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First published online 30 May 2007
doi: 10.1242/dev.005074
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1 IGBMC, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP.10142, 67400 Illkirch,
France.
2 MPI for Medical Research, Jahnstr. 29, 69120 Heidelberg, Germany.
3 Institut des Neurosciences Cellulaires et Intégratives, UMR 7168 CNRS,
67084 Strasbourg, France.
Author for correspondence (e-mail:
lmichel{at}igbmc.u-strasbg.fr)
Accepted 11 April 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: ARHGAP20, C. elegans, Rho-kinase, RhoGAP, Epithelial, Morphogenesis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
absence of overt variability in the general shape of individuals from a given
species indicates that these events proceed with great precision and
robustness. Such reproducibility is remarkable because morphogenesis involves
many cells and, within cells, many molecular complexes whose activity must be
accurately coordinated. In particular, morphogenesis involves multiple forces
representing cell adhesion, internal contraction and external traction
(Keller, 2006), which must be
tightly balanced to avoid producing misshapen embryos.
Most morphogenetic processes are orchestrated by small GTPases, which
ultimately induce cytoskeleton remodelling through the activity of various
effectors (Burridge and Wennerberg,
2004; Van Aelst and Symons,
2002). Among Rho GTPase effectors, the Rho-binding kinase (ROCK)
and two of its targets, the myosin regulatory light chain and the
myosin-binding phosphatase regulatory subunit (called MBS or MYPT), play a
crucial role in smooth cell contraction, cytokinesis and various morphogenetic
processes (Franke et al.,
2005; Jacinto and Martin,
2001; Matsumura,
2005; Piekny et al.,
2003; Wissmann et al.,
1999; Wissmann et al.,
1997; Young et al.,
1993). ROCK activates myosin II by phosphorylating either the
myosin regulatory light chain, or the MBS, a negative regulator of myosin II
(Riento and Ridley, 2003).
How this pathway is regulated in vivo remains unclear. Some observations
underscore the necessity to achieve precision in controlling myosin II
activity. During Drosophila dorsal closure, myosin II generates the
forces that allow cells of the leading edge to extend and amnioserosa cells to
constrict (Franke et al.,
2005; Jacinto et al.,
2002; Young et al.,
1993). When myosin II activity is not uniform along the leading
edge, the lateral-epidermal sheets become misaligned at the dorsal midline,
which impairs dorsal closure (Franke et
al., 2005). Likewise, excessive myosin activity during
Drosophila eye morphogenesis causes photoreceptor to move out of the
eye disc epithelium (Lee and Treisman,
2004).
We study elongation of the C. elegans embryo as a paradigm for
tube elongation. During this process, epidermal cells surrounding the embryo
extend along the anterior-posterior axis of the embryo and constrict along its
circumference (Chisholm and Hardin,
2005). Remodelling of circumferentially oriented actin filaments
is thought to drive elongation, because treating embryos with cytochalasin D
blocks elongation (Priess and Hirsh,
1986). Genetic analysis has outlined the central role played by
the ROCK-myosin II pathway in elongation. Mutations affecting the myosin II
heavy chain NMY-1, its regulatory light chain MLC-4, or the ROCK homologue
LET-502, result in hypoelongation with embryos arresting at the 2-fold stage
(twice the length of the eggshell; normal embryos elongate 4-fold)
(Piekny et al., 2003;
Shelton et al., 1999;
Wissmann et al., 1997).
Conversely, mutants of the MBS homologue MEL-11 are characterised by the
presence of a bulge resulting from rupture of the embryo
(Wissmann et al., 1999). This
phenotype presumably results from myosin II hyperactivity, as let-502
and nmy-1 mutations prevent the formation of a bulge, and because
mel-11 and let-502 mutations suppress each other
(Piekny et al., 2003;
Wissmann et al., 1999).
LET-502/ROCK and the myosin II heavy chain have a similar filamentous
distribution, whereas MEL-11/MBS is localised at membranes during elongation,
where it may no longer inhibit the contractile apparatus, and becomes
cytoplasmic at the end of elongation
(Piekny et al., 2003). This
differential localisation, the observation that MEL-11/MBS is cytoplasmic in
let-502 mutants and the genetic interactions between both genes have
suggested that MEL-11/MBS acts at the end of elongation to block any further
myosin II contractility (Piekny et al.,
2003).
Here we characterise a new component of the C. elegans elongation
pathway, RGA-2, as a member of the RhoGAP family (GTPase-activating protein).
Two classes of regulatory proteins influence the state of small GTPases:
guanine exchange factors (GEFs) promote conversion to the GTP-bound active
form that is able to interact with various effectors, whereas GAPs accelerate
the return to the GDP-bound inactive state
(Moon and Zheng, 2003). Using
fast time-lapse analysis, we show that excessive tension builds up in
rga-2 mutant embryos. Using a genetic approach we show that RGA-2
acts as a negative regulator of LET-502/ROCK in a subset of epidermal cells.
Altogether, our data suggest that different epidermal cells have different
roles in elongation and underline the need to moderate actomyosin tension in
dorsal/ventral cells. Moreover, our data provide genetic evidence supporting
the current models of C. elegans embryonic morphogenesis as a seam
cell-driven process.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
following strains were used: NL2099, rrf-3(pk1426); CB698,
vab-10(e698); HR483, mel-11(sb56) unc-4(e120)/mnC1[dpy-10(e128)
unc-52(e444)]; KK332, mel-11(it26) unc-4(e120)
sqt-1(sc13)/mnC1[dpy-10(e128) unc-52(e444)]; HR1184,
+/szT1[lon-2(e678)]; nmy-1(sb115) dpy-8(e130)/szT1. Other alleles
were: let-502(ca201sb54)
(Wissmann et al., 1997);
let-502(sb118ts) (gift from P. Mains, University of Calgary, Calgary,
Canada); nzIs1[hsp::rho-1(G14V) ttx-3::gfp] (LGI)
(McMullan et al., 2006).
let-502(sb118) is a missense mutation in a conserved residue of the
catalytic domain (P. Mains, personal communication). Recombinant
let-502(sb118ts) nzIs1 animals were recovered based on the presence
of the ttx-3::gfp marker. The allele rga-2(hd102) was
obtained by screening a frozen mutagenesis library (which was generated by
H.H. and his colleagues). It was outcrossed six times to N2 and balanced with
hIn1[unc-54(h1040)]. let-502(sb118ts) dpy-5(e61) rga-2(hd102) animals
were recovered as Dpy recombinants from the progeny of let-502(sb118ts)
dpy-5(e61)/rga-2(hd102) heterozygotes, based on temperature sensitivity
(for sb118) and PCR tests (for hd102). The integrated
transgene mcIs46[dlg-1::rfp; unc-119(+)] was obtained by biolistic
transformation using a dlg-1::rfp (gift from J. Hardin, University of
Wisconsin, Madison, WI) plasmid
(Köppen et al., 2001;
Praitis et al., 2001) and was
crossed into rga-2(hd102) or mel-11(it26) backgrounds.
RNA interference and identification of rga-2
We performed an RNA interference (RNAi) screen using the Ahringer-MRC
feeding RNAi library (Kamath et al.,
2003), testing in parallel at 20°C the strains N2 and CB698,
which we serendipitously found to contain a mutation conferring enhanced
sensitivity to RNAi. L3-L4 stage larvae were placed for 36-40 hours on
double-stranded RNA-producing bacteria; lethality was scored 24 hours after
removing adults. RNAi against Y53C10A.4 (clone I-6A17, spanning exons
6-7) induced embryonic lethality in CB698 and NL2099, but not in N2. Since
RNAi by feeding against this gene, which we named rga-2, did not
induce lethality in a six-time outcrossed vab-10(e698) derivative of
CB698, we conclude that rga-2 should act in a process unrelated to
vab-10. For RNAi by injection
(McMahon et al., 2001), we
prepared double-stranded RNA from a PCR-amplified genomic fragment spanning
rga-2 exons 6-8; embryos laid during the first 12 hours after
injection were discarded.
Production of GFP-tagged rga-2 transgenes
The rga-2::gfp fusion constructs F1-F5 (see
Fig. 3; the GFP was C-terminal)
were generated through a PCR-fusion protocol
(Hobert, 2002), using a
5' primer located 2.9 kb upstream of rga-2 exon 1. They
correspond to full-length rga-2 (F1); residues 1-161 (F2), 1-444
(F3), 242-444 (F4) and 437-908 (F5). The plasmid constructs P6-P10 were
obtained by cloning into the GFP-expression vector pPD95.75 (gift from A.
Fire, Stanford University, Stanford, CA) a 3.9 kb fragment starting 2.9 kb
upstream of rga-2 and extending until exon 2, and subsequently
inserting genomic (P6, P7) or cDNA (P8-P10) rga-2 fragments encoding
residues 482-700 (P6), 701-908 (P7), 701-785 (P8), 736-861 (P9) or 822-908
(P10). Deletion constructs were obtained by cloning rga-2 genomic
fragments encoding residues 1-908 (P11), 1-700 (P12) and 1-433 (P13) into
pPD95.75. Tissue-specific expression constructs were generated by cloning the
rga-2 open reading frame cDNA (taken from yk660; gift from
Y. Kohara, National Institute of Genetics, Shizuoka, Japan) downstream of a
1.9 kb or 2.9 kb fragment corresponding to the elt-3
(Gilleard et al., 1999) or
ceh-16 promoter (Cassata et al.,
2005), respectively, leaving 7 bp of the rga-2
5'UTR upstream of its initiation codon. Plasmids encoding a CAAX box- or
SAAX box-modified RGA-2::GFP were obtained as follows: the GFP coding sequence
of plasmid pPD95.75 was modified by a PCR-fusion strategy to insert residues
KPQKKKKS(C/S)NIM downstream of the GFP, and then substituted for that in
construct P13 (see above). Expression was assessed in N2 animals after
injection at 10 ng/µl, along with pRF4 [rol-6(su1006)] as a
selection marker (100 ng/µl) and pBSKII at (80 ng/µl). Rescue was
assessed after injecting plasmids at 1 ng/µl into
rga-2(hd102)/hIn1[unc-54(h1040)] animals (rescued animals did not
segregate Unc progeny and had the mutant rga-2 genotype in PCR assays
using a 5' primer absent from the injected plasmid).
Temperature-shift experiments and interactions with hs::rho-1(G14V)
L4 larvae were picked and put at 20°C or 25.5°C overnight. The next
morning, to roughly synchronise embryos, mothers were allowed to lay eggs for
1-hour time intervals on plates preheated to 20°C or 25.5°C. Wild-type
embryos were collected in parallel. At regular intervals after egg-laying,
embryos were picked at the desired stage under a binocular microscope and
transferred to preheated plates. Unhatched embryos, arrested, dumpyish and
normal larvae were counted the next day and 3 days after transfer.
Interactions with the hs::rho-1(G14V) construct were performed in a
similar way, and embryos then subjected to a 30-minute heat shock at 30°C.
Plates were returned to 25.5°C for assays involving
let-502(sb118ts), or to 20°C for assays involving RNAi against
rga-2. Heat shock was performed either 3 or 4 hours after removing
mothers. Harsher heat shock (45 minutes at 31°C) induced some L1 larval
lethality in the nzIs1 background, but still almost no embryonic
lethality with the characteristic ventral bulge of rga-2 embryos
(data not shown).
Biochemical analysis of RGA-2
Recombinant C. elegans RHO-1, CED-10 and CDC-42 GTPases with a
C-terminal 6xHis tag
(Jantsch-Plunger et al., 2000)
were expressed overnight at 25°C and purified at 4°C using BD-Talon
Resin (Clontech). A fragment encoding the GAP domain of RGA-2 (residues
219-475; the GAP domain itself corresponds to residues 261-429) was cloned
into pGEX4T-1, expressed at 25°C and purified using Glutathione Sepharose
4B (Pharmacia Biotech). Proteins were dialysed overnight at 4°C into 10 mM
Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM MgCl2, 0.1 mM DTT,
concentrated with a Centricon column (Millipore) and quickly frozen at
-80°C. GAP activity assays were performed as described elsewhere
(Jantsch-Plunger et al.,
2000).
Embryo staining
C. elegans embryos carrying the rga-2::gfp F1 transgene
(see above) were stained with bodipy-TRX-phallacidin (Invitrogen, Molecular
Probes) diluted at 1:10 in PBS (Costa et
al., 1997), without prior chitinase digestion. Stacks of 40 images
every 0.25 µm (GFP fluorescence) or 6-10 images every 0.15 µm
(phallacidin staining) were captured using a Leica SP2 AOBS RS confocal
microscope, then projected using the Tcstk software
(McMahon et al., 2001) and
processed with Adobe Photoshop.
Time-lapse videomicroscopy
DIC time-lapse was performed using a Leica DMRXA2 microscope equipped with
a PE120 Peltier heating stage set at 25.5°C by a PE94 controller (Linkam).
Images were captured with a Coolsnap HQ camera (Roper Scientifics) and
analysed using MetaMorph (Universal Imaging). Fluorescent videomicroscopy was
performed in animals carrying the integrated transgene mcIs46 using a
Leica TCS SP5 confocal microscope (using a 100x 1.4 Plan Apochromat HCX
CS objective), controlled by Leica LAS AF imaging software; illumination was
via a 561nm DPSS yellow laser (10 mW). Acquisition was performed in resonant
mode (fast scan), with a line average of four. Embryos were mounted on an
agarose pad in M9 buffer and the coverslip was sealed with paraffin oil.
z-stacks (up to ten planes) through the upper 2-6 µm of embryos
were acquired every 10 seconds. Images were computationally projected using
MetaMorph.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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). To confirm that the aforementioned RNAi phenotype corresponds to loss of rga-2 function, we recovered a deletion, named hd102, removing 831 bp around the first exon (Fig. 1G). rga-2(hd102) is a strong recessive loss-of-function or null allele because its phenotype did not become more severe in trans to the deficiency hDf17 (Table 1A). DIC microscopy (Fig. 1C,D) and time-lapse analyses (Fig. 1J) showed that rga-2(hd102) embryos elongated normally until the 1.8-fold stage (Fig. 1H); then, their head generally failed to constrict, and most of them ruptured ventrally before the 2.3-fold stage, causing a highly penetrant embryonic lethality. For comparison, mel-11(it26) mutants usually ruptured around the 1.6- to 1.7-fold stage (Fig. 1H,K) and let-502 embryos elongated slightly beyond 2-fold but slower than wild type (Fig. 1F,H,L).
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).
Indeed, actin staining in a strain expressing the RGA-2::GFP construct showed
that the two networks coincide (Fig.
2D). In larvae and adults, the circumferential filamentous pattern
did not persist, but expression was seen in a subset of head
(Fig. 2F) and tail (not shown)
socket cells, the vulva (Fig.
2H), more faintly the uterus (not shown), and the rectum
(Fig. 2J).
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|
To assess the biological significance of RGA-2 association with actin
microfilaments, we first examined whether constructs lacking its C-terminal
domain could rescue the lethality of rga-2(hd102) embryos. Such
constructs (see P12 and P13, Fig.
3A) could rescue rga-2(hd102) almost as efficiently as
the full-length rga-2::gfp construct P11 (data not shown). This
observation could mean either that the terminal microfilament-association
domain is dispensable for viability, or, as indicated above, that the second
weaker microfilament-association domain is sufficient for activity. Since the
latter might correspond to the GAP domain, we could not directly remove it
without affecting RGA-2 function. To test whether association with
microfilaments is crucial for function, we inserted the prenylation signal
(CAAX box) of MIG-2 (Portereiko and Mango,
2001) at the C-terminus of the P13 construct to force membrane
binding. Presence of the CAAX box, but not of a control SAAX box
(Hochholdinger et al., 1999),
caused the protein to adopt an irregular peripheral distribution and abolished
the rescuing activity of the P13 construct
(Fig. 3F,G,
Table 2). Thus, association
with microfilaments is probably essential for RGA-2 function.
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; Bossinger et al.,
2001; Costa et al.,
1998; Köppen et al.,
2001; McMahon et al.,
2001), or from a disruption of the epidermal cytoskeleton as
observed in genes affecting microfilament anchoring
(Bosher et al., 2003;
Costa et al., 1998;
Norman and Moerman, 2002).
Alternatively, it could be due to hypercontraction of the actomyosin network
during embryogenesis, as occurs in mel-11 mutants
(Wissmann et al., 1999). We
did not detect any major integrity defect of epidermal junctions nor of the
actin cytoskeleton (see Fig. S2 in the supplementary material), arguing
against the first possibility.
Two sets of experiments showed that rga-2 controls actomyosin
activity. First, to examine the architecture of rga-2(hd102) embryos
when they rupture, we performed fast time-lapse imaging of the adherens
junction marker dlg-1::rfp
(Köppen et al., 2001;
McMahon et al., 2001).
Starting at the 1.9-fold stage of elongation (time point 1500 seconds in
Fig. 4A), deep indentations of
the DLG-1::RFP signal could be seen exclusively in dorsal and ventral cells of
rga-2(hd102) but not wild-type embryos
(Fig. 4C,D). We interpret these
indentations as a manifestation of an extreme actomyosin tension pulling on
the plasma membrane at a position where actin bundles are tethered by a
cadherin-catenin complex. Interestingly, we could partially rescue the
rupturing phenotype of rga-2(hd102) mutants by increasing the DNA
concentration of the dlg-1::rfp construct from 1 to 10 ng/µl in
injection mixes, indicating that more DLG-1 can strengthen junctions against
tension (Table 2). For
comparison, we examined mel-11(it26) embryos
(Fig. 4B), and noticed that
seam cells were often deformed at the 1.5-fold stage: instead of being
rectangular they were W-shaped, indicating that there was an imbalance of
tension between dorsal/ventral and seam cells. In addition, although we
observed fewer indentations than in rga-2(hd102) embryos, they
protruded both within dorsal/ventral and seam cells and were often larger
(Fig. 4C,D). Therefore, we
suggest that mel-11 acts earlier than rga-2 and in all
epidermal cells.
Second, we examined whether rga-2 could act in the ROCK and myosin II pathway defined by let-502, nmy-1 and mel-11. Since rga-2 and mel-11 mutations lead to similar defects, lowering RGA-2 and MEL-11 activities together might result in synergistic effects. Conversely, as rga-2 and let-502 mutations result in seemingly opposite phenotypes, if they act in the same pathway then lowering gene activity for both genes might restore normal elongation and viability. Using RNAi by feeding, which does not induce a phenotype in wild-type animals, we found that RNAi against rga-2 increased the embryonic lethality of mel-11(sb56) and mel-11(it26) mutants to almost 100% (Table 1A). At the cellular level, we observed the sum of defects resulting from loss of each gene separately (Fig. 4B), causing embryos to rupture earlier with more severe head defects. These observations suggest that mel-11 and rga-2 affect a similar process.
To test for genetic interactions between rga-2 and
let-502, we used let-502(sb118ts), a thermosensitive allele
(P. Mains, personal communication) that shows no defect at 20°C, but at
25°C displays the strong elongation defects and L1-stage lethality
characteristic of other let-502 alleles
(Piekny et al., 2000)
(Fig. 1F,
Table 1A). We found that
rga-2 and let-502 partially suppressed each other. For
instance, RNAi against rga-2 by feeding reduced the lethality of
let-502(sb118ts) at 25°C from 95% to 33%, resulting in slightly
shorter animals than normal, probably because elongation was slightly
impaired. These observations were confirmed with another let-502
allele (Table 1A),
let-502(ca201sb54) (Piekny et
al., 2000), showing that suppression is not allele-specific.
Moreover, using the nearly 100% lethal allele rga-2(hd102), we could
build a let-502(sb118ts) rga-2(hd102) double-mutant, which was 68%
viable at 20°C but non-viable at 25°C. At 20°C, non-viable
let-502(sb118ts) rga-2(hd102) embryos ruptured like
rga-2(hd102) embryos, whereas at 25°C they did not rupture and
displayed the hypoelongation phenotype of let-502(sb118ts) embryos
alone. Thus, partial reduction of LET-502/ROCK activity can suppress
rga-2 lethality and, conversely, let-502 lethality can only
be suppressed by partial reduction of rga-2 function.
|
|
; Leung et al.,
1996; Matsui et al.,
1996). To test this prediction, we performed in vitro GAP activity
assays using a recombinant protein spanning the RGA-2 GAP domain (residues
219-475) and C. elegans RHO-1, CED-10 (RAC1) and CDC-42 recombinant
fusion proteins. The GAP domain of RGA-2 activated GTP hydrolysis only in the
presence of RHO-1 and CDC-42 (Fig.
5, and see Fig. S3 in the supplementary material), and required a
40 times lower RGA-2 GAP concentration to activate RHO-1 than CDC-42 (10
versus 400 nM). Interestingly, the mammalian homolog ARHGAP20 also displays
stronger specificity towards RHOA (Yamada
et al., 2005).
The data presented above are compatible with a model whereby RGA-2 activity
maintains RHO-1 in the GDP-bound state, thereby preventing LET-502 activation.
To test this model, we could not use RNAi against rho-1 because it
blocks the first embryonic division
(Jantsch-Plunger et al.,
2000), nor could we use a rho-1 mutation as there is none
available. Instead, we used a strain with an integrated transgene expressing a
constitutively-active form of RHO-1 (G14V) under a heat-shock promoter
(McMullan et al., 2006),
expecting that it should enhance the lethality of a partial reduction of
rga-2 function or reduce the lethality of let-502(sb118ts)
embryos. In a wild-type background, expression of the RHO-1(G14V) protein
after heat shock had only a moderate effect, presumably because the wild-type
RHO-1 was still present (Table
1B). RNAi against rga-2 in this strain more than doubled
the rate of lethality, even in the absence of heat shock
(Table 1B). We do not think
that this strain is hypersensitive to RNAi because RNAi against rga-2
by feeding was as poorly efficient as in wild-type animals
(Table 1B). Instead we reason
that the heat-shock promoter is slightly active even at 20°C. Heat-shock
expression of RHO-1(G14V) significantly reduced the lethality of
let-502(sb118ts) animals (Table
1B). Taken together, our data are consistent with the RhoGAP RGA-2
acting through RHO-1 in vivo, although we cannot exclude the possibility that
it can also act through another GTPase, and support the notion that RHO-1
activates LET-502/ROCK during elongation.
RGA-2 acts mainly in dorsal/ventral epidermal cells
Our data suggest that the RhoGAP RGA-2 acts to negatively regulate RHO-1
and LET-502/ROCK, and ultimately myosin II. Since another negative regulator
of myosin II, MEL-11, was already known, the identification of RGA-2 raises
the issue of whether it acts in the same cells and at the same time as
LET-502.
The lateral epidermal seam cells are thought to play a central role in
embryonic elongation (Piekny et al.,
2003; Priess and Hirsh,
1986; Shelton et al.,
1999; Wissmann et al.,
1999). In this framework, we asked whether rga-2 acts in
seam and/or dorsal/ventral cells. We expressed a rga-2 cDNA under the
control of promoters active only in seam cells (ceh-16)
(Cassata et al., 2005), or in
dorsal/ventral epidermal cells (elt-3)
(Gilleard et al., 1999). The
ceh-16p::rga-2 could not rescue the lethality of
rga-2(hd102) embryos, whereas the elt-3p::rga-2
construct could rescue about 75% of transgenic embryos after correcting for
transgene transmission frequency (Table
2). Joint presence of both constructs did not significantly
improve rescue. We conclude that rga-2 acts mainly in dorsal/ventral
epidermal cells, presumably to moderate contraction as it is occurring.
RGA-2 and LET-502 act during the same embryonic period
Several observations indicate that rga-2 acts during the first
part of elongation: our time-lapse recordings show that membrane indentations
appear around the 1.9-fold stage and that embryos rupture by the 2.2-fold
stage (Figs 1,
4). To determine until what
stage rga-2 is required, we took advantage of the
let-502(sb118ts) rga-2(hd102) double mutant, which is mostly viable
at 20°C but non-viable above 25°C
(Table 1A). We first determined
when LET-502/ROCK is essential during embryonic development. We found that
let-502(sb118ts) embryos up-shifted to the non-permissive temperature
once they had reached the 2-fold stage elongated normally
(Fig. 6A). Conversely, embryos
down-shifted to the permissive temperature at the lima-bean stage elongated
normally, whereas they did not recover when transferred beyond that stage.
These observations strongly suggest that LET-502/ROCK is required for
elongation between the lima-bean and 2-fold stages. We thus reasoned that the
double mutant let-502(sb118ts) rga-2(hd102) should allow us to test
whether RGA-2 is essential beyond the 2-fold stage. Temperature-shift
experiments showed that let-502(sb118ts) rga-2(hd102) embryos are
viable at the non-permissive temperature once they have reached the 2-fold
stage (Fig. 6A), which we
interpret to mean that RGA-2 is dispensable beyond the 2-fold stage. Thus,
RGA-2 and LET-502 are likely to act during the same developmental period, at
the beginning of elongation.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Priess and Hirsh,
1986; Shelton et al.,
1999). Two proteins are known to regulate myosin II contractility
during this process, the ROCK homologue LET-502, and MEL-11, the regulatory
myosin-binding subunit of PP1 phosphatase
(Piekny et al., 2003;
Piekny et al., 2000;
Wissmann et al., 1999;
Wissmann et al., 1997). Our
work identifies a microfilament-associated RhoGAP, RGA-2, as a new key
regulator of LET-502 and brings novel insights into the balance of forces
controlling elongation.
|
) and
LET-502 is interspersed with these structures, consistent with the idea that
RGA-2, LET-502 and myosin II act together to control actomyosin
contractility.
Some RhoGAP proteins can act as effectors rather than downregulators of
small GTPases (Kozma et al.,
1996; Zheng et al.,
1994). In particular, the RGA-2 homologue ARHGAP20 behaves as a
RAP1 GTPase effector during neurite outgrowth
(Yamada et al., 2005). We do
not believe that RGA-2 acts like this, as it does not possess a clear RA
domain and because the C. elegans RAP1 homologues mainly affect
larval secretion (Pellis-van Berkel et
al., 2005).
Our observations suggest that RGA-2 contains two domains for colocalisation
with epidermal actin: a major C-terminal domain that appears dispensable for
function, and a minor domain that coincides with the GAP domain. Forcing
membrane localisation of RGA-2 to sequester it away from actin affects its
activity, suggesting that RGA-2 association with microfilaments is important
for function. Whether RGA-2 colocalisation with actin reflects direct or
indirect binding to actin remains to be determined. The colocalisation of
other RhoGAPs to actin stress fibres, focal contacts, filopodia or
lamellipodia has been reported before
(Fauchereau et al., 2003;
Hildebrand et al., 1996;
Kozma et al., 1996;
Lavelin and Geiger, 2005). The
targeting of RhoGAPs to diverse subcellular localisations might provide
specificity and explain the requirement for more GAPs than GTPases (22 GAPs
versus six Rho family GTPases in the C. elegans genome).
Functional studies in tissue culture cells indicate that RhoGAPs can affect
the actin cytoskeleton, leading to loss of stress fibres or the formation of
filopodia and/or lamellipodia (Kozma et
al., 1996; Lavelin and Geiger,
2005; Yamada et al.,
2005). Our genetic analysis did not reveal a role for
rga-2 in maintaining cytoskeleton integrity. Rather, the finding that
rga-2 acts as a negative regulator of ROCK strongly suggests that
RGA-2 activity influences actomyosin contractility. It will be interesting to
determine whether its closest vertebrate homologue, ARHGAP20, also acts in the
ROCK pathway, particularly in cancer, because ROCK has been recognised to
promote transcellular invasion of tumour cells
(Itoh et al., 1999).
RGA-2 inhibits actomyosin tension in dorsal/ventral epidermal cells
Twenty years ago, Priess and Hirsh proposed that the lateral seam cells
should play the leading role in embryonic elongation
(Priess and Hirsh, 1986).
Since then, the identification of let-502 and mlc-4
(encoding the myosin regulatory light chain), and the demonstration using GFP
reporters that they are primarily expressed in seam cells, provided molecular
data to reinforce the seam-based elongation model
(Shelton et al., 1999;
Wissmann et al., 1999).
However, let-502 and mlc-4 have not yet been shown to act
only in seam cells. In particular, a caveat of GFP reporters is that they only
reveal the zygotic expression of genes with a maternal requirement (which is
the case for mlc-4 and let-502). Furthermore, dorsal/ventral
epidermal cells might also play a role in elongation, as they are linked to
muscles, which are essential to progress beyond the 2-fold stage
(Williams and Waterston,
1994). Indeed, immunofluorescence analysis clearly indicates that
LET-502 and NMY-1 are also present in dorsal and ventral cells
(Piekny et al., 2003).
Our work provides the strongest genetic evidence so far that the seam-based
model of elongation is accurate, at least until the 2-fold stage. Three
observations support this notion: (1) LET-502/ROCK and RGA-2 have opposite
functions; (2) they act during the same developmental period, as they both
proved dispensable beyond the 2-fold stage; (3) RGA-2 is functionally required
in dorsal and ventral epidermal cells, and excessive pulling on junctional
complexes of rga-2 mutants is only observed in these cells. We
interpret the pulling as a result of excessive actomyosin tension, as
let-502 and nmy-1 are epistatic to rga-2, and
imagine that it eventually causes junctions to rupture. Interestingly, we did
not observe indentations in let-502(sb118) rga-2(hd102) embryos (not
shown), suggesting that let-502 activity in dorsal/ventral cells is
needed for their occurrence. The simplest model is that RGA-2 negatively
regulates LET-502 in dorsal/ventral cells by keeping RHO-1 in the GDP-bound
form (Fig. 6C). Hence, we
suggest that to reach the 2-fold stage the embryo must activate actomyosin in
seam cells and repress it in dorsal/ventral cells to moderate tension. Whether
RGA-2 completely inhibits or simply moderates actomyosin tension in
dorsal/ventral cells is unclear yet. RGA-2 is also present in seam cells,
although at lower levels, and we do not know whether it is inactive therein or
whether it has a minor role to ensure appropriate contraction. Interestingly,
the RhoGAP crossveinless c asymmetrically inactivates actomyosin along the
apicobasal axis of tracheal and spiracle cells during Drosophila
morphogenesis (Brodu and Casanova,
2006; Simoes et al.,
2006). A general feature of morphogenesis might thus be the
asymmetric activation of myosin II, either within a cell or among different
cells.
Our findings have implications for the roles of LET-502 and MEL-11 during elongation. Temperature-shift experiments indicate that LET-502 is not required beyond the 2-fold stage, the stage at which muscle mutants arrest. Although this conclusion relies on a single allele, let-502(sb118ts), affecting a conserved residue in the kinase domain (P. Mains, personal communication), it should reflect when LET-502 is active. Hence, beyond the 2-fold stage, either LET-502 acts redundantly with other proteins, or the shortening of actin microfilaments does not entirely depend on anti-parallel sliding through myosin II activity and might involve other molecular mechanisms possibly implying a muscle-dependent pathway.
Our identification of a second negative regulator of ROCK raises the issue
of why embryos would need two negative regulators of actomyosin activity with
distinct distributions. It is thought that LET-502 phosphorylates MEL-11
during elongation, inducing its sequestration to membranes, away from the
actomyosin apparatus (Piekny et al.,
2003). Our time-lapse recordings indicate that defects in
mel-11 and rga-2 mutants become visible at slightly
different stages (Fig. 1).
Since we observed tension in all epidermal cells of mel-11 embryos,
MEL-11 should act, in part, in the same cells as RGA-2. One possibility could
be that MEL-11 and RGA-2 act sequentially: MEL-11 could initially prevent the
premature onset of elongation and then become inactive when recruited to
junctions (Piekny et al.,
2003), whereas RGA-2 would subsequently take the relay.
Alternatively, MEL-11 could remain active when junctional, implying that
MEL-11 and RGA-2 would ultimately affect different pools of myosin II located
either at junctions (MEL-11) or along circumferential actin filaments (RGA-2).
We note that in vertebrate epithelia, disproportionate ROCK activity can
disrupt adherens junctions (Sahai and
Marshall, 2002). By analogy, the presence at junctions of an
inhibitor of actomyosin, such as MEL-11, could help maintain junction
integrity during C. elegans elongation. Further analysis of MEL-11
and RGA-2 should determine which model or models (they are not mutually
exclusive) is the most likely.
In conclusion, we have identified a microfilament-associated RhoGAP that acts as a negative regulator of ROCK to moderate tension in a subset of epidermal cells during morphogenesis. It will be interesting to see whether the RGA-2 homologue in vertebrates also acts in the ROCK pathway.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/13/2469/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
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These authors contributed equally to this work
Present address: Simon Fraser University, Burnaby, BC V5A 1S6, Canada
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REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bosher, J. M., Hahn, B. S., Legouis, R., Sookhareea, S., Weimer,
R. M., Gansmuller, A., Chisholm, A. D., Rose, A. M., Bessereau, J. L. and
Labouesse, M. (2003). The Caenorhabditis elegans
vab-10 spectraplakin isoforms protect the epidermis against internal and
external forces. J. Cell Biol.
161,757
-768.
Bossinger, O., Klebes, A., Segbert, C., Theres, C. and Knust, E. (2001). Zonula adherens formation in Caenorhabditis elegans requires dlg-1, the homologue of the Drosophila gene discs large. Dev. Biol. 230, 29-42.[CrossRef][Medline]
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Brodu, V. and Casanova, J. (2006). The RhoGAP
crossveinless-c links trachealess and EGFR signaling to cell shape remodeling
in Drosophila tracheal invagination. Genes Dev.
20,1817
-1828.
Burridge, K. and Wennerberg, K. (2004). Rho and Rac take center stage. Cell 116,167 -179.[CrossRef][Medline]
Cassata, G., Shemer, G., Morandi, P., Donhauser, R.,
Podbilewicz, B. and Baumeister, R. (2005).
ceh-16/engrailed patterns the embryonic epidermis of
Caenorhabditis elegans. Development
132,739
-749.
Chisholm, A. D. and Hardin, J. (2005). Epidermal morphogenesis. In WormBook (ed. The C. elegans Research Community), doi/10.1895/wormbook.1.35.1, http://www.wormbook.org.
Costa, M., Draper, B. W. and Priess, J. R. (1997). The role of actin filaments in patterning the Caenorhabditis elegans cuticle. Dev. Biol. 184,373 -384.[CrossRef][Medline]
Costa, M., Raich, W., Agbunag, C., Leung, B., Hardin, J. and
Priess, J. R. (1998). A putative catenin-cadherin system
mediates morphogenesis of the Caenorhabditis elegans embryo.
J. Cell Biol. 141,297
-308.
Fauchereau, F., Herbrand, U., Chafey, P., Eberth, A., Koulakoff, A., Vinet, M. C., Ahmadian, M. R., Chelly, J. and Billuart, P. (2003). The RhoGAP activity of OPHN1, a new F-actin-binding protein, is negatively controlled by its amino-terminal domain. Mol. Cell. Neurosci. 23,574 -586.[CrossRef][Medline]
Franke, J. D., Montague, R. A. and Kiehart, D. P. (2005). Nonmuscle myosin II generates forces that transmit tension and drive contraction in multiple tissues during dorsal closure. Curr. Biol. 15,2208 -2221.[CrossRef][Medline]
Gilleard, J. S., Shafi, Y., Barry, J. D. and McGhee, J. D. (1999). ELT-3: A Caenorhabditis elegans GATA factor expressed in the embryonic epidermis during morphogenesis. Dev. Biol. 208,265 -280.[CrossRef][Medline]
Hildebrand, J. D., Taylor, J. M. and Parsons, J. T. (1996). An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase. Mol. Cell. Biol. 16,3169 -3178.[Abstract]
Hobert, O. (2002). PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans. Biotechniques 32,728 -730.[Medline]
Hochholdinger, F., Baier, G., Nogalo, A., Bauer, B., Grunicke,
H. H. and Uberall, F. (1999). Novel membrane-targeted ERK1
and ERK2 chimeras which act as dominant negative, isotype-specific
mitogen-activated protein kinase inhibitors of Ras-Raf-mediated
transcriptional activation of c-fos in NIH 3T3 cells. Mol. Cell.
Biol. 19,8052
-8065.
Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatsu, A., Fujita, A., Watanabe, N., Saito, Y., Kakizuka, A., Morii, N. et al. (1996). The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J. 15,1885 -1893.[Medline]
Itoh, K., Yoshioka, K., Akedo, H., Uehata, M., Ishizaki, T. and Narumiya, S. (1999). An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat. Med. 5,221 -225.[CrossRef][Medline]
Jacinto, A. and Martin, P. (2001). Morphogenesis: unravelling the cell biology of hole closure. Curr. Biol. 11,R705 -R707.[CrossRef][Medline]
Jacinto, A., Wood, W., Woolner, S., Hiley, C., Turner, L., Wilson, C., Martinez-Arias, A. and Martin, P. (2002). Dynamic analysis of actin cable function during Drosophila dorsal closure. Curr. Biol. 12,1245 -1250.[CrossRef][Medline]
Jantsch-Plunger, V., Gönczy, P., Romano, A., Schnabel, H.,
Hamill, D., Schnabel, R., Hyman, A. A. and Glotzer, M.
(2000). CYK-4: A Rho family gtpase activating protein (GAP)
required for central spindle formation and cytokinesis. J. Cell
Biol. 149,1391
-1404.
Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M. et al. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421,231 -237.[CrossRef][Medline]
Keller, R. (2006). Mechanisms of elongation in
embryogenesis. Development
133,2291
-2302.
Köppen, M., Simske, J. S., Sims, P. A., Firestein, B. L., Hall, D. H., Radice, A. D., Rongo, C. and Hardin, J. D. (2001). Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia. Nat. Cell Biol. 3,983 -991.[CrossRef][Medline]
Kozma, R., Ahmed, S., Best, A. and Lim, L. (1996). The GTPase-activating protein n-chimaerin cooperates with Rac1 and Cdc42Hs to induce the formation of lamellipodia and filopodia. Mol. Cell. Biol. 16,5069 -5080.[Abstract]
Lavelin, I. and Geiger, B. (2005).
Characterization of a novel GTPase-activating protein associated with focal
adhesions and the actin cytoskeleton. J. Biol. Chem.
280,7178
-7185.
Lee, A. and Treisman, J. E. (2004). Excessive
Myosin activity in mbs mutants causes photoreceptor movement out of the
Drosophila eye disc epithelium. Mol. Biol.
Cell 15,3285
-3295.
Lee, S. and Kolodziej, P. A. (2002). Short Stop
provides an essential link between F-actin and microtubules during axon
extension. Development
129,1195
-1204.
Leung, T., Chen, X. Q., Manser, E. and Lim, L. (1996). The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol. Cell. Biol. 16,5313 -5327.[Abstract]
Matsui, T., Amano, M., Yamamoto, T., Chihara, K., Nakafuku, M., Ito, M., Nakano, T., Okawa, K., Iwamatsu, A. and Kaibuchi, K. (1996). Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J. 15,2208 -2216.[Medline]
Matsumura, F. (2005). Regulation of myosin II during cytokinesis in higher eukaryotes. Trends Cell Biol. 15,371 -377.[CrossRef][Medline]
McMahon, L., Legouis, R., Vonesch, J. L. and Labouesse, M. (2001). Assembly of C. elegans apical junctions involves positioning and compaction by LET-413 and protein aggregation by the MAGUK protein DLG-1. J. Cell Sci. 114,2265 -2277.[Medline]
McMullan, R., Hiley, E., Morrison, P. and Nurrish, S. J.
(2006). Rho is a presynaptic activator of neurotransmitter
release at pre-existing synapses in C. elegans. Genes
Dev. 20,65
-76.
Moon, S. Y. and Zheng, Y. (2003). Rho GTPase-activating proteins in cell regulation. Trends Cell Biol. 13,13 -22.[CrossRef][Medline]
Norman, K. R. and Moerman, D. G. (2002). Alpha
spectrin is essential for morphogenesis and body wall muscle formation in
Caenorhabditis elegans. J. Cell Biol.
157,665
-677.
Pellis-van Berkel, W., Verheijen, M. H., Cuppen, E., Asahina,
M., de Rooij, J., Jansen, G., Plasterk, R. H., Bos, J. L. and Zwartkruis, F.
J. (2005). Requirement of the Caenorhabditis elegans
RapGEF pxf-1 and rap-1 for epithelial integrity.
Mol. Biol. Cell 16,106
-116.
Piekny, A. J., Wissmann, A. and Mains, P. E.
(2000). Embryonic morphogenesis in Caenorhabditis
elegans integrates the activity of LET-502 Rho-binding kinase, MEL-11
myosin phosphatase, DAF-2 insulin receptor and FEM-2 PP2c phosphatase.
Genetics 156,1671
-1689.
Piekny, A. J., Johnson, J. L., Cham, G. D. and Mains, P. E.
(2003). The Caenorhabditis elegans nonmuscle myosin
genes nmy-1 and nmy-2 function as redundant components of
the let-502/Rho-binding kinase and mel-11/myosin phosphatase
pathway during embryonic morphogenesis. Development
130,5695
-5704.
Pilot, F. and Lecuit, T. (2005). Compartmentalized morphogenesis in epithelia: from cell to tissue shape. Dev. Dyn. 232,685 -694.[CrossRef][Medline]
Portereiko, M. F. and Mango, S. E. (2001). Early morphogenesis of the Caenorhabditis elegans pharynx. Dev. Biol. 233,482 -494.[CrossRef][Medline]
Praitis, V., Casey, E., Collar, D. and Austin, J.
(2001). Creation of low-copy integrated transgenic lines in
Caenorhabditis elegans. Genetics
157,1217
-1226.
Priess, J. R. and Hirsh, D. I. (1986). Caenorhabditis elegans morphogenesis: the role of the cytoskeleton in elongation of the embryo. Dev. Biol. 117,156 -173.[CrossRef][Medline]
Riento, K. and Ridley, A. J. (2003). Rocks: multifunctional kinases in cell behaviour. Nat. Rev. Mol. Cell Biol. 4,446 -456.[CrossRef][Medline]
Sahai, E. and Marshall, C. J. (2002). ROCK and Dia have opposing effects on adherens junctions downstream of Rho. Nat. Cell Biol. 4,408 -415.[CrossRef][Medline]
Shelton, C. A., Carter, J. C., Ellis, G. C. and Bowerman, B.
(1999). The nonmuscle myosin regulatory light chain gene
mlc-4 is required for cytokinesis, anterior-posterior polarity, and
body morphology during Caenorhabditis elegans embryogenesis.
J. Cell Biol. 146,439
-451.
Simoes, S., Denholm, B., Azevedo, D., Sotillos, S., Martin, P.,
Skaer, H., Hombria, J. C. and Jacinto, A. (2006).
Compartmentalisation of Rho regulators directs cell invagination during tissue
morphogenesis. Development
133,4257
-4267.
Sun, D., Leung, C. L. and Liem, R. K. (2001). Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins. J. Cell Sci. 114,161 -172.[Abstract]
Van Aelst, L. and Symons, M. (2002). Role of
Rho family GTPases in epithelial morphogenesis. Genes
Dev. 16,1032
-1054.
Williams, B. D. and Waterston, R. H. (1994).
Genes critical for muscle development and function in Caenorhabditis
elegans identified through lethal mutations. J. Cell
Biol. 124,475
-490.
Wissmann, A., Ingles, J., McGhee, J. D. and Mains, P. E.
(1997). Caenorhabditis elegans LET-502 is related to
Rho-binding kinases and human myotonic dystrophy kinase and interacts
genetically with a homolog of the regulatory subunit of smooth muscle myosin
phosphatase to affect cell shape. Genes Dev.
11,409
-422.
Wissmann, A., Ingles, J. and Mains, P. E. (1999). The Caenorhabditis elegans mel-11 myosin phosphatase regulatory subunit affects tissue contraction in the somatic gonad and the embryonic epidermis and genetically interacts with the Rac signaling pathway. Dev. Biol. 209,111 -127.
32P]GTP (see Materials and methods), resolving the reaction
products by thin layer chromatography (see Fig. S3 in the supplementary
material), and quantifying the GTP/GDP fraction with a phosphorimager. Each
time point represents the fraction of GTP hydrolysed after 2 minutes as a
function of RGA-2GAP concentration. The graph is representative of
four independent experiments.