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First published online May 23, 2006
doi: 10.1242/10.1242/dev.02367
1 Department of Neurobiology and Anatomy, University of Utah School of Medicine,
Salt Lake City, UT 84112, USA.
2 Department of Pediatrics, University of Utah School of Medicine, Salt Lake
City, UT 84112, USA.
3 Institute of Molecular Medicine, Department of Medicine, University of
California, San Diego, La Jolla, CA 92093, USA.
4 Cardiovascular Research Institute, University of California, San Francisco, CA
94143, USA.
5 Children's Health Research Center, University of Utah School of Medicine, Salt
Lake City, UT 84112, USA.
6 Program in Human Molecular Biology and Genetics, University of Utah School of
Medicine, Salt Lake City, UT 84112, USA.
* Author for correspondence (e-mail: anne.moon{at}genetics.utah.edu)
Accepted 16 March 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Fgf8, Cardiovascular, Outflow tract, Congenital heart disease, Truncus Arteriosus, Transposition, 22q11 deletion syndrome, Pharyngeal arches, Neural crest, DiGeorge syndrome
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Harvey,
2002). Given the duration and complexity of heart development, it
is not surprising that nearly one percent of humans are born with congenital
cardiovascular defects (Creazzo et al.,
1998; Harvey,
2002; Olson and Schneider,
2003).
Myocardial precursors leave the primitive streak and reside briefly in a
`cardiac crescent' of bilateral mesoderm where their fate is influenced by
adjacent endoderm (Lough and Sugi,
2000; Schultheiss et al.,
1995; Yutzey and Kirby,
2002). Clonal analyses revealed that two distinct lineages
contribute to the heart (Zaffran et al.,
2004), and studies in mouse and chick indicate that crescent
mesoderm contains two precursor populations: a `primary heart field' (PHF)
contains the earliest cells to undergo myocardial differentiation, which
contribute to the primitive tubular heart and ultimately to the left ventricle
(LV), whereas the `anterior heart field' (AHF) contributes right ventricular
(RV) and outflow tract (OFT) myocardium
(Buckingham et al., 2005;
Zaffran et al., 2004;
Cai et al., 2003;
de la Cruz et al., 1977;
Kelly et al., 2001;
Mjaatvedt et al., 2001;
Waldo et al., 2001). Although
Isl1 is expressed by undifferentiated cells in the AHF, it is also expressed
by many LV precursors (Cai et al.,
2003) (this manuscript). To date, no proteins have been identified
whose expression specifically identifies either field and it is unknown if
cells are developmentally restricted within each field. Rather than indicating
a distinct molecular identity, the concept of the AHF is currently a
teleologic one, reflecting the relative position of a cell in the crescent,
the time it contributes to the heart, and its ultimate location within the
heart. For clarity, in this manuscript we employ AHF to refer to mesodermal
cells residing in the dorsomedial crescent and, subsequently, in anterior
splanchnic mesoderm (SM) ventral to the foregut, which are required to form
the RV and the OFT.
Transcription factor networks regulate cardiomyocyte specification (Gata4
and Nkx2.5), differentiation (Mef2c), and regional identity within the heart
(Brand, 2003;
Bruneau, 2002;
Cripps and Olson, 2002;
Firulli and Thattaliyath,
2002; Fishman and Chien,
1997; Srivastava and Olson,
2000). AHF expression of Mef2c is regulated by at least
two intronic elements, one of which confers responsiveness to Isl1 and Gata
(Dodou et al., 2004) and the
other to a Tgfß/Foxh1/Nkx2.5 pathway
(von Both et al., 2004).
Although expression of Mef2c, Isl1 and Foxh1 is not
restricted to the AHF, their inactivation primarily disrupts the formation of
AHF-derived structures, suggesting that this network regulates the
proliferation and survival of undifferentiated AHF cells
(Cai et al., 2003;
Lin et al., 1997;
von Both et al., 2004).
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), and members of
these families have later roles in OFT and valve morphogenesis
(Abu-Issa et al., 2002;
Armstrong and Bischoff, 2004;
Delot et al., 2003;
Frank et al., 2002;
Liu et al., 2004;
Sanford et al., 1997).
Fgf8 and Fgf10 are secreted signaling proteins expressed in the cardiac
crescent and in SM (Crossley and Martin,
1995; Kelly et al.,
2001). Although Fgf10-/- mice survive
embryogenesis without cardiac defects (Min
et al., 1998; Sekine et al.,
1999), germline ablation of Fgf8 is embryonic lethal
(Sun et al., 1999). The
complex cardiac and OFT phenotypes seen in Fgf8-deficient hypomorphic mice
indicate that this protein has required functions at different times and
locations during cardiogenesis and the subsequent OFT remodeling. Severe
hypomorphs have looping defects, reflecting a role for Fgf8 in embryonic
and/or thoracic left-right axis determination
(Meyers and Martin, 1999).
Milder hypomorphs have hypoplastic OFTs and defects reflecting disrupted
ventriculoarterial alignment (double outlet right ventricle, transposition of
the great arteries) or failed OFT septation (persistent Truncus Arteriosus)
(Abu-Issa et al., 2002;
Frank et al., 2002).
The source(s) of Fgf8 required for outflow tract formation and subsequent
remodeling are unknown. OFT development is normal after Fgf8
loss-of-function in pharyngeal ectoderm
(Macatee et al., 2003). Early
widespread Fgf8 ablation recapitulates the entire spectrum of OFT
defects observed in hypomorphs (Brown et
al., 2004), whereas later ablation throughout the caudal pharynx
causes only a low incidence of an alignment-type OFT defect
(Macatee et al., 2003)
(A.M.M., unpublished).
In the wake of these studies, several important questions remain. Does the Fgf8 expression domain in the cardiac crescent include both PHF and AHF cells, and what are the cellular targets of Fgf8 signaling in the crescent? What are the source(s) and timing of Fgf8 signals required for OFT formation and for Fgf8-dependent morphogenetic events during subsequent OFT remodeling? Is the source of Fgf8 required for OFT septation separable from that supporting rotation/alignment?
In order to address these questions, we determined the onset and breadth of Fgf8 expression in the cardiac crescent, heart tube and pharynx. By precisely characterizing the activity of a series of Cre drivers relative to these Fgf8 expression domains, we were able to examine and interpret the effects of Fgf8 loss-of-function at specific times, and in particular cell populations, during the earliest stages of cardiogenesis and subsequent OFT remodeling. We found that autocrine Fgf8 signaling is required at the crescent stage to support formation of the heart tube and the RV/OFT, and for looping. Unlike the variable, complex phenotypes of previously reported Fgf8 mutants, our approach generated discrete OFT defects in the face of normal embryonic LR axis specification and revealed specific, required roles for endoderm and mesoderm-derived Fgf8 in different aspects of OFT remodeling.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Macatee et al.,
2003; Moon and Capecchi,
2000; Saga et al.,
1999; Verzi et al.,
2005). A novel Fgf8C conditional allele was
generated by inserting a loxP site 5' to exon 4, and frt-flanked
neomycinr and a loxP site into the 3' untranslated
region. Construction of Isl1Cre will be detailed elsewhere
(C.-L.C. and S.E., unpublished); Cre was inserted in frame into exon
1 of Isl1, creating a Cre knock-in, Isl1
knockout.
Immunohistochemistry and TUNEL analysis
Primary and secondary antibodies, and immunohistochemical methods have been
previously described (Macatee et al.,
2003). Anti-Isl1 antibody was described previously
(Pfaff et al., 1996).
Experiments were repeated a minimum of three times per somite stage.
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) using standard
protocols; counterstaining was with nuclear Fast Red.
Whole mount in situ RNA hybridization
Embryos were harvested, fixed and hybridized using a standard protocol
(Moon et al., 2006). Mutants
and controls were processed in the same vial throughout. Experiments were
repeated a minimum of three times; representative results are shown.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and a
universally expressed Cre recombinase, HPRTCre
(Tang et al., 2002).
Production of Fgf8GFP in Fgf8-expressing cells requires Cre-mediated
recombination of Fgf8GFP and Fgf8GFP faithfully
recapitulates the expression of the endogenous Fgf8 locus throughout
embryogenesis (Boulet et al.,
2004; Ladher et al.,
2005; Macatee et al.,
2003).
In Fgf8GFP/+;HPRTCre/+ embryos,
myocardial precursors migrating anterolaterally from the primitive streak
initiate Fgf8 expression as they accumulate in the cardiac crescent
at late headfold (LHF) to 1-somite stage (ss)
(Fig. 1A,B). It has been
proposed that Fgf8 expression is limited to the AHF
(Buckingham et al., 2005;
Kelly and Buckingham, 2002);
however, many LV precursors in the ventral heart tube express Fgf8GFP
(Fig. 1C-E). At 10ss, Fgf8GFP
persists in splanchnic mesoderm (SM) and nascent RV/OFT, but not in the LV or
inflow (Fig. 1F,G). Rapid loss
of GFP in the LV and inflow (already waning by 6ss, not shown) indicates that
the half-life of the Fgf8GFP fusion protein is <10 hours in these cells. In
the pharyngeal epithelia, Fgf8GFP is detectable in pouch endoderm and surface
ectoderm by 5ss (Fig.
1D,E).
Given the broad expression of Fgf8 in the crescent and early pharynx, and the complex phenotypes of previously reported Fgf8 mutants, we assembled a panel of Cre-expressing `drivers' to systematically generate Fgf8 loss-of-function in subsets of myocardial precursor mesoderm and early endoderm. A detailed comparison of the activity of these drivers relative to Fgf8 expression is crucial to interpreting and understanding the consequences of differential ablation of Fgf8; as will become apparent, subtle differences in the timing and location of Fgf8 function profoundly influence ultimate phenotype.
MesP1Cre ablates Fgf8 in myocardial precursors prior to crescent formation
We used MesP1Cre
(Saga et al., 1999) to ablate
Fgf8 in all cardiac precursor mesoderm, as evidenced by lacZ
expression from the Rosa26-Cre reporter throughout the cardiac crescent of
presomite embryos, in the unlooped HT, and in all myocardium of embryonic day
(E) 10.5 hearts (Fig. 1H-K)
(Saga et al., 2000;
Saga et al., 1999). In
Fgf8GFP/+;MesP1Cre/+ embryos,
Fgf8GFP is broadly detected in the crescent at LHF (1ss;
Fig. 1A1,B1), in dorsal and
ventral cells of the 5ss heart tube (Fig.
1C1,D1), and in AHF-derived RV/OFT
(Fig. 1F1,G1). Mesodermal
Fgf8GFP expression resulting from HPRTCre and MesP1Cre activity is
indistinguishable (compare Fig. 1A-G with
1A1-1G1), indicating that MesP1Cre completely ablates
Fgf8 in the precardiac mesoderm.
Isl1Cre ablates Fgf8 in a subset of myocardial precursors and in endoderm
Fgf8 is expressed in the pharyngeal endodermal pouches, but its
role in this tissue is unknown. Because Isl1 is expressed broadly in
pharyngeal endoderm and throughout the AHF
(Cai et al., 2003), we
generated a new, highly efficient `Isl1Cre'-expressing line to determine
whether the loss of endodermal Fgf8 contributes to the cardiac or OFT defects
observed in previously described Fgf8 mutants. Isl1Cre-mediated
recombination of the Rosa26-lacZ reporter is first evident in pharyngeal
endoderm and crescent mesoderm at the 1-2ss
(Fig. 1L). Its activity expands
rapidly such that, by 5ss, the first pouch and adjacent endoderm, and many
cells in the ventral heart tube, are lacZ-positive
(Fig. 1M-O2).
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Mef2C-AHFCre ablates Fgf8 in the AHF at the 2- to 3-somite stage
To assess whether Fgf8 is required independently in AHF mesoderm,
we employed Mef2c-AHFCre. Consistent with published activity of this driver
(Verzi et al., 2005), Fgf8GFP
is present in few AHF cells of Fgf8GFP/+;Mef2c-AHFCre
embryos at 2-3ss, but throughout the AHF by 5ss
(Fig. 1A3-E3). In contrast to
MesP1Cre and Isl1Cre, no Fgf8GFP-positive cells were present in LV precursors
in the ventral heart tube of 5ss Fgf8GFP/+;Mef2c-AHFCre
embryos (Fig. 1D3,R).
The later onset and restricted domains of recombination in cardiac mesodermal precursors of Mef2c-AHFCre and Isl1Cre relative to MesP1Cre are summarized in Fig. 1P-S: Fgf8GFP is present in few crescent cells of Fgf8GFP/+;Isl1Cre/+ or Fgf8GFP/+;Mef2c-AHFCre embryos prior to 2-3ss, which represents at least six hours of ongoing mesodermal Fgf8 signaling in these mutants relative to MesP1Cre (Fig. 1A1,B1 compare with A2,B2 and A3,B3). By 5ss, all three drivers are active throughout the SM/AHF, but only Isl1Cre has endodermal activity (Fig. 1R,S).
To generate Fgf8 conditional mutants in the absence of confounding hypomorphic effects, we used a new loxP-containing allele with minimal exogenous sequence (abbreviated Fgf8C, see Materials and methods). Fgf8C/C and Fgf8C/- animals are phenotypically normal and are indistinguishable from Fgf8C/+ littermates.
Because MesP1Cre and Isl1Cre are loss-of-function alleles, we looked for evidence of genetic interaction with Fgf8. Fgf8C/+;MesP1Cre/+ embryos were morphologically and molecularly indistinguishable from Fgf8C/+ or Fgf8C/- littermates and were present at the expected Mendelian ratios (E8-E11.5: n=79, 105% of predicted; E18.5/newborn: n=30,107% of predicted; Fig. 2A-C,L). Nor did we detect evidence of a genetic interaction between Fgf8 and Isl1 in Fgf8C/+;Isl1Cre/+ embryos (Fig. 2F-H,N; E8-E11.5: n=68, 100% of predicted; E18.5/newborn: n=26, 100% of predicted).
Autocrine Fgf8 activity in the crescent mesoderm is required for heart tube and outflow tract formation
Early, complete ablation of Fgf8 function in both heart fields in
Fgf8C/-;MesP1Cre/+ conditional mutants
(Fgf8;MesP1Cre) disrupted HT formation, looping, and accretion of
OFT/RV myocardium. These severely affected embryos (66/103, 65%) had
hypoplastic heart tubes (Fig.
2E), short or absent OFTs, and a single dilated ventricle and
atrium (Fig. 2E'), and
died by E10.0 with anasarca and pericardial effusion
(Fig. 2M'). This
phenotype is also seen after ablation of Fgf8 with Nkx2.5Cre (E.
Meyers, unpublished). The lower incidence of this severe phenotype in
Fgf8C/-;Isl1Cre/+ mutants (26/73, 35%;
Fig. 2J,J',O')
correlates with a later onset and a restricted domain of Isl1Cre activity in
crescent mesoderm. By contrast, AHF-limited Fgf8 ablation driven by
Mef2c-AHFCre permitted all Fgf8;Mef2c-AHFCre mutants to survive to
birth (n=16, 105% of expected) after normal initial formation of the
heart tube, OFT and RV (Fig.
2K,K',P').
Fgf8 in AHF mesoderm supports normal outflow tract alignment
Differences in Fgf8 signaling obtained after MesP1Cre, Isl1Cre and
Mef2c-AHFCre ablation were not only manifest in the frequency of cardiac
hypoplasia and embryonic death, but also in the OFT remodeling phenotypes of
surviving mutants. Thirty-five percent of Fgf8;MesP1Cre mutants
survived but had short, narrow OFTs and small RVs at midgestation
(Fig. 2D,D',M). Based on
their embryonic phenotypes, we expected these mild mutants to have hypoplastic
RVs at birth, but they did not; rather, 30% had D-transposition of the great
arteries (TGA, n=15; Fig.
2R). This indicates that OFT rotation/alignment was perturbed by
the loss of mesodermal Fgf8 and the resulting deficiency of RV/OFT myocardium
at the time of OFT remodeling. Intrathoracic and intrabdominal LR axes
appeared to be normal. Forty percent of Fgf8;MesP1Cre newborns also
had a bicuspid aortic or pulmonary valve (the LV-associated valve;
Fig. 2R'). No OFT or
valve defects were observed in Fgf8C/- or
Fgf8C/+;MesP1Cre/+ controls
(n=28). The importance of post-crescent stage mesodermal Fgf8 for
subsequent OFT alignment was confirmed by the occurrence of TGA and double
outlet right ventricle (DORV) in Fgf8C/-;Mef2c-AHFCre
mutants (25%, n=16; Fig.
2S,S'). These findings allow us to attribute the OFT
rotation/alignment and valve defects previously described in Fgf8
hypomorphs, Fgf8;Hoxa3IresCre mutants and Fgf8;Tbx1Cre
mutants (Abu-Issa et al., 2002;
Frank et al., 2002;
Macatee et al., 2003;
Brown et al., 2004)
specifically to disrupted autocrine Fgf8 function in AHF mesoderm.
Outflow tract septation requires Fgf8 function in the pharyngeal endoderm
Rather than alignment-type OFT defects, some Fgf8 hypomorphs have
persistent Truncus Arteriosus (PTA), in which failed OFT septation leaves a
single vessel arising from both ventricles
(Abu-Issa et al., 2002;
Frank et al., 2002). Septated
OFTs in all surviving Fgf8;MesP1Cre and Fgf8;Mef2c-AHFCre
mesodermal mutants, and after ablation of Fgf8 from pharyngeal ectoderm
(Macatee et al., 2003),
indicates that septation is independent of these sources and is likely to
depend on endodermal Fgf8.
Most Fgf8C/-;Isl1Cre/+ mutants survived embryogenesis (E18.5/newborn: n=18, 65% of predicted). At E8.75-E10.5 they had small pharyngeal arches and severe RV hypoplasia, with a markedly altered relationship between the LV and OFT (Fig. 2I,I',O). Fgf8C/-;Isl1Cre/+ conotruncal cushions were hypocellular relative to controls; fusion of the conotruncal cushions to form the AP septum was already underway in E10.5 Fgf8C/+;Isl1Cre/+ controls, but not in Fgf8C/-;Isl1Cre/+ mutants (Fig. 2N,O). Remarkably, 100% of E18.5/newborn Fgf8;Isl1Cre mutants had PTA. The truncal vessel was abnormally positioned over the RV (Fig. 2T-T''), which is surprising given the severity of RV hypoplasia observed at midgestation.
Loss of Fgf8 in crescent mesoderm disrupts Erm and Isl1 expression in the AHF, and alters the balance between proliferation and cell death in the nascent heart
In order to understand the etiology of the early shared phenotype of
MesP1Cre and Isl1Cre mutants, namely the disrupted formation of primary heart
tube and OFT/RV, we sought to identify Fgf8-sensitive tissues at crescent and
early somite stages and thus examined the expression of Erm. Erm is
regulated by Fgf8 and encodes an Ets-domain transcription factor that is an
effector of Fgf/Fgf receptor signaling
(Brent and Tabin, 2004;
Firnberg and Neubuser, 2002;
Raible and Brand, 2001). In
severe Fgf8;MesP1Cre and Fgf8;Isl1Cre mutants, decreased
size and lower intensity of the Erm expression domain are apparent
relative to controls and to mildly affected Fgf8;MesP1Cre mutants
(Fig. 3A-F). Erm was
absent in proximal OFT myocardium, and decreased in SM of both classes of
mutants (Fig.
3A'-F'). Endodermal Erm expression was more
severely decreased in Fgf8;Isl1Cre mutants, suggesting autocrine Fgf8
activity in the endoderm (Fig.
3F').
Isl1 is a LIM-homeodomain transcription factor required for the survival
and proliferation of AHF mesoderm (Cai et
al., 2003). We examined Isl1 expression by in situ
hybridization and Isl1 protein production with an anti-Isl1 antibody
(Pfaff et al., 1996). Early
disruption of Isl1 expression in Fgf8;MesP1Cre and
Fgf8;Isl1Cre mutants mirrored those observed in Erm
(Fig. 3G-L). This is not
attributable to the Isl1 loss-of-function allele in
Fgf8C/-;Isl1Cre/+ mutants because Isl1 mRNA and
protein production are decreased in MesP1Cre mutants
(Fig. 3I), and are more severe
in Fgf8C/-;Isl1Cre/+ mutants than in
Fgf8C/+;Isl1Cre/+ controls
(Fig. 3L,K). Analysis of Isl1
protein revealed both fewer Isl1-producing cells in SM, and less intense Isl1
immunostaining in cells in SM, proximal OFT and endoderm of conditional
mutants relative to controls (Fig.
3M-P''). Levels of Isl1 mRNA correlated with the
severity of OFT defects observed in E9.5 MesP1Cre or Isl1Cre mutants: embryos
dying with severe OFT/RV hypoplasia and incomplete looping had a markedly
decreased intensity of Isl1 expression in SM and endoderm
(Fig. 3Q-V').
We investigated whether the survival or expansion of cardiac precursors was decreased by a loss of Fgf8 in the crescent mesoderm and reasoned that PHF/AHF dysfunction must occur early in cardiogenesis in mutants, because abnormal heart tube and OFT morphology, and decreased Isl1 immunostaining were evident at 7-9ss. No reproducible increase in apoptosis was observed in mutant mesoderm at 0-4ss (Fig. 4A-F; data not shown), although apoptosis in neuroectoderm and ventral endoderm of mutants was increased relative to controls after 0ss (Fig. 4C-F). At 7-9ss, we detected excess apoptotic cells in ventral endoderm and adjacent midline SM accumulating to the OFT in both MesP1Cre and Isl1Cre mutants (Fig. 3M-P', Fig. 4E-J), relative to double heterozygote controls (P=0.004, paired t-test; controls mean=9 cells/high-power field, n=6; mutants mean=22 cells/high-power field, n=4 Fgf8;MesP1Cre and n=5 Fgf8;Isl1Cre mutants).
Anti-PHH3 labels cells in all phases of mitosis
(Li et al., 2005). At 0ss,
high levels of proliferation were detected in both mutants and controls;
notably the mutants had more PHH3-positive cells in the neuroectoderm, but
fewer in the mesoderm and endoderm than controls
(Fig. 4A,B). 4ss mutants had an
average of 46% fewer proliferating cells in crescent mesoderm
(Fig. 4E,F; P=0.005,
paired t-test; controls mean=66%, n=4;
Fgf8;MesP1Cre mutants mean=20%, n=4) and this difference
persisted at 9ss. Fewer PHH3-positive cells were also noted in the proximal
OFT and pharyngeal epithelia of 9ss conditional mutants
(Fig. 4G-J).
Ablation of Fgf8 in mesoderm disrupts required transcription factor and signaling pathways in the anterior heart field and outflow tract without perturbing the left/right axis
To characterize the molecular events associated with disrupted growth of
the OFT/RV in Fgf8;MesP1Cre and Fgf8;Isl1Cre conditional
mutants, we examined gene expression in the AHF/nascent OFT. Expression of
Mef2c is regulated by Isl1 and Foxh1 via AHF-specific enhancers
(Dodou et al., 2004;
von Both et al., 2004). When
RV/OFT-fated myocardium first accrues to the heart (7-8ss in this system),
expression of Mef2c in the AHF localizes to the right SM and nascent
RV/OFT myocardium. This localization occurred in conditional mutants, but the
level of Mef2c mRNA in OFT and SM was markedly decreased, whereas
expression in the first pharyngeal arch and inflow was intact
(Fig. 5A,A'). We
attribute this alteration in Mef2c expression to the demonstrated
decrease in Isl1 activity, because Foxh1 expression was intact in
Fgf8;MesP1Cre and Fgf8;Isl1Cre mutants (not shown).
Wnt11 influences cell adhesion, migration and polarity
(McEwen and Peifer, 2000); it
is expressed in truncal myocardium (Cai et
al., 2003) and is required for OFT alignment and septation in mice
(W. Zhou, L. Lin, A. Majumdar, X. Li, X. Zhang, W. Liu, L. Etheridge, Y. Shi,
J. Martin, W. Van de Ven, V. Kaartinen, A. Wynshaw-Boris, A. McMahon, M. G.
Rosenfeld and S.E., unpublished). Wnt11 expression correlated with
the severity of RV/OFT dysplasia: it was undetectable in severe
Fgf8;MesP1Cre and Fgf8;Isl1Cre mutants, decreased in mild
variants, and preserved in Fgf8;Mef2c-AHFCre mutants
(Fig. 5B,B').
|
). Fgf8 functions upstream of nodal and Pitx2c in
embryonic LR axis determination (Boettger
et al., 1999; Fischer et al.,
2002; Kioussi et al.,
2002; Liu et al.,
2002; Meyers and Martin,
1999; Schneider et al.,
1999; Welsh and O'Brien,
2000). We examined Pitx2 expression to assess whether the
looping and OFT defects observed in MesP1Cre and Isl1Cre Fgf8
conditional mutants reflect the altered specification/maintenance of the
embryonic LR axis and found that Pitx2 localized appropriately to the
left SM, OFT and atrial myocardium in mild and severely affected mutants at
the 10-16ss (Fig. 5C-C'';
data not shown). Normal expression of Ventricular myosin light chain
2 (Franco et al., 1999)
indicated that myocardial differentiation occurred in the hypoplastic right
ventricles of conditional mutants (not shown).
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;
Xu et al., 2004). As
Tbx1 and Fgf10 expression were intact in
Fgf8;MesP1Cre and Fgf8;Isl1Cre mutants
(Fig. 5D,E), disruption of
these factors does not cause the observed OFT defects.
Based on the shared phenotypes of Fgf receptor 1 (Fgfr1) and
Fgf8 mutants, on recent data indicating that Fgfr1 is activated by
Fgf8 in vivo (Moon et al.,
2006), and on our unpublished observation that OFT septation is
sensitive to Fgfr1 function, Fgfr1 probably mediates Fgf8 signals in target
cells required for OFT septation. Moreover, decreased Erm expression
in the endoderm of Fgf8;Isl1Cre mutants, which develop PTA, suggests
that Fgf8 has an autocrine role in the endoderm required for OFT septation. It
has previously been demonstrated that Fgf8 expression is markedly
decreased in the endoderm of Tbx1-/- mutants, in which PTA
is attributable to loss of Tbx1 in the endoderm
(Xu et al., 2004); thus, we
questioned whether loss of Tbx1 also disrupts Fgfr1
expression and found a dosage-sensitive decrease in the expression of
Fgfr1 in the endoderm and mesenchyme, specifically in the caudal
pharyngeal arches of Tbx1 heterozygotes and null mutants
(Fig. 6).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Autocrine Fgf8 signals in cardiac crescent mesoderm regulate heart tube and OFT formation, and the Isl1/Mef2c pathway in the AHF
Although no markers exist for the PHF, greater numbers of Fgf8GFP-positive
LV precursors in the ventral heart tubes of
Fgf8GFP/+;MesP1Cre/+ and
Fgf8GFP/+;HPRTCre/+ embryos than
Fgf8GFP/+;Isl1Cre/+ embryos strongly
suggest that Fgf8 is expressed in both the PHF and the AHF. MesP1Cre
reliably ablates early Fgf8 function throughout the cardiac crescent,
generating a high percentage of mutants with severe heart tube and RV/OFT
hypoplasia (Fig. 7B). Although
a small subset of Fgf8;Isl1Cre mutants display this severe phenotype,
AHF-restricted mesodermal mutants (Fgf8;Mef2c-AHFCre) do not. The
differences in timing and domain of these Cre drivers thus reveal a previously
unknown role for autocrine Fgf8 signaling in the crescent mesoderm to support
normal formation of the heart tube and AHF-derived tissues. Although we
attribute the death of severe mutants to cardiac insufficiency from a
decreased pump size and OFT obstruction, abnormal Fgf8 signaling may also
disrupt myocardial calcium handling and cardiac function
(Farrell et al., 2001).
Loss of Fgf8 in the AHF/SM resulted in decreased Erm
expression within the SM itself, providing additional evidence for autocrine
Fgf8 activity in the AHF. Furthermore, we detected altered
apoptosis/proliferation and decreased numbers of Isl1- and
Mef2c-expressing cells in the AHF of mutants, suggesting that Fgf8
maintains the Isl1/Mef2c transcriptional cascade required for normal survival
and accrual of AHF-derived cells to the heart. As Wnt11 expression is
undetectable in the OFT myocardium of severe MesP1Cre and
Isl1Cre mutants (and in Fgf8;Nkx2.5Cre conditional mutants)
(Ilagan et al., 2006), but is
preserved in Fgf8;Mef2cAHFCre mutants, we conclude that
Wnt11 expression in AHF-derived myocardium depends on autocrine Fgf8
function in the AHF prior to the 2-3 ss.
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|
), this driver is active in all anterior primitive
streak-derived mesoderm. Thus, we interpret the survival of some
Fgf8;MesP1Cre mutants to indicate that other factor(s) can support
heart tube and RV/OFT formation. Fgf10 is a candidate for functional
redundancy, as its expression was intact in the mesoderm of these mutants.
Experiments testing Fgf8/Fgf10 functional redundancy are underway.
Outflow tract alignment and septation are regulated by different Fgf8 tissue sources
The distinct OFT phenotypes of MesP1Cre, Isl1Cre and
Mef2c-AHFCre mutants also reveal domain-specific functions of Fgf8
during OFT remodeling. TGA and DORV, observed in Fgf8;MesP1Cre and
Fgf8;Mef2c-AHFCre mesoderm-only mutants,
(Fig. 7D), exist along a
spectrum of OFT alignment/rotation defects in which the Truncus is septated,
but the great vessels are not aligned correctly with the ventricles.
Conotruncal septal spiraling, conal involution and positioning, and OFT
rotation (Lamers and Moorman,
2002) may be affected directly by loss of Fgf8 signaling, or
indirectly by an insufficient quantity of OFT myocardium
(Yelbuz et al., 2002).
In contrast to TGA/DORV, PTA (observed in 100% of Fgf8;Isl1Cre mutants, Fig. 7E) reflects a complete failure of OFT septation. Because PTA was observed in Fgf8 hypomorphs and Fgf8;Tbx1Cre mutants (both of which have intact Isl1 loci), it is unlikely that the failed septation in Fgf8;Isl1Cre mutants is due to the additive effects of Fgf8 loss-of-function and Isl1 heterozygosity. Furthermore, Isl1Cre/+ and Fgf8C/+;Isl1Cre/+ OFTs are normal. However, formal assessment of the contribution of Isl1 haploinsufficiency to OFT defects observed in Fgf8;Isl1Cre mutants requires a Cre-expressor that ablates Fgf8 in the same temporospatial domains as Isl1Cre.
Notably, in most human and mouse examples of PTA, the Truncus is normally
aligned, straddling both ventricles
(Bartelings and Gittenberger-de Groot,
1989; Xu et al.,
2004; Yelbuz et al.,
2002). However in Fgf8;Isl1Cre mutants (and
Tbx1-/-) (Xu et al.,
2004; Zhang et al.,
2005) the Truncus is abnormally aligned, 100% committed to the RV,
indicating that the mechanism(s) that disrupt the alignment/rotation after
loss of mesodermal Fgf8 in Fgf8;MesP1Cre mutants also operate in
Fgf8;Isl1Cre endodermal/mesodermal mutants.
Cellular targets of Fgf8
Our conclusion that the endoderm mediates crucial Fgf8 signals to regulate
OFT septation is consistent with studies indicating that PTA in
Tbx1-/- mutants results from a loss of endodermal Tbx1,
accompanied by a loss of Fgf8 in this tissue
(Arnold et al., 2006;
Xu et al., 2004). Our finding
of decreased Fgfr1 expression in the endoderm of
Tbx1-/- mutants indicates that Tbx1 function influences
Fgf signaling at multiple levels, and provides new evidence that such
crosstalk regulates OFT septation.
The OFT septum is formed by contributions from the endothelium (by
epithelial to mesenchymal transformation) and the cardiac neural crest (CNC),
so these are potential paracrine targets of endodermal Fgf8
(Fig. 7D,E). However, ablation
of Fgfr1 and/or Fgfr2 in premigratory CNC does not cause OFT
septation defects, so CNC is not a direct target of endodermal Fgf8 (L.
Francis and A.M.M., unpublished) (Ilagan
et al., 2006). By contrast, the ablation of Fgfr1 and one
copy of Fgfr2 with Isl1Cre generates PTA at high penetrance (L.
Francis, S.E. and A.M.M., unpublished). Because the Isl1Cre expression domain
includes the mesodermal precursors of OFT endothelium
(Fig. 1O1 and data not shown)
and endoderm, further tissue-restricted receptor ablation analyses are
required to determine whether PTA in Fgf8 and Fgfr mutants results
from disrupting paracrine and/or autocrine endodermal Fgf8 signaling.
When considered in the context of earlier studies, these findings provide
new insight into the requisite timing of Fgf8 signals in different expression
domains. Ablation of Fgf8 with Nkx2.5Cre
(Ilagan et al., 2006), or in
precardiac mesoderm at E7.75-E8.0 (LHF-1ss), disrupts PHF and AHF function,
causing severe phenotypes in Fgf8;MesP1Cre and some
Fgf8;Isl1Cre mutants. Later ablation of Fgf8 with a
Tbx1Cre transgene (expressed in multiple domains from E8.5) generated
both septation and alignment-type OFT defects
(Brown et al., 2004). By
contrast, only a low incidence of alignment-type defects occurred with
Fgf8 ablation after E9.0 using Hoxa3IresCre
(Macatee et al., 2003). Thus,
Fgf8 signals at E7.75-E8.0 in precardiac mesoderm are required for normal PHF
and AHF function, whereas signaling from pharyngeal endoderm and splanchnic
mesoderm, required for OFT septation and alignment/rotation, respectively,
occurs prior to E9.0. This is an important finding, as invasion of the AP
septum and OFT cushion fusion begin at least one day later
(Lamers and Moorman,
2002).
Fgf8-related cardiovascular malformations and the embryonic left-right axis
Pitx2 mutants revealed that local, organ-specific LR axes can be
determined independently of the embryonic LR axis and at different
morphogenetic stages (Kioussi et al.,
2002; Kitamura et al.,
1999; Liu et al.,
2001; Liu et al.,
2002). It has been unclear whether looping defects and pulmonary
isomerism of severe Fgf8 hypomorphs
(Meyers and Martin, 1999)
reflect embryonic LR-axis disruption due to Fgf8 deficiency in the primitive
streak or node, or later dysfunction in the SM that perturbs the intrathoracic
LR axis. Preserved Pitx2 lateralization in early stage
Fgf8;MesP1Cre and Fgf8;Isl1Cre mutants (including those with
altered looping) supports the former hypothesis. It remains to be determined
whether disruption of a later, cardiac-specific LR pathway contributes to OFT
remodeling defects in these conditional mutants.
Conclusion
The survival/proliferation and molecular analyses presented here focused on
heart tube formation and the accumulation of RV/OFT myocardium in the absence
of mesodermal Fgf8. Further studies are underway to examine these processes
during OFT remodeling and to determine the bases of the distinct OFT defects
observed in mesodermal versus endodermal Fgf8 mutants. The primitive
cardiovascular system must support the entire organism while crucial aspects
of its development are still underway and vulnerable to a variety of genetic
or environmental insults. Genetic systems that reliably create specific
cardiovascular defects are an important step towards ultimately dissecting how
structural and hemodynamic inputs interact with, and modify, the genetic and
molecular programs that control cardiac morphogenesis in normal and
pathological conditions.
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ACKNOWLEDGMENTS |
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REFERENCES |
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