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First published online 30 January 2008
doi: 10.1242/dev.011940
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Department of Molecular and Cell Biology, Division of Genetics, Genomics and Development, Center for Integrative Genomics, University of California, Berkeley, CA 94720, USA.
* Author for correspondence (e-mail: wshi{at}berkeley.edu)
Accepted 9 December 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Ciona, Ephrin, MAPK, Endomesoderm
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Martindale et al.,
2004). Indeed, portions of the mesoderm arise from a bipotential
cell type, the endomesoderm, in a variety of metazoan embryos
(Rodaway and Patient, 2001).
For example, in C. elegans, the endomesoderm blastomere (EMS) is
polarized by a Wnt/MAPK signal that inhibits mesoderm fate in the posterior E
lineage (Maduro, 2006),
whereas the anterior blastomere (MS) forms mesoderm by default. In sea
urchins, Notch signaling promotes secondary mesenchyme cell formation while
repressing endoderm fate (Sherwood and
McClay, 1999). Heart, blood and some muscle tissues are derived
from endomesoderm precursors at the marginal zone of zebrafish and frog
embryos (Rodaway et al., 1999;
Kimelman and Griffin, 2000;
Mathieu et al., 2004).
In this study, we examine the basis for distinct endoderm and mesoderm
derivatives arising from the A6.3 endomesoderm cell in the early embryo of
Ciona intestinalis, a simple chordate. A6.3 divides at the 32-cell
stage to form A7.5 and A7.6 daughter cells. A7.6 gives rise to the embryonic
trunk lateral cells (TLCs), which generate blood cells and additional mesoderm
derivatives during metamorphosis (Tokuoka
et al., 2005). Its sibling cell, A7.5, gives rise to anterior gut
endoderm. Blastomere isolation experiments suggest that the A7.6 mesoderm fate
is induced by a signal emanating from ectodermal cells in the animal
hemisphere (Kawaminani and Nishida,
1997). Here, evidence is presented that Ci-ephrin-Ad is
essential for the asymmetric cell fates arising from A6.3 through local
inhibition of MAPK signaling. We discuss these findings with regard to the
recent demonstration that Ephrin signaling is also required for the asymmetric
specification of the notochord and nerve cord
(Picco et al., 2007).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). For
microinjection, morpholino oligos or mRNA was co-injected with 1 mM Texas-Red
Dextran 10 kDa (Molecular Probes) and only fluorescent embryos were analyzed.
For MAPK inhibition assays, embryos were grown in seawater supplemented with 2
µM U0126 (Promega). For cell division arrest experiments, seawater was
supplemented with cytochalasin B to a final concentration of 2 µg/ml.
In situ hybridization and immunohistochemistry
Embryos were fixed in 4% paraformaldehyde, 0.5 M NaCl, 0.1 M MOPS pH 7.5, 2
mM MgSO4, 1 mM EDTA at 4°C overnight. In situ hybridization was
performed as described previously (Corbo et
al., 1997b). Digoxigenin-labeled antisense RNA probes were
synthesized from the following cDNA clones: Ci-Hand-like (citb018l16,
Ciona intestinalis Gene Collection Release 1), Ci-FGF8/17/18
(citb002j04), Ci-Delta-like (cieg005o22), Ci-MyTF
(citb040p06), Ci-Brachyury (Corbo
et al., 1997a), Ci-ephrin-Ad (ciad008n17),
Ci-TTF-1 (ciad042d09), Ci-Snail (cibd020p17). Alkaline
phosphatase (AP) enzyme assays were performed as follows. Fixed embryos were
rinsed three times in PBS, 0.1% Tween and stained in AP staining buffer
(Whittaker and Meedel, 1989);
staining was enhanced using 10% polyvinyl alcohol. For immunohistochemistry,
rehydrated embryos were treated with 0.3% H2O2 for 30
minutes to quench endogenous peroxidase activity, blocked for endogenous
biotin (SP-2001, Vector Labs, Burlingame, CA) and then incubated overnight in
mouse anti-dpERK (1:1000, Sigma, M9692). dpERK was visualized with the
VECTASTAIN Elite ABC System (Vector Labs).
Molecular biology
A constitutively active form of the Ciona FGF receptor (Ci-FGFR) was
created by fusing the intracellular kinase domain of Ci-FGFR (the last 437
amino acid residues) to the first 455 amino acids from the Drosophila
torso4021 mutant. The torso4021 peptide
includes the extracellular and transmembrane domain, and results in the
constitutive activation of the RTK receptor
(Sprenger and Nusslein-Volhard,
1992). The torso-FGFR fusion gene was cloned into the
pCS2+ vector, and capped mRNA was synthesized using the mMESSAGE mMACHINE SP6
Kit (Ambion, Austin, TX). RNA was microinjected into 1-cell embryos at a final
concentration of 0.5 µg/µl. The torso-FGFR fusion gene was also attached
to 
2 kb of the ZicL 5' flanking regulatory sequence, which
was amplified from genomic DNA using the following primers: forward,
5'-CATATTGTAAGGTGGAGATG-3' and reverse,
5'-TGCATAGGGACGATCAACC-3'. The resulting fusion gene is expressed
in the progenitors of the notochord and nerve cord.
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4 kb upstream regulatory sequence from the FoxD
gene was cloned using the following primers: forward,
5'-GAAACGATCTTCGGCGGATC-3' and reverse,
5'-ATATTGCACACAACACTGCAC-3'. The resulting DNA fragment was cloned
into the pCESA vector (Harafuji et al.,
2002) to drive Ci-ephrin-Ad throughout the vegetal
hemisphere. A Ci-ephrin-Ad morpholino
(5'-GGTAGTAGGTAAATTGAGTTGCCAT-3')
(Picco et al., 2007) was
purchased from GeneTools LLC (Eugene, OR) and injected at 0.5 mM. |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Imai et al., 2004). None of
these genes is significantly expressed in the A7.5 sister cell or other
regions of the endoderm (Fig.
1A), and we therefore refer to them as the A7.6 group genes
(Hudson and Yasuo, 2006;
Imai et al., 2006).
Conversely, transcription factors such as TTF-1 exhibit restricted
expression in the endoderm, including A7.5, and are excluded from A7.6
(Fig. 1B). The A7.6 group genes
and TTF-1 were used as markers to evaluate A7.6/A7.5 specification
upon experimental perturbation.
Transformation of anterior endoderm into supernumerary A7.6 cells
The presumptive endoderm provides a localized source of FGF signaling that
induces adjacent mesoderm tissues such as the notochord and mesenchyme
(Kim et al., 2000;
Imai et al., 2002c).
Inhibition of this signal causes a loss of Brachyury
(Ci-Bra) expression in the presumptive primary notochord and loss of
Twist-like genes in the mesenchyme. To determine whether FGF
signaling is also important for the induction of the A7.6 fate, we employed in
situ hybridization assays to examine the expression of marker genes in embryos
treated with the MEK inhibitor, U0126 (Fig.
2A-E).
To our surprise, the treated embryos exhibited a striking expansion, not loss, of A7.6 group gene expression. Hand-like, FGF8, Delta-like and MyTF (Fig. 2A-D) displayed expression in both A7.6 and anterior endoderm cells, including the A7.5 blastomeres. The ectopic endoderm expression was somewhat variable, but in the most extreme cases, all anterior endoderm cells expressed A7.6 group genes. Conversely, expression of the TTF-1 gut marker gene was completely lost upon drug inhibition (Fig. 2E). These observations suggest that MAPK signaling is essential for inhibiting A7.6 group genes within the endoderm, and thereby restricting their activities to the A7.6 lineage. FGF signaling in the anterior endoderm might inhibit the expression of A7.6 determinants (see Discussion).
Anti-dpERK antibody staining was used to visualize sites of active receptor tyrosine kinase (RTK) signaling, including FGF signaling. On the vegetal side of the embryo, staining was detected throughout the presumptive endoderm, primary notochord and posterior mesenchyme of 64-cell embryos (Fig. 2F). Staining was excluded from the A7.6 blastomere, but detected in the sister A7.5 endoderm cell. This pattern - dpERK staining in A7.5 but not A7.6 - persisted during the 110-cell stage (Fig. 2G). As expected, there was a complete loss of dpERK staining in embryos treated with the MEK inhibitor drug U0126 (Fig. 2H).
In summary, differential MAPK activation is observed in the descendants of the A6.3 endomesoderm blastomere. A7.6 group genes are expressed in the lineage that lacks MAPK activity, whereas endoderm marker genes are expressed in the sister lineage exhibiting dpERK staining. Inhibition of MAPK signaling causes expansion of A7.6 group genes throughout the anterior endoderm.
Ephrin attenuates MAPK signaling in the A7.6 lineage
The asymmetric activation of MAPK in A7.5 cannot be explained by the
localization of FGF signaling, as Ci-FGF9/16/20 is expressed in the
A6.3 progenitor cell at the 32-cell stage
(Hudson et al., 2003;
Nishida, 2003). Thus, some
inhibitory signals from neighboring cells might be required to specifically
inhibit MAPK activity in A7.6. A recent study demonstrated that Ephrin-Eph
signaling is essential for the asymmetric specification of the A6.2 and A6.4
blastomeres, which form notochord and nerve cord
(Picco et al., 2007).
FGF9/16/20 emanating from the endoderm induces two of the daughter cells, A7.3
and A7.7, to form primary notochord cells via Brachyury gene
activation. By contrast, Ci-ephrin-Ad inhibits FGF signaling in the sister
cells, A7.4 and A7.8, that produce the future nerve cord. Disruption of
Ci-ephrin-Ad gene activity via morpholino (MO) injection led to
ectopic MAPK activation in the neurogenic lineage, ectopic activation of
Brachyury gene expression, and a corresponding transformation of
these cells into supernumerary notochord cells.
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). Thus,
Ci-ephrin-Ad appears to be expressed in the right place and time to
serve as a crucial signal for A7.6 specification during the division of the
A6.3 blastomere.
To test whether Ci-ephrin-Ad is required to inhibit MAPK signaling
and thereby establish mesoderm fate in A7.6, its function was disrupted via
microinjection of a specific inhibitory MO. Embryos were allowed to develop to
the 64- or 110-cell stage, and then hybridized with a variety of markers
(Fig. 4). Upon MO injection,
there was a severe loss of Hand-like, FGF8, Delta-like, and
MyTF expression (Fig.
4A-D). By contrast, endoderm marker genes such as TTF-1
exhibited ectopic expression in the A7.6 blastomere
(Fig. 4G, arrow). As shown
previously (Picco et al.,
2007), Brachyury expression expands in
Ci-ephrin-Ad MO embryos owing to loss of MAPK inhibition in the
presumptive A-line CNS (Fig.
4F).
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).
110-cell embryos were arrested by treatment with the cytokinesis inhibitor,
cytochalasin B, and allowed to develop to the equivalent of the tailbud stage.
Endogenous AP enzyme activity was visualized via standard colorimetric methods
(see Materials and methods). In wild-type arrested embryos, AP activity was
restricted to the endoderm, including the A7.5 blastomere, and was absent from
A7.6 (Fig. 4J). However, mutant
embryos injected with the Ci-ephrin-Ad MO exhibited AP staining in
both the endoderm and A7.6 blastomere (Fig.
4I). These results raise the possibility that
Ci-ephrin-Ad normally inhibits FGF signaling in A7.6, thereby
fostering a mesoderm identity as opposed to endoderm fate.
Support for this model was obtained by examining the dpERK staining pattern
in mutant embryos injected with Ci-ephrin-Ad MO. As shown earlier
(see Fig. 2F), MAPK activity is
not normally detected in the A7.6 blastomere. By contrast, mutant embryos
displayed dpERK staining in A7.6, in addition to the normal sites of
expression in the endoderm, primary notochord cells and mesenchyme
(Fig. 4E, arrows). Thus, it
would appear that the specification of asymmetric cell fates from the A6.3
lineage is similar to that seen for A6.2 and A6.4
(Picco et al., 2007). In each
case, Ephrin-Ad inhibits RTK signaling in the daughter cells that are in
direct contact with animal blastomeres.
FGF signaling inhibits A7.6 specification
The preceding analysis suggests that RTK signaling, as visualized by dpERK
staining, is inhibited by Ephrin-Ad upon specification of the A7.6 mesoderm
lineage. In the case of notochord formation, FGF signaling directly activates
Ci-Bra expression through ETS-binding sites in the Ci-Bra
enhancer (Matsumoto et al.,
2007), and recombinant FGF protein is sufficient to induce
Ci-Bra expression in cultured explants
(Nakatani et al., 1996).
Ephrin normally attenuates FGF signaling and MAPK activity in the neuronal
descendants of the A6.2 and A6.4 progenitors.
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). This strategy has been used to create constitutively active
forms of a number of receptors (Galindo et
al., 1995).
The torso-FGFR fusion gene was attached to Ci-ZicL
regulatory sequences, which mediate expression in the progenitors of the
notochord/nerve cord and muscle/mesenchyme lineages
(Imai et al., 2002b). As
expected, ectopic Ci-Bra expression was observed in the A-line nerve
cord, suggesting a transformation of neural fate to notochord due to ectopic
activation of MAPK signaling (Fig.
5A, compare with B). This confirms that the torso-FGFR is
constitutively active and can overcome Ephrin-mediated MAPK inhibition in
A-line nerve cord precursors. We then used an 4 kb cis-regulatory
sequence from the Ci-FoxD gene to ectopically express
torso-FGFR in all vegetal blastomeres, beginning at the 16-cell
stage. Unfortunately, the FoxD::torso-FGFR fusion gene did not appear
to diminish expression of the A7.6 group genes, as might be predicted by
sustained MAPK activity (data not shown). Perhaps the transgene is not
expressed sufficiently early to interfere with Ephrin-mediated inhibition of
MAPK in the A7.6 lineage.
To circumvent this potential problem, we injected full-length, capped torso-FGFR mRNA into 1-cell embryos, and then reared the injected embryos to the 110-cell stage. In situ hybridization assays revealed consistent reductions in Hand-like, FGF8 and MyTF expression in A7.6 (Fig. 5C,D,F), although Delta-like expression was only sporadically reduced (Fig. 5E). These results are consistent with those of the Ci-ephrin-Ad MO experiments (Fig. 4A-D).
The incomplete inhibition of A7.6 group genes by activated FGFR is likely
to be due to the repression of MAPK activation by endogenous Ephrin-Ad. To
test this, we co-injected ephrin-Ad MO and torso-FGFR mRNA
into 1-cell embryos and scored the expression of Delta-like in
110-cell embryos. Delta-like expression was now repressed in the A7.6
blastomere in 80% of the embryos (Fig.
6A), whereas the eprhin-Ad MO or activated FGFR
mRNA alone caused just 40% or 7% repression, respectively. As
expected, the genes that are more sensitive to the loss of MAPK activity
exhibited a complete loss in expression upon injection of the Ephrin-Ad MO and
activated FGFR mRNA (Hand-like, 0/34 embryos exhibited
expression in A7.6; FGF8, 1/31 embryos; MyTF, 0/32 embryos;
data not shown).
The synergistic disruptions obtained by inhibiting Ci-ephrin-Ad and activating MAPK strongly suggest that the major role of Ephrin-Eph signaling is the suppression of FGF/MAPK in A7.6. If true, then the Ephrin-Ad MO mutant phenotype should be suppressed by the inhibition of MAPK activity. To test this prediction, we treated ephrin-Ad MO-injected embryos with the U0126 inhibitor at the 32-cell stage and assayed for the expression of the A7.6 group marker genes (Fig. 6B-E). The expression patterns resembled the situation seen when wild-type embryos were treated with U0126 alone (see Fig. 2A-D). A7.6 group genes were expressed in both the A7.6 blastomere and the anterior endoderm (Fig. 6B-E). These observations are consistent with the possibility that the sole role of Ephrin-Ad in A7.6 specification is the inhibition of MAPK activation.
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The Ci-FoxD 5' regulatory sequence was used to ectopically
express the full-length Ci-ephrin-Ad coding region. Ci-FoxD
is activated in the progenitors of A6.1 and A6.3 at the 16-cell stage of
embryogenesis (Imai et al.,
2002a). In principle, the Ci-FoxD enhancer should direct
Ci-ephrin-Ad expression in the A6.3 endomesoderm cell just before its
division into separate endoderm and mesoderm lineages.
Electroporated embryos expressing the FoxD::Ci-ephrin-Ad transgene were harvested at the 110-cell stage for in situ hybridization (Fig. 7). There was sporadic expansion of the A7.6 group genes in the anterior endoderm (Fig. 7A-D, arrows), whereas expression of TTF-1 was lost in the anterior endoderm, including A7.5 (Fig. 7E, arrows). The mosaic expansion of A7.6 marker genes and incomplete loss of TTF-1 probably reflect the timing of transgene expression. Expression of the Ci-ephrin-Ad transgene at the 16-cell stage might lead to the secretion of ectopic Ephrin-Ad protein just at the critical time when MAPK is incompletely inhibited, resulting in partial transformation of endoderm to mesoderm fate.
In summary, it appears that misexpression of Ci-ephrin-Ad in the vegetal blastomeres leads to inhibition of FGF signaling and MAPK activity in the endoderm. This results in a mutant phenotype similar to that obtained upon application of the U0126 MEK inhibitor (see Fig. 2).
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).
Indeed, Nodal MO or a drug inhibitor (SB431542) of the Nodal
receptor completely blocks expression of the A7.6 group genes
(Imai et al., 2006) (data not
shown). However, it is difficult to reconcile this role for Nodal signaling
with the ectopic activation of A7.6 group genes in the endoderm upon MAPK
inhibition (e.g. Fig. 2A-D). To
clarify this issue, we blocked Nodal expression in b6.5 using the
MAPK U0126 inhibitor at different time points during development. It
has been shown that Nodal expression in b6.5 is activated by an FGF
signal from the endoderm at the 32-cell stage
(Hudson and Yasuo, 2005).
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40%), but the remaining embryos exhibited expression in both
A7.6 and the endoderm (Fig.
8L). Thus, loss of the Nodal signal in b6.5 does not significantly
alter the induction of the A7.6 group genes in the A7.6 lineage.
The preceding observations suggest that MAPK signaling in the endoderm
prevents endogenous Nodal from activating the A7.6 group genes. To determine
the epistatic relationship between Nodal and MAPK signaling, we ectopically
expressed Nodal in the vegetal hemisphere using the Ci-FoxD
enhancer. We used the Snail gene as a control because it is induced
in the lateral neural plate by Nodal signals in b6.5
(Hudson and Yasuo, 2005). In
wild-type embryos, Snail is expressed in the A8.15/A8.16 neural
precursors (Fig. 8M). By
contrast, embryos electroporated with the FoxD::Nodal transgene displayed an
expanded Snail expression pattern that encompassed the medial rows of
the A-line neural precursors, A8.7/A8.8
(Fig. 8N, compare with M). This
result suggests that the FoxD::Nodal transgene is able to drive
functional Nodal expression throughout the vegetal hemisphere.
We then examined the expression of A7.6 marker genes in embryos expressing the FoxD::Nodal transgene (Fig. 8O-Q). There was no ectopic expression in the endoderm blastomeres, suggesting that the augmented levels of Nodal signaling are unable to overcome repression by endogenous MAPK. Hand-like and Delta-like sometimes displayed ectopic expression in the A-line neural precursors (Fig. 8O,P), suggesting that high levels of Nodal are sufficient to activate their expression when MAPK signaling is absent. No ectopic FGF8 expression was observed, whereas MyTF was sometimes ectopically expressed in the B7.7 lineage (Fig. 8Q, arrow).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In both cases, a localized Ephrin-Ad signal produced by the
primitive ectoderm (the animal blastomeres) competes with FGF9 signals from
the primitive gut. Those cells in extended contact with the ectoderm lack MAPK
activation, whereas those cells in contact with the endoderm experience MAPK
activation and follow a different fate. It is conceivable that this interplay
of Ephrin and FGF signaling is used in other systems to produce asymmetric
cell fates.
Ephrin-FGF competition establishes multiple Ciona tissues
Ephrins have been implicated in a variety of cellular processes, including
axonal guidance, repulsive cell-cell interactions, and adhesion
(Poliakov et al., 2004;
Pasquale, 2005). Different
Ephrin family members can activate (Zisch
et al., 2000) or inhibit RTK signaling
(Elowe et al., 2001;
Miao et al., 2001) in
different cellular contexts. The present study, along with the recent analysis
of Ci-Bra regulation (Picco et
al., 2007), suggest that Ci-ephrin-Ad functions as a
localized inhibitor of FGF signaling to produce asymmetric cell fates in
Ciona. The presumptive endoderm/endomesoderm produces a localized
source of FGF9/16/20, which induces the specification of diverse mesoderm
lineages, including the notochord and mesenchyme. We have presented evidence
that Ephrin also controls the subdivision of the A6.3 endomesoderm.
A model for the specification of the A7.6 blastomere is summarized in
Fig. 9. Previous studies have
shown that Nodal is essential for the expression of several A7.6
marker genes, including Hand-like (also known as NoTrlc),
FGF8 and Delta-like
(Hudson and Yasuo, 2006;
Imai et al., 2006).
Nodal is expressed in the A6.3 blastomere of 32-cell embryos, as well
as in the other progenitors of the endoderm. FGF/MAPK signaling is also active
in the A6.3 at this stage, as judged by anti-dpERK staining (data not shown).
Ephrin-Ad produced by b6.5 (and other animal blastomeres) inhibits MAPK in
A7.6, thereby permitting Nodal to activate the A7.6 group genes. Nodal
signaling in A7.6 might be reinforced by Nodal expression in the b6.5 lineage.
Thus, the inhibition of FGF signaling by Eprhin-Ad, along with augmented
levels of Nodal signal, might be responsible for the activation of A7.6 group
genes. However, we have presented evidence that Nodal in b6.5 is not essential
for A7.6 group gene expression. Instead, it would appear that the combination
of endogenous Nodal in the A6.3 progenitor, along with the localized
inhibition of MAPK in A7.6 by Eprhin-Ad, is the decisive determinant of A7.6
specification.
Inhibition of MAPK signaling via drug treatment or ectopic expression of
Ephrin-Ad leads to misexpression of A7.6 marker genes in the anterior
endoderm, where Nodal is normally inactive owing to FGF/MAPK signaling.
Posterior endoderm cells also contain Nodal but fail to express A7.6 marker
genes upon inhibition of MAPK. This might reflect the restricted distribution
of additional activators required for A7.6 gene expression. For example,
Hand-like is activated by the combination of Nodal signaling and the
FoxA transcription factor (Imai
et al., 2006). FoxA expression is restricted to the
anterior endoderm, dorsal mesoderm and future CNS floorplate, but is absent
from the posterior endoderm (Corbo et al.,
1997b). This is consistent with the result of ectopic
Hand-like and Delta-like activation in the A-line neural
lineage by expression of the FoxD::Nodal transgene.
A7.6 serves as a signaling center
A7.6 expresses a number of localized determinants, including two crucial
signaling molecules, FGF8 and Delta-like. A7.6 is located in a strategically
important position within the vegetal hemisphere. It contacts components of
all three germ layers: the endoderm, ectoderm and mesenchyme. The Delta-like
ligand expressed in A7.6 induces the secondary notochord lineage via Notch
signaling, and also induces the lateralmost neural fate
(Hudson et al., 2007).
Similarly, FGF8 expression is required for maintaining the primary notochord
fate (Yasuo and Hudson, 2007).
Because these signaling pathways require either direct cell-cell contact
(Notch) or act over one or two cell diameters (FGF), it is crucial to activate
the expression of Delta-like and FGF8 to A7.6, but not in its sibling A7.5
endoderm blastomere. The activities of three pathways, Ephrin, MAPK and Nodal
signaling, are employed to achieve this precise asymmetric cell-fate
specification event.
Specification and subdivision of the Ciona endomesoderm
Recent phylogenetic analysis suggests that tunicates (e.g. Ciona)
are the closest living relatives of the vertebrates
(Delsuc et al., 2006). As a
result, it is possible that vertebrates employ a mechanism for the
specification and subdivision of the endomesoderm that is similar to the one
used in Ciona. The A6.3 endomesoderm cell is established by the
action of a localized maternal determinant, β-Catenin
(Imai et al., 2000), which
activates the expression of multiple signaling molecules including
Nodal and FGF9. Nodal is required to activate A7.6-specific
genes such as Hand-like, FGF8 and Delta-like. The failure of
Nodal to activate A7.6 group genes in the endoderm is due to MAPK signaling.
FGF signaling either directly or indirectly inhibits Nodal (e.g.
Kretzschmar et al., 1999;
Grimm and Gurdon, 2002). As a
result, Nodal signaling is blocked in A6.3, but is activated in A7.6 owing to
the localized inhibition of FGF signaling by Eprhin-Ad.
Most or all metazoan embryos possess a transient endomesoderm that
generates specific mesodermal derivatives
(Rodaway and Patient, 2001).
In vertebrates, the presumptive endomesoderm gives rise to blood, heart and
muscle (Kimelman and Griffin,
2000). Formation of the vertebrate endomesoderm depends on
TGF-β signaling molecules such as Xnrs in Xenopus and Squint and
Cyclops (Nodal-related 1 and 2, respectively - ZFIN) in zebrafish. The
subsequent subdivision of the endomesoderm is not clearly understood, but
might depend on FGF signaling (Rodaway et
al., 1999). It remains to be seen if competitive interactions
between Nodal (or some other TGF-β signaling molecule) and FGF lead to
the subdivision of endomesoderm in vertebrate embryos.
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ACKNOWLEDGMENTS |
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