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First published online 16 May 2007
doi: 10.1242/dev.005108
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Developmental Biology Unit, University Pierre et Marie Curie (Paris 6) and CNRS, Observatoire Océanologique, 06230 Villefranche-sur-Mer, France.
* Author for correspondence (e-mail: christian.gache{at}obs-vlfr.fr)
Accepted 10 April 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Sea urchin, Embryo, Primary mesenchyme, Cell migration, Guidance, Differentiation, VEGF, Skeleton, Morphogenesis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Directed cell movement is controlled by external signals that guide
migrating cells to their target. Primordial germ cells and leucocytes receive
signals through G protein-coupled 7-TM receptors
(Raz, 2004
;
Parent, 2004). Axon guidance
relies on specific ligands (netrin, slit, ephrin and semaphorin) and their
receptors (Dickson, 2002),
some of which are also used by endothelial cells during vascular development
(Carmeliet and Tessier-Lavigne,
2005). Recently, gradients of morphogens such as members of the
BMP, SHH and WNT families were also shown to act as positional cues in axon
guidance (Yoshikawa and Thomas,
2004). Many different cell types respond through tyrosine kinase
receptors to signaling molecules controlling migration, such as PDGF in
mesoderm cells in Xenopus (Nagel
et al., 2004), GDNF in enteric neurons in mammals
(Natarajan et al., 2002), FGFs
in mesoderm cells during gastrulation in mouse and chick
(Sun et al., 1999;
Yang et al., 2002) and during
trachea formation in Drosophila (where FGF is also known as BNL -
Flybase) (Ribeiro et al.,
2002; Sato and Kornberg,
2002), VEGFA in precursors of hematopoietic and neural cells
(Hiratsuka et al., 2005;
Zhang et al., 2003), PVF1
(VEGF-PDGF) in hemocytes (Cho et al.,
2002) and together with EGF-family ligands, Keren and Spitz, in
border cells in Drosophila
(Duchek et al., 2001;
Duchek and Rorth, 2001;
McDonald et al., 2006).
The primary mesenchyme cells (PMCs) of the sea urchin embryo offer a remarkable model to study the mechanism of cell migration and its involvement in morphogenesis (Fig. 1A). The PMCs synthesize the spicules, which are the mineralized rods that constitute the skeleton of the embryo. At the mesenchyme blastula stage, the PMC precursors (32 cells in most species) leave the blastula wall to become the PMCs, which migrate within the blastocoel and become positioned along the inner ectoderm wall following a stereotypical pattern. At the early gastrula stage they form a characteristic ring with two symmetrical ventrolateral clusters. Spiculogenesis begins by the formation of a tri-radiate spicule rudiment in each ventrolateral cluster. Later, the two rudiments elongate and branch to form the two symmetrical halves of the skeleton. From specification to terminal differentiation, the process takes about two days, and although the number of cells implicated is small, the spatial patterning is stereotypical and the transparency of the embryo allows easy observation of cell behavior.
In the sea urchin embryo, several lines of evidence indicate that ectoderm
influences many aspects of skeleton formation, including timing
(Ettensohn and McClay, 1986),
growth rate (Guss and Ettensohn,
1997), number of spicules and final size
(Armstrong et al., 1993). The
three-dimensional structure of the skeleton and its bilateral symmetry are
foreshadowed and determined by the spatial organization that PMCs can achieve
only within the embryo, in intimate contact with the ectoderm wall, suggesting
guidance cues from the ectoderm control the well-defined PMC spatial pattern
(Gustafson and Wolpert, 1967;
Ettensohn, 1990;
Malinda and Ettensohn, 1994).
However, little is known at the molecular level about the interaction between
ectoderm and the PMCs. Perturbation of the function of several secreted
molecules or transcription factors expressed in the ectoderm affects skeleton
formation, including nectin (Zito et al.,
2000), nodal and BMP2/4 (Duboc
et al., 2004), Msx (Tan et
al., 1998), coquillette (Tbx2-3)
(Croce et al., 2003;
Gross et al., 2003), Dri
(Amore et al., 2003) and Otp
(Di Bernardo et al., 1999;
Cavalieri et al., 2003).
However, with the exception of Otp (the targets and function of this
transcription factor are not known), these molecules appear to be implicated
primarily in patterning the oral-aboral (AO) axis, rather than specifically
controlling PMC organization, Therefore, the molecular basis of the
interaction between ectoderm and PMCs is still not understood, as the
signaling molecules and guidance cues implicated in this interaction have not
yet been identified.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Treatment with LiCl was performed according to Ghiglione et
al. (Ghiglione et al., 1993)
and treatment with NiCl2 as described by Croce et al.
(Croce et al., 2003) following
Hardin et al. (Hardin et al.,
1992).
Cloning VEGFR and VEGF cDNAs
During a systematic search for receptor tyrosine kinases by RT-PCR using
degenerate oligonucleotides, we isolated a fragment encoding part of a protein
displaying similarity with VEGFR. This fragment was used to probe a P.
lividus mesenchyme blastula cDNA library. The longest clone isolated was
5832 bp, with a 300 bp 5' UTR, an ORF encoding a 1802 amino acid
protein, and a 126 bp 3' UTR.
VEGF cDNA was isolated by RT-PCR using information from published sequences including genomic sequences from the California species Strongylocentrotus purpuratus, available from the Baylor College of Medicine (http://www.hgsc.bcm.tmc.edu/blast/blast.cgi?organism=Spurpuratus), followed by screening a P. lividus pluteus cDNA library. We isolated a 5790 bp cDNA, with a 1161 bp 5' UTR, an ORF coding for a 291 amino acid protein, and a 3756 bp 3' UTR.
EMBL/GenBank accession numbers are AM419057 for VEGFR and AM419058 for VEGF nucleotide sequences.
Northern blots
Total RNA was extracted by the method of Cathala et al.
(Cathala et al., 1983).
Northern blots were carried out by standard methods as described by Lepage and
Gache (Lepage and Gache, 1990)
and Croce et al. (Croce et al.,
2003). All probes were 32P-labelled by random priming
using the Prime-a-Gene Labelling System (Promega). The VEGF probe was
a 1.4 kb fragment corresponding to nucleotides 1596-1997 of the cDNA. The 2.2
kb VEGFR probe corresponded to nucleotides 2851-5122 of the cDNA.
Whole-mount in situ hybridization
Whole-mount in situ hybridization (WMISH) was performed following Harland
(Harland, 1991) and Lepage et
al. (Lepage et al., 1992).
DIG-labelled probes were revealed using an alkaline phosphatase-conjugated
antibody with NBT-BCIP as chromogenic substrates.
The probe for VEGFR was a 2920 bp RNA derived from position
2913-5833 of the cDNA. The VEGF probe corresponds to the entire cDNA.
The probe for Ske-T has been described by Croce et al.
(Croce et al., 2001). The
MSP130 probe (1.5 kb), which derives from the coding sequence of the
MSP130 cDNA, was a gift from T. Lepage (Université Pierre et
Marie Curie and CNRS, Villefranche-sur-Mer, France). The SM30 (0.9
kb) and SM50 (1.3 kb) probes were gifts from M. Di Bernardo (Istituto
di Biologia dello Sviluppo del CNR, Palermo, Italy).
Microinjection of mRNA and antisense morpholino oligonucleotides
Microinjection into eggs was performed as described by Emily-Fenouil et al.
(Emily-Fenouil et al., 1998).
The VEGF ORF, plus six nucleotides upstream of the initiation codon
and the stop codon, was amplified by PCR using Pfu polymerase. The PCR
fragment was inserted at the BamHI-ClaI sites of pCS2+
(Turner and Weintraub, 1994).
The plasmid was linearized with NotI. The pmar1 clone was a
gift of P. Oliveri and has been described by Oliveri et al.
(Oliveri et al., 2002). Capped
mRNAs were synthesized in vitro with the SP6-mMessage-mMachine Kit (Ambion)
and injected at 400 and 10 ng/µl for VEGF and pmar1,
respectively. Embryos expressing pmar1 or pmar1 and VEGF were transferred at
the blastula stage to Petri dishes coated with 2% agarose in sea water.
Morpholino antisense oligonucleotides (Mos) against splice sites were designed as follows. The sequences of the P. lividus VEGF and VEGFR cDNAs were aligned against the genomic sequences from S. purpuratus (Baylor College of Medicine, see above) to identify intron positions. The 5'-most introns identified were selected. Using primers from adjacent exons, introns were amplified from P. lividus genomic DNA. Mos were designed by Gene Tools from sequences of the intron-exon junctions. Mo-VEGF was 5'-CGTGTAACTACTCACATTCATCATAGTC-3' and covers nucleotide positions 1659-1668 of the cDNA (codons 165-168) and the first 15 bases from the adjacent intron. Mo-VEGFR was 5'-TTAAAGTACAACTTACCTGGCGAGC-3', corresponding to positions 683-691 of the cDNA (codons 127-129) and the following 16 bases from the adjacent intron. Before injection, Mos were dissolved at 1 mM for Mo-VEGF and 0.7 mM for Mo-VEGFR.
For both mRNA and Mo microinjections, more than 100 injected embryos were observed in each experiment and the experiments were repeated at least four times with different egg batches.
Double-injection experiments
In experiments relying on double injection, Mo-VEGF (1 mM) was injected
into eggs together with RLDX tracer (rhodamine coupled to dextran 10 kDa,
Molecular Probes) to select injected eggs. Later, VEGF mRNA (400
ng/µl) was injected into a single blastomere of the eight-cell stage embryo
together with FLDX lineage marker (fluorescein coupled to dextran 70 kDa,
Molecular Probes). FLDX labelling was observed on living or fixed embryos by
fluorescence microscopy. To reveal expression of both the marker gene and the
lineage marker we followed the procedure of Thisse et al.
(Thisse et al., 2004). Gene
expression was first revealed by WMISH as described above. After washing
steps, FLDX was revealed using an anti-fluorescein antibody coupled to
alkaline phosphatase and Fast Red as substrate (Roche). The substrate NBT-BCIP
gives a purple-brown color, whereas Fast Red appears reddish. About 20
double-injected embryos were observed in each experiment, and the experiments
were repeated three to four times with different egg batches.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) using
sequences from the Strongylocentrotus purpuratus genome shows that
this domain groups with kinase domains from the VEGFR family and not with
those from the PDGF family. The protein is, however, atypical in that the
extracellular domain is predicted to contain ten Ig-like domains instead of
seven and, as far as we know, it is unique to sea urchins.
In addition, we isolated a cDNA coding for a ligand of the VEGF family. The
predicted protein consists of a poorly conserved N-terminal domain containing
a serine-rich stretch, followed by the conserved typical VEGF/PDGF domain with
the cysteine knot and a cysteine-rich C-terminal domain. The corresponding
gene was later found in the S. purpuratus genome and designated
Sp-VEGF-3 (Lapraz et al.,
2006).
Other related genes have been found in the genome of S. purpuratus
(Sea Urchin Genome Sequencing Consortium, 2006;
Lapraz et al., 2006), but
their cDNAs have not been cloned. Another VEGFR-family gene codes for a
classic VEGFR receptor with seven Ig-like domains. Quantitative PCR
measurements (F. Rizzo and M. Arnone, personal communication) on S.
purpuratus mRNA indicate that VEGFR-7-Ig is expressed during
embryogenesis, although at a lower level than VEGFR-10-Ig. Both VEGFR
genes are expressed in coelomocytes of the adult (Sea Urchin Genome Sequencing
Consortium, 2006). Two other VEGF-family genes, Sp-VEGF and
Sp-VEGF-2 were predicted. In S. purpuratus, the expression
of Sp-VEGF2 has been shown to be absent or barely detectable during
the first 52 hours of development (J. Rast, personal communication). Tilling
data suggest that Sp-VEGF is also poorly expressed during
embryogenesis
(http://www.genboree.org).
Our study focuses on the ten Ig-domain receptor and on the VEGF-3 ligand, referred to here simply as VEGFR and VEGF, respectively.
The VEGFR and VEGF genes are expressed during the same period in distinct domains
The expression patterns of the VEGFR and VEGF genes were
characterized by northern blot and whole-mount in situ hybridization (WMISH)
(Fig. 1B,C). Northern analysis
showed that each gene is expressed as a single transcript, around 8 kb for
VEGFR and 6 kb for VEGF. The VEGFR and
VEGF genes are not maternally expressed, but instead have a single
phase of zygotic expression that takes place after hatching. VEGFR
was found to be expressed from mesenchyme blastula to pluteus, with a peak at
the gastrula stage. VEGF expression began earlier, at the swimming
blastula stage, and was maintained at a nearly constant level until the
pluteus stage. Thus, the temporal expression profiles of both genes are very
similar and largely overlapping. By contrast, the two genes have distinct
domains of expression (Fig.
1C). The VEGFR transcripts were detected exclusively in
the PMC lineage, and in agreement with the northern data, only after
ingression of these cells into the blastocoel and up to the pluteus stage. At
the mesenchyme blastula and gastrula stages (i.e. during PMC migration)
VEGFR was expressed in all PMCs. Later, at the prism stage,
expression decreased in cells of the aboral chain. In the pluteus,
VEGFR transcripts were present in only a few PMCs at the tip of the
oral and post-oral arms and at the aboral apex, close to the sites of spicule
elongation. The VEGF domain of expression did not correspond to a
known lineage or territory. Although northern data indicated that they were
present, VEGF transcripts could not be detected by WMISH at the
swimming blastula stage, suggesting that they might be expressed transiently
in a diffuse area. From mesenchyme blastula and throughout gastrulation, the
VEGF transcripts were restricted to two small areas of the ectoderm,
close to the ectoderm/endoderm border and to the limit between the oral and
aboral ectoderm. This symmetrical ventrolateral localization is co-incident
with the localization of the PMC clusters that form at the early gastrula
stage along the ectoderm wall. At the pluteus stage, the VEGF
transcripts were detected only in a few ectodermal cells at the tip of the arm
buds, close to the VEGFR-expressing cells.
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Organization of the PMCs and of the skeleton correlate with the expression of VEGF in embryos mispatterned along the AV and OA axes
VEGF is expressed in very restricted areas precisely positioned
along the animal-vegetal (AV) and oral-aboral (OA) axes. Alterations of
patterning along these axes are known to produce abnormal skeleton. Patterning
of the AV axis is controlled by the maternal components of the canonical WNT
pathway (Logan et al., 1999;
Emily-Fenouil et al., 1998;
Huang et al., 2000;
Vonica et al., 2000) and can
be perturbed by LiCl. Treatment with LiCl vegetalizes the embryo, i.e. it
alters cell fate specification along the AV axis by shifting the
ectoderm/endoderm border towards the animal pole, resulting in overdevelopment
of endoderm at the expense of ectoderm and a morphological radialization of
the embryo. The perturbation of the skeleton depends on the degree of
vegetalization (e.g. Emily-Fenouil et al.,
1998). Organization along the OA axis is controlled by the nodal
and BMP2/4 pathways (Duboc et al.,
2004). The OA polarity is strongly affected by treatment with
NiCl2, the target of which is not yet identified. Treated embryos
are oralized and produce multiple tri-radiate spicule rudiments along the
circumference (Hardin et al.,
1992). The phenotype of embryos treated with NiCl2 is
similar to those overexpressing nodal
(Duboc et al., 2004).
The expression of the VEGFR and VEGF genes in perturbed
embryos is shown in Fig. 2. In
all cases, the PMC precursors were apparently not affected and the PMCs
ingressed on schedule and expressed VEGFR at a normal level. However,
the expression of VEGF and the behavior of the PMCs were modified. In
embryos treated with LiCl, the enlarged endoderm was exogastrulated and the
ectoderm was reduced to a small area around the animal pole. VEGF was
expressed in this area at the tip of the embryo, and the PMCs, which expressed
VEGFR, formed a single cluster close to the expression domain of
VEGF. In some cases, a single abnormal spicule formed
(Fig. 2A)
(Emily-Fenouil et al.,
1998).
In embryos treated with NiCl2, the PMCs did not form the two
ventrolateral clusters and did not line up to form the oral and aboral chains.
Two different phenotypes were observed depending on egg batches: PMCs were
either arranged around the circumference of the embryo and formed a variable
number of spicule rudiments as already described
(Hardin et al., 1992); or the
PMCs formed a single vegetal aggregate that was later carried to the center of
the blastocoel by the invaginating archenteron and eventually made a spicule.
In both cases, the bilateral symmetry of the skeleton was lost. In all cases,
WMISH showed that the expression domain of VEGF was not limited to
the two ventrolateral patches, but formed a ring around the embryo
(Fig. 2B).
Thus, when the VEGF domain was shifted along the AV axis and reduced to a single spot at the animal pole of the embryo, all the PMCs aggregated into a single cluster adjacent to the VEGF source. In radialized embryos, the absence of symmetrical bilateral clusters of PMCs and the skeletogenic defects observed correlate with the radialization of the VEGF expression domain. Therefore, PMC positioning and cluster formation appear to be closely linked to the localization, extent and shape of the VEGF expression domain.
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Monitoring expression of marker genes (see below) revealed that the number
of PMCs was approximately twice that in control embryos
(Fig. 3E,J). In the early
pluteus, a probe for SM30 transcripts marked about 70 PMCs, as
opposed to 29 in the control. At the late pluteus stage, MSP130
transcripts were detected in two spots at the tip of the arms of normal
embryos and in four spots in embryos overexpressing VEGF. This
observation reveals a potential influence of VEGF on cell division that might
have a physiological role in species in which PMCs undergo their last round of
division shortly after ingression. However, it is unlikely that the
artifactual increase in cell number observed here leads by itself to
additional aggregation of the PMCs and to the skeleton abnormalities observed,
as it has been shown by Ettensohn
(Ettensohn, 1990) that embryos
with three times the normal complement of PMCs form a normal skeleton.
Therefore, perturbation of skeletogenesis by overexpression of VEGF
suggests that VEGF plays a direct role in spiculogenesis by the PMCs.
Loss-of-function of VEGF or VEGFR perturbs PMC positioning, downregulates differentiation genes and blocks skeleton formation
Loss-of-function for the VEGF or VEGFR gene was achieved
by microinjection of morpholino antisense oligonucleotides (Mos). The
phenotypes obtained in more than 90% of the embryos were specific to PMC
development and furthermore were identical for both gene knockdowns
(Fig. 4). This suggests that
the effects are specific and that VEGF and VEGFR are interacting partners.
In embryos that were left to develop, the morphological defects were first detected at the early gastrula stage. The normal complement of PMCs formed and ingressed, but did not lead to the classic PMC ring. Neither the ventrolateral clusters nor the oral or aboral chain formed. At the pluteus stage, instead of the elongated shape typical of the P. lividus pluteus, the embryos were of a squatty appearance with poorly extended arms. The AV and OA polarities were present, the digestive tripartite gut had developed and the mouth opened, and pigmented cells were visible within the ectoderm. However, from early gastrula to prism, no spicule rudiment was ever observed and at the pluteus stage bright spicule rods could not be seen, indicating that the skeleton was totally absent.
To further follow the behavior of the PMCs, we monitored expression of
Ske-T (also known as T-brain) and MSP130, two
PMC-specific genes that begin to be expressed before PMC ingression and thus
before VEGF signaling could operate. Ske-T encodes a T-box
transcription factor that is expected to play a regulatory role at an
intermediate level between specification and the terminal differentiation
genes (Croce et al., 2001;
Fuchikami et al., 2002;
Oliveri et al., 2002) (C.G.,
unpublished). MSP130 codes for a cell surface protein implicated in
the biomineralization process, probably by regulating calcium transport
(Carson et al., 1985;
Leaf et al., 1987). As shown
in Fig. 4, both genes were
expressed by the PMCs in control embryos and in embryos injected with Mos
against VEGF or VEGFR. However, whereas in control embryos
the PMCs were positioned following the stereotypical pattern, in about 90% of
the embryos in which VEGF/VEGFR signaling has been impaired, the PMCs were
disorganized. In most embryo batches (80%), they were randomly dispersed
within the blastocoel, whereas in some embryo batches (20%) they formed
several small aggregates randomly positioned within the vegetal part of the
embryo and were often found later near the aboral apex.
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; George et al.,
1991; Wilt, 2002).
The SM30 and SM50 genes are turned on after PMC ingression
and although they are expressed with slightly different spatial patterns
(Guss and Ettensohn, 1997),
their transcripts can be detected in most PMCs
(Fig. 5). In embryos in which
expression of VEGF or VEGFR was blocked, both SM30
and SM50 were strongly downregulated. SM30 transcripts were
never detected, whereas SM50 transcripts were sometimes detectable in
a few cells (Fig. 5). Because VEGFR has a special function among PMC-specific genes, we next asked whether continuous expression of VEGFR itself might depend upon VEGF signaling. In normal embryos, VEGFR was expressed in all PMCs from ingression to the gastrula stage (Fig. 1C and Fig. 5). In embryos in which VEGF signaling had been impaired by injection of Mo-VEGF, VEGFR transcripts were detected in PMCs at the mesenchyme blastula stage but were undetectable at the gastrula stage (Fig. 5). This indicates that after an autonomous early phase, VEGFR expression is maintained by VEGF signaling. The late expression domains of VEGF and VEGFR are very restricted, closely adjacent and localized at all skeletal extension sites (Fig. 1C), suggesting that this coupling between VEGF and expression of VEGFR might participate in the control of skeletal growth.
These results show that VEGF/VEGFR signaling is required for the proper positioning of the PMCs, the formation of the ventrolateral clusters, the maintenance of VEGFR expression, the activation or maintenance of terminal differentiation genes functioning in spiculogenesis, and the production of spicules.
Ectopic VEGF expression can direct spicule formation in embryos in which endogenous VEGF function is blocked
We next addressed whether an exogenous source of VEGF expression
in embryos in which the endogenous pathway has been blocked was capable of
directing spicule formation. As the Mo-VEGF is targeted against a splice
junction, it should not inhibit synthetic VEGF mRNA. In order to
carry out this experiment, we choose embryonic batches in which injection of
Mo-VEGF alone led to a strong dispersion of the PMCs. In these batches,
co-injection of Mo-VEGF and VEGF mRNA led to a recovery of spicule
formation in about 50% of the embryos.
In the first series of experiments, we co-injected Mo-VEGF and VEGF mRNA into unfertilized eggs (Fig. 6A). The resulting pluteus-stage embryos displayed AV and OA polarities with a tripartite gut, but their overall shape was abnormal. This shape resulted from the presence of spicule elements with abnormal shapes and variable and incorrect positioning. Thus, spicule rods, sometimes with branching, could be rescued by broadly supplied exogenous VEGF, but skeletal morphology could not.
In the preceding experiments, VEGF was uniformly expressed in the embryo. In order to create a more localized source of VEGF, Mo-VEGF was first microinjected into the egg and then at the 8-cell stage VEGF mRNA was microinjected into a single blastomere together with a fluorescent lineage marker (Fig. 6E). The injected embryos were allowed to develop until non-injected embryos reached the pluteus stage. Injection into one blastomere at the 8-cell stage was random in terms of whether it was in an animal or vegetal blastomere. As VEGF is normally expressed in ectoderm, embryos displaying lineage marker in the ectoderm were selected. Control embryos injected with Mo-VEGF developed without forming any spicules as described above. Differential interference contrast (DIC) and fluorescence microscopy showed that double-injected embryos formed a single spicule rod of variable shape, close to the lineage tracer and thus to the VEGF-expressing cells (Fig. 6F-H). Moreover, SM30 was expressed in the PMCs that were found in aggregates adjacent to the ectoderm patches expressing VEGF (Fig. 6I-K).
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VEGF triggers formation of spicule primordia within a population of PMCs
It is known that isolated micromeres cultured in sea water do not produce
spicules but can do so if the medium is supplemented with serum or
blastocoelar fluid (Okazaki,
1975; Kiyomoto and Tsukahara,
1991). To address whether exposure to VEGF could induce PMCs to
synthesize spicules, we produced a population of PMCs by taking advantage of
the powerful ability of the transcriptional regulator pmar1 to specify PMC
fate (Oliveri et al., 2002).
When pmar1 mRNA is injected into an egg, most cells of the embryo are
converted to a PMC fate, forming a ball of loosely connected mesenchyme cells
that express some PMC markers but do not form spicules. To obtain such PMC
balls and to expose them to VEGF, we coexpressed pmar1 and
VEGF. Embryos derived from eggs injected with pmar1 mRNA or
with pmar1 and VEGF mRNAs are shown in
Fig. 7. Embryos expressing only
pmar1 developed as previously described
(Oliveri et al., 2002) and did
not form any spicule or spicule primordium. However, embryos expressing both
pmar1 and VEGF displayed the same morphology, but many
refractive dots typical of spicule material and some spicule primordia were
visible. Therefore, VEGF is sufficient to trigger formation of spicule
primordia in a population of aggregated cells reoriented towards a PMC fate.
This indicates that VEGF is a differentiation signal sufficient to initiate
skeletogenesis by PMCs.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This VEGF/VEGFR signaling system provides a molecular basis for the
interaction between ectoderm and the PMCs that has been outlined in several
studies (for reviews, see Gustafson and
Wolpert, 1967; Decker and
Lennarz, 1988; Ettensohn et
al., 1997; Wilt,
2002). The complementary expression of the ligand in the ectoderm
and of the receptor in the PMCs demonstrates a unidirectional interaction
between these two tissues. The symmetrically localized expression of VEGF
constitutes a spatial cue that is required for the formation of the two
ventrolateral clusters and the formation of the spicule rudiments.
Furthermore, our findings fit with previous important observations. First,
restriction of VEGFR to the PMCs explains why only PMCs are competent to
respond to the guidance signal (Ettensohn
and McClay, 1986). Second, PMCs from mesenchyme blastula injected
into the blastocoel of younger blastula form a pattern only when the host
reaches the mesenchyme blastula stage
(Ettensohn and McClay, 1986),
which corresponds to the onset of VEGF expression. Conversely, the
permanent expression of VEGF and VEGFR during gastrulation
explains why late PMCs, having already completed their migration, are still
capable of responding to ectoderm directional cues when transplanted to a new
host (Ettensohn, 1990). Third,
by using embryos of different sizes and manipulating the OA axis, Armstrong et
al. (Armstrong et al., 1993)
have shown that normal embryos always form two rudiments, whatever their size,
whereas radialized embryos display supernumerary spicule rudiments in
proportion with their size. This follows from the transformation of the
VEGF expression domain from two symmetrical patches into a continuous
ring the size of which is proportional to the size of the embryo. Fourth, it
has been predicted that signaling from the ectoderm should regulate expression
of PMC-specific products (Guss and
Ettensohn, 1997). Indeed, when VEGF/VEGFR signaling is impaired,
the two PMC-specific genes SM50 and SM30 are repressed,
although a third one, MSP130, is not significantly affected. The
expression patterns of these three genes are different
(Guss and Ettensohn, 1997).
MSP130 begins to be expressed before PMC ingression, whereas
SM50 and SM30 are turned on at early and late mesenchyme
blastula, respectively. During the initial phase of PMC patterning,
MSP130 and SM50 are expressed uniformly in all PMCs, whereas
SM30 is expressed at a higher level in the ventrolateral clusters.
Furthermore, in PMC cultures, all cells express SM50, whereas
SM30 is expressed only by cells that participate in spicule
synthesis, and the expression of SM30 is extremely sensitive to the
presence of serum (see Guss and Ettensohn,
1997). In addition, misexpression of pmar1 upregulates
MSP130 strongly and SM50 moderately, but does not
significantly affect SM30 expression
(Oliveri et al., 2002). Thus,
it appears that MSP130 is not controlled by VEGF as it is probably
part of the autonomous program of the PMCs. SM50 might be turned on
autonomously, but VEGF signaling is required to maintain a high level of
expression. SM30, which is expressed in the PMC clusters and
sensitive to the ectoderm signal, requires VEGF signaling. Finally, our
results support the view that the endogenous signal from ectoderm and the
signal provided by serum or blastocoelar fluid might be similar
(Ettensohn et al., 1997), but
this remains to be demonstrated through serum fractionation. In summary,
VEGF/VEGFR signaling appears to be a major element of the interaction between
ectoderm and PMCs.
Identification of the function of VEGF opens the way to a better understanding of where this signaling event fits in the regulatory interactions that built the embryo, and of its influence upon the migratory behavior of the PMCs.
The sea urchin embryo is particularly well suited to unravel gene
regulatory networks such as the network that controls mesendoderm formation
(Davidson et al., 2002).
VEGF/VEGFR signaling is a bridge between the ectoderm network, which is
largely unknown, and the PMC network
(Oliveri et al., 2002).
Although PMCs have a largely autonomous program of differentiation, the PMC
network cannot go to completion without an input from the ectoderm. VEGF is a
spatial cue that directs the formation of the ventrolateral clusters, controls
expression of VEGFR and of spiculogenic genes and probably regulates
skeletal growth. VEGF, therefore, establishes a coupling between morphogenesis
and differentiation. It will be important for future studies to integrate
these signaling factors within their respective networks. In contrast to
VEGFR expression, VEGF expression is not lineage-restricted
and thus results from fine patterning of the ectoderm. The spatial pattern of
VEGF is reminiscent of the pattern of the homeobox gene Otp
(Di Bernardo et al., 1999),
but it is unlikely that Otp controls VEGF as Otp
seems to be activated later than VEGF and in a territory much smaller
than the VEGF domain. The VEGF expression domain overlaps that of
another signaling molecule: in an independent study on the function of FGF
during gastrulation, E. Röttinger and T. Lepage (personal communication)
have shown that FGF signaling plays a role in PMC patterning. The spatial
expression of the two ligands is linked to the main embryonic axes, but
otherwise VEGF and FGF signals are independent and functionally nonredundant
(our unpublished results and T. Lepage, personal communication) and are thus
both required for correct morphogenesis of the primary mesenchyme.
How migrating mesenchyme cells detect the signal transmitted from the
ectoderm and modulate their behavior is another essential question. Pioneering
studies (Gustafson and Wolpert,
1961) and a more recent work
(Malinda et al., 1995) have
shown that PMCs contact the blastocoel wall through long filopodia and move
through contraction of these filopodia. Gustafson and Wolpert
(Gustafson and Wolpert, 1961)
observed that PMCs randomly explore the blastula wall with dynamic filopodia,
and suggested that they became trapped as the ectoderm displays areas with
differential adhesive properties. No such patterning of the ectoderm has been
discovered so far. Instead, our findings point to a role for VEGF, as in other
systems. For example, during angiogenesis of the mouse retina, endothelial tip
cells expressing VEGFR2 (KDR - Mouse Genome Informatics) extend long filopodia
that detect steep gradients of VEGF and guide their migration
(Gerhardt et al., 2003). In
the sea urchin embryo, VEGF signaling might stabilize PMC filopodia that
randomly explore the ectoderm wall and contact the VEGF expression
domains or closely approach a local VEGF gradient. Thus, PMCs would reach
their target through short-range contact guidance.
VEGF, first identified as a regulator of vascular permeability, was later
shown to have a very important role in vascularization and angiogenesis.
Recently, VEGF was shown to be a guidance cue for endothelial, hematopoietic
and neural precursors in vertebrates
(Gerhardt et al., 2003;
Hiratsuka et al., 2005;
Zhang et al., 2003). VEGF
(PVF1) is also a guidance cue for border cells and blood cells in
Drosophila. Border cells are follicular cells that migrate towards
the oocyte during oogenesis. Border cells express the receptor (PVR, similar
to both VEGFR and PDGFR), whereas the oocyte expresses VEGF
(Duchek et al., 2001).
Hemocytes, which are produced in the head and migrate throughout the body,
express VEGFR (PVR), whereas VEGF is expressed along their migration pathways
(Cho et al., 2002;
Wood et al., 2006). As
Drosophila has no blood vessels, Cho et al.
(Cho et al., 2002) suggested
that the VEGF pathway might have originally functioned in blood cells and was
later recruited for vascular development. In the sea urchin embryo, which
lacks a vascular system and blood cells, VEGF might carry out an ancestral
function. We showed that the guidance function of VEGF, already described in
protostomes and in vertebrate deuterostomes, is also present in sea urchin, a
nonchordate deuterostome, and thus may indeed constitute a primordial function
conserved during evolution.
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ACKNOWLEDGMENTS |
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