AMER. ZOOL., 17:565-576 (1977).
Determination During Early Embryogenesis in Drosophila melanogaster
GEROLD SCHUBIGER AND W E N D E J . WOOD
Department of Zoology, University of Washington, Seattle, Washington 98195
SYNOPSIS Ligation of developing embryos of Drosophila melanogaster was performed at
three different stages of nuclear multiplication and at the cellular blastoderm stage. Egg
fragments of variable sizes are able to continue development up to the hatching stage.
Partial embryos differentiate larval structures, anterior fragments forming larval head and
posterior fragments larval abdominal structures. These fragments differentiate a variable
number of the twelve larval cuticular bands formed by intact embryos. We found that
ligation at cellular blastoderm can lead to anterior and posterior fragments which
differentiate together all the twelve bands, indicating that at this stage the embryo develops
these patterns in a mosaic fashion. Ligation of younger embryos prevents the differentiation of some intermediate larval cuticular bands, while the terminal ones are consistently
differentiated. The number and position of the deleted bands is correlated with the time
and position of ligation. This indicates that the mosaic pattern present in the egg at
blastoderm is not fully formed at earlier stages in development.
processes. We are interested in the question:
To what extent does oogenesis fix the
The fertilized egg is probably the most
in a mosaic egg as expressed by
patterns
discussed and the most puzzling cell in
determination
of the early embryo? Folevery organism. Its contents, and in parlowing
the
development
of isolated parts
ticular its cytoplasmic organization, are a
product of maternal gene expression. The of early embryos should indicate to what
morphogenetic function of the egg cyto- degree patterns of determination have
plasm has been the subject in recent years been laid down in the mature egg, and
of renewed interest and has led to new ex- whether further refinement of the determiperimental investigations (for review see nation system follows during the early
Davidson, 1968, and Gurdon, 1975) as well stages of development.
as theoretical analyses (Kauffman, 1975).
A genetic approach to analysis of the THE EARLY DEVELOPMENTAL EVENTS IN
DROSOPHILA
morphogenetic functions of the Drosophila
melanogaster egg has led in the last years to I would like to describe briefly some of
a widespread search for maternal effect the early developmental events in embryos
mutants affecting embryonic patterns of Drosophila melanogaster (for details of the
(Bakken, 1973; Rice and Garen, 1975, description see Sonnenblick, 1950; Scriba,
Gans el al., 1975). One of the goals of these 1964; Bownes, 1975a; Zalokar and Erk,
investigations is to identify genes active 1976; Turner and Mahowald, 1976). In
during oogenesis and involved in the for- the majority of the insects, including
mation of the normal architecture of the Drosophila, the zygote nucleus divides
egg cytoplasm. In this indirect way muta- without the formation of cells. These nutions interfering with early development clear multiplications are synchronous and
may be discovered, leading to a better occur every 8-10 min at 25°C. The zygote
understanding of the early morphogenetic nucleus is always located in the anterior
half of the egg, and even after thefirsttwo
Thanks are due to Drs. John L., Haynie, Martin J. nuclear divisions, the posterior egg half in
Milner and Ms. S. L. Tobin for suggestions and most cases does not include any nuclei
criticisms during writing of this manuscript.
(Parks, 1936). Later during nuclear mulThis work was supported by N.S.F. Grant PCM73tiplication the nuclei are more evenly dis06968 to G. S.
INTRODUCTION
565
566
GEROLD SCHCBIGER AND WENDE J. WOOD
tributed. Between the 8th and 9th divisions, the majority of nuclei appear at the
periphery of the egg and enter the cortical
cytoplasm, forming the syncytial blastoderm (Fig. 1). Some of the nuclei, called
vitellophages, remain in the yolky egg
center. The number of nuclei increases to
about 6000 at 2 hrs and 30 min (25°C)
after egg deposition. At this stage cell
membranes start to form, giving rise to a
single layered cellular blastoderm. Prior to
formation of the cellular blastoderm the
pole cells, the primordial germ cells, bud
off at the posterior end of the egg. The
formation of the cellular blastoderm is
followed by germ band extension and gastrulation, processes which have recently
been analyzed with the electron microscope (Rickoll, 1976). It is not within the
scope of this paper to discuss the complicated morphogenetic movements leading
to the differentiated larva 19 hrs after
fertilization. However, not all the blastoderm cells differentiate into larval structures, some being designated to form the
imaginal discs. These folded, single
layered epithelial tissues are the precursors of various cuticular structures of the
adult fly.
TIME OF THE EARLY DETERMINATION EVENTS
During nuclear mutiplication the nuclei
are surrounded by islands of cytoplasm.
Illmensee (1972, 1973) has shown that
successfully transplanted nuclei at this
stage are at least pluripotent by isolating
single nuclei with their surrounding
cytoplasm and transplanting them into unfertilized eggs. These embryos with transplanted nuclei can develop into complete
larvae, indicating that the developmental
potencies of these nuclei are not restricted.
Chan and Gehring (1971) have presented
evidence that the imaginal cells of the
cellular blastoderm are restricted in their
adult developmental potencies at least to
the regions of anterior and posterior.
Other experiments involving injuring the
egg by microcautery, pricking and UVmicrobeam irradiation have shown that if
cells are damaged at blastoderm stage or
later, the positions of the resulting larval
and adult abnormalities correlate with the
positions of the experimental defects
(Howland and Child, 1935; Bownes and
Kalthoff, 1974; Bownes and Sang, 1974 a,
b; Bownes, 19756). However, neither the
mechanism nor the extent and specificity
of larval and adult determination occurring at this time is understood. We would
like to know whether specific qualities such
as legness, wingness, etc. are fixed at this
time or whether more general qualities
such as "anterior" or segment specificity
are the only ones determined.
Wieschaus and Gehring (1976a) and
Steiner (1976) were able to show that mitotic recombination induced at blastoderm
stage can lead to clones of marked cells
which extend from structures derived
from the wing disc into those derived from
the second leg disc; other such clones can
overlap third leg and balancer structures.
However, they did not find clones that
included structures belonging to different
body segments, such as second and third
leg, even though the physical distance between the anlage of midleg and hindleg in
the blastoderm was estimated to be about
the same as between midleg and wing
(Wieschaus and Gehring, 19766). These
findings indicate that determination for a
single disc is not fixed at blastoderm stage,
but supports the idea that determination
might be segment specific at this time.
Similar segmental restrictions during blastoderm formation are also reported to
occur in the abdomen of Oncopelttis (Lawrence, 1971, 1973, 1975).
Treatment of wild-type blastoderm
stage embryos with heat or ether shock
(Henke and Maas, 1946; Gloor, 1947) results in phenocopies of the mutant tetraptera and bithorax respectively (Lindsley and
Grell, 1968). These phenocopies transform the dorsal metathorax into a second
copy of the dorsal mesothorax, giving rise
in the most extreme cases to animals with
four wings. These phenocopies are only
inducible during the cellular blastoderm
stage. If one assumes that the phenocopying agent is interfering directly with the
normal process of determination, then
these results are in agreement with the
notion that segment specific determination
567
EARLY DETERMINATION IN DROSOPHILA
10
18
•V
27
>^
36
(3000)
FIG. 1. Normal development of Drosophila
melanogaster between fertilization and onset of gastrulation. Numbers on top left of each egg refer to
minutes after deposition, large numbers to the right
of each egg refer to stages and small numbers to the
left on the bottom to number of nuclei. Stages 1 to 9;
nuclear multiplication 10: pole cells, 14b: cellular
blastoderm (from Zalokar and Erk 1976).
568
GEROLD SCHUBIGERAND WENDE J. WOOD
takes place at the cellular blastoderm stage.
Thus we see that the pluripotent cleavage
nuclei are probably not actively involved in
the process of determination and that the
interaction of these nuclei with the cortical
cytoplasm of the egg at the time of blastoderm formation is the most likely explanation for the determination which can be
experimentally demonstrated.
ESTABLISHMENT OF THE SYSTEM OF EMBRYONIC
DETERMINATION
Although the above results all indicate
that a restriction of developmental potencies may occur when the nuclei populate
the cortical cytoplasm, there are very few
direct experimental data confirming the
existence of discrete localized determinants in the cortex. Unfortunately the nature of the determining factors, cues or
signals possibly localized in the egg. cortex
are not well known. Nevertheless, it is
possible to imagine at least two different
modes by which such factors may be laid
down.
We would like to discuss two possibilities
of how factors involved in determination
at blastoderm are laid down. One comes
from a generalization of the way pole cells
become determined. The cortical cytoplasm in the most posterior part of the egg,
the pole plasm, which includes the polar
granules is responsible for the determination of the germ cells and some cells of the
midgut. UV-irradiation of this area results
in sterile but otherwise normal flies (Geigy,
1931). Okada et al. (1974) and Warn
(1975) demonstrated that the adult fertility
of embryos treated in this manner could be
restored by injecting pole plasm including
polar granules from non-treated eggs.
Recently Illmensee et al. (1976) were
able to show that germ cell determining
factors already exist and are functional
prior to fertilization. They implanted
polar plasm including polar granules isolated from oocytes of stages 13 and 14
(King, 1970) into the anterior halves of
fertilized eggs. As a consequence of this
manipulation, at the blastoderm stage, additional "pole cells" were formed in the
anterior part of the egg at the site of the
injected pole plasm. After transplantation
of such ectopic pole cells into the posterior
region of another embryo, the authors
were able to prove that these transplanted
cells differentiated into functional germ
cells. However, no germ cells were recovered with polar plasm transplanted from
earlier oogenetic stages. From these results
a generalization for the determination of
somatic structures may be made which
suggests that other regions of the unfertilized egg cortex could have morphological and/or functional differences comparable to those of the polar cytoplasm and
that these differences would be responsible
for the determination at blastoderm stage.
However, cytological analysis has as yet
failed to detect such differences.
A generally different mode of determination is proposed for the early development of the sea urchin. Runnstrom (1928,
1929), and Horstadius (1928) suggested
that two opposing gradients of unknown
nature arise in the developing embryo.
The determination of different body parts
results from concentration differences of
these morphogenetic substances along the
egg axis. Such a mode implies that the
middle region is specified by interaction of
the two ends. This model has been given
renewed attention and clarification by
Wolpert (1969).
Let me assume that two such gradients
present in the Drosophila egg, one in an
anterior-posterior direction, the other in a
posterior-anterior direction, are responsible for the segmental determination which
probably takes place at blastoderm. The
formation of such gradients might have
started at the two ends of the egg and their
interaction might be a process which extends over a period of time. Such an idea
implies that cues or determining factors
which specify the final pattern responsible
for determination are laid down in a continuous process starting at the two ends
and as the two gradients grow, more and
more intermediate positions are specified.
Such a mode of determination has already
been proposed by Sander (1960, 1975). It
predicts that regions closer to the middle
of the embryo would become instructed at
successively later times during develop-
569
EARLY DETERMINATION IN DROSOPHILA
ment, and that the presence of both ends
of the egg would be required for the
normal process to be completed, an assumption not made in the first model
discussed.
To decide which of these two modes of
determination is more likely to apply to the
early development of the Drosophila egg we
have tried to demonstrate in different egg
regions, prior to the blastoderm stage, the
existence of factors which cause development to proceed in different directions.
Furthermore, we attempted to determine
whether the physical arrangement of such
factors changes during the earliest developmental period following fertilization,
the nuclear multiplication stages. From a
single type of experiment we have obtained results which bear on both of these
questions.
We have ligated eggs during different
stages of nuclear multiplication and cellular blastoderm at different levels along the
long axes of the eggs. Such experiments
were performed at 15-30, 45-60, 75-90
and 175-190 min after egg deposition.
These time periods correspond to the following stages (Fig. 1): 2-8 nuclei, 32-128
nuclei, 512-800 (pole nuclei first visible)
and cellular blastoderm stage (Zalokar,
1976). Eggs of the same age were arranged
with their long axes perpendicular to a line
cut into double-sided Scotch tape mounted
on a slide. The slide was then positioned
under a blunted razor blade which could
be lowered using two screws, ligating the
aligned eggs. This technique was developed by Sander (1971) for ligation of
leaf hopper eggs. We calculated the level
of ligation by dividing the distance of the
blade from the posterior end by the total
egg length (% EL). Zero percent EL designates the posterior-most end and 100% EL
the anterior-most end. Thus high % EL
refers to ligation in the anterior half,
whereas low % EL indicates ligation in the
posterior egg half (Fig. 2).
When we ligated 30 min after egg deposition, more than 95% of the surviving
eggs formed a cellular blastoderm only in
one egg fragment. This was expected since
the nuclei in a 15-30 min egg are located
predominantly in a small area in the an-
%EL
1OO
-
80
-
20
-1-
0
posterior
FIG. 2. Blastoderm formation of a 3-hr embryo ligated 60 min after egg deposition at 50% egg length.
%EL Refers to the level of ligation (from Schubiger,
1976). X240.
terior half of the egg. Ligation in front of
the nuclei allowed the posterior fragment
to develop, whereas ligation behind the
nuclei restricted development to the anterior fragment. When older eggs were
ligated, development in both fragments
occurred more frequently (Fig. 3). Table 1
summarizes the data of blastoderm formation after ligation at various stages. Anterior fragments only differentiated anterior structures such as mouthparts, brain
and cranial segments, whereas posterior
structures of the caudal segments, including posterior spiracles and Malpighian
570
GEROLD SCHUBIGERAND W E N D E J . WOOD
FIG. 3. Blastoderm formation of embryos ligated at
30 min (a and b) and 60 min (c) (from Schubiger,
1976). xl40.
tubules, developed only from posterior
fragments (Fig. 7). We did not observe any
complete embryos from half of an egg. We
therefore can conclude that in a 15-30 min
egg different determinative information
already exists in different egg regions
which specifies anterior and posterior development. These findings cannot be the
result of differences in the nuclei as shown
by Illmensee (p. 566). Moreover, are the
signals determining the different larval
segments already laid down in particular
positions in a 15-30 min old egg?
To score the larval segments differen-
tiated after ligation we used the cuticular
hair pattern, mandibular hook and the
posterior spiracles as markers. A normal
larva has 12 parallel transverse rows of
small hairs which are easily visible on the
ventral side (Fig. 4). These belts are arranged in three bands of lighter hairs on
the thoracic segments followed by nine
dark abdominal belts while the most posterior one is a small patch of dark hairs.
The results of ligation at different
levels and at different times during the
nuclear multiplication and cellular blastoderm stages are summarized in Figure 5.
°"l
FIG. 4. Normal cuticular banding pattern on the
ventral side of a fully differentiated larva (19 hr after
fertilization). M = mandibular hook; S = posterior
spiracular openings; numbers refer to segmental
band numbers, x 145.
571
EARLY DETERMINATION IN DROSOPHILA
CM
p to oq
-T
CM T
win
• * US
o CJ r» —<
O —' o in
•a.
e
a.
s
0
V
r»_ p •* oq
O 00 O CO
V
•a
oni
1c
dur
We have scored the extent of different
larval structures differentiated by anterior
(dark squares) and by posterior egg fragments (open circles). A particular dark
square stands for the most posterior band
differentiated in a given fragment and the
open circle represents the most anterior
band from a posterior fragment. The ordinates give the levels of ligation in % EL;
larval bands in anterior to posterior direction are arranged on the abscissa. Band
#4, for example, refers to the first dark or
abdominal band.
Following ligation 15-30 min after egg
deposition posterior fragments can give
rise to all the dark belts (4-12) if the egg is
ligated at about 70% length or higher.
Anterior fragments ligated at the same
time differentiate the dark anterior-most
abdominal belt in addition to the light ones
only when ligated at 38% egg length or
less.
These results show that small anterior
fragments develop into head and thorax,
and larger fragments in addition form
abdominal belts. On the other hand, only
large posterior fragments differentiate
thoracic structures in addition to all the
abdominal segments.
Similar results were obtained when ligation was performed later during nuclear
multiplication (60 and 90 min), or at the
cellular blastoderm stage. In these experiments again posterior fragments of all
sizes always differentiate abdominal belts,
and in anterior fragments the structure
which always appears is the mandibular
hook or part of it.
We now would like to compare the array
of bands developed from fragments ligated at the same level at different times in
order to see whether the number varies
with the time of ligation. An anterior
fragment ligated at 30% egg length at 30
min differentiates bands 1 to 5, at 60 min
bands 1 to 6, at 90 min bands 1 to 8, and at
190 min bands 1 to 9. The behavior of the
posterior fragments shows a similar tendency (Fig. 5). For example, an egg ligated
at 60% EL 30 min after oviposition differentiates after 19 hrs belts 12 to 8, at 60
min 12 to 7, at 90 min 12 to 5 and at 190
min 12 to 2. Fragments identical in size
(N W CO 00
<N 1O • * O
O O —< 00
iri GO o S ( O
bc
C
00
bo
•2
00
00
1O O — CM
O 60
Z. OO
•fi
1
4^
2 5
J3 re
H
572
GEROLD SCHUBIGER AND WENDE J. WOOD
therefore differentiate additional larval
segments when ligated later during development.
In Figure 6 we have selected all cases
which differentiated larval structures in
anterior as well as posterior fragments
from the same ligated egg. These pairs
show a similar behavior as shown in Figure
5. Furthermore, they show that ligation at
blastoderm stage leads to pairs which
taken together either differentiated all or
all but one of the belts. Therefore, in terms
of belt differentiation, the fragments at
blastoderm show mosaic development.
Separation at the earliest time of nuclear
multiplication (30 min) never leads to a full
80%
EL
30n
set of belts in two fragments (one anterior,
one posterior) ligated at the same level,
which would be expected if they develop
mosaically. If the fragments could regulate
we would expect to find a complete or
nearly complete larval pattern in each of
the fragments. However, we find that a
fragment of a given size ligated earlier in
development produces fewer segments
than does the same size fragment from
ligation at cellular blastoderm stage.
Figure 7 illustrates two cases of posterior
differentiation after ligation at about the
same level (55%EL) but at different times.
In Figure 7a belts 9-12 and the posterior
spiracles developed after ligation at 30
60 min
80-
EL
50-
50-
20-
20-
90min
180 mm
80-i
%
EL
50-
50-
•
•
&r=
•~s °
-
•>
m o
20-
20-
12
FIG. 5. Origin of larval bands (1-12) obtained from
embryos ligated at 30, 60, 90 and 190 min after egg
deposition. • = differentiated by anterior fragments,
o = differentiated by posterior fragments. Ordinate
12
gives the level of ligation in percent egg length
(%EL). Abscissa: M = mandibular hook. 1 to 12 larval segmental band numbers.
573
EARLY DETERMINATION IKDROSOPHILA
30 min
80%
EL
60 m in
80O
§° &
°
EL
8
o
-
50-
° £°
°
50•
o
o
-
•
1
m
20-
12
M 1
90mm
80%
EL
°
i
•:
20-
-
I
m
•
12
M I
190min
80- 8
EL
3
-o a,
oo 8
m
50-
o
« o
-
-
i
°
~ S
50-
-
a
s
•
0
o
8
o
r 1&
c-
o
SS8 B °
Sr L
"
-
0
^ o
I
m
20-
m
20-
M1
12
M 1
12
FIG. 6. Origin of larval bands (1-12) obtained from
complementary fragments of a given embryo, which
differentiated larval structures in anterior as well as
posterior fragments, after ligation at different times
and levels (see Fig. 5 for details).
min, whereas in Figure 7b bands 7-12
differentiated after ligation at 90 min after
egg deposition. Note that the distance between single bands in Figure 7a is much
larger than in Figure 7b (e.g., between
bands 9 and 10). Therefore at different
times different structures develop at the
same position. These results do not support the hypothesis that all pattern determining elements are localized in their final
positions at the time of fertilization. One
could still argue that these elements are
laid down at this time, but subsequently
shift around during nuclear multiplication. However, the observation that in the
paired cases up to 7 belts can be missing
after early ligation rules out this argument.
It is still possible, however, that the
youngest eggs are much more susceptible
to damage and therefore make a poor
574
GEROLD SCHUBIGER AND WENDE J. WOOD
found no differences in these results compared to the previous ones.
We have seen that ligation during nuclear multiplication (e.g., 60 min) leads to a
deficiency of 6 out of the 12 bands. Let us
assume that the young egg is very sensitive
to damage and that this damage affects
normal blastoderm formation. Therefore
histological sections of blastoderms of ligated eggs at 60 min (Fig. 8b) were compared with those ligated at 150 min (Fig.
8c). Cell number and cell density were
identical in both cases suggesting that ligaFIG. 7. Differentiation of larval bands after ligation tion of younger embryos did not cause
at different times (a = 30 min; b = 90 min) at about additional damage.
the same level (a = 56% egg length; b = 55% egg
In addition we ligated 60 min embryos
length). L = Ligation mark, marks anterior end of
larval fragment, S = posterior spiracular openings. and immediately released the blade. In six
cases the two egg parts reunited and
X80.
formed a uniform blastoderm. These emrecovery from ligation, leading to missing bryos later developed into larvae, which
belts. We have repeated the ligation exper- differentiated all the segments. If the proiments with a blade 2-3 times as thick as in cess of ligation had caused major damage
the previous one, attempting to increase these results should not have been obthe damage. In this manner we ligated tained.
15-30 min and 175-190 min eggs and
We also have studied the ability of egg
FIG. 8. Sections of cellular blastoderm (3 hrs) developed from a) nonligated b) after ligation at 60 min
c) after ligation at 150 min. x 170.
EARLY DETERMINATION IN DROSOPHILA
fragments to differentiate adult structures
with the same ligation technique. After
ligation at the same times as in the previous experiments, the larval fragments
where injected into adult females where
the imaginal disc tissues contained in the
fragment proliferated. Imaginal disc tissues differentiated into adult structures
after a second transfer into larval hosts.
Here again we found that structures were
missing when the embryo was ligated early, but that a complete inventory of adult
structures differentiated when the embryos were ligated at blastoderm
(Schubiger, 1976).
Our resuts are in good agreement with
similar ligation experiments performed on
the blowfly Protophormia (Herth and Sander, 1973). Sander and his collaboraters
performed a series of ligation experiments
on different species: the beetle Bruchidius
(Jung, 1966), the leaf hopper Euscelis
(Sander 1959) and the midge Smittia, a
"lower" dipteran (Sander 1975). Herth and
Sander (1973) pointed out that in Protophormia, ligation at blastoderm stage
leads to two partner fragments which show
mosaic behavior. In Smittia only after ligation at a later stage—shortly before the
formation of the germ anlage—were all
segments differentiated, as was also found
for Euscelis (at the stage of germ anlage)
(Herth and Sander, 1973). Furthermore
the authors pointed out that "mosaic egg
type" and "regulative egg type" resemble
the two extreme ends of the spectrum and
are simply manifestations of the difference
in the timing of the mosaic behavior.
The mosaic nature of a Drosophila egg
has been pointed out by several authors
(e.g., Waddington, 1956; Anderson, 1966).
We believe that our results are not due to
an experimental artifact and show mosaic
behavior of egg fragments only at the
blastoderm stage.
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