1 Samuel Lunenfeld Research Institute of Mount Sinai Hospital, 600 University
Avenue, Toronto M5G 1X5, Canada
2 Department of Molecular and Medical Genetics, University of Toronto, Toronto
M5S 1A8, Canada
* Author for correspondence (e-mail: culotti{at}mshri.on.ca)
Accepted 23 December 2004
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SUMMARY |
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Key words: C. elegans, SMP-1, PLX-1, Morphogenesis, Guided migration
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Introduction |
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Vulva morphogenesis in C. elegans is a process that depends on
tight control over cell lineage and fate
(Sternberg and Horvitz, 1986;
Sulston and Horvitz, 1981
), as
well as stereotypical patterns of cell shape changes and movements
(Sharma-Kishore et al., 1999
;
Sulston and Horvitz, 1977
).
Vulva formation in C. elegans encompasses many aspects of
morphogenesis observed in animal development, and therefore is likely to
embody molecular aspects of organ formation conserved throughout
evolution.
Although genetic approaches have revealed major molecular mechanisms that
underlie vulva cell fate determination
(Greenwald and Rubin, 1992;
Horvitz and Sternberg, 1991
),
little is known about the molecular mechanisms involved in vulva cell shape
changes and movements that form the vulva proper. Fortunately, the sequence of
cellular events taking place during vulva morphogenesis have been described in
detail (Sharma-Kishore et al.,
1999
), and this description provides a blueprint for using
genetics to understand vulva morphogenesis at a molecular level.
The C. elegans vulva comprises seven ring-shaped cells stacked
precisely one on top of the other. Vulva development begins during larval
stage 3 (L3) when three vulva precursor cells (VPCs), P5.p, P6.p and P7.p, are
induced by a somatic gonad cell, the anchor cell (AC) (normally located
immediately dorsal to P6.p), to divide by mirror image sublineages
(Greenwald and Rubin, 1992;
Kimble, 1981
;
Sternberg and Horvitz, 1989
;
Sulston and White, 1980
).
These sublineages ultimately form a longitudinally oriented row of 22 ventral
midline epithelial cells comprising the primordial vulva. These are arranged
in a palindrome of homologous cell types (vulA, vulB1, vulB2, vulC, vulD,
vulE, vulF, vulF', vulE', vulD', vulC', vulB2',
vulB1' and vulA') (Greenwald
and Rubin, 1992
; Sternberg and
Horvitz, 1989
). The position between F and F' in this
sequence represents the position of the vulva midline, the future position of
the vulva lumen surrounded by ring-shaped vulva cells.
Based on laser-ablation and genetic studies, it has been shown that vulva
morphogenesis can occur independently for each anteroposterior mirror-image
vulva half (Sharma-Kishore et al.,
1999), suggesting that the guidance mechanisms used to position
the ring-forming homologs function autonomously from within each half
palindrome. Each half ring may comprise one or two cells, depending on the
ring (Fig. 1A). Each homologous
opposite half ring undergoes similar mirror image shape changes and movements.
In the first step of vulva morphogenesis during early L3, the four midline
flanking cells (daughters of P6.pap and P6.ppa) first arrange as a four-cell
rectangle with the anchor cell nestled into a pocket in the middle
(Fig. 1A). The anchor cell
later breaks through the center of the rectangle and opens a pore that
comprises the most dorsal part of the vulva lumen as the four cells fuse to
form the vulF ring (Sharma-Kishore et al.,
1999
).
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Eventually, the two extending processes of each crescent shaped half ring (Fig. 1A) meet and adhere to similar processes from mirror-image homologs projecting towards the presumptive vulva midline from the opposite half palindrome. As the lateral extensions insert between the hypodermis and the previously formed ring, the cells entering the vulva tend to migrate beneath their inner neighbors and push them upwards (in a dorsal direction, see Fig. 1A). The adherence of opposite extensions from mirror image half-ring homologs forms a full ring of cells, which encircle the vulva lumen. Ultimately, these adhering homologs fuse (with the exception of vulB1 and vulB2) to form a mature vulva.
As the lateral processes extend towards the vulva midline, the primordial vulva cell bodies also begin to move towards the vulva midline. Because the shape changes and extensions of lateral processes from vulva halves resemble cell extensions that cause cell movements and because the presumptive vulva cell bodies become displaced toward the vulva midline, we collectively refer to these shape changes and movements as vulva cell migrations.
The first cells to form extensions and to move are the precursors of vulE
(daughters of P6.paa and P6.ppp). Although the lateral vulva cell extensions
lead the way, the concave surface of these crescent-shaped cells also seems to
actively migrate towards the midline of the vulva. By moving along the ventral
membrane of the vulF ring, the four vulE cells (two cells per half vulva)
eventually form a ring of four cells connected to each other by adherens
junctions and align precisely along the DV axis attached to the ventral
surface of the vulF ring. In a similar fashion, more concentric rings of vulva
cells are formed by sequential recruitment of the next outer group of mirror
image homologous half rings to the vulva midline until seven precisely stacked
rings of cells have formed the vulva
(Sharma-Kishore et al.,
1999).
Normally during this process, all primordial vulva cells appear contiguously connected to one another through adherens junctions that are constantly remodeled as primordial vulva cells change shape and move. At no stage does a primordial cell normally become obviously dissociated from its neighbor(s).
During C. elegans development, different cell shape changes and
movements require a combination of Rac GTPases MIG-2 and CED-10, and their GEF
activator (UNC-73) (Lundquist et al.,
2001; Spencer et al.,
2001
; Steven et al.,
1998
; Wu et al.,
2002
), suggesting that Rac signaling could be required downstream
of different tissue-specific guidance receptors for controlling cellular
movements. Vulva morphogenesis has been shown to require the activity of
MIG-2, CED-10 and UNC-73. These GTPases are required primarily for shape
changes and movements of these cells during vulva morphogenesis
(Kishore and Sundaram, 2002
),
although they also redundantly regulate, to a minor extent, the axis of cell
divisions that form the primordial vulva cells. These results suggest that Rac
signaling promotes rearrangement of the cytoskeleton required for primordial
vulva cell migration.
Here, we provide evidence for a primordial vulva cell migration system in which PLX-1-expressing vulva cells that are poised to enter the forming stack of vulva rings are attracted towards SMP-1 expressed on the surface of their inner neighbors that have already entered the stack. SMP-1 expression occurs in a sequence that progresses from cells of ring 1 to ring 7. Using this model, the sequential expression of SMP-1 in each vulva cell as it forms a vulva ring explains the sequential attraction of outer neighbors towards inner neighbors and the orderly formation and alignment of concentric rings of cells that comprise the mature vulva.
Although the absence of SMP-1 and PLX-1 signaling causes vulva cell migration defects, the defects are not fully penetrant. This indicates that other mechanisms act in parallel with SMP-1 and PLX-1 to guide primordial cell migrations. The genetic data presented here suggest that CED-10 acts in the same pathway as SMP-1 and PLX-1, and that MIG-2 and UNC-73 act in a parallel pathway for vulva morphogenesis.
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Materials and methods |
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Microscopy and vulva morphogenesis observation
Vulva morphogenesis defects were scored by mounting 50 mM sodium
azide-treated animals on 2% agarose pads for observation using DIC and
fluorescence optics. Young adult hermaphrodites carrying the
ajm-1::GFP reporter for adherens junctions
(Simske and Hardin, 2001) were
scored for vulva precursor cell body migration defects and for vulva ring
formation defects (see Results and Tables). The ajm-1::GFP
translational reporter was visualized with a Leica DMRXA microscope to assess
epithelial cell morphologies. Confocal microscopy was performed using a Leica
DMFLS laser confocal microscope equipped with a 63 x PC APO CS lens
(1.40-0.60). Serial optical sections in the z-axis were collected
every 0.15 µm. Three-dimensional image reconstructions were obtained by
processing confocal z-axis series using Volocity (Improvision,
version 2.6.1) or the Leica Confocal software (version 11.04). Cell fate
analysis was carried out with an egl-17p::gfp (ayIs4)
reporter (Burdine et al.,
1998
).
Standard errors for percentages of vulva defects were calculated assuming a
binomial distribution with the observed percentage value and the actual sample
size. Statistical tests were carried out using a standard (two-tailed)
comparison of two proportions (Moore and
McCabe, 1998). All P values represent the probability
that the measured penetrance of the phenotype is significantly different
between two strains. A P value of less than 0.05 is considered to be
significant.
Molecular biology
Standard molecular biology methods
(Sambrook et al., 1989) were
used unless otherwise noted.
Transgenic constructs
The transcriptional and translational reporters of plx-1
(plx-1::gfp and plx-1p::PLX-1::GFP, respectively), the
plx-1(+) minigene, the plx-1p::UNC-73(+) minigene, the
smp-1p::gfp and the smp-1::GFP (here referred to as
smp-1p::SMP-1::GFP reporters) have been described previously
(Dalpe et al., 2004;
Ginzburg et al., 2002
).
For ectopic expression of SMP-1, we amplified a cDNA encoding the
extracellular and transmembrane regions of SMP-1 and subcloned it into the
SalI/PstI cut pPD95_77cplx plasmid
(Dalpe et al., 2004). The
resulting plasmid, pECTSMP_plx-1p::SMP-1(+), encodes a functional SMP-1
protein (as demonstrated in the Results), encompassing amino acids 1-616
(deleted for a portion of the cytoplasmic domain), under the control of the
plx-1 5' regulatory region.
To make a construct encoding PLX-1 with its cytoplasmic region deleted
(plx-1p::PLX-1delC::GFP), we used modified PCR primers to amplify a 670 bp
fragment of the plx-1(+) rescuing minigene
(Dalpe et al., 2004), digested
it with SphI and KpnI and, ligated the fragment into the
original plx-1 minigene plasmid cut with the same enzymes. The
resulting plasmid encodes the extracellular and the TM domains of PLX-1,
encompassing amino acids 1-1317 inclusively and an inframe GFP.
Germline transformation
Transgenic strains were as follows:
evIs140 [pPD95_77cplx plx-1::gfp); rol-6(su1006)] (plx-1 transcriptional reporter);
evEx162 [pZH127 plx-1p::PLX-1(+); rol-6(su1006)]; (cDNA rescues plx-1 mutant);
evIs162 [pZH127 plx-1p::PLX-1(+); rol-6(su1006)] (cDNA rescues plx-1 mutant);
evEx168 [pZH163 plx-1p::UNC-73(+); rol-6(su1006)] (unc-73 expressed by plx-1 5' regulatory region);
evEx169 [pZH157 plx-1p::PLX-1(+)::GFP; rol-6(su1006)] (functional plx-1 translational reporter);
evEx170 [pVGS1a smp-1p::SMP-1delC(+)::GFP; rol-6(su1006)] (functional smp-1 translational reporter);
evEx183 [pECTSMP_plx-1p::SMP-1; rol-6(su1006)] (ectopically expressed smp-1 cDNA); and
evEx184 [plx-1p::PLX-1(+)delC::GFP; rol-6(su1006)] (plx-1 minigene deleted for cytoplasmic domain).
Transgenic strains were generated by co-microinjection of the DNA mix into
the distal gonad arms of N2 or him-5(e1490) hermaphrodites
(Mello and Fire, 1995). DNA
mixes consisted of a test construct at a concentration of 50 µg/µl or 30
µg/ml, and a co-injection marker to create a final DNA concentration of 100
µg/µl. Transgenic extra-chromosomal arrays were integrated using a UV
irradiation based method (Mitani,
1995
). Integrated alleles were backcrossed five times to N2
Bristol (wild type) before phenotypic analysis.
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Results |
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In wild-type animals carrying the AJM-1::GFP reporter, the intermediate steps of ring formation during vulva morphogenesis are readily observed (in temporal order: early, intermediate and completed morphogenesis in Fig. 1G,H,I, respectively). During the third larval stage of wild-type animals, lateral processes from crescent shaped half-vulvae extend underneath the lateral edges of the next innermost primordial vulva cells (Fig. 1A,B). In smp-1(ev715), plx-1(ev724) and plx-1(nc37) mutants of the same stage, cells are occasionally found detached and mispositioned within the array of primordial cells (Fig. 1C). In wild-type adult hermaphrodites, the vulva appears as seven concentric rings (Fig. 1D). In adult smp-1 and plx-1 mutants, the primordial vulva cells frequently fail to assume a crescent shape. Instead, smp-1 and plx-1 mutant vulva cells either stay round or abnormally change their shape without generating lateral processes that extend towards the vulva midline (Fig. 1E,F,J). We observe cells that are detached and others that are still positioned as a contiguous row of abnormally shaped cells, flanking a vulva with abnormally shaped rings in adult smp-1 and plx-1 mutants (Fig. 1E,F,J). These defects are variably observed on only one (Fig. 1E,F) or on both sides (Fig. 1J) of the mutant vulvae.
In smp-1 and plx-1 mutants, not all guidance functions
are absent, as exemplified by migration defects that affect only one half of
the vulva (Fig. 1E,F; below).
In addition, when mutant vulva cells (i.e. smp-1 and plx-1
mutants) from one half of the vulva fail to generate two normally migrating
lateral extensions, these cells rarely, if ever, meet the extensions from
their mirror-image homologs in the opposite half of the vulva. However, vulva
cells that fail to contact their appropriate homologs at the vulva midline do
not fuse with non-homologous cells of another cell fate (see different
examples in Fig. 1E,F,J) (see
Shemer et al., 2000). These
results suggest that the mechanisms of target recognition for homotypic cell
fusion are still intact in plx-1 and smp-1 mutants, but
fusion is prevented because process extensions are not appropriately guided
for homologs to make contact.
The vulva cell migration and spreading defects of smp-1 and plx-1 mutants typically involve the most external cells (i.e. vulA, B1, B2, C, D) (Fig. 1C,F), but at a lower frequency many vulva cells from an entire half vulva are involved (Fig. 1J). In the latter case, the vulva cells that are detached from the forming vulva sometimes form a separate invagination, because some maintain their own ability to form torroids (Fig. 1J, asterisks).
In principle, the vulva cell lineages could be modified in plx-1 mutants, increasing or reducing the number of vulva cells and in this way perturb their normal migration pattern. To address this possibility, we carefully examined several larval stage 4 (L4) mutant animals without finding any alteration in the number of vulva cells (0%, n=52). However, we found occasional changes in the axis of cell division from longitudinal to transverse (4%, n=52).
To determine more precisely if primordial vulva cells are made in excess or
if cell fates are being altered in the mutants in ways that may not affect
axis of division, we examined the vulva cell reporter egl-17::gfp
(Burdine et al., 1998) for
dividing P6.p cells in early L3s. Dividing P6.p cells were readily observed in
all of these strains and there was never an excess of P6.p-derived vulva cells
(vulE and F). Only six out of 90 unc-73(rh40) and four of 95
plx-1(ev724) animals were missing one or two
egl-17::gfp-expressing cells. In late L3 early L4 larvae,
egl-17::gfp expression decreases in P6.p-derived cells and increases
dramatically in P5.p- and P7.p-derived cells (vulC and vulD). Only seven out
of 105 unc-73(rh40) and six of 162 plx-1(ev724) animals were
missing one or two egl-17::gfp late L3-expressing P5.p- or
P7.p-derived cells. In the few animals that lacked egl-17::gfp
expression in two cells, the two non-expressing cells were always from the
same half of the vulva. Extra expressing cells were never observed in the
mutants or wild type. Cell-fate defects (monitored by egl-17::gfp)
are therefore minor compared with primordial vulva cell migration defects,
suggesting that the SMP-1, PLX-1 and UNC-73/Rac pathway are functioning
primarily in guidance of these cells, rather than in determining their fates.
Moreover, it is conceivable that an early migration defect in these strains
[e.g. plx-1(ev724) and unc-73(rh40)] might disturb vulva
cell fate specification [e.g. perturbing the LIN-3 availability for one vulva
half or affecting LET-23 localization
(Kim, 1997
)].
Based on their highly related mutant vulva phenotypes, our data suggest that smp-1 and plx-1 are required for normally oriented vulva cell extension and stereotypical movements that take place during vulva morphogenesis. In smp-1 and plx-1 presumptive null mutants, the fact that not all guidance functions are absent indicates that unidentified guidance mechanisms can sometimes compensate for the absence of SMP-1 and PLX-1 signaling within each half vulva.
plx-1 and smp-1 function in the same pathway for vulva morphogenesis
Using the criteria defined above, we evaluated the penetrance of vulva ring
formation defects in plx-1 and smp-1 mutants. The
plx-1(ev724) mutant is predicted to encode a truncated receptor that
is missing its transmembrane and cytoplasmic domains
(Dalpe et al., 2004), and is
therefore predicted to lack signaling activity. In young plx-1(ev724)
mutant adults, we observe a penetrance of
52% vulva defects
(Table 1, row 2). For a
previously described male tail phenotype, the plx-1(ev724) allele is
genetically equivalent to another putative null deletion allele,
plx-1(nc37), in which the initiation codon and the first four exons
have been deleted (Dalpe et al.,
2004
; Fujii et al.,
2002
). plx-1(nc37) has vulva morphogenesis defects
essentially equivalent in penetrance and expressivity to those observed in
plx-1(ev724).
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As some guidance functions are preserved in the absence of smp-1 or plx-1 function, or both, a vulva morphogenesis defect can occur on the anterior half of the vulva without affecting the posterior half, and vice versa. If both vulva halves act independently in response to smp-1 and plx-1 functions, the frequency of animals with defects in both vulva halves should roughly equal the product of the frequencies of animals with defects in one or the other vulva half. Indeed, we find that 19% of plx-1(ev724) mutant animals (n=124) have a defect in the anterior half of the vulva, 21% have a defect in the posterior half and 7% have a defect in both halves. The observed 7% of animals with defects in both vulva halves is not significantly different from the expected frequency of 4% (P>0.05), suggesting that smp-1- and plx-1-mediated morphogenesis functions operate autonomously within each half of the vulva.
The second smp-1 gene in C. elegans
[semaphorin-1b or smp-2
(Ginzburg et al., 2002)] may
play a very limited role in vulva morphogenesis as only
3% of
smp-2(ev709) mutant animals have vulva cell migration defects
(Table 1, row 10), and
smp-1(ev715); smp-2(ev709) double mutants do not display any
enhancement of the smp-1(ev715) mutant penetrance (data not shown).
This also corroborates the lack of smp-2::gfp transcriptional
reporter expression in these cells (Dalpe
et al., 2004
; Ginzburg et al.,
2002
). Thus, plx-1 and smp-1 largely function in
the same pathway for the proper formation and guidance of vulva cell
migrations, while SMP-2, another putative ligand for PLX-1 in other
semaphorin-regulated mechanisms (Dalpe et
al., 2004
; Ginzburg et al.,
2002
), appears to play little, if any, role in vulva
morphogenesis.
PLX-1 is expressed on the vulva midline-facing membrane in migrating vulva cells and on the lumen membrane within the forming vulva
To determine in which cell types PLX-1 exerts its function, we used
previously described transcriptional and rescuing translational reporters for
plx-1 (Dalpe et al.,
2004) [e.g. evIs140[plx-1p::gfp] (transcriptional) and
evEx169[plx-1p::PLX-1(+)::GFP] (rescuing)]. Before the beginning of
vulva morphogenesis, both reporters are expressed in all the descendants of
P5.p and P7.p, and are expressed weakly in the descendants of P6.p
(Fig. 2A,B). The GFP signal of
the transcriptional plx-1::gfp reporter fills the cytoplasm and
nuclei of expressing cells at this stage
(Fig. 2A). However, the GFP
signal of the plx-1p::PLX-1::GFP translational reporter is found
predominantly at the cell membrane of the same cells, as expected if PLX-1 is
a transmembrane receptor (Fig.
2B). The cell expression pattern (described below), is the same as
the one observed for a previously described N-terminal translational reporter
(plx-1::egfp) (Fujii et al.,
2002
) and is consistent with the expression of our
evIs140[plx-1p::gfp] transcriptional reporter (data not shown).
|
At the beginning of vulva morphogenesis, a strong expression from the plx-1::gfp transcriptional reporter is found in all migrating vulva cells (Fig. 2C). As vulva morphogenesis progresses, expression from the plx-1p::PLX-1::GFP translational reporter increases at the plasma membrane of migrating vulva cell (Fig. 2B,D-F). However, although some signal is found on the entire cell membrane, PLX-1::GFP appears to be predominantly localized on the vulva center facing membrane (future lumen surface) of primordial vulva cells destined to enter the vulva proper (Fig. 2D,E).
Through analysis of reconstructed 3D confocal images, we observe that, at the end of morphogenesis, PLX-1::GFP is predominantly expressed in the most ventral vulva rings [vulA, vulB1, vulB2, vulC and vulD (which are P5.p and P7.p derived)] and the signal is localized along the lumen formed by these cells (Fig. 2 M,N).
The subcellular localization of the PLX-1::GFP signal is surprisingly not concentrated at the tip of processes but is rather localized on a more central segment of the concave cell surface contacting its inner neighbor, this segment being shorter in cells poised to enter the vulva proper and longer in cells that have already entered the vulva stack (Fig. 2M,N). This suggests that the zone of PLX-1 localization increases as the zone of contact between a migrating cell and its inner neighbor increases.
PLX-1 subcellular localization partially depends on SMP-1 and UNC-73
To evaluate whether PLX-1 subcellular localization is dependent upon its
predicted ligand SMP-1, we introduced the PLX-1::GFP translational reporter
into smp-1(ev715) mutants. In smp-1(ev715), as in the wild
type, the PLX-1::GFP signal is observed predominantly on the vulva
midline-facing side of wild-type crescent shaped migrating primordial vulva
cells (Fig. 2G,H). However, on
vulva cells from smp-1 mutants that display a non-crescent-shaped
migration phenotype, plx-1p::PLX-1::GFP is more uniformly distributed
on the whole cell membrane, rather than just the midline-facing side
(Fig. 2I,J).
We also evaluated whether unc-73, which displays genetic interaction with plx-1 (see results below), could affect the localization of PLX-1::GFP. In unc-73(rh40) (the strongest allele that does not display a severely lethal phenotype), we obtained results that were essentially identical to those observed in the smp-1(ev715) background (Fig. 2K,L). These results suggest that the specific subcellular localization of PLX-1 on the presumptive lumen of ring-forming vulva cells is partially dependent on SMP-1 and UNC-73, perhaps indicating that some PLX-1 clustering on the future lumen membrane may require SMP-1 ligand on an inner neighboring cell and a cell-autonomous intracellular polarizing function of UNC-73.
SMP-1 is expressed sequentially in ring-forming vulva cells
The previously described transcriptional (smp-1::gfp) and genomic
translational (smp-1::GFP; here referred to as
smp-1p::SMP-1::GFP) reporter genes
(Dalpe et al., 2004;
Ginzburg et al., 2002
) were
used to evaluate smp-1 expression during vulva development. At the
beginning of the third larval stage, both types of reporters are expressed in
dividing VPCs. The smp-1::gfp transcriptional reporter is
predominantly expressed in P6.p-derived cells, and is more weakly expressed in
P5.p and P7.p daughters. Before the beginning of morphogenesis, the expression
in P6.p-derived cells diminishes over time, while the expression in P5.p and
P7.p daughters increases and later decreases.
In order to follow SMP-1 protein expression during vulva morphogenesis, we
focused on the translational smp-1p::SMP-1::GFP reporter
(Dalpe et al., 2004;
Ginzburg et al., 2002
), because
it encodes a functional SMP-1 protein with the ability to rescue the
smp-1(ev715) vulva morphogenesis defect (see above; compare, rows 9
and 8 in Table 1). Expression
from the smp-1p::SMP-1::GFP translational reporter is dynamic. Early
during vulva morphogenesis, the protein is observed only in the first
effective ring of cells (vulF) that in principle can serve as a template for
aligning other cells that will form the next ring of the vulva proper.
SMP-1::GFP signal appears localized to vulF cell membranes facing the anchor
cell and also on their ventral surface
(Fig. 3A,B). At this time,
other primordial vulva cells, the processes of which have not completed their
migration to the vulva midline, do not exhibit any detectable expression
(Fig. 3A,B). Later on, vulva
cells and their processes entering the forming vulva upregulate SMP-1::GFP on
the lumen side of the newly forming ring-shaped cell
(Fig. 3C-F), then attach to the
ventral side of the previously formed vulva ring, pushing it upwards. This
cycle of SMP-1 expression repeats until all 22 primordial cells have migrated,
aligned and attached to one another to form the vulva proper (shown for the
beginning, intermediate and late stages of vulva morphogenesis in
Fig. 3A-J).
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In principle, sequential expression of smp-1 from cells already in or poised to enter the forming vulva could depend on a cell-autonomous or non-cell-autonomous program. In a cell-autonomous situation, vulva cells in plx-1 mutants that fail to migrate towards the vulva midline express SMP-1::GFP at the same time as those that migrate normally. However, if SMP-1 expression is activated non-cell autonomously by a cell position-dependent mechanism (e.g. dependent on reaching and contacting the forming vulva or dependent on contiguous contacts between inner neighbors and the forming vulva, or dependent on a certain position within a morphogen gradient that radiates from the vulva midline), its expression should not be activated in mutant cells that fail to migrate towards the midline. In plx-1(ev724) mutants that show migration defects specific to one side of the presumptive vulva (i.e. anterior only), we observe a correlation between lack of SMP-1::GFP expression and cells that do not migrate properly towards the vulva midline, in contrast to cells that migrate correctly (Fig. 3K,L). These observations favor a position-dependent model of smp-1 activation: those cells that do not migrate to the forming vulva are not autonomously programmed to express detectable levels of the SMP-1::GFP translational reporter.
Our results strongly support the idea that SMP-1 is induced in primordial vulva cells as they begin to form the vulva and this expression recruits (by attraction) the next outermost PLX-1-expressing cells into the vulva.
A gain of function in Ras GTPase [let-60(n1046gf)] requires plx-1 to form ectopic pseudovulvae and pseudovulvae express smp-1 reporters
Gain-of-function mutations in Ras let-60(1046gf) cause VPCs that
would normally adopt 3° fates to now adopt 2° and 1° fates. This
produces ectopic pseudovulvae in addition to a largely normal vulva proper
(multivulva phenotype or Muv) (Ferguson and
Horvitz, 1985; Han and
Sternberg, 1990
). The number of cells in each pseudovulva varies.
Some pseudovulvae have vulF cells as centers of attraction for neighboring
cells, whereas others may lack vulE and vulF, in which case vulD could serve
as a center of attraction for vul neighbors. In each case, the order of
attraction appears preserved as in the wild type, so some pseudovulvae may
have all the vul cell types (vulF, vulE, vulD, vulC, vulB, vulA in proper
order) and others may only comprise rings vulD, vulC, vulB, vulA in proper
order (Shemer et al., 2000
).
In light of our smp-1- and plx-1-mediated model of vulva
morphogenesis, this suggests to us that the most dorsal vulva cell fate in a
pseudovulva could function as an organizer by expressing SMP-1 to serve as an
attractive guidance cue for neighboring PLX-1-expressing cells, just as
happens in normal vulva formation.
This prompted us to examine the expression of the smp-1::gfp transcriptional reporter in let-60(n1046gf) strain. The smp-1::gfp is initially expressed in dividing P6.p cells in wild-type L3 hermaphrodites (see Fig. S1 in supplementary material). In L3 stage let-60(n1046gf) mutants, we observe what appears to be multiple vulva cell clusters that express the smp-1::gfp. The signal corresponds in intensity to the one we normally observe in P6.p-derived cells of wild-type hermaphrodites. Later on, we observe the smp-1::gfp expression in ring cells of pseudovulvae. This suggests that smp-1 is expressed in cells that serve as centers of attraction for other cells that will form a vulva or a pseudovulva.
Torroids tend to form normally in a half vulva autonomous manner for both
the pseudo and normal vulvae of let-60(n1046gf) mutants, with the
exception of vulA cells that tend to be simultaneously attracted toward the
midline of neighboring primordial clusters and therefore never enter either
cluster because of inter-vulva competition
(Shemer et al., 2000).
However, we frequently observe severe torroid formation defects in both the
pseudo and normal vulvae of plx-1(ev724);let-60(n1046gf) double
mutants (see Fig. S1 in supplementary material). In
plx-1(ev724);let-60(n1046gf), torroid formation is dramatically
impaired when compared with the control let-60(n1046gf) strain (a
fusion between vulA cells of two different vulvae was not considered to be a
defect for this comparison). The type of vulva morphogenesis defects are
similar to the ones we previously observed in plx-1(ev724)
mutants.
All together, these results suggests that pseudovulvae form torroids by means of initiating sequential smp-1::gfp expression in cells of the most dorsal vulva cell fate in let-60(n1046gf) mutants. Not surprisingly, we also find a role for plx-1 in guiding torroid formation in pseudovulvae. The fact that we also observe normal torroid development in plx-1(ev724);let-60(n1046gf) suggests that plx-1 is not the only mechanism at work for proper vulva cell migration in pseudovulvae.
SMP-1 expression is instructive and PLX-1 expression is permissive for guiding vulva cell movements during morphogenesis
If SMP-1 has an instructive guidance function, disturbing its precise
temporal expression pattern during vulva morphogenesis should, in theory,
misguide migrating presumptive vulva cell extensions. By contrast, no effects
would necessarily be expected if SMP-1 has a purely permissive role in vulva
cell movements. To further examine these possibilities, we placed a functional
smp-1 cDNA (functional in spite of being truncated for its
cytodomain-encoding portion, see Table
1, row 9 versus row 8) under the control of the plx-1
5' regulatory region to drive expression in all vulva cells with a
predominant expression in P5.p- and P7.p-derived cells (see above). We observe
that animals carrying this plx-1p::SMP-1 transgene on an
extra-chromosomal array show variable body morphology defects (data not
shown), a result that is not surprising considering the previously described
role for smp-1 and smp-2 in embryonic morphogenesis
(Ginzburg et al., 2002). These
transgenic animals also exhibit frequent vulva cell migration defects
[Table 1, row 12; 11% at
20°C (n=35) and 38% at 25°C (n=42)] like those
observed in plx-1 and smp-1 mutants
(Fig. 4A-C). The defects are
frequently observed with vulA and vulB cells, consistent with the strong
plx-1 regulatory region activity in these cells (see PLX-1::GFP
expression pattern above).
|
C. elegans Rac GTPases MIG-2 and CED-10, and their putative GEF activator UNC-73 function in a pathway parallel to plx-1
Vulva cell migration defects similar to those described in the
smp-1 and plx-1 mutants have also been described in mutants
for the C. elegans genes mig-2 and ced-10, encoding
homologs of mammalian Rac GTPases (Kishore
and Sundaram, 2002; Lundquist
et al., 2001
; Zipkin et al.,
1997
). Similar defects have also been described for mutants of the
C. elegans unc-73 gene (Kishore
and Sundaram, 2002
), which encodes a guanine exchange factor that
functions upstream of MIG-2 and CED-10 for many guided cell migrations
(Lundquist et al., 2001
;
Steven et al., 1998
;
Wu et al., 2002
).
We also recently described a role for unc-73, mig-2 and
ced-10 in a pathway that functions in parallel to smp-1 and
plx-1 for preventing anterior displacement of ray 1 cells during male
tail development (Dalpe et al.,
2004). This led us to examine whether unc-73, mig-2 and
ced-10 might act in the same or in a pathway parallel to
smp-1 and plx-1 for vulva morphogenesis. As for male ray 1
cell movements, both mig-2 and ced-10 single mutants display
few vulva defects on their own (Table
2, rows 3,4) when compared with wild type
(Table 2, row 1) or
plx-1 mutants (Table
2, row 2). However, as reported previously
(Kishore and Sundaram, 2002
),
the mig-2(mu28); ced-10(n1993) double mutants show a considerably
enhanced expressivity and penetrance of vulva cell migration defects
(Table 2, row 5) when compared
with either single mutant. Although ced-10(n1993) is not a null
allele (Lundquist et al.,
2001
), mig-2(mu28) is a null
(Zipkin et al., 1997
);
therefore, these results strongly suggest that the two genes act in parallel
to guide vulva cell migrations and positioning.
|
Two non-null mutant alleles of unc-73 also show vulva
morphogenesis defects like those observed in mutants of plx-1 and
smp-1. unc-73(rh40) behaved as expected for a strong loss-of-function
allele for this phenotype compared with the weaker hypomorph
unc-73(e936) (R. Steven, personal communication)
(Table 2, row 8 versus row 9).
Based on the enhancement phenotype observed between plx-1(ev724) and
mig-2(mu28), and on the likely possibility that unc-73
functions upstream of both mig-2 and ced-10 in many cell
migration processes (Lundquist et al.,
2001; Wu et al.,
2002
), one would predict a strong enhancement in the
plx-1(ev724); unc-73(rh40) double mutant compared with either single
mutant. Unfortunately, this double mutant is embryonic lethal
(Dalpe et al., 2004
); however,
plx-1(ev724); unc-73(e936) double mutants survive and show a
synergistically enhanced vulva morphogenesis phenotype when compared with
either plx-1(ev724) or unc-73(e936) single mutants
(Table 2, row 10 versus rows 9
and 2). Taken together, our results suggest that unc-73 and
mig-2 have functions that parallel plx-1 functions during
vulva formation. However, the genetic data do not exclude the possibility that
unc-73, mig-2 and ced-10 also have a related function in the
PLX-1 pathway (Fig. 5C).
|
Interestingly, when the region of cDNA encoding the cytodomain of PLX-1 is deleted from PLX-1::GFP (PLX-1delC::GFP), the construct no longer rescues the plx-1(ev724) vulva cell defects (see results above), but enhances it considerably when compared with the null on its own (78% versus 52%, Table 1, row 7 versus row 2). The finding that PLX-1delC::GFP induces more severe defects than a putative plx-1-null allele suggests that in the absence of the endogenous plx-1(+), the PLX-1delC::GFP functions as a dominant negative, possibly by interfering with a signaling component that takes part in a pathway functioning in parallel to PLX-1 for vulva morphogenesis.
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Discussion |
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In smp-1 and plx-1 mutants, primordial vulva cells fail
to extend two normal processes towards the vulva midline, suggesting that a
mechanism guiding these processes is lost in the mutants, which prevents the
formation of normally stacked vulva rings. Our genetic analyses show that both
single smp-1(ev715) and plx-1(ev724) null mutants display
approximately the same penetrance for this phenotype. The fact that a
smp-1(ev715); plx-1(ev724) double mutants do not show any enhancement
over either single mutant indicates that SMP-1 and PLX-1 function in the same
pathway for vulva formation (Fig.
5C). This is entirely consistent with the finding that SMP-1 binds
PLX-1 in vitro (Fujii et al.,
2002).
Rac GTPase- and UNC-73-dependant mechanisms of vulva cell migration
The incomplete penetrance of the null alleles and the lack of enhancement
in double mutants indicates that there must be other mechanisms that act in
parallel with SMP-1 signaling to regulate vulva cell migrations and
morphogenesis. Similar vulva cell migration defects were previously reported
for mutants of genes encoding C. elegans homologs of the Rac GTPases,
MIG-2 and CED-10, and a gene encoding the Rac activator UNC-73. As observed by
others (Kishore and Sundaram,
2002), we also find that loss-of-function mutations in
mig-2 and ced-10 alone cause few vulva cell migration
defects, but mig-2(mu28); ced-10(n1993) double mutants have highly
penetrant vulva cell migration defects. This suggests that mig-2 and
ced-10 function redundantly in vulva morphogenesis
(Fig. 5C).
Partial loss-of-function mutations in unc-73 also cause a
significant penetrance of vulva cell migration defects, which is entirely
consistent with the finding that this gene is required for the function of
mig-2, ced-10 and rac-2 in other types of guided cell
migrations (Lundquist et al.,
2001; Wu et al.,
2002
). Our finding that mutations in mig-2 and
unc-73 enhance the smp-1 or plx-1 null mutant vulva
defects suggests that they function in a pathway parallel to smp-1
and plx-1 (Table 2;
Fig. 5C). Mutations in
ced-10 are unable to enhance the plx-1 mutant vulva defects,
suggesting that CED-10 acts redundantly with MIG-2 because they act in
parallel mechanisms (CED-10 in the PLX-1 mechanism and MIG-2 in an unknown
mechanism). However, CED-10 is probably not the only pathway through which
PLX-1 functions, because if it were, plx-1 mutants are not expected
to have a major vulva phenotype. Considering the synergistic enhancement of
vulva morphogenesis defects we observe in the mig-2(mu28);
ced-10(n1993) double mutants (77%
Table 2, row 5), there is a
high probability that MIG-2 and, by implication, UNC-73 also functions in the
same pathway as PLX-1 (Fig.
5C). This interpretation takes into account that
ced-10(n1993) and unc-73(rh40) are probably not null alleles
as nulls of these genes are lethal
(Lundquist et al., 2001
;
Steven et al., 1998
).
The involvement of Rac GTPases in PLX-1 signaling is consistent with the
classical biochemical view of plexin signaling in which activated
RacGTP binds the cytoplasmic portion of plexin receptors
(Driessens et al., 2001;
Hu et al., 2001
;
Pasterkamp and Kolodkin, 2003
;
Turner et al., 2004
).
The nearly full enhancement (90% Table 2, row 10) of a plx-1 putative null allele by a partial loss-of-function in unc-73 indicates that PLX-1 and UNC-73 signaling mechanisms can account for most and possibly all of the guided cell migrations involved in vulva morphogenesis. However, as PLX-1 and UNC-73 act in parallel, it is possible that they act in different cell types. Our results show that plx-1 mutant animals carrying a plx-1p::PLX-1(+) minigene and unc-73 mutants carrying a plx-1p::UNC-73(+) minigene are both rescued for their respective vulva phenotypes. Thus, our results suggest that PLX-1 and UNC-73, and by implication the GTPases activated by UNC-73 (e.g. MIG-2 and CED-10) function cell-autonomously in all vulva cells to guide their movements.
The observed interference by the cytodomain-deleted PLX-1 transgene in a
plx-1 null mutant suggests that components acting in parallel to and
redundantly with PLX-1 for vulva cell migration are probably interacting with
the extracellular or transmembrane domains of PLX-1. Based on the intermediate
penetrance of the plx-1(ev724) phenotype, this redundant mechanism
must have at least one function that is independent of PLX-1. Nevertheless,
the dominant-negative effect we observe with the cytodomain-deleted PLX-1
transgene indicates that components of this unknown parallel mechanism might
take part in a protein complex that includes PLX-1. Plexins usually function
in conjunction with co-receptors
(Pasterkamp and Kolodkin,
2003). The proposed dominant-negative effects of
plx-1p::PLX-1delC::GFP may result from an effect on the ability of a
co-receptor to bind and be activated by a non-semaphorin ligand involved in
vulva morphogenesis.
A model for vulva morphogenesis based on sequential SMP-1 expression
Seven vulva cell types are formed from 22 epithelial cells (primordial
vulva cells) arranged in a longitudinal row along the ventral epidermis.
During vulva formation, these cells are sequentially recruited to the midline
position of the primordial vulva, starting with cells closest to the center of
the primordium and extending outwards. First the four innermost cells (vulF
cells) are born surrounding the anchor cell at the midline of the primordial
vulva and eventually fuse to form a single ring-shaped cell (vulF ring). Next,
the four vulE cells (outer neighbors to vulF cells) are recruited to the vulva
midline, align just ventral to the vulF ring and eventually fuse to each other
to form the vulE ring. This process continues for vulD (two cells recruited),
vulC (four cells recruited, two cells per half ring), vulB2 (two cells
recruited), vulB1 (two cells recruited) and vulA cells (four cells recruited,
two cells per half ring) until the longitudinal row of 22 primordial cells are
converted into seven rings of cells aligned along the dorsoventral axis that
comprise the mature vulva (Fig.
5A,B).
The spatial and temporal expression of plx-1 and smp-1 in the primordial vulva cells is consistent with a role for these proteins in mediating orderly attraction of these cells to the midline of the forming vulva and can also explain their precise alignment to form a contiguous vulva lumen. As observed with our PLX-1::GFP translational reporter, PLX-1 is found at the cell membrane of vulva cells undergoing morphogenesis, with a much greater localization on membrane facing the vulva midline (the presumptive lumen membrane of the vulva). The SMP-1::GFP reporter is also highly expressed on vulva cell membrane facing the midline but this expression is dynamic. SMP-1::GFP expression is first observed on the cell membranes of each presumptive vulF cell on the side that faces the anchor cell (Fig. 3), which marks the vulva center and presumptive vulva lumen. Expression on the other primordial vulva cells is first evident on the midline facing membrane (presumptive lumen side) of these cells as they acquire a ring shape and as they dock onto the ventral end of the forming vulva cell stack. Expression continues on the lumen membrane of these cells even after they have formed a ring and become an integral part of the forming vulva. Sequentially, the next set of vulva cells start expressing SMP-1 on their presumptive lumen membrane as they form a ring ventral to the previously formed ring. This process repeats until the seven precisely stacked vulva rings are formed (Fig. 5A,B).
SMP-1 is involved in lateral process extension and migration of primordial vulva cells to the vulva midline, and of positioning vulva rings precisely one on top of the other. Theoretically, SMP-1 could function as a diffusible cue that emanates from the vulva midline and attracts PLX-1-expressing vulva cell processes; however, this is unlikely given that SMP-1 is a predicted transmembrane protein. We propose that SMP-1 guides vulva cell morphogenesis by means of its precise spatiotemporal expression pattern. In so doing, poised PLX-1-expressing vulva cells that are adjacent to a SMP-1 expressing half ring, extend lateral processes that crawl underneath the half ring, presumably to reach high concentration of SMP-1 present at the ring lumen membrane of its inner neighbor.
The sequential expression of the SMP-1 on the lumen membrane of ring cells ensures that only the PLX-1-expressing outer neighbors will extend processes and move towards the midline before other primordial vulva cells even further away from the midline extend lateral processes in the same direction. This is most probably what accounts for the ordered stacking of vulva cell types during vulva morphogenesis. Second, sequential SMP-1 expression from ring shaped cells of the vulva proper dictates a polarity of migration, ensuring that cell processes from vulva cells neighboring a SMP-1 expressing ring cell (in the vulva proper) extend towards the forming vulva lumen. Third, the processes of forming half rings that spread underneath the previously formed half ring and follow the outline of the presumptive vulva lumen ensures that each new ring aligns itself according to the shape of the previously formed ring.
In principle, if a precise SMP-1 expression pattern is necessary for the orderly migration of vulva cell processes, then disturbing the spatiotemporal sequence should greatly affect the polarity of migration within the vulva primordium. Consistent with this idea, a more ubiquitous SMP-1 expression, which is driven by the plx-1 5' regulatory region (with higher expression from the P5.p- and P7.p-derived cells), causes vulva migration defects even when competing with the endogenous smp-1 gene. Furthermore, we observe a dramatic enhancement of vulva cell migration defects when ectopic SMP-1 expression operates in the absence of all endogenous smp-1(+) function [i.e. plx-1p::SMP-1 in a smp-1(ev715) background], indicating that new centers of vulva cell recruitment can be created by inducing SMP-1 expression in different vulva cells. By implication and based on the smp-1 loss-of-function phenotype, SMP-1 expression in the wild type initiated at the midline has an instructive role for the orderly recruitment of vulva cells (Fig. 5A,B).
Interestingly, we observe cell detachment from externally positioned vulva primordium cells in smp-1 and plx-1 mutants as they begin vulva morphogenesis. This suggests that low levels of SMP-1, undetectable by the smp-1::GFP translational reporter, might also guide these cells in a cell-autonomous or non-autonomous manner. This interpretation would be consistent with our observation that P5.p- and P7.p-derived cells initially express low levels of the smp-1::gfp transcriptional reporter.
Interestingly, PLX-1 localizes to the midline-facing side of the
crescent-shaped migrating vulva cells at first to a patch at the region that
first enters the vulva proper then spreads along the entire leading concave
edge as the cell aligns with the previously formed vulva ring. This suggests
that the vulva cells use this entire leading edge for transducing the SMP-1
signaling into a migration and adhesion response. This adds to the concept
that the tips of lateral cell extensions constitute the only motile sudomains
of vulva cells that guide their migration, as suggested by direct microscopic
observation (Sharma-Kishore et al.,
1999). Based on the SMP-1- and PLX-1-dependent guidance function,
we propose that the leading edge (i.e. the midline-facing membrane expressing
PLX-1) of vulva cells senses SMP-1 on the lumen of the neighboring ring cell,
then modifies the shape of that leading edge to spread and adhere to the lumen
surface. PLX-1 might even guide migration by means of a spreading mechanism
using the well-known adhesion functions of activated Rac signaling
(Luo, 2000
;
Mueller, 1999
;
Suter and Forscher, 1998
;
Yuan et al., 2003
), which is
required in a cell-autonomous manner for vulva morphogenesis
(Kishore and Sundaram,
2002
).
PLX-1 localization to the lumen-facing side suggests that migrating vulva
cells could use this PLX-1 subcellular domain as a structure that leads their
migrations the same way axons use growth cones or the gonad primordium in
C. elegans uses distal tip cells
(Hedgecock et al., 1987).
Consistent with this, we have observed the accumulation of actin at the
leading edge of migrating vulva cells is correlated with the greater
accumulation of PLX-1 at the leading edge (data not shown).
Observations of abnormally migrating cells demonstrate that the subcellular
localization of PLX-1 partially depends on SMP-1 and UNC-73, suggesting that
PLX-1 is probably recruited to the midline facing side of the migrating cell
by the ligand it recognizes and by intracellular signaling events triggered by
UNC-73, which could involve docking of PLX-1 to activated Rac (e.g. activated
MIG-2 and CED-10 could be localized to the leading edge). Particularly, UNC-73
has been shown to be required for polarizing neuroblast migration in C.
elegans, along with UNC-40/DCC and DPY-19
(Honigberg and Kenyon, 2000).
Other unknown factors may also help localize PLX-1 to this membrane
compartment.
Homotypic recognition between homologous vulva cells is not affected by SMP-1 or PLX-1 signaling
The tips of misguided cell processes do not fuse in a heterotypical manner
in an smp-1 or a plx-1 mutant, indicating that recognition
of the homolog target processes is independent of the plx-1 and
smp-1 functions. Correspondingly, Sharma-Kishore and colleagues
(Sharma-Kishore et al., 1999)
have shown that, in laser-ablation experiments targeting only one vulva half,
the cells from the non-ablated half send out processes that migrate correctly,
but instead of meeting their contralateral homolog target pairs, they meet
processes from the same half ring at the presumptive vulva midline
(Sharma-Kishore et al., 1999
).
Taken together, these findings suggest that homologous target cell recognition
is a specific event that normally occurs in a homotypical manner and is
independent of PLX-1 signaling. Considering the fact that each vulva half
begins morphogenesis non-simultaneously
(Sharma-Kishore et al., 1999
),
these findings also indicate that the migration guidance mechanisms are
autonomous within each vulva half. Moreover, pseudovulvae from the
let-60(n1046) Muv mutant lacking half of the primordial cells form
torroids autonomously. Characterization of many pseudovulvae in these Muv
mutants revealed asymmetry between vulva halves, evidence for the model in
which vulva halves develop autonomously
(Shemer et al., 2000
).
The independence of each vulva half could arise by two separate signals that regulate individually the independent formation of the anterior and posterior half vulva. However, our data showing that the effects of PLX-1 are independent for each half vulva indicate that SMP-1 and PLX-1 can function independently in each half vulva. Thus, a single signal, SMP-1, possibly emanating from a single midline source, can have independent effects on cells destined to form each vulva half.
The dorsal vulva ring organizer initiates SMP-1 expression
VPC-derived cells destined for a primary fate make vulF cells, which appear
programmed to express SMP-1 as they form the first ring during normal vulva
development. In let-60(gf) Muv mutants, there are cell fate changes
such that not only P6.p but also other VPCs can form primordial vulva cells
developing into pseudovulvae. The cells forming the most dorsal ring in
pseudovulvae, whether they are of vulF or vulD fate, act as organizer for
torroid formation and are able to express the SMP-1, which presumably attracts
PLX-1-expressing neighbors almost to the same level as vulF-fated cells of the
normal vulva. As let-60(gf) mutations effect complex fate changes
among the VPCs that allow them to bypass the anchor cell mediated induction,
we believe it likely that pseudovulva midline cells that serve as an organizer
for torroid formation may adopt some, but not all, of the same properties of
vulF cells from the normal vulva. One of these properties is the ability to
express SMP-1 and thereby attract PLX-1 expressing vulva primordial cells to
form torroids.
Whether an inductive signal is involved in inducing SMP-1 expression in the normal vulva remains to be examined. However, the correlation we observe between cells that did not migrate towards the midline in plx-1 mutants and the lack of SMP-1 expression suggests that there might be a position-dependent stimulation of smp-1 expression. In this manner, cells that reach their normal position and are about to enter the vulva proper are instructed to express SMP-1 at the lumen membrane, and, by so doing, a cell with proper shape and position serves as template for the next round of vulva cell migration. This way of establishing sequential semaphorin expression would also ensure that smp-1- and plx-1-dependent guidance functions are autonomous within each half of the vulva.
SMP-1 appears to be localized in an active subcellular region of the cell
membrane of ring cells. These ring cell protrusions express membrane-anchored
SMP-1 and contain higher concentrations of actin (data not shown). They extend
away from the vulva lumen, suggesting that cells expressing PLX-1 that are
poised to enter the vulva proper could sense a low SMP-1 concentration on the
thin membrane protrusions and initiate migration towards a higher SMP-1
concentration on the lumen side of the forming vulva. Likewise, in
Drosophila, long-range cell-cell signaling can be established between
developing wing imaginal disc cells and signaling centers through actin-based
extensions called cytonemes that project from disc cells
(Ramirez-Weber and Kornberg,
1999), indicating that both morphogens and guidance molecules
could, in this way, increase their range of action.
Summary
Multicellular tube morphogenesis, such as the development of the
Drosophila tracheal system and the vascular system in vertebrates,
requires regulatory mechanisms coordinating the complex cell shape changes and
movements involved (Nelson,
2003). The stereotypical series of cellular shape changes and
movements taking place during C. elegans vulva morphogenesis makes it
a powerful model system for a genetic approach to understanding these
processes. Following a cell differentiation phase, linearly arranged
primordial vulva cells tend to send extensions and move in a mirror image
fashion toward the vulva midline, and in so doing form an aligned stack of
vulva ring cells. We propose that sequential SMP-1 expression from the ring
cells formed at the vulva midline ensures coherent movements of the
PLX-1-expressing cells as they enter the vulva proper. The apparent long-range
attraction of some of the outer primordial vulva cells can therefore be
explained by a series of short-range attractions involving the sequential
establishment of local semaphorin gradients. Interestingly, the molecular
pathways involving semaphorin signaling and Rac function are required in a
strikingly similar manner for guided cell migrations taking place in the
C. elegans male tail (Dalpe et
al., 2004
) and in hermaphrodite vulva morphogenesis. Conceivably,
the two C. elegans sexual organs could have co-evolved a common
mechanism for their morphogenesis.
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ACKNOWLEDGMENTS |
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