Download Requirements for autosomal gene activity during

495
Development 1M, 495-509 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Requirements for autosomal gene activity during precellular stages of
Drosophila melanogaster
PAULINE T. MERRILL, DARI SWEETON and ERIC WIESCHAUS
Department of Biology, Princeton University, Princeton, NJ 08540, USA
Summary
To identify early requirements for zygotic gene
activity in Drosophila, we used compound autosomes
and autosome-Y translocations to generate embryos
deficient for cytologically defined portions of the
genome. No obvious gross morphological defects were
observed in any deficiency class until the beginning of
cycle 14. Only seven autosomal regions were identified
with discrete effects visible prior to the onset of
gastrulation. These regions include genes with locusspecific effects on the clearing of the cortical cytoplasm
during early cycle 14, (22AB), the initiation of the slow
and fast phases of cellularization (26BF and 40AC,
respectively), the apical-basal distribution of nuclei
during cycle 14 (71C-7SC) and the closing off of
furrow canals during cellularization (100AC). The
distal tip of the third chromosome also contains two
loci (99DF and 100AC) whose deletion causes multiple
nuclei to be cellularized into single cells, a phenotype
similar to that produced in embryos totally lacking the
X-chromosome.
Introduction
of RNA begin to be made in Drosophila during the
syncytial blastoderm stage and the levels of newly
synthesized RNA are substantial by cellularization
(Zalokar, 1976; McKnight & Miller, 1976; Anderson
& Lengyel, 1981; Edgar & Schubiger, 1986). Few
such early-acting genes have been identified genetically, however, and very little is known about their
function.
In a preceding paper (Wieschaus & Sweeton,
1988), we have studied the role of X-chromosomal
gene activity in early development and have identified a single locus at 6F1-2 that is required for the
establishment of the hexagonal actin arrays associated with cytokinesis in early cycle 14. To identify
other loci involved in the cellularization process, we
have examined embryos that lack a substantial portion
of each autosome, but are derived from mothers that
are genetically normal. In Drosophila, such embryos
can be generated using compound chromosomes and
translocations (Rasmussen, 1960; Scriba, 1967, 1969).
In the following paper, we use such chromosomes to
create overlapping deletions for the entire Drosophila genome. We show that even very large deletions develop normally to the late syncytical stages
and only begin to show discrete abnormalities during
The cellular transformations that occur in most early
embryos are rapid and synchronous, and they occur
on a scale not duplicated later in development.
Embryos from a variety of different species have
therefore been used to study basic cellular phenomena like DNA replication, cytokinesis and cytoplasmic localization. Moreover, since the genotype of an
individual is established only at fertilization, the very
early stages of embryogenesis provide a useful system
for identifying specific gene activities required for
these events.
In Drosophila, most of the gene products required
for cleavage and blastoderm formation are already
present as RNA or proteins in the unfertilized egg
(Zalokar & Erk, 1976; Arking & Parente, 1980;
Gutzeit, 1980). The egg cell can thus be likened to a
complex machine poised for a series of morphological
transformations that must occur at precise times and
often in precise regions of the egg. What triggers
these events or causes them to occur at particular
times is not known. One obvious candidate would be
the controlled synthesis of zygotic gene products at
particular stages of development. Significant amounts
Key words: Drosophila melanogaster, autosomal gene
activity, precellular stages.
4%
P. T. Merrill, D. Sweeton and E. Wieschaus
A 2LW
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2L ~
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2R minus ><
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2L ^
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2L *
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2L
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Fig. 1. Generation of embryos deficient for large regions of the second chromosome. (A) Compound autosome stocks.
Instead of the normal diploid arrangement in which the left and right arms of a given chromosome are attached to the
same centromere, compound autosomes have both left arms attached to one centromere and both right arms attached
to the other (Rasmussen, 1960). These individuals contain the normal genetic complement; their gametes, however,
contain either two left or two right arms of the attached autosome, or all four arms or none. Segregational analysis
using embryonic lethals (Materials and methods) suggest that virtually all the female gametes on the compound-2 stock
are either 2R + 2L" or 2R"2L + , but that 44% of the male gametes are 2R + 2L + or 2R~2L". Similar values are obtained
for the compound-3 stock (95% and 48%, respectively). In either stock, inbreeding produces about 50% normal
embryos, 25 % with no right arm of the attached autosome and 25 % with no left arm of that autosome. The latter two
classes of embryos will show defects as soon as a zygotic gene product from the missing arm is required for normal
development. (B) Crosses between compound autosome stocks and 2Y translocations. To obtain smaller deletions,
compound autosome females were crossed to males bearing Y-autosome translocations with breakpoints at various
points along the chromosome. As long as the required region is distal to the translocation breakpoint, one eighth of the
embryos from the cross will lack the relevant gene and exhibit the abnormal phenotype; a cross in which no embryos
show the abnormality indicates that the gene is proximal to this breakpoint.
cycle 14. We identify seven early-acting regions responsible for these abnormalities and analyse the
phenotypes the corresponding deletions produce during cycle-14 cellularization.
Materials and methods
Genotypes and stocks used
Two attached autosome stocks were used to generate
embryos deficient for entire chromosome arms:
RM(2L);RM(2R), marked with vermillion (= C(2)v, Bowling Green Stock Center no. nl6); and C(3L)st;C(3R)e, a
compound third chromosome obtained from the University
of Indiana, Bloomington. The meiotic segregation pattern
of each compound chromosome was determined by mating
males or females from the compound stock to flies heterozygous for four different embryonic lethals (NiissleinVolhard & Wieschaus (1980); i.e. odd-skipped• evenskipped/paired • Kruppel in the case of the second chromosome, or hairy hedgehog/hunchback knirps in the case of
the third chromosome). Aneuploid embryos from such
crosses die but make cuticle, which can be scored for
segmentation phenotypes. Embryos with normally segmented cuticle will arise only when the parent from the
compound stock contributes both halves of the compound
to the gamete. The fraction of the total embryos derived
from the cross with normal segmentation thus provides a
measure for the frequency with which the right and left
arms of the compound segregate to the same pole during
meiosis (see Fig. 1).
The autosome-Y translocations that were used to produce embryos with smaller genomic deficiencies are listed
in Table 1. When necessary, the segregation pattern of
individual translocations was determined by mating translocation males to females heterozygous for lethal mutations
that had been mapped previously to the right or left arm of
the chromosome affected by the translocation (generally
odd-skipped and even-skipped on the second chromosome,
or hairy and hedgehog on the third chromosome). The
frequency with which the zygotes displayed the segmental
phenotype associated with the mutant on the given arm was
used to determine the segregation pattern of the particular
translocation tested.
Phenotypic characterization of living embryos
Living embryos were covered with Voltalef 3S Halocarbon
oil to make the egg shell transparent and their development
was followed in bright-field optics using a stereomicroscope
(x50). About 100-200 eggs were collected from a given
stock or cross, observed at 10-15 min intervals and classified according to phenotype. A fraction of the eggs from
most collections showed no sign of development and
appeared to be unfertilized. While it is possible that some
abnormal embryos might show this appearance, the large
fluctuations in the frequency of apparently unfertilized eggs
in different collections (19%, 13%, 15%, 7%, 5 % , 7%
for the second chromosomal stocks and 9 %, 5 %, 12 % and
2% for the third chromosomal stock) can best be interpreted as due to variable fertilization in the compound
stocks; abnormalities associated with a particular embryonic genotype would be expected to give more consistent
Autosomal gene activity in precellular Drosophila
497
Table 1. Chromosomal aberrations used to generate deficiency embryos
Cytology
Name
T(Y;2)H165
T(Y;2)J69
T(Y;2)H56
Df(2)ast-1
Df(2)ast-2
Df(2)ast-4
Df(2)ast-5
Df(2)Star-2
T(Y;2)B137
T(Y;2)D211
T(Y;2)H121
T(Y;2)J136
T(Y;2)H52
T(Y;2)A145
T(Y;2)A80
T(Y;2)L138
Df(2)SD-Romapr67
Df(2L)65
Df(2L)161
T(Y;2)G
Df(2L)Cl,PR31
T(Y;2)B199
T(Y;2)R116?)
T(Y;2)H118
T(Y;2)G10
T(Y;2)B135
T(Y;2)B26
T(Y;2)H136
T(Y;2)L23
T(Y;2)G53
T(Y;2)R124
T(Y;2)B202
T(Y;2)H143
T(Y;3)P50
T(Y;2)J112
T(Y;3)J100
T(Y;3)J44
T(Y;3)B132
T(Y;3)H147
T(Y;3)J44
T(Y;3)L132
T(Y;3)G8
T(Y;3)B116
T(Y;3)H173
T(Y;3)B226
T(Y;3)B152
T(Y;3)R133
T(Y;3)R130
Df(3R)X3F
Df(3R)tlP
Df(3R)tir
21E
22A
22B
21C7-8;23Al-2
21Dl-2;22B2-3
21Dl-2;21El-2
21E1-2;21F3-22A1
21C6-D1:22A6-B1
25D
26A
26B
26F
27E
29F
35B
39C
38A5-8:39CE
37F5-38A1;39E2-F1
38A6-B1;4OA4-B1
36B5-C1;4OF
40, heterochromatin
40, heterochromatin
40, heterochromatin
40, heterochromatin
41
42AB
43EF
44C
45F
50A
55E
59A
59F
65D
71BC
75C
75CD
76A
77B
78CD
83 CD
85F
90E
95E
98B
98F
99E
100A
99D1-2;99E1
99Fl-2;100B4-5
100Al-2;Cl-2
Ref.
Early Phenotype*
b
b
b
c
c
c
c
c
b
b
b
b
b
b
b
b
d
f
f
a
a
b
b
b
b
b
b
b
b
b
b
b
b
none (0 halos/43 total)
15/111 halos
8/130 halos
32/120 halos
30/101 halos
none (0 halos/89 total)
none (0 halos/100 total)
none (0 halos/90 total)
28/220 halos, short germband
10/109 halos, short germband
5/25 halos, short germband
3/15 halos, slow phase block
10/100 halos, slow phase block
51/404 halos, slow phase block
33/262 halos, slow phase block
46/316 halos, slow phase block
none (38/131 ventralized gastrula)
none (0/72 abnormal)
none (0/60 abnormal!)
shallow cellulanzation
none (0/60 abnormal)
no cellularization
no cellularization
no cellularization
16/122 gastnilation defects
20/114 gastnilation defects
29/252 gastnilation defects
10/111 gastnilation defects
60/373 gastnilation defects
16/186 gastnilation defects
3/12 no ventral furrow
3/23 no ventral furrow
none (all normal)
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
g
e
e
none (0/209 cellularization defects)
10/279 cellularization defects
11/114 cellularization defects
22/171 cellularization defects
12/110 cellularization defects
43/426 cellularization defects
38/346 cellularization defects
76/568 cellularization defects
66/479 cellularization defects
69/430 cellularization defects
78/591 cellularization defects
42/206 cellularization defects
14/114 cellulanzation defects
10/84 cellularization defects
27/229 cellularization defects
22/89 cellularization defects
0/45 cellularization defects
7/28 cellularization defects,
no posterior midgutt
References: A, Lindsley & Grell (1968); b, Lindsley et al. (1972); c, Roberts et al. (1985); d, Brittnacher & Ganetsky (1983); e,
Strecker et al. 1988; f, Wright et al. 1976; g, Kongsuwan, Merriam and Lengyel, personal communication.
* For translocations, 'Early Phenotype' refers to the phenotype in the distally deficient embryos obtained when translocation males
are crossed with compound females. 'Slow phase block' refers to the failure to cellularize during the first 40min of cycle 14. The early
cycle 14 defects in 3L~ and 3R~ embryos are difficult to distinguish from each other under a stereomicroscope and are collectively
referred to as 'cellularization defects' in the table. At the onset of gastnilation, deficiencies generated by more proximal translocation
breakpoints can often be distinguished by the failure to initiate normal posterior midgut formation in 3L~ embryos.
t Because of a significant dominant maternal effect in heterozygous females, this deficiency was tested by mating deficiency
heterozygous males to compound females.
percentages. When these 'unfertilized' eggs are not considered, all expected deficiency classes are observed and at
frequencies (25-29%) expected for normal meiotic segre-
gation. This argues that, whether or not these eggs are in
fact unfertilized, they do not represent the particular
deficiency class whose members are the subject of these
498
P. T. Merrill, D. Sweeton and E. Wieschaus
experiments.
Photomicrographs and time-lapse video tapes of optical
sections of individual embryos at x200 or X400 were made
following previously published procedures (Wieschaus &
Niisslein Volhard, 1986). The conclusions presented in the
results section are based on videotapes of 66 normal
embryos, 9 2L~ embryos, 4 2R~ embryos, 19 3L~ and 18
3R~ embryos.
To follow the density and spacing of surface nuclei during
cleavage cycles 10 through 14, we used a compound
microscope and epi-illumination created by directing the
light beam from a fibre optic source across the surface of an
otherwise unilluminated embryo. For maximum resolution,
embryos were dechorionated with dilute sodium hypochlorite, viewed with a xlOO oil immersion objective and
the image enhanced by transmission through the video
camera/time-lapse recorder system.
Whole-mount preparations
The F-actin and nuclear distributions during late cleavage
stages were analysed by staining fixed whole-mount embryos with rhodamine-labelled (RH) phalloidin and
Hoechst 33258 dye, following previously described procedures (Wieschaus & Nusslein Volhard, 1986). The
stained embryos were examined at xl6, x25 and xlOO
using a Zeiss compound microscope with fluorescence
optics.
In most cases, embryos were stained with both Hoechst
and RH-phalloidin. However, in experiments where the
frequency of mitotic abnormalities in different stocks were
to be compared, embryos from both stocks were stained
with Hoechst 33258 but only one of the stocks was stained
with RH-phalloidin. The embryos were then mixed and
transferred to glass slides. The cleavage pattern of each
embryo was scored using the u.v. wave length (based on the
Hoechst stain) and the stock origin of the embryo was
determined using the RH-phalloidin only after the embryos had been classified.
Sectioned material for light microscopic and electron
microscopic analysis was prepared following previously
published protocols for fixation and embedding (Wieschaus
& Nusslein Volhard, 1986).
Results
To identify early requirements for zygotic gene activity, we have followed the development of embryos
deficient for defined portions of the Drosophila
genome. Many of the most characteristic events of
early Drosophila development can be scored directly
in living embryos (Foe & Alberts, 1983; Wieschaus &
Nusslein-Volhard, 1986). These include (1) the
emergence of the polar buds and pole cells following
the eighth and ninth nuclear division; (2) the
synchronous contractions of the cortical cytoplasm as
the nuclei undergo their tenth through thirteenth
cleavage divisions; (3) the clearing of the cytoplasm at
the outer edge of the syncytial blastoderm as yolk and
lipid droplets are transported basally and (4) the even
descent of the membranes between regularly aligned
nuclei during cellularization.
No single region on either the second or the third
chromosome is required for gross normal
morphology up to the onset of cycle 14
The second and third chromosome each represent
about 40 % of the genome in Drosophila and judging
from the number of bands on polytene chromosomes
should each contain about 2000 lethal complementation groups. Both are metacentric in morphology,
with similarly sized right and left arms attached at the
centromere. To determine whether activity of either
chromosome is required during the cleavage or cellularization, we generated deficiency embryos using
attached autosomes in which the two right arms and
the two left arms, normally present in a diploid, were
fused to each other (Fig. 1A). When such females
undergo meiosis, half the resulting gametes lack all
copies of the right arm and half lack all copies of the
left. Meiotic segregation in males gives rise to these
gametes, but also produces sperm that have both
right and left arms or neither arm (Baldwin &
Chovnick, 1967; Wright, 1970). Ultimately about one
quarter of the embryos derived from a cross between
compound males and females will lack the right arm
and one quarter will lack the left arm (Fig. 1).
Although each compound stock produced two
morphologically distinct classes of deficiency embryos
by gastrulation, virtually all embryos from the stocks
develop normally until cycle 14, at least judging from
their appearance under a stereomicroscope. To analyse early development in greater detail, we made
time-lapse video tapes of developing embryos from
compound stocks at stages between pole cell formation and gastrulation. Embryos were videotaped
using either optical cross-sections at x40 magnification and epi-illumination to follow the dividing
nuclei in the surface cortex (Fig. 2, see also Materials
and methods). Based on their subsequent phenotypes
at gastrulation (see below), it was possible to distinguish the deficiency embryos from their heterozygous siblings. The data in Table 2 show that wildtype and deficiency embryos have cleavage cycles of
similar length and duration. The density of the nuclei
in deficiency embryos at interphase of each cycle is
basically normal, although some deviation from a
uniform spacing was observed in 3L~ embryos (and
possibly 2RT, see below). Deficiency embryos also
begin the major morphogenetic movements of gastrulation at approximately the same times as wild-type
embryos. Taken together, these results identify no
region on either the second or the third chromosome
required for gross normal morphology prior to cycle
14. They argue in turn that the major morphological
transformations that occur during cleavage rely
Autosomal gene activity in precellular Drosophila
499
Fig. 2. Epi-illumination photomicrographs of surface nuclei during cleavage cycle 10-14. Normal embryo (left panel);
2R~ embryo (middle panel); 3L~ embryo (right panel). Note the irregular spacing and reduced number of nuclei in the
3L~ embryo, compared to the more regular distributions in normal and 2R~ embryos.
almost entirely on maternal gene products already in
the egg at fertilization. A similar conclusion is suggested by the observation that embryos injected with aamanitin develop normally to the beginning of cycle
14 (Edgar et al. 1986). The early cleavage defects
described for various deficiency embryos by other
authors (Scriba, 1967, 1969: reviewed in Wright,
1970) may be attributed to dominant maternal effects
of the heterozygous mothers (Garcia-Bellido et al.
1983); or to the difficulty in staging abnormal embryos that have been fixed and sectioned (see Wieschaus & Sweeton, 1988).
Embryos deficient for the left arm of the second
chromosome are defective in transport of lipid
droplets from the periphery
28% of the embryos from the compound-2 stock
showed a three-layered ('halo') appearance early in
cycle 14 (Fig. 3A,C), due to an altered distribution of
the cortical cytoplasm prior to cellularization. In wildtype embryos, the depth of the cortical cytoplasm
increases after the thirteenth cleavage as lipid and
yolk are transported basally (Fullilove & Jacobson,
1971; Lundquist & Emanuelsson, 1979; Foe &
Alberts, 1983). In 'halo' embryos from the com-
500
P. T. Merrill, D. Sweeton and E. Wieschaus
Table 2. Length of cleavage cycles and density of surface nuclei between cycles 10 and 14 of embryos derived
from the compound-2 and compound-3 stocks
Cycle length (min cycle l)
Genotype
Cycle
11
10±2
Normal
2L11±2
2R10±l
3L~
[10]
10±3
3RNormal
10±l
(Foe & Alberts, 1983)
Cycle
12
Cycle
13
Cycle
14*
12±2
13±2
10±l
11±1
12±3
12+1
18±3
18±2
17±2
19±3
17±3
21±2
53 ±10
63±7
61±3
72±13
65±15
(50)
Density (nuclei 1600/an 2)
PB/I4t
52±8
53±7
48±5
[46]
47 ±10
(56)
Cycle
11
Cycle
12
Cycle
13
Cycle
14
7±1
6±1
7±2
6±1
6±0
11±2
22±3
24±1
25±2
19±3
21±2
20-32
44±3
40±3
43±4
25±5
43 ±15
43-60
5-7
n±\
12±1
11±1
10±l
9-13
Cycle times were measured in minutes between that point in each cell cycle when the nuclei again become distinct after mitosis.
Values given are arithmetic averages plus/minus standard deviations. Values in brackets were obtained from measurements on a single
embryo. Published values (Foe & Alberts, 1983) for cycle times were given as average plus/minus standard deviation, whereas nuclear
densities were given as ranges. Values in parenthesis are derived from the text (Foe & Alberts, 1983).
* Total time from the reappearance of distinct nuclei at the onset of cycle 14 to the initial surface changes at gastrulation.
t Total time from polar bud formation to the reappearance of distinct nuclei at the onset of cycle 14.
pound-2 stock, yolk transport occurs normally but the
lipid droplets remain in the peripheral cytoplasm
immediately below the nuclei (Fig. 4A,B)- The refraction of these lipid droplets darkens the cytoplasm
when embryos are viewed with transillumination,
causing a halo of shaded cytoplasm below the nuclei.
Below the lipid halo, the cytoplasm next to the yolk is
poor in lipid, producing a second clear layer immediately adjacent to the yolk. The phenotype becomes
unambiguous in time-lapse videos 10 min after the
beginning of cycle 14, but since the cytoplasmic
clearing begins much earlier, it is likely that a
quantitatively less striking phenotype may be detectable in early cycles.
The zygotically active gene whose absence causes
halo formation was localized on the left arm of the
second chromosome by crossing C(2)v females with
T(Y;2) translocation males with breakpoints in that
arm. One eighth of the embryos from such crosses
will be deficient for all genes distal to the translocation breakpoint (Fig. IB). Halo embryos were
produced by males with translocation breakpoints
from 39C to 22 A, but not by males with breakpoints
in 21E (Table 1). These results suggest that the locus
responsible for the halo phenotype lies between 21E
and 22A. To localize further the gene, embryos were
collected from stocks heterozygous for deficiencies in
the 21-23 region (Table 1). The definitive halo
phenotype (Fig. 3F, Fig. 4C) was observed only in
embryos from the ast-1 (21C7-8,23Al-2) and ast-2
(21Dl-2,22B2-3) stocks, where they represented
one quarter of the total embryos from the stock. They
were not observed in embryos homozygous for other
deficiencies that substantially overlap the distal regions of ast-1 and ast-2 (Table 1), localizing the gene
to the 22A6-B2 region.
In spite of their abnormal lipid transport, ast-2
homozygotes cellularize and gastrulate normally. At
24 h, mouth parts and segmentation are visible, but
none of these embryos hatch. These later phenotypes
can be attributed to other genes within the ast-2
deficiency since they are also produced by slightly
overlapping deficiencies which do not lack bands in
22A6-B2 responsible for the 'halo' phenotype.
Genes affecting cellularization on the second
chromosome
Cellularization in Drosophila normally begins immediately after the nuclei enter interphase of cycle 14
(Mahowald, 1963; Fullilove & Jacobson, 1976;
Turner & Mahowald, 1976; Lundquist & Emanuelsson, 1979) and is completed in about 55 min at room
temperature. It occurs in two phases: an initial slow
phase thought to proceed solely by addition of new
membrane (Turner & Mahowald, 1976; Warn &
MacGrath, 1983), followed by a fast contractile phase
in which membrane from the surface microvilli appears to be pulled down into the cleavage furrows.
The 2L~ halo embryos derived by inbreeding C(2)v
males and females show normal nuclear and actin
localization through early stage 14, but they do not
form cells. Since Df(2)ast-2 homozygotes cellularize
and gastrulate normally, other regions on 2L must be
responsible for the abnormalities observed in embryos that lack the entire arm. By crossing C(2)v
females to various translocation males (Table 1), the
cellularization defects observed in 2L~ embryos
could be localized to two additional zygotically active
regions, one between 26B and 26F and the other
between 39C and the centromere.
The 2L-deficient embryos from translocation
crosses with breakpoints at or distal to 26B form
AutOsomal gene activity in precellular Drosophila
501
B
Fig. 3. Living embryos deficient for large regions of the second chromosome. (A) Normal embryo during cellularization
and early gastmlation; (B) 2R~ embryo during late cellularization and early gastmlation. Note absence of the ventral
furrow and presence of normal posterior midgut; (C) 2L~ embryos during midcycle 14 and gastmlation. Note obvious
'halo' of lipid-rich cytoplasm and abnormal attempt at posterior midgut formation and gastmlation (—>); (D) Embryo
deficient for the tip of the second chromosome to 26A, during early and late cellularization. Note 'halo' and uniform
cellularization ring; (E) Embryo deficient for the tip of the second chromosome to 39C, during late cellularization. Note
'halo' and irregular cellularization ring; (F) Embryo homozygous for Df(2)ast-2 (=21Dl-2; 22B2-3). Note 'halo'.
502
P. T. Merrill, D. Sweeton and E. Wieschaus
videos demonstrate that this is largely due to the
failure of the tnvaginating membranes to accelerate
their rate of elongation during the last 15 min of
cellularization (Fig. 5B). The phenotype is consistent
with a role of the proximal gene in the fast contractile
phase of cellularization, but judging from the rates of
cellularization depicted in Fig. 5B, the underlying
defect may extend into the earlier phase of cellularization as well.
Fig. 4. Electron micrographs of lipid clearing during
early cycle 14. (A) Normal embryo; (B) deficiency 2L~
embryo (segregant from the C(2)v stock) and (C)
Df(2)ast-2 homozygous embryo.
normal cellular blastoderms (Fig. 3D); embryos lacking regions distal to 26F only begin to cellularize
about 40min into the cycle (Fig. 3E; Fig. 5). The
resulting cellularization is not uniform over the entire
surface of the embryo, possibly because the embryo
begins to gastrulate only about 15 min after the visible
onset of cellularization, at a time when the membranes have reached less than half their normal
extent. The staging and rapidity with which the
membranes advance suggest that the abnormal cellularization in these embryos corresponds to 'fast',
contractile, phase. The absence of cellularization
during the first 40 min of cycle 14 indicates the
existence of a gene in 26B-F which is required for the
initial slow phase involving membrane addition.
The gene in 26B-F does not, however, account for
the entire nullo-2L phenotype, since no cellularization at all is observed in nuUo-2L embryos and halo
embryos derived from translocation males with
breakpoints in the centromeric heterochromatin.
Since translocation males with breakpoints in 39C
produce the same cellularization phenotype as those
with breakpoints in 26F, the gene required for the late
cellularization occurring in both genotypes must lie
between 39C and the centromere. To obtain embryos
deficient for the proximal regions of 2L, we mated
compound-2 females to males carrying an insertional
translocation T(Y;2)G (36B6-C; 40F). The most
obvious abnormality in embryos deficient for the
proximal 2L is the shallowness of the blastoderm cell
layer as the animal begins to gastrulate. Time-lapse
Embryos deficient for 2R cellularize normally but
fail to make ventral furrows at gastrulation
The compound-2 stock produces a second class of
abnormal embryos that fail to form a ventral furrow
at gastrulation (Fig. 3D). Such embryos begin a
posterior midgut imagination normally and form
cephalic furrows, but those structures also soon
become abnormal. Identical phenotypes are produced in one eighth of the embryos when C(2)v
females are mated to translocation males with breakpoints in proximal 2R, confirming that this abnormal
class results from deficiencies of the right arm of the
second chromosome.
The normal appearance of 2R~ embryos through
cellularization was unexpected, given the observation
by Karr et al. (1985) that embryos homozygous for a
mutation on 2R (engrailed) become abnormal during
cleavage divisions. Using our mounting procedures,
we could not detect the reported right-left asymmetry of the pole cells in living embryos shown
subsequently deficient for 2R, nor have we observed
any other consistent pregastrulation defects in the
embryos we videotaped (Fig. 2, Table 2). To detect
more subtle defects, we compared the frequency of
mitotic irregularities in fixed embryos from the C(2)v
stock with those found in wild-type stocks. Because
the phenotype reported for engrailed overlaps patterns we have seen in wild-type stocks (Fig. 6), we
used procedures that allowed us to score individual
embryos without knowing their genotype or stock of
origin (Materials and methods). When summed over
cycles 10-14 (Table 3), the C(2)v stock embryos
showed approximately the same overall percentages
of asynchronous divisions and irregular patterns as
wild type. There was, however, a significant increase
of abnormal patterns in embryos from the C(2)v stock
fixed during cycles 12 and 13 (Table 3). The increased
frequency of abnormalities during these cycles is
compatible with their being a deficiency phenotype, if
one assumes that only one of the two second chromosomal arms affects mitotic synchrony. Overall the
effect is rather subtle and we have not used translocations to localize it further. In the context of the
present paper, it does indicate a lower limit in the
ability of our procedures to detect early defects.
Autosomal gene activity in precellular Drosophila
GASTRULATION
/
10
20
30
40
50
Length of invaginated membrane
60
10
20
30
40
50
Length of invaginated membrane
60
Fig. 5. CeUularization rates in normal embryos and embryos deficient for either the proximal or distal regions of 2R.
(A) Embryos deficient for the distal portion of the left arm of the second chromosome (=O), obtained by mating
compound-2 females to either T(Y;2)L138,39C or T(Y;2)A80,35A males. Two of the three embryos recorded were
selected based on their halos phenotype. Since the first 15min of cycle 14 were not videotaped, the data from these
embryos have been aligned with the third embryo using the point when they began gastrulation. The data for normal
embryos ( = • ) were derived from six sibling embryos from the same cross. (B) Embryos deficient for the proximal
regions of the left arm of the second chromosome (=O), obtained by mating compound-t2 females to
T(Y;2)G,26B6-C1 males. Three embryos were measured. Values for normal embryos ( = • ) are based on five sibling
embryos from the same cross. The depth of the CeUularization furrow was measured on the dorsal side where it is not
distorted by ventral furrow formation. Error bars indicate one standard deviation.
Fig. 6. Abnormal cleavage patterns observed in embryos from wild-type and compound-2 stocks. Wave-like mitotic
patterns observed in Oregon-R stock (A) and in compound stock (B). Asynchronous mitotic pattern observed in
Oregon-R embryo (C) and in compound stock (D). Variable irregular spacing of nuclei in Oregon R stock (E) and in
compound stock (F,G). Dense internalized nuclei at posterior pole of Oregon R embryo (H) and in compound stock
(I)-
503
504
P. T. Merrill, D. Sweeton and E. Wieschaus
Table 3. Cleavage patterns in embryos derived from the compound-2 stock and thus potentially deficient for
either the right or left arm of the second chromosome
Abnormal class
Mitotic patterns
embryos
scored
Asynchronous
Wave
Normal
internal1
nuclei
Spacing
variable
Other
C(2)v
+
C(2)v
+
C(2)v
+
C(2)v
+
C(2)v
+
C(2)v
+
C(2)v
+
122
104
146
167
539
126
120
161
131
538
84
75
37
43
57
77
68
54
50
62
9
10
8
0
6
11
10
11
0
8
7
12
27
7
8
10
17
1
9
0
5
12
8
7
0
0
0
44
14
0
0
1
41
10
1
2
3
1
2
5
8
B
0
2
25
4
8
Cycle
<10
10/11
12/13
14
Total
6
0
4
For each cycle, the frequency (%) of the embryos from the compound-2 stock (C(2)v) showing a given kind of abnormalities is
compared to the frequency measured using the Ore-R wild-type stock. Photogtaphs of each kind of abnormality are given in Fig. 6.
Embryos deficient for 3L show an abnormal
distribution of nuclei to the central cytoplasm
Approximately a quarter of the embryos from the
compound-3 stock show a 'fuzzy' appearance at the
interface between the yolk and cortical cytoplasm in
the earliest stages of cycle 14 (Fig. 7). In videotapes
made of the posterior pole at x40, the abnormality is
apparent as soon as the nuclei become again visible
after the 13th division. When viewed using epiillumination, the spacing of the nuclei in the surface is
irregular and not all nuclei can be resolved in the
same focal plane. In sectioned embryos at this stage,
many nuclei are observed below the surface, apparently being transported to the central yolky core
(Fig. 8A,B). A large fraction (85%) of the 'fuzzy'
embryos undergo an additional round of division
midway through the cellularization of cycle 14 (Fig. 7,
middle panel, second frame), perhaps in response to
the reduced nuclear density at the surface. After the
extra division is completed, the embryos continue
cellularization and attempt to make a ventral furrow
and a posterior midgut, but the basic appearance of a
'fuzzy' yolk mass remains.
When compound-3 females were crossed with
translocation males with breakpoints from 75C or
78CD, one eighth of the progeny showed the 'fuzzy'
phenotype, localizing the required gene to the left
arm of the third chromosome (Table 1). Under the
dissecting microscope, the embryos derived from
crosses using more distal breakpoints (71C. 65D)
were phenotypically normal until the onset of gastrulation. This argues against the existence of any loci
with early zygotic effect on gross morphology distal to
71C and suggests that the 'fuzzy' locus must lie
somewhere between 71C and 75CD.
Development of embryos deficient for 3R
A second class of embryos from the compound-3
stock becomes distinct only as cell membranes descend between nuclei about 15min into cycle 14. At
this point, the nuclei in mutant embryos become
disorganized and some are shifted out of the uniform
single layer (Fig. 7C). In sectioned material, these
nuclei appear constricted in the middle (Fig. 8C,G),
as though they are being carried by the advancing
cellularization front into the interior of the embryo.
When the embryo has reached mid-cellularization
stages, two layers of nuclei can be observed, the more
basal layer coinciding with the advancing front of
cellularization (Fig. 8E). When cell membranes have
reached their normal depth, the embryos begin an
abnormal gastrulation and soon assume a characteristic morphology due to their failure to form a
posterior midgut (Fig. 7, right panel, last frame).
Normally during cellularization, the cleavage furrows only begin to widen and pinch off cells as they
approach the prospective yolk sack. In 3RT embryos,
this pinching-off occurs early during the cellularization process, trapping nuclei and dragging them
from the surface (Fig. 8G-J). All of the crosses
between C(3)se females and T(Y;3R) males produced
embryos that showed the double-layered nuclear
phenotype, a result that localized the gene responsible beyond the most distal of the translocation
breakpoints we used (100A5-6). By examining the
development of embryos homozygous for various
deficiencies in that region, we found that the nuclear
layering was produced in embryos homozygous for
Df(3)tW (100A1-2; 100C1), but not in Df(3)tlF
(99F1-2; 100B4-5), putting the locus in the six bands
between 100B4 and 100C1. A cellularization defect
mapping to Df(3)tlle but not Df(3)tll8 has been
described previously (Mahoney & Lengyel, 1987).
Autosomal gene activity in precellular Drosophila
505
Fig. 7. Development of cycle-14 embryos deficient for various portions of the third chromosome. Posterior third of
embryo, photograph taken directly from the time-lapse videomonitor during playback. Normal embryo (left panel); 3L~
embryo (middle panel); 3R~ (right panel).
3R embryos show a second phenotype very similar to that observed in nullo-X embryos (Wieschaus &
Sweeton, 1988). Early during cellularization multiple
nuclei are often enclosed within the same cell membranes. Subsequently, the ventral side of 3R~ embryos becomes particularly irregular and, like nullo-X
embryos, the yolk sac begins 'oozing' out to the
surface at the posterior ventral pole (Fig. 7).
Rh-phalloidin-stained whole mounts of early cycle14 embryos show the same irregular patterns of actin
and nuclei observed in nullo-X embryos, arguing that
the abnormal cellularization may arise by similar
disruptions in the hexagonal actin arrays responsible
for the initial positioning of the cell membranes.
This phenotype is produced in all crosses using
translocations with breakpoints proximal to 98F
(Table 1) and in somewhat diminished form by even
the most distal translocations used in this study.
Analysis of deficiencies for the distal region of 3R
identify at least two regions required for that aspect
of normal cellularization (Df(3)XF3, 99D1-2 to
99E1, Fig. 8K,L; and Df(3)tlf, 100A1-2 to 100C,
Fig. 8G-J). The 6F1-2 region of the X-chromosome
and the distal tip of the third chromosome seem to be
506
P. T. Merrill, D. Sweeton and E. Wieschaus
Autosomal gene activity in precellular Drosophila
the only regions of the Drosophila genome required
for this aspect of cellularization since embryos deficient for 2L (21A-39C), 2R (41-60F) and 3L
(61-80) show normal actin arrays at the beginning of
cycle 14 (data not shown).
Discussion
By generating synthetic deficiencies for cytologically
defined portions of the Drosophila genome, we have
examined the early requirements for zygotic gene
activity during early embryogenesis. The largest deficiencies represented a single chromosomal arm,
about 20% of the entire genome. All such deficiencies allowed development to the beginning of cycle
14, arguing that the first thirteen cleavage cycles run
predominantly, if not exclusively, using gene products supplied by the mother during oogenesis. We
could reliably detect early defects only during the
initial stages of cycle 14 and several fairly large
deletions (e.g. 41A to 60F; 61A to 71CD) allowed
normal development until the onset of gastrulation.
Even in those cases where the deficient embryos
became abnormal, the defects were initially fairly
discrete, an observation that can be most easily
explained if they represent the requirement for only a
single gene. In most cases, the translocation mapping
allowed localizing the required gene to small regions
of the chromosome, often only a few bands in length.
Overall our data argue for the existence of seven
autosomal genes whose activities are required prior to
the onset of gastrulation. The reliability of that
Fig. 8. Distribution of nuclei and cortical actin during
cycle 14 in embryos deficient for either the right or the
left arm of the third chromosome. Sections through
surface cortex of normal (A), 3L~ (B) and 3R~ (C)
embryos, showing the transport of nuclei from the surface
of 3L~ embryos, and the entrapment of nuclei at the
cellularization furrow in 3R~ embryos. Surface views of
3L~ embryo (upper) and 3R~ embryo (lower) stained to
show nuclei (D) and actin (E) distributions. Note the
abnormal clustering of nuclei and irregular actin arrays in
the 3R~ embryo, and the less-densely spaced nuclei, but
more regular actin arrays in the 3L~ embryo. In optical
cross-sections (F), the nuclei of the 3R~ embryo appear
trapped at the cell interface, whereas many of the nuclei
in the 3L~ embryo have been displaced to the
cytoplasm-yolk interface. Embryos hemizygous for the
DF(3)tlf show the same pinched nuclear morphology (G)
at the cellularization front (H) observed in 3R~ embryos.
Surface views at (I) and immediately above (J) the
cellularization front show the small pinched off openings
at the front. Note the occasional larger openings when
more than one nucleus is cellularized into the same cell.
Abnormal distributions of actin (K) and clustered nuclei
(L) in embryos homozygous for Df(3R)X3F.
507
number obviously depends on the resolution of our
morphological techniques. Regions causing more
subtle effects (e.g. those reported for engrailed, Karr
et al. 1985) would have been missed. Moreover,
because of the manner in which smaller deletions
were generated, more proximal regions on a given
arm could usually only be examined in embryos also
deficient for all distally located genes. Given the
cluster of early-acting genes on the tip of 3R, for
example, more proximal loci might be masked by the
cellularization phenotypes associated with the distal
genes. On the other hand, embryos totally deficient
for 3R from the compound stock do not seem
significantly more abnormal during cellularization
than embryos deficient from 98F to the tip.
Although the primary goal of the present study was
to identify requirements for zygotic gene activity
during cellularization, the relative scarcity of such
early-acting loci allowed us to examine many of the
large deficiency class embryos for defects during
gastrulation. In general, we have not identified any
new loci in this way; i.e. most of the abnormalities we
observed during gastrulation could be attributed to
genes previously identified in our EMS screens (Niisslein-Volhard et al. 1984; Jiirgens et al. 1984; Wieschaus et al. 1984). For example, using various
translocations (Table 1), we have shown that the
failure of nullo-2R embryos to make a ventral furrow
is due to a gene between 59A and 59F, most likely the
twist locus (Niisslein-Volhard et al. 1984). Other
gastrulation effects in the compound-2 stock could be
similarly attributed to the decapentaplegic (22D) and
the snail (35CD) loci. The only case where a gastrulation phenotype could not be explained using previously known point mutants is the failure of embryos
deficient for distal 3R to make a posterior midgut
(Fig. 7C). This might be a secondary consequence of
the cellularization defects associated with that region,
but it is also possible that the phenotype is due to a
previously undescribed gastrulation locus. In general,
however, our results confirm the scarcity of earlyacting loci in Drosophila, both for cellularization and
gastrulation.
Perhaps the most remarkable thing is not that the
number of early loci is so small but that their
phenotypes are so unique and specific for particular
aspects of cellularization. The discrete nature of the
phenotypes argue that the wild-type functions associated with each locus may be distinct and therefore
useful in dissecting early cellularization into its component parts. An analysis of the slow and fast phases
of membrane elongation is particularly promising in
this respect, but general usefulness of the approach
may extend to other aspects of cellularization as well.
In contrast to the mutations identified in our
previous EMS mutagenesis experiments (Niisslein-
508
P. T. Merrill, D. Sweeton and E. Wieschaus
Volhard et al. 1984; Jiirgens et al. 1984; Wieschaus et
al. 1984), the defects described in this paper are not
localized to spatially defined regions of the developing embryo. Given the low requirements for zygotic
gene activity in Drosophila, it had seemed possible
that the embryo might use early transcription predominantly to localize gene products to particular
cells or primordia. This view was consistent with the
localized defects and transcript distributions described for many previously analysed embryonic
lethals. We were surprised to find zygotic requirements during cellularization, given that the process
occurs uniformly over the entire surface of the
embryo. A priori, it is not obvious why these gene
products, like most other components of cellularization, would not be supplied maternally. One possibility is that their requirements are localized, but
localized in time rather than space. The sequence of
events during cellularization is complex enough that
the individual phases may require specific zygotic
initiators. Given the stage-specific nature of their
phenotypes, the early-acting loci we have identified
are good candidates for zygotic initiators.
We are grateful to Jim Kennison for supplying most of
the translocation stocks used in this study. Judy Lengyel
gave us the deficiencies in distal 3R and Paul Mahoney
shared unpublished information with us. David Roberts
and Barry Ganetzky gave us stocks for the distal and
proximal regions of 2L respectively. We are also grateful for
our colleagues at Princeton for comments and criticisms
during the course of the experiments. These experiments
were supported by NIH grant no. HD155587 to E.W.
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{Accepted 25 July 1988)