Download Zygotic expression of the pebble locus is required for cytokinesis

Development 114, 165-171 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
165
Zygotic expression of the pebble locus is required for cytokinesis during
the postbiastoderm mitoses of Drosophila
GARY fflME and ROBERT SAINT*
Department of Biochemistry, University of Adelaide, GPO Box 498, Adelaide, South Australia 5001, Australia
* To whom correspondence should be addressed
Summary
Mutations at the pebble locus of Drosophila melanogaster
result in embryonic lethality. Examination of homozygous mutant embryos at the end of embryogenesis
revealed the presence of fewer and larger cells which
contained enlarged nuclei. Characterization of the
embryonic cell cycles using DAPI, propidium iodide,
anti-tubulin and anti-spectrin staining showed that the
first thirteen rapid syncytial nuclear divisions proceeded
normally in pebble mutant embryos. Following cellularization, the postbiastoderm nuclear divisions occurred
(mitoses 14, 15 and 16), but cytokinesis was never
observed. Multinucleate cells and duplicate mitotic
figures were seen within single cells at the time of the
cycle 15 mitoses. We conclude that zygotic expression of
the pebble gene is required for cytokinesis following
cellularization during Drosophila embryogenesis. We
postulate that developmental regulation of zygotic
transcription of the pebble gene is a consequence of the
transition from syncytial to cellular mitoses during cycle
14 of embryogenesis.
Introduction
this factor triggered entry into mitosis (Masui and
Markert, 1971; Smith and Ecker, 1971). This factor was
subsequently shown to contain the p34cdc2 protein
(Dunphy et al., 1988; Gautier et al., 1988) which is
required for entry into mitosis in all eukaryotes
examined to date. The relationship between growth
arrest in mammalian tissue culture cells and cell cycle
regulation is poorly understood, but the recent demonstration that activation of macrophages by the CSF1
growth factor results in the expression of cyclin-like
molecules (Matsushime et al., 1991) may be the first
clue to such regulation. These newly identified cyclins
may be members of the Gl cyclin group required for
progression past "start" in the Saccharomyces cerevisiae
cell cycle (Richardson et al., 1989).
Development of Drosophila melanogaster provides
an excellent system for the study of the developmental
regulation of cell proliferation. The cellular basis of
Drosophila embryogenesis has been well characterized
and molecular genetic studies have led to the identification of regulatory genes that generate the complex
patterns of cell and tissue types during organogenesis
(Ingham, 1988). The embryonic cell cycles of Drosophila have been characterized in detail. With the
exception of the vitellophages and germ cells, which are
not discussed in this paper, the first thirteen mitoses are
parasynchonous and rapid, occurring at approximately
10 minute intervals (Rabinowitz, 1941; Foe and
Recent dramatic advances in the molecular genetic
analysis of the eukaryotic cell cycle have revealed
highly conserved mechanisms that regulate progress
through the cell cycle (for reviews, see Nurse, 1990;
Reed, 1991). Much less is known about the usage of cell
cycle control points in the developmental regulation of
cell proliferation. Developmental regulation of the cell
cycle in eukaryotes has been documented in a number
of cases. In the budding yeast, Saccharomyces cerevisiae, cells arrest at the Gl phase of the cell cycle in
response to the mating pheromone, a-factor, (for a
review, see Thorner, 1981). During embryogenesis in
Drosophila melanogaster, a developmental^ controlled
period of cell cycle arrest occurs in the G2 phase of
cycle 14. In this case, cell cycle arrest has been shown to
be the result of the regulated expression of string
mRNA (Edgar and O'Farrell, 1989). string is a
Drosophila homologue of the cdc25 mitotic initiator of
Schizosaccharomyces pombe, which encodes a product
required for the activation, by dephosphorylation, of
the p34cdc2 protein kinase (Gould and Nurse, 1989).
Maturation of Xenopus laevis oocytes also involves a
period of cell cycle arrest at the G2-M transition. The
arrested oocytes provided an assay for a factor, termed
maturation or M-phase promoting factor (MPF) that is
found in mitotic cells. When added to arrested oocytes
Key words: cytokinesis, mitosis, Drosophila, zygotic
expression, pebble.
166
G. Hime and R. Saint
Alberts, 1983). During cycles 7 to 10, a series of nuclear
movements towards the periphery of the embryo results
in the formation of a syncytial blastoderm.
Following the 13 parasynchronous mitoses, cell
proliferation ceases for at least 70 minutes (Foe, 1989).
During this period, cellularization proceeds by the
growth of membranes from the periphery of the embryo
to enclose the nuclei, resulting in the formation of the
cellular blastoderm.
The 14th mitoses are radically different from all
preceding mitoses. They are the first cellular mitoses
and they occur asynchronously, groups of cells dividing
in a complex spatio-temporal pattern. Each group of
synchronously dividing cells, termed a mitotic domain,
has been accurately mapped with respect to its position
in the embryo and the time at which it undergoes
mitosis (Foe, 1989). The domains frequently coincide
with organ primordia, so this pattern of mitosis is one of
the earliest manifestations of organ formation.
As noted above, zygotic transcription of one gene,
string, has been shown to regulate the timing and spatial
occurrence of the 14th mitoses (Edgar and O'Farrell,
1989, 1990). Here we describe a second gene, pebble
ipbt) the transcription of which is required for cytokinesis during the postblastoderm mitoses.
propidium iodide staining of cytoplasmic RNA, allowing
nuclei to be clearly observed, p-phenylenediamine was added
to the mounting medium (90% glycerol/PBS) to prevent
fluorescence quenching (Johnson and Nogueira Araujo,
1981). Alternatively, nuclei were visualized by incubating
fixed embryos in 10 /ig/ml DAPI for 3 minutes. Antibodies
used were MC10-2, an anti-a^spectrin monoclonal antibody
(Pesacreta et al., 1989) and YLl/2, a rat anti-tyrosinated atubulin antibody (Kilmartin et al., 1982). MC10-2 was used at
1:1; YLl/2 (SeraLabs) at 1:5 and 22C10 at 1:100. Secondary
and tertiary antibodies used were 1:30 biotinylated antimouse (Amersham); 1:100 streptavidin Texas Red (Amersham) and 1:30 FITC-anti-mouse (Silenus). In all experiments, the progeny of flies heterozygous for particular pebble
mutations were examined. Approximately one-quarter of the
embryos showed the mutant phenotype in all cases. Although
we could not discriminate between the pbl/+ and +/+
embryos, we refer to this class of phenotypically wild-type
embryos as "wild-type" throughout this paper.
Microscopy
Materials and methods
Fluorescence microscopy was performed on a Zeiss Axioplan
microscope equipped with objectives Plan-Neofluar 2Ox/0.5
and 100x/l.3 oil immersion and Planapochromat 40x/l.O oil
immersion. DAPI staining was observed using a Zeiss No. 2
filter. Confocal microscopy was performed with a Bio-Rad
MRC 600 Confocal Imaging System fitted to an Olympus
BH2-RFCA microscope, using objective SPlan 100x/l.25 oil
immersion. Images were recorded by photographing the
screen of a Mitsubishi FA3435KE/V colour monitor.
Strains, media and growth conditions
Results
Fly stocks were grown on standard media at 25°C or room
temperature. Egg lays were performed at 25°C on apple juice
agar plates (Wieschaus and Niisslein-Volhard, 1986). Egg lays
were usually for one hour with the eggs then allowed to age
for the desired time at 25°C before removal of the chorion
membranes with 4% hypochlorite.
The EMS alleles used in this study were pbl50, pbf°,
pbl"D and pbl58 (Jiirgens et al., 1984). Df(3L)pbrR and
Df(3L)pblxf deficiency strains were generated during this
study and are described in the text, w; ve TE 217/TM3Sb was
obtained from G. Ising. The wild-type strain used was
Canton-S.
Recombination mapping and X-ray mutagenesis
Recombination percentages were calculated as the percentage
of recombinant progeny scored for the markers indicated in
Fig. 1. X-ray mutagenesis was performed by irradiating
batches of 100 w; ve TE 217/TM3Sb males with 4800 rad and
mating them to 100 w; TM3Sb/TM6b or w; rusteca females.
Putative deficiencies were scored by the reversion of TE 217
to w~.
Polytene chromosome spreads
Polytene spreads were performed using the method of Pardue
(1986).
Immunohistochemistry
Dechorionated embryos were fixed using the method of Karr
and Alberts (1986) and antibody staining was performed as
described by Foe (1989) except that the secondary antibody
incubations were performed at 37°C for 2 hours in the
presence of 1 mg/ml RNAaseA. Nuclei were observed by
the addition to the mounting media of 10 /ig/ml propidium
iodide. RNAase treatment of embryos was used to avoid
Genetic characterization of the pebble region
Recombination mapping of pbl placed it between the
genes Henna and hairy on chromosome 3 (Table 1, Fig.
1A.). Deficiencies covering this region were not
available, so attempts were made to generate them
using a transposable element insertion, TE217, previously localized to 66B1-8 (G. Ising, personal communication). Recombination between pbl and TE 217
showed these to be less than 0.5 map units apart. TE
elements are very large transposable elements, several
hundred kilobases in size, carrying a region of the Xchromosome including the genes roughest and white
(Ising and Block, 1984). This large size may result in the
suppression of recombination, and therefore underestimation of the genetic distance between the point of
insertion and the pbl locus. Despite this uncertainty,
TE217 was used to detect potential deficiencies by the
loss of the w+ phenotype. The screening of 16797 flies
recovered 19 revertants to w~, from which 13 lines were
Table 1. Recombination between markers in
subdivision 66
Markers
pbl 5D v
Hn v h
pbl 7 0 v
pbl 5D v
pbl 7 0 v
h
P(w+]30
TE217
TE217
Recombinants
Total
%
Recombination
Standard
error
23
34
8
3
0
2178
1502
1306
1748
2561
3.2
4.6
1.8
0.5
0.0
±0.8
±0.4
±0.8
±0.4
—
Drosophila pebble gene
4.6ft
3.2)1
Hn
<0.5R
««—• - ^
TE217
Pbl
i
\
\
\
/
/
18ft
[Pw+]
/
Y
66A
66B
I
I
66C
I
66D
txtt0f%%ff%f??}0!(ft%?%?%%00?%%ff>00{fii D f ( 3 D pbl
NR
Df(3L) pbl
Fig. 1. (A) Location of the pbl locus, pbl has been
localized to subdivision 66B of chromosome 3.
Recombination percentages are indicated above arrows
connecting the appropriate markers. The extent of
deficiencies, Df(3L)pblxl, 65F3;66B9 and Df(3L)pblNR,
66B1;B2, are indicated by hatched boxes. Abbreviations
used are Hn; Henna, [Pw+]; P[w]30 (G.Rubin, personal
communication), h; hairy. (B and C) Cytological
preparations of Df(3L)pbt*''/'+ and Df(3L)pblNR/+
respectively. Arrows indicate the loopout in the wild-type
chromosome.
established. Two deficiencies were generated. Xirradiation
generated
a
deficiency,
termed
Df(3L)pblXI,
which failed to complement any pbl
mutant alleles. Df(3L)pblxl was mapped cytologically
as a deficiency from 65F3 to 66B9 of the Drosophila
salivary gland polytene chromosome map described by
Bridges (1941) (Fig. IB). A spontaneous revertant to
w~ was also found among the TE217 stock and shown
to be the result of a deficiency. This deficiency,
Df(3L)pblNR, was found to delete bands 66B1-2, (Fig.
1C) and failed to complement any pbl mutant allele. We
have therefore generated deficiencies used in the
further analysis of the pbl phenotype described below
and assigned pbl to the cytological interval, 66B1-2.
Mutations at the pbl locus result in an abnormal
nuclear phenotype
pbl mutations result in recessive embryonic lethality
167
with a characteristic mutant cuticle phenotype (Jiirgens
et al., 1984). Approximately one quarter of offspring
from heterozygous parents exhibited a mutant phenotype as visualized by DAPI staining of 15-20 hours
AED (after egg deposition) embryos. Mutant embryos
derived from parents carrying the EMS-induced pbl70
allele showed nuclei that were reduced in number and
exhibited gross morphological alterations (Fig. 2B)
I relative to normal embryos in which nuclei were
organized into clearly definable organ structures (Fig.
2A). The pbl50, pblUD and pb?S alleles yielded an
identical mutant phenotype to that of pbl70 (results not
shown), as did embryos rraAW-heterozygous for the
deficiencies Df(3L)pblNR and Df(3L)pblxl (Fig. 2C).
Apart from those nuclei undergoing mitosis, nuclei of
wild-type 5-7 hours AED embryos were evenly distributed and of a constant size (Fig. 2D). Homozygous
pbl70 (Fig. 2E) and DftfQpbl™ (Fig. 2F) embryos
displayed nuclei of uneven distribution and varying
size. Large diffusely stained structures were present
together with smaller brightly stained nuclei in clusters
of two and four. From this preliminary characterization, we conclude that the pbl mutation results in
disruption of cell proliferation during Drosophila
embryogenesis.
Cells of pbl mutant embryos are multinucleate after
the postblastoderm mitoses
Examination of mutant embryos, using DAPI staining
of nuclei and Differential Interference Contrast microscopy, failed to reveal any disruption either of the
first 13 rapid syncytial divisions or of cellularization
(results not shown). The appearance of abnormal nuclei
was first noted after the 14th mitosis, the first
postblastoderm mitosis. This is also the first mitosis
after cellularization of the peripheral nuclei. To
determine if the nuclear clustering, observed in the
DAPI-stained embryos, was the result of multinucleate
cells, 4-5 hours AED embryos were double stained with
an antibody directed against a-spectrin, which is
distributed on the inner surface of plasma membranes
(Pesacreta et al., 1989), and with propidium iodide.
This allowed co-visualization of nuclei and the surrounding cell membrane. Embryos were viewed by
laser scanning confocal microscopy, thereby generating
an optical slice through the whole-mount embryos and
showing the relationship of nuclei to plasma membranes. In the photomicrographs shown in Fig. 3 and
described below, the occurrence of apparently
anucleate cells is due to the nucleus of that cell not
being in the plane of the image.
Cells in stained wild-type embryos contained single
mitotic figures or single interphase nuclei of uniform
size. The example shown is at the germ band retraction
stage with all nuclei in interphase (Fig. 3A). The cells of
homozygous pbl70 embryos were much larger in size
and were variable in shape. Cells containing four nuclei
were observed (Figs 3B, 3D) with some undergoing
mitosis (Fig. 3C). The cell at the top of Figure3B shows
a binucleate cell in telophase of cycle 14 whilst the cell
in the centre is tetranucleate, having just undergone
168
G. Hime and R. Saint
Fig. 2. Nuclear
morphology as revealed byRDAPI staining
and fluorescence. (A)-(C) 16-20 hours AED embryos. (A) wildhomozygote (C) Df(3L)pbfNR
lDf(3L)pbri- Bar=200 microns. (D-F) 5-7 hours AED embryos. (D) wildtype (B) pbl70
70
type (E) pbl homozygote (F) Df(3L)pbl homozygote. Bar=400 microns.
mitosis 15. After cessation of the postblastoderm
mitoses, most nuclei were observed as large diffuse
interphase structures (Fig. 3E).
The presence of mitotic figures at the appropriate
postblastoderm stages and of enlarged multinucleate
cells subsequent to this suggest that cells of pbl embryos
proceed through the nuclear divisions of the postblastoderm mitoses but do not undergo cytokinesis to
produce two daughter cells with individual nuclei.
Cells in pbl mutant embryos can have more than one
mitotic spindle
As described above, the cycle 14 nuclear mitoses occur
normally in pbl mutant embryos, but apparently in the
absence of cytokinesis. To determine whether spindle
formation was normal in the mutant mitoses, 5-7 hours
AED embryos were stained with YLl/2 antiserum
directed against tyrosinated a-tubulin (Kilmartin et al.,
1982). Wild-type and heterozygous embryos exhibited a
wild-type pattern of mitotic spindles arranged in
domains as described by Foe (1989) (Fig. 4A,C).
Observation of a large number of embryos, one quarter
of which must have been homozygous for the pbl70
mutation showed that the mutant embryos were
indistinguishable from wild-type or heterozygous embryos during the cycle 14 mitoses prior to cytokinesis
(results not shown). Mitoses at the time expected for
the 15th mitoses, however, were observed to have
duplicate mitotic apparatuses. Figs 4B and 4D-4F show
several examples of the presence of two mitotic spindles
in cells undergoing the 15th mitosis. Fig. 4F is from an
embryo rraAW-heterozygous for the deficiencies
Df(3L)pblNR and Df(3L)pblxl and shows a gTOup of
Fig. 3. Nuclear phenotype of pbl embryos. Cell membranes were stained with anti-a'-spectrin Ab (red) and nuclei with
propidium iodide. (yellow-gTeen) (A) wild-type (B-D) pbl70 homozygotes showing multinucleate cells and multiple mitotic
figures. (E) pbl70 homozygote showing large diffuse nuclei. Bar=5 microns.
Fig. 4. Mitotic spindles of (A) wild-type and (B) pbl70 embryos revealed by staining with a rat anti-tyrosinated-a^tubulin
Ab. Bar=25 microns. C and D are enlargements of A and B. D and E are examples of pbl70 homozygotes showing cells
with multiple spindles. (F) Df(3L)pblNR/DflSLjpbt*1 showing four overlapping spindles within a single cell. Bar=
5 microns.
Drosophila pebble gene
169
Fig. 5. Differentiated structures present in pbl embryos. (A) pbl70 homozygote that has completed germ band retraction
and has partially segmented, exhibiting rudimentary denticle bands (small arrowhead) and cephalopharyngeal skeleton
present (large arrowhead). (B) DAPI stain of the same embryo shown in A demonstrating pbl phenotype. Bar=200
microns.
four overlapping spindles. These spindles were observed to be disordered, presumably because of the
interference of the establishment or stability of each
spindle by the presence of the other. The presence of an
apparent tripolar spindle in Fig. 4D is due to a cell
containing two orthogonal spindles in which one spindle
pole was out of the plane of the image.
The appearance of duplicate mitotic figures within
single cells following the 14th mitosis is further evidence
that the mutant phenotype is due to normal postblastoderm nuclear divisions in the absence of cytokinesis,
resulting in polyploid cells.
Developmental effects of the pbl mutation
Although normal cell proliferation was seen to be
disrupted at the end of cycle 14 in pbl mutant embryos,
postblastoderm morphogenetic movements and tissue
differentiation still occurred, pbl embryos gastrulated
and germ-band extension occurred to varying degrees.
A few embryos extended the germ band fully and
formed some segmental structures. For example, the
embryo shown in Fig. 5A contained an almost complete
cephalopharyngeal skeleton and four rudimentary denticle bands. Thus cells of pbl mutant embryos were seen
to differentiate. Differentiation of other tissue types
was demonstrated. mAb 22C10, a monoclonal antibody
directed against differentiating cells of the peripheral
nervous system (Zipursky et al., 1984), stained subsets
of cells that had the general distribution expected of the
developing PNS but were disorganized in comparison to
the wild-type pattern (result not shown). Thus the
mitotic disruption caused by the pbl mutation does not
prevent the cells from receiving and interpreting, at
least to some degree, the positional information that
directs pattern formation and cell differentiation in
normal embryos. A similar observation has been made
in cells mutant for the string gene which blocks all
mitotic activity beyond that of the G2 phase of cycle 14
(Hartenstein and Posakony, 1990).
Discussion
We have used a variety of cell biological techniques to
characterize the phenotype of pbl mutant embryos. The
key observations were (1) the normal development of
embryos up to the cellular blastoderm stage, (2) the
appearance of mitotic figures with normal mitotic
spindles and with normal chromosome segregation at
the times expected for the cycle 14 mitoses, (3) the
presence of multinucleate cells following these mitoses,
(4) the appearance at later stages of multiple mitotic
figures within single cells and (5) the presence of fewer
cells with enlarged nuclei at the end of embryogenesis.
There are several possible explanations for these
observations. One is that the pbl gene encodes a factor
required for cytokinesis so that nuclear mitoses proceed
in the absence of cytokinesis mpbl mutants. A second is
that cell fusion follows the normal occurrence of the
postblastoderm mitoses. A third is that a novel cell
cycle, such as the "endo-cell cycle", proposed by Smith
and Orr-Weaver (1991) for the generation of polytene
cells later in development, is prematurely triggered in
pbl mutant embryos. The cell fusion explanation is
unlikely as we never see a quantal decrease or increase
in cell size at any stage during embryogenesis, as would
be expected if cytokinesis occurred normally and was
followed by fusion of the cells. Premature activation of
the endo-cell cycle is also unlikely to account for the
mutant phenotype, as these cycles involve endoreduplication of the DNA rather than nuclear mitoses
followed by nuclear fusion. A role for pebble in
cytokinesis accounts for all the observations described
in this paper and is the most likely explanation for the
pebble mutant phenotype.
While the phenotype of pbl mutant embryos clearly
implies a role for the pbl gene product in cytokinesis, a
more subtle phenotype, that of the irregularity of cell
shape, may indicate an additional role for the pbl gene
product. Although the irregular cell shape most
probably reflects physical forces acting on the large
multinucleate cells during morphogenesis, it could
result from a role for the pbl product in cytoskeletal
interactions.
The large polyploid diffuse interphase nuclei seen
late in development in most cells is likely to be an
indirect effect of the disruption of cytokinesis. They
170
G. Hime and R. Saint
could result either from a failure of chromosome
segregation (disordered and intersecting spindles were
frequently observed in cells with duplicate mitotic
figures) or nuclear fusion during development.
Little is known about the process of cytokinesis,
although a number of mutants affecting cytokinesis
have been described. These include the Drosophila
mutants spaghetti-squash, a mutation in the non-muscle
myosin light chain (Karess et al., 1991), zipper (also
called mhc-c), a mutation in the non-muscle myosin
heavy chain that effects cell shape change, morphogenesis and possibly cytokinesis (D. Kiehart, personal
communication), four wheel drive, a mutation causing
failure of cytokinesis during male meiosis, resulting in
early spermatids with four nuclei (N. Wolf and M.
Fuller, manuscript in preparation) and abnormal
spindle, a mutation in a non-tubulin component of the
meiotic spindle (Casal et al., 1990). The C. elegansspell mutant (Hill et al., 1989) and mutations at five
Tetrahymena loci (Frankel et al., 1977) have also been
"shown to result in cytokinetic defects. One of the
Tetrahymena loci encodes a protein that is concentrated
at the cleavage furrow (Ohba et al., 1986).
We do not yet know whether a relationship exists
between the pbl gene and any of these genes.
Furthermore, we cannot as yet assign a specific role for
pebble in cytokinesis. An actinomyosin motor generates
the force necessary for furrowing (Otto and Schroeder,
1990), but the positions of actin and myosin genes of
Drosophila do not correspond to the pbl gene (Lindsley
and Zimm, 1985, 1990). Candidates for the pbl gene
product include accessory proteins to myosin and actin,
such as the barbed end-capping protein radixin (Sato et
al., 1991), some of which must be involved in the
initiation and constriction of the contractile ring, or the
association of the contractile ring with the plasma
membrane. The INCENP proteins (Cooke et al., 1987;
Earnshaw and Cooke, 1991) also behave in a fashion
that suggests a possible role in the process of cytokinesis.
A striking feature of the pbl gene that sets it apart
from other Drosophila genes implicated in cytokinesis
is the requirement for zygotic expression of this gene
prior to cytokinesis at the 14th mitosis. It is clear from
genetic analysis of cell cycle genes in Drosophila (Gatti
and Baker, 1989) that the majority of genes required for
cell cycle progression are provided as maternal products
in sufficient quantities to allow the embryo to proceed
through all 16 embryonic mitoses. Thus, cell cycle
mutants are most frequently mutants that disrupt
imaginal disc and neural growth, resulting in late larval
or pupal lethality, or they are maternal lethal mutants
(Glover, 1989). The only other gene characterized to
date for which zygotic expression is required for
progression through the 14th cell cycle is the string
gene. Even in this case, a large amount of maternal
mRNA is present initially and is actively degraded
during cycle 14, establishing the need for zygotic
transcription of the gene (Edgar and O'Farrell, 1989).
Why should the pbl mutant specifically affect the cycle
14 cytokineses and why should there be a requirement
for zygotic pbl transcription early in embryogenesis?
The answer to the first question is likely to be that the
14th mitosis is the first cellular mitosis and therefore the
first mitosis in which there is a requirement for cellular
cytokinesis. Thus, the pbl product may not be required
during the syncytial mitoses prior to cycle 14.
The reason for the requirement for zygotic transcription is less clear. One possibility is that there is no
maternal pbl mRNA provided, so that the mutant
phenotype arises at the first cellular mitosis at which
time the pbl gene product is first required. An
alternative is that the maternal products are present
during the first 13 mitoses but are actively degraded
during cycle 14, as is the case for the stg mRNA.
Regardless of the reason, maternal pbl products are not
present when the gene product is required at the 14th
mitosis. Near-saturating mutageneses of Drosophila
have identified most of the genes whose zygotic
transcription is required for normal cuticle formation
during embryogenesis. pbl is the only such zygotic gene
to have been shown to be required for cytokinesis, so
we presume all other necessary factors are present as
maternal products. The requirement for zygotic expression may result from developmental events that
convert the syncytial blastoderm to a cellular blastoderm immediately prior to the 14th mitosis. Cellularization proceeds by the inward migration of furrow canals
from the periphery of the embryo in a process that can
be considered a modified cytokinesis (Warn et al.,
1990). We postulate that pebble plays a key role in the
process of mitotic cytokinesis and that the presence of
the pebble product earlier than the 14th mitosis may
interfere with the modified cytokinesis that occurs
during cellularization. Transcriptional regulation of the
pebble gene would ensure that the product is absent
during cellularization, while expression during cycle 14
would generate the product in time for the first cellular
division. This is a particularly exciting possibility, as it
suggests that the pbl gene product may play a key role
in the regulation of postblastoderm cytokineses. We are
currently attempting to test this postulate by the
isolation and further characterization of this gene.
We would like to thank Dr. C. Nusslein-Volhard for
bringing the pebble mutation to our attention and for
providing fly strains, Dr W. Francis and Dr D. Turner for
assistance with X-irradiation of flies, Dr T.J. Lockett, Dr B.
Smith and Professor M. Vadas for assistance and use of the
confocal microscope, Dr G. Ising and Dr G. Rubin for fly
strains, Dr D. Branton and Dr N. Brink for providing
antibodies, Dr M. Green for valuable suggestions on the work
and to the other members of our laboratory for helpful
discussions. This work was supported by the National Health
and Medical Research Council of Australia (Grant No.
890191 to R.S.) and by the CSIRO-University of Adelaide
Collaborative Research Fund. G.H. was supported by an
Australian Postgraduate Research Award.
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