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First published online August 24, 2007
doi: 10.1242/10.1242/dev.010140
Center for Integrative Genomics, Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA 94720, USA.
Authors for correspondence (e-mails:
bjd18{at}email.arizona.edu;
lionelchristiaen{at}berkeley.edu)
Accepted 5 July 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: FGF signaling, Ascidian, Cardiac morphogenesis, Directed cell migration, Forkhead
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Cardiogenesis is controlled by a highly conserved cassette of regulatory
genes, which includes tinman/Nkx2.5, pannier/Gata4/6, Hand
and T-box genes (Davidson,
2007; Davidson and Erwin,
2006; Olson,
2006). How this network coordinates cardiac morphogenesis and
heart cell differentiation remains poorly understood. Here and elsewhere we
have studied the earliest events in heart formation in an emerging model
system, the ascidian Ciona intestinalis.
Despite their simplicity, tunicates, which include ascidians, are the
closest living relatives of the vertebrates
(Delsuc et al., 2006). The
ascidian heart consists of a single layer of striated cardiomyocytes,
surrounded by a pericardial sheath. This simple single-compartment heart beats
rhythmically and can undergo reversible contractions (for details, see
Davidson, 2007).
Lineage studies showed that the adult heart derives from two founder cells
(the B7.5 blastomeres in the early gastrula embryo), which also form the
larval anterior tail muscles (Davidson and
Levine, 2003; Hirano and
Nishida, 1997). By the end of neurulation, the B7.5 daughter cells
constitute bilateral clusters of two small anterior and two large posterior
cells occupying an anterior location in the tail. Shortly thereafter, the
small anterior B7.5 lineage cells migrate to the ventral side of the trunk,
hence their designation as trunk ventral cells (TVCs). The large posterior
daughter cells remain in the tail, where they differentiate into muscle cells.
The TVCs are the heart precursor cells in ascidian embryos. During subsequent
stages of embryogenesis, they migrate into the trunk and fuse at the ventral
midline in a fashion reminiscent of vertebrate heart precursor cells
(Davidson and Levine,
2003).
Previous studies illuminated some aspects of the genetic regulation of
early heart development in ascidians. The Mesp basic helix-loop-helix (bHLH)
transcription factor is expressed exclusively in B7.5 cells from the 110-cell
stage until the end of gastrulation (Imai
et al., 2004; Satou et al.,
2004). Morpholino knock-down showed that Mesp activity is required
for both TVC migration in the embryo and cardiomyocyte differentiation during
metamorphosis (Satou et al.,
2004). In addition, ascidian orthologs of the conserved heart
specification genes NK-4 (tinman/Nkx2.5), GATA-a
(pannier/GATA4/5/6), Hand and Hand-like (initially
termed NoTrlc) (Imai et al.,
2003) are expressed in the TVCs
(Davidson, 2007;
Davidson and Levine, 2003;
Satou et al., 2004). NK-4,
Hand and Hand-like are downregulated upon Mesp knock-down
(Satou et al., 2004). Mesp is
also thought to upregulate Ets1/2 expression in the B7.5 lineage,
thus conferring competence to respond to an unknown extrinsic FGF signal,
possibly FGF9/16/20 (Davidson et al.,
2006) (B.D., unpublished).
FGF signaling, transduced via the MAPK pathway, was recently shown to
induce both heart tissue specification and TVC migration
(Davidson et al., 2006). This
induction event takes place only in the anterior B7.5 daughters, thus
distinguishing the heart precursors from their sister tail-muscle cells. Heart
specification and cell migration are both transcriptionally regulated by
Ets1/2 in response to FGF signaling.
Migration and specification are thus tightly linked by common molecular
determinants. However, it has been possible to uncouple TVC migration from
cardiac muscle induction by targeted expression of a constitutively activated
form of Mesp, the Mesp:VP16 fusion protein, in the B7.5 lineage
(Davidson et al., 2005). TVCs
are sometimes arrested in the anterior tail, and differentiate into
disorganized aggregates of contractile cardiomyocytes at an ectopic location
in the juvenile. Although the molecular basis of this uncoupling is not known,
this result suggests that distinct, but interconnected, genetic pathways
differentially regulate the acquisition of cardiac tissue identity and
migratory behavior.
Here, we present evidence that the forkhead/winged helix transcription
factor FoxF is essential for TVC migration, but not for heart muscle
specification. FoxF orthologs are highly conserved among diverse
metazoans, and have been analyzed in vertebrates and Drosophila
(Adell and Muller, 2004;
Kaestner et al., 2000;
Yagi et al., 2003).
FoxF is one of the first genes to be activated in the anterior B7.5
lineage following FGF induction. We identified a FoxF minimal heart enhancer
and used a cis-trans complementation test to show that Ets1/2 is an immediate
activator of the enhancer in vivo. A dominant-negative form of FoxF inhibited
cell migration but not subsequent heart differentiation, resulting in a
striking phenotype: a beating heart in an ectopic position within the body
cavity. These observations suggest that FoxF is a direct downstream effector
of the FGF/MAPK/Ets signaling pathway and is required to induce the migratory
behavior of heart precursors.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A FoxF morpholino oligonucleotide
(5'-TGGTGTCCTGTCACTTCCATACTTC-3') was purchased from Gene Tools.
Morpholino injection was performed as described (
40 pl, 0.3-0.5 mM)
(Imai et al., 2000). Juveniles
were obtained by transferring electroporated embryos to uncoated Petri dishes
filled with filtered artificial seawater with ampicillin (80 µg/ml). Water
was changed 48 hours later, and every 24 hours subsequently. Heart phenotypes
were scored from first water change and ongoing for the following 5 days.
Histochemistry and in situ hybridization
X-gal staining was performed as described
(Locascio et al., 1999),
except that embryos were fixed in MEM-GA (0.1 M MOPS, pH 7.4, 0.5 M NaCl, 2 mM
MgSO4, 1 mM EGTA, pH 8.0, 0.2% glutaraldehyde, 0.05% tween-20) for
30 minutes at room temperature and staining was performed at 37°C. Stained
embryos were mounted in glycerol. Double fluorescent in situ hybridizations
and immunohistochemistry were performed as described by Dufour et al.
(Dufour et al., 2006).
ß-galactosidase was detected using a mouse monoclonal antibody (dilution
1:1000, Promega, Z378A); antisense RNA probes for either FoxF, Hand-like,
GATA-a or NK-4 were visualized with TSA-fluorescein and the
amplification kit according to the manufacturer's recommendations
(PerkinElmer, NEL741). Embryos were mounted in ProLong Gold (Invitrogen,
P36931) and analyzed with a LEICA TCS SP2 confocal microscope.
Molecular cloning
The coding sequence for the FoxF DNA binding domain (FoxF-DBD) was
amplified from the Ciona Gene Collection library clone cicl007c02.
The Mesp>FoxF:VP16 and Mesp>FoxF:WRPW fusion genes
were derived from the previously reported Mesp>Mesp:VP16 and
Mesp>Mesp:WRPW fusion genes
(Davidson et al., 2005), by
replacing the Mesp bHLH domain with the FoxF-DBD fragment.
Approximately 3 kb of the FoxF 5' flanking sequence was
PCR-amplified from genomic DNA and cloned into the pCESA vector containing a
lacZ reporter gene (Harafuji et
al., 2002). The minimal FoxF TVC enhancer (-1135 to -840)
was PCR amplified and cloned upstream of the Ci-FoxAa basal promoter
included in pCESA. Small deletions and point mutations were introduced in the
FoxF regulatory sequences using the QuickChange site-directed mutagenesis kit
(Stratagene, 200519-5).
Migration phenotype analysis
Both the Mesp>GFP and Mesp>lacZ reporters were used
to assess migration phenotypes. Transformed embryos and larvae expressing the
Mesp>GFP reporter gene were fixed in 4% formaldehyde overnight and
mounted in glycerol or ProLong Gold (Invitrogen, P36931). Migration defects
were grouped into five distinct phenotypic classes based on the relative
position of the B7.5 lineage cells within the embryo
(Fig. 3G). At least two
independent experiments were performed for each condition. The proportions of
each phenotypic class were compared between conditions using a
2 test.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Selective disruption of Mesp gene activity causes
reduced expression of several downstream genes, including Tolloid, NK-4,
Hand-like and FoxF, in the B7.5 lineage. Previous studies
suggested a possible role for FoxF1 (also known as Foxf1a)
in mesenchyme migration during the development of the lung, gall bladder and
liver in mouse embryos (Kalinichenko et
al., 2001; Kalinichenko et
al., 2002; Mahlapuu et al.,
2001). We therefore explored the possibility that FoxF
plays a role in heart migration during Ciona embryogenesis.
FoxF expression correlates with heart migration
The FoxF expression pattern was determined by in situ
hybridization (Fig. 1A-C). At
the late neurula stage, FoxF transcripts were detected in the trunk
epidermis, and in the two TVCs at the anterior end of the tail
(Fig. 1A,B, arrowheads). At the
tailbud stage, epidermal expression persisted, while the TVCs had moved to a
ventro-posterior position in the trunk
(Fig. 1C, arrowheads). Hence,
FoxF is expressed in both the trunk epidermis and TVCs.
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Previous studies demonstrated that heart induction occurs at the late
neurula stage in the anterior B7.5 cells in response to an FGF signal, which
induces the activity of the transcription factor Ets1/2
(Davidson et al., 2006). In an
initial attempt to determine whether FoxF expression requires Ets1/2
activity, embryos were co-electroporated with constitutively active (Ets:VP16)
and dominant-negative (Ets:WRPW) forms of Ets1/2 under the control of the
Mesp enhancer. Co-electroporation of the Mesp>Ets:VP16 fusion gene
caused the posterior B7.5 lineage to migrate into the trunk and form
supernumerary heart precursors (Fig.
1E-E'') (for details, see
Davidson et al., 2006). All
four cells, the normal heart cells and transformed muscle cells, expressed
FoxF upon Ets:VP16 overexpression. By contrast, FoxF
expression was lost in embryos that were co-electroporated with a
Mesp>Ets:WRPW fusion gene, which blocked heart induction and converted the
entire B7.5 lineage into tail muscles (Fig.
1F-F'').
The preceding analysis suggests that FoxF expression is downstream
of Ets1/2-mediated heart specification. To determine whether FoxF
expression correlates with either migration or tissue specification, we
examined tadpoles that express the Mesp>Mesp:VP16 fusion gene.
Targeted expression of Mesp:VP16 caused sporadic inhibition of migration, but
did not prevent heart tissue differentiation in juveniles
(Davidson et al., 2005). Cells
that failed to migrate into the trunk upon Mesp:VP16 over-expression did not
express FoxF (Fig.
1G-G''). By contrast, these same non-migrating B7.5 lineage
cells do express Hand-like and undergo heart tissue differentiation
in the juvenile (Davidson et al.,
2005). Thus, FoxF and Hand-like expression
differentially correlate with migration and tissue specification,
respectively.
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FoxF is an immediate target of the FGF/MAPK/Ets pathway
The preceding results raise the possibility that FoxF is a direct
transcriptional target of the FGF/MAPK/Ets pathway in the B7.5 lineage. To
test this hypothesis, we isolated and characterized FoxF
cis-regulatory sequences. The genomic region upstream of the first
FoxF exon is highly conserved between Ciona intestinalis and
Ciona savignyi (Fig.
2A), which often points to functional non-coding DNA. We found
that 3 kb of the 5' flanking region drives lacZ reporter gene
expression in the trunk epidermis and heart precursors, thus recapitulating
the endogenous FoxF expression pattern in electroporated tadpoles
(Fig. 2B).
A series of 14 truncated constructs were generated and analyzed in an effort to identify a minimal heart enhancer (Fig. 2A and J.B., unpublished). We mapped a 295 bp TVC-specific enhancer between 1135 and 840 bp upstream of the translation initiation codon (Fig. 2A,C). When fused to a Ci-FoxAa basal promoter, reporter gene expression driven by this enhancer was restricted to the heart precursor cells (Fig. 2D). FoxF expression in the trunk epidermis depends on separate elements that map within the proximal 845 bp of the 5' flanking region (Fig. 2A and J.B., unpublished).
Close examination of the 295 bp TVC enhancer revealed the presence of three
putative Ets1/2-binding sites matching the consensus recognition sequence
MGGAWNY (Choi and Sinha, 2006)
(Fig. 2C). To test whether
these sites are required for enhancer activity, point mutations were
introduced in the minimal FoxF TVC enhancer and assayed by
electroporation and X-gal staining (Fig.
2E). Point mutations in each individual putative Ets1/2 site
significantly reduced TVC expression of the transgene. Because no single
alteration completely eliminated reporter gene expression; we combined
mutations of the two sites that showed the greatest effects
(Fig. 2E, EtsA and EtsB sites).
Strikingly, combined mutations of the EtsA and EtsB sequences completely
abolished reporter gene expression (Fig.
2E). These results show that putative Ets1/2-binding sites are
required for FoxF minimal TVC enhancer activity.
Further evidence that Ets1/2 can directly transactivate the minimal TVC
enhancer of FoxF stems from a cis-trans complementation test. This test was
based on the requirement for a second sequence motif, CACTTG, which was also
found to be essential for the activity of the FoxF cardiac enhancer. This
motif conforms to an E-box (CANNTG consensus). Deletion of this sequence from
either the full-length FoxF>lacZ fusion gene or the minimal TVC
enhancer abolished reporter gene expression in heart precursor cells
(Fig. 2E-G; 
E-box
construct). In addition, ectopic expression of the constitutively activated
form of Ets1/2 induces FoxF>lacZ expression in both the anterior
and posterior B7.5 lineages (Davidson et
al., 2006). Therefore, we reasoned that, if Ets is a direct
activator, the hyper-active Ets:VP16 fusion protein should be able to restore
the activity of a defective FoxF enhancer lacking the E-box
motif.
Indeed, co-electroporation of the Mesp>Ets:VP16 fusion gene with the damaged FoxF enhancer resulted in robust lacZ expression in the entire B7.5 lineage, as compared with an empty-vector control (Fig. 2H,I). To further test whether this cis-trans complementation results from direct activation by Ets:VP16, we repeated the experiment using a mutant FoxF enhancer lacking all three putative Ets1/2-binding sites as well as the E-box motif. These additional mutations abolished specific trans-activation of the enhancer by Ets:VP16.
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FoxF is essential for TVC migration
Several methods were used to interfere with FoxF function in heart
precursor cells, including morpholino injection and targeted expression of a
constitutive repressor form of FoxF, obtained by attaching the FoxF DNA
binding domain to the Drosophila Hairy WRPW repressor motif
(FoxF:WRPW construct). Mesp>GFP or Mesp>lacZ reporter
constructs were used to visualize the B7.5 lineage cells and assess migration
defects in tailbud embryos, after normal TVC migration to a ventro-lateral
position in the trunk (Fig.
3A). A migration scoring scheme was developed to take into account
the observed phenotypic variability (Fig.
3G).
Injection of a FoxF morpholino led to an inhibition of TVC migration in 80% of the examined embryos (n=32/40; Fig. 3B,H). However, only 37.5% of these embryos (n=15/40) showed normal morphology, suggesting that gross morphological defects - and possibly migration inhibition - might arise from FoxF disruption in the trunk epidermis, consistent with the dual expression of FoxF in both tissues (see Fig. 1A-C).
To circumvent potential indirect effects arising from disruption of the trunk epidermis via morpholino injection, we targeted expression of FoxF:WRPW in the B7.5 lineage using the Mesp enhancer. This allowed us to assess the cell-autonomous effects of FoxF gene activity in heart precursor cells. Sporadic defects were observed in embryos electroporated with the Mesp>GFP construct alone (5.5%, n=17/306; no significant difference was observed with the Mesp>lacZ reporter construct). By contrast, targeted expression of the constitutive repressor FoxF:WRPW fusion protein severely inhibited migration (77.6%, n=422/544; Fig. 3D,H). These results are consistent with the morpholino gene-disruption assays (Fig. 3H), suggesting that transcriptional activation by FoxF promotes TVC migration.
An epistasis experiment was conducted in order to establish a more
definitive link between FoxF gene activity and heart cell migration.
As shown previously, targeted expression of the constitutively active form of
Ets1/2 (Ets:VP16) causes both anterior and posterior B7.5 lineage cells to
migrate into the trunk and form cardiac tissues
(Fig. 3E,H)
(Davidson et al., 2006).
Co-expression of FoxF:WRPW with Ets:VP16 appears to reverse the Ets:VP16
effect, inhibiting cell migration (Fig.
3F). Although B7.5 lineage cells migrated in some embryos, the
proportion of tadpoles showing inhibited migration was indistinguishable from
that observed with FoxF:WRPW alone (Fig.
3H; 2 test, P=0.108). This result shows
that normal FoxF function is required downstream of the FGF/MAPK/Ets cascade
to promote cardiac cell migration.
Because a dominant-negative form of FoxF inhibits Ets:VP16-induced cell migration, we asked whether FoxF activity would be sufficient for the heart cells to migrate in the absence of Ets1/2 activity. To this aim, we engineered a constitutive activator form of FoxF, by fusing its forkhead domain to the VP16 trans-activation domain. The Mesp>FoxF:VP16 transgene seemed to enhance the migration of B7.5 lineage cells in 47.3% of the observed tadpoles (Fig. 3C,I). However, only 9.4% (n=67/712) of the observed embryos showed complete migration of the entire B7.5 lineage into the trunk (versus 61.6%, n=122/198 with Ets:VP16; Fig. 3H,I), and normal TVC migration was slightly inhibited in 13.8% of the embryos (Fig. 3I, `mostly tail' class, n=98/712). These observations suggest that FoxF activity alone can mediate some aspects of heart cell migration. However, these results are not conclusive regarding sufficiency because Ets1/2 is potentially still active in B7.5 lineage cells.
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). The
Mesp>Ets:WRPW fusion gene inhibited TVC migration in 91.7%
(n=628/685) of the electroporated embryos
(Fig. 3I). Co-expression of
FoxF:VP16 did not rescue the migration defects induced by Ets:WRPW, which were
indistinguishable from the effects of Ets:WRPW alone (2 test,
P=0.258; Fig. 3I).
These results confirm that FoxF is not sufficient for the migration of B7.5
lineage cells. Instead, it appears that the FGF/MAPK/Ets pathway is required
in parallel with FoxF to induce cardiac cell migration.
FoxF function is not absolutely required for early heart specification
Previous studies established that Ets1/2 activity induces both cell
migration and cardiac fate specification during early heart development
(Davidson et al., 2006). We
therefore tested the possibility that FoxF plays an additional,
cell-autonomous role in cardiac fate specification downstream of FGF
signaling. To this aim, we first analyzed cardiac gene expression in embryos
electroporated with the dominant-negative FoxF fusion genes.
As indicated previously, Hand-like expression correlates with heart muscle specification. Injection of the FoxF morpholino or expression of the dominant-negative FoxF transgene (Mesp>FoxF:WRPW) in the entire B7.5 lineage did not seem to alter the normal Hand-like expression pattern. Indeed, in embryos showing inhibited cell migration, Hand-like exhibited normal expression in the anterior, but not posterior, B7.5 lineage cells (Fig. 4B-C'').
To gain further insight into the regulatory relationship between FoxF and the heart specification cassette, we investigated NK-4 and GATA-a expression in embryos electroporated with the Mesp>FoxF:WRPW construct. Both GATA-a and NK-4 were silent in the absence of FGF signaling, but were ectopically expressed in extra migrating cells upon targeted expression of Ets:VP16 (see Fig. S1 in the supplementary material).
The dominant-negative FoxF construct had variable effects on GATA-a and NK-4 expression (Fig. 5 and see Fig. S1 in the supplementary material). We focused our attention on embryos showing inhibited migration, and found that both GATA-a and NK-4 expression were either unaffected (Fig. 5C,D) or lost (Fig. 5E,F) in embryos displaying conspicuous migration defects. GATA-a and NK-4 expression was maintained in 35-50% of the FoxF:WRPW-expressing embryos (see Fig. S1 in the supplementary material). Taken together, these observations suggest that disruption of FoxF function has limited effects on cardiac specification, because expression of core heart differentiation genes (Hand-like, GATA-a and NK-4) can persist in embryos showing severely inhibited migration (see Discussion).
Disruption of FoxF function causes a dramatic repositioning of the beating heart
Our observations raise the possibility that FoxF regulates cell migration,
but not heart tissue specification. To further evaluate this possibility, we
repeated the Ets:VP16/FoxF:WRPW epistasis test and assessed Hand-like
gene expression in tailbud embryos. Our previous studies showed that the
Mesp>Ets:VP16 transgene induces the complete B7.5 lineage to migrate and
express Hand-like (Davidson et
al., 2006). As shown earlier, co-expression of the
dominant-negative FoxF:WRPW protein inhibited migration, so that both the
anterior and posterior B7.5 lineage cells remained in the tail
(Fig. 3F). However, all of the
cells expressed Hand-like (Fig.
4D-D''), suggesting that Ets:VP16 is still able to induce
cardiac muscle fate in the absence of migration and FoxF function. In this
experiment, heart tissue specification and cell migration were more completely
uncoupled, with the entire B7.5 lineage being converted into heart muscle
precursors that remained at the anterior end of the tail.
In order to determine whether these misplaced cells can form a beating
heart, tadpoles expressing various combinations of the Mesp>lacZ,
Mesp>Ets:VP16 and Mesp>FoxF:WRPW transgenes were grown through
metamorphosis and allowed to develop into juveniles. Heart phenotypes were
grouped in four distinct classes based on morphology, size and position
(Table 1). In all conditions,
we observed general heart defects, which might result from the dechorionation
procedure, which was shown to interfere with metamorphosis
(Sato and Morisawa, 1999).
Most of the remaining Mesp>lacZ-electroporated juveniles displayed
normal heart morphology and position. As shown previously, overexpression of
the Ets:VP16 chimera led to an expansion of the heart tissue, sometimes
resulting in the striking `two-compartments' heart phenotype, whereas Ets:WRPW
strongly impaired heart formation (Davidson
et al., 2006). In Mesp>FoxF:WRPW-electroporated
juveniles, we found twitching heart-like tissue mis-positioned at the base of
the resorbed tail in 7.8% of animals (n=15/191,
Table 1). In 5.2% of cases
(Table 1), the heart-like
tissue appeared disorganized, but the other 2.6% showed the so-called
`tail-heart' phenotype (see below).
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These observations provide striking evidence that targeted disruption of FoxF function in the B7.5 lineage specifically blocks cell migration during embryogenesis, with little to no impact on subsequent cardiac tissue differentiation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Making a heart without moving
In ascidian embryos, heart specification in the rostral B7.5 lineage
requires Mesp activity and Ets1/2 activation in response to the FGF/MAPK
pathway, which induces all aspects of cardiac specification, including
migration and subsequent differentiation. Previous observations suggested that
heart muscle specification and cell migration could be uncoupled to some
extent (Davidson et al., 2005).
Here, we showed that the transcription factor FoxF is expressed in response to
FGF signaling and is required for cell migration. However, FoxF function
appears dispensable for heart muscle differentiation, because embryos
expressing the dominant-negative FoxF:WRPW could develop into juveniles with
mis-positioned heart tissue.
Where does the `tail-heart' come from? Our analysis of heart differentiation genes shows that Hand-like expression is independent of FoxF function, because it was retained in non-migrating TVCs after FoxF-morpholino injection or targeted expression of the FoxF:WRPW fusion protein.
On the other hand, GATA-a and NK-4 expression was reduced upon FoxF:WRPW overexpression. The cellular and molecular basis for this effect is unknown, but suggests that additional linkages might connect the core heart regulatory network to cell migration in ascidian embryos (summarized in Fig. 7).
In line with the uncoupling hypothesis, we found that GATA-a and NK-4 expression persisted in 50% and 36%, respectively, of the embryos with inhibited TVC migration upon FoxF:WRPW overexpression. Hence, expression of three core heart-differentiation genes, Hand-like, GATA-a and NK-4, is maintained in 15-35% of embryos in which heart migration is inhibited. It seems likely that juveniles showing mis-positioned heart tissue develop from these embryos, in which the early heart-specification network appears unaffected.
An essential role for Ets1/2 co-factors in heart cell migration
FGF signaling can regulate fate decision and morphogenesis via distinct
intracellular pathways (e.g. Sivak et al.,
2005). In Ciona, FGF/MAPK/Ets signaling controls cardiac
fate specification predominantly via Ets1/2-mediated transcriptional
activation; the constitutively active Ets:VP16 fusion protein can fully
restore cardiac specification in cells expressing a dominant-negative form of
the FGF receptor (Davidson et al.,
2006) (B.D., unpublished).
|
In ascidian embryos, distinct Ets1/2 co-factors might account for tissue-
or process-specific gene activation in response to FGF (e.g.
Bertrand et al., 2003;
Kumano et al., 2006). For
instance, early Mesp activity is required for all aspects of cardiogenesis in
ascidians (Satou et al.,
2004). Mesp presumably upregulates Ets1/2 expression, but
might also function in parallel to the FGF/MAPK/Ets pathway. Indeed, targeted
expression of the Mesp:VP16 fusion protein downregulates FoxF,
whereas Hand-like expression persists in the non-migrating anterior
B7.5 daughter cells (Davidson et al.,
2005). By contrast, the FGF/MAPK/Ets cascade is required for TVC
expression of both FoxF and Hand-like. Thus, the Mesp:VP16
chimera does not seem to interfere with FGF signal transduction through
Ets1/2. Instead, we propose that Mesp:VP16 interferes with the ability of Mesp
to indirectly regulate Ets1/2 co-factors upstream of FoxF
(Fig. 7).
We found that a CACTTG motif is required for FoxF minimal TVC
enhancer activity, which could be restored by co-expression of the
hyper-active Ets:VP16 chimera. This further supports the hypothesis that an
Ets1/2 co-factor is required for TVC-specific activation of FoxF in
response to FGF signaling. As mentioned above, this motif matches the E-box
consensus (CANNTG). It might therefore bind cardiac bHLH transcription factors
in vivo, but alternative possibilities can be envisioned. Indeed, we found
that the CACTTG motif also matches the Nkx2.5/tinman consensus sequence CAMTTR
(Sandelin et al., 2004;
Zaffran and Frasch, 2002).
Further investigation will be required to identify Ets1/2 co-factors and
determine their precise roles in the selective regulation of cell migration
via FoxF regulation.
An Ets/FoxF circuit specifically regulates cell migration
FGF signaling has extensively documented roles in regulating cell type
specification and morphogenesis of mesoderm derivatives (reviewed in
Thisse and Thisse, 2005). Our
results point to an essential role of FoxF in the transcriptional control of
cardiac cell migration downstream of FGF/MAPK/Ets signaling. In vertebrates
and Drosophila, FGF signaling and FoxF orthologs have been
implicated in a variety of morphogenetic processes involving mesenchyme cells
derived from the lateral plate mesoderm, consistent with their widespread
expression in these tissues (e.g. Mahlapuu
et al., 2001; Malin et al.,
2007; Mandal et al.,
2004; Michelson et al.,
1998; Zaffran et al.,
2001). Here, we found that a hyper-active form of FoxF slightly
enhanced migration of the B7.5 lineage cells, but failed to rescue the
migration defect caused by the dominant-negative Ets1/2. These results suggest
that Ets1/2 activity is also required in parallel with FoxF to regulate the
full spectrum of genes required for TVC migration (summarized in
Fig. 7).
Most of our internal organs arise from primordia that undergo directed migration to ensure that they are positioned in an orderly fashion within the body cavity. It seems likely that similar principles seen for migration of the ascidian heart primordium will also apply to additional organ systems. In particular, we speculate that genes such as FoxF will serve to connect gene regulatory cassettes controlling organogenesis to the process of directed cell migration.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/18/3297/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adell, T. and Muller, W. E. (2004). Isolation and characterization of five Fox (Forkhead) genes from the sponge Suberites domuncula. Gene 334,35 -46.[CrossRef][Medline]
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