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First published online 12 December 2007
doi: 10.1242/dev.014282
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1 UMR 7009 CNRS, Université Pierre et Marie Curie (Paris 6) Observatoire
Océanologique, 06230 Villefranche sur mer, France.
2 Department of Biology, French Family Science Center, Duke University Durham,
NC 27708, USA.
* Author for correspondence (e-mail: lepage{at}obs-vlfr.fr)
Accepted 30 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Fgf, Sea urchin, Gastrulation, Skeletogenesis, Cell migration, PMCs, FGF, Sprouty, FGFR1, FGFR2, Pea3, Pax
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; McClay et al.,
1992; McClay et al.,
2004). During early development of the sea urchin embryo, a
well-defined series of morphogenetic events transforms the hollow epithelial
ball of the blastula into a three-layered bilaterally organized pluteus larva
with its characteristic organization of mesodermally derived spicules.
Spicules are constructed by a population of mesodermal cells called the
primary mesenchyme cells (PMCs). At the blastula stage, PMCs precursors
delaminate into the blastocoel and then undergo a dramatic sequence of
morphogenetic behaviors (Decker and
Lennarz, 1988). Immediately following ingression, they stop
dividing and begin to migrate along the extracellular matrix that lines the
blastocoel wall. During migration, the PMCs constantly extend and retract long
and thin filopodia that survey the inner surface of the ectoderm
(Malinda et al., 1995;
Miller et al., 1995). Then,
before the onset of gastrulation, they gradually adopt a stereotyped pattern
consisting of two ventrolateral clusters joined by a ring of cells surrounding
the archenteron. PMCs within the clusters subsequently fuse through their
filopodial processes, forming a syncytium that begins to accumulate calcium
and magnesium carbonates, leading to formation of a small tri-radiate
crystalline structure: the spicule rudiment, deposited within an organic
matrix. Each rudiment then increases in size and complexity
(Decker and Lennarz, 1988) and
branches in a specific pattern to form the extraordinary polycrystalline
skeleton that gives the pluteus larva its easel-like shape.
It has been shown experimentally that the ectoderm plays a crucial role in
guiding PMC migration as well as in regulating the skeletogenic gene
expression program (Armstrong et al.,
1993a; Ettensohn,
1990b; Ettensohn and Malinda,
1993; Ettensohn and McClay,
1986; Katow et al.,
2000; Malinda and Ettensohn,
1994; McClay et al.,
1992; Peterson and McClay,
2003; Wilt, 1997).
For instance, PMCs taken from embryos treated with the ventralizing agent
NiCl2 form a normal skeleton when transplanted into untreated host
embryos, whereas normal PMCs recombined with a nickel-treated ectoderm form
multiple spicule rudiments (Hardin et al.,
1992). The molecular nature of the spatial cues provided by the
ectoderm to the underlying PMCs remained elusive for a long time, but one
signal has recently been identified as VEGF
(Duloquin et al., 2007). VEGF
ligands emitted from two small regions of the ectoderm are specifically
required for the oriented migration and differentiation of the PMCs, which
specifically express VEGFR.
While the formation of spicules from PMCs is a process unique to
echinoderms, the invagination of the archenteron and delamination of the
secondary mesenchyme cells (SMCs) are clearly comparable to those driving
vertebrate gastrulation. In the sea urchin, internalization of the endodermal
precursors occurs by buckling of the vegetal plate rather than by ingression
or involution (McClay et al.,
1992). Formation of the blastopore is thought to involve the
coordinated constriction of the apical surface of cells at the center of the
vegetal plate. Although recent studies have demonstrated the crucial role
played by a subpopulation of SMCs and by non-canonical Frizzled 5/8 and Rho
signaling in triggering primary invagination of the gut, archenteron
morphogenesis remains poorly understood at the molecular level
(Beane et al., 2006;
Croce et al., 2006;
Kominami and Takata,
2004).
In this study we describe a molecular pathway that is initiated before gastrulation within the ectoderm of the blastula and involves the sequential and spatially restricted expression of FGFA and of transcription factors in the Ets and Pax families in discrete regions of the ectoderm to which migrating mesenchymal cells are attracted. We report that this pathway is required not only for oriented migration of the PMCs, which is required for construction of the skeleton, but also that it regulates invagination of the archenteron. Therefore, this study has identified a regulatory module essential for morphogenesis in the sea urchin embryo and the FGF pathway as a key player of the gene regulatory network directing patterning of the PMCs and gastrulation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Accession numbers for novel sequences used in this study are: fgfA, EF157978; sprouty, EF157979; FGFR2, EF439654; pea3, EF439653.
Constructs, RNA and morpholino injections, treatments
The coding sequence of fgfA was amplified by PCR using the Pfx DNA
polymerase and inserted at the BamHI-XhoI sites of pCS2+
(Turner and Weintraub, 1994).
To construct the dominant-negative FGFR1, a portion of the cDNA coding for the
signal peptide, extracellular and transmembrane domains but truncated of the
intracellular kinase domain was amplified by PCR and cloned into pCS2. Capped
RNA was injected at the following concentrations: fgfA:600 µg/ml;
dn-FGFR1, 780 µg/ml.
The exon2-intron2 boundary located within the FGF core domain was selected for morpholino targeting. A genomic DNA fragment containing intron 2 was first amplified using PCR primers within exon2 and exon3 and its sequence was determined. The in-vivo specificity and efficiency of the splice-blocking morpholino oligonucleotide was monitored via semi-quantitative RT-PCR. RNA was extracted at the early gastrula stage from batches of 150 embryos injected with increasing doses of the morpholino and reverse transcribed. PCR primers located within exon1 and exon3 were used to amplify the mRNA products generated in the presence of the splice-blocking oligonucleotide. The PCR products were gel eluted, cloned and deletion of exon 2 was confirmed by sequencing. The FGFA-splice-Mo was effective when injected at relatively low doses (0.5 mM), and it did not cause any toxicity even when injected at high concentrations (up to 2 mM). Similarly, the FGFR2-Mo could be injected at doses up to 1.5 mM without causing toxicity. By contrast, when injected at doses above 1 mM, the FGFA-ATG and the FGFR1 morpholinos started to be toxic. Therefore, these morpholinos were used below 1 mM (0.6-0.9 mM).
Sequence for morpholino oligonucleotides are: FGFA-splice: 5'-ACACATTTTGGATACTTACAGCTCC; FGFA-ATG: 5'-ACTTTCATCCATTTTCGCTTTCATG; FGFA 5'-UTR: 5'-ATGGATGCCGCGTCGTACACACGAG; FGFR1-Mo1: 5'-CATCATGCCGTGGCTGCCTTGAGCA; FGFR1-Mo2: 5'-AGTTCCAGCAAAAGATGACGAAAAG; FGFR2-Mo1: 5'-TAAAGCATCGGATCGCCATTTCCAT; and FGFR2-Mo2: 5'-TTCCGTTTAATTTTCTCCAAATCAC.
All the injections were repeated many times with different batches of embryos and for each experiment, 100-150 embryos were analysed. Only representative phenotypes present in at least 80% of the injected embryos are presented.
Immunocytochemistry and U0126 treatment
Detection of phosphorylated ERK and treatments with U0126 were performed as
described previously (Röttinger et
al., 2004). Treatments with human bFGF (a kind gift from Hitoyoshi
Yasuo) were performed at 50 ng/ml starting at early blastula stage. LiCl was
used at 30 mM and NiCl2 at 0.5 mM.
Sequence analysis and phylogenetic analysis
To predict the signal sequence of FGFA, we used the SignalP software
available at
http://www.cbs.dtu.dk/services/SignalP/,
and to predict the secondary structure we used the Porter software available
at
http://distill.ucd.ie/porter/.
For phylogenetic analysis, the amino acid sequences of the core domain of FGF
ligands and of the tyrosine kinase domains of FGFRs were collected from
GenBank using the SMART software and aligned with ClustalW with default
parameters
(http://www.ebi.ac.uk/clustalw/).
The tree was calculated using the maximum likelihood method with PhyML using
the WAG substitution model
(http://atgc.lirmm.fr/phyml/)
(Guindon et al., 2005). A
consensus tree with 50% cut off value was derived from bootstrap analysis (500
iterations) using Mega 3.1
(http://www.megasoftware.net/).
Numbers above branches represent posterior probabilities, calculated from this
consensus.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Ornitz,
2000; Ornitz and Itoh,
2001) (see Fig. S1B in the supplementary material). The predicted
protein sequence shows similarities with vertebrate FGF9, FGF16 and FGF20, as
well as with FGF8, FGF17 and FGF18. However, phylogenetic analysis gave
ambiguous results, and therefore, in the absence of strong evidence for
grouping this sequence with other known FGF subfamilies, we decided to name
this FGFA (see Fig. S1C in the supplementary material).
A sea urchin FGF receptor named FGFR1 has been characterized previously in
Strongylocentrotus purpuratus
(McCoon et al., 1996;
McCoon et al., 1998), but its
function has not been studied. The expression pattern of FGFR1 has been
re-examined recently in Paracentrotus lividus, revealing a highly
dynamic expression of FGFR1 in various territories, including mesodermal and
endodermal precursors (Lapraz et al.,
2006). By screening a P. lividus EST database for
sequences related to FGFR1, we identified a second putative FGFR receptor,
which we named FGFR2 (Lapraz et al.,
2006). FGFR2 has the typical architecture of FGFRs, with 3Ig
domains in the extracellular ligand-binding domain, a Fibronectin III (FN III)
domain, a transmembrane region and a tyrosine kinase domain (see Fig. S1E in
the supplementary material). The organization of these domains is slightly
different from that of FGFR1 (in which the FN III domain is upstream of the Ig
domains rather than downstream). Also, FGFR2 apparently lacks the acidic box
usually found between Ig domains 1 and 2, required for interaction between
FGFR and N-CAM and/or cadherins
(Sanchez-Heras et al., 2006).
The kinase domain of FGFR2 contains all the structural motifs required for
coordinating Mg2+ATP, including the GTGSFGKV, VAIK and DFG motifs,
as well as the HRDLXXXN motif that constitutes the catalytic loop of the
kinase. Blast analysis indicated that the amino acid sequence of FGFR2 is
mostly related to FGFRs from insects and cephalochordates (see Fig. S1F in the
supplementary material). However, phylogenetic analysis using a large set of
sea urchin RTKs and FGFRs from various species indicated that the tyrosine
kinase domain of FGFR2 is divergent compared with that of other FGFRs (see
Fig. S1G in the supplementary material). Taken together, these results suggest
that this receptor kinase encodes a putative FGF receptor with a divergent
kinase domain.
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The temporal expression of FGFR2 is largely parallel to that of fgfA. Two zygotic fgfr2 transcripts started to accumulate in the embryo at the mesenchyme blastula stage (Fig. 1B). Their abundance peaked during gastrulation, both transcripts being still expressed at a high level at the prism and pluteus stages.
fgfA expression prefigures formation of the bilateral clusters of PMCs and foreshadows the branching pattern of the skeleton
Expression of fgfA was first detected at the hatching blastula
stage in an equatorial belt of ectodermal cells surrounding the embryo
(Fig. 1E). Immediately after
ingression of the primary mesenchyme cells, while there is yet no
morphological sign of bilateral symmetry, the expression of fgfA was
downregulated in part of this equatorial belt and progressively restricted to
two broad domains of the ectoderm (Fig.
1F,G). Double in situ hybridization with probes for fgfA
and nodal, which is expressed on the presumptive ventral side, showed
that the bilateral ectodermal regions expressing fgfA are located
between the dorsal and ventral regions and therefore coincide with the lateral
domains where the PMCs aggregate to form bilateral clusters before
differentiation (Fig. 1C and
data not shown). Furthermore, at the early gastrula stage, intense expression
of fgfA was observed in the ectodermal regions in which the PMCs had
accumulated to form the bilateral clusters
(Fig. 1H).
|
). At this
stage, intense expression of fgfA was detected within the PMCs
located in the center of the newly formed PMC clusters
(Fig. 1I-K). During
gastrulation, novel domains of expression appeared near the thickened animal
plate, on either side of the neurogenic ectoderm
(Fig. 1I,K). These are regions
in which another of the three arms of the tri-radiate rudiment (the
dorsoventral connecting rod) will branch to give rise to the anterolateral
rods. Expression of the fgfA gene in these four ectodermal domains
persisted at the late gastrula and prism stages but became undetectable in
plutei (Fig. 1L,M). In the
early pluteus larva, expression of fgfA transcripts was restricted to
the region surrounding the stomodeum and to four clusters of PMCs comprising
three to four cells located at the rod tip of the growing spicules that
sustain the oral arms (Fig.
1L,M). These clusters probably correspond to the plug of PMCs
associated with the growth zones of the two pairs of arm rods
(Ettensohn and Malinda, 1993;
Wolpert and Gustafson,
1961).
FGFR2 is expressed in migrating PMCs
Whereas fgfA (Fig.
1E,F) and FGFR1 (Lapraz et
al., 2006; McCoon et al.,
1996; McCoon et al.,
1998) were abundantly expressed in the ectoderm at blastula and
gastrula stages, fgfr2 transcripts were expressed exclusively within
the PMCs (Fig. 1Q-V).
Consistent with the northern blot analysis, expression of fgfr2
started at the beginning of PMC ingression and continued to be restricted to
these cells during gastrulation and morphogenesis. At the gastrula stage,
fgfr2 transcripts were detected in all subpopulations of PMCs,
including the bilateral clusters, and the oral and aboral chains
(Fig. 1S,T and data not shown).
However, at the prism stage, a spatial restriction of fgfr2
transcripts was observed in PMCs located in regions in which the oral rods of
the skeleton will branch (Fig.
1U), close to regions expressing fgfA
(Fig. 1L,M). fgfr2
expression was no longer detected at the pluteus stage
(Fig. 1W). Experiments using
dissociated embryos (data not shown) showed that this gene, like most other
PMC marker genes studied so far, was expressed in a cell-autonomous manner and
therefore that its expression is part of the cell-autonomous program of the
PMCs. Thus, fgfA and FGFR2 are expressed during similar periods and
partially overlapping regions, suggesting that they may act as a couple
ligand-receptor.
An fgfA synexpression group comprising genes encoding the FGF/MAP kinase modulator Sprouty and the transcription factors Pea3 and Pax2/5/8
In the course of an in situ hybridization screen, we identified three
additional genes expressed at specific stages in bilateral regions of the
ectoderm in a pattern strikingly similar to that of fgfA
(Fig. 2). The first gene
encodes a protein related to the FGF/MAP kinase modulator Sprouty, which is
thought to act in a negative-feedback regulatory loop during FGF and EGF
signaling (Casci et al., 1999;
Hacohen et al., 1998;
Kramer et al., 1999;
Sivak et al., 2005). The
second encodes the Ets domain transcription factor Pea3 (Polyoma enhancer
activator 3) (Raible and Brand,
2001; Roehl and
Nusslein-Volhard, 2001) and the third is the paired domain
transcription factor pax2/5/8
(Czerny et al., 1997). From
late mesenchyme blastula/early gastrula to pluteus stages, expression of
sprouty largely followed that of fgfA in bilateral regions
of the ectoderm, in the PMC clusters, and at the tip of the growing arms of
the larva (Fig. 2A-H),
consistent with the idea that sprouty expression is dependent on FGF
signaling, as shown in other systems
(Mason et al., 2006).
Similarly, during gastrulation, pea3 was expressed in four ectodermal
domains that can be superimposed over the four domains of fgfA at
this stage (Fig. 2K). However,
both pea3 and sprouty were strongly expressed in the
endoderm, whereas fgfA transcripts were absent from this tissue.
Expression of pax2/5/8, like that of fgfA, was strictly
zygotic and started abruptly at late mesenchyme blastula/early gastrula stage
in two lateral domains of the ectoderm, at the intersection between the
ectoderm-endoderm and oral-aboral boundaries
(Fig. 2M,N). Thus,
pax2/5/8 together with pea3 and sprouty, belongs to
the fgfA synexpression group. However, in contrast to fgfA,
sprouty and pea3, which are expressed both in the ectoderm and
in the PMC clusters, pax2/5/8 was expressed exclusively in the
ectoderm overlying the PMC clusters.
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;
Hacohen et al., 1998;
Kramer et al., 1999), Pax2/5/8
(Hans et al., 2004;
Reifers et al., 1998) and Pea3
(Raible and Brand, 2001;
Roehl and Nusslein-Volhard,
2001) are probable downstream targets of FGF signaling during
morphogenesis of the skeleton in the sea urchin.
fgfA and pax2/5/8 are de-repressed in most of the ectoderm in the absence of Nodal signaling
To gain insight into the function and regulation of fgfA
expression, we first examined its expression in embryos treated with reagents
that perturb patterning along the animal-vegetal and dorsal-ventral axes
(Fig. 2Q-Zb). In embryos
treated with lithium, the PMCs migrated towards the animal pole and arranged
into a ring at the level of the displaced ectoderm-endoderm boundary
(Fig. 2Q). In these embryos,
fgfA and pax2/5/8 were expressed in the animal pole region
immediately above the aggregated PMCs (Fig.
2U,Y). By contrast, in embryos radialized by treatment with NiCl2,
which causes circumferential expression of Nodal
(Duboc et al., 2004), the PMCs
formed multiple clusters around the basis of the archenteron
(Fig. 2R)
(Armstrong et al., 1993b;
Hardin et al., 1992). In all
the NiCl2-treated embryos, fgfA was expressed radially in two rings
located at the level of the ectoderm-endoderm boundary and below the animal
pole region (Fig. 2V,
n>200), whereas pax2/5/8 was expressed in a single belt
of cells at the level of the endoderm-ectoderm boundary
(Fig. 2Z, n>200).
These observations show that treatments that affect the bilateral symmetry of
the embryo, such as vegetalization by lithium or ventralization by NiCl2, are
associated with dramatic perturbations of the expression of fgfA and
pax2/5/8, causing them to be expressed radially near the sites where
the PMCs differentiate into spicule rudiments.
The TGF-β Nodal is a crucial determinant of dorsal-ventral polarity of
the sea urchin embryo and is required for establishment of bilateral symmetry
(Duboc et al., 2005;
Duboc et al., 2004). We
therefore examined fgfA and pax2/5/8 expression in embryos
in which Nodal signaling was exacerbated or abrogated. Strikingly,
overexpression of nodal by microinjection of mRNA into the egg did
not promote but abolished fgfA and pax2/5/8 expression
(Fig. 2W,Za,
n>100), suggesting that Nodal signaling leads to repression of
fgfA expression. As described above, in NiCl2-treated embryos,
fgfA was excluded from the equatorial region located between the two
rings of cells expressing fgfA
(Fig. 2V). This medial region
that does not express fgfA following NiCl2 treatment most likely
corresponds to the belt of cells that ectopically expresses Nodal in these
embryos (see Duboc et al.,
2004) (Fig. 2Cn),
in agreement with the finding that misexpression of Nodal represses
fgfA expression. Reciprocally, in most embryos in which translation
of nodal mRNA was blocked by microinjection of a morpholino
oligonucleotide, fgfA and pax2/5/8 were expressed radially
in a large equatorial belt of cells that surrounded the embryo but not in the
animal pole territory (Fig.
2X,Zb, n=70). These results show that in the absence of
Nodal, fgfA and pax2/5/8 are expressed ectopically in most
of the ectoderm. They suggest that Nodal establishes the bilateral expression
pattern of fgfA by repressing its expression in the ventral ectoderm
and probably by inducing a repressor of fgfA expression in the dorsal
territory.
Overexpression of fgfA disrupts morphogenesis
To test the potential of FGFA to regulate morphogenesis of the sea urchin
embryo, we overexpressed it by microinjection of mRNA into the egg.
Microinjection of fgfA transcripts strongly perturbed morphogenesis
and differentiation of mesenchymal cells
(Fig. 3). At the gastrula
stage, whereas in the control embryos the PMCs had formed a regular ring with
two clusters, in the FGFA-injected embryos the pattern of PMCs was much more
irregular, some embryos displaying a radial organization
(Fig. 3A,B). Also, we noted
that the number of PMCs was frequently above the number of 32 cells normally
present in P. lividus (Fig.
3A,B). At 48 hours, a supernumerary spicule was present in about
30% of the embryos (n>500), and the remaining embryos frequently
displayed abnormal branching of the spicules
(Fig. 3D-F). Abnormal
skeletogenesis was correlated with ectopic expression of pax2/5/8 in
the injected embryos (Fig. 3J-L
n=35/45), suggesting that pax2/5/8 is a downstream target of
FGFA signaling.
Overexpression of fgfA also caused exogastrulation in a small fraction (1 to 10%, depending on the batch) of the injected embryos (Fig. 3G, n>500). The same defects in skeletogenesis and gastrulation were observed following treatment of embryos with purified recombinant human bFGF (also known as NUDT6 - Human Gene Nomenclature Database) (treatment at 50 ng/ml caused exogastrulation in 5-10% of the larvae and abnormal skeletogenesis in 20-40% of the larvae (Fig. 3H,I, n>200). These results suggest that FGFA signaling may regulate differentiation of mesenchymal cells as well as cellular movements during gastrulation.
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Expression of FGFA in the vegetal hemisphere is required for skeleton formation
As fgfA is expressed early in part of the animal hemisphere and
later in tissues derived from the vegetal pole, such as the PMC clusters and
overlying ectoderm, we analysed the respective roles of this ligand in the
animal and vegetal domains by making chimeras. Eggs were injected with the
fgfA-spliceMo morpholino together with a lineage tracer and allowed
to develop up to the 16-32-cell stage; then the animal and vegetal regions
were separated and recombined with their complementary halves derived from
wild-type embryos (Fig. 5A).
Chimeras in which the fgfA morpholino was present in the animal
hemisphere developed into almost normal pluteus larvae, with a skeleton and
tripartite archenteron (100%, n=7). The only defect observed in these
larvae was incomplete development of the anterolateral arms and spicules
consistent with previous expression of fgfA in two regions of the
animal hemisphere where the anterolateral arms and spicules form
(Fig. 5Bc-Bf). Thus
fgfA is not required in the ectoderm derived from the animal region
for gastrulation or formation of most of the skeletal rudiments. By contrast,
when the function of fgfA was inhibited in the vegetal hemisphere,
the resulting chimeras displayed a phenotype very similar to that observed
following injection of the morpholino into the egg
(Fig. 5Bg-Bj): skeletal
differentiation was blocked, the archenteron remained poorly differentiated
and the stomodeum did not form (n=6/6). These findings indicate that
fgfA function is required in tissues derived from the vegetal
hemisphere for skeletogenesis and morphogenesis of the gut.
FGFA is required for expression of pax2/5/8 and pea3 in the ectoderm and of genes encoding spicule matrix proteins in the PMCs
As inhibition of fgfA function interferes with invagination and
differentiation of the archenteron, we examined the expression of
nova and bhmt, two endodermal markers that start to be
expressed in the vegetal plate at mesenchyme blastula stage
(Röttinger et al., 2006),
of brachyury, which is expressed dynamically at the ectoderm-endoderm
boundary (Croce et al., 2001a;
Gross and McClay, 2001), and
of papss, which is expressed in the SMC territory
(Röttinger et al., 2004),
in the fgfA morphants (Fig.
6). Apparently normal levels of expression of brachyury
(Fig. 6A,B,G,H,
n>70) and slightly reduced expression of nova
(Fig. 6C,D,I,J,
n>100) and bhmt (data not shown) were still observed at
the mesenchyme blastula and gastrula stage in most embryos in which FGF
function had been blocked. These results indicate that FGF signaling is
probably not required for mesendoderm specification per se, but most likely
for the cellular movements of gastrulation and for invagination of the
archenteron. Similarly, the expression of papss
(Fig. 6E,K, n>200)
and the early expression of ets1, skeT and alx1,
(Fig. 6F,L and data not shown,
n>70 for each marker) three transcription factors required for PMC
specification (Croce et al.,
2001b; Ettensohn et al.,
2003; Kurokawa et al.,
1999), were unaffected by FGFA morpholinos, suggesting that FGFA
signaling participates in regulating PMC differentiation, although not their
specification. We then examined the expression of gene markers in the
ectoderm, such as otp (Cavalieri
et al., 2003; Di Bernardo et
al., 1999), fgfA, pax2/5/8 and pea3, in the FGF
morphants. Expression of the homeobox gene otp, which has been
implicated previously in skeletogenesis, was largely unaffected (data not
shown, n>40) following downregulation of FGFA. Remarkably, FGFA
expression was always much stronger in the FGFA morphants compared with
controls, suggesting that FGFA regulates its own expression through a
negative-feedback loop (Fig.
6M,S, n>100). By contrast, expression of
pax2/5/8 and pea3 in bilateral regions of the ectoderm
(Fig. 6N,T,O,U) was abolished
in most (95% n>200) of the FGFA morphants. Furthermore, the
expression of two out of three genes examined encoding spicule matrix
glycoproteins synthesized during skeletogenesis, msp130, SM30 and
SM50 (Anstrom et al.,
1987; Benson et al.,
1987; George et al.,
1991; Guss and Ettensohn,
1997), was either abolished or barely detectable
(n>200), whereas the third remained expressed at a high level
(Fig. 6P-R,V-X)
(n>150). Most of these experiments were initially performed using
the FGFA-ATG morpholino and subsequently repeated using the FGFA-splice
morpholino with identical results.
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|
).
The domains of activation of ERK coincide with the territories expressing
fgfA and sprouty during gastrulation, raising the
possibility that FGFA signaling may be responsible for activation of ERK in
these bilateral domains. In most embryos, blocking translation of the
fgfA transcript strongly reduced activation of ERK within the lateral
ectoderm at gastrula stages (Fig.
7A,C). Similarly, expression of sprouty within the
lateral ectoderm was abolished in most of the FGFA morphants whereas
expression in the endoderm was still present
(Fig. 7B,D; n>200).
To ask if the FGFA-dependent expression of sprouty and
pax2/5/8 requires the ERK signaling pathway, we treated embryos with
the MEK inhibitor U0126 immediately following ingression of the PMCs and
analysed expression of these genes at the early gastrula stage. fgfA
was strongly expressed in the lateral ectoderm following treatment with the
inhibitor (Fig. 7E,H;
n>50). By contrast, expression of sprouty
(Fig. 7F,I; n>150)
and pax2/5/8 (Fig.
7G,J; n>200) was abolished, which is consistent with
these genes being downstream transcriptional targets of FGF signaling.
|
). Surprisingly, at 48 hours, a significant recovery
of archenteron formation and differentiation of the spicules was observed in
most of the embryos, but these spicules remained rudimentary and the embryos
did not acquire the typical elongated pluteus shape
(Fig. 8H,L). In embryos
injected with antisense morpholino oligonucleotides against fgfr2
transcripts, PMC ingression and archenteron invagination occurred on schedule
(Fig. 8M,N,Q,R), but in most
cases these PMCs did not acquire the regular spatial organization
characteristic of these cells (Fig.
8N,R). Small skeletal rudiments nevertheless formed in these
embryos, but in most of the larvae they did not elongate as much as in the
controls and remained as small tri-radiated spicules
(Fig. 8O,P,S,T). FGFR2
signaling thus appears to be required, in addition to FGFR1, for normal
patterning and differentiation of the PMCs. When the morpholino
oligonucleotides against FGFR1 and FGFR2 were co-injected, the resulting
phenotype was more severe than with each morpholino: PMC ingression and
gastrulation were strongly delayed, and no spicule rudiment formed
(Fig. 8U-X), as described above
for the fgfA morphants. To characterize the respective roles of FGFR1 and FGFR2 in gastrulation and skeletogenesis, we examined the expression of fz5/8, bhmt, pax2/5/8 and SM30 in embryos injected with MoFGFR1 and MoFGFR2, singly or together (Fig. 9). Vegetal pole expression of fz5/8 was perturbed following interference with FGFR1 but not with FGFR2 (Fig. 9A-D). Expression of bhmt in the endoderm was only slightly affected by inhibition of FGFR1 or of FGFR2, consistent with the idea that FGF signaling does not regulate endomesoderm specification (Fig. 9E-H). By contrast, and as anticipated from the FGFR1 expression pattern, pax2/5/8 expression in the ectoderm was abolished in all the embryos injected with FGFR-Mo (Fig. 9J). Unexpectedly, pax2/5/8 expression was also lost in all embryos injected with FGFR2-Mo (Fig. 9K). This result could be explained if reciprocal FGF signaling between the PMCs and the ectoderm is required to maintain ectodermal pax2/5/8 expression.
In 48 hour larvae in which either FGFR1 or FGFR2 function was blocked, expression of the biomineralization marker SM30 was downregulated but still detectable (Fig. 9N,O), consistent with the presence of small spicule rudiments in these embryos. By contrast, co-injection of the two morpholinos resulted in a complete loss of SM30 expression (Fig. 9P). We conclude that FGFR1 and FGFR2 both participate in the regulation of skeletogenesis and have partially redundant function in regulating the gene expression in the ectoderm and in the skeletogenic differentiation program of the PMCs.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies had shown that the ectoderm plays a crucial role in
steering the PMCs after their ingression and that it regulates the
skeletogenic gene expression program in these cells (Ettensohnn and
Winklbauer, 2004; Hardin,
1996; McClay et al.,
1992; McClay et al.,
2004). Micromeres isolated at the 16-cell stage can differentiate
in vitro into spicules, and this property has been interpreted as reflecting
the cell-autonomous programming of the PMCs
(Okazaki, 1975). However,
normal seawater cannot support spicule formation unless it is supplemented
with horse serum, suggesting that isolated PMCs lack some components normally
provided by the other cells. FGFA is an excellent candidate for the elusive
growth factor present in the serum. When extra PMCs are transplanted into the
blastocoel of recipient embryos, resulting in two or three times the normal
number of skeletogenic precursors being present, a skeleton of normal size
forms, indicating that the size and scale of the spicules depends on spatial
cues provided by other cell types
(Armstrong et al., 1993b;
Ettensohn, 1990b). The finding
that fgfA regulates morphogenesis of the spicules and is first
expressed in the ectoderm overlying the PMC clusters is consistent with these
observations. Finally, treatments with NiCl2, which radialize sea urchin
embryos, cause development of supernumerary spicule primordial, probably by
extending the spatial cues normally present in ventrolateral regions of the
embryo (McClay et al., 1992).
The radial extension of fgfA expression in NiCl2-treated embryos
could explain these effects.
|
), and
these same factors act as attractants for mesenchymal cells during limb bud
development (Webb et al.,
1997). During gastrulation in the chick, mesodermal cells emerging
from the primitive streak follow different trajectories guided by FGF signals
from neighboring tissues (Yang et al.,
2002). Experiments using implantation of beads soaked with
different FGFs showed that streak cells are attracted by FGF4 and repelled by
FGF8. Axonal navigation and angiogenesis provide some of the best examples of
FGF-dependent regulation of cell migration. Axonal navigation and angiogenesis
depend upon attractive and repulsive guidance cues provided by the cellular
environment and interpreted by the growth cones of axons or the migrating
endothelial precursors (Dickson,
2002; Tessier-Lavigne and
Goodman, 1996). A classical example of axonal navigation is
provided by the axons of the spinal motoneurons that leave the spinal cord.
After following a common ventral path, these axons subsequently diverge and
follow specific pathways to innervate different organs, such as the limbs,
sympathetic ganglia or axial musculature. Recent studies have demonstrated
that FGF family members including FGF9 are produced by the dermomyotome and
selectively attract the axons of a subpopulation of spinal cord motoneurons
called MMCm (Shirasaki et al.,
2006), which selectively express FGFR1, whereas the other
subclasses of neurons lack this receptor. The role of FGFs in this process is
remarkably similar to the role of FGF in guidance of primary mesenchyme cells
in the sea urchin embryo. In both cases, the ligand is produced by the target
cells (ectodermal cells in the sea urchin embryo and dermomyotome in mouse)
and it appears to act on a long range to attract the migrating cells, which
specifically express the receptor.
Another interesting parallel could be drawn between skeletogenesis in the
sea urchin embryo and angiogenesis in vertebrates. Neovascularization is a
complex process that involves guided migration of endothelial cells and the
combined activities of different RTKs
(Cross and Claesson-Welsh,
2001). FGF was the first growth factor purified with angiogenic
properties (Shing et al.,
1984), a finding rapidly followed by the discovery of VEGF. A
recent study has described the essential role of VEGF/VEGFR signaling in PMC
patterning and skeletal morphogenesis
(Duloquin et al., 2007). The
spatial expression of the FGFA and VEGF and of FGFR2 and VEGFR are largely
congruent. However, these two signals are independent and not functionally
redundant and thus both are required for correct migration and differentiation
of the primary mesenchyme.
FGF signaling and branching morphogenesis
FGF signaling plays a key role in regulating the formation of the ramified
respiratory system of Drosophila and vertebrates. In
Drosophila, a core FGF signaling pathway including Btl/FGFR and its
FGF ligand, Branchless (Bnl), is used repeatedly to control the successive
rounds of branching (Metzger and Krasnow,
1999). In this process, FGF expressed by clusters of cells in the
tracheal sac plays the role of a chemoattractant for the migrating tracheal
cells. Similarly, in vertebrates, FGF10 regulates the branching pattern of the
lung, as shown by the absence of lung in the Fgf10-knockout mice
(Min et al., 1998) and by the
severe inhibition of branching observed following expression of a
dominant-negative FGF receptor in the bronchial epithelium
(Peters et al., 1994).
In sea urchin embryos, previous studies had suggested that signals from the
ectoderm are required to direct both the growth, the branching pattern and the
final size of the spicules (Armstrong et
al., 1993a; Ettensohn,
1990b; Ettensohn and Malinda,
1993; Ettensohn and McClay,
1986; Hardin et al.,
1992; Katow et al.,
2000; Malinda and Ettensohn,
1994; McClay et al.,
1992; Peterson and McClay,
2003). For example, spicules formed in vitro from isolated
micromeres are linear and lack the complex branches observed in vivo
(Okazaki, 1975). They are also
much longer than the spicules present in normal embryos, suggesting that
isolated PMCs lack signals that regulate growth of the spicules.
Despite the fact that the skeleton of the sea urchin larva does not form from an epithelium as the lung of vertebrates and the tracheae of flies, but from a syncytium, we found that, fgfA, sprouty, pea, ets1 and fgfr2 are expressed selectively in all regions where the spicules will branch, allowing formation of the oral arms of the pluteus larva. This suggests that the FGF signaling pathway regulates formation of the complex three-dimensional structure that serves as a skeleton for the embryo and reinforces the idea that the function of this pathway in regulating formation of ramified organs and structures is highly conserved between species.
FGF as a signal regulating growth of the spicules
Local PMC-ectoderm interactions are required for skeletal rod elongation,
as shown by photoablation experiments of the growing arms
(Ettensohn, 1990a). Curiously,
fgfA, ets1, pax2/5/8 and sprouty are all expressed in the
regions where the arms of the pluteus larva will form but in a different
register. Expression of fgfA and ets1 is mainly restricted
to the isolated PMCs located just at the tip of the growing arms, while
pax2/5/8 and sprouty are detected in the ectoderm
surrounding the tip of the arm rods. This suggests that the interplay between
ectoderm and mesenchyme cells continues at later stages and that FGFA and
Pax2/5/8 may also be involved in regulating skeletal rod elongation.
FGFR2 and reciprocal FGF signaling between the PMCs and the ectoderm
Studies on lung morphogenesis and outgrowth of the limb bud suggest that
FGF signaling between the epithelial and mesenchymal compartments is
reciprocal (Lewandoski et al.,
2000; White et al.,
2006; Zhang et al.,
2006). In other words, the epithelium signals to the mesenchyme,
which signals back to the epithelium. For example, during limb bud outgrowth
in the mouse embryo, both FGF8 in the mesenchyme and FGF9 in the apical
ectodermal ridge regulate outgrowth of the bud. Reciprocal signaling between
the ectoderm and mesenchymal cells probably also occurs in the sea urchin
embryo, as we found that blocking FGFR2 signaling in the PMCs eliminates
pax2/5/8 expression from the overlying ectoderm. The signal released
by the PMCs that is required to maintain pax2/5/8 in the overlying
ectoderm is presently not known. As fgfA is first expressed within
the ectoderm, then within the PMCs of the ventrolateral clusters, this signal
could be fgfA itself. Alternatively, it could be another member of
the FGF family, as in the case of vertebrates. Experiments using chimeras in
which micromeres derived from FGFA morpholino-injected embryos will be
transplanted into control embryos could help to address this issue.
FGF signaling and the regulation of gastrulation
The role of FGF signals in gastrulation is also well documented
(Bottcher and Niehrs, 2005;
Sivak and Amaya, 2004;
Thisse and Thisse, 2005).
Studies in Xenopus have shown that FGF signaling regulates cell
motility, lamellipodia formation and cellular polarization. In addition, it is
required for gastrulation through the expression of downstream genes such as
sprouty and of the neurotrophin-receptor-like molecule Nrh
(Christofori, 2003;
Chung et al., 2005;
Nutt et al., 2001;
Sivak et al., 2005). Our
finding that FGF signaling is crucially required for gastrulation is in line
with previous observations showing that FGFR1 is expressed at high levels in
the vegetal pole region before and during gastrulation
(Lapraz et al., 2006). It is
also consistent with previous findings showing that treatments with the growth
factor inhibitor suramin severely interfere with gastrulation when applied
during blastula stages onward (Katow and
Aizu, 2002) and that MEK inhibition by U0126 causes
exogastrulation in the Strongylocentrotus
(Fernandez-Serra et al., 2004;
Kumano and Foltz, 2003).
However, FGFA is probably not the only FGF ligand involved in the regulation
of gastrulation, as its inhibition does not eliminate expression of
sprouty and pea3 within the vegetal plate and
archenteron.
Taken together, our findings show that FGF signals are essential components of the developmental program of the sea urchin embryo, regulating several essential processes such as oriented cell migration and differentiation of skeleton, as well as gastrulation. The sea urchin embryo, which is simple, readily accessible to experimental perturbations and transparent, will hopefully help to unravel the fundamental regulatory mechanisms underlying these processes.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/2/353/DC1
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ACKNOWLEDGMENTS |
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