/. Embryol. exp. Morph. Vol. 32, 1, pp. 253-272, 1974
Printed in Great Britain
253
Experimental manipulations of early
Drosophila embryos
I. Adult and embryonic defects resulting from microcautery at
nuclear multiplication and blastoderm stages
By MARY BOWNES 1 AND J. H. SANG2
From the School of Biological Sciences, University of Sussex
SUMMARY
Localized regions of Drosophila eggs were damaged at both nuclear multiplication and
blastoderm stages, using the technique of microcautery. The resulting defects in embryos and
adults were recorded in order to study the effects of microcautery on determination of larval
and adult structures. Using 70 °C microcautery, few specific defects were found in either
embryos or adults. Sensitivity maps relating embryonic mortality to sites of damage were
prepared for both egg stages and compared. Microcautery at 75 °C was used to check experimentally the putative localization in the egg of adult disc cells, as indicated by genetic mosaic
studies. Although some of the expected adult defects were found, there was also evidence for
regulation. Localized embryonic defects were found using this higher temperature, such that
anterior damage produced anterior defects and posterior damage led to posterior defects.
Mid-region microcautery resulted in both anterior and mid-region defects, the latter being
most common after damage to the blastoderm. Comparisons are made with results obtained
using other experimental manipulations and with the development of some embryonic lethal
mutants.
INTRODUCTION
One of the outstanding questions concerning the development of Drosophila
is whether or not the egg is truly mosaic, i.e. if the fates of all parts of the embryo
are fixed at fertilization such that localized damage will subsequently manifest
itself as loss of parts. Early work suggested that this was the case, and 'fate
mapping' of the egg using genetic mosaics might be similarly interpreted.
However, the latter experiments do not prove this, and there is some evidence
(see below) of regulation after damage to early egg stages. It is therefore possible
that mosaicism is established only after the egg becomes cellular, or that different areas of the egg become determined at different times. Certainly if there is
any period of lability it must be early and short. The experiments described here
compare the consequences of treating two early stages, the acellular and cellular
cortex, using microcautery of small areas to damage them.
1
Author's address: Center for Pathobiology, University of California, Irvine, California
92664, U.S.A.
2
Author's address: School of Biological Sciences, University of Sussex, Falmer, Brighton,
Sussex BN1 9QG, U.K.
254
M. BOWNES AND J. H. SANG
Previous experiments of this sort have given conflicting results. Howland &
Child (1935) removed cells from blastoderm stage eggs and noted defects in
adults correlated with the site of damage, but they found very few defective
flies, and many that might have regulated. Howland & Sonnenblick (1936),
who pricked eggs at nuclear multiplication stages (pre-blastoderm), found no
defective adults, and concluded that regulation was then possible. Ilmensee
(1972), on the other hand, found adult defects roughly correlated with the site of
damage after similar, but more extensive, pricking experiments. Nothiger &
Strub (1972) found adult defects after irradiating early embryos with u.v., but
these defects were not correlated with the sites of irradiation. All these experiments are concerned with the cells which will form the adult discs, and these
are certainly determined shortly after blastoderm formation since dissociated
cells from this stage can give rise to adult structures after culture in vivo (Chan &
Gehring, 1971). But the problem-was the mosaic established before this ? —
cannot be resolved by this transplantation technique.
Surprisingly, only Hathaway & Selman (1961) appear to have looked at the
determination of larval structures. They found that u.v. damage to blastoderm
stages produced a range of embryonic and larval defects, with some regularity in
their pattern related to the site of initial injury. We have also looked at this aspect
of determination.
MATERIALS AND METHODS
Origin and preparation of eggs
The eggs used for microcautery at 70 °C were Oregon-Samarkand hybrids,
and the 75 °C microcautery eggs were Oregon K stock. Eggs were collected at
25 °C on agar plates coated with yeast paste. After a 1 h prelay, eggs were
collected at 20 min intervals and used immediately, or incubated at 25 °C until
they were of the required age. The eggs were washed from the agar with 0-9 %
sodium chloride, then dechorionated with 3 % sodium hypochlorite for 5 min.
They were then washed again with sodium chloride solution before treatment.
For the first set of microcautery experiments eggs were used from accurately
timed collections. Nuclear multiplication (NM) stage eggs were 1 hr ± 15 min,
and blastoderm (Bl) stage were 2 | h ± 15 min. Eggs for the second set of experiments were selected using the binocular microscope. Stage NM was when the
nuclei were dividing inside the yolk of the egg. This stage can be easily recognized
from the lack of pole cells. Stage Bl was when the outer layer of blastoderm cells
was clearly visible, but before any morphogenetic movements had begun.
Experimental technique
A few eggs were placed on a piece of lens tissue lying on black polythene. This
prevents the eggs from drying out (which reduces their viability) and makes them
clearly visible against the black background. The eggs were orientated as required
under a dissecting microscope using a fine needle. A very fine tungsten needle
Microcautery of Drosophila eggs
255
ANTERIOR
VENTRAL
DORSAL
Abdomen
A
POSTERIOR
Fig. 1. (A) Grid reference of the egg, used for microcautery experiments at 70 °C and
for describing regions of the egg with reference numbers. (B) An adaptation of the
fate map produced by Hotta & Benzer (1972) showing the location of the presumptive adult disc cells. These points were damaged by microcautery at 75 °C.
was mounted on a micromanipulator and connected to a variable a.c. heat
source. The needle was heated to 70 or 75 °C (for the 1st and 2nd set of experiments respectively), and calibrated by using paraffin waxes of known melting
point. Each egg was touched at the relevant spot with the tip of the needle. Control
eggs were treated in the same way, but not touched with the needle. For the first
set of experiments one side of the egg was divided into reference points shown in
Fig. 1 A. Samples of 10-20 eggs were cauterized at each of these points, at each
of the two developmental stages (NM and Bl). For the second set of experiments
the egg was treated at the points in Fig. 1B, which is an adaptation of Hotta &
Benzer's (1972) map showing the location of the presumptive adult disc cells
on the surface of the egg.
There are a number of variable factors inherent in using this technique of
thermocautery. The site damaged is judged by eye, and as the areas are small
there is bound to be some inaccuracy in this. There will also be some variation
in the actual temperature of the needle due to the amount of water left on the
surface of the egg, which will conduct away heat; this will lead to variations in
the degree of damage. The length of time the needle is in contact with the egg
will also be subject to human error. By dissecting adult females and looking at
the development of the ovary, it is possible to show that even with these variations the area damaged by this technique is fairly limited. The only areas of the
egg leading to defective ovaries were in positions 10 and 11, both at the posterior
256
M. BOWNES AND J. H. SANG
of the egg; and flies were often found with an ovary one side of the body, but
not on the other.
The treated eggs were picked up with a Pasteur pipette and put on an agar
plate in a small amount of 0-9 % saline. The plate was covered with a Petri dish
lid and incubated at 25 °C for 24 h in the dark.
Analysis of defective embryos
Those embryos which failed to hatch after 24 h were picked up in a saline
solution, put on a slide and covered with a coverslip supported by broken
pieces of coverslip. This arrangement prevents the eggs from bursting and also
flattens them somewhat, making the internal organs more easily visible. The
eggs were looked at under high magnification, described and photographed.
Analysis of adults
The hatched larvae from the agar plates were picked up with a paper spoon
and put into vials containing yeasted Lewis medium, and then kept at 25 °C to
continue development. These adults were observed at the time of emergence 10
days later, and scored for abnormalities in external morphology. Any pupae
failing to hatch after some days were dissected and all abnormalities noted.
Adults were kept for at least 5 days to see if any died early due to internal deficiencies. Preparations of relevant parts of the fly were made. Adults were left
overnight in 10 % potassium hydroxide, which destroyed soft internal tissues,
but not the chitinized parts of the body. The potassium hydroxide was rinsed
away with water and the flies blotted dry. They were then dehydrated in glacial
acetic acid for 5 min and blotted dry before placing them in clove oil to clear.
Each fly was then put on a slide in a drop of oil and the relevant part dissected
and arranged as required. The clove oil was then removed and the parts mounted
in Canada balsam.
Statistical analysis of microcautery data
Chi-square tests at the 5 % significance level were used to compare the
mortalities and abnormalities at different stages of development - embryonic,
larval, pupal and adult. The graphs of percentage embryonic abnormality
against the position along the embryo (anterior-posterior) show the standard
error of the percentage.
RESULTS AND DISCUSSION
Microcautery at 70 °C
General results
Microcautery at both NM and Bl increased the proportion of eggs failing to
show any differentiation, and also the number of embryos developing abnormally
(Table 1). Pupal death was unaffected, but more larvae died after treatment at
Bl than at NM. The number of adult defects was increased, though only just
Microcautery
257
of Drosophila eggs
Table 1. Effects of 70 °C treatment on survival
Defects (%)i
c
Microcautery at
Control
Eggs dying
Larvae which failed to pupate
Unhatched pupae
Eggs failing to differentiate
Eggs showing abnormal development
Eggs developing abnormally of those
Nuclear
multiplication
Blastoderm
8-3 %
27-5 %
1-3%
7-6 %
0-7 %
0-6%
55-8 %
29-5%
38-3%
17-5 %
28-4%
32-1%
34-5 %
1-6%
20-6%
11-6%
14-6%
1"1 %
20%
3-7%
i-o%
which began to develop
Morphologically abnormal adults and
pupae, excluding ovary defects
Total number eggs
701
1122
1516
significantly, at Bl. The NM treatment caused more embryonic death than the
Bl treatment, with the greatest difference being at the poles (Fig. 2).
Embryonic mortality
The difference between the effects of microcautery at NM and Bl can best be
seen in Fig. 2, which records the mortalities found when microcautery in dorsal
(Fig. 2 A), in ventral (Fig. 2B) and in central (Fig. 2C) regions are plotted
against position along the anterior axis of the egg. From the combination of
these data, sensitivity maps for the nuclear multiplication and blastoderm stages
were made to provide a visual summary of the results (Fig. 3). Clearly, the
nuclear multiplication stage embryo is more easily damaged by microcautery, and
the two stages show notably different sensitive areas.
In the ventral areas ABC (Fig. 2) the anterior is very sensitive at NM, but is
considerably less so by Bl. Similarly region 10, and probably region 5, show this
marked decrease in sensitivity between NM and Bl. The high mortality response
to the treatment moves from the two poles of the egg at NM to the midline by
Bl, essentially due to a decline in the sensitivity at the poles during this period.
The dorsal regions DEF (Fig. 2B) are different and, generally, all regions are
more sensitive at NM than Bl to about the same extent, the exceptions being
areas around the posterior pole.
Nothiger & Strub (1972) studied the effects of u.v. microbeam irradiation of
NM eggs. Their irradiated area was approximately equivalent to the combined
regions CDE. The relevant data is summarized in Fig. 2C, which shows virtually
no difference between the two techniques of damaging the periplasm, except at
the posterior pole, where thermocautery causes higher mortality than u.v.
17
EMB
32
258
M. BOWNES AND J. H. SANG
100 i—
80
60
40
20
J
100
I
I
I
I
J
I
J
I
I
I
I
I
I
I
I
9
10
11
(B) Regions DEF
80
o 60
%
40
w
20
100 r
I
I
(C) Regions CDE
80
60
40
20
2
3
4
5
6
7
8
Position along anterior-posterior axis of embryo
Microcautery o/Drosophila eggs
259
10
11
11
(a) Nuclear multiplication
(b) Blastoderm
Fig. 3. Diagrammatic representation of the sensitivity of the embryo to microcautery, ranging from most (ED) to least ( • ) sensitive. These drawings are approximate and calculated from the graphs shown in Fig. 2. The grid references are as
shown in Fig. 1 A.
irradiation. Jlmensee (1972) tested for the most suitable region for injection of
nuclei into eggs 17-20 min old (NM) by pricking the egg surface and subsequently scoring the sensitivity of the area by the number of eggs hatching. He
found the least sensitive region was a lateral one, midway between the anterior
and posterior poles; a similar region can be seen in Fig. 2C. These similarities
in the sensitivity of early eggs suggest that u.v. irradiation, pricking and thermo-
FIGURE 2
—, Bl; —, NM. x - x , Nothiger & Strubb (1972) u.v. irradiation
(A) Graph of percentage embryonic mortality for the combined results of ventral
regions ABC against the anterior-posterior position along the embryo 1-11.
Standard errors of the percentages are shown by bars.
(B) Graph of percentage embryonic mortality for the combined results of dorsal
regions DEF against the anterior-posterior position along the embryo 1-11.
Standard errors of the percentages are shown by bars.
(C) Graph of the percentage embryonic mortality for the combined results CDE
against the anterior-posterior position along the embryo 1-11. Also the percentage
embryonic mortality after u.v. irradiation of an area equivalent to CDE (Nothiger
& Strub, 1972) plotted against their percentage values along the anterior-posterior
axis of the egg. No results were given for the anterior 20 % of the egg. Standard
errors of the percentage are shown by bars.
17-2
260
M. BOWNES AND J. H. SANG
cautery all damage the same 'targets', involved in embryonic development and
located in the periplasm.
The sensitivity differences in the two maps at NM and Bl (Fig. 3) may be due
to a distinct change when the nuclei migrate into the periplasm and cells form,
or they may be two steps in a pattern which is constantly changing as the embryo
develops. The fragmentation experiments of Herth & Sander (1973) with
Protoformia and with Drosophila suggest that the latter hypothesis is more likely
to be true. The segment pattern is gradually established from fertilization to
blastoderm formation, so one might expect the sensitivity of different regions of
the egg to change correspondingly.
Embryonic abnormalities and the formation of larvae
Several organs may be abnormally formed after microcautery at one particular
region. There are two possible explanations for this kind of result.
(1) Normal morphogenesis may be impeded as a consequence of the primary
defect, leading to a general deformity in which the primary cause may not readily
be identified.
(2) Some cells, or groups of cells, may influence differentiation in other groups
of cells, and damage may thus interfere with a sequential series of relationships.
It follows that only particular and specific abnormalities are useful in the
context of mapping the egg for larval structures, although the frequent multiple
effects of microcautery may tell us about some of the general mechanisms underlying the development of larval structures.
It was not possible to classify these embryonic defects in a sensible fashion;
almost every egg was slightly different from the next. Many eggs (61-2 % at NM
and 57-8 % at Bl) reached the stage of having some segmentation, some gut
formation and often some tracheal formation. There were often small defects in
several systems, and because of possibilities (1) or (2) above, these were not
correlated to the initial site of damage. At NM, 18-3 % of eggs, and 13-9 % at Bl,
died during early development before the formation of segments or tracheals.
The gut was usually disarranged, a general defect found after microcautery at
many sites. At Bl a few embryos formed specifically anterior or posterior defects.
Those with the posterior well formed and the anterior undifferentiated or containing a contracting gut mass resulted from damage to regions 1-9, and embryos
with anteriors well formed and posteriors containing a gut mass arose from
damage to posterior regions 6-11. At NM this type of defect was rare, but was
always the result of damage to sites causing the same defects at Bl. Such defects
did not arise more frequently from particular regions on the dorso-ventral axis.
Abnormalities of the head and of mouthpart formation usually arose from
damage in the anterior half of the egg at Bl; at NM damage at almost any point
on the egg's surface produced these defects, although with greater frequency
from anterior microcautery.
Microcautery of Drosophila eggs
261
Adult defects
Very few imaginal defects were found amongst the hatching adults and unemerged pupae. The kinds of abnormalities found were as follows:
(1) Defects of mouthparts: proboscis or labial palps absent or deformed.
(2) Wings absent, incomplete or deformed, usually accompanied by an
abnormally shaped thorax and scutellum.
(3) Abnormalities of the legs: parts of the leg may be absent, joints crippled
or too long; a leg may be completely absent, or fail to evert correctly.
(4) Abnormal abdomens: fusion of tergites of different segments, missing
halves of tergites, distorted shape of abdomen as a whole.
No defects were found of eyes, antennae or halteres in these experiments.
When the treated embryos were less than 1-hr-old (NM) there were so few
adult abnormalities that no conclusions can be drawn from them.
After microcautery at blastoderm formation, defects were on the expected
side of the adult, except occasionally when both prothoracic legs were abnormal
or the mouthparts were abnormal on both sides of the proboscis. Not enough
defects were found to make an accurate map related to the surface of the egg,
but defects found did show some correlation with the initial site of damage.
Mouthparts were only abnormal after treatment of the anterior third of the
embryo, and the abdomen was affected much more frequently with posterior
than with anterior microcautery. The wing and leg abnormalities were spread
over quite a large area of damage.
Microcautery at 75 °C
General results
As the eggs were selected to be in the correct developmental stage, the nuclear
multiplication control sample showed a higher frequency of embryonic death than
blastoderm controls. When corrections were made for this, a larger number of eggs
failed to continue development after treatment at NM than at Bl, as shown in
Table 2. More embryos developed abnormally, more larvae died, and a greater
frequency of morphologically abnormal adults and pupae were found, after treatment at Bl. The only adult defects in the controls were abnormal abdomens.
Embryonic defects
Defective embryos arising from thermocautery at the sites shown in Fig. 1B
were classified according to the scheme below, and the results were tabulated in
relation to the area of the egg damaged (Table 3).
Class 1. Anterior defects. Embryos with abnormal head formation and with
mouthparts abnormal or absent (Fig. 4); embryos where the posterior has
some segmentation and the spiracles are present, the gut and yolk often extruded
at the anterior of the embryo. This class includes all embryos from those with
just a few abdominal segments partially or abnormally formed, to those with all
1036
983
NM
Bl
NM
Bl
NM
Bl
NM
Bl
NM
Bl
NM
Bl
NM
Bl
NM
Bl
NM
Bl
NM
Bl
Control
Experimental
Total
Abdomen
Wing
Thorax
Leg 3
Leg 2
Legl
Proboscis
Head
208
111
109
105
110
136
146
142
129
137
145
130
145
94
136
112
116
127
Stage
Area
Number
treated
508
269
26
3
51
11
64
53
76
48
52
49
75
28
57
9
91
22
42
49
Undifferentiated
eggs
0
2
3
14
12
18
14
14
3
20
12
15
19
12
11
48
7
27
81
168
447
546
Abnormal
embryos
182
106
55
80
34
65
56
80
74
68
58
87
69
73
34
42
67
51
Larvae
hatched
267
335
45
29
38
61
18
41
28
49
36
39
27
50
34
35
14
25
22
35
Larvae
dead
10
14
6
3
1
3
0
2
2
4
2
2
0
2
4
1
1
1
0
0
3
3
1
0
0
2
0
0
0
0
0
1
0
0
2
0
0
1
0
0
220
197
131
82
16
16
16
22
26
27
46
27
31
36
31
37
19
16
45
16
12
17
1
2
0
0
0
2
0
1
2
2
1
4
0
5
1
1
0
2
Pup
Pupae Abnormal Adults Abnormal
dee
pupae
hatched
adults
Table 2. Effects of microcautery at 75 °C on specific areas of the egg
0
X
«—i
0
m
w
o
to
Os
Microcautery of Drosophila eggs
263
Table 3. Classification of embryonic abnormalities resulting from
microcautery at 75 °C
Class
Head
Proboscis
Legl
Leg 2
Leg 3
Thorax
Wing
Abdomen
A
Stage
Total
1
NM
Bl
NM
Bl
NM
Bl
NM
Bl
NM
Bl
3
14
12
18
14
14
3
20
12
15
19
12
11
48
7
27
11
9
13
11
11
3
7
4
2
16
6
3
27
1
NM:
Bl
NM
Bl
NM
Bl
2
3
4
5
—
—
—
1
1
—
—
—
2
2
3
5
3
2
3
2
2
2
1
1
to to
Treated
area
3
1
1
2
z
4
3
4
2
1
6
2
—
2
2
5
—
3
2
Other
1
1
4
2
1
1
18
11
—
4
5
segments, a well-formed tracheal system and some head formation (Fig. 5). In
some embryos the anterior contains a large yolk patch and the posterior has a
contracting mass of gut tissue; they sometimes also have a few abnormally
arranged bristle rows on the surface, and one or two spiracles present (Fig. 6).
Class 2. Mid-region defects. Embryos where there is some formation of segments both at the anterior and posterior, but with an abnormality in the central
region which does not usually extend right across the embryo (Fig. 7). In some
embryos the abdominal segmentation is irregular, the bands running into one
another as in 'abnormal abdomen' adults (Fig. 8).
Class 3. Posterior defects. Embryos where the head and thorax have formed
abnormally, but there are signs of mouthpart formation. The posterior of the
embryo contains a mass of yolk and gut (Fig. 9). In other embryos the head and
thoracic segments are formed perfectly and the mouthparts are complete. There
may be abdominal segments of varying number, either complete or abnormally
formed behind the thorax; the gut is extruded at the posterior (Fig. 10). More
damaged embryos may have a large yolk patch at the posterior with the anterior
containing a gut mass (Fig. 11).
Class 4. Mouthpart formation amongst anterior defects. Embryos with a
contracting gut mass, where there is often irregular segmentation but there has
at some stage been abnormal mouthpart formation amongst the gut tissue. Some
embryos have a well-formed posterior and gut extruded at the anterior, but also
264
M. BOWNES AND J. H. SANG
Microcautery o/Drosophila eggs
265
have a set of well-formed mouthparts facing into the embryo or to the side
of it; there is no head formation or pharynx associated with this structure
(Fig. 12 A, B).
Class 5. Non-specific defects. Embryos with a contracting mass of gut tissue
(Fig. 13). Others are extremely transparent in appearance, with the gut
formed, but the segmentation very faint. These embryos appear to have died
before tracheal formation or mouthpart chitinization (Fig. 14), but not due to a
defect in a specific region.
Class 6. Others. Embryos which did not fit into any class and which were not
found more than once.
In contrast to the previous set of microcautery data, the embryonic defects
were easy to classify and closely correlated with the initial site of damage. In
many cases they agreed well with the kinds of defects found after u.v. irradiation
and pricking of similar areas. This is discussed further below, as each treated
region is looked at in detail.
Treatment in head, proboscis and leg 1 regions. In all these regions treatment at
both stages produced either anterior (Class 1) defects, or non-specific defects of
contracting gut masses (Class 5), as with u.v. irradiation at Bl and NM (Bownes
& Kalthoff, 1974). Class 4 embryos which were transparent in appearance
(Fig. 14) were also found after microcautery in these regions at NM.
Treatment at leg 2 region. At NM, leg 2 region damage produced anterior
defects. After treatment at Bl Classes 2 and 4 were found as well as anterior
defects. These were not found after u.v. irradiation, possibly because the area
damaged by microcautery is smaller, so that anterior morphogenesis is not
always impaired, as may occur after u.v. irradiation of a central band of the
egg. One embryo was found with anterior and posterior development and an
abnormal mid-region after pricking the embryo at blastoderm formation, but
usually anterior defects were found.
Treatment at leg 3 region. After treatment at NM mostly anterior and nonspecific defects were found, though examples of posterior and mid-region defects
Fig. 4. An embryo showing abnormal head formation and also some abnormal
mouthpart (m) formation. (Class 1 in Table 3.) The anterior is at the top of the
figure in this, and subsequent, photographs. The length of the egg is 0-42 mm.
Fig. 5. This embryo contains all the abdominal segments at the posterior, but the
gut (g) is extruded at the anterior. (Class 1 in Table 3.)
Fig. 6. At the anterior is a yolk mass, and at the posterior a contracting gut mass (g)
with signs of spiracles (s) and one or two abdominal segments. (Class 1 in Table 3.)
Fig. 7. Both the anterior and posterior show clear segmentation. Centrally there is a
large yolk mass (y). (Class 2 in Table 3.)
Fig. 8. The abdominal segment boundaries are abnormal, two running into one as in
'abnormal abdomen' adults. (Class 2 in Table 3.)
Fig. 9. The posterior contains a yolk mass (y) and the anterior a contracting gut mass
(g). (Class 3 in Table 3.)
266
M. BOWNES AND J. H. SANG
Microcautery o/Drosophila eggs
267
were also seen. Bl treatment produced anterior defects, non-specific posterior and
mid-region defects, and also embryos with inverted mouthparts. The abnormal
abdominal segmentation was found after treatment at Bl and NM. It should be
noted that hatched larvae were not checked for defects, so it is possible that
more defective embryos of this class were formed, which hatched and were thus
omitted from the classification of abnormal embryo.
Treatment of wing and thorax regions. Damage at both Bl and NM produced
mostly anterior defects and non-specific defects. After Bl treatment Classes 2
and 4 were also found, but these were generally absent after NM treatment.
Embryos with inverted mouthparts occur most frequently after treatment of this
region. It seems that mouthparts are able to form correctly amongst a mass of
gut tissue when there is no head formation, and their formation does not seem
to be dependent on induction from the pharynx, or on being in the correct
position in the head; yet when the head forms abnormally the mouthparts are
always absent or defective. It is difficult to imagine a scheme of differentiation
and determination of mouthpart material which can encompass both these
types of defect.
Treatment of the abdominal region. Non-specific and posterior defects, Classes
3 and 5, were most frequent after damage to this area at both stages. Some Class
2 defects were also found after damage at Bl. This is in agreement with the results
of u.v. irradiation and pricking of the posterior egg regions (Bownes & Sang,
1974).
In general the areas located nearest to the anterior or posterior reacted exactly
as in experiments using u.v. irradiation and pricking. Damage to more central
regions sometimes produced anterior defects, as with u.v. irradiation and pricking, but occasionally produced central defects not found in previous experiments.
These new defects arose much more frequently from treatment at blastoderm
rather than at nuclear proliferation stages. This suggests that the axes for the
development of the embryo are more completely established at blastoderm
Fig. 10. At the posterior there is a yolk mass (y), but at the anterior there are
perfectly formed head and mouthparts and all eight abdominal segments, including
one spiracle. This almost complete embryo occupies little more than half of the egg.
(Class 3 in Table 3.)
Fig. 11. At the anterior the mouthparts (m) and a number of abdominal segments
are perfectly formed. At the posterior the gut (g) is extruded. (Class 3 in Table 3.)
Fig. 12. (A) The posterior two-thirds of this embryo contains all the abdominal
segments and part of some thoracic segments. A large mass of undifferentiated
material is present at the anterior, yet amongst it is a set of perfectly formed mouthparts (w) facing into the embryo. (Class 4 in Table 3.) (B) High-power view of (A) to
show detail of mouthparts.
Fig. 13. A contracting gut mass with no visible segmentation. (Class 5 in Table 3.)
Fig. 14. Embryo which died in early development; the midgut is misplaced. (Class 5
in Table 3.)
268
M. BOWNES AND J. H. SANG
formation than they are at proliferation stages. This is in agreement with the
findings of Herth & Sander (1973), who found that the segmentation pattern of
Drosophila is gradually established with time, and is eventually complete at
blastoderm formation. The fact that mid-region defects are occasionally found
after treatment at NM might be due to the damaged area of cortex preventing
this region reacting as part of a gradient system.
The higher temperature, producing more damage to the embryo, gives results
rather different to the first set of microcautery experiments. Embryonic defects
are well correlated with the area damaged, and are usually large defects.
By looking at particular embryonic abnormalities it is possible to deduce some
facts concerning the interactions between certain areas of the embryo and the
autonomy of development of some organ systems. Tt is quite possible for the
anterior of the egg to develop normally when the other half is completely undifferentiated or contains a gut mass. It seems that the physical relationships
between the anterior and posterior are not necessary for at least some morphogenetic movements to occur. It is also clear that the egg has some capacity to
modify size. All abdominal segments and sometimes up to three thoracic segments and some head formation can be present after microcautery, but they
occupy only half of the egg, the segments being much more closely crowded
together than is usual.
Irregular segmentation has been observed several times around undifferentiated tissue. Segments occur on one side of the mass and not on the other in
some cases, and in others may radiate out from a single point on the egg surface.
Tracheal tubes are also able to form over a mass of tissue and are not dependent
on normal segmentation or organization before they can form. Spiracles have
been seen at the posterior of the egg without any segmentation being present, or
any other recognizable organ formation in the embryo. We cannot deduce from
these results that the egg is a mosaic of small areas determined to be specific
structures. Although there is some ability for organs to develop autonomously,
they are not always able to do this, suggesting that there are normally many
interactions between cells and developing organ systems during embryogenesis.
Adult defects
The great majority of formed adults were normal (Table 4), but the higher
temperature of microcautery did produce more specific defects in the adults than
were found in the first experimental series. There is a greater frequency of adult
defects with blastoderm damage than from damage to the periplasm (10 % of Bl
formed adults and 5-7 % of NM formed adults). In both cases the defects found
tend to be those expected within the limits of accuracy of the technique (see
Materials and Methods). Where more than one structure is affected, the damage
is to closely adjacent disc areas.
There could be four explanations of the high frequency of normal adults.
Either the technique is so inaccurate that the necessary localization of the damage
Microcautery
of Drosophila eggs
269
Table 4. Morphogenetic abnormalities of adults and pupae resulting
from microcautery at 75 °C
Treatment at
nuclear multiplication
Treatment at
blastoderm stage
Head
—
Proboscis
Legl
Leg 2
—
—
Antenna and eye missing from
1 side of head (pupa)
Proboscis damaged, 2 examples
Leg 3 damaged
Leg 2 and haltere missing, leg 3
abnormal, thorax abnormal with
leg and wing absent
Leg 3 abnormal (3 examples),
abnormal abdomen
Abnormal abdomen, wing and
thorax abnormal, scutellen
abnormal, wing abnormal, leg
and haltere missing
Area damaged
Leg 3
Thorax
Wing
Abdomen
Leg 1 abnormal, leg 1 and
leg 2 abnormal
Leg 3 abnormal and haltere
missing
Abnormal abdomen, thorax
abnormal with leg and wing
absent, wing leg and haltere
abnormal, leg 2 abnormal,
thorax abnormal with leg, wing
and haltere missing
Abnormal abdomen, wing
Abnormal abdomen
abnormal
Abnormal abdomen, 2 examples
is not achieved, or some form of repair, regeneration, or regulation is possible.
There is no certain way of eliminating the first possibility, but since damage to
each area greatly reduces the chances of survival to the adult stage by causing
malformations of the embryo or larva, it seems improbable that such a high
proportion (21 % of all eggs) of normal adults would survive. It is possible that
molecular repair processes may exist within the egg which can repair some of the
microcautery damage, but as yet no information is available to decide if this
unlikely event could happen. Several forms of cellular compensation are possible:
either other cells take over the function of disc cells, or any surviving disc cells
are capable of regenerating the whole (see Schubiger, 1971), or the undamaged
disc cells become reorganized and regulate to form a whole disc.
Although defects correlated to the initial site of damage are produced after
treatment at NM, this does not mean that there are localized regions in the
periplasm which are committed to-a specific fate in development, since areas
may be unable to respond to a later developmental signal to become committed
regions, or nuclei may be unable to move into this damaged region and the disc
cells would then not form.
Ilmensee (1972) also found imaginal defects correlated with the site of damage
after pricking NM stage embryos, although Howland & Sonnenblick (1936)
found no defective adults after similar experiments, and concluded that the
Drosophila egg was capable of limited regulation at this stage. Nothiger & Strub
270
M. BOWNES AND J. H. SANG
(1972) found adult defects, not correlated to the initial site of damage, after
partial u.v. irradiation of nuclear multiplication eggs. This could be because they
irradiated dorso-laterally instead of ventro-laterally where the presumptive disc
cells are located; damage to the discs probably resulted from u.v. which had been
scattered within the embryo.
The defective adults resulting from treatment at Bl do not necessarily show
that the cells are determined to be adult disc cells at this time, because the
damaged cells may remain in position and prevent the migration of nearby cells
into this region so that no compensation occurs. Although possible, this seems
unlikely since so many adults which hatch have normal morphology. The data
agree on the other hand with the results of Chan and Gehring (1971) which
show that cells are committed to become an adult disc after blastoderm formation. Chan and Gehring cut blastoderm eggs in half, disaggregated and reaggregated them. After culture in adults the fragments were injected into larvae and
underwent metamorphosis. Head and thoracic structures formed from anterior
fragments and thoracic and abdominal structures from posterior fragments,
thus showing there was at least an anterior-posterior determination of adult
disc cells at blastoderm formation. Combined with these results, microcautery
suggests that the specification is for individual discs, i.e. the adult disc cells are
determined to be a specific cuticular structure at blastoderm formation. The
abnormalities in the adults were always large; structures were missing or grossly
abnormal. There were never small defects such as a few bristles absent, or a small
defect in the wings or legs. This suggests that there is no specificity within the
group of cells determined to be a disc for specific parts of that disc. This 'all or
nothing' type of response to microcautery suggests that if only a small number of
disc cells are damaged they can be repaired or regenerated by the remaining cells,
and that regulation will eventually produce a normal adult.
DISCUSSION
Microcautery at 75 °C gives a greater frequency of more precise defects than
microcautery at 70 °C, probably because it produces a greater degree of damage
at a similarly small site. Nonetheless there is a considerable variability in the
effects produced by damage at any one site, and it is worth noting that this is not
necessarily a consequence of the technique, but has also been found with
mutants affecting embryogenesis. For instance, the dominant vestigial (VgD)
mutant of Bull (1952) causes abnormal head formation during embryogenesis
very similar to that described above (Fig. 4). Further study of the mutant (M.
Bownes, unpublished observations) shows that the whole range of defects
comprising Class 1 were produced by this small deletion of the second chromosome. This suggests that the large variety of defects found after experimental
manipulations is a feature inherent in the system of a developing egg, and not
totally due to small variations in the technique. Further, the variety of defects
Microcautery o/Drosophila eggs
271
found amongst VgD homozygotes alters with the genetic background (A. L.
Bull, personal communication; M. Bownes, unpublished observations) showing
that the genetic background of the organism can also affect the type of aberrant
development which results from this small deletion.
Shannon (1973) has described abnormal posterior development in eggs produced by the female sterile mutant almondex (amx) of Drosophila. Some embryos
which are genetically heterozygous resemble embryos experimentally damaged
at the posterior (Fig. 11). The occasional heterozygous females which hatch
often show thoracic and abdominal defects, like those resulting from mid-region
and posterior microcautery. It seems that both larval and adult structures may
be affected by the same gene, the defect being dependent on the location in the
posterior of the embryo of the presumptive cells of these structures. Similarly,
microcautery produces adult and embryonic defects dependent on the site initially
damaged.
The two stages of the embryo included in this study respond differently to
microcautery not only with respect to survival but also in their patterns of larval
and adult defects. In particular, mid-region defects and reversal of mouthparts
were commonest, as were particular adult defects after microcautery of blastoderm eggs (Table 3), and we have the impression that the state of determination is
more specific then than earlier. However, there is considerable variability between
regions of the egg, and there is no clear evidence that the egg is a mosaic, sensu
stricto, at either stage. Here we must note, however, that damage must also be
affecting embryonic cell movement, and that our results may be confused by
these secondary effects, which require further study. Since mapping with genetic
mosaics (Hotta & Benzer, 1972) localizes adult structures very precisely, the
implication is that mosaicism is established somewhat later than blastoderm
formation. This certainly seems to be the case for larval cells removed from4-6 h
embryos, some of which differentiate to their final state when cultured in vitro
(Shields & Sang 1970, and unpublished observations). We conclude, therefore,
that the distinction between regulative and mosaic eggs is one which depends
on the time of determination, and is not absolute. Our evidence for regulation,
particularly with respect to adult structures, is certainly open to the criticism
that it may be a consequence of other mechanisms, particularly molecular repair,
and this will be dealt with in a subsequent paper.
This work was supported by a Medical Research Council Studentship to Mary Bownes, and
the support of the Science Research Council is also gratefully acknowledged. We would like
to thank Mrs J. Atherton for preparing Figs. 1-3.
272
M. BOWNES AND J. H. SANG
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{Received 14 January 1974)