src64 and tec29 are required for microfilament contraction during Drosophila cellularization
Jeffrey H. Thomas and
Eric Wieschaus*
Howard Hughes Medical Institute, Molecular Biology Department, Washington
Road, Princeton University, Princeton, NJ 08544, USA
*
Author for correspondence (e-mail:
ewieschaus{at}princeton.edu)
Accepted 17 November 2003
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SUMMARY
|
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Formation of the Drosophila cellular blastoderm involves both
membrane invagination and cytoskeletal regulation. Mutations in src64
and tec29 reveal a novel role for these genes in controlling
contraction of the actin-myosin microfilament ring during this process.
Although membrane invagination still proceeds in mutant embryos, its depth is
not uniform, and basal closure of the cells does not occur during late
cellularization. Double-mutant analysis between scraps, a mutation in
anillin that eliminates microfilament rings, and bottleneck suggests
that microfilaments can still contract even though they are not organized into
rings. However, the failure of rings to contract in the src64
bottleneck double mutant suggests that src64 is required for
microfilament ring contraction even in the absence of Bottleneck protein. Our
results suggest that src64-dependent microfilament ring contraction
is resisted by Bottleneck to create tension and coordinate membrane
invagination during early cellularization. The absence of Bottleneck during
late cellularization allows src64-dependent microfilament ring
constriction to drive basal closure.
Key words: Drosophila, Cellularization, Blastoderm formation, Src64, Tec29, Scraps, Anillin, Bottleneck, Microfilament, Contractile
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Introduction
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The mechanisms that establish the cellular blastoderm of the early
Drosophila embryo are as yet incompletely understood. During
cellularization, the peripheral syncytial nuclei of the wild-type embryo are
surrounded by membrane to form an epithelial blastoderm. This is accomplished
by the simultaneous and uniform invagination of membrane between the
peripheral nuclei (Mazumdar and Mazumdar,
2002
; Schejter and Wieschaus,
1993a
). At the leading edge of membrane invagination, known as the
cellularization front, are stable infoldings of plasma membrane known as
furrow canals (Lecuit and Wieschaus,
2000
; Fullilove and Jacobson,
1971
). The base of each furrow canal is rich in the cytoskeletal
proteins F-actin and myosin II, and may provide a contractile force that helps
pull membrane inward (Schejter and
Wieschaus, 1993a
; Young et
al., 1991
; Warn and
Robert-Nicoud, 1990
). To identify additional cytoskeleton
components involved in early embryogenesis, we used deficiencies to screen
part of the third chromosome (J.H.T. and E.W., unpublished). Analysis of the
genes that mapped to a region identified in this screen revealed a
cellularization phenotype associated with deletions in a Drosophila
src homolog, src64 (Src64B - FlyBase).
Src proteins have been shown to be involved in the regulation of the
cytoskeleton and its components during the reorganization of microfilaments in
both lamellipodia and filopodia in fibroblasts
(Frame et al., 2002
;
Thomas and Brugge, 1997
;
Thomas et al., 1995
). The
structure of members of the Src family of consists of an N-terminal
myristoylation site, an SH3 domain, an SH2 domain, a tyrosine kinase domain
and a C-terminal regulatory domain. The myristoylation of Src protein allows
it to be tethered to the plasma membrane, whereas the SH3 and SH2 domains
serve to bind proline-rich recognition sequences and phosphotyrosine residues,
respectively (Harrison, 2003
;
Frame, 2002
;
Thomas and Brugge, 1997
). In
vertebrates, Src has been shown to activate Tec family non-receptor tyrosine
kinases, which are similar to Src in that they have an SH3, an SH2 and a
kinase domain, and are also thought to interact with gene products associated
with the cytoskeleton (Smith et al.,
2001
; Thomas and Brugge,
1997
).
In Drosophila, there are two src homologs, src42
(Src42A - FlyBase) and src64, and one Tec family kinase
encoded by tec29 (Btk29A - FlyBase)
(Takahashi et al., 1996
;
Katzen et al., 1990
;
Vincent et al., 1989
;
Gregory et al., 1987
;
Wadsworth et al., 1985
;
Simon et al., 1985
;
Simon et al., 1983
). There is
evidence that these genes play a role in cytoskeletal regulation. During
embryogenesis, for example, tec29 src42 double mutants and src42;
src64 double mutants have dorsal closure defects associated with reduced
quantities of phosphotyrosine and filamentous actin
(Tateno et al., 2000
).
However, the best-studied example of src64 and tec29
involvement in cytoskeletal regulation is found in the formation and growth of
the ring canals during oogenesis. The ring canals are formed by the sequential
addition of several different proteins, including Src64 and Tec29, to an
actin- and anillin-rich arrested cleavage furrow left from the incomplete
divisions of the female germ cells (Sokol
and Cooley, 1999
; Dodson et
al., 1998
; Guarnieri et al.,
1998
; Roulier et al.,
1998
; Robinson and Cooley,
1996
; Majajan-Miklos and Cooley, 1994;
Robinson et al., 1994
). After
assembly and the loss of anillin localization, the ring canal enters a growth
phase (Robinson and Cooley,
1996
). In src64 and tec29 mutants, ring canal
growth is stunted so that fully grown ring canals are never formed
(Dodson et al., 1998
;
Guarnieri et al., 1998
;
Roulier et al., 1998
). The
ultimate consequence of Src activity may be the phosphorylation of Kelch, an
actin bundling protein that regulates actin polymerization by reversible
cross-linking (Kelso et al.,
2002
; Tilney et al.,
1996
).
Here we report that src64 and tec29 play a role in the
cytoskeletal dynamics that occur during cellularization of the
Drosophila embryo. src64 and tec29 are essential
for the contraction of the microfilament rings that are present in the
cellularization front, and appear to play a role in membrane invagination and
in subsequent basal closure. This role is distinct from that of
scraps (anillin), which is required for the formation of these rings
but is not directly required for their contraction. Using double-mutant
analysis, we show that a previously identified regulator of cellularization,
bottleneck (bnk), acts by countering the
src64-dependent contraction of the microfilament rings, but shows
only an additive effect in combination with scraps. Finally, we
propose a mechanical model for cellularization, taking into account the
similar and different roles played by microfilament contraction and
src64-independent forces.
 |
Materials and methods
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Fly strains and genetics
OreR was used as the wild-type strain. Unless otherwise noted, other
strains are described by Lindsley and Zimm
(Lindsley and Zimm, 1992
) or
The Flybase Consortium (The Flybase
Consortium, 2003
). src64
17 was
used as the src64 mutation in these experiments; it is a strong
reduction-of-function allele that eliminates most of the Src64 protein
(Dodson et al., 1998
).
Df(3L)10H and Df(3L)Flex14
(Nose et al., 1994
) were used
as the deficiencies for src64. tec29k00206 was
used as the tec29 allele as it eliminates the tec29
transcript, as assayed by in situ hybridization
(Roulier et al., 1998
). Baba
et al. (Baba et al., 1999
) and
Sinka et al. (Sinka et al.,
2002
) report some tec29 activity in this mutant
suggesting that it is a strong reduction-of-function allele. Other alleles
used include scrapsRS and
scrapsPQ
(Schüpbach and Wieschaus,
1989
), and Df(3R)tll-e to delete bnk
(Schejter and Wieschaus,
1993b
).
tec29 germline clones were constructed essentially as previously
described (Roulier et al.,
1998
; Guarnieri et al.,
1998
). OreR males were crossed into tec29 germline clone
females to generate embryos.
Histology and image analysis
To visualize myosin, Even-skipped, Armadillo, Anillin and Bottleneck
proteins, embryos were methanol heat-fixed
(Wieschaus and Nusslein-Volhard,
1998
) and stained with rabbit anti-myosin (a gift from C. Field,
Harvard Medical School, Boston, MA), guinea pig anti-Eve, mouse anti-Arm
(N27A1), rabbit anti-anillin (a gift from C. Field, Harvard Medical School,
Boston, MA) or rat anti-Bottleneck (5)
(Schejter and Wieschaus,
1993b
) antibody, respectively. To visualize Tec29 protein and
phosphotyrosine-containing proteins, embryos were fixed in a
formaldehyde/phosphate buffer in the presence of heptane
(Oda et al., 1994
) and stained
with either mouse anti-Tec29 (I19) antibody
(Roulier et al., 1998
;
Vincent et al., 1989
) or mouse
anti-phosphotyrosine (PY20) antibody (Transduction Laboratories, BD
Biosciences). Src64 protein was visualized by fixing embryos as described for
Tec29 protein, or by using 4% paraformaldehyde in lieu of formaldehyde and
staining with rabbit anti-Src64 antibody (a gift from T. Xu, Yale University,
New Haven, CT). Primary antibodies were detected with Alexa 488- and Alexa
546-conjugated goat antisera (Molecular Probes). Nuclei were visualized by
staining with Hoechst dye. Sagittal sections were obtained optically.
Cross-sections were made by using a 26-gauge hypodermic needle to manually cut
fixed and stained embryos. Embryos were mounted in Aquapolymount
(Polysciences), and were observed using a Nikon E800 fluorescence microscope
and a Zeiss LSM-510 confocal microscope.
Image analyses were performed using ImageJ software for Macintosh (W.
Rasband, NIH;
http://rsb.info.nih.gov/ij/).
Circularity was calculated by Image J software as the normalized ratio of area
to perimeter (c=4
A/p2, where c=circularity, A=area and
p=perimeter) so that in a true circle this ratio is one. The mean circularity
is reported as the circularity index. Samples were analyzed by calculating the
circularities of approximately 25 contiguous basal openings of embryos of the
same age and the results were compared using a t-test assuming
unequal sample variances. Subsamples of 20 contiguous basal openings were also
compared using a Wilcoxon-Mann-Whitney test. Genotypes were considered
different only if both tests produced P values of less than
0.001.
To analyze cellularization dynamics, six wild-type and six src64
embryos were mounted on biofoil membrane (Kendro) in halocarbon oil 27
(Sigma), covered with a coverslip supported by another coverslip on either
side of the embryo and examined under bright field illumination using a Nikon
E800 microscope. Time-lapse images were collected every 60 seconds using a
CoolSNAP cf camera (Photometrics) and IPLab 3.6.3 image processing software
for Macintosh (Scanalytics). Cellularization front depth was measured using
ImageJ software.
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Results
|
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Src64 mediates cytoskeletal contraction at the cellularization front
Mutations in src64 that eliminate Src64 protein expression during
oogenesis cause defects in the ring canals formed in the nurse cell-oocyte
complex. Despite these structural defects, a significant fraction of
homozygous mutant females produce eggs that are fertilized and give rise to
viable embryos (Dodson et al.,
1998
). In addition to the reduced egg production, reduced hatch
rate and increased incidence of short embryos observed by Dodson et al.
(Dodson et al., 1998
), we
observed that syncytial blastoderm stage src64 embryos also display a
weak nuclear fallout phenotype, such that some nuclei have dropped out of the
periphery before cellularization. This phenotype is much weaker than that
found in mutants such as nuf, SCAR and Arpc1
(Rothwell et al., 1998
;
Zallen et al., 2002
). We
examined older embryos of src64
17
homozygous females for the level of Src64 expression and its intracellular
localization during cellularization. In wild-type embryos, Src64 protein
localizes most intensely to the cellularization front at the beginning of
cycle 14, in a domain that roughly overlaps that of other cytoskeletal
proteins such as anillin and myosin. Embryos from
src64
17 homozygous females stained for
Src64 protein show no specific staining of the cellularization front and
essentially lack all non-background staining
(Fig. 1A,B).

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Fig. 1. Src64, Tec29 and anillin localization in wild-type and src64
mutants during cellularization. Cross-sections of a wild-type (A) and a
src64 17 mutant (B) embryo, and sagittal
sections of wild-type (C,E,G) and src64 17
mutant (D,F,H) embryos during cellularization. Embryos were stained with
antibodies to Src64 (A,B), Tec29 (C,D), phosphotyrosine (E,F) and anillin
(G,H). (A) Src64 protein localizes predominantly to the cellularization front
in wild-type embryos but is absent in
src64 17 mutant embryos (B). (C,D) Tec29
localizes strongly to the cellularization front and less strongly to the
apico-lateral membrane in both wild-type and src64 embryos. (E,F)
Phosphotyrosine-containing proteins localize to the cellularization front in
both wild-type embryos and src64 embryos. (G,H) Anillin localizes to
the cellularization front in both wild-type and src64 embryos. Scale
bar: 10 µm.
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In wild-type embryos, the cellularization front marks the site of membrane
invagination between adjacent nuclei of the syncytial blastoderm. Actin and
myosin at the base of each furrow canal may provide a contractile force that
helps pull invaginating membrane inward
(Schejter and Wieschaus,
1993a
). In cross-sections of early cellularization stage wild-type
embryos, each furrow canal has a rounded appearance and adjacent furrow canals
are at a similar depth, giving the cellularization front a uniform and taut
appearance around the circumference of the embryo
(Fig. 2A). In
src64
17 mutant embryos, the furrow canals
are less rounded, and adjacent furrow canals extend to different depths in the
embryo, giving the cellularization front a wavy, slack appearance as if it
were no longer under tension (Fig.
2B). Viewed from the surface, the early cellularization front in
wild-type embryos appears as a network of tightly apposed, densely staining
myosin microfilament rings surrounding the nuclei
(Fig. 2C). In
src64
17 embryos, the microfilament rings
are not rounded but instead are irregular in shape and sometimes sharply
angular (Fig. 2D). We have used
the ImageJ circularity assay to estimate the tension of the microfilament
ring, based on the assumption that rings under tension will more closely
resemble a circle and will therefore have a circularity index close to 1.0 (W.
Rasband, NIH; see Materials and methods). During early cellularization, the
microfilament rings of the wild-type embryo have a circularity of 0.93
(Table 1). At the same stage in
src64
17 embryos, the microfilament rings
enclose roughly the same area as those of wild type, but have a longer
perimeter such that the circularity ratio is 0.80
(Table 1), a significant
deviation from that of wild type (P<0.001). The longer perimeter
is the result of the microfilament ring having a convoluted and meandering
shape, such that indentations occur in the rings. The deviation from
circularity suggests that the microfilament rings are not under tension and
are held together in a loose mesh.

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Fig. 2. src64 is required for microfilament ring contraction during
cellularization. Cross-sections (A,B,E,F) and projections of confocal sections
of the cellularization front (C,D,G,H) of wild-type (A,C,E,G) and
src64 17 mutant (B,D,F,H) embryos, before
(A-D) and after (E-H) the cellularization front has passed the bases of the
nuclei. Embryos were stained with antibodies to myosin (A-H) and Armadillo
(E,F). The early cellularization front, shown by myosin localization, is of
uniform depth along the circumference of wild-type embryos (A), but is of
non-uniform depth in src64 mutant embryos (B). The newly formed
microfilament rings are round during early cellularization in wild-type
embryos (C), but are less rounded in src64 mutant embryos (D). The
late cellularization front is of uniform depth along the circumference of
wild-type embryos (E) and the furrow canals are expanded into a flask-like
shape, whereas in src64 mutant embryos, the late cellularization
front is also of uniform depth but the furrow canals are unexpanded (F). The
microfilament rings of wild-type embryos during late cellularization are round
and constricted (G), whereas the microfilament rings of src64 mutant
embryos are less rounded and are not constricted (H), similar to the
microfilament rings of src64 mutant embryos during early
cellularization (D). Scale bar: 10 µm.
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As the cellularization front passes the bases of the peripheral nuclei, the
actin-myosin cytoskeleton undergoes a reorganization such that the
microfilament rings are no longer linked. Each microfilament ring now
contracts in diameter so as to lead to the gradual basal closure of the cells
into a narrow stalk (Schejter and
Wieschaus, 1993b
). This contraction causes the lumen of the furrow
canals in wild-type embryos to expand into a flask-like shape
(Fig. 2E). The microfilament
rings are round and constricted (Fig.
2G), and have a circularity of 0.94
(Table 1). In
src64
17 embryos the furrow canals do not
expand (Fig. 2F). The
microfilament rings are large, less rounded and convoluted
(Fig. 2H), they enclose a
greater area than the wild type (P<0.001) and have a circularity
index of 0.81 (Table 1), a
significant deviation from wild type (P<0.001).
Membrane insertion during cellularization still proceeds in src64
embryos. The depth of membrane invagination and the dynamics of
cellularization front ingression is similar to that of wild-type embryos
(Fig. 3). These data suggest
that src64-mediated microfilament contraction does not play a
significant role in cellularization front invagination.

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Fig. 3. src64 is not required for membrane invagination during
cellularization. For both wild-type and src64 embryos, the progress
of membrane invagination was measured from the cell apices to the
cellularization front and plotted at five minute intervals starting at the
beginning of cycle 14 (time=0 minutes) at 25°C. Maximum cellularization
depth is obtained just before gastrulation begins in the interval between 55
minutes and 60 minutes. During early cellularization, the s.e.m. values are
between 0.1 µm and 0.6 µm, whereas during late cellularization, the
s.e.m. values are between 0.5 µm and 1.0 µm.
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tec29 mutants are similar to src64 mutants, but src64 is not required to localize Tec29 protein
tec29, a non-receptor tyrosine kinase, is also required for the
morphogenesis of ovarian ring canals and interacts with Src64 protein to
control ovarian ring canal growth (Dodson
et al., 1998
; Guarnieri et
al., 1998
; Roulier et al.,
1998
). Cellularizing embryos derived from
tec29k00206 germline clones have large and
non-rounded microfilament rings like those of src64 embryos
(Fig. 4). tec29
microfilament rings have a circularity index of 0.82
(Table 1), similar to that of
src64 but very different from that of wild-type microfilament rings
(P<0.001). Tec29 protein is expressed at the cellularization front
and more weakly along the lateral cellular membrane in both wild-type embryos
and src64
17 embryos, suggesting that
src64 does not act to localize Tec29 protein during cellularization
(Fig. 1C,D). This is in
contrast to the situation in the ovary where Src64 protein acts to localize
Tec29 protein to the ring canal (Guarnieri
et al., 1998
; Roulier et al.,
1998
). Phosphotyrosine staining is observed at the cellularization
front in both wild-type and src64 mutant embryos
(Fig. 1E,F). This suggests that
Src64 is not the major source of phosphotyrosine during cellularization as it
is in the egg chamber (Dodson et al.,
1998
; Roulier et al.,
1998
). Thus both src64 and tec29 are required
for microfilament ring contraction during cellularization, but tec29
is localized to the cellularization front independently of src64
activity.

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Fig. 4. tec29 is required for microfilament ring contraction during
cellularization. (A,B) Projections of confocal sections of the cellularization
front after it has passed the nuclear bases of a wild-type (A) and a
tec29k00206 germline clone (B) embryo. Embryos
were stained with antibody to myosin. The microfilament rings of wild-type
embryos during late cellularization are round and constricted (A), whereas the
microfilament rings of tec29 germline clone mutant embryos are less
rounded and are not constricted (B). Scale bar: 10 µm.
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scraps (anillin) is required for the formation of actinmyosin contractile rings
To determine whether other actin-binding proteins affect microfilament ring
contraction in a manner similar to src64, we examined the
actin-binding protein anillin, which is expressed at the cellularization front
in a domain similar that of Src64 (Field
and Alberts, 1995
; The Flybase
Consortium, 2003
) (Fig.
1E,F). During later development the anillin protein also localizes
to contractile rings during cytokinesis
(Field and Alberts, 1995
).
Anillin is encoded by the gene scraps, which is defined by a maternal
effect lethal mutation (Schüpbach and
Wieschaus, 1989
). We analyzed the phenotype of embryos from
mothers trans-heterozygous for two strong reduction-of-function mutations of
scraps (Schüpbach and
Wieschaus, 1989
). The most informative phenotype of
scraps mutants is the absence of microfilament rings. This can be
readily seen by observing the density and continuity of myosin staining around
the basal openings in the cellularization front (compare
Fig. 5B with
Fig. 2C, and
Fig. 5D with
Fig. 2G). The furrow canals are
collapsed and lack the early bulb-like or the late flask-like morphology of
the wild type. Only some furrow canals show strong myosin staining
(Fig. 5A,C). Myosin is seen in
dense rod-like clumps lying between some of the basal cellular openings
(Fig. 5B,D). A similar
phenotype has been observed by C. Field (C. Field, personal communication).
This myosin distribution is similar to that of F-actin in the septin mutant
peanut during cellularization
(Adam et al., 2000
), suggesting
that both anillin and certain septins play a role in the assembly or
maintenance of the microfilament rings.

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Fig. 5. scraps (anillin) is required for the formation of microfilament
rings during cellularization. (A-D) Sagittal sections (A,C) and projections of
confocal sections of the cellularization front (B,D) of a
scrapsRS/scrapsPQ
mutant embryo shortly before (A,B) and after (C,D) the cellularization front
has passed the nuclear bases. Embryos were stained with antibody to myosin.
(A) The furrow canals of scraps mutant embryos during early
cellularization are only slightly abnormal. (B) Microfilament rings are not
present in scraps mutant embryos during early cellularization and the
basal lumens are less rounded than in wild-type embryos. Myosin is found in
aggregates scattered along the cellularization front (compare with
Fig. 2C). (C) The furrow canals
of scraps mutant embryos during late cellularization are collapsed
and lack a flask-like morphology. (D) Microfilament rings are not present in
scraps mutant embryos during late cellularization; the basal lumens
are angular and are less rounded than those during early cellularization in
scraps mutant embryos (compare with B). Myosin is found in aggregates
scattered along the cellularization front (compare with
Fig. 2G). Scale bar: 10
µm.
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During early cellularization in scraps mutant embryos, the basal
cytoplasmic openings are angular and resemble polygons with relatively
straight sides (Fig. 5B).
Because the sides of these polygons are somewhat uniform in length, they
approximate circles and have a circularity index of 0.89
(Table 1), differing only
slightly from wild-type microfilament rings. Unlike in src64 mutants,
they are not convoluted or wavy, and indentations are not observed. During
late cellularization, the scraps phenotype becomes more severe
(Fig. 5D) with a circularity
index of 0.73 (Table 1),
significantly differing from wild type (P<0.001). This decrease in
circularity reflects an increase in the length of some of the sides of each
polygon and a decrease in the length of others so that the basal openings more
closely resemble polygons with fewer sides. The sides remain straight and show
no waviness that would indicate a lack of tension. Instead, the gradual
distortion of the polygons in scraps mutants is consistent with
stretching due to microfilament contraction in the absence of an organizing
structure. The scraps phenotype is therefore distinct from that of
src64 mutants, which appear to lack microfilament tension and
maintain similar, but deviant, circularities throughout cellularization. This
interpretation implies that microfilament rings are not required for
microfilament contraction. The low circularity index of later basal openings
in scraps mutants suggests that microfilament ring structure provides
a stabilizing framework for microfilament contraction during the late phase of
cellularization.
The premature contraction in bottleneck embryos does not require scraps (anillin)
We have used mutations in bottleneck (bnk) to define more
clearly the roles that src64 and scraps play in
cellularization. Bnk is a small, highly basic protein that regulates the
dynamic restructuring of the actin cytoskeleton so as to control the timing of
microfilament ring contraction during late cellularization. It is expressed
during early cellularization and its level drops precipitously during the
transition to the late phase (Schejter and
Wieschaus, 1993b
). During early cellularization, Bnk co-localizes
with myosin, but extends further apically in the furrow canal
(Fig. 6).

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Fig. 6. Bottleneck protein co-localizes to the cellularization front with myosin.
Cross-sections (A,C,E) and projections of confocal sections of the
cellularization front (B,D,F) before the cellularization front has passed the
nuclear bases of wild-type embryos. Embryos were stained with antibodies to
Bnk and myosin, and images have been arranged to show Bnk protein (A,B),
Myosin (C,D), and merged images of both Bnk and myosin (E,F). (A,B) Bnk is
expressed along the entire furrow canal and in microfilament rings during
early cellularization. (C,D) Myosin is expressed basal-laterally in the furrow
canal and in microfilament rings. (E,F) Bnk and myosin localization overlaps
in microfilament rings and overlaps basal-laterally in the furrow canal, but
Bnk localization extends farther apically. Scale bar: 10 µm.
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The bnk phenotype is distinct to that of src64 or
scraps in that embryos homozygous for a bnk deficiency have
a hypercontractile phenotype. The microfilament rings are prematurely
constricted during early cellularization
(Fig. 7A)
(Schejter and Wieschaus,
1993b
). The rings squeeze the nuclei into dumbbell shapes during
early cellularization, trapping and dragging some of them along with the
advancing cellularization front during late cellularization
(Fig. 7D) (Schejter and Wieschaus,
1993b
). The microfilament rings of early cellularization and late
cellularization bnk embryos have circularity indices of 0.92 and 0.91
(Table 1), respectively, values
that do not differ from those of similarly staged wild-type embryos, even
though initially they enclose a much smaller area of open cytoplasm
(P<0.001).

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Fig. 7. Phenotypes of bnk and scraps are co-expressed.
Projections of confocal sections of the cellularization front (A-C) and
sagittal sections (D-F) of the same bnk mutant (A,D),
scrapsRS/scrapsPQ
mutant (B,E) and scrapsRS/scrapsPQ; bnk
double-mutant (C,F) embryos. Embryos were stained with antibody to myosin
(A-F) and with Hoechst dye (D-F). (A) Microfilament rings are hypercontracted
in bnk mutant embryos; some nuclei are constricted into dumbbell
shapes by the hyperconstricted microfilament rings and carried out of the
periphery by the cellularization front in bnk embryos (D). (B)
scraps mutant embryos showing the absence of microfilament rings,
angular basal lumens and a normal nuclear morphology (E). (C) Microfilament
rings are not formed in scraps; bnk mutant embryos, but the
cellularization front still exhibits increased contraction and large gaps in
the microfilament network. (F) Despite the absence of microfilament rings in
scraps; bnk mutant embryos, some nuclei are constricted into dumbbell
shapes by the contracted microfilaments and carried out of the periphery.
Scale bar: 10 µm.
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scraps; bnk double-mutant embryos show a mixture of the phenotypes
of both scraps and bnk embryos
(Fig. 7). Like scraps
embryos, scraps; bnk embryos fail to form actinmyosin rings, and
instead show dense rod-like aggregates of myosin II lying between some of the
non-rounded basal cellular openings. In spite of the absence of contractile
rings, scraps; bnk embryos still display the premature
hypercontraction phenotype characteristic of bnk embryos
(Fig. 7C). The cytoskeleton
surrounding cells in scraps; bnk embryos is more contracted, in terms
of both area and circularity, than the cytoskeleton surrounding cells in
scraps embryos. Microfilaments are constricted around dumbbell-shaped
nuclei, which are trapped and dragged out of the periphery of the embryo by
the cellularization front in scraps; bnk double-mutant embryos, as is
characteristic of bnk embryos
(Fig. 7F). The area enclosed by
the microfilaments of scraps; bnk embryos is significantly less than
that of scraps embryos (P<0.001), but is still larger
than that of bnk embryos (P<0.001). During late
cellularization, the circularity index of the basal openings of scraps;
bnk embryos is 0.90 (Table
1), similar to that of bnk embryos, but significantly
different from the 0.73 value of scraps embryos
(P<0.001). This difference suggests that the actin-myosin network
can still contract in the absence of microfilament rings, but without the
efficiency that is conferred by the organization of the cytoskeleton into
rings.
The hypercontraction caused by the absence of Bnk protein, coupled with the
loss of structural integrity of the cellularization network caused by the
absence of anillin and microfilament rings, leads to the apparent tearing of
parts of the cellularization network. Several regions of the cytoskeleton are
either stretched thin or broken, leaving large gaps in the cellularization
front (Fig. 7C). This suggests
that the loss of anillin and microfilament rings results in a fragile
cytoskeletal structure that unravels in the absence of Bnk. These
double-mutant results suggest that Bnk and anillin both play structural roles
in the cellularization front, but that neither are necessary for microfilament
contraction itself.
src64 is required for the premature contraction of bnk embryos
In restructuring the cytoskeleton during cellularization, Bnk controls the
timing of microfilament ring contraction so that basal closure does not occur
until after the cellularization front has passed the bases of the nuclei.
bnk mutants have a prematurely hyperconstricted ring phenotype
opposite to the non-constricted ring phenotype of src64 mutants.
src64 bnk double-mutant embryos look like src64 mutant
embryos. The src64 bnk embryos have the large, non-constricted
microfilament rings that appear to be under no tension
(Fig. 8). They have a
circularity index of 0.85 (Table
1), similar to that of src64 embryos but different from
that of bnk embryos (P<0.001). A few double-mutant
embryos showed some degree of microfilament ring contraction during late
cellularization; it is likely that these embryos are the result of some
residual activity of the reduction-of-function
src64
17 allele. The analysis of src64
bnk double-mutant embryos demonstrates that the premature
hypercontraction of bnk requires src64 activity. The
interaction of bnk mutation with src64 and scraps
reveals the difference between the two genes: src64 is required for
microfilament contraction and scraps (anillin) is not. This suggests
that bnk regulates cytoskeletal contractility during cellularization
by counteracting the src64-mediated contraction of the microfilament
rings.

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Fig. 8. src64 activity is required for bnk hypercontraction.
Projections of confocal sections of the cellularization front (A-C) after it
has passed the nuclear bases of a bnk mutant (A), a src64
mutant (B) and a src64 bnk double-mutant (C) embryo. Embryos were
stained with antibody to myosin (A-C). (A) bnk mutant embryo showing
hypercontracted microfilament ring phenotype. (B) src64 mutant embryo
showing non-contracted microfilament ring phenotype. (C) src64 bnk
double-mutant embryo showing a non-contracted microfilament ring phenotype
similar to that of src64 mutant embryos (B). Scale bar: 10 µm.
|
|
 |
Discussion
|
---|
src64 mediates microfilament contraction during cellularization
Our analyses suggest that src64 and tec29 are required
for tension in the cellularization front during early cellularization, and for
the constriction of the basal microfilament rings during late cellularization.
Src64 and Tec29, which are present at higher levels in the microfilament
rings, might activate actin-myosin contraction or be essential for the ability
of the actin-myosin network to contract. Despite a general similarity of form,
the cellularization microfilament ring and the oocyte-nurse cell complex ring
canal differ substantially in structure and dynamics. In the ovary,
src64, and presumably tec29, control ring canal expansion by
regulating actin polymerization and cross-linking
(Kelso et al., 2002
;
Tilney et al., 1996
). It is
unlikely that myosin can play a role in this process as myosin-driven sliding
of actin filaments would lead to contraction rather than expansion
(Tilney et al., 1996
).
Although myosin is localized to the ring canal, and null mutations in
regulatory myosin light chain cause defects in the ring canals, these defects
are not severe and do not prevent ring canal assembly or expansion
(Hudson and Cooley, 2002
;
Jordan and Karess, 1997
;
Edwards and Kiehart, 1996
).
Thus, despite a similar involvement of src64 and tec29, it
is unlikely that microfilament ring constriction and ring canal expansion are
mechanistically similar.
scraps (anillin) is required for the formation of stable contractile microfilament rings
Anillin, which localizes to the cellularization front and shows higher
concentration in the contractile microfilament rings, is required for proper
cellularization. Anillin bundles actin filaments and may stabilize these
filaments during actin-myosin contraction
(Field and Alberts, 1995
). On
the basis of these observations, we conclude that in the absence of anillin,
stable contractile microfilament rings do not form; instead the contractile
protein myosin is irregularly distributed in aggregates throughout the
cellularization front. Strikingly, loss of anillin in bnk embryos
does not suppress the severe early contraction defect seen in bnk
embryos. In the absence of the structure provided by these rings, the
contraction of the microfilaments is uneven, leading to increasing defects in
the shape of the basal openings as cellularization progresses. This suggests
that anillin is not required for the ability of the microfilaments of the
cellularization network to contract, only for their organization into stable
rings.
src64 and bnk oppose each other during early cellularization
The phenotypes presented in this paper support a model in which
src64 and bnk oppose each other to control contraction of
the early cellularization network. Double-mutant analysis reveals that
src64 is epistatic to bnk
(Fig. 9A). Bnk acts only to
restrain and partially redirect Src64-mediated ring constriction. The fact
that cellularization proceeds in src64 and tec29 mutants
suggests that a force other than microfilament ring contraction is sufficient
to drive cellularization front invagination. This force may be a result of the
insertion of membrane (Lecuit and
Wieschaus, 2000
; Sisson et
al., 2000
), or may be due to the action of plus-end directed
microtubular motors (Mazumdar and
Mazumdar, 2002
; Foe et al.,
2000
; Foe et al.,
1993
), or some combination of both.

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|
Fig. 9. Model for src64-dependent and src64-independent forces
acting during cellularization. (A) Epistasis pathway for microfilament
contraction during cellularization. src64 is epistatic to
bnk in the pathway controlling microfilament ring contraction.
tec29 is speculatively placed in the pathway with src64 as
Tec kinases are generally activated by Src kinases, although this has not been
experimentally confirmed for Drosophila cellularization.
scraps (anillin) has been placed in a parallel pathway to
bnk and src64 because it plays an important, but indirect,
role in microfilament ring contraction by stabilizing microfilament rings. In
the absence of anillin, microfilament rings are not formed, but the
disorganized microfilaments still have the ability to contract in the absence
of bnk activity. peanut (pnut) also acts in this
process. (B) Mechanical model for the interaction of Bnk and Src64 protein
during early cellularization. See Discussion for details. (C) Mechanical model
for the interaction of Bnk and Src64 protein during late cellularization. See
Discussion for details.
|
|
Most models for cellularization invoke a role for myosin contraction during
the ring constriction and basal closure that occur during late stages of the
process; a role during early stages is more controversial. The early phenotype
of src64 mutants, if our interpretation is correct, suggests a role
for microfilament ring contraction in the early stages as well, acting both to
coordinate the invagination of the furrow canals by maintaining tension along
the cellularization front and to direct their invagination inward. This force
is a product of the interaction of src64-dependent, myosin-mediated
contraction of the microfilament rings and resistance to this contraction
exerted by Bnk protein, which acts as a linker between the rings. These forces
oppose each other at all points along the contractile microfilament ring
network, generating a dynamic tension over the entire network, keeping it taut
and driving the minimization of its surface area. The addition of these force
vectors acting on a cross-section of one ring on a curved surface produces a
resultant vector directed both toward the interior of the embryo (the center
of the circular cross-section) and toward the center of the microfilament
ring. The first component of the resultant force vector is the
src64-mediated force that provides direction to the invagination that
follows the increase in surface area produced by membrane insertion during
early cellularization (Fig.
9B). The other component of the resultant force vector is in the
plane of the microfilament ring, coordinating constriction about the entire
circumference of the embryo and driving a small degree of constriction
consistent with the decrease in cellularization front surface area during
invagination (Fig. 9B).
As the cellularization front passes the bases of the nuclei and
cellularization shifts into its late phase of rapid progression
(Lecuit and Wieschaus, 2000
),
Bnk expression is shut off and the protein is rapidly degraded and removed
from the cellularization network (Schejter
and Wieschaus, 1993b
). In the absence of Bnk protein, there is no
force resisting microfilament ring contraction, so it no longer contributes to
driving cellularization front invagination. The src64-mediated force
is now directed along the radii of the rings, leading to their constriction.
This constriction pulls the membrane toward the center of the base of the
cell, expanding the furrow canals and leading to basal closure
(Fig. 9C). The
src64-independent force (membrane addition or microtubular motors)
may be the only force now driving the inward invagination of the
cellularization front.
In conclusion, our data define the differing roles that src64,
tec29 and anillin play in the cytoskeletal dynamics of
Drosophila cellularization, and reveal more precisely the role that
the cytoskeleton plays in the formation of the cellular blastoderm. These data
establish that microfilament ring organization and contraction are crucial to
basal closure of the blastoderm cells during cellularization. However, these
data also suggest that membrane invagination can proceed, though abnormally
and less efficiently, in the absence of microfilament organization or
contraction. It will be interesting to determine what the comparative roles
and contributions of membrane insertion and microtubular motors are to the
progression of the cellularization front.
 |
ACKNOWLEDGMENTS
|
---|
We thank T. Xu for the gift of anti-Src64 antibodies, C. Field for the gift
of anti-anillin and anti-myosin antibodies, and M. Stern and S. Beckendorf for
the gift of anti-Tec29 antibodies and tec29 FRT40 fly stocks. We
thank R. Samanta for histology advice and assistance, and J. Goodhouse for
confocal microscopy advice and assistance. We thank I. Clark and G. Deshpande
for helpful comments on the manuscript. J.H.T. was supported by a National
Institutes of Health postdoctoral fellowship and by the Howard Hughes Medical
Institute. This work was supported by the Howard Hughes Medical Institute and
by National Institute of Child Health and Human Development grant 5R37HD15587
to E.W.
 |
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