a genome-wide screen for suppressors of par-2 uncovers ......2006/07/02 · protein activators and...

Potential regulators of cell polarity in C. elegans Labbé et al.

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A genome-wide screen for suppressors of par-2 uncovers potential regulatorsof PAR protein-dependent cell polarity in Caenorhabditis elegans

Jean-Claude Labbé1,3, Anne Pacquelet3, Thomas Marty2,3 and Monica Gotta

Swiss Federal Institute of Technology, Institute of Biochemistry, Schafmattstrasse 18, ETHHönggerberg HPM G16, 8093 Zürich, Switzerland

Footnotes:

1 Current address: Institut de recherche en immunologie et cancérologieUniversité de Montréal, Pav. Marcelle-CoutuC.P. 6128, Succursale Centre-villeMontréal, QC H3C 3J7Canada

2 Current address: Swiss Contact Office for Research and Higher Education (SwissCore)Rue du Trône 98B-1050 BruxellesBelgium

3 These authors contributed equally to this work.

Genetics: Published Articles Ahead of Print, published on July 2, 2006 as 10.1534/genetics.106.060517


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Running title: Novel polarity genes in C. elegans

Keywords: Cell polarity; Asymmetric cell division; PAR proteins; RNA interference; C. elegans

embryogenesis

Corresponding author:

Monica Gotta

Swiss Federal Institute of Technology

Institute of Biochemistry

Schafmattstrasse 18, ETH Hönggerberg HPM G16

8093 Zürich, Switzerland

Tel: +41 44 633 6575

Fax: +41 44 632 1298

Email: [email protected]


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ABSTRACT

The PAR proteins play an essential role in establishing and maintaining cell polarity.

While their function is conserved across species, little is known about their regulators and

effectors. Here we report the identification of 13 potential components of the C. elegans PAR

polarity pathway, identified in an RNAi-based, systematic screen to find suppressors of par-

2(it5ts) lethality. Most of these genes are conserved in other species. Phenotypic analysis of

double mutant animals revealed that some of the suppressors can suppress lethality associated

with the strong loss-of-function allele par-2(lw32), indicating that they might impinge on the

PAR pathway independently of the PAR-2 protein. One of these is the gene nos-3, which

encodes a homologue of Drosophila Nanos. We find that nos-3 suppresses most of the

phenotypes associated with loss of par-2 function, including early cell division defects and

maternal effect sterility. Strikingly, while PAR-1 activity was essential in nos-3; par-2 double

mutants, its asymmetric localization at the posterior cortex was not restored suggesting that the

function of PAR-1 is independent of its cortical localization. Taken together, our results identify

conserved components that regulate PAR protein function and also suggest a role for NOS-3 in

PAR protein-dependent cell polarity.

INTRODUCTION

Asymmetric cell division is a key process for the generation of cell diversity during the

development of metazoans (reviewed in (BETSCHINGER and KNOBLICH 2004; ROEGIERS and JAN

2004). Stem cells, for instance, divide asymmetrically to generate two daughter cells with

distinct fates: one daughter committed to differentiation, and another that retains the totipotent

characteristics of the mother. Asymmetric cell division relies on the partitioning of cell fate


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determinants and orientation of the mitotic spindle prior to cytokinesis to generate cells of

different fates. Accordingly, polarity cues in these cells must be interpreted accurately to ensure

proper partitioning of these determinants at cytokinesis. Although establishment and

maintenance of cell polarity is crucial for asymmetric cell division in all organisms, the nature of

the molecular interactions leading to these processes remains elusive.

In recent years, work in several systems demonstrated that the conserved PAR proteins

play a fundamental role in establishing and maintaining polarity in many asymmetrically

dividing cell types (reviewed in (ETIENNE-MANNEVILLE and HALL 2003; NANCE 2005). In

Drosophila, PAR proteins are essential for the asymmetric division of neuroblasts and sensory

organ precursor cells (BETSCHINGER and KNOBLICH 2004). Mutations in any gene encoding a

PAR protein result in loss of cell polarity and failure to divide asymmetrically. PAR proteins are

also required to establish and maintain polarity in the developing oocyte (COX et al. 2001;

HUYNH et al. 2001). In mammalian cells, PAR proteins were shown to be localized at tight

junctions and participate in epithelial cell polarity (HURD et al. 2003; LIN et al. 2000; QIU et al.

2000). They are also required for the correct migration of astrocytes after wound healing and for

specifying axonal fate in rat hippocampal neurons (ETIENNE-MANNEVILLE and HALL 2001; SHI

et al. 2003).

The C. elegans embryo is an excellent model system to study polarity and spindle

positioning (KEMPHUES and STROME 1997). The first division of the embryo is asymmetric

resulting in two cells that are different in size and fate. The seven PAR proteins (PAR-1 to -6 and

PKC-3) are found at the cortex of the zygote and are responsible to specify the antero-posterior

axis of polarity (KEMPHUES and STROME 1997; TABUSE et al. 1998) (CUENCA et al. 2003).

Embryos mutant for any par gene do not properly establish polarity, undergo symmetric cell


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divisions and fail to hatch. The PAR-3, PAR-6 and PKC-3 (hereafter referred to as anterior

PARs) proteins are localized at the anterior cortex of the embryo (ETEMAD-MOGHADAM et al.

1995; HUNG and KEMPHUES 1999; TABUSE et al. 1998), while PAR-1 and PAR-2 are restricted

to the posterior cortex (BOYD et al. 1996; GUO and KEMPHUES 1995). This localization along the

antero-posterior axis is mutually exclusive, as members of the anterior and posterior groups show

little overlap in their respective localization and the PAR-2 and PAR-3 proteins exclude each

other from their respective cortices (BOYD et al. 1996; CUENCA et al. 2003; ET E M A D-

MOGHADAM et al. 1995). Therefore, PAR proteins define anterior and posterior cortical domains

in the zygote and specify cell polarity and asymmetric cell divisions in the early C. elegans

embryo.

While PAR proteins are conserved across species, little is known about the precise

molecular mechanisms by which they contribute to the establishment and maintenance of cell

polarity. This lack of understanding is partly due to the fact that only a limited number of PAR

protein activators and effectors that function during these processes have been identified thus far

(MACARA 2004). Interestingly, previous work demonstrated that while removing both copies of

par-2 leads to the mislocalization of the anterior PARs to the posterior of the embryo, and thus to

defects in polarity and embryonic lethality, reducing by half the amounts of PAR-6 in a par-2

mutant background is sufficient to restore viability (WATTS et al. 1996) (Supplemental Figure 1).

Similarly, RNAi disruption of cdc-42, a regulator of the anterior PAR activity, in a par-2 mutant

also restores viability (GOTTA et al. 2001). These results indicate that genes that are required to

regulate the levels, the localization and/or the activity of anterior PAR components can be

identified based on their ability to restore viability in a par-2 mutant background. We therefore

sought to find additional genes that are required for polarity by screening for suppressors of the


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lethality caused by disruption of the polarity gene par-2. Using a systematic, genome-wide,

RNAi-based screening approach we identified several potential components of the PAR polarity

pathway in C. elegans. Many of these components are present in other species, suggesting that

their role in cell polarity might also be conserved. To gain further insights into possible

suppression mechanism(s), we studied in more detail the phenotype of embryos mutant for the

suppressor gene nos-3, which encodes one of the three C. elegans homologues of the protein

Nanos (KRAEMER et al. 1999; SUBRAMANIAM and SEYDOUX 1999). In Drosophila, Nanos has

been shown to control embryonic polarity by repressing translation of the hunchback mRNA in

the posterior of the embryo (HULSKAMP et al. 1989; IRISH et al. 1989). We find that nos-3 can

suppress a strong loss-of-function allele of par-2 indicating that it could function independently

of the PAR-2 protein. Surprisingly, PAR-1 was localized in the cytoplasm of these embryos,

suggesting that the essential function of PAR-1 is independent of its asymmetric cortical

localization. Taken together, these results indicate that the par-2 suppressor screen revealed

genes involved in regulating PAR protein function and further demonstrate that nos-3

participates in PAR protein-dependent cell polarity in C. elegans embryos.

MATERIALS AND METHODS

Strains

All strains were maintained as described by Brenner (BRENNER 1974) and were grown at

15ºC unless otherwise stated. The alleles used in this study were LGI: rpn-10(tm1180), rpn-

10(tm1349); LGII: nos-3(q650), fbf-1(ok91); LGIII: par-2(lw32), par-2(it5ts); LGX: spat-

3(gk22). The wild-type strain was the N2 (Bristol) strain.


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The generation of nos-3(q650); par-2(it5ts) double mutants was done as follows. Males

mutant for nos-3(q650) were mated with par-2(it5ts) mutant hermaphrodites at permissive

temperature and five animals of the resulting F1 cross progeny were singled. These F1 animals

were allowed to lay eggs for 24h at permissive temperature before being lysed and the presence

of the nos-3(q650) allele was confirmed by PCR analysis, using pairs of primers specific for the

nos-3 locus. Twenty F2 animals were then singled and allowed to lay eggs for 24h at permissive

temperature before being transferred to new plates and shifted to restrictive temperature.

Animals producing dead embryos at restrictive temperature were considered homozygote for

par-2(it5ts). These animals were lysed and genotyped by PCR analysis to identify those bearing

the nos-3(q650) allele. F3 animals that had hatched at permissive temperature were then singled,

allowed to lay eggs and genotyped to find those homozygote for nos-3(q650). The presence of

par-2(it5ts) was further confirmed by non-complementation analysis, by mating par-2(it5ts)

males with nos-3(q650); par-2(it5ts) hermaphrodites at permissive temperature, shifting the F1

cross progeny at restrictive temperature and scoring for absence of hatched larvae. Double

mutant strains with other par-2 suppressors were done by the same approach and genotyping was

done by PCR using pairs of primers specific for each locus.

The generation of nos-3(q650); par-2(lw32) double mutants was done as follows. Males

mutant for nos-3(q650) were mated with unc-45(e286) par-2(lw32)/qC1[dpy-19(e1259) glp-

1(q339)] mutant hermaphrodites. Males resulting from the F1 cross progeny were then mated

with their F1 sister hermaphrodites and 30 F1 wild-type animals resulting from this second cross

were singled to individual plates. The animals giving rise to wild-type, Unc and Dpy animals

were lysed and genotyped by PCR analysis to identify those homozygote for the nos-3(q650)

allele. The presence of the par-2(lw32) was confirmed by sequence analysis of the par-2 locus.


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Double mutant strains with other par-2 suppressors were done by the same approach and

genotyping was done by PCR using pairs of primers specific for each locus.

Genome-wide RNAi screen

The screen for par-2 suppressors was done using the following procedure. par-2(it5ts)

animals were grown in large quantities on solid media at 15ºC and bleached to collect embryos.

These embryos were incubated at 15ºC with rocking in M9 buffer to allow hatching of L1 larvae.

Assays were done in plates containing 96 well. RNAi clones from the available collection

(KAMATH et al. 2003) were seeded in individual wells and grown overnight at 37ºC in LB

medium containing 100 µg/ml of carbenicillin. Each well in 96-well plates was then filled with

75 µl of 3xNGM and 2 µl of overnight bacterial culture was added. The plates were incubated at

37ºC for 2.5 hours without shaking. Twenty-five µl of 3xNGM containing 24 mM IPTG was

then added to each well (6 mM IPTG final) and the plates were incubated at 37ºC for 5 hours

without shaking. Five to ten L1 worms were then added to each well (15 µl, from a suspension

containing ~8 worms per 15 µl of M9 buffer). The plates were incubated for 2.5 days at the

permissive temperature of 15ºC without agitation, and were then shifted to the semi-restrictive

temperature of 22ºC for 3-4 days, until food was depleted and F1 progeny had hatched. The

suppression level was estimated by visual inspection under a dissecting scope, and the presence

of more swimming L1 larvae compared to the control was scored as positive. Liquid handling

was performed using a Beckman Biomek FX robotic system equipped with a 96-channel

pipetting head.

Each RNAi clone was scored in duplicate in two independent screens. The first screen

identified 1847 RNAi clones showing suppression while the second screen identified 952 RNAi


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clones. Of these RNAi clones, 136 were identified in both screens and were kept for further

analysis. The high number of false positives in these screens might reflect the fact that these

were carried out at semi-permissive temperature for embryonic lethality, where minute

fluctuations of temperature might have important consequences on the physiology of the

animals. RNAi to these 136 clones was then performed on par-2(it5ts) animals in duplicate on

solid assays as previously described (KAMATH et al. 2003), and 18 clones were found to have a

higher proportion of hatching progeny compared with the control at 20ºC (Supplemental Figure 1

and Supplemental table 1). RNAi to these 18 clones was then repeated 8 additional times at 20ºC

and the 8 clones with a percentage of hatching progeny significantly higher than the controls

after counting were considered as suppressors of par-2(it5ts) (Supplemental table 2). The

molecular identity of these clones was confirmed by DNA sequencing.

Suppression assays

For mutant animals, L4 animals from each strain were shifted to semi-restrictive (20ºC)

or restrictive (25ºC) temperature for 24h. Nine worms were transferred to three plates (three

worms per plate) and allowed to lay eggs at the same temperature for 16h before being removed.

The viability of the progeny was determined after incubating another 24h at the same

temperature, dividing the number of unhatched embryos by the total number of progeny.

To assess the level of par-2(RNAi) suppression, mutant animals were injected with

dsRNA to par-2 or wild-type animals were co-injected with dsRNA to both par-2 and a given

suppressor as described previously, and worms were subsequently incubated for 16-24h at 20ºC.

Twelve injected animals were then transferred to three plates (four worms per plate) and allowed

to lay eggs for 10h. The viability of the progeny was assessed 24h after removing the worms.


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The following alleles were tested and did not suppress par-2(RNAi): LGI: cey-2(ok902), rpn-

10(tm1180), rpn-10(tm1349); LGII: lat-2(tm463), lat-2(ok301), nos-1(ok250), fbf-1(ok91);

LGIII: dgk-3(gk110); LGIV: fat-2(ok873); LGV: nhr-84(ok792), egl-3(ok979); LGX: ceh-

18(mg57), sad-1(ky289), hen-1(tm501), zig-3(tm924).

To test for the involvement of fbf-1 and fbf-2 in par-2 suppression, par-2(it5ts) mutant or

fbf-1(ok91); par-2(it5ts) double mutant L4 animals were placed on plates containing bacteria

expressing dsRNAs for either fbf-1 or fbf-2 and incubated at 20ºC for 48h. Nine worms were

transferred to three plates (three worms per plate) containing the appropriate dsRNA-expressing

bacteria and allowed to lay eggs at the same temperature for 12h before being removed. The

viability of the progeny was determined as described above.

To assay for sterility, L4 animals from each strain were shifted to permissive (15ºC) or

restrictive (25ºC) temperature and allowed to develop until some eggs had been laid. These eggs

were allowed to hatch and develop at their respective temperature until animals reached

adulthood. Sterility was then determined by the presence or absence of embryos in the uterus of

these animals, dividing the number of animals without progeny by the total number of animals.

Microscopy

For the visualization of early embryonic development in live specimens, embryos were

obtained by cutting open gravid hermaphrodites using two 25-gauge needles. Embryos were

handled individually and mounted on a coverslip coated with 1% poly-L-lysine in 20 µl of egg

buffer (EDGAR 1995). The coverslip was placed on a 3% agarose pad and the edge was sealed

with petroleum jelly. Time-lapse images were acquired by an Orca ER Hamamatsu 16-bit cooled

CCD camera (Hamamatsu Photonics, Bridgewater, NJ) mounted on a Zeiss Axiovert 200


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microscope (Carl Zeiss AG, Germany), and the acquisition system was controlled by Openlab

software (Improvision Ltd, Coventry, England). Images were acquired at 10 sec intervals using

a Plan Apochromat 63X/1.4 NA objective.

For immunofluorescence analysis, embryos were fixed in methanol and stained according

to standard procedures. The following primary antibodies were used: rabbit anti-PAR-1

(GONCZY et al. 2001) (1/1000), rabbit anti-PAR-2 (N-terminal antibody generated as described

(BOYD et al. 1996), 1/20), rabbit anti-PAR-3 (generated as described (ETEMAD-MOGHADAM et

al. 1995), 1/100), rabbit anti-PAR-6 (generated as described (HUNG and KEMPHUES 1999), 1/50),

mouse OIC1D4 (Developmental Studies Hybridoma Bank, University of Iowa, USA, 1/1) for P

granule staining. Secondary antibodies were Cy3-coupled goat-anti-rabbit (Jackson

Immunoresearch, 1/100) and Alexa488-coupled goat-anti-mouse (Molecular Probes, 1/500).

Images were acquired using a Leica SP2 confocal microscope.

RESULTS

A screen for suppressors of par-2(it5ts) uncovers 11 loci

Previous reports have demonstrated that the lethal phenotype of par-2 can be suppressed

by reducing the levels of either par-6 or cdc-42 (GOTTA et al. 2001; WATTS et al. 1996)

(Supplemental Figure 1). We first tested whether this effect is specific to these genes or can be

obtained by reducing the levels of any of the anterior PAR proteins. We found that disruption by

RNA interference (RNAi; (FIRE et al. 1998)) of either par-3, par-6, pkc-3 or cdc-42 can

effectively suppress the lethal phenotype of the temperature sensitive allele par-2(it5ts) at a

semi-restrictive temperature (Table 1). This indicates that genes encoding proteins that regulate


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anterior PARs function can be identified based on their ability to suppress par-2(it5ts) embryonic

lethality.

We therefore devised a method, based on RNAi, to screen for genes that suppress the

lethal phenotype of par-2(it5ts) (see the Materials and Methods section). We used this method to

assay whether individual bacterial clones from an available collection (KAMATH et al. 2003),

each expressing a unique dsRNA molecule, can restore viability to the progeny of par-2(it5ts)

animals at a semi-restrictive temperature. Using an RNAi assay in liquid, we tested 16,458

bacterial clones in two independent screens and found 136 clones that qualitatively showed

partial suppression of par-2(it5ts) embryonic lethality in both assays (Supplemental Figure 1 and

Supplemental Table 1). We then quantitatively tested each of the 136 clones on solid assay and

found that embryonic viability following disruption by RNAi of 8 of them was significantly

higher than the viability of par-2(it5ts) embryos when tested multiple times at a semi-restrictive

temperature (Supplemental Tables 1 and 2). The description of these 8 suppressors of par-

2(it5ts) embryonic lethality can be found in Table 1. We collectively refer to the genes identified

as spat genes (suppressor of par-two genes).

As an independent assay, we tested whether available alleles for the genes disrupted by

the 136 RNAi clones identified in the primary screen could suppress the lethal phenotype of par-

2 when the par-2 gene itself is disrupted by RNAi. Among the available strains mutant for the

136 clones identified, we found that lethality resulting from par-2(RNAi) was efficiently

suppressed by nos-3(q650) and by spat-3(gk22) (Supplemental Table 3). Interestingly, while

nos-3 was recovered in the primary screen, we found that nos-3(RNAi) did not significantly

suppress par-2(it5ts) lethality in subsequent assays (Table 1). This suggests that our solid assay

for RNAi was not exhaustive and that other suppressors of par-2(it5ts) lethality might be present


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among the RNAi clones that were negative in the subsequent validation steps (see the Materials

and Methods section).

Finally, we scored embryonic viability in animals mutant for both par-2(it5ts) and either

nos-3(q650) or spat-3(gk22). At semi-restrictive temperature, the progeny of all of the double

mutant strains has a significantly increased embryonic viability compared to par-2(it5ts) (Table

2). Taken together, these results suggest that the spat genes that we identified are bona fide

suppressors of par-2.

Sequence analysis of the spat genes suggests that they are quite diverse in their function.

The most common domain identified consists of zinc-binding motifs, which are found in 3 of our

suppressor proteins (NOS-3, ZTF-1 and SPAT-3). Two of these proteins (NOS-3 and SPAT-3)

also contain Q/N-rich domains, which have been found in prions and implicated in

neurodegenerative disorders (FORMAN et al. 2004; MICHELITSCH and WEISSMAN 2000). We also

identified RNAi clone Y43E12A.1, which is predicted to disrupt the function of the two cyclin B

homologues cyb-2.1 and cyb-2.2 (together referred to as cyb-2). Since these two genes are >95%

identical at the nucleic acid level, we could not assess whether RNAi disruption of only one of

them is sufficient to suppress par-2(it5ts) embryonic lethality. One of the suppressors, ceh-18,

encodes a homeodomain-containing transcription factor that was previously shown to function

during oocyte maturation (GREENSTEIN et al. 1994; MILLER et al. 2003). Another suppressor, fat-

2, encodes a fatty acid desaturase that is required to maintain normal levels and composition of

polyunsaturated fatty acids in the animal (WATTS and BROWSE 2002). Two of the suppressors

encode proteins that do not have well defined homologues in other species (SPAT-1 and SPAT-

2).


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One of the suppressors identified consists of the gene encoding the non-ATPase

proteasome subunit RPN-12. In various systems, RPN-12 has been shown to function in the

regulatory particle of the proteasome and was proposed to share some activities with the subunit

RPN-10 (GLICKMAN and CIECHANOVER 2002; TAKAHASHI et al. 2002; WILKINSON et al. 2000).

In C. elegans, only 3 of the ~30 genes shown so far to encode proteasome subunits (DAVY et al.

2001) do not result in embryonic lethality upon disruption by RNAi: rpn-9, rpn-10 and rpn-12

(KAMATH et al. 2003; SONNICHSEN et al. 2005; TAKAHASHI et al. 2002). While we did not

identify rpn-9 or rpn-10 in the genome-wide screen, subsequent analysis revealed that RNAi to

either of these two genes can also suppress par-2(it5ts) embryonic lethality (Table 1), albeit less

efficiently than RNAi to rpn-12. Furthermore, we found that embryos double mutant for par-

2(it5ts) and either allele of rpn-10 (tm1180 or 1349) are viable at a semi-permissive temperature

(Table 2), indicating that rpn-10 is a suppressor of par-2. This demonstrates that additional

suppressors of par-2(it5ts) can be found by looking for genes that are related in sequence or

function to the identified suppressors.

Taken together, our results describe several suppressors of par-2 lethality which, based

on their protein sequence, are likely to play different roles in polarity.

Some of the par-2 suppressors could function independently of par-2.

The spat genes identified could conceivably suppress the phenotypes associated with a

loss of function of par-2 by at least two distinct but non-exclusive mechanisms: 1) by

downregulating the function of the anterior PARs (as in the case of RNAi to par-3, par-6, pkc-3

or cdc-42), or 2) by upregulating residual PAR-2 function in par-2(it5ts) mutants. To distinguish

between these two possibilities, we first tested whether some of our suppressors can suppress the


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lethal phenotype of par-2(it5ts) at a fully restrictive temperature. Except for par-3 and par-6,

none of the RNAi clones listed in Table 1 could significantly suppress par-2(it5ts) lethality at a

fully restrictive temperature in our assay (data not shown). However, we found that the progeny

of animals double mutant for par-2(it5ts) and either nos-3(q650) or spat-3(gk22) have a much

higher viability at fully restrictive temperature than the progeny of par-2(it5ts) single mutants

(Table 2). On the contrary, viability of the progeny of animals double mutant for par-2(it5ts) and

either allele of rpn-10 (tm1180 or tm1349) is only slightly higher than that of par-2(it5ts) in the

same conditions (Table 2). Furthermore the lethality of par-2(RNAi) is not suppressed in animals

mutant for either allele of rpn-10 (Supplemental Table 3). This suggests that nos-3 and spat-3

function independently of PAR-2 and that rpn-10 requires some residual PAR-2 activity to

suppress the embryonic lethal phenotype of par-2.

To assess whether nos-3 and spat-3 could exert their function independently of PAR-2,

we tested whether nos-3 or spat-3 can suppress the embryonic lethality associated with par-

2(lw32). The lw32 allele contains a stop codon in the middle of the par-2 coding sequence and

was shown to behave as a strong loss-of-function (BOYD et al. 1996; LEVITAN et al. 1994). We

found that mutations in both nos-3 and spat-3 can suppress par-2(lw32): the viability of embryos

double mutant for par-2(lw32) and either nos-3(q650) or spat-3(gk22) was much higher

compared with par-2(lw32) single mutant embryos (Table 2). Furthermore, the viability of nos-

3(q650); par-2(it5ts); par-2(RNAi) embryos at restrictive temperature was also increased

compared to par-2(RNAi) (Supplemental Table 3). Taken together, these results suggest that

some of the spat genes, such as nos-3 and spat-3, could suppress par-2 embryonic lethality

independently of PAR-2 activity, possibly by modulating the function of the anterior PARs.


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Co-disruption of both fbf-1 and fbf-2 suppresses par-2 lethality

The gene nos-3 encodes a C. elegans homologue of the Drosophila protein Nanos. NOS-

3 functions in the germline and is also found associated with P granules in the early embryo

(KRAEMER et al. 1999; SUBRAMANIAM and SEYDOUX 1999). There are two other paralogs of nos-

3 in C. elegans, nos-1 and nos-2, and all three genes have been shown to have redundant

functions in the germline (KRAEMER et al. 1999; SUBRAMANIAM and SEYDOUX 1999). We

therefore tested whether any of these Nanos orthologs can also suppress par-2(it5ts) lethality.

Contrary to nos-3, we found that individual disruption of nos-1 or nos-2 by RNAi does not

suppress par-2(it5ts) lethality (data not shown). Furthermore, co-disruption of nos-1 or nos-2 by

RNAi in nos-3(q650); par-2(it5ts) animals did not result in any increase in embryonic viability

(data not shown). These results suggest that, in contrast to nos-3, nos-1 and nos-2 do not

modulate PAR protein function.

In Drosophila, Nanos functions as a negative regulator of translation for various mRNAs

in conjunction with the ribonucleoprotein Pumilio (reviewed in (WICKENS et al. 2002; WILHELM

and SMIBERT 2005). There are eleven Pumilio homologues predicted in the C. elegans genome

(WICKENS et al. 2002). We found that RNAi disruption of none of these genes individually can

suppress par-2(it5ts) lethality (Table 3 and data not shown). Furthermore, embryos double

mutant for par-2(it5ts) and fbf-1(ok91), a presumptive null allele of the pumilio homologue fbf-1

(CRITTENDEN et al. 2002), also largely fail to hatch (Table 3), indicating that disruption of fbf-1

alone is not sufficient to suppress par-2 lethality. However, fbf-1 has been shown to function

redundantly with fbf-2 in the C. elegans germline (CRITTENDEN et al. 2002). These two genes are

required, along with nos-3, to regulate the synthesis of the sex determining FEM-3 protein and

the translational repressor GLD-1 protein (HANSEN et al. 2004; KRAEMER et al. 1999; ZHANG et


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al. 1997). We therefore tested whether co-disruption of fbf-1 and fbf-2 can suppress par-2(it5ts)

lethality. We found that the viability of fbf-1(ok91); par-2(it5ts); fbf-2(RNAi) triply disrupted

embryos is significantly higher than that of any double disruption combination at semi-restrictive

temperature (Table 3). This indicates that FBF-1 and FBF-2 can both modulate the function of

the C. elegans PAR protein pathway.

nos-3 suppresses the embryonic phenotypes associated with disruption of par-2

To further understand the mechanism of par-2 suppression, we characterized in more

detail the par-2 phenotypes, besides embryonic lethality, that are suppressed by nos-3. We first

monitored early patterns of cell division by generating time-lapse images of early embryonic

development and compared the phenotype of nos-3; par-2 double mutant embryos to that of

wild-type, nos-3 and par-2 mutant embryos (Figure 1). In nos-3 mutants, early embryonic

development proceeds as in wild type and most embryos develop into adults at all temperatures

(Figure 1 and Tables 2 and 4). In 1-cell stage par-2 mutant embryos, the normal posterior

displacement of the first mitotic spindle does not occur, resulting in two cells of equal size after

cytokinesis (KEMPHUES and STROME 1997). Furthermore, while blastomeres of wild-type 2-cell

stage embryos have perpendicular spindle orientations and asynchronous cytokineses, the two

spindles align parallel to each other and the division of both blastomeres is synchronous in par-2

mutant embryos (KEMPHUES and STROME 1997). We found that all of these par-2 phenotypes are

suppressed in embryos double mutant for nos-3 and par-2 (Figure 1). This indicates that nos-3

can efficiently suppress the early cell division phenotypes of par-2 mutants, and that the NOS-3

protein can regulate some early embryonic development processes.


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We then used immunofluorescence to monitor the localization of PAR proteins in early

embryos. PAR-3 and PAR-6 localize to the anterior cortex in wild-type embryos (ETEMAD-

MOGHADAM et al. 1995; HUNG and KEMPHUES 1999). In par-2 mutants, polarity cannot be

maintained and the anterior PARs expand towards the posterior (CUENCA et al. 2003). We

observed that PAR-3 and PAR-6 proteins are restricted to the anterior cortex of most nos-

3(q650); par-2(it5ts) double mutant embryos (Figure 2). Therefore, disruption of nos-3 restores

the asymmetric localization pattern of anterior PARs in embryos mutant for par-2.

The posterior localization of PAR-2 was not restored by disrupting NOS-3 function: nos-

3(q650); par-2(it5ts) double mutants showed no detectable PAR-2 staining at the posterior

embryonic cortex (Figure 2). This is consistent with the notion that nos-3 can suppress par-2

lethality independently of PAR-2 function. Taken together, these results indicate that nos-

3(q650) can suppress most of the early phenotypes associated with loss of PAR-2 function.

PAR-1 is undetectable at the cortex of nos-3; par-2 mutant embryos

A surprising result was obtained when we monitored the localization of the protein PAR-

1. PAR-1 is a Ser/Thr kinase that is asymmetrically localized at the posterior cortex of wild-type

1-cell embryos (GUO and KEMPHUES 1995). Upon disruption of par-2, PAR-1 cortical

localization is lost and PAR-1 is found in the cytoplasm (BOYD et al. 1996). Interestingly, we

observed that PAR-1 was undetectable at the cortex of 20 out of 22 nos-3(q650); par-2(it5ts)

double mutant embryos at restrictive temperature (Figure 2). A very weak posterior cortical

localization of PAR-1 was observed in 2 out of 22 embryos. Because 54% of nos-3(q650); par-

2(it5ts) double mutant embryos hatch at restrictive temperature (Table 2), this suggests that the

asymmetric localization of PAR-1 at the posterior cortex is not essential in these double mutants.


19

PAR-1 has been shown to be important to stabilize P granules in the early embryo

(CHEEKS et al. 2004). P granules consist of large ribonucleoprotein complexes that localize to the

posterior end of the embryo and that have been proposed to play a role in germline specification

(KAWASAKI et al. 2004; KEMPHUES and STROME 1997). In par-2 mutant embryos, in which

PAR-1 is cytoplasmic, P granules localization is largely normal in one- and two-cell embryos,

but becomes abnormal in four-cell stage embryos (BOYD et al. 1996). Interestingly, we found

that P granules are correctly localized in 12 out of 18 nos-3(q650); par-2(it5ts) double mutant, 4-

cell stage embryos (Figure 3). Since P granules are required for germline development, we then

monitored maternal effect sterility in single and double mutant animals at both permissive and

restrictive temperatures for par-2(it5ts). As shown in Figure 3, nos-3(q650) can efficiently

suppress the maternal effect sterility of par-2(it5ts) animals at permissive and restrictive

temperature. This suggests that the P granules that are localized at the posterior of nos-3(q650);

par-2(it5ts) embryos are functional, despite the observation that PAR-1 is not detectably

enriched at the posterior cortex in these double mutant embryos at the restrictive temperature.

One possibility was that PAR-1 was not functional in these embryos and that its function

was compensated by a secondary, redundant activity. To test this, we disrupted par-1 by RNAi in

embryos double mutant for nos-3(q650); par-2(it5ts) and found that none of these triply

disrupted embryos were viable at restrictive temperature (0% viability, n=200 embryos counted

in 3 assays ). This indicates that PAR-1 is active in nos-3(q650); par-2(it5ts) double mutants and

that posterior cortical enrichment of PAR-1 is not required for its essential function(s).

DISCUSSION


20

In this work, we described a screen for suppressors of par-2(it5ts) lethality in which we

uncovered 13 genes that modulate the function of the PAR polarity pathway. Of these, the three

suppressors that we characterized in more details fall into distinct classes: those that require

residual PAR-2 activity to suppress par-2 lethality (rpn-10) and those that can suppress a strong

loss-of-function allele of par-2 and could therefore exert their activity independently of PAR-2,

perhaps by modulating the function of the anterior PARs (nos-3 and spat-3). We also find that

while nos-3 can suppress most of the early embryonic phenotypes of par-2(it5ts), it does not

restore cortical asymmetry of PAR-1. PAR-1 is active in these embryos suggesting that its

essential function is independent of its asymmetric enrichment at the posterior cortex. Taken

together, these results identify several putative regulators of PAR protein function and further

implicate NOS-3 in PAR protein-dependent cell polarity.

Some of the suppressors that we identified could function upstream of anterior PAR

signaling. Upstream components could include genes involved in regulating the levels of the

anterior PARs or in the activation or the anchoring of anterior PARs at the cell cortex. Both nos-

3 and spat-3 could fall in this class: NOS-3 is a translational regulator and SPAT-3 contains a

RING finger-domain, which have been implicated in ubiquitin-dependent degradation in other

systems (PETROSKI and DESHAIES 2005). Since both suppressors function independently of par-

2, they might be involved in regulating protein levels of components of the anterior PARs. Some

other suppressors could also function downstream of PAR proteins. For example, disruption of a

gene required to transduce the signal of the anterior PARs could result in weaker signaling, and

thus suppression of lethality. It is unclear at the moment whether any of the candidates that we

identified fall in this class. Such candidates could conceivably be potential targets for

phosphorylation by PKC-3, and might further cooperate with each other to suppress the various


21

phenotypes associated with disruption of par-2. It will be interesting in the future to determine

the functional and biochemical relationships between the PAR proteins and the suppressors

identified in this work.

Some of the suppressors could also function via par-2 itself. For instance, our results

demonstrated that rpn-10 can suppress par-2(it5ts) lethality in double mutant animals at a semi-

restrictive temperature, indicating that rpn-10 is a suppressor of par-2. However, embryonic

lethality in double mutants was not suppressed at a fully restrictive temperature, or when par-2

dsRNA was injected in rpn-10 mutant animals. This suggests that rpn-10 can only suppress par-

2 lethality in embryos that have residual PAR-2 levels or activity. RPN-10 is a subunit of the 26S

proteasome and could directly regulate the levels of PAR-2 protein. PAR-2 contains a RING

finger domain, which has often been shown to function in E3 ubiquitin ligase complexes

(PETROSKI and DESHAIES 2005). Interestingly, this RING-finger domain was recently proposed

to function in regulating PAR-2 protein levels in the embryo (HAO et al. 2006). However, while

the levels of PAR-2 are decreased in par-2(it5ts) embryos, we could not observe a detectable

increase in PAR-2 levels in either rpn-10 mutants or rpn-10; par-2(it5ts) double mutants (data

not shown). It is possible that an undetectable increase in PAR-2 levels is sufficient to suppress

lethality. Alternatively, RPN-10 may regulate the levels of an upstream or downstream

component of the PAR-2 pathway that has yet to be identified.

We find that disruption of nos-3 or fbf-1/fbf-2 suppresses par-2 lethality. Nanos

homologues, together with Pumilio homologues, have been best characterized for their role in

repressing mRNA translation (WICKENS et al. 2002; WILHELM and SMIBERT 2005). In C. elegans

for instance, nos-3, fbf-1 and fbf-2 were shown to repress the translation of the fem-3 mRNA at

the sperm-to-oocyte transition during gamete production (KRAEMER et al. 1999; ZHANG et al.


22

1997). These genes also participate in the regulation of GLD-1 function during meiotic

progression (HANSEN et al. 2004). While all three C. elegans orthologs of Drosophila Nanos

have redundant roles in the germline, we find that nos-1 or nos-2 do not seem to have a role in

cell polarity. The protein NOS-3 contains an N-terminal Q/N-rich domain that is not present in

the other two C. elegans Nanos proteins. It is possible that this domain confers different

functions to NOS-3, perhaps allowing it to modulate the polarity pathway. Many proteins in C.

elegans contain a Q/N-rich domain and preliminary analysis indicated that mutations in many of

these are unable to suppress par-2(RNAi) lethality. This indicates that proteins bearing this

domain are not systematically playing a role in cell polarity. Nanos and Pumilio were previously

shown to repress the translation of Cyclin B mRNA in both Drosophila and Xenopus

(NAKAHATA et al. 2003; SONODA and WHARTON 2001). Interestingly, we found that disruption

of cyb-2 can also suppress par-2 lethality. However, given that both nos-3 and cyb-2 suppress

par-2 lethality, it is unlikely that NOS-3 acts as a translational repressor of cyb-2 in this pathway.

Strikingly, we observed that PAR-1 enrichment at the posterior cortex is not restored in

nos-3(q650); par-2(it5ts) mutant embryos at restrictive temperature. Since the anterior

localization of the anterior PARs is normal in these embryos, this indicates that PAR-2 is

required to recruit PAR-1 at high levels at the cortex. A high proportion of nos-3(q650); par-

2(it5ts) mutant embryos hatch and P granules localization is largely normal in these embryos.

The activity of PAR-1 is required in these double mutants because disrupting PAR-1 function

results in embryonic lethality. This indicates that PAR-1 can exert its functions independently of

its asymmetric localization at the posterior cortex and that PAR-2 is not required for PAR-1

activity. A possible explanation is that cytoplasmic PAR-1 activity could be asymmetrically

regulated, for instance by the activity of the anterior PARs. In support of this latter hypothesis,


23

previous reports demonstrated that phosphorylation of mammalian PAR-1 by a PKC-3

homologue negatively regulates its activity in vivo. Consistent with our findings, Boyd et al.

have shown that P granules are enriched at the posterior of one-cell par-2 mutant embryos where

PAR-1 is not cortical, also suggesting that PAR-1 can function independently of its cortical

localization (BOYD et al. 1996).

Finally, it is interesting to note that many of the par-2 suppressors that we have identified

have homologues in other species. Since the function of PAR proteins is conserved in many

polarized cell types, it will be of interest to investigate whether some of the suppressors

identified also participate in cell polarity in other organisms. While Nanos has already been

implicated in establishing polarity of the Drosophila embryo, its role in cell polarity had not been

described in other systems. Our results suggest that the conservation of Nanos function in cell

polarity might be more general than previously appreciated.

ACKNOWLEDGEMENTS

We are indebted to Matthias Peter for providing us with access to the robotic equipment

and Jan Riemer for help with northern blot analysis. We are also grateful to Yves Barral, Damien

D’Amours, Patrick Meraldi and Matthias Peter for comments on the manuscript, Ulrike

Margelisch, Astrid Volmer and Anton Lehmann for technical assistance, as well as the Gotta and

Peter labs for helpful advice and stimulating discussions. We also thank the Light Microscopy

Center of the ETH for providing state of the art microscopes and Gabor Csucs for technical help

with image acquisition. We thank Pierre Gönczy, Ken Kemphues, Shohei Mitani (Japanese

National Bioresource Project for C. elegans), the C. elegans Gene Knockout Consortium and the

Caenorhabditis Genetics Center (which is funded by the National Center for Research Resources


24

of the NIH) for strains and reagents. This work was supported by postdoctoral fellowships from

the European Molecular Biology Organization to J.-C. L., the Human Frontier Science Program

to T. M. and the Fondation Pour la Recherche Medicale to A.P., as well as a grant from the Swiss

National Foundation and from Novartis to M. G.

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FIGURE LEGENDS

Figure 1: nos-3(q650) suppresses the early embryonic phenotypes of par-2 mutants. A.

While nos-3(q650) embryos have a wild-type pattern of early development (n=22), such pattern

is rarely observed in par-2(it5ts) (n=24) or par-2(lw32) (n=19) mutant embryos. This phenotype

is suppressed in nos-3(q650); par-2(it5ts) (n=22) and nos-3(q650); par-2(lw32) (n=31) double

mutant embryos. B-I. Images from time-lapse movies of embryos undergoing first (B, D, F, H)

or second (C, E, G, I) division. Phenotypes were scored in wild-type (B, C), nos-3(q650) (D, E),

par-2(it5ts) (F, G) or nos-3(q650); par-2(it5ts) (H, I) embryos at 25°C. In each panel, anterior is

to the left and arrowheads indicate centrosome positions.

Figure 2: PAR-3 and PAR-6, but not PAR-2 and PAR-1, localization is restored in nos-

3(q650); par-2(it5ts) mutants. A-D. In wild-type embryos, PAR-3 (A) and PAR-6 (B) localize

at the anterior cortex while PAR-2 (C) and PAR-1 (D) localize at the posterior cortex. E-H. In

nos-3 mutants, PAR protein localization is as in wild-type: 100% of embryos showed anterior

PAR-3 (E, n=32), anterior PAR-6 (F, n=24), posterior PAR-2 (G, n=20) and posterior PAR-1 (H,

n=27). I-L. In par-2(it5ts) mutants, the localization of PAR-3 (I) and PAR-6 (J) is expanded

towards the posterior pole (in 90% of embryos, n=19 for PAR-3 and n=29 for PAR-6) whereas

PAR-2 (K) and PAR-1 (L) are absent from the cortex (in 100% of embryos, n=15). M-P. In nos-

3(q650); par-2(it5ts) mutants, wild-type localization of PAR-3 (M) and PAR-6 (N) is restored

(in 68% of embryos for PAR-3 (n=22) and 74% of embryos for PAR-6 (n=19)) whereas PAR-2

(O) and PAR-1 (P) are absent from the cortex (in 100% of embryos for PAR-2 (n=20) and 91%

of embryos for PAR-1 (n=22)). All animals were shifted to 25ºC at 24h before dissection and

staining. In all panels, anterior is to the left.


29

Figure 3: Fertility and P granule localization are restored in nos-3(q650); par-2(it5ts)

mutants. A. Proportion of sterile animals determined for the indicated genotype at 15ºC and

25ºC. Maternal effect sterility was scored in all cases except in par-2 mutant animals at 25ºC

because of embryonic lethality. However, escapers were previously shown to have a fully

penetrant sterile phenotype (WATTS et al. 1996). B-E. P granule localization in wild-type (B),

nos-3(q650) (C), par-2(it5ts) (D) and nos-3(q650); par-2(it5ts) (E) four-cell stage embryos. In

every wild-type and nos-3 embryo observed, P granules were restricted to the posterior-most cell

(n=10) whereas they were localized in several cells in all embryos mutant for par-2(it5ts) (n=10).

P granules were restricted to the posterior-most cell in 66% of nos-3(q650); par-2(it5ts) embryos

(n=18). All animals were shifted to 25ºC at 24h before dissection and staining. In all panels,

anterior is to the left.


30

Table 1 Suppressors of par-2(it5ts) lethality1

RNAi clone Gene Embryonic

viability (%)2

Domains & description

Vector Neg.

control

5.5 ± 2.6 -

C32E12.2 Neg.

control

8.4 ± 4.5 -

F54E7.3 par-3 75.1 ± 5.6 PDZ domains, component of the anterior PARs

T26E3.3 par-6 29.2 ± 16.1 PDZ and CRIB domains, component of the anterior PARs

F09E5.1 pkc-3 38.3 ± 19.8 Kinase domain, component of the anterior PARs

R07G3.1 cdc-42 48.2 ± 27.5 GTPase domain, activator of the anterior PARs

Y53C12B.3 nos-3 10.0 ± 6.33 Q/N-rich domain, zf-nanos-type Zn finger domain,

Drosophila Nanos and Xenopus Xcat-2 homologue

F57C2.6 spat-1 15.4 ± 5.5 Conserved in C. briggsae only

F54F2.5 ztf-1 16.3 ± 5.2 C2H2-type Zn fingers

Y48A6B.13 spat-2 32.0 ± 8.8 Conserved in C. briggsae only

Y43E12A.14 cyb-2.1

cyb-2.2

59.3 ± 6.1 Cyclin B homologues

W02A2.1 fat-2 22.7 ± 9.6 Fatty acid desaturase

ZC64.3 ceh-18 20.9 ± 7.0 POU-class homeodomain transcription factor

T13H2.5 spat-3 50.9 ± 12.1 C3HC4-type RING finger domain and Q/N-rich domain

ZK20.5 rpn-12 48.0 ± 18.5 Component of the 26S proteasome non-ATPase regulatory

particle

T06D8.8 rpn-9 22.9 ± 6.6 Component of the 26S proteasome non-ATPase regulatory

particle

B0205.3 rpn-10 25.4 ± 10.3 Component of the 26S proteasome non-ATPase regulatory

particle


31

1 As determined by feeding RNAi clones to animals at the L1 stage. All of these clones were not identified

in the genome-wide screen; see main text and Materials and Methods for details.

2 The value corresponds to the average percentage of embryos that hatched over the total number of

embryos ± standard deviation over 8 assays.

3 Although nos-3 was not found to suppress par-2 in this particular assay, it was included in this table as a

suppressor of par-2 since it suppressed all of the par-2 phenotypes in other assays.

4 Clone Y43E12A.1 might disrupt the function of both cyb-2.1 and cyb-2.2 since these two genes are 95%

identical at the nucleic acid level.


32

Table 2 Mutations in nos-3, spat-3 or rpn-10 can suppress par-2 lethality

Genotype Embryonic

viability at

20ºC (%)1

Embryonic

viability at

25ºC (%)1

Wild type 99.8 ± 0.0 99.3 ± 0.5

par-2(it5ts) 16.9 ± 3.7 0.8 ± 0.8

par-2(lw32) 1.7 ± 1.02 N.D.

nos-3(q650) 99.4 ± 0.3 75.2 ± 3.9

spat-3(gk22) 99.8 ± 0.0 98.7 ± 0.1

rpn-10(tm1180) 99.0 ± 0.1 99.2 ± 0.3

rpn-10(tm1349) 99.0 ± 0.2 97.3 ± 0.8

nos-3(q650); par-2(it5ts) 86.5 ± 2.0 53.6 ± 1.6

nos-3(q650); par-2(lw32) 54.6 ± 4.72 N.D.

par-2(it5ts); spat-3(gk22) 86.6 ± 2.2 22.9 ± 5.9

par-2(lw32); spat-3(gk22) 43.1 ± 6.82 N.D.

rpn-10(tm1180); par-2(it5ts) 62.2 ± 2.9 10.3 ± 1.8

rpn-10(tm1349); par-2(it5ts) 73.5 ± 2.7 8.8 ± 0.8


embryos ± standard error of the mean over 3 independent assays; see Materials and Methods for details.

2 Viability for these particular assays was determined at 15ºC instead of 20ºC because par-2(lw32)

animals also contain the mutation unc-45(e286ts), which confers an egg-laying defective phenotype at

higher temperatures.


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Table 3 Co-disruption of fbf-1 and fbf-2 can suppress par-2(it5ts) lethality1

RNAi

clone

Gene Embryonic viability in

par-2(it5ts) (%)2

Embryonic viability in

fbf-1(ok91); par-2(it5ts) (%)2

Vector Neg.

control

4.6 ± 3.1 12.9 ± 7.3

C32E12.2 Neg.

control

7.3 ± 7.5 19.8 ± 4.9

ZK20.5 rpn-123 50.8 ± 11.5 64.1 ± 8.5

H12I13.a fbf-1 7.5 ± 4.3 30.9 ± 5.84

F21H12.5 fbf-2 5.2 ± 5.2 45.5 ± 14.6

1 As determined by feeding RNAi clones to animals at the L4 stage; see Materials and Methods for

details.


embryos ± standard deviation over 3 assays.

3 RNAi to rpn-12 was used as a positive control in this assay.

4 This fbf-1 RNAi clone might partially disrupts the function of fbf-2 since these two genes are 93%

identical at the nucleic acid level.

Novel polarity genes in C. elegans Labbé et al.

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Table 4 Mutations in nos-3 can suppress the early embryonic phenotypes associated with mutations in

par-2

Genotype

Posterior spindle

displacement at

1st mitosis

Perpendicular

spindle orientation

at 2nd mitosis

Asynchronous

cytokinesis at

2nd mitosis

Number

recorded

Wild type 100% 100% 100% 20

nos-3(q650) 100% 100% 100% 22

par-2(it5ts) 17% 0% 4% 24

par-2(lw32) 16% 5% 16% 19

nos-3(q650); par-2(it5ts) 86% 45% 73% 22

nos-3(q650); par-2(lw32) 48% 39% 45% 31


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a genome-wide screen for suppressors of par-2 uncovers ......2006/07/02 · protein activators and...

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