1 Division of Developmental Biology, Cincinnati Children's Research Foundation,
3333 Burnet Avenue, Cincinnati, OH 45229, USA
2 Physician Scientist Training Program, University of Cincinnati College of
Medicine, University of Cincinnati College of Medicine, PO Box 670555,
Cincinnati, OH 45267, USA
Author for correspondence (e-mail:
christopher.wylie{at}cchmc.org)
Accepted 18 April 2005
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SUMMARY |
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Key words: Cortical actin, G protein coupled receptor, Xenopus
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Introduction |
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We have previously shown that the Arm repeat protein Plakoglobin
( catenin) is both necessary and sufficient for assembly of the
cortical actin skeleton in early Xenopus embryos, and is downstream
of the signaling intermediate cdc42
(Kofron et al., 2002
). In
order to identify more fully the pathways leading to cortical actin assembly,
an arrayed two- to four-cell cDNA library was divided into mRNA pools, which
were expressed in Xenopus embryos by microinjection at the two-cell
stage. Pools that caused effects similar to plakoglobin/cdc42 overexpression
were split in a matrix fashion (Grammer et
al., 2000
) to identify the active mRNAs. One of these mRNAs was
found to encode a novel G protein-coupled receptor (GPCR), whose sequence is
related to a subfamily of GPCRs, the OGR1 (Ovarian cancer G protein-coupled
Receptor 1) subfamily. Overexpression of this mRNA dramatically increased the
density of the actin skeleton in the early Xenopus embryo, whereas
oligodeoxynucleotide-mediated depletion of the endogenous mRNA had the reverse
effect, and caused embryos to lose their shape and rigidity. On the basis of
the phenotype caused by its depletion, we have given this protein the
provisional name of XFlop. In a previous paper, we showed that intercellular
signaling is required to maintain the normal density of the cortical actin
skeleton in Xenopus, and that the expression of LPA receptors is both
necessary and sufficient to maintain the normal density of cortical actin
filaments in interphase cells (Lloyd et
al., 2005
). Here, we compare the roles of these two ligand
receptors, and conclude that they each play independent roles in generating
the normal pattern of actin filaments in the embryo. Thus, multiple cell
signaling pathways are involved in this process.
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Materials and methods |
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Cortical actin-assembly assays and pixel-intensity analysis
Cortical actin assembly assays were carried out exactly as described
previously (Kofron et al.,
2002; Lloyd et al.,
2005
). The size of each confocal image was set as 512x512
pixels. Z stacks, that include cortical actin of all the superficial cells of
the blastocoelic surface of the cap, are shown in all low power (20x)
pictures of entire caps. The intensity of each pixel was calculated by Zeiss
510 software in a linear range of 128-4095 (2-256 for the older version of
software). The overall amount of the cortical actin for each cap is expressed
as mean pixel intensity across the whole cap. Five to eight caps from each
treatment were analyzed in all experiments, and the results presented as mean
pixel intensity ±s.d. Treatments were compared for statistical
significance by Student's t-test.
mRNA depletion and host transfers
These were carried out as described previously
(Kofron et al., 2002).
Briefly, stage VI oocytes were obtained by manual defolliculation and cultured
in oocyte culture medium (OCM). To deplete the maternal store of XFlop mRNA,
8-12 ng of HPLC-purified antisense phosphorothioate-modified
oligodeoxynucleotides (18-mer) were injected into each oocyte. After 2-3 days
of culture at 18°C, oocytes were matured and transferred using host
transfer techniques (Holwill,
1987
). The sequences of two antisense oligonucleotides used to
deplete maternal XFlop mRNA are: oligo 1s,
5'-A*A*-G*GGAACACTGTAG*C*C*A-3';
oligo 5s,
5'-G*T*T*GTACGTTTTGGC*T*G*G-3',
where * indicates the phosphorothioate diester bond
substitution.
mRNA synthesis and injection
Plasmids from the expression library, and those encoding pCS2+
XFlop C and FL were linearized with NotI and transcribed in
vitro using SP6. XFlop mRNA (10-250 pg) was injected into the animal
cytoplasm at the two- or eight-cell stage. mRNA-injected embryos were reared
in 0.2xMMR containing 2% Ficoll, and transferred to 0.1xMMR at the
mid-blastula stage.
Total RNA isolation and real-time RT-PCR
These were performed exactly as described previously
(Kofron et al., 2002).
Immunostaining
Microtubules and cytokeratin filaments in control and XFlop-depleted animal
caps were detected as described previously
(Kofron et al., 2002).
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Results |
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Based upon the effect of its depletion in the early embryo (see below), we temporarily named this protein XFlop, until a more systematic name, based upon function and structure, can be allocated.
Characterization and expression pattern of XFlop
Three independent colonies from 12D10D were sequenced in both strands. The
sequence has been deposited in GenBank (AY766161). The XFlop cDNA
sequence is 1.5 kb long. The predicted open reading frame encodes a protein of
348 amino acids (Fig. 2A),
which contains a seven-pass transmembrane domain, suggesting that XFlop is a G
protein-coupled receptor (GPCR). Furthermore, XFlop contains a putative
N-linked glycosylation site (N*QSC, aa4-7) in the N-terminal
extracellular domain (aa1-aa18), and a conserved acidic-arginine-aromatic
triplet (D-R-F) in the N-terminal extremity of the second cytoplasmic loop
(aa111-aa113), each characteristic of GPCRs
(Bause, 1983;
Wheatley and Hawtin, 1999
).
The acidic-R-aromatic triplet has been implicated in G-protein coupling
(Oliveira et al., 1994
). XFlop
shows 46-53% sequence homology with members of a recently proposed subfamily
including mammalian ovarian cancer G protein-coupled receptor 1 (OGR1), G
protein-coupled receptor 4 (GPR4), G2 accumulation (G2A) and T cell
death-associated gene 8 (TDAG8) (Fig.
2B) (Heiber et al.,
1995
; Xu,
2002
).
Recently, another member of the GPR4 family was identified in
Xenopus (Xgpcr4) (Chung et al.,
2004). Fig. 2A
shows that the putative amino acid sequences are very divergent in the C
termini of Xgpcr4 and XFlop, which suggests that they are encoded by different
genes that are not pseudoalleles of the same gene. We also found that Xgpcr4
and XFlop are encoded by different genomic sequences even though they reside
in the same chromosome (Fig.
2C). Comparison with the genomic scaffold sequence (Scaffold 3407,
JGI) shows that the ORF of XFlop is encoded by a single exon, a feature shared
by mammalian GPR4 proteins (Heiber et al.,
1995
).
Comparison of the sequence of the original 12D10D clone with the contig
sequence from the NIBB database suggested that there might be a single
adenosine insertion at position 947 in the open reading frame of the 12D10D
clone (Fig. 2D, highlighted in
green), which would result in a premature translation stop, affecting the
C-terminal 30 amino acids (Fig.
2A). To establish which coding sequence is expressed in the
embryo, we used the contig-coding sequence to PCR-amplify the putative
full-length coding sequence from a late blastula cDNA preparation. Sequencing
of the amplified cDNA showed that the original 12D10D clone did indeed contain
a single adenosine insertion at nucleotide 947. We further compared this cDNA
sequence with the Xenopus tropicalis genomic sequence (scaffold 3407)
available from the JGI database, which further supported the notion that the
adenosine insertion in the 12D10D clone is an artifact of library
construction. Fig. 2D compares
the coding sequences of the 12D10D clone and the full-length cDNA isolated
from late blastula mRNA, and shows that the 12D10D clone has a stop codon
earlier than in the full-length sequence. We therefore denoted the original
12D10D sequence as XFlop C. Interestingly, these two
forms of the protein had exactly the same activities in regulating actin
assembly (see below), suggesting that the C-terminal 30 amino acids are not
required for the action of XFlop on the actin skeleton.
To assay the spatial and temporal expression pattern of XFlop mRNA during early development, we made cDNA from embryos frozen at different stages, and assayed the level of XFlop mRNA by quantitative real-time PCR. XFlop was expressed maternally, and ubiquitous expression was observed in the blastula throughout early development (Fig. 2E).
Overexpression of XFlop accelerated wound healing and enhanced actin assembly
We transcribed both XFlop C and full-length
XFlop mRNA and microinjected them, in doses of between 10 and 250 pg
per embryo, into the animal cytoplasm at the two-cell stage. After excision of
animal caps at the late blastula stage, the remainder of the embryos (the
bases) were found to heal significantly faster than controls
(Fig. 3A,E, compare blue and
yellow bars), in a dose-dependent fashion. The blastocoel roofs of
XFlop-injected embryos were also thicker than those of controls
(Fig. 3B). The mean
(±s.d.) thickness of the blastocoel roofs (the distance from the middle
of the roof to the animal pole) was measured in ten embryos from photographs
of fixed, bisected embryos. The mean roof thickness (±s.d.) of
XFlop-overexpressing embryos was 195±37 µm, which compared with
73±25 µm for control embryos.
The effect of XFlop overexpression on cortical actin is shown in
Fig. 3C,D. At low
magnification, the level of staining in the whole cap is dramatically
increased, as shown by the pixel intensity charts
(Fig. 3C). The mean pixel
intensity of five to eight caps (±s.d.) is shown in
Fig. 3D, together with the
P-value from the comparison by Student's t-test of the
control, and mean and standard deviation of a 250 pg dose. Increasing doses of
XFlop mRNA caused a corresponding increase of phalloidin binding, and
therefore of F-actin concentration in the cell cortices. Examination of
individual caps showed that not all cells have increased actin staining (arrow
in Fig. 3C). This could be due
to one of two possibilities. First, the injected mRNA may not have filled this
area of the injected embryo. This is unlikely, as cells surrounding the
injected one have increased actin staining. Second, and more likely, we have
shown previously that cells undergoing cytokinesis change their cortical actin
to a less-dense network (Lloyd et al.,
2005). As cells divide normally in XFlop-overexpressing embryos,
it is likely that they go through the same cycles of actin assembly and
disassembly during the cell cycle. However, we have not experimentally
discriminated between these two possibilities.
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At the blastula stage, XFlop-depleted embryos looked morphologically normal, but when the vitelline membranes were removed, they became less spherical than control embryos (Fig. 5B,C), which made them look a little larger when viewed from above (Fig. 5B, compare the lengths of the lines of control and depleted embryos). When fixed and bisected along the animal/vegetal axis, and viewed from the side, the less spherical architecture becomes more obvious (Fig. 5C). After excision of the animal caps, the bases healed more slowly than control bases did. After 45 minutes, the wound margins of the control bases were smooth, and the wound diameter had contracted. By contrast, the wound margins of XFlop-depleted embryos were still open (Fig. 5D). These data are quantitated and compared with the effect of XFlop overexpression in Fig. 3E. Animal caps from these embryos were cultured for 10 minutes, fixed, stained with Alexa 488-coupled phalloidin, and their blastocoelic surfaces examined by confocal microscopy. Fig. 5E shows low magnification images of whole caps (upper panels), and high magnification images (lower panels) of control and XFlop-depleted animal caps. The overall level of cortical actin staining was significantly reduced by XFlop depletion. Quantitation was performed by comparing the mean pixel intensity (±s.d.) of six caps from each treatment (Fig. 5F). In addition, none of the XFlop-depleted caps had actin-rich purse strings around their circumferences (arrow in Fig. 5E). High magnification images (Fig. 5E) show the reverse of XFlop overexpression; staining of all actin-containing structures (except for the contractile rings of dividing cells) was reduced. There were fewer cell processes, and those that were present had less actin staining than did those of controls. The cortical actin network was also reduced. As is the case for overexpression, this suggests that XFlop signaling is required to maintain the normal amount of F-actin in all actin-containing structures, except those required for cell division.
We next examined the cortical actin network in dissociated cells from
control and XFlop-depleted embryos (Fig.
5G). Animal caps were dissociated and cultured in
calcium/magnesium-free saline for 10 minutes before fixation and staining.
This culture time is not sufficient for the cortical network to transform from
the dense to the coarse network type, a process that takes at least 30 minutes
(Lloyd et al., 2005). Actin
staining was significantly reduced in individual dissociated cells that were
XFlop depleted. This was quantitated by the comparison of mean pixel intensity
(±s.d.) from 20 control and 28 XFlop-depleted cells, which showed that
the decrease is statistically significant (P<0.01,
Fig. 5H).
Fig. 5G (middle panels) shows
the images in rainbow scale, in which the Zeiss LSM510 software assigns
different spectral colors to different pixel intensities. This illustrates the
fact that, even though staining levels are reduced in XFlop-depleted embryos,
there is still an actin network present, but it contains less actin. In some
areas (see arrow, Fig. 5G),
areas denuded of actin filaments can be seen, through which underlying yolk
platelets can be seen.
In order to check whether microtubule and intermediate filament assemblies were also affected by XFlop depletion, we stained animal caps from XFlop-depleted and control late blastulae with antibodies against tubulin or cytokeratin. In the absence of maternal XFlop, the microtubule and cytokeratin networks in the animal caps were both intact, although the F-actin network was significantly reduced (Fig. 6A,B), suggesting that the cytoarchitectural defects seen at the blastula stage are primarily due to the loss of the cortical actin skeleton.
In order to show that these defects are specific for the loss of the maternal store of XFlop mRNA, and were not caused by non-specific degradation of other mRNAs by the oligo, or by random toxicity, we injected XFlop mRNA-depleted embryos at the two-cell stage with 50 pg of full-length XFlop mRNA. The injected mRNA rescued the rigidity and spherical architecture of the late blastula (Fig. 6C), as well as their ability to heal wounds (Fig. 6C) and the density of the cortical actin skeleton (Fig. 6D,E). This rescue was seen in four repeat experiments. As an additional control for oligo specificity, we found that two antisense oligos targeting different regions of XFlop mRNA gave identical phenotypes, and that a 1:1 mixture of these two oligos had additive effects (data not shown). The XFlop-depletion phenotypes have been reproduced in five independent experiments. In wound healing assays, we analyzed five to six embryos from each treatment at late blastula stage. Taken together, these data show that XFlop is necessary, as well as sufficient, for maintenance of the correct levels of cortical actin and the maintenance of the spherical architecture of the embryo.
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XFlop and LPA signaling play distinct roles in cortical actin assembly
We have recently shown that a different signaling pathway, mediated by the
phospholipid ligand lysophosphatidic acid (LPA), and its two receptors XLPA1
and XLPA2, also controls the cortical actin skeleton of the early
Xenopus embryo, so it was important to establish the relationship
between these two signaling pathways. In particular, we wanted to test whether
each pathway independently initiates actin assembly, or whether they are in
the same pathway.
A comparison of XFlop overexpression and depletion (Figs
3,
4,
5,
6) with previously published
data on LPA1 and LPA2 depletion (Lloyd et
al., 2005) suggests that the two signaling pathways control
different aspects of actin assembly. LPA depletion causes the dense actin
network characteristic of interphase blastomeres in vivo to become replaced by
the coarser network seen in dividing blastomeres in vivo, or in dissociated
blastomeres (Lloyd et al.,
2005
). Overexpression of XFlop, however, caused a general increase
in actin in all actin-containing structures (Figs
3,
4), whereas its depletion
caused an overall decrease in the actin present in all actin-containing
structures (Figs 5,
6) without causing a shift to
the looser network characteristic of dividing or dissociated cells. This would
suggest that LPA1/2 and XFlop play different roles in actin assembly.
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To test further whether XFlop and LPA lie in the same pathway, we investigated whether depletion of one would block the effect of overexpression of the other. If they lie in the same pathway, then they will be epistatic; one will rescue the other, but the reverse will not be the case. Oocytes depleted of either LPA1 and LPA2, or XFlop, were fertilized, and mRNA encoding either XFlop or LPA1 was injected into the animal cytoplasm at the two-cell stage. Both LPA1 overexpression in XFlop-depleted embryos, and XFlop overexpression in LPA1 and LPA2-depleted embryos, increased the levels of cortical actin (Fig. 8C). This shows that neither receptor requires the other for its action, and strongly suggests that they have separate roles in cortical actin assembly.
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Discussion |
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XFlop is most closely related to mammalian GPR4, a member of a recently
proposed sub-family of G protein-coupled receptors, the OGR1 sub-family
(Xu, 2002). Comprising OGR1
(ovarian cancer G protein-coupled receptor 1), G2A (G2 accumulation), TDAG8 (T
cell death-associated gene 8) and GPR4 (G protein-coupled receptor 4), the
sub-family is moderately conserved, with 36-51% amino acid identity. Ligands
for this group are still being identified, but include related signaling
lipids. TDAG8 has been reported to bind psychosine (galactosyl sphingosine)
(Im et al., 2001
), whilst OGR1
binds sphingosylphosphoryl choline (SPC)
(Xu et al., 2000
), and GPR4
and 2GA bind both SPC and lysophosphatidyl choline (LPC)
(Bektas et al., 2003
;
Kabarowski et al., 2001
;
Zhu et al., 2001
). More
recently, it has been reported that OGR1 and GPR4 respond differentially to
changes in extracellular proton concentration
(Ludwig et al., 2003
). OGR1
increases inositol phosphate formation, whilst GPR4 increases cyclic AMP
formation, in response to increased proton concentration. Ligand specificities
have generally been identified by overexpressing the receptor in cultured
cells, so it is not yet clear which ligands are used in vivo, and whether
these change spatially and temporally during tissue differentiation, nor are
the in vivo functions of the OGR1 sub-family fully established. G2A null mice
have an abnormal expansion of both B and T lymphocytes, and an autoimmune
syndrome, suggesting that G2A acts as a repressor in the immune system
(Le et al., 2001
). GPR4 is
thought to mediate the growth stimulatory effects of SPC on Swiss 3T3 cells,
whilst OGR1 may mediate the inhibitory growth effects of this same ligand
(Xu et al., 2000
;
Zhu et al., 2001
), suggesting
complementary functions of these related receptors. We show here that a
related receptor, XFlop, in Xenopus, plays an essential in vivo role
in the assembly of the correct amount of cortical actin. It will be
interesting to study the related mammalian receptors in this respect, to see
whether this function is conserved throughout the vertebrates.
Signaling through G protein-coupled receptors has been implicated
previously in the control of actin assembly, particularly in cell responses to
chemotactic stimuli. Ligands for these can be small molecules such as cAMP
(reviewed by Hereld and Devreotes,
1992), peptide ligands such as stromal derived factor 1 (SDF1)
(Nagasawa et al., 1999
), or
N-formyl methionine peptides (Panaro and
Mitolo, 1999
). However, the role of intercellular signaling in
establishing the amount, and pattern, of cortical actin that maintains the
correct shape and rigidity of the early embryo is less well understood. During
the egg to blastula stages, the number of cells in the embryo doubles with
each cell cycle. However, after each division cycle, each cell assembles the
cortical actin network appropriate to the shape and rigidity of the whole
embryo. In general, this could be accomplished by intracellular components of
the actin polymerization machinery inherited from the egg, or by intercellular
signaling. This work, and a previous paper
(Lloyd et al., 2005
), shows
that at least two intercellular signaling pathways control this process in
early Xenopus embryos. The role of the XFlop-mediated pathway appears
to be to control the overall amount of F-actin in most, but not all,
actin-containing structures, as XFlop depletion causes reduced F-actin in all
structures except the contractile rings of cytokinesis, which must be
controlled by other signals. By contrast, the role of LPA signaling seems to
be to control the switch from dense cortical actin during interphase to a less
dense network during cytokinesis (Lloyd et
al., 2005
). The two pathways can also be distinguished by the fact
that each can be initiated in the absence of the other.
The ligand for XFlop has yet to be identified, as have details of its downstream signaling pathways. The multiple effects of XFlop depletion (reduction in the cortical network and cell processes, and absence of wound healing and purse-string formation), and the fact that overexpression increases the amount of F-actin without amplifying only one type of actin-containing structure, suggest that it acts through more than one downstream signaling pathway. It will be important to identify which cytoplasmic signaling intermediates, and which of their effectors, act downstream of LPA and XFlop signaling at the cell surface in this in vivo developing system.
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ACKNOWLEDGMENTS |
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Footnotes |
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