerably reduced owing to binding of many into tightly integrated
organ systems which are eliminated in preparation of the suspensions), actual
mature neuron-like cells are present from the beginning. These have the typical
neuron-like body, consisting of a nucleus with only a very thin layer of cytoplasm, but usually no, or very weak, threads. They remain active, however,
extending their sparse cytoplasm into characteristic though limited thread
systems, and show the pulling together of isolated elements earlier described
(Fig. 1D,E).
Muscle cells
The muscle cells (Fig. 2 A) are long spindle-shaped elements, with a narrow,
elongated attachment process at either end and a somewhat widened central
region containing an ovoid nucleus with quite a prominent nucleolus. Their
cytoplasm is quite clear, containing relatively few large, dark granules. Some
have more than two attachment processes, the cell body then tending to be
pulled out into triangular or polygonal form, and the processes may be somewhat flattened and expanded. They occur free or in strings and sheets of a
number of cells, often with the individual elements orientated to lie alongside
one another.
As with the neuron-like cells, they begin to appear between 6 and 12 h, the
majority seeming to derive from cells which are perhaps slightly more darkly
granulated than the average embryonic cell, and which develop by flattening
down yet further and extending long, slender processes that attach to the coverslip and pull the cell out into the elongated form. By about 15 h most have
attained the typical form, and there is little further increase in numbers after
this; the cells are then quite numerous, though not as many as the neuron-like
cells. Contractile movement is slight or absent to begin with (15 h), but becomes
stronger and more widespread up to about 36 h.
60
G. SHIELDS AND J. H. SANG
With continued incubation, most of the cells become leaner and stringierlooking, with the nucleus then appearing more prominent, but they survive
with good movement for up to about a week. Increasing numbers then begin to
lose their movement and deteriorate more seriously, and few last longer than
2-3 weeks. However, some cells show further development from about 24 h on,
expanding rather than shrinking, and coming to form broad, sheet-like ele-
FIGURE 2
(A) Normally developed muscle cells at 24 h of culture.
(B) Sheeted muscle cells at 8 days of culture. Central cell binucleate; right-hand
cell with a binucleolate nucleus.
Drosophila embryonic cells
61
ments (Fig. 2B). These are usually apparent by the 3rd day and, although
never very numerous, increase in numbers up to about 7 days. In them, the cytoplasm is very much flattened and almost granule-free (seeming to lose many
of its granules as it develops), and the nucleus contains a very prominent
nucleolus.
From about the 5th day, binucleate forms begin to appear among these
sheet elements, and a few tri- and multinucleate ones, and the nuclei become
increasingly binucleolate (with one of the nucleoli usually slightly smaller and
fainter than the other). The nuclei are generally situated closely together, in the
centre of the cell, as if nuclear division had just taken place, but it seems likely
that most of the binucleate forms derive from fusion of cells, with the nuclei
then moving together, as has been seen to happen.
The expanded muscle cells survive well, maintaining both their condition
and their movement up to about 2 weeks, when they begin to shrink a little,
and take on rather a worn look, before they decline slowly. Most have gone
by the end of the 3rd week after their appearance.
Epithelial-type cells
In contrast to the muscle cells, the epithelial cells are large, flat polygonal
cells, with a centrally placed nucleus containing a very prominent nucleolus,
and cytoplasm heavily charged with large, dark granules (Fig. 3B). They
develop free or in small groups, but owing to their wide expansion in the mature
form usually come to form continuous monolayer sheets. Perhaps owing to their
greater sensitivity to cultural conditions, they are rather variable in the frequency of their appearance, and though they can be very numerous—much
more than the muscle cells—they are usually about as common as the latter.
The epithelial-type cells develop rather more slowly than the neuron-like or
muscle type, and, though a few mature ones are present at 12 h, they do not
become common until between 24 and 48 h. From 12 h on, they seem mostly
to derive from largish, dense, rather rounded cells, with a large nucleus and
quite prominent nucleolus, the origin of which from earlier embryonic cells is
not yet clear. These cells develop by flattening down and spreading out to a
recognizable, but still rather compact, polygonal form by around 24 h, and
then continuing to expand to the full size by about 48 h, with the cytoplasm
becoming more thickly dark-granulated and the nucleolus more prominent
during the whole period.
Where the original elements are closely clustered together, the developing
cells often early assume a broad-spindle form, and shift out from their initial
site as they expand, travelling in the direction in which they are pointing. This
movement may partly be due to pressure from the expanding inner cells of the
cluster, but seems also to involve slight motility of the cells, which put out a
single slender process ahead of them, and perhaps pull themselves out by it.
They never move very far, however, remaining in close contact with one another.
G. SHIELDS AND J. H. SANG
FIGURE 3
(A) Very early appearing epithelial-typecells at 3 h of culture. Cells at an early stage
in their development, with long and short processes extended.
(B) Epithelial-type cells of Fig. 3 A at 13 h of culture. Cells, now in normally developed form, have pulled together to give a continuous sheet.
(C) Epithelial-type cells of Fig. 3 A at 36 h of culture. Some of cells developing
beyond normal form to enlarged forms.
(D) Enlarged epithelial-type cells at 3 days of culture, with non-granulated cytoplasm and rhizoidal extensions.
Drosophila embryonic cells
63
Some very early maturing cells develop by flattening out to an angular form
with a number of processes, which may help to pull the cell out into its polygonal shape, and here the cells may actually pull together as they expand
(Fig. 3A, B).
Having attained their full size, most of the epithelial-type cells begin to decline
at the same rate as they developed, thus seeming to go through a continuous
cycle of development and decline. From 48 h on, they shrink back from their
expanded state to a rounder and denser form, fill increasingly with highly
refractile spheres (perhaps oil-droplets), and eventually detach. Few good cells
remain beyond the 5th day.
However, as with the muscle cells, some show development beyond the
normal common form, continuing to expand, elongating in certain directions,
and exhibiting a more irregular cell border (Fig. 3C). The cytoplasm then
becomes clearer, owing to dispersion of the dark granules, and the nucleolus
even more prominent (close observation of the free edges of the cells in this
condition reveals them to be in a constant state of slow movement due to continuous extension and retraction of short, finger-like projections). With yet
further development, elongation of the cytoplasm may extend it into broad
arms, these may become rhizoidal-looking owing to the production of short,
branched processes, and the cell may become practically granule-free (actually
appearing to lose most of the dark granules) (Fig. 3D). In a few cases, the expansion and flattening reaches such an extent that the cell becomes barely visible
in outline against the background.
These very large cells generally reach their maximum extension at about 5 days,
then remain seemingly unchanged up to about the 8th day, and contract and
are lost by around the 11th day.
Macrophage-like
cells
Like the epithelial cells these are wide, very much flattened cells, of circular
outline, with a centrally placed nucleus containing a prominent nucleolus, and
with cytoplasm which is granular immediately around the nucleus, but otherwise clear (Fig. 4B). They are much less numerous than the three previous
types of cell, and occur singly; only in very thick cultures do they become
sufficiently common to form small continuous monolayer sheets.
They are also slow to develop, not usually appearing before 24 h, and then
increasing slightly in numbers over several days. Their early derivation is not
known, but from 24 h on they seem mostly to develop from bulky, rather
shapeless cells, often with large vacuoles in the cytoplasm, which flatten down
on to the coverslip and put out a narrow peripheral border of clear cytoplasm
which slowly widens to the full expansion over 5-6 h. The development is not
continuous, however, and cells may stop or partly retract the border, and then
carry on widening. In the fully expanded state also, the border may be retracted
and re-extended—all round or locally (to give irregularity of outline), with the
64
G. SHIELDS AND J. H. SANG
FIGURE 4
(A) Early macrophage-like cells at 24 h of culture. Lower one in pseudopodal
form, upper one beginning to spread out and assume disc form.
(B) Macrophage-like cells at 3 days of culture. Somewhat larger than normal, and
with more clearly defined and regular outline.
(C) Fibroblast-type cells at 8 days of culture.
(D) Same cells as Fig. 4C 1 h later, to show the movement and change of form
of the cells.
Drosophila embryonic cells
65
leading edge thickening and darkening or thinning and lightening—being in
fact in a continuous state of slow movement; and the passage of wave-like
thickenings over the body of the cell may be seen. When fully expanded, as with
the developed epithelial-type cells, the outline of the cell may be difficult to
define.
The macrophage-like cells are variable in their survival, some shrinking
back and degenerating almost as soon as they have formed but others maintaining themselves well for 2-3 weeks. Many melanize—sometimes very heavily—
from the beginning, with or without prior rounding up of the cells, this seeming
particularly to occur where culture conditions are bad (e.g. crowded).
With the first medium change (at 3 days), there may be a further increase in
the number of the cells, and larger ones may begin to appear. Towards the end
of the first week, usually in thick cultures, giant ones, of 2 x to 3 x the normal
diameter, sometimes form. These are often bi- or multinucleate, and then
perhaps arise by fusion of normal cells, as with the bi- or multinucleate sheet
muscle cells. Very occasionally super-giant, or syncytial, forms are seen, with a
diameter of about 2 x the giant ones, and containing up to 10 nuclei, and these
probably derive from fusion of the giant forms.
Medusoid types—with the cell fringed all round with filaments of varying
length—may also appear at the same time as the giant forms. These possibly
result from shrinkage of large cells, with points on the cell periphery left
attached and so drawn out into the filaments, and cases have been seen of cells
expanding immediately after a medium change to obliterate the filaments and
become again smooth in outline, but in some instances the filaments appear to
be too long to have been formed in this way, and they seem able to alter their
length and position to some extent.
The giant and medusoid forms usually survive well for about 2 weeks, and
then tend to contract and round up to large, greyish, dead-looking bodies, which
ultimately detach by the end of the third week.
Fibroblast-type cells
The fibroblast-type cells differ from the other types in their very delayed
appearance in the cultures. They do not usually begin to show until about the
12th day of incubation, then arising by emergence from undissociated cell
clumps. Their development is best followed using non-trypsinized cell-suspensions, and seems to proceed better in hanging-drop rather than column-drop
preparations.
The cells are characterized by extreme mobility and plasticity. Thus, although
in general of fairly uniform medium size—with a large nucleus containing a
prominent nucleolus, and cytoplasm dense-looking but granule-free—and tending to assume an elongated form, they show wide variety in degree of extension
and shape, and are constantly changing these (Fig. 4C, D). They usually have
one or more processes, of variable thickness and length, and sometimes simply
66
G. S H I E L D S A N D J. H. S A N G
branched, extended at any one time. They appear in variable, though usually
small, numbers (some cultures never develop them at all), and form constantly
altering strands, open networks and occasionally epithelial-looking sheets.
In their emergence, the cells appear either as individual elements projected
radially all round the clump of origin, or as a tongue of compacted cells adhering closely together—so closely as to appear syncytial. The latter may remain
intact, creeping forward as a unit, but usually the cells soon begin to separate
and become more expanded. They generally retain some contact with one
another as they extend out, however—hence the strand and network patterns—
and only occasionally do individual cells break right away and migrate out
independently into the medium, then usually assuming a long, slender, lanceolate form, and often soon degenerating.
Cell movement, and change of shape, is by extension of broad or narrow
processes, into which the cells flow or by which they are drawn forward, at a
r a t e visible u n d e r the m i c r o s c o p e , a n d occasionally cells send o u t very l o n g
(4-5 cell-lengths), fine, seemingly probing processes along which the nucleus and
cell contents may sometimes be seen to pass as a wide bulge.
With regular medium change the cells survive well—over 10 weeks in some
cases—but they seem very sensitive to changes in their environment and may
contract back and be lost soon after their emergence.
DISCUSSION
The studies described above relate mainly to 6-8 h embryos, and further
work with younger and older embryos is needed before definite conclusions can
be reached regarding the relationship of the initial embryonic cells to one another
and to the cell types that derive from them. We have therefore concerned
ourselves primarily with an account of the developed cell types as they first
appear and as they later develop, and with the identification of these with differentiated cells in the larva.
The neuron-like cells are presumably the 'nerve cells' of Seecof & Unanue
(1968), and are perhaps identical with the small, multilayered cells of Gvozdev
& Kakpakov (1968) and the small embryonic cells of Horikawa & Fox (1964).
The retractile behaviour of their threads does not preclude them from being
nerve, since developing vertebrate nerve cells show the same activity (Nakai,
1964). However, other similar cells have been described, e.g. the primary
mesenchyme cells of embryonic sea-urchin (Gustafson & Wolpert, 1961) and
the neoblasts associated with regeneration in Planaria (Betchaku, 1967). In the
intact Drosophila embryo, neuroblasts differentiate early (4-4£ h at 22-23 °C),
and show intense mitotic activity with a rapid division cycle between 5 and 9 h.
The first nerve fibres are found at about 10 h (Poulson, 1950). The neuroblasts
are large cells which divide with unequal cytoplasmic division to 'bud off'
small cells which may undergo at least one further mitosis before becoming
Drosophila embryonic cells
67
ganglion cells (Poulson, 1950). These changes correspond to what we might
expect to find if our dividing large cells were neuroblasts (proportionately
commoner in early embryos) and the small cells were pre-ganglion and ganglion
cells.
Horikawa & Fox (1964) and Gvozdev & Kakpakov (1968) report multiplication of the small cells in their cultures. As we have noted above, some of our
initial large cells probably undergo division to give clusters of the small cells
which differentiate into neuron-like cells, but this does not last beyond about
the first 12 h, and the small cells do not show continued multiplication.
Development of muscle cells in embryonic Drosophila cultures was noted by
Echalier et al. (1965) and by Seecof & Unanue (1968), and the latter authors
reported the change from spindle forms to enlarged multinucleate or syncytial
types. In the intact embryo, muscle cells appear by about 8-9 h, when cell
groups which will form the muscle blocks are set aside by subdivision of the
mesoderm. Elongated myogenic elements are recognizable by 10 h, when they
attach to developing apodemes, fuse to give multinucleate muscle fibres and, by
13 h, show co-ordinated muscular activity. We should expect our initial cultures
to contain cells close to the differentiated state: they develop through all the
expected stages of differentiation, attachment and association, and show prolonged muscular activity. We have no evidence that the primary muscle cells
divide, as occurs with similar vertebrate cells. Syncytial muscle sheets from late
(10-12 h) embryos do not adapt to the culture medium, and degenerate within
a day or so.
Echalier et al (1965) and Gvozdev & Kakpakov (1968) describe epithelialtype cells, but we are uncertain if these are the same elements as we have found.
These authors have not noted the later expansion of some of the cells, which
has not been referred to in the general literature on insect cells in culture. This
change may be an artifact due to culture conditions. We have been unable to
relate the epithelial-type cells to any late embryonic or larval structures, but
similar-looking cells are found in formed tubules (tracheae?) from preparations
of 14-16 h embryos, and they also somewhat resemble the cells of the posterior
mid-gut.
The macrophage-like cells have not been reported previously in Drosophila
tissue culture work. Hirumi & Maramorosch (1964) found similar cells emerging
from in vitro expiants of embryonic leaf-hopper tissue, and they may also resemble the 'veil' cells which Wyatt (1956) found to emerge from larval ovarian
tissue of Bombyx mori. In their form and behaviour they seem very like vertebrate macrophages, which can also assume a flat disc shape in culture, combine in epithelium-like sheets, show waving movements of the membrane and
give giant forms which may become multinucleate through cell fusion (Jacoby,
1965), and this similarity is further apparent in the early stages (first 24 h) of
development of the cells, when some of them pass through a more typically
macrophagic form with pseudopodia (Fig. 4 A) before assuming the common
5
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68
G. SHIELDS AND J. H. SANG
disc appearance. In Drosophila, the only cells that at all resemble them in the
later organism are the lamellocytes of the haemolymph (Rizki, 1957), and
possibly the nephrocytes associated with the dorsal aorta and diaphragm
(Miller, 1950).
The fibroblast-type cells are similar to the cells which Echalier et al. (1965)
describe as emerging at around 15 days in their embryonic cultures. They
believe these cells spread by division, although they had difficulty in detecting
mitoses. We also find that these very active cells appear late, apparently by the
differentiation of pre-existing elements present in the cell masses. Careful
search of film records shows no sign of division of free cells. We have been
unable to relate the cells to any larval tissue.
No attempt was made to follow cell division in our cultures using radioactive
labelling techniques. In the early stages of culture a number of cells divide for a
short period, and would be labelled, but later differentiation of many seems to
occur without further division, as we have described. Since the fate of any
particular early cell is not easily followed, we cannot be certain that division
always immediately precedes differentiation. Certainly differentiation is often
long delayed (e.g. fibroblast-type cells), but it is not clear how far this is a
consequence of the culture conditions.
SUMMARY
1. A culture medium is described which permits differentiation of Drosophila
embryonic cells.
2. The primary form and behaviour of five distinct types of cell appearing in
in vitro cultures of material from 6-8 h embryos of Drosophila melanogaster is
reported. The cell types are: neuron-like, muscle, epithelial, macrophage-like
and a fibroblastic type.
3. The initial appearance of the embryonic cells from which these cell types
derive is indicated, together with their later development during culture for a
period of weeks.
RÉSUMÉ
Caractéristiques de cinq types cellulaires qui apparaissent au cours de la
culture in vitro de matériel embryonnaire de Drosophila melanogaster
1. Un milieu de culture permettant la différenciation de cellules embryonnaires de Drosophila est défini.
2. La forme primaire et le comportement de cinq différents types de cellules
qui apparaissent in vitro à partir d'expiants prélevés dans des embryons de
Drosophila melanogaster à l'âge de 6-8 h sont décrits. Ces types cellulaires
sont: en forme de neurone, musculaire, epithelial, en forme de macrophage,
ainsi qu'un type fibroblastique.
Drosophila embryonic cells
69
3. L'aspect initial des cellules embryonnaires dont dérivent ces types cellulaires est indiqué, ainsi que leur développement ultérieur dans la culture au
cours d'une période s'étendant sur des semaines.
This work was supported by grants from the Agricultural Research Council and the
Science Research Council of Great Britain. We are indebted to Professor D. F. Poulson for
reading the manuscript and offering a number of useful comments and suggestions.
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{Manuscript received 10 March 1969)
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