/ . Embryol. exp. Morph. Vol. 45, pp. 173-181, 1978
Printed in Great Britain © Company of Biologists Limited 1978
173
Cell culture of individual Drosophila embryos
II. Culture of X-linked embryonic lethals
By DAVID P. CROSS 1 AND JAMES H. SANG 2
From the School of Biological Sciences, Sussex University
SUMMARY
Results are reported from the culturing in vitro of cells from individual early gastrulae of the
following four groups of X-linked embryonic lethal mutants of Drosophila melanogaster. (1)
Notch lethals. Five Notch mutants were studied which have been reported to give similar abnormalities in whole embryos: the nervous system displays a three-fold hypertrophy as part of a
shift in the pattern of differentiation within ectodermal derivatives, and mesodermal derivatives
do not differentiate. An hypertrophy of nerve was found in cell cultures prepared from
embryos of all five mutants. In addition, four of the five alleles consistently gave abnormalities
of muscle differentiation: when compared to controls, Notch cultures had a reduced frequency of myotubes, and displayed unusual clusters of myocytes which had either failed to
fuse or had fused incompletely. Results from mixed cultures prepared from two embryos
were consistent with the autonomous expression of nerve and muscle abnormalities by
Notch-8 cells in the presence of wild-type cells. It is argued that the Notch locus has a direct
role in the differentiation of both nerve and muscle. (2) white deficiencies. Cells carrying
either of two deficiencies gave a clear-cut pattern of abnormalities: initial cellular differentiations were normal, but nerve, muscle and fat-body cells progressively deteriorated during
the culture period. Mixed cultures showed that wild-type cells could not 'rescue' mutant
muscle and fat-body cells; however, the status of the autonomy of mutant nerve abnormalities in these cultures was unclear. Both white deficiencies remove cytological band 3C1, and
this permits a comparison of results with those from cultures of cells from Notch-8 embryos
(also deficient for 3C1). Abnormalities displayed in cultures of the two types of mutant show
no overlap. Therefore no consistent cellular abnormality can be attributed to absence of
band 3C1. (3) lethal(l)myospheroid. In contrast to earlier observations on in vitro cell cultures
(Donady & Seecof, 1972) muscle was seen to differentiate, though its morphology was
extremely abnormal. Observations indicated that all cell types within the cultures had poor
properties of adhesion to a glass substrate. It is argued that the observed abnormalities are
not consistent with a mutant lesion which is restricted to the basement membrane (contra
Wright, 1960), and that all cell types carry a basic defect which may reside in the cell membrane. (4) shibirets alleles. Cultures of two temperature-sensitive lethal shibire alleles (shilsl,
shim) were normal at the permissive temperature of 22 °C. At the restrictive temperature
(29° C) early cell differentiation was normal but subsequent development was blocked. This
blockage could be partially reversed by shifting cultures to the permissive temperature after
as much as 10 days exposure to the high temperature. It is suggested that shits cells are mutant
in a process which is basic to several cell types.
1
Author's address: Department of Zoology, University of British Columbia, Vancouver,
Canada.
2
Author's address for reprints: School of Biological Sciences, University of Sussex, Falmer,
Brighton, England.
12
EMB 45
174
D. P. CROSS AND J. H. SANG
INTRODUCTION
The intention of this report is to demonstrate the utility of in vitro culture for
the characterization and analysis of cellular abnormalities in embryonic lethal
mutants of Drosophila melanogaster. The rationale behind this work has been
set out fully elsewhere (Cross & Sang, 1978). Results are described from the
culturing in vitro of cells from embryos bearing various X-linked lethal mutations. These mutations fall into four groups which are dealt with in turn below.
(1) Notch lethals
The chief abnormalities of embryos bearing extreme Notch alleles are
dramatic, and involve a three-fold hypertrophy of the nervous system at the
expense of several other ectodermal derivatives (chiefly the hypoderm) and a
complete failure of the differentiation of mesodermal derivatives (Poulson,
1940, 1945).
The results presented here show that for each of five Notch mutants which
were studied, an hypertrophy of neural tissue occurred within cell cultures of
single embryos. Four of the five mutants which were tested also gave muscle
abnormalities: at the most extreme (the cytologically deficient Notch-S) functional myoblasts were formed, yet normal myotubes were not observed. Mixed
cultures of Notch-8 and wild-type cells gave results which were consistent with
the autonomous expression of nerve and muscle abnormalities. Based on these
observations it is suggested that the Notch locus has a direct role in derivatives
of both ectodermal and mesodermal rudiments.
(2) White deficiencies
There have been differing opinions concerning the ontogeny and the cytology
of the embryonic lethal 'white deficiencies' (reviewed by Kaufman, Shannon,
Shen & Judd, 1975).
The results demonstrate that nerve, muscle and fat-body cells show abnormalities in single embryo cell cultures of both of two tested white deficiencies.
These abnormalities do not overlap those displayed by Notch-S cells which, in
common with white deficiency cells, lack cytological band 3C1. Therefore, no
consistent cellular phenotype has been shown to be associated with deficiency
for band 3C1, in contrast to the conclusions of Poulson (1940, 1945) derived
from studies of whole embryos. Abnormalities of muscle and fat-body cells are
autonomously expressed in the presence of wild-type cells, and, therefore, these
are probably direct effects of the mutations.
(3) lethal(l)myospheroid
Wright (1960) undertook the conventional embryological analysis oUethal(l)myospheroid (1(1) mys) and suggested that the notable pleiotropic effects of the
mutation were due to a delay in the formation of the basement membrane such
Cell cultures from lethal Drosophila embryos
175
that normal muscular contractions in its absence led to these multiple effects,
essentially for mechanical reasons. The results reported here show that, when
cultured in vitro l{l)mys cells show abnormalities which are consistent with
those observed in both sectioned material and in vivo cultures. However, the
observation that l{l)mys cells adhere poorly to a glass substrate provides further
information that suggests a new interpretation of the l(l)mys syndrome which
emphasizes a general cell membrane defect rather than one specific to the
basement membrane. It should be stressed that contractile muscle was observed
in our l{l)mys cultures since Donady & Seecof (1972) prepared cultures from
l{l)mys cells and found that muscle did not differentiate; this latter result was,
however at variance with observations made on whole embryos, which show
that functional muscle is clearly formed.
(4) shibirets lethals
Grigliatti, Hall, Rosebluth & Suzuki (1973) isolated six alleles of the shibire
(sin) locus (general designation shiis). Homozygous stocks of all the mutant
alleles are essentially normal at 22 °C, whereas at 29 °C the most severe alleles
give reversible larval and adult paralysis; in addition exposure to 29 °C during
critical periods of development causes lethality (including embryonic lethality).
The present studies were intended to reveal the long-term effects of exposure
to the restrictive temperature on individual shits cell types. The results show
that cultures of shitsX and shim cells display similar and dramatic abnormalities
when exposed to the restrictive temperature. The early differentiation of shits
cells at 29 °C is qualitatively normal, but subsequent development of all identifiable cell types is blocked. For example, the continued proliferation of nerve
axons and the full maturation of fat-body cells is impeded at the restrictive
temperature. This blockage may be reversed, at least partially, by shifting
cultures to the permissive temperature after as much as 10 days exposure to
the high temperature. It is suggested that shits cells are mutant in a process which
is basic to several, and possibly all, cell types.
MATERIALS AND METHODS
Flies were raised on a standard cornmeal, yeast, sugar and agar medium.
Unless otherwise noted, full details of all mutations and balancer chromosomes are given in Lindsley & Grell (1968). The following embryonic lethals
and special X chromosomes were used:
(1) Notch mutants: Notch-S {Ns), an 18-band deficiency; N264'39, of doubtful cytology, it may be a one-band Notch locus deficiency (Slizynska, 1938), or
else may not be visibly deficient (Welshons, 1965); N264-40, N264-47, cytology
indicates no visible deficiency.
(2) white deficiencies: w25S~42, a 13- to 15-band deficiency; w25S~45, probably
176
D. P. CROSS AND J. H. SANG
associated with two deficiencies which together remove seven bands (Kaufman
et al. 1975).
(3) lethal{l)myospheroid (l(l)mys).
(4) shibirets mutants: shitsl and shits3, extreme shits alleles with similar properties (Grigliatti et al. 1973; Poodry, Hall & Suzuki, 1973; Kelly, 1974; Kelly &
Suzuki, 1974); shits alleles were kept as homozygous stocks.
(5) Various X chromosome balancers: FM1, FM4, FM7b (Merriam &
Duffy, 1972), dl-49, suppress crossing over in heterozygous females, male
fertile and homozygous female sterile.
In balanced stocks females were heterozygous for a lethal mutation and a
balancer, and males were hemizygous for a balancer; only one quarter of the
eggs produced by such stocks are expected to be lethal (hemizygous lethal
males). The hatchability of eggs produced by each lethal stock was determined
(Wright, 1973). In no case was a significant deviation from the expected
frequency of lethality observed: 25 % in the case of balanced lethals, and 100 %
for shits eggs at 29 °C.
Eggs generally were collected by standard procedures from stocks kept at
22° C. However, the normal methods of egg collection produced very poor
results with shits females. In this case eggs were collected at 29 °C from paralysed females (Cross, 1975); this procedure did not affect embryonic viability.
All eggs were incubated at 22 °C, after collection and prior to further handling.
Whole lethal embryos of all balanced stocks were allowed to develop for
26-30 h after laying (at 25 °C), and were then examined under phase-contrast
microscopy using standard procedures. Hemizygous l{l)mys embryos displayed
a pattern of abnormalities which was as described previously (Wright, 1960).
Notch lethal embryos were as expected (Poulson, 1940, 1945), except that
individual embryos bearing each of the five alleles occasionally showed muscular
contraction in the region of both the dorsal hypoderm and the rudimentary
fore- and hindgut structures. The white deficiency embryos were as described
by Kaufman et al. (1975): the great majority of embryos died as apparently
complete larvae which failed to hatch.
Standard single embryo cell cultures were prepared from early gastrulae as
described by Cross & Sang (1978). All cultures, with the exception of those of
shits (see Results), were incubated at 25 °C and examined at daily intervals for
the first 5 or 6 days, and thereafter every 2 days.
Cell counts were undertaken in cultures of Notch embryos: nerve and muscle
cells were counted at 24 h of culture, fat-body cells at 48 h. All distinct clusters
of neuron cell bodies were regarded as nerve units and, with the aid of an ocular
grid, were counted at 100 x magnification over an area of 0-31 mm2 (chosen
blind). This method gave equal weight to isolated cells and very large clusters,
but was quick and easy to perform. The number of large clusters of neuron cell
bodies (arbitrarily defined as having some dimension greater than 35 jam) also
was noted separately within the same field. All distinct muscle figures were
Cell cultures from lethal Drosophila embryos
111
Table 1. Mean values of counts in cultures made from the stock
N8/FM1
Phenotype of culturefj
Type of element
Notch
Normal
Neuron clusters
Large neuron clusters
Muscle figures
Myotubes
Fat-body cells
154± 13
14-3 ±1-9**
18 1 ± 19*
0**
28-8 ±5-5
152 + 7
5-4 ±0-7
110 ± 2 0
7-7 ±1-4
310±6-6
I Eleven phenotypically Notch cultures were scored together with an equal random sample
of normal cultures; mean values are given ( +standard errors).
\ The non-parametric Mann-Whitney ranking test (Snedecor & Cochran, 1967) was
applied to the data summarized in this and all subsequent Tables.
* Difference between Notch and normal cultures significant at the 5 % level.
** Significant difference at the 1 % level.
scored as muscle units. Again this method gave equal weight to isolated cells
and large clusters. Muscle counts were made at 200 x magnification and an area
of 016 mm2 was scored for each culture. The same area of the culture was also
scored for distinct myotubes or groups of myotubes. Fat-body cells were scored
at 200 x magnification over an area of 016 mm2.
For cell cultures prepared from two embryos, the culture volume was doubled.
Such cultures were given an aeration at 5 days by briefly removing the coverslip
and then replacing it. Counts were made of nerve and muscle cells and the procedures used for single embryos were followed. Neurons were scored in an area
of 0-62 mm2, and muscles in an area of 0-31 mm2.
In one experiment, shitflX cells were cultured according to a bulk embryo
procedure (Shields, Dubendorfer & Sang, 1975), employing column-drops
prepared from an homogenate of approximately 500 embryos in medium
M3(BF) (Cross & Sang, 1978). Embryos were dissociated at 5-6 h after laying
and all cultures incubated at 22 or 29 °C from 6 to 7 h.
RESULTS
Notch lethals
Detailed observations were made on cultures prepared from stocks of all five
Notch lethals. In order to limit the data presented here, oniy the abnormalities
of the identified cell types which were consistently observed will be mentioned.
Full details may be found elsewhere (Cross, 1975). Results from four of the
lethals are grouped together, N€0°n being dealt with separately.
A total of 341 single embryo cultures were prepared from stocks of the main
group of four lethals and this number was made up as follows: NS/FM1, 120;
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D. P. CROSS AND J. H. SANG
Table 2. Mean values of counts in cultures made from the stock
Phenotype of culturef
Type of element
Notch
Normal
Neuron clusters
Large neuron clusters
Muscle figures
Myotubes
Fat-body cells
188± 12
13-5 ±1-0**
230 ± 2 0
6-7 ±1-6**
22-8
158 ± 13
5-5 ±0-9
201 ±1-2
12-4± 1-1
29-5
t Ten phenotypically Notch cultures were scored together with an equal random sample
of normal cultures.
Ns/dl-49, 20; Ns/FM7b, 39; N264-39/FM4, 68; N264-*°/FM7b, 50; N264~47/FM7b,
44. In the case of each stock a class of abnormal cultures was observed and the
frequency of such cultures never significantly differed from 25 %. Notch cultures
at 24 h displayed a two- to three-fold increase in the number of large clusters
of neuron cell bodies (Fig. 1): example data for Ns and N264~39 is presented in
Tables 1 and 2. In no case was there any indication of an increase in the overall
number of neuron clusters. The development of nerve in Notch cultures showed
no qualitative variation from that of normal cultures.
Muscular abnormalities provided the most reliable criterion for the identification of Notch cultures at 24 h. Ns cultures were the most abnormal in this
respect and did not display any muscle figures which could be positively identified as myotubes, whereas they did have a number of apparent myocyte' bundles'
which were not observed in normal cultures (Fig. 2). These myocyte 'bundles'
were, in fact, used as the identifying criterion for Notch cultures. Numbers of
muscle figures were higher in N8 than in normal cultures (Table 1) and this was
probably a consequence of the failure of individual muscle cells to form myotubes. Although organized myotubes were not seen in Ns cultures, it was
impossible to rule out myoblast fusion at 24 h. In fact, at 2 days when the
muscle cells tended to spread out, groups which showed signs of fusion were
often noted. Ns muscle contracted normally and showed no other departure
from the normal pattern of development.
Cultures ofN264~39, N264~40 and Nm~47 showed muscle development which was
between that of Ns and normal cultures. At 24 h small numbers of myotubes
were present, but the unusual 'bundles' of myotubes were consistently noted.
When compared to controls, myotubes were about half as frequent in Notch
cultures (example data for N2U~39 in Table 2).
Fat-body cells were present in normal numbers in Notch cultures at 48 h
(Tables 1 and 2). A delay in fat-body maturation was displayed by Ns cultures
but this was not consistently true in cultures of the other three lethals.
Cell cultures from lethal Drosophila embryos
179
Fig. 1. A large cluster of neuron cell bodies in a N8 culture at 24 h. The bar in this
and other Figures represents 20 fim.
Fig. 2. (A) A myocyte 'bundle' in a N8 culture at 24 h. Three nuclei are arrowed.
(B) Three myotubes in a control culture at 24 h. Three nuclei are arrowed.
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D. P. CROSS AND J. H. SANG
Table 3. Mean values of counts in cultures made from pairs of
the stock N 8 /FM7b
Phenotype of culture!
Type of element
Neuron clusters
Large neuron clusters
Muscle
figures
Myotubes
Mixed Afote/j/Normal
323 + 18
17-2±20*
28-8 + 2-4*
9-9 ±1-2*
Normal
341 ± 19
1 1 0 ± 1-2
21-0 ± 2 1
15-2 ±1-6
t Eleven phenotypically mixed Notch/normal cultures were scored together with an equal
random sample of normal cultures.
In experiments with the stock N60oll/FM7b it was not possible to unequivocally identify an abnormal class of cultures, since no myocyte 'bundles' were
observed. Some cultures were similar to those of the other Notch lethals in the
development of nerve, but no other abnormalities were detected. Pooling all
the data on neuron development, 76 % of Ns, N264-39, N264'40 and N264~47
cultures gave a count of 10 or more large neuron clusters, whereas only 8-5 %
of normal cultures gave a score of 10 or more. Of the 65 cultures from N60°n/
FM7b, 14 (21-5%) gave a count of 10 or more large neuron clusters. It
therefore seems likely that about 25 % of the cultures were derived from
jyeogii hemizygotes, and that these cultures did show abnormally large quantities
of nervous tissue.
In order to investigate the autonomy of Ns abnormalities in the presence of
wild-type cells, 26 cultures of two embryos were made from the stock Ns/FM7b.
One-sixteenth of the cultures were expected to involve two hemizygous Ns
embryos, three-eighths to involve one Nshemizygote and one non-lethal embryo,
and the remainder two non-lethal embryos.
All of the cultures which were set up had clear examples of myotubes, and it
seems likely that no culture involved two Ns embryos. Each of 11 cultures
displayed 'bundles' of myocytes and these were the mixtures of Ns and wildtype embryos. In these cultures large neuron clusters were increased and myotubes reduced in number relative to a sample of apparently normal cultures
(Table 3); and increase in overall muscle counts was again observed.
white deficiency lethals
A total of 41 cultures of the stock w25S~45/FM4 gave 8 (19-5 %), showing a
clear pattern of abnormalities (i.e. derived from w25S~45 hemizygotes); similar
abnormalities were displayed by 6 out of 24 (25 %) cultures of the stock
w25S~42/dl-49.
white deficiency cultures appeared to be relatively normal at 24 h, with the
exception that muscular contractions were weak and very infrequent. By 3 days
Cell cultures from lethal Drosophila embryos
181
Fig. 3. Three muscle cells in a l(l)mys culture at 24 h. Note the unusual granular
appearance. All these cells showed weak pulsations.
the three major cell types, nerve, muscle and fat-body, showed clear signs of
deterioration. Over the next 5 days this deterioration progressed steadily until
all neuron cell bodies had a swollen appearance and muscle and fat-body cells
had completely degenerated. Indeed the lethal cultures showed no positive
developments after the second day: for instance, there was no sign of either new
axon production by the nerve cells or accumulation of fat by the fat-body cells.
Twenty-two cultures were made from the mixed cells of two embryos of the
stock w25S-45/FM4. In one of them, all nerves, muscle and fat-body cells developed abnormally, and this was presumably a culture of two mutant embryos.
Nine cultures showed classes of both normal and abnormal muscle and fat-body
cells, and were presumed to consist of mixed wild-type and mutant cells. The
remaining cultures were completely normal. In the mixed mutant/wild-type
cultures the abnormal muscle and fat-body cells displayed a development which
exactly paralleled that observed in cultures of single mutant embryos. Nerve
cells, on the other hand, showed little variation within a particular culture, but
a good deal of variation between the nine mixed mutant/wild-type cultures:
development ranged from a completely normal to a completely mutant pattern.
lethal( 1 )myospheroid
Seventy-two cultures were prepared from the stock l{l)mys/FM7b and
15 (21 %) were abnormal. The most striking abnormality was in the muscle cells,
which could be identified at 24 h by their weak pulsations: they had an unusual
form with a rather granular cytoplasm (Fig. 3) and were at first confused with the
earliest form of haemocytes. Rather stronger pulsations were noted in a few cell
clumps at 48 h, but the muscle cells were hidden and could not be visualized;
contractions in these clumps persisted for 6-7 days. Another consistent abnormality in l{l)mys cultures was the apparently poor attachment of cells to the
coverslip substrate: in the latter half of the 10-day culture period the coverslip
cell density reduced as all types of cell fell away to the bottom of the culture
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D. P. CROSS AND J. H. SANG
drop. Aside from this poor attachment, nerve, fat-body and chitin-secreting
cells were capable of extensive development which in the optimal cases was
indistinguishable from that observed in wild-type controls.
shibirets lethals
In the experiments with shits cells control cultures were prepared from
embryos of the wild-type Oregon-K stock. Development of Oregon-K cultures
at 29 °C was similar to that at 22 °C, with the exception that it proceeded more
rapidly at the higher temperature. Cultures of shits cells kept at 22 °C were
indistinguishable from controls. When kept at 29 °C, cultures of both shitsl and
shitsZ, produced either by the single embryo or bulk embryo methods, displayed
a consistent pattern of abnormalities. A proportion of cells degenerated in the
first 24 h of culture, but nevertheless, nerve, muscle, fat-body and haemocyte
cells did appear in their normal forms. However, beyond 24 h no positive
developments were observed in the cultures: no new nerve axons were produced,
fat-body cells did not mature and haemocytes did not enter cell division. In
fact, the cultures progressively deteriorated: muscle cells, for instance, took on
a highly condensed form and by 7 days had beome unrecognizable; the cell
density of the cultures progressively reduced, through a steady falling away
from the coverslip.
A group of 20 cultures of shits, prepared by the bulk embryo method, were
initially put at 29 °C and shifted down in pairs to 22 °C every day from day
1 to day 10. All these shift-down cultures showed some degree of recovery from
the effects of exposure to the higher temperature. The earliest downshifts (at 1,
2 and 3 days) showed the most dramatic recovery. New nerve axons were clearly
developing within 1 day of the shift down and continued to appear. Muscle
cells reversed the tendency to condense and showed considerable activity. The
later downshifts (4 days and on) showed reduced recovery, which almost
certainly reflected the progressive deterioration of the cultures as length of
exposure to the restrictive temperature increased. Cultures shifted down at
10 days did, however, show some improvement, with a few fresh axons, active
muscles and mature fat-body cells eventually appearing.
shits cultures which were shifted up to the restrictive temperature after 1 day
at 22 °C at first showed an arrested development and then a deterioration which
was similar to that described for cultures kept continuously at 29 °C.
DISCUSSION
The specific conclusions to be drawn from experiments on each group of
mutants will be set out first, leaving generalizations about the work until the
end of this section.
Cell cultures from lethal Drosophila embryos
183
(1) Notch lethals
Notch mutations have been the most thoroughly studied embryonic lethals of
Drosophila: their embryology and genetics have been comprehensively reviewed
by Wright (1970). The question as to which are the primary abnormalities in the
lethal Notch syndrome has interested a number of authors (Poulson, 1945;
Counce, 1961; Wright, 1970). The differing views that have been taken both
provoked the present work and amply illustrate the difficulties faced in interpreting the abnormalities of lethal Drosophila embryos (see also Cross & Sang,
1978).
The most straightforward hypothesis to account for the data from culturing
experiments is that the Notch locus has a direct role in derivatives of both
ectodermal and mesodermal rudiments. There can be little doubt that the
hypertrophy of nerve, reported for whole embryos, is part of the 'Notch
syndrome' in vitro. Though our measurements of the quantity of nerve were
crude, they gave consistent results for four of the mutants: in the case of the
fifth mutant, N60°n, it was not possible to positively identify Notch cultures
within a segregating group, but neuron cluster counts strongly suggest that
about 25 % of the cultures showed neural hypertrophy. Furthermore, the consistent observation of reduced myotube frequency and presence of myocyte
bundles in cultures of four of the five mutations suggests that these defects, too,
are part of this Notch syndrome. The last assertion raises two obvious questions:
(1) why did N6mi cultures give only nerve abnormalities?, and (2) why did the
other four alleles vary with respect to the degree of the muscle abnormalities?
j^cogii m u s t ^ e sef a p a r t from the other alleles because it has temperaturesensitive properties (Foster & Suzuki, 1970) and hemizygotes are likely, therefore, to retain some sort of functional gene product. The available embryological
(Poulson, 1939, 1968) and genetic (Welshons, 1965) data suggest that N264-30,
NJG4-40 and N264-°7 behave as do Notch deficiencies and they are presumed to be
amorphs. However, in culture these three alleles did not behave as did Notch-%,
which is obviously a Notch amorph. This particular difference could be interpreted to mean either that the three alleles are not amorphs, or that the Notch-%
abnormalities are rendered extreme by lesions of genes other than Notch
(Notch-% is an 18-band deficiency). Further experiments with deficiencies restricted to the Notch locus would be fruitful in this regard.
The data from mixed cultures of Notch-8 and wild-type cells are consistent
with the autonomous expression of muscle and nerve abnormalities and this
suggests that these abnormalities are both primary. The mixed cultures showed
about 60 % more large neuron clusters and about 35 % fewer myotubes when
compared to control cultures from two wild-type embryos (Table 3). However,
a mixed culture experiment with reliable genotypic markers would be highly
desirable and interesting in this connexion. Indeed, one may speculate that
Notch nerve acts to cause the muscle defects since there is evidence that the
184
D. P. CROSS AND J. H. SANG
nervous system has an important role in the development of muscle in insects
(see review of Nuesch, 1968).
(2) white deficiencies
Abnormalities of nerve, muscle and fat-body cells were observed in cultures
prepared from white deficiency embryos. Initial cellular differentiations were
normal but defects became readily detectable after 3 days of culture; this 'late'
appearance of abnormalities reflects the fact that the mutants die as late,
apparently complete, embryos. A central issue has been the possibility of
localization of lethal defects to a particular cytological region. Poulson (1940,
1945) found that a series of six white deficiencies gave similar embryonic defects
which, on the basis of existing cytology, could be localized to band 3C1. However, Kaufman et al. (1975) revised the cytological data and considered that an
embryo lethal effect could not be localized to 3C1. Similar abnormalities were
observed for two white deficiencies in culture and this strongly suggests that
defects can be localized to the regions common to the two deficiencies: tentatively 3A9-3B1 (3 bands) and 3B3-3C2 (4 bands). Further, abnormalities of
white deficiency cells cannot be caused by the lack of bands 3C1-2 since they
showed no overlap with those observed in Notch-S cultures.
Mixed cultures of wild-type and mutant cells gave sharply distinct normal
and abnormal classes of both muscle and fat-body cells, thus abnormalities of
these two cell types seem likely to be autonomous by this test. The pattern of
development of nerve tissue varied among the individual mixed cultures, but
was homogeneous within each one, sometimes appearing normal and sometimes
typically mutant. Neural defects did not show a clear autonomous expression
and this could lead to the conclusion that they were secondary effects in culture.
However, since it is not clear to what extent neurons interact with one another
in vitro, the homogeneous appearance of nerve in the mixed cultures could be
due to some influence of normal on mutant nerve, or vice versa.
(3) lethal(l)myospheroid
Muscle of abnormal morphology was identified in l(J)mys cultures (contra
Donady & Seecof, 1972), consistent with the whole embryo data of Wright
(1960). We should expect, for instance, that within the embryo these abnormal
cells would give rise, as in the case of the somatic muscles, to spheroidal structures which would not attach to the hypoderm. The results from the in vitro
cultures do, however, suggest (contra Wright, 1960) that at least some of the
abnormalities of muscle which occur in whole embryos are intrinsic to the
muscle cells themselves, and not related to failures in the basement membrane.
It should be noted that electron micrographs show neurons and myocytes
cultured in vitro to be free of extracellular material, and therefore suggest that
basement membranes are not necessary for the differentiation and survival of
wild-type cells in culture (Donady & Seecof, 1972). The observation that all cell
Cell cultures from lethal Drosophila embryos
185
types adhered poorly to the glass substrate in the l(l)mys cultures suggests to
us that the mutant factor acts directly to alter the picture of the mutant embryo
which Wright has suggested; it still remains one of a rather unstable organization which is disrupted by the first muscular contractions.
Amongst the more positive findings is that nerve, fat-body and chitinsecreting cells were capable of considerable growth and development in the
in vitro cultures, and this agrees well with Wright's observation that mutant
tissues continue to grow, within the vitelline membrane, for at least 10 h (at
25 °C) beyond the normal hatching time, and for several days if transplanted
into larval hosts.
(4) shibirets Jethals
Several studies point to a specific neural defect as the cause of the shits
paralysis phenotype (Kelly, 1974; Kelly & Suzuki, 1974; Ikeda, Ozawa &
Hagiwara, 1976); however, it is difficult to reconcile several of the shitH phenotypes with a purely neural defect (Poodry et al. 1973; Suzuki, 1974; Swanson &
Poodry, 1976).
The shitH cultures showed abnormalities of all cell types at 29 °C, but were
normal at 22 °C. The results are consistent with the idea that normal shi locus
function is important in all tissues. With continuous culture at 29 °C, the initial
differentiation of nerve, muscle, fat-body and haemocyte cells was not blocked,
but their continuing development and maintenance was. Taken in isolation, the
poor condition of s/rits cultures at 29 °C could make for difficulties of interpretation, since it could be argued that cell degeneration would have the effect of
generally depressing the quality of the cultures, making the identification of
specific genetic effects difficult. However, the reversal of many of the observed
abnormalities upon downshift to 22 °C even after several days at the restrictive
temperature, argues both that any adverse conditioning of the medium at 29 °C
was not too serious, and that exposure to the restrictive temperature was not
disastrous to cellular integrity.
The 'bulk embryo' procedure will be convenient for the culturing of cells
from temperature-sensitive mutants; and it is clear that results will be comparable to those from the single embryo culture method: the two procedures
gave similar results with both wild-type cells (see also Cross & Sang, 1978) and
s/nt8 alleles.
(5) Overall conclusions
From the outset of this work it was hoped that the culture system would allow
cells from mutant embryos to realize more developmental potential than would
have been possible in situ: firstly, by limiting the possibility for deleterious interactions between cell types; and secondly, by allowing long-term survival of
cells. The success of the approach has been demonstrated in the case of each
mutant which was studied, and is well-illustrated by the example of the problem
186
D. P. CROSS AND J. H. SANG
of comparing abnormalities ofNotch-% and white deficiency cells. Whole Notch-%
embryos show very limited differentiation of muscle and fat-body cells in situ,
and abnormalities of these cell types are developmentally epistatic to those
reported in white deficiency embryos; whereas in vitro Notch-% cells give extensive
differentiation and long-term survival of these two cell types. Furthermore,
defects revealed in the white deficiency cell cultures are of the sort that might be
expected to be observed well after the normal hatching time in whole mutant
embryos. In situ such cellular defects would probably be difficult to distinguish
from the degenerative events which must eventually take place in any embryo
which fails to hatch. In view of these facts it is apparent that a direct comparison
of the abnormalities shown by Notch-S and white deficiency cells is not possible
in situ.
This research was supported by the Science Research Council, which is gratefully acknowledged. D.P.C. was in receipt of an S.R.C. Research Studentship.
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