1 Department of MCD Biology, University of Colorado, Boulder, CO 80309,
USA
2 Section of Molecular and Cellular Biology, University of California, Davis, CA
95616, USA
* Author for correspondence (e-mail: wood{at}colorado.edu)
Accepted 29 August 2003
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SUMMARY |
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Key words: C. elegans, Asymmetry, G protein
![]() |
Introduction |
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Although much is now known about how LR asymmetry is elaborated and
maintained in various embryos (for a review, see
Mercola and Levin, 2001), the
mechanism of the initial symmetry-breaking event has remained mysterious.
Until recently, it appeared that the initial event in vertebrate embryos
involved the chirality and asymmetric beating of nodal cilia
(Nonaka et al., 1998
;
Essner et al., 2002
;
Nonaka et al., 2002
), and must
therefore be quite different from the much earlier decision made in
invertebrates such as snails (Crampton,
1894
) and nematodes (Wood,
1998
) (see below). However, evidence has accumulated during the
past year that vertebrates also exhibit LR asymmetries during cleavage stage
(Kramer et al., 2002
;
Levin et al., 2002
), raising
the possibility that there are common aspects of the handedness choice
mechanism in both classes of animals.
In C. elegans embryos, LR asymmetry first becomes apparent between
the four-cell and six-cell stages. The anterior (ABa) and dorsal (ABp)
blastomeres of the four-cell embryo enter mitosis in synchrony, with their
spindles oriented parallel to the LR axis, orthogonal to the AP and DV axes.
During telophase, just before cytokinesis begins, they skew about 20°,
always in a counter-clockwise direction (as viewed ventrally), with the result
that the two daughter cells formed on the left (ABal and ABpl) lie anterior to
their respective sisters (ABar and ABpr) on the right (see
Fig. 1B and Movie 1 at
http://dev.biologists.org/supplemental/).
From this time onwards, the embryo is LR asymmetric
(Sulston et al., 1983), and
this invariant choice of handedness dictates the polarity of all subsequent LR
asymmetries during embryonic and larval development
(Wood, 1991
). We have
arbitrarily designated this normal handedness as dextral. Under standard
laboratory growth conditions (Sulston and
Hodgkin, 1988
), neither sinistral wild-type C. elegans
embryos nor adults have ever been observed in our laboratory
(n>15,000) or elsewhere to our knowledge. However, sinistral
embryos can be obtained by micromanipulation
(Wood, 1991
), early removal of
the egg shell (Wood and Kershaw,
1991
), cold treatment (Wood et
al., 1996
) and as we show below, as the result of maternal-effect
mutations. Sinistral embryos develop normally into healthy fertile adults with
all of their normal LR asymmetries reversed (for a review, see
Wood, 1998
).
|
Control of spindle orientations during the first two cleavages also
involves heterotrimeric G proteins (Zwaal
et al., 1996; Jansen et al.,
1999
) and the product of the let-99 gene
(Rose and Kemphues, 1998
;
Tsou et al., 2002
). In early
cleavages, the Gß protein GPB-1 is associated with centrosomes in
prophase and metaphase (Zwaal et al.,
1996
), and there is evidence that G
, Gß and G
proteins are all involved in controlling centrosomal behavior, in particular
the plane of centrosomal migration around the interphase nucleus that
determines orientation of the subsequently forming spindle
(Gotta and Ahringer, 2001
).
Aberrant third cleavage spindle orientations are also observed in
G-protein-deficient embryos, but it is not clear whether these effects are a
direct consequence of G-protein defects or a secondary consequence of earlier
anomalies (see Discussion).
With the knowledge that sinistral embryos are viable, we undertook screens
for mutations that would increase the frequency of sinistral animals, in
attempts to identify factors involved in the initial handedness choice. Such
screens are cumbersome, because mutations affecting third cleavage are
expected to have maternal effects, and because the animals always lie on their
sides, so that viewing LR asymmetries with a dissecting microscope is
impractical without manually rolling individual worms 90° to obtain a
dorsal or ventral view. In a small screen of about 2000 genomes, in which we
scored gonad handedness in the F3 generation following EMS mutagenesis, we
found no handedness reversal mutants. As an alternative strategy, we screened
known maternal-effect mutants with early cleavage defects, looking for
survivors with sinistral handedness. We describe the results of this screen
and characterization of a gene originally designated spn-1, which can
mutate to cause near randomization of handedness choice at the four- to
six-cell stage (Wood, 1998).
We show that spn-1 is the same as a molecularly defined gene encoding
the G
protein GPA-16 (Jansen et
al., 1999
), and we will subsequently refer to this gene as
gpa-16.
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Materials and methods |
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Screen of maternal-effect lethals for handedness reversal
Alleles were selected on the basis of reported cleavage-plane-defects and
where possible, incomplete penetrance so that adult animals could be obtained.
Each strain was cultured at 20 or 25°C, and adults were scored for
handedness reversal under a dissecting microscope by rolling 90° to give a
dorsal or ventral view and observing the position of the anterior gonad
relative to the gut [gonad handedness is a reliable predictor of overall
handedness (Wood, 1991;
Wood et al., 1996
)]. For two
mutations that resulted in lack of any viable embryos from homozygous mutant
mothers, the embryonic spindle orientations in ABa and ABp were scored for
handedness using Nomarski optics (* in the list below). For three mutations
that caused low penetrance gonad abnormalities, handedness was checked by
observation of ventral nerve cord and coelomocyte positions (**in the list
below).
For the following strains, no reversed animals were seen at either
temperature (n50 for each strain): N2 (n>15,000),
emb-5(g16ts), emb-5(g65ts), emb-8(hc69ts), emb-11(g1), emb-12(g5ts),
emb-14 (g14ts), emb-18(g21ts), emb-21(g31ts), emb-26(g47), emb-27(g48),
emb-34(g62), gad-1(ct226)**, glp-1(q224ts) (n=11*),
glp-1(e2141) (n=8*), let-99(it141), let-99(s1201), par-2(it5)**,
par-4(it33)**, par-4(it47), spd-1(oj5), spd-2(oj29),
spn-2(it149), stu-10(oj14), stu-11(oj18), zyg-2(g57), zyg-10(b261ts),
zyg-11(b273ts), zyg-11(b2) and zyg-11(mn40).
A few percent of reversed animals were seen in strains of genotypes: goa-1(n363), 2%; gpb-1(pk44);Ex170, 4%; spn-3(it151), 3% (n>300); zyg-8(b235ts), 1% at 25°C only (n=100); zyg-9(b288ts), 2% at 25°C only; zyg-11(b271ts), 2% at 25°C only. In the gpa-16(it143) strain, 20% of animals were reversed at 20°C (n>10,000) and 39% at 25°C (n>6000).
Genetic mapping and complementation testing
All phenotypic analysis was performed with a gpa-16(it143) strain
that had been outcrossed to wild-type (N2) at least ten times. This mutation
was located on Linkage Group (LG) I using STS mapping
(Williams et al., 1992) and
was further mapped by standard 2- and 3-factor crosses relative to dpy-5,
unc-13, egl-30, src-1 and mex-3 (see
Fig. 4A). The it143
mutation mapped close to the left telomere of linkage group (LG) I in a region
poor in known genetic markers and cosmid coverage. The free duplication
sDp2 complemented it143; the deletion hDf10 deletes
the gpa-16 locus.
|
Temperature-sensitive period (TSP) of it143
The TSP for the it143ts mutant phenotypes was determined by
shifting homozygous it143 hermaphrodites or early embryos dissected
from them between the permissive (16°C) and non-permissive (25°C)
temperatures. Onset of the TSP was determined by rearing homozygous
i143 hermaphrodites at 25°C, then shifting them to 16°C,
collecting laid eggs at 2 hour intervals and estimating the stage of oogenesis
at which each batch of eggs was shifted from the known time course of oocyte
maturation, fertilization, early embryogenesis and egg-laying at these two
temperatures (Wood et al.,
1980), as described elsewhere
(Wood et al., 1996
). To
determine the end of the TSP, early embryos from homozygous it143
hermaphrodites reared at 16°C were removed at the one- to two-cell stage
in a 16°C constant temperature room. The initial cell divisions were
observed with a dissecting microscope and embryos at various stages (up to
eight cells) were transferred to pre-warmed 25°C plates to mature. In both
protocols, embryos were assayed for viability (hatching within 24 hours), and
the adults that developed from viable embryos were scored for dextral or
sinistral handedness as described in the text. The range of embryonic arrest
phenotypes for all shifted embryos was indistinguishable from that observed in
embryos developing entirely at 25°C.
Microscopy
For observations of early spindle orientations by Nomarski microscopy, one-
to two-cell embryos were dissected from gravid hermaphrodites reared at either
25 or 20°C. To mount embryos in a suitable orientation for observing the
LR cleavages of ABa and ABp (dorsal or ventral aspect), embryos were allowed
to settle through a small drop of 25% EGM/75% egg salts onto a poly-lysine
coated coverslip and covered with a drop of silicone oil. The coverslip was
inverted over a slide onto two pieces of double-sided tape (3M core series
2-0300) for support. Development of suitably oriented embryos was recorded
using a multi-focal-plane, time-lapse video microscopy system (4D microscope)
as described elsewhere (Powell-Coffman et
al., 1996; Knight and Wood,
1998
). Recordings were made at 20-22°C on embryos raised at
20°C except where noted. A through-focus series of optical sections in 2
µm steps was recorded every 30 seconds until embryos reached the 28-cell
stage, and a final series was taken 14 hours later to record terminal
phenotype. Handedness was scored in the adults developing from hatched
first-stage (L1) larvae that could be recovered to NGM plates from the hanging
drop mounts.
For immunofluorescence microscopy, embryos were prepared by standard
procedures for C. elegans (Miller
and Shakes, 1995), with the following modifications. All PBS
washes were supplemented with 0.5% Triton X-100. The following antibodies were
used: 3A5 mouse monoclonal anti-tubulin (gift of J. R. McIntosh); rabbit
polyclonal anti-GPB-1 (gift of R. Plasterk), rabbit polyclonal anti-PAR-3
(gift of K. Kemphues). Secondary FITC- and TRITC-conjugated antibodies were
from Molecular Bioprobes. Embryos were also stained with DAPI to visualize
nuclei. Immunofluorescence was observed on the Leica microscope described for
4D recording, and images were deconvoluted using SlideBook v.2.6 software (3I,
Denver, CO).
Blastomere isolations
Isolations were performed as described elsewhere
(Edgar, 1995), with a few
modifications. Embryos were cultured in a different medium
(Shelton and Bowerman, 1996
),
and because at 25°C it143 mutant embryos lysed easily, blastomere
isolations were performed at room temperature (22°C) on embryos from
hermaphrodites reared at 20°C. In addition, the time of incubation in
hypochlorite was reduced to 1 minute to increase survival of sensitive mutant
embryos.
Laser ablations
Laser ablations were performed with a VSL-337 (Laser Science)
nitrogen-pumped dye laser through a Zeiss microscope equipped with Nomarski
optics. Embryos were mounted in hanging drops as described above, but in egg
salts. Each target cell was irradiated until debris accumulated in the nucleus
and the cell subsequently divided no more than once.
RNA interference (RNAi)
To silence gpa-16 expression by RNAi, gpa-16 dsRNA was
prepared in vitro and administered either by injection
(Fire et al., 1998) or by
soaking (Tabara et al., 1998
)
as described elsewhere (Van Auken et al.,
2002
). RNAi effects on first and second cleavage and on lethality
and handedness reversal were analyzed using a 425 bp fragment and full-length
gpa-16 dsRNA, respectively. In some RNAi experiments, worms were
reared on a feeding vector strain of E. coli carrying a
gpa-16-derived construct (kindly provided by Monica Gotta) that could
be induced by IPTG to produce both gpa-16 sense and antisense
transcripts (Timmons and Fire,
1998
).
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Results |
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The it143 mutation was originally isolated in a screen for
maternal-effect lethal mutants with defects in early spindle orientations
(Rose and Kemphues, 1998), and
its effect on handedness choice has been mentioned previously
(Bergmann et al., 1998
;
Wood, 1998
). It causes
temperature-sensitive, partially penetrant maternal-effect lethality (Mel
phenotype) as well as LR reversal, as shown in
Table 1. When it143
homozygous hermaphrodites are reared at 25°C, about 70% of their embryos
are inviable. The survivors grow to adulthood, and about 40% of these animals
are sinistral. At 16°C, about 98% of the embryos survive, and only a few
percent develop into sinistral animals, while at 20°C, the results are
intermediate.
|
Like the LR reversed animals produced by micromanipulation of four- to
six-cell embryos (Wood, 1991;
Wood and Kershaw, 1991
),
sinistral it143 mutant animals exhibit reversal of all LR asymmetric
anatomical features examined, including positions of the gonad and intestine,
positions of the coelomocytes, organization of the ventral nerve cord,
migration of the Q neuroblasts, and patterning of the motor axons from the
ventral cord that innervate dorsal muscles (D.C.B., unpublished). The only
exception is chirality of the cuticle, which remains the same in dextral and
sinistral animals (Bergmann et al.,
1998
).
Characterization of the it143 phenotype
To define the temperature-sensitive period (TSP) of it143 for
embryonic lethality and handedness reversal, homozygous mutant hermaphrodites
or their embryos were shifted from 16°C to 25°C or vice versa at
different stages (see Fig. S1 at
http://dev.biologists.org/supplemental/).
Downshifting of hermaphrodites during different stages of oocyte maturation
defined the start of the TSP as after oocyte maturation for reversal and
mid-oogenesis for lethality. Upshifting of individual embryos isolated and
staged at the two- to eight-cell stages showed that the TSP for reversal was
over by the four-cell stage and for lethality by the eight-cell stage.
Therefore, as it143 is a loss-of-function mutation (see below),
action of the gene it defines is required at the four-cell stage or before to
dictate the handedness of all subsequent LR asymmetries. These results are
consistent with the previous demonstration that reversal of handedness by
micromanipulation during the ABa and ABp cleavages is sufficient to reverse
all LR asymmetries during development
(Wood, 1991).
Effects of the mutation on spindle orientations could be directly observed by DIC microscopy of it143 mutant embryos. When produced and observed at 25°C, these embryos exhibited aberrant behavior of mitotic spindles in each of the first three cleavages.
In one-cell mutant embryos, nuclear rotation occurred, and first cleavage was asymmetric (as in the wild type), producing a larger AB and a smaller P1 cell. However, the oscillations of the posterior spindle pole during anaphase were absent or greatly reduced (9/13 embryos) compared with wild-type embryos. During second cleavage, nuclear rotation in P1 during prophase failed in most it143 mutant embryos (14/18), although late rotations during anaphase were sometimes observed (5/15).
Dramatic effects were observed in the third-cleavage behavior of the ABa and ABp spindles, whose orientations are critical for establishing embryonic handedness. To analyze LR asymmetries, 27 mutant embryos from hermaphrodites reared at 20°C (the temperature at which the highest percentage of LR reversals was seen among all, including inviable, embryos), were observed in dorsal or ventral view. Under these conditions, spindle misorientations in cells other than ABa and ABp were rare (4/27). At third cleavage, most of the remaining embryos initially elongated their ABa and ABp spindles correctly along the LR axis (e.g. Fig. 2H). In nine out of these 23 embryos, the ABa and ABp spindles skewed approximately normally (not shown; similar to Fig. 2C) to generate dextral embryos and adults. However, in about 40% of the embryos (10/23), the elongated spindles skewed in the opposite direction (Fig. 2I), causing the right daughters to be anterior at the six-cell stage. The result was a sinistral embryo, and for three that were recovered and allowed to mature, a sinistral adult. The remaining three animals exhibited highly abnormal A/P or D/V skewing (Fig. 2F,L), and these embryos were inviable. Lack of P1 nuclear rotation did not appear to correlate with third-cleavage spindle behavior. The third-cleavage spindle orientations and terminal phenotypes for the 27 embryos described above are summarized in Table S1 at http://dev.biologists.org/supplemental/.
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|
gpa-16(it143) is a loss-of-function mutation
The gpa-16(it143) mutation behaved genetically as a fully
recessive, loss-of-function (lf) allele at 25°
(Table 1). Homozygous
gpa-16 animals hermaphrodites carrying one copy of the wild-type gene
on a free duplication exhibited no embryonic lethality or reversal. The
defective phenotypes of a hemizygous strain carrying gpa-16(it143) in
trans to a deficiency that uncovers the gpa-16 locus were
only slightly more severe than those of gpa-16(it143) homozygotes at
all three temperatures tested.
To test whether gpa-16(RNAi) mimics the it143 phenotype, as predicted if it143 is a lf mutation, gpa-16 double-stranded (ds) RNA was injected into wild-type N2 adult hermaphrodites. The resulting gpa-16(RNAi) embryos observed at 25°C exhibited spindle abnormalities similar to those described above for gpa-16(it143) at 25°C: reduction or absence of posterior spindle pole oscillations preceding first cleavage (9/10), and failure of P1 nuclear rotation prior to spindle elongation during second cleavage (7/9).
Embryonic lethality and handedness reversals were analyzed on larger numbers of embryos following soaking of N2 hermaphrodites in gpa-16 dsRNA. Soaking at 16°C or 20°C had little effect, but soaking at 25°C caused up to 50% embryonic lethality, with a handedness reversal rate of about 10% among survivors (n=50). gpa-16(RNAi) in the gpa-16(1t143) background caused no increase in embryonic lethality at 25° over the 70% observed to result from gpa-16(it143) alone. Therefore, gpa-16(RNAi) and gpa-16(it143) cause similar Mel and reversal phenotypes, supporting the conclusion that this allele results in loss of gene function at 25°C.
The it143 allele did not cause sterility of surviving animals at
25°C (n>100). By contrast, the pk481 strain, after 10
backcrosses to wild type, exhibited no zygotic lethality among progeny of
pk481/+ hermaphrodites, but nearly complete sterility of the progeny
homozygotes, regardless of temperature. This sterility could not be overcome
by mating with wild-type males. These results suggested that the
gpa-16-null phenotype might be a gonadogenesis or oogenesis defect
resulting in hermaphrodite sterility. However, further characterization of
pk481 indicated that interpretation of the resulting phenotype might
be problematic, for two reasons. First, about 10% of pk481/+
heterozygotes were found to be sterile (see supplemental text at
http://dev.biologists.org/supplemental/),
suggesting that the allele is semidominant. Second, analysis by PCR using
appropriate primers showed that in addition to the reported deletion mutation,
pk481 homozygotes also carried a linked, wild-type copy of the
gpa-16 gene, suggesting that pk481 is a complex
rearrangement (see Fig. S2 at
http://dev.biologists.org/supplemental/).
Based on these findings, plus the prediction that the pk481 deletion
should result in synthesis of a GPA-16 protein lacking the GTP binding domain
but otherwise complete (Jansen et al.,
1999), it seemed possible that pk481 might be an
antimorphic rather than a null allele.
To obtain a further indication of the null phenotype, we asked whether gpa-16(RNAi) also caused sterility. Wild-type worms were reared from hatching on an appropriate feeding vector expressing gpa-16 dsRNA or on a control strain expressing the empty vector, at 16, 20 and 25°C. The effects on maternal-effect embryonic lethality were weaker than in the soaking experiments, and again were seen only at 25°C. The rate of maternal-effect lethality increased from 13% (n=597) on control plates to 23% (n=250) on the feeding vector, while sterility increased from 2% (n=60) to 98% (n=58). These results suggest that the gpa-16 null phenotype is maternal-effect lethality if gene function is lacking during oogenesis, and hermaphrodite sterility if gene function is lacking during larval development. The basis for sterility has not been investigated, except that in each of four pk481 homozygous adult hermaphrodites examined the gonad morphology appeared abnormal.
Loss of gpa-16 function reduces normal centrosomal
association of the Gß protein GBP-1
Earlier results indicated that the Gß protein of C. elegans,
encoded by the gpb-1 gene (Zwaal
et al., 1996), is required for correct orientations of early
blastomere cleavages and that GPB-1 associates with the centrosomes of these
cells during mitosis. It was further shown that maternal silencing of the two
G
genes goa-1 and gpa-16 by RNAi caused loss of both
cortical and centrosomal localization of GPB-1
(Gotta and Ahringer, 2001
). Our
screen (Materials and methods), showed that reduction of gpb-1
function in early embryos also caused a low frequency of handedness reversals,
although its effect was not as strong as that of gpa-16(it143). Tests
for effects of the gpa-16(it143) mutation on localization of GPB-1
were done using an anti-GPB-1 antibody
(Zwaal et al., 1996
) to stain
wild-type and gpa-16(it143) mutant embryos of two to 28 cells.
Staining was seen around the cortex of each blastomere in both genetic
backgrounds at all temperatures tested. In wild-type embryos at 25°C,
centrosomal staining was seen in 74% of cells in mitosis (n=58; see
Fig. 5). In mutant embryos from
hermaphrodites reared at 16°C, the centrosomal association did not appear
significantly affected (69%; n=13). However, in mutant embryos from
animals reared at 20°C or 25°C, centrosomal staining was significantly
reduced to 28% (n=40) and 25% (n=36), respectively. These
results suggest that functional G
protein GPA-16 may be required for
the association of Gß protein with centrosomes in early embryos.
|
In gpa-16/+; par/+ double heterozygotes, a significant frequency
of embryonic lethality was observed with the par-4(it33) allele but
not with another par-4(lf) allele or the alleles of other
par genes that were tested. However, in animals of genotype
gpa-16; par/+, a single copy of par-3(it162), par-4(it147),
or par-6(zu222) in the homozygous gpa-16(it143) background
caused significant enhancement of the gpa-16 embryonic lethality
(Table 2). The
par-6(zu222) allele, but not the others tested, also enhanced the
gpa-16 handedness reversal frequency: embryos from gpa-16(it143);
par-6(zu222)/+ hermaphrodites at 16°C were 16% viable with a reversal
frequency of 51% among survivors (n=60). Finally, using two
ts alleles, it was possible to make the doubly homozygous strain
gpa-16(it143); par-4(it47). At 16°, this strain exhibited a
similar handedness reversal frequency to the gpa-16(it143) single
mutant, but enhanced embryonic lethality (from 2% to 70%). In addition,
the gpa-16(it143) mutation, which was never observed to affect
production of differentiated intestinal cells in inviable embryos (D.C.B.,
unpublished) as assayed by presence of gut granules
(Laufer et al., 1980
),
appeared to largely suppress the lack of gut granules seen in 70% of
par-4(it47) single mutant embryos
(Morton et al., 1992
), to 8%
(n=92) in the double mutant. Thus, it appears that
gpa-16(it143) can interact genetically with mutations in par-3,
par-4 and par-6, all of which encode components of the AB-cell
cortex.
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Discussion |
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There are also two possible interpretations of the temperature effects we
have observed. First, the gpa-16 functions in early cleavages could
be required only at higher temperatures, so that any loss-of-function
condition would show temperature sensitivity. Second, the it143
mutation could cause loss of function only at higher temperatures, and the
temperature dependence of the gpa-16(RNAi) phenotypes could result
from temperature-sensitivity of the RNAi soaking method for this gene. As
temperature effects on RNAi efficiency have been noted previously (e.g.
Winston et al., 2002), we
cannot yet distinguish between these alternatives.
The embryonic lethality and handedness reversal resulting from loss of gpa-16 function results from the apparent randomization of AB-cell spindle orientations during the third cleavage in gpa-16 mutant embryos. At 25°C, gpa-16 defects also affect spindle behavior during second cleavage as discussed further below. However, our observations at 20°C, where second cleavage is normal, suggest that the misorientation at third cleavage is a specific effect of the gpa-16 defect rather than an indirect consequence of abnormal second cleavage.
There are some indications that GPA-16 may play a special role,
particularly in controlling the third-cleavage AB-cell spindle orientations
required for normal establishment of LR asymmetry and handedness choice.
Although GPA-16 and GOA-1 functions are at least partially redundant for
spindle orientation during first and second cleavages
(Miller et al., 2000;
Gotta and Ahringer, 2001
) (this
report), we have shown that gpa-16 loss of function alone also causes
aberrant spindle behaviors in one-cell and two-cell embryos. Moreover, while
maternal goa-1 and gpb-1 loss of function cause only low
percentages of embryonic lethality and very little handedness reversal
(Materials and methods), gpa-16 loss of function causes up to 70%
embryonic lethality and nearly complete randomization of handedness. Our
results suggest that in the absence of gpa-16 function, embryos are
more often reversed but otherwise normal than when other genes affecting
spindle orientation are defective. The basis for this difference is not clear;
at this point we can conclude only that there is some specific requirement for
GPA-16 in orientation of the AB-cell spindles during third cleavage.
Possible roles of G-proteins in spindle orientation and handedness
choice
The function of the GPA-16 G protein in control of third-cleavage
AB-cell spindle orientations remains to be determined, but possible roles can
be suggested based on previous results with G-protein-defective embryos. The
C. elegans Gß protein GPB-1 was shown to associate with
centrosomes and the cortex in early cleavages, and lack of maternal GPB-1
caused spindle orientation defects and embryonic lethality
(Zwaal et al., 1996
).
G
(RNAi), that is, simultaneous silencing of the two
embryonic G
-protein genes goa-1 and gpa-16, was
reported to prevent the normal migration of centrosomes around the nucleus
prior to second cleavage, leading to abnormal spindle orientations
(Gotta and Ahringer, 2001
).
Silencing of gpb-1 appeared to disrupt normal centrosomal steering
prior to second cleavage, with similar effects. In addition, both G
and
Gß silencing caused defects in P1 nuclear rotation, which
depends on interactions between astral microtubules and the cortex. Finally,
several recent studies have implicated G
proteins in the
force-generating mechanisms that asymmetrically position the first-cleavage
spindle to produce daughter cells of different sizes
(Colombo et al., 2003
;
Gotta et al., 2003
;
Grill et al., 2003
;
Srinivasan et al., 2003
;
Tsou et al., 2003a
), although
the roles of G-proteins in these mechanisms remain unclear.
Thus, G and Gß proteins appear to function together in
dictating early spindle orientations, by controlling centrosomal migration as
well as cortical interactions with astral microtubules in force generation.
Consistent with an important role for GPA-16 in such interactions, we have
presented evidence that this protein is required at high temperature for
centrosomal association of the GPB-1 Gß protein and for normal
P1 nuclear rotation, as well as the normal positioning of the ABa
and ABp spindles preceding the skewing that results in handedness choice.
GPA-16 could also be involved in generating the forces that cause this
asymmetric skewing. Finally, a role in astral-cortical interaction is
supported by the genetic interactions of gpa-16(it143) with
par genes that encode cortical components of both the ABa and ABp
cells.
Our results demonstrate for the first time that heterotrimeric G proteins play a role in establishment of embryonic LR asymmetry and handedness choice. However, it is too early to conclude whether the initial symmetry-breaking event occurs before, during, or after the GPA-16-mediated process of AB-cell spindle orientation parallel to the LR axis. The initial establishment of LR asymmetry could occur subsequently, as the ABa and ABp spindles skew from their initial LR orientation. Alternatively, it could already have occurred in the four-cell embryo prior to ABa and ABp cleavage, causing a so-far undetected asymmetry that becomes apparent only when these cells divide. Searches for earlier asymmetries using molecular markers and further elucidation of the G-protein requirements for spindle orientation will help to distinguish these alternatives.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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