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First published online 2 October 2008
doi: 10.1242/dev.025437
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1 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT
84112, USA.
2 Department of Developmental Biology, CNRS URA 2578, Pasteur Institute, Paris
75015, France.
3 Department of Cell Biology, Duke University, Durham, NC 27710, USA.
4 Department of Pediatric Cardiology, Tokyo Women's Medical University, Tokyo
162-8666, Japan.
5 Department of Pharmacology and Toxicology, University of Arizona, Tuscon, AZ
85721, USA.
6 Department of Pediatrics, University of Utah, Salt Lake City, UT 84112,
USA.
7 Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake
City, UT 84112, USA.
Author for correspondence (e-mail:
anne.moon{at}genetics.utah.edu)
Accepted 4 September 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: FGF, Heart development, Outflow tract, Second heart field, Autocrine signaling, Epithelial-mesenchymal transformation, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
After looping of the heart, the myocardium secretes a specialized
extracellular matrix (ECM) called the cardiac jelly that forms internal
cushions; these cushions ultimately form the septa and the valves. The cushion
jelly is invaded by endothelial cells lining the OFT myocardium that undergo
an endothelial-to-mesenchymal transformation (EMT), and by a subset of neural
crest cells called the cardiac neural crest (CNC)
(Kirby, 2006). These events
require BMP, TGFβ, WNT and semaphorin signaling from the OFT myocardium,
in addition to proper cardiac jelly composition
(Armstrong and Bischoff, 2004;
High and Epstein, 2007). Thus,
dysfunction of many different cell types and signaling pathways can contribute
to abnormal OFT morphogenesis. At midgestation, the OFT is remodeled by spiraling and fusion of the cushions to septate the OFT into the aorta and pulmonary artery; this occurs concurrent with rotation and alignment of the OFT relative to the ventricles. When alignment/rotation of the septated OFT is disrupted, transposition of the great arteries (TGA) or double-outlet right ventricle (DORV) occurs. Complete failure of OFT septation results in persistent truncus arteriosus (PTA), in which the primitive OFT (the truncus arteriosus) is not divided into the aorta and pulmonary artery. These defects disrupt the partitioning of blood flow required for adequate oxygenation and are lethal in mice and humans.
The importance of FGF8 signaling for heart development is clear.
Fgf8-null mutants die at gastrulation
(Sun et al., 1999). However,
Fgf8 hypomorphs survive to birth with OFT septation and
alignment/rotation defects (Abu-Issa et
al., 2002; Frank et al.,
2002). Conditional mutagenesis has been used to determine the
spatiotemporal requirements for Fgf8 function in different aspects of
OFT morphogenesis (Ilagan et al.,
2006; Macatee et al.,
2003; Park et al.,
2006). Mutation of Fgf8 in mesodermal heart precursors in
the primitive streak can prevent OFT formation and cause embryonic death;
however, survivors display OFT rotation/alignment defects. When Fgf8
is inactivated later in development, in OFT precursors in the SHF and in
pharyngeal endoderm, the mutants survive, but all have PTA
(Park et al., 2006). Although
Fgf10 is expressed in the SHF, and Fgf15 in multiple tissues
in the pharyngeal arches, only Fgf15-null mutants have OFT defects
(Marguerie et al., 2006;
Vincentz et al., 2005).
We have made progress in identifying the sources and FGF ligands required
for OFT development, but their cellular targets remain unknown. The accepted
paradigm is that FGFs signal in a paracrine manner to target cells via the FGF
receptor tyrosine kinases (RTKs, FGFR1-4). Mitogenic assays suggest that
ligand-receptor preferences exist (Zhang,
X. et al., 2006), but overlapping expression patterns and ablation
studies reveal functional redundancy (Sun
et al., 2002). Variations in ECM composition are likely to modify
ligand-receptor interactions and signaling in a context-dependent manner.
Thus, defining functionally relevant ligand-receptor interactions on target
cells that regulate specific morphogenetic events in vivo is an important
endeavor.
Some insight into these crucial in vivo interactions can be obtained by
comparing phenotypes of FGF receptor and ligand mutants. Fgfr1-null
mutants die at gastrulation (Deng et al.,
1994), whereas Fgfr2-null mutants die post-implantation
(Arman et al., 1998). Previous
manipulations of Fgfr1/2 function that bypass lethality reveal
crucial roles for these receptors in many processes and suggest that they are
important for cardiovascular development
(Marguerie et al., 2006;
Moon et al., 2006;
Trokovic et al., 2003). By
contrast, Fgfr3 and Fgfr4 double-null mutant mice survive
without cardiovascular defects (Weinstein
et al., 1998).
Here, we conditionally inactivate Fgfr1 and Fgfr2 and
conditionally overexpress sprouty 2 (Spry2, which encodes an FGF
signaling antagonist) in different cell types to identify the direct cellular
targets of FGF signals required for OFT remodeling. Our results reveal that
although published evidence points to CNC and/or OFT endothelial cells as
crucial paracrine targets (Kirby,
2006; Presta et al.,
2005), disrupting FGF signaling to these populations does not
prevent OFT remodeling. Rather, we show that interrupting autocrine FGF
signaling in SHF mesoderm causes an OFT myocardial secretory defect; this
secondarily perturbs endothelial EMT and CNC invasion in association with
altered BMP and TGFβ signaling. Graded alterations in OFT development
with varying FGF receptor gene dosage reveal a marked sensitivity to FGF
signaling in target cells. We conclude that the specialized secretory and
signaling properties of the primitive OFT that drive OFT morphogenesis are
regulated by an autocrine FGF signaling loop.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Trokovic et al., 2003;
Xu et al., 2002;
Yu et al., 2003). Generation
and characterization of Wnt1Cre, Ap2
IRESCre, Pax3Cre,
Mesp1Cre, Isl1Cre, Tie2Cre (Tie2 is also known as Tek -
Mouse Genome Informatics), Foxa2MCM and Spry2-GOF alleles
have also been reported previously (Basson
et al., 2008; Danielian et
al., 1998; Engleka et al.,
2005; Jiang et al.,
2000; Kisanuki et al.,
2001; Macatee et al.,
2003; Park et al.,
2006; Park et al.,
2008; Saga et al.,
2000). The Isl1Cre and Mesp1Cre males used to
generate the mutants are in mixed backgrounds: Isl1Cre is 50% C57Bl6
and 25% each BlSw and Sv129; Mesp1Cre is 50% C57Bl6 and 25% each ICR
and SV129. These males were bred to Fgfr1/2 conditional females that
are in a complex mixed background (including C57Bl6, Sv129 and others). Thus,
the embryos arising from each cross are outbred. To generate the
Fgfr1;IRESCFP and Fgfr2;IRESYFP alleles in mice, we inserted
IRES;FP;frt-flanked neomycin cassettes into the 3' untranslated
regions of each gene upstream of the polyadenylation signal. All experiments
were in accordance with institutional and national guidelines and
regulations.
OFT explant culture and co-culture on collagen gels
Collagen gels were made as previously described
(Camenisch et al., 2002). OFT
segments were isolated, opened to expose endocardium, placed on the gel and
allowed to attach for 12 hours without addition of culture media. Then
1xM199 containing 1% fetal bovine serum (FBS), 0.01%
insulin-transferrin-selenium (ITS) and 1% PenStrep (Gibco-BRL) was added and
the explants cultured for 48 hours. For co-cultures,
Fgf8c/c;Rosa26lacZ/lacZ and
Fgf8c/c females were bred with
Fgf8+/-;Isl1Cre males to obtain labeled
Fgf8;Isl1Cre mutants and controls. OFTs from mutants and controls
were placed immediately adjacent to one another. Explants were fixed and
stained using standard protocols.
Immunohistochemistry and in situ hybridization
YFP and CFP detection was performed using rabbit anti-GFP and Texas
Red-conjugated anti-rabbit secondary antibodies (Molecular Probes) on
cryosectioned specimens. Anti-phosphohistone H3 (Ser10, Cell Signaling
Technology), anti-ISL1 (Developmental Studies Hybridoma Bank), anti-AP2
(TCFAP2 - Mouse Genome Informatics) (3B5, Developmental Studies
Hybridoma Bank), anti-phosphorylated (p) ERK1/2 and SMAD1/5/8 (Cell Signaling
Technology) and FITC-conjugated anti-mouse secondary (Molecular Probes)
antibodies were used. In situ hybridization was performed with a standard
protocol (Grove et al., 1998)
and digoxigenin-labeled antisense RNA probes. Alkaline phosphatase and
β-galactosidase staining were performed using published protocols
(Frank et al., 2007;
Lobe et al., 1999).
Preparation of RNA and cDNA for microarray and quantitative RT-PCR
E9.5 OFTs were dissected and stored in RLT buffer (Qiagen) at -80°C.
Seven specimens of each genotype (Fgf8c/+, control;
Fgf8c/-;Isl1Cre, mutant) were pooled to generate each
sample. Total RNA was extracted from four samples (RNeasy Micro Kit, Qiagen).
Agilent two-color LRILAK labeling, the Agilent two-color GE hybridization/wash
protocol, and the Agilent 5-micron XDR scanning protocols were carried out by
the University of Utah Microarray Core Facility. RNA (100 ng) was
reverse-transcribed to cDNA using the SuperScript III First-Strand Synthesis
System (Invitrogen). Quantitative RT-PCR was performed with iQ SYBR Green
Supermix on the iCycler system (Bio-Rad). Hprt transcript was used as
the reference level.
Microarray data analysis
This experiment was run in quadruplicate on Agilent mouse whole-genome
expression arrays. The array image data were quantitated using Agilent Feature
Extraction software (version 9.5.1.1). Subtle intensity-dependent bias was
corrected with LOWESS normalization, with no background subtraction.
Statistical analysis of normalized log-transformed data was performed in Gene
Sifter
(www.genesifter.net).
Differentially expressed transcripts were defined (adjusted for multiple
testing using the Benjamini and Hochberg method) as P<0.05. Spots
with an intensity below background were removed prior to statistical analysis.
Data were hierarchically clustered with Spotfire (TIBCO) and heat maps for
selected genes were generated. Log-transformed, normalized gene intensity data
were clustered by the UPGMA method; Pearson's correlation was the distance
metric.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We found that the OFT cushions of these mutants
contain markedly less cardiac jelly and fewer mesenchymal cells than controls
(Fig. 1A-H; see Fig. S1 in the
supplementary material). The mutant OFTs were short and aberrantly
angulated.
To distinguish the sources of cushion mesenchymal cells at early stages of
OFT septation, we used the conditional Rosa26lacZ reporter
(Soriano, 1999) and
Tie2Cre (Kisanuki et al.,
2001) to label endothelial cells (which constitute the endocardial
lining of the OFT). Tie2Cre activity was uniform in the OFT
endothelium by E8.25 (Fig.
1I-K). This experiment shows that most cells in the proximal
cushions at E11.5 are of endothelial origin; although CNC cells have invaded
the distal OFT, few have as yet migrated into the proximal cushions
(Fig. 1E,G,J and data not
shown). We explanted proximal cushions from E9.25
Rosa26lacZ;Tie2Cre embryos onto collagen gels and found
that the cells from the explant that undergo EMT to invade and migrate into
the gel are β-galactosidase-positive
(Fig. 1K,L), confirming their
endothelial origin. Since the OFT cushions of Fgf8;Isl1Cre mutants
were hypoplastic along their entire proximodistal extent (the proximal
cushions were both thinner and had fewer cells, whereas the distal cushions
were thinner but with no change in cell density)
(Fig. 1; see Fig. S1 in the
supplementary material), both endothelial and CNC cells are affected in these
mutants.
OFT remodeling is independent of direct FGF signaling to cardiac neural crest and endothelial cells
In order to understand how FGF8 regulates endothelial and CNC invasion of
the OFT cushions, we sought to identify the direct cellular targets of FGF8
and of other FGF ligands required for OFT remodeling. The overlapping
phenotypes reported in different types of Fgf8, Fgfr1 and
Fgfr2 mutant mice suggest that FGF8 signals through these receptors
during embryogenesis. We first determined which cells within the pharyngeal
arches and SHF express these receptors. We generated novel alleles to
fluorescently label cells expressing Fgfr1 and Fgfr2 by
targeting an IRES (internal ribosome entry site) and CFP to Fgfr1,
and an IRES and YFP to Fgfr2. We detected ubiquitous expression of
both receptors in the anterior embryo, including all cells in the pharynx at
E8.5-9.5 (see Fig. S2 in the supplementary material), with variable levels of
signal in different cell types.
|
). Each of
the Cre drivers used has an onset of Cre-mediated recombination in the desired
tissue that is prior to the requisite window of FGF8 signaling at E8.5 (8- to
10-somite stage) that we previously identified for OFT remodeling
(Park et al., 2006).
In complementary experiments, we employed conditional Spry2
gain-of-function (Spry2-GOF) to inhibit ERK phosphorylation
downstream of activated RTK signaling, including that of FGFRs
(Hanafusa et al., 2002). After
Cre-mediated recombination, the Spry2-GOF transgene constitutively
expresses Spry2 (Fig.
2) (Basson et al.,
2008). Activation resulted in markedly increased production of
Spry2 mRNA relative to controls
(Fig. 2F,H), and effective
antagonism of FGF signaling was evident by decreased ERK activation
(Fig. 2J,J') and
decreased expression of the FGF8 target gene Erm (Etv5)
(Fig. 2L)
(Park et al., 2006;
Raible and Brand, 2001;
Roehl and Nusslein-Volhard,
2001). Pea3 (Etv4) expression was also decreased
at E8.5 (not shown).
To test whether CNC cells are direct targets of the FGF signaling required
for OFT remodeling, we used the well-characterized Wnt1Cre and
Ap2IRESCre drivers to ablate Fgfr1 and
Fgfr2 function in premigratory neural crest, and in premigratory
neural crest and pharyngeal ectoderm, respectively
(Jiang et al., 2000;
Macatee et al., 2003).
Surprisingly, ablation of either receptor, independently or in combination,
did not disrupt OFT remodeling (see Table S1 in the supplementary material).
As expected, these embryos had disrupted craniofacial development (see Fig. S3
in the supplementary material). Furthermore, when we overexpressed
Spry2 in neural crest, 100% of mutants had abnormal craniofacial
structures but normal OFT morphology (see Table S1 and Fig. S3 in the
supplementary material). This indicates that OFT septation is independent of
FGF signaling directly to CNC. PDGF signaling, which is known to occur in
neural crest (Richarte et al.,
2007), is therefore not a major target of inhibition by sprouty 2
in these cells.
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). Since the proximal OFT cushions of
Fgf8;Isl1Cre mutants have few endothelium-derived mesenchymal cells,
we examined the ability of endothelial cells from Fgf8;Isl1Cre
mutants to migrate from an explanted OFT, undergo EMT, and invade a collagen
gel. Remarkably, 100% of mutant OFT explants exhibited a dramatic failure of
EMT (Fig. 3B, n=11).
In control explants, only an occasional cell appeared rounded and on the gel
surface (Fig. 3A), whereas in
the mutants, the few cells that migrated away from the explant were rounded up
on the surface or fragmented into debris
(Fig. 3B). For quantitation of
these findings, see Fig. S4 in the supplementary material.
We then tested whether loss of FGF signaling directly to endothelial cells
causes OFT defects using Tie2Cre
(Kisanuki et al., 2001) to
ablate Fgfr1 and Fgfr2. Tie2Cre conditional mutants survived
and had normal OFTs (see Table S1 in the supplementary material; data not
shown). This suggests that the endothelial EMT defect seen in
Fgf8;Isl1Cre mutants in vivo (Fig.
1), and in explants (Fig.
3), is endothelial cell-non-autonomous. To test this, we
co-cultured Fgf8;Isl1Cre mutant explants adjacent to those from
controls. To identify the origin of invading cells in co-culture, we used the
conditional Rosa26lacZ line and Isl1Cre to label
endothelial cells from control explants, or carried the
Rosa26lacZ allele in the females used to generate
conditional mutant explants [the Isl1Cre expression domain includes
OFT endothelium (Park et al.,
2006) (data not shown)]. We found that control explants rescued
the ability of Fgf8;Isl1Cre mutant endothelial cells to undergo EMT
(Fig. 3D,E), with no adverse
effect of lacZ expression in controls
(Fig. 3C). In addition to
confirming that the EMT defect in Fgf8;Isl1Cre mutants is not due to
primary endothelial dysfunction, this finding suggests that the essential
defect in the mutant OFTs resides within the myocardium itself.
SHF mesoderm is a direct target of FGF signals required for OFT remodeling
Based on the co-culture results, we predicted that loss of FGF signaling to
mesodermal precursors of the OFT would phenocopy the defects of Fgf8
conditional mutants. We ablated Fgfr1 and Fgfr2 using the
mesoderm-specific driver Mesp1Cre. This driver is active in the
anterior primitive streak and ablates gene function in all myocardial and
endocardial precursors (Park et al.,
2006; Saga et al.,
1999). Fourteen percent of
Fgfr1c/c;Fgfr2+/+;Mesp1Cre mutants had
alignment defects (TGA and DORV, Table
1). The frequency of alignment defects increased to 40% with
decreasing Fgfr1/2 gene dosage in
Fgfr1c/c;Fgfr2c/+;Mesp1Cre mutants
(Fig. 4C,
Table 1). Normally, persistence
of myocardium in the right ventricular subvalvar outflow region (called the
conus, derived from the SHF) and regression of this myocardium on the left
side causes the pulmonary valve to ultimately reside above (distal to the
ventricle) the aortic valve (Fig.
4A'). Hypoplasia of the right conus in Fgfr1/2
mutants was apparent externally as a flattening of this region
(Fig. 4C), and resulted in a
side-by-side valve position (Fig.
4C'). Linkage of Mesp1 and Fgfr2 on
chromosome 7, with a predicted recombination frequency of 23%, prevented
analysis of large numbers of
Fgfr1c/+;Fgfr2c/c;Mesp1Cre and
Fgfr1c/c;Fgfr2c/c; Mesp1Cre mutants. However,
there were no OFT defects in the
Fgfr1c/+;Fgfr2c/c;Mesp1Cre mutants we obtained
(Table 1). By contrast, four
out of five of Fgfr1c/c;Fgfr2c/c;Mesp1Cre
mutants had PTA type III, a severe grade of PTA in which the OFT is unseptated
along its entire proximodistal extent (Fig.
4D,D'). The truncal OFT valve was also abnormally aligned.
Since the incidence and severity of OFT defects increase with decreasing
Fgfr1/2 gene dosage in the mesoderm, the ability to align and septate
the OFT depends on the amount of FGF signaling received by these cells. Our
data also indicate that the dominant receptor transducing the required signals
in this process is FGFR1, as FGFR2 has only partial compensatory activity.
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)] increased the frequency of type III PTA to 100% in
Fgfr1c/c;Fgfr2c/c;Isl1Cre mutants
(Table 1), as compared with 66%
in mesoderm-only double mutants. Defects in
Fgfr1c/c;Fgfr2c/+;Isl1Cre mutants were also
more severe than those in their mesoderm-only counterparts because proximal
OFT septation frequently failed, resulting in type I PTA
(Fig. 4G,
Table 1). In this form of PTA
(which is common in human patients), there is a single truncal valve, but the
distal OFT is septated (Fig.
4G,G'). Consistent with our hypothesis that FGFR1 is the
principal receptor, Fgfr1c/+;Fgfr2c/c;Isl1Cre
mutants had milder defects than
Fgfr1c/c;Fgfr2c/+;Isl1Cre mutants
(Fig. 4F,
Table 1).
These data suggest that FGF signaling to the pharyngeal endoderm plays a
role in OFT remodeling. We used a novel Cre driver, Foxa2IRES
mER-Cre-mER (Park et al.,
2008), to ablate Fgf8 or Fgfr1 and
Fgfr2 in pharyngeal endoderm by E8.0 and test whether independent
effects of endodermal FGF signaling on OFT septation could be detected.
Although loss of endodermal receptor function disrupts vascular development
(E.J.P. and A.M.M., unpublished), endodermal ligand and receptor mutants
survived to birth and had normal OFTs (see Table S1 in the supplementary
material).
Antagonism of FGFR-mediated signaling in SHF mesoderm in
Spry2-GOF;Mesp1Cre embryos caused phenotypes consistent with the
receptor ablation results: 75% of these mutants had heart defects, most of
which were arterial pole abnormalities (PTA and DORV,
Fig. 5A-I,
Table 1). Additionally, as in
Fgf8;Isl1Cre mutants (Fig.
1), the OFTs of E9.5 Spry2-GOF;Mesp1Cre mutants were
significantly shorter than in controls and were at an obtuse angle to the
right ventricle (Fig. 5J-O and
data not shown). Wnt11 transcripts in the OFT myocardium were reduced
in E9.5 Spry2-GOF;Mesp1Cre mutants
(Fig. 5J,K) and Bmp4
expression was also notably downregulated in the OFT and SHF
(Fig. 5L,M). Both
Spry2-GOF;Mesp1Cre and FGFR mutants phenocopied the early defects in
endothelial EMT and CNC invasion seen in Fgf8;Isl1Cre mutants
(Fig. 5P,Q; see Fig. S5 in the
supplementary material). Reduction in EMT was demonstrated in
Spry2-GOF;Mesp1Cre mutant explants
(Fig. 5R,S), similar to that
shown for Fgf8;Isl1Cre mutants
(Fig. 3A,B). Proliferation of
ISL1-expressing cells in the SHF and OFT myocardium was significantly
decreased in Spry2-GOF;Mesp1Cre mutants at multiple stages
(Fig. 5T-V and data not shown);
this is also seen in Fgf8 mutants
(Park et al., 2006).
Mesodermal Spry2-GOF phenotypes are similar to those of the FGFR
mutants; since SPRY2 inhibits signaling downstream of other RTKs, this
demonstrates the primacy of FGF signaling in SHF mesoderm for subsequent OFT
morphogenesis. However, the effects on Wnt11 and Bmp4
transcripts suggest that downstream modulation of other signaling pathways is
likely to affect CNC and endothelial cell behavior in the OFT. Detection of
these OFT markers, albeit at a lower level, demonstrates that OFT progenitors
continue to be added from the SHF when FGF signaling is compromised.
Crucial signaling pathways that regulate EMT and cardiac neural crest behavior are disrupted by loss of FGF signaling in the SHF and pharyngeal endoderm
In order to examine downstream effects of loss of FGF8 on myocardial
signaling, we examined gene expression in isolated E9.5 OFTs from
Fgf8;Isl1Cre mutants and controls using genome-wide microarray
analysis. This timing allowed us to examine myocardial and endocardial gene
activity at the onset of endocardial EMT and CNC invasion. In the FGF pathway
(Fig. 6A), few genes were
dysregulated greater than 2-fold. Expression of the known FGF8 target genes
Pea3 and Erm was significantly decreased, as expected.
Reductions seen in the expression of genes encoding FGF counter-regulatory
factors [sprouty, Spred, Il17rd (Sef) and Dusp genes] were
similar.
When FGF signaling to the SHF and endoderm was perturbed, there were
striking changes in the level of transcripts of components of the BMP,
TGFβ and semaphorin/plexin pathways, which are known to be essential to
both endocardial EMT and CNC survival and invasion of the OFT cushions
(Barnett and Desgrosellier,
2003; High and Epstein,
2007) (Fig. 6B,D,E
and data not shown). Quantitative RT-PCR confirmed these findings and revealed
that the array frequently underestimated the magnitude of the expression
changes (see Table S2 in the supplementary material). Bmp4 and
Bmp2 transcripts were decreased, as was the expression of genes in
the Bmp4 synexpression group
(Karaulanov et al., 2004),
including the transcriptional effectors Msx1 and Msx2, and
target genes with roles in OFT endocardial EMT (Has2, Gata4, Tbx3, Twist1,
Snai1) (Liu et al.,
2004). Transcripts for some BMP antagonists (noggin,
Bambi) and inhibitory Smads (Smad6, Smad7) were also
decreased, and increased expression of the BMP antagonist gremlin 2 in the
face of decreased Bmp4/2 would further decrease BMP signaling.
Tgfb1 and Tgfbr1 were also downregulated, together with
Tgfbrap1, a protein induced by TGFβ that transmits signal from
TGFβ receptors to SMAD2 and 4
(Wurthner et al., 2001).
|
); latent TGFβ-binding protein 1 (Ltbp1), a
regulator of TGFβ bioavailability required for OFT septation
(Todorovic et al., 2007); and
the proteoglycans decorin (Dcn), biglycan (Bgn) and versican
(Vcan; Cspg2), which influence the fibrous structure of
collagen in the ECM and its physical interaction with cells, as well as the
bioavailability and cellular responses to TGFβ and BMPs
(Fig. 6B)
(Macri et al., 2007).
|
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), in which
ablation of the FGFR adaptor protein FRS2 (FRS2 - Mouse Genome
Informatics) in the SHF and pharyngeal endoderm (but not neural crest or
endothelium) causes arterial pole defects.
|
;
Ilagan et al., 2006;
Park et al., 2006). This
temporal window indicates that the required FGF/FGFR signaling event occurs in
the mesodermal precursors of the OFT in the SHF. Similar temporal requirements
were identified for FRS2-mediated signaling
(Zhang et al., 2008).
Furthermore, decreased cell proliferation, increased apoptosis and alterations
in the expression of Isl1 and known FGF8 target genes are already
marked in the SHF mesoderm of Fgf8;Mesp1Cre and Fgf8;Isl1Cre
mutants by the 5- to 8-somite stage (Park
et al., 2006); decreased proliferation is also striking in
Spry2-GOF;Mesp1Cre embryos (Fig.
5), all of which point to primary effects on the progenitor cell
population resulting in decreased OFT size and other defects.
An NKX2.5-BMP2/SMAD1 negative-feedback loop has been documented in the SHF
in which diminution of BMP2/SMAD1 signaling increases progenitor cell
proliferation and myocardial specification
(Prall et al., 2007). Notably,
Fgf8 function was preserved in this system. Our findings suggest that
BMP4/FGF8 participate in a different regulatory pathway because compromising
FGF signaling resulted in decreased proliferation in the SHF associated with
decreased Bmp4 transcript levels in the SHF and reduced BMP signaling
in the OFT. Indeed, differences between BMP2 and BMP4 function in the context
of myocardial progenitor specification/proliferation, and in the OFT itself,
have been documented (Armstrong and
Bischoff, 2004; Klaus et al.,
2007; Ma et al.,
2005). In addition to effects on BMP signaling, which may affect
the recruitment of differentiating myocardial cells
(Waldo et al., 2001), we show
that disrupted FGF signaling leads to reduced expression of Wnt11 in
OFT myocardium (Fig. 5), which
was also seen in our microarray analysis (not shown), consistent with previous
observations on Fgf8;Isl1Cre mutants
(Park et al., 2006).
Perturbation of non-canonical WNT11 signaling in the myocardium affects OFT
development and interferes with Tgfb2 transcription and ECM
composition (Zhou et al.,
2007).
The cushion defects in our FGF mutants focused our attention on the
recruitment of neural crest. CNC cells do not arrive in the pharynx until
E9.0. Thus, our finding that these cells are not the direct target of the
early FGF signals is consistent with the temporal window identified using FGF
ligand mutants in the SHF (Ilagan et al.,
2006; Park et al.,
2006). Although we did not specifically rule out a role for
Fgfr3 or Fgfr4 in CNC during OFT remodeling,
Fgfr3/4 mutants have no cardiovascular defects
(Weinstein et al., 1998).
Spry2-GOF expression in these cells would antagonize signal
transduction downstream of all four FGFRs, and Spry2-GOF neural crest
cells have normal OFTs. Factors that antagonize SPRY2 activity could also
influence the phenotype of these mutants. However, in an accompanying report,
Zhang et al. show that ablation of Frs2 in CNC does not
disrupt OFT development (Zhang et al.,
2008), which is consistent with our findings. Indeed, the severe
OFT defects in Mesp1Cre and Isl1Cre receptor mutants, in
which Fgfr1/2 function remains intact in CNC, indicate that disrupted
FGF signaling to CNC is not required to generate these phenotypes.
Several lines of evidence indicate that secondary effects on CNC after loss
of FGF signaling in the SHF play a role in the OFT phenotypes obtained in our
mesodermal FGFR and Spry2 gain-of-function mutants. The finding of
aortic arch malformations in these mutants (Figs
4 and
5), but not in the neural crest
conditional mutants (see Fig. S3 in the supplementary material), is consistent
with downstream effects on neural crest. Excessive neural crest apoptosis is
observed in Fgf8 mutants (Ilagan
et al., 2006; Macatee et al.,
2003; Park et al.,
2006), and Spry2-GOF;Mesp1Cre mutants have decreased
neural crest Crabp1 expression. Aortic arch defects have also been
shown to be the result of secondary neural crest dysfunction due to
Tbx1 loss-of-function in the mesoderm of the SHF
(Xu et al., 2004;
Zhang, Z. et al., 2006). BMP
and TGFβ signaling are essential for CNC invasion of the OFT cushions and
for pharyngeal arch artery development
(High and Epstein, 2007;
Liu et al., 2004;
Stottmann et al., 2004); the
changes that we document in these pathways in the face of compromised FGF
signaling will impact these processes. Modifications to ECM components, which
also affect BMP/TGFβ signaling (Macri
et al., 2007), further contribute to effects on the neural crest.
In addition to the ECM and other signaling defects discussed above,
transcription of Acvr1 (Alk2), semaphorin 3c, plexin A3 and
neuropilin 2 is decreased in Fgf8;Isl1Cre mutants (not shown). Each
of these proteins critically impacts CNC function
(High and Epstein, 2007).
Neural crest cells modulate FGF8 signaling in the pharynx and influence not
only the addition of myocardium to the OFT from the SHF
(Hutson et al., 2006), but
also the contractile and secretory function of the myocardium itself,
including its ability to produce cardiac jelly
(Stottmann et al., 2004;
Waldo et al., 1999). Thus, in
affected Fgf8, FGFR and Spry2 gain-of-function mutants,
initial myocardial dysfunction and subsequent abnormal CNC behavior might
interact in a cycle that progressively impairs OFT morphogenesis.
Since FGFR ablation in the endothelial precursors of the OFT endocardium
does not perturb OFT remodeling, and EMT defects in Fgf8;Isl1Cre
mutants can be rescued by wild-type myocardium, direct signaling between FGF
ligands produced by the SHF and the endocardium is not required for OFT
septation. Furthermore, ablation of Frs2 in the endothelium
does not disrupt OFT morphogenesis (Zhang
et al., 2008). However, defects in the expression of ECM
components and signal modulators downstream of the BMP/TGFβ pathways
provide insight into the molecular basis of the secondary endothelial
dysfunction we observe (Barnett and
Desgrosellier, 2003; Liu et
al., 2004). Effects on these signaling pathways resulting from
loss of FGF8 compromise the expression of numerous target genes with roles in
endocardial EMT (Has2, Gata4, Tbx3, Twist1, Snai1)
(Armstrong and Bischoff, 2004;
Liu et al., 2004). TGFβi
(TGFβ induced) stimulates endothelial migration by altering the structure
of VE-cadherin intercellular junctions and integrin activity
(Ma et al., 2008), and its
downregulation might contribute to defective EMT in the mutant OFT.
In contrast to the paradigm of paracrine signaling established in other
tissues, our data show that in the SHF, the cellular source of the ligand
(signal) is also the target. Such an autocrine pathway can be easily
understood in terms of a feedback loop that maintains FGF production within a
tight range (E.J.P. and A.M.M., unpublished), which is crucial for FGF8
function (Hutson et al.,
2006). Secondary effects on other signaling pathways that we
observe may also be integrated into this regulatory loop. Fgf8 and
FGFR mutant analyses establish that the autocrine pathway not only regulates
survival and proliferation of SHF cells (a common response to FGFs), but also
the secretory and signaling capacities of their derivatives in the OFT (this
study) (Zhang et al., 2008;
Ilagan et al., 2006;
Park et al., 2006). The few
transcriptional targets of the PEA3 family of FGF8 effector proteins thus far
identified are ECM components, ECM-modifying enzymes and cell adhesion
molecules (de Launoit et al.,
2006), suggesting that an autocrine pathway might provide a means
of regulating the ECM and microenvironment to ensure uniform signal reception
and response within a specialized cell population. Our findings are of
biomedical importance, not only in the context of understanding the causes of
congenital malformations of the OFT, but also because the crucial role we
demonstrate for an autocrine FGF signaling pathway has broad implications for
understanding fundamental properties of FGF signaling in different
developmental and pathological contexts.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/21/3599/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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