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First published online May 23, 2006
doi: 10.1242/10.1242/dev.02408
1 Department of Pediatrics, Duke University Medical Center, Durham, NC 27710,
USA.
2 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710,
USA.
3 Department of Anatomy, School of Medicine, University of California at San
Francisco, San Francisco, CA 94143, USA.
4 Department of Genetics, University of Alabama at Birmingham, Birmingham, AL
35294, USA.
5 Institute of Biosciences and Technology, Texas A&M University Health
Science Center, Houston, TX 77030, USA.
* Author for correspondence (e-mail: e.meyers{at}cellbio.duke.edu)
Accepted 18 April 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Fgf8, Anterior heart field, Cardiogenesis, Cell Survival, Proliferation, Pea3, Bmp4, Erk, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Harvey,
2002). The primary heart tube gives rise to the cardiac inflow
tract (atria) and the left ventricle. The remaining chambers of the heart,
specifically the cardiac outflow tract (OT) and the right ventricle (RV) are
derived from the pharyngeal mesoderm contiguous with the primary heart tube.
Recent studies in chick and mouse demonstrate that cells within the developing
splanchnic mesoderm (SM), as well as possibly the pharyngeal arch core
mesoderm (CM), migrate from the pharynx and contribute to the myocardial wall
of the OT and RV during early stages of development
(Kelly et al., 2001;
Mjaatvedt et al., 2001;
Waldo et al., 2001). These
myocardial precursors have been defined as the anterior heart field (AHF),
which we use to describe specifically the SM and CM that contribute to the
myocardium of the OT and RV.
In more recent studies, several transcription factors (Isl1, Tbx1,
Nkx2.5, Foxh1, Mef2c) and extracellular signaling molecules (SHH, FGF10,
BMP4) that are expressed within or adjacent to the SM and CM have been
functionally implicated in AHF development
(Baldini, 2004;
Cai et al., 2003;
Dodou et al., 2004;
Lyons et al., 1995;
Hu et al., 2004;
Jiao et al., 2003;
Kelly et al., 2001;
Liu et al., 2004;
von Both et al., 2004;
Xu et al., 2004)
(Washington Smoak et al.,
2005). Moreover, extensive genetic analyses have shed some light
on the regulatory network that guides its development. For example,
Shh has been implicated as a regulator of Tbx1 expression
through the Foxa1, Foxc1 and Foxc2 transcription factors
(Yamagishi et al., 2003), and
Tbx1 in turn may regulate Fgf8 expression
(Hu et al., 2004).
Interestingly, heterozygosity for Fgf8 increases the incidence of
cardiac defects of Tbx1+/- embryos
(Hu et al., 2004;
Vitelli et al., 2002) and
Fgf8 hypomorphic mutants have OT defects, such as double outlet right
ventricle and persistent truncus arteriosus
(Abu-Issa et al., 2002;
Frank et al., 2002).
Fgf8 is a member of a large gene family encoding extracellular
ligands that have a wide range of cellular effects depending on the
developmental context. Several studies have indicated that there are at least
four non-mutually exclusive roles for Fgf8 in the development of OT
defects. First, there is a primary requirement for Fgf8 during
gastrulation, and loss of Fgf8 results in failure to form a crescent
of heart precursor cells (Sun et al.,
1999). Second, Fgf8 is required for establishing the
left-right axis and cardiac looping. Abnormalities in this process may affect
OT development (Albertson and Yelick,
2005; Meyers and Martin,
1999). Third, Fgf8 is required for migratory cardiac
neural crest cell survival, and therefore OT septation defects could arise
from neural crest cell deficiencies
(Abu-Issa et al., 2002;
Macatee et al., 2003).
Finally, Fgf8 is expressed in the precardiac SM as early as E7.75
(Crossley and Martin, 1995;
Kelly et al., 2001) and could
have a direct role in AHF maintenance and/or specification. Reduction of the
AHF population, and subsequent shortening of the OT, could result in OT
septation defects. In zebrafish, it has been demonstrated that the loss of
fgf8 results in the severe truncation of the ventricular myocardial
segment (Reifers et al.,
2000).
In this study, we have used genetic manipulations to distinguish between these possibilities to better understand how Fgf8 regulates OT development. We find that Fgf8 is required for AHF proliferation and survival; furthermore, we define the responsive tissues as well as the expression domains of Fgf8 that are crucial for AHF development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
Fgf8lacZ `knock in' line was used to follow Fgf8
gene expression and was used interchangeably as a null allele (D.B. and G. R.
Martin, unpublished). Nkx2.5Cre
(Moses et al., 2001) and
TnT-Cre (Jiao et al.,
2003) were crossed to the Cre-reporter line
Rosa26R (R26R) (Soriano,
1999) to determine the domains of DNA recombination as shown by
ß-galactosidase (ß-gal) activity in the progeny.
Bmp4lacZ and Ptch1lacZ lines have been
described previously (Goodrich et al.,
1996; Lawson et al.,
1999).
Generation of Cre-mediated Fgf8-null mutants
To generate Nkx2.5Cre/+; Fgf8flox/-
embryos, male mice heterozygous for Nkx2.5Cre and
Fgf8 were mated to females homozygous for the Fgf8 floxed
allele (Fgf8flox/flox). The embryos were genotyped for
Cre and Fgf8 alleles as previously described
(Meyers et al., 1998;
Sun et al., 2002). Similar
strategies were used to generate Nkx2.5Cre/+;
Fgf8flox/lacZ and TnT-Cre;
Fgf8flox/lacZ mutant embryos. For Cre lineage
trace analysis in the conditional mutant background,
Nkx2.5Cre/+; Fgf8+/- males were
crossed to females homozygous for both Fgf8flox and
R26R. Fgf8lacZ, Ptch1lacZ and
Bmp4lacZ embryos were genotyped for lacZ by
staining for ß-gal activity as described below.
lacZ staining
For embryos containing the R26R reporter allele or
Bmp4lacZ, tissues were fixed for 20 minutes at room
temperature in 2% formaldehyde/0.2% glutaraldehyde in PBS with 0.02% NP40,
then stained overnight at 37°C in standard X-gal stain. For
Fgf8lacZ and
Ptch1lacZ embryos, tissues were fixed
for 2-3 minutes at room temperature, followed by standard X-gal stain for 48
to 72 hours at 37°C.
In situ hybridization
Digoxigenin antisense riboprobes were synthesized using template plasmids
described in the following: Fgf8
(Crossley and Martin, 1995),
Wnt11 (Majumdar et al.,
2003), Anf, Mhc, Mlc2v
(Christoffels et al., 2000)
and Pea3 (Firnberg and Neubuser,
2002). Whole-mount mRNA in situ hybridization was performed
essentially as previously described
(Neubuser et al., 1997). All
results represent at least three mutants and three control embryos.
Immunohistochemistry
All antibodies were diluted in blocking solution containing 3% milk, 0.1%
Triton X in PBS. Antibodies were diluted as indicated: phospho-Histone H3
(1:500; Upstate Biotechnology), phospho-Erk (1:200; Cell Signaling
Technologies) and AP2
(1:100; Developmental Studies Hybridoma Bank).
Alexa Fluor 594 anti-rabbit IgG (Molecular Probes) and Cy2 anti-mouse IgG
(Jackson Immuno) secondary antibodies were diluted 1:500. pErk staining was
amplified with 1:200 biotinylated anti-rabbit IgG (Vector Laboratories),
followed by 1:50 HRP-streptavidin (Jackson Immuno). For peroxidase
colorimetric detection, embryos were bleached in 3% H2O2
in PBS, and incubated for 10 minutes at room temperature in 0.67 mg/ml
3,3'-diaminobenzidine (DAB, Sigma) in PBS containing 0.1% Tween 20.
Cell death
LysoTracker Red (Molecular Probes) staining has been previously described
as a marker for cells death (Zucker et
al., 1999). We have performed LysoTracker Red staining as
described by Abu-Issa et al. (Abu-Issa et
al., 2002), with the exception that DMEM was substituted for
Lactated Ringer's Solution.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Frank et al., 2002).
Eighty-five percent of Fgf8neo/- hypomorphic mutants
survive to term with various cardiac defects. Approximately 50% of these
embryos have abnormal left-right axis establishment (data not shown), based on
earlier ratios of laterality markers as well as organ laterality at term
(Meyers and Martin, 1999). The
remaining survivors exhibit severe craniofacial and cardiovascular defects,
such as double outlet right ventricle (data not shown). These defects are
reminiscent of DiGeorge/Velocardiofacial Syndrome and appear statistically and
phenotypically independent of left-right defects. Instead, these defects are
consistent with significant losses of neural crest cells
(Abu-Issa et al., 2002;
Macatee et al., 2003) and
possibly deficiencies in AHF development. In order to determine whether defects in the AHF contribute to these observed phenotypes, we examined Fgf8neo/- and Fgf8neo/lacZ embryos for OT or RV abnormalities at embryonic day (E) 9.5, prior to any substantial contribution of neural crest cell (NCC)-derived populations. Most of the mutant embryos that survive early gastrulation defects exhibit moderate (Fig. 1C) or severe (Fig. 1D) truncations of the OT and RV when compared with stage-matched controls (Fig. 1A). Most of these moderate and severe mutants presumably die by E10.5 owing to circulation failure, as evidenced by pericardial edema (data not shown). The remaining hypomorphic mutants demonstrate mild truncation of the OT/RV (Fig. 1B). These mild Fgf8neo/- mutants presumably progress to term with a variety of OT defects such as double outlet right ventricle, which may be the result of the OT/RV hypoplasia. No defects in the atria or left ventricle were evident in our analysis at this stage.
Surprisingly, we noted strong ß-gal activity in the developing OT and
RV of Fgf8+/lacZ embryos at E9.5-E10.5, where
Fgf8 mRNA has not previously been detected. This expression pattern
is consistent with a transgenic Fgf8-lacZ line which has detectable
ß-gal activity in the OT (Hu et al.,
2004). Given the abnormal OT/RV phenotype and this unexpected
expression, we compared the expression of both Fgf8 mRNA and
Fgf8lacZ relative to AHF development.
Fgf8lacZ faithfully recapitulates Fgf8 mRNA
expression pattern in all areas examined between E7.5 and E10.5 with some
notable differences described below.
Analysis of Fgf8 expression relative to heart development
At E7.75, both Fgf8 mRNA and Fgf8lacZ
expression are present in bilateral fields immediately caudal to the primary
heart field (Crossley and Martin,
1995). These cells ingress inward with the foregut pocket, moving
both dorsally and rostrally in relation to the primary heart field. Consistent
with this model, both Fgf8 mRNA and Fgf8lacZ are
expressed in the SM contiguous with the developing OT between E8.0 and E8.5.
(Fig. 2C,C' and
2D,D', respectively).
Later, between E9.0 and E10.5, Fgf8 mRNA is expressed in the pharyngeal endoderm and ectoderm in close proximity to the AHF, but not detected in the SM or OT (Fig. 2E,E',G,I). By contrast, ß-gal activity is still detected within the SM and extensively in the OT and RV (Fig. 2F,F',H). To better define which cells are expressing lacZ, we sectioned whole-mount stained embryos at multiple stages. Transverse sections at E8.0 demonstrate expression within the SM extending from the OT myocardium caudally to where the foregut has not closed (Fig. 2K1-K3). In addition, we detect expression at E9.5-E10.0 in the central area of the developing first and second pharyngeal arch, consistent with non-neural crest CM (Fig. 2F,L,L'). These Fgf8lacZ-positive CM cells are contiguous with the developing OT wall.
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Given the expression of Fgf8lacZ within the SM and CM, and the cardiac defects associated with Fgf8 hypomorphic mutants, we hypothesized that Fgf8 expression is required within the AHF for the development of the OT and RV. To test this hypothesis, we performed genetic ablation of Fgf8 using the tissue-specific Cre lines Nkx2.5Cre and TnT-Cre.
Nkx2.5Cre, but not TnT-Cre, mediates ablation of Fgf8 in the AHF
The Nkx2.5 locus targets most cardiogenic populations when
expressing Cre (Moses et al.,
2001; Stanley et al.,
2002); however, early cardiac crescent expression of the
Nkx2.5Cre allele used in this study is somewhat mosaic
(data not shown). Nevertheless, complete recombination in the cardiogenic
fields is observed at E8.0-E8.5 prior to heart tube elongation
(Fig. 4B,B'). Although
mesodermal expression of Fgf8 precedes robust
Nkx2.5Cre expression, we can use the
Nkx2.5Cre allele to ablate Fgf8 in the AHF prior
to the elongation process. Nkx2.5Cre also targets cells in
the heart tube, the ventral pharyngeal endoderm and the anterior ectoderm of
the first pharyngeal arch (Fig.
4B,B',E,E',E''). As revealed by the
Fgf8lacZ line, Fgf8 is expressed in the
pharyngeal endoderm, ectoderm, the AHF and the lineage of the AHF
(Fig.
4A,A',D,D',D''). Therefore, Fgf8 will
functionally be ablated from the AHF, endoderm, first pharyngeal arch ectoderm
and subsequent derivatives of these tissues.
By contrast, TnT-Cre drives DNA recombination in the primary heart
field cardiac crescent as early as E7.5, but not in the AHF precursors
(Jiao et al., 2003). Between
E8.5 and E11.5, the expression domain is exclusive to the entire myocardium
including OT, ventricles and atria. Importantly, TnT-Cre is expressed
in AHF derivatives only after these cells have migrated into the heart tube
proper and differentiated to form the OT and RV myocardium
(Fig. 4C,C',F-F'').
TnT-Cre is not expressed in the endoderm. Comparisons of both lateral
and medial sections of Fgf8lacZ,
Nkx2.5Cre; R26R and TnT-Cre;
R26R at E9.5 demonstrate the relevant overlapping domains of
expression (Fig.
4D-F,D'-F',D''-F'').
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Nkx2.5Cre, but not TnT-Cre, mediated deletion of Fgf8 results in OT/RV truncation and pharyngeal arch hypoplasia
We obtained the expected ratio of Nkx2.5Cre/+;
Fgf8flox/- class embryos at stages up to E9.5. However, at
later time points, we observed a marked reduction in the number of mutant
class embryos with no surviving mutants after E10.5. By gross morphology at
E9.5, mutant embryos exhibit severely truncated heart tubes and hypoplastic
pharyngeal arches (Fig. 5).
Using a general marker of specified myocardium (myosin heavy chain,
Mhc), the left ventricle and both atria appear grossly normal,
although slightly dilated, while the presumptive OT and RV are almost
completely absent (Fig. 5A,B).
Only a small segment of myocardium joins the left ventricle to the aortic sac.
To better define the regions of heart tube that are missing, we performed in
situ hybridization analysis on the Nkx2.5Cre/+;
Fgf8flox/- mutants and littermate controls with a number
of regionally specific markers (Fig.
5C-J). Wnt11, previously described as a marker specific
for the truncus of the OT of the heart (Cai
et al., 2003), is completely absent, indicating that mutants are
deficient in this region of the OT (Fig.
5C,D). We used the chamber-specific markers atrial natriuretic
factor (Anf) and myosin light chain 2v (Mlc2v) to further
characterize the remnant anterior heart segment. Anf specifically
marks the left ventricle and both atria at E9.5
(Fig. 5E)
(Christoffels et al., 2000),
while Mlc2v is specifically expressed by ventricular myocardium
(Fig. 5G)
(Cai et al., 2003). We observe
in our Nkx2.5Cre/+; Fgf8flox/- mutants
that the small anterior heart segment does not express Anf
(Fig. 5F), but does express
Mlc2v (Fig. 5H). This
analysis suggests that the remnant region is primarily residual right
ventricular myocardium, with very little contribution by OT myocardium.
As a final means to characterize the mutant heart tubes, we bred the
Bmp4lacZ `knock in' allele onto wild-type and mutant
backgrounds, and stained the embryos for ß-gal activity. In wild-type
controls, Bmp4lacZ is expressed in the SM, the OT and at
low levels in the RV, but is not expressed in left ventricle or the atria
(Fig. 5I)
(Jiao et al., 2003). In
Nkx2.5Cre/+; Fgf8flox/- mutants, very
few lacZ-expressing cells are present in the small heart segment,
supporting the hypothesis that this region is reduced residual RV
(Fig. 5J). The sum of these
data indicates that primarily the OT and much of the RV are deficient in these
mutant embryos. These are the tissues that derive from the AHF.
Our earlier analysis of both Fgf8 mRNA and lacZ expression reveals low levels in the OT of the heart tube itself. To ascertain any functional role for Fgf8 expression within the specified myocardium itself rather than the AHF cells, we used the heart tube-specific TnT-Cre line. As predicted by our hypothesis, TnT-Cre mediated ablation of Fgf8 does not result in any discernable OT or RV defects when examined at E10.5 (n=15) or OT septation defects at term. In fact, TnT-Cre; Fgf8flox/- embryos are viable. This finding not only indicates that Fgf8 expression is dispensable in the myocardium of the differentiated heart tube, but it further suggests that the crucial regions of Fgf8 expression for heart tube elongation include the AHF and/or the pharyngeal endoderm. These are regions of Nkx2.5Cre expression not inclusive of the TnT-Cre domain. However, these data do not rule out that OT myocardium could still be responding to FGF8 signals originating from the AHF or pharyngeal endoderm.
The Nkx2.5Cre lineage is reduced in the Nkx2.5Cre/+; Fgf8flox/- mutants
As Nkx2.5Cre is expressed in the AHF, we can use the
Cre reporter line R26R
(Soriano, 1999) to trace the
fate of these cells. Using this strategy, we analyzed the
Nkx2.5Cre lineage in the Fgf8 conditional mutant
background. At stages prior to heart tube elongation (E8.0), we do not observe
any change in the number of R26R lacZ-expressing cells of
Nkx2.5Cre/+; Fgf8flox/-; R26R
mutants. However, differences become noticeable by E8.5-E9.0 as the AHF cells
move from the SM and into the heart tube
(Fig. 6A,B). By E9.0-E9.5, we
observe a significant reduction in ß-gal-positive cells in the SM
(Fig. 6C-H) and the pharyngeal
endoderm (Fig.
6E',F'), evidenced by the severe thinning of these
cell layers (Fig.
6E',F'). ß-gal-positive cells of the CM are also
significantly decreased in mutants (Fig.
6G,H). The overall reduction in the Nkx2.5Cre
lineage strongly suggests that loss of these cells results in the severe heart
tube truncation phenotype that is observed in Fgf8 conditional
mutants.
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We also observe that the OT/RV cells in Nkx2.5Cre/+; Fgf8lacZ/flox mutants are Fgf8lacZ positive. Although consistent with our data indicating that this segment is remnant RV, this finding is surprising in that we expected a lineage autonomous function of Fgf8, i.e. that deletion of Fgf8 would result in a loss of all the Fgf8lacZ-expressing cells of the heart tube. There are several explanations for the persistence of Fgf8lacZ-expressing cells in Nkx2.5Cre/+; Fgf8lacZ/flox embryos. First, this subset of AHF cells may develop independently of Fgf8. Second, deletion of Fgf8 by Nkx2.5Cre may not be early enough to affect a small population of AHF as Fgf8 expression precedes Nkx2.5 expression. Alternatively, residual RNA or protein after genetic deletion can result in some functional rescue of the earliest AHF cells, which form the RV. The latter two explanations are more likely given that at early stages, R26R data clearly demonstrates a relatively normal number of Nkx2.5-positive cells of the AHF, and residual Fgf8 mRNA is observed in mutants (Fig. 4H, Fig. 6B) despite complete recombination between E8.0 and 9.5 (Fig. 4C',E,E' and data not shown).
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;
Katz and McCormick, 1997;
Rommel and Hafen, 1998;
Sato and Nakamura, 2004).
Distribution of phospho-Erk (pErk) can therefore identify tissue targets
undergoing active signaling as a result of Fgf8 activity in anterior heart
development. We first observe pErk staining in the SM at E8.5 (data not
shown). pErk persists until E9.0, where it is observed in the SM as well as
the emerging OT, but not significantly in the pharyngeal endoderm
(Fig. 7A,A'). After E9.5,
when AHF cells have been added and looping is complete, there is an absence in
pErk staining in the SM and OT, indicating absence of active signaling through
this pathway (data not shown). This decrease in Erk phosphorylation temporally
coincides with the observed downregulation of Fgf8 mRNA, suggesting
that Fgf8 signals through Erk in the AHF. If this hypothesis is correct, then
loss of Fgf8 will result in the loss of Erk phosphorylation in the
SM. We therefore compared the pErk levels in wild-type and
Nkx2.5Cre/+; Fgf8flox/- mutant
embryos. In early cardiac crescent stages, there is no detectable difference in pErk levels between wild-type and mutant embryos (data not shown). This finding is consistent with the fact that at these early stages, Nkx2.5Cre expression appears low and incomplete, as revealed by the R26R reporter gene. Between E8.5 and E9.5 as the heart tube undergoes elongation, Nkx2.5Cre/+; Fgf8flox/- embryos do not stain positively for pErk in the SM, indicating a loss of signal specifically within these AHF cells (Fig. 7B,B'). It therefore appears that Fgf8 signaling through Erk phosphorylation within the SM is necessary for AHF addition to the myocardium.
Genetic targets Pea3 and Bmp4 are reduced in Nkx2.5Cre/+; Fgf8flox/- mutants
To further test our hypothesis that FGF8 signals in an autocrine fashion to
the AHF, we analyzed downstream genetic components of the Fgf8 pathway.
Members of the ETS family of transcription factors, Pea3, Erm and
Er81, are transcriptional targets of FGF8 signaling
(Corson et al., 2003;
Munchberg and Steinbeisser,
1999; Raible and Brand,
2001; Roehl and
Nusslein-Volhard, 2001). Pea3 is expressed in the SM, the
CM and the pharyngeal ecto- and endoderm, suggesting a role for pharyngeal
arch and AHF development (Fig.
7C). Genetic ablation of Fgf8 within the
Nkx2.5Cre domain results in a significant decrease in
Pea3 expression in the first arch
(Fig. 7C,D) as well as
undetectable expression within the SM and pharyngeal endoderm
(Fig. 7C',D'). This
result provides functional evidence that this Ets transcription
factor is a downstream target of FGF8 within these tissues. Interestingly, we
did not detect expression of activated pErk within the endoderm in wild-type
embryos. Pea3 is expressed in both tissues in wild-type embryos, and
is absent in both tissues after Fgf8 ablation. These data suggest
that FGF8 may signal directly to the SM to activate Erk, and that a secondary
signal (independent of Erk) may be transduced both in an autocrine fashion to
the SM and by a paracrine mechanism to the endoderm to initiate Pea3
expression. Alternatively, FGF8 could regulate Pea3 expression
independently of Erk activation.
In consideration of the potential crosstalk between the AHF and the
pharyngeal endoderm, we determined if Fgf8 ablation altered other
extracellular signaling pathways (Shh and Bmp4) known to be
involved in OT development (Liu et al.,
2004) (Washington Smoak et
al., 2005). We do not observe any alterations of Shh
expression in the endoderm of Nkx2.5Cre/+;
Fgf8flox/- mutants
(Fig. 7G,H).
Ptch1lacZ expression, which is upregulated in response to
activation of the hedgehog pathway
(Goodrich et al., 1996), is
likewise unaltered in the endoderm and SM
(Fig. 7E,E',F,F').
However, examination of Bmp4lacZ expression in the SM of
Nkx2.5Cre/+; Fgf8flox/- mutants
(Fig. 7I,J and
Fig. 5J) indicates a reduction
in gene expression. These data reveal that Fgf8 does not regulate the
hedgehog pathway in the SM, but may regulate Bmp4. In addition, they
suggest that the loss of pErk and Pea3 expression are indeed the
result of Fgf8 ablation and not the result of dysplastic SM and
endoderm, as these tissues are still capable of expressing Ptch1 and
Shh, respectively.
Fgf8 is required for proliferation and survival of the precardiac mesoderm and pharyngeal endoderm
Lineage tracing of the Nkx2.5Cre domain has revealed a
reduction in both the endoderm and SM populations. Therefore, we analyzed cell
proliferation in the pharyngeal regions from E8.5 to E9.5 using antibodies for
phosphorylated histone H3 (pHH3), which marks mitotic cells. In
Nkx2.5Cre/+; Fgf8flox/- mutants, we
have observed a significant decrease in cell proliferation in both the SM and
pharyngeal endoderm (Fig.
8A,B). A comparison of wild-type and mutant embryos reveals that
mitotic indices are reduced by 
50% in the SM and by 60% in the pharyngeal
endoderm, although no significant differences in mitotic indices were noted in
the outflow region, the left ventricle, atria
(Fig. 8G) or the pharyngeal
arches (data not shown).
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Neural crest cells undergo excess cell death in Nkx2.5Cre/+; Fgf8flox/- mutants
We and others have previously described abnormal cell death of neural crest
cells (NCCs) in Fgf8 hypomorphs
(Abu-Issa et al., 2002;
Frank et al., 2002).
Interestingly, tissue-specific ablation of Fgf8 in the
Nkx2.5Cre domain also results in excess cell death of NCCs
in the pharyngeal arch mesenchyme at E9.5
(Fig. 8E,F). The excess cell
death in the pharyngeal arch mesenchyme encompasses much of the migratory
streams of NCCs up to the dorsal neural tube. These migratory NCCs are
specified and migrate in relatively normal streams from the dorsal neural tube
as seen by the markers Ap2 and Crabp1 (data not
shown). In addition, we see increased cell death of presumptive cardiac NCCs
in close proximity to the OT (Fig.
8D).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Although these data suggest that Fgf8 expression in the SM alone is
required for heart tube elongation, this phenotype (driven by
Mesp1Cre) could represent an earlier defect in mesodermal
specification and/or maintenance. Erk phosphorylation is restricted to the SM,
and not endoderm, demonstrating that FGF8 signaling to the AHF is crucial for
OT/RV outgrowth and development.
Other studies have also used tissue-specific gene deletion to determine the
specific functions of the various Fgf8 expression domains in the
pharynx. Tbx1-Cre, like Nkx2.5Cre, is expressed
in both pharyngeal endoderm and mesoderm. Tbx1-Cre ablation of
Fgf8 results in DiGeorge-like defects reminiscent of late term
Fgf8 hypomorphs (Brown et al.,
2004), but the majority of these mutants do not exhibit the early
heart tube truncation phenotype seen in Nkx2.5Cre/+;
Fgf8flox/- embryos. As Nkx2.5Cre is a
null allele, and Nkx2.5 may lie genetically downstream of
Fgf8 (Alsan and Schulthesis,
2002), Nkx2.5 heterozygosity may contribute to early
OT/RV truncation. However, the Nkx2.5Cre/+;
Fgf8flox/- mutants can be phenocopied through the use of
other Cre lines, such as Isl1Cre
(Park et al., 2006).
Furthermore, we observe the heart tube truncation phenotype in Fgf8
hypomorphs where Nkx2.5 gene dose is unaltered.
Phenotypic differences between Nkx2.5Cre/+;
Fgf8flox/- embryos and Tbx1-Cre;
Fgf8flox/- embryos may be the result of slightly different
Cre expression domains. Brown et al. have shown that DNA
recombination in the endoderm is complete at E10.5, but the extent of
endodermal Tbx1-Cre expression at earlier stages may not be complete
(C. Brown, personal communication). Incomplete deletion of Fgf8 in
the endoderm earlier in development may explain the discrepancy between
Tbx1-Cre; Fgf8flox/- and
Nkx2.5Cre/+; Fgf8flox/- mutants. This
explanation suggests a crucial role for early endodermal Fgf8
expression and AHF expression in the development and outgrowth of the OT/RV.
In support of this hypothesis, Fgf8 ablation specifically in the AHF
does not phenocopy the early heart tube truncation phenotype
(Park et al., 2006). These
data, therefore, suggest a requirement for both domains of Fgf8
expression for heart tube elongation. Although it remains unclear whether or
not endodermal and mesodermal sources of FGF8 have separate and distinct
functions, we have nevertheless shown that Fgf8 has a role in determining the
length of the OT/RV through proliferation and survival of the AHF.
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;
Yelbuz et al., 2002).
Distinguishing between such possibilities will be difficult because of
potential interactions between the two fields. We are currently investigating
the requirement for the Fgf receptor genes Fgfr1 and Fgfr2
in the AHF to determine if Fgf signaling has a direct influence on AHF
development. Preliminary data indicate that loss of these receptors from the
Nkx2.5Cre domain results in OT septation defects, but not
OT/RV truncation (G. Smyth and E.N.M., unpublished).
Neural crest cells and the anterior heart field
Expansion of the pharyngeal arches is the result of NCCs migrating into the
pharyngeal region, forming much of the mesenchyme. Loss of Fgf8 from
the AHF and endoderm results in marked cell death of migratory NCCs
(Fig. 8F), similar to that seen
in Fgf8neo/- embryos
(Abu-Issa et al., 2002;
Macatee et al., 2003). This
expansive cell death can account for the pharyngeal arch hypoplasia observed
in Fgf8 mutants. It was surprising to observe that NCCs located as
far as the dorsal neural tube undergo excess cell death in
Nkx2.5Cre/+; Fgf8flox/- embryos. This
observation leads to two hypotheses. First, given the close apposition and
function of the AHF and NCCs in OT development, it is possible that the AHF
and NCCs are interdependent. That is, the development of the NCCs depends on
signals from the AHF (perhaps through Bmp4). Defective AHF, owing to loss of
Fgf8, negatively impacts NCC development indirectly. It follows then
that pharyngeal arch hypoplasia would result from the loss of NCCs.
The second possibility is that FGF8 protein from the AHF is able to diffuse long distances to directly influence NCC development. However, NCC-specific deletion of both Fgfr1 and Fgfr2 does not affect cardiac NCC development, suggesting an indirect effect of FGF8 on these cells in the lower arches (G. Smyth and E. Meyers, unpublished). Alternatively, FGF8 may signal directly to the NCCs through other Fgf receptors.
Role of the pharyngeal arch core mesoderm
Interestingly at E9.5, Fgf8lacZ ß-gal activity
persists within the CM of the pharyngeal arches, but neither Fgf8 nor
lacZ mRNA transcripts are detected (data not shown). Previous work
has shown that this population of cells is derived from the paraxial mesoderm
(Trainor et al., 1994), and
Fgf8 appears to be expressed in this region as early as E8.0
(Crossley and Martin, 1995).
The persistence of ß-gal activity again could represent a lineage trace
of this mesoderm into the core of the pharyngeal arches rather than nascent
gene expression.
Gross analyses of the AHF markers Fgf10lacZ,
Tbx1lacZ and Fgf8lacZ, as well the
Nkx2.5Cre lineage trace, reveal that the CM cells are
contiguous with the OT (Brown et al.,
2004; Kelly et al.,
2001). DiI-labeling studies suggest that these CM cells are part
of the AHF, and contribute cardiomyocytes to the OT at early stages
(Kelly et al., 2001). We have
found that like in the SM, there is a marked reduction of the
Nkx2.5Cre-positive cells in the CM of conditional
Fgf8 mutants. The reduction of CM may not only contribute to OT/RV
truncation, but also to pharyngeal arch hypoplasia. Additionally,
Fgf8 expression may be required in the CM for the survival of the
NCCs that populate the pharyngeal arches.
At E9.5, when heart tube elongation has been completed, we observe a
persistence of Fgf8lacZ and
Nkx2.5Cre-positive cells in the CM. Although these cells
remain contiguous with the OT myocardium, it is unlikely that at this stage
they continue to contribute to the OT. Instead, these cells are known to give
rise to the muscles of the jaw (Trainor et
al., 1994). It is intriguing to speculate that OT/RV myocardium
and the muscles of the jaw arise from a common lineage represented by the
Fgf8lacZ allele and Nkx2.5Cre line. At
the least, the two populations share common genetic regulators required for
myogenesis and/or for proper development, such as Tbx1, Isl1 and
Nkx2.5 (Cai et al.,
2003; Kelly et al.,
2004; Lyons et al.,
1995).
Cellular functions of Fgf8 in the anterior heart field
Aside from cell proliferation and survival, FGF8 signaling has a very
specific role in gene regulation. FGF8 signaling has been shown to modulate
the expression of Wnt (Grieshammer et al.,
2005) and Bmp (Lewandoski et
al., 2000) genes to pattern the embryo. Fgf8 has also
been shown in vivo to be necessary for expression of various transcription
factors such as Pea3, Erm and Tbx2
(Firnberg and Neubuser, 2002;
Munchberg and Steinbeisser,
1999; Raible and Brand,
2001; Roehl and
Nusslein-Volhard, 2001). Here, we have found that Fgf8 is
necessary for Pea3 expression within the AHF itself, and may also
modulate Bmp4 expression levels.
Many ETS and Fgf proteins have roles in epithelial-to-mesenchymal
transition and cell migration (reviewed by
Hsu et al., 2004;
Thiery, 2002). Fgf1 and Fgf2
have been shown in vitro to induce the internalization and nuclear
localization of the cell-adhesion molecule E-cadherin
(Bryant et al., 2005). In
addition, Fgf signals can activate cell motility through the small GTPase Rho,
as seen in Drosophila (Schumacher
et al., 2004), and through activated Mapk to promote myosin light
chain kinase phosphorylation (Klemke et
al., 1997). Developmentally, the loss of the Fgf ligand
heartless in Drosophila demonstrates defects in mesodermal
migration (Beiman et al.,
1996). In mouse, Fgf8-null
(Sun et al., 1999) and
Fgfr1-null embryos (Ciruna and
Rossant, 2001) have altered cadherin expression, and exhibit
migrational defects during gastrulation. We speculate that Fgf8 signaling in
the AHF increases cell motility at both the transcriptional and cellular
levels, allowing for the migration of cells into the heart tube. Loss of this
motility signal may disrupt the AHF program, resulting in increased cell death
and decreased proliferation. Studies are currently under way to address this
possibility.
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ACKNOWLEDGMENTS |
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REFERENCES |
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