1 Program in Human Molecular Biology and Genetics, University of Utah School of
Medicine, Salt Lake City, UT 84112, USA
2 Childrens Health Research Center, University of Utah School of Medicine, Salt
Lake City, UT 84112, USA
3 Program in Molecular Biology, University of Utah School of Medicine, Salt Lake
City, UT 84112, USA
4 Program in Neuroscience, University of Utah School of Medicine, Salt Lake
City, UT 84112, USA
5 Department of Pediatrics, University of Utah School of Medicine, Salt Lake
City, UT 84112, USA
6 Department of Neurobiology and Anatomy, University of Utah School of Medicine,
Salt Lake City, UT 84112, USA
* Author for correspondence (e-mail: anne.moon{at}genetics.utah.edu)
Accepted 4 September 2003
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SUMMARY |
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To test our hypothesis, and to determine whether the pharyngeal ectoderm and endoderm Fgf8 expression domains have discrete functional roles, we performed conditional mutagenesis of Fgf8 using novel Crerecombinase drivers to achieve domain-specific ablation of Fgf8 gene function in the pharyngeal arch ectoderm and endoderm.
Remarkably, ablating FGF8 protein in the pharyngeal arch ectoderm causes failure of formation of the fourth pharyngeal arch artery that results in aortic arch and subclavian artery anomalies in 95% of mutants; these defects recapitulate the spectrum and frequency of vascular defects reported in Fgf8 hypomorphs. Surprisingly, no cardiac, outflow tract or glandular defects were found in ectodermal-domain mutants, indicating that ectodermally derived FGF8 has essential roles during pharyngeal arch vascular development distinct from those in cardiac, outflow tract and pharyngeal gland morphogenesis. By contrast, ablation of FGF8 in the third and fourth pharyngeal endoderm and ectoderm caused glandular defects and bicuspid aortic valve, which indicates that the FGF8 endodermal domain has discrete roles in pharyngeal and valvar development. These results support our hypotheses that local FGF8 signaling from the pharyngeal epithelia is required for pharyngeal vascular and glandular development, and that the pharyngeal ectodermal and endodermal domains of FGF8 have separate functions.
Key words: Cardiovascular development, Pharyngeal arch, FGF8, Endoderm, Heart field, Pharyngeal arch artery, Congenital heart disease, Vasculogenesis, Aortic arch, Outflow tract, Coronary artery, Thymus, Parathyroid, 22q11 deletion syndrome, DiGeorge Syndrome
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Introduction |
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Fgf8 encodes a crucial member of the FGF family
(MacArthur et al., 1995).
Secreted FGF8 protein provides survival, mitogenic, anti/pro-differentiation
and patterning signals to adjacent tissues, and may also have autocrine
activity. Complete ablation of Fgf8 function in mice results in early
embryonic lethality at approximately embryonic day (E) 8.5
(Meyers et al., 1998
;
Moon and Capecchi, 2000
;
Sun et al., 1999
). We and
others have thus employed hypomorphic and conditional alleles to study its
role in limb, face, brain, cardiovascular and pharyngeal development
(Abu-Issa et al., 2002
;
Frank et al., 2002
;
Garel et al., 2003
;
Meyers et al., 1998
;
Meyers and Martin, 1999
;
Moon et al., 2000
;
Moon and Capecchi, 2000
;
Storm et al., 2003
;
Sun et al., 2000
;
Trumpp et al., 1999
).
Fgf8 mutations in several species demonstrate its role(s) in early
cardiovascular and PA development. Zebrafish acerebellar Fgf8 mutants
have abnormal cardiogenesis, ventricular hypoplasia and circulatory failure
(Reifers et al., 2000).
Removal of Fgf8-expressing endoderm adjacent to precardiac mesoderm
in chicks alters expression of cardiac markers such as Nkx2.5
(Alsan and Schultheiss,
2002
).
In the mouse, Fgf8 is expressed in several temporospatial domains
that are potentially relevant to cardiovascular and pharyngeal development.
These include the precardiac mesoderm
(Crossley and Martin, 1995),
the early foregut endoderm and later, in restricted regions of PA endoderm and
ectoderm. Murine Fgf8 hypomorphic mutants (mice with globally
decreased FGF8 signaling throughout embryogenesis) have severe cardiovascular
and pharyngeal defects, including altered cardiac outflow tract (OFT)
alignment and septation, disrupted pharyngeal vascular development, and
abnormal formation of the thymus and parathyroids
(Abu-Issa et al., 2002
;
Frank et al., 2002
). These
Fgf8 hypomorphs phenocopy human syndromes associated with deletion of
chromosome 22q11 (del22q11) such as DiGeorge syndrome
(Epstein, 2001
;
Frank et al., 2002
;
Lindsay, 2001
;
Scambler, 2000
). Furthermore,
Fgf8 genetically interacts with Tbx1
(Vitelli et al., 2002
), a gene
located in the human del22q11 region known to play a crucial role in
generating human del22q11 phenotypes
(Jerome and Papaioannou, 2001
;
Lindsay et al., 2001
;
Merscher et al., 2001
). Thus,
delineating the function of specific FGF8 signaling domains and downstream
pathways will provide insight into how their dysfunction results in the
spectrum of birth defects seen in human del22q11syndromes.
Although the Fgf8 hypomorphic model provides enormous insight into the importance of FGF8 signaling during cardiovascular and pharyngeal development, it does not allow us to test the role of local (pharyngeally produced) FGF8 in development of the pharynx or cardiovascular system, or to dissect the respective role(s) of different Fgf8 expression domains that are relevant to these morphogenetic processes. Furthermore, analyses of the molecular and cellular pathways that are disrupted by loss of a specific Fgf8 expression domain cannot be assessed with this system.
We have postulated that the Fgf8 hypomorph cardiovascular and
pharyngeal phenotypes result from disrupted local FGF8 signaling from the
epithelia of PAs 3-6 to mesenchymal cells populating and migrating through
these arches, including cardiac neural crest en route to the OFT
(Frank et al., 2002). To test
this hypothesis, and to determine whether FGF8 signals emanating from the
pharyngeal ectoderm and endoderm perform discrete functions, we generated a
unique series of Fgf8 conditional alleles and Cre
recombinase-expressing drivers designed to ablate FGF8 in different pharyngeal
epithelial domains.
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Materials and methods |
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Conditional alleles of Fgf8
The conditional alleles employed in this study are shown schematically in
Fig. 1. Insertion of the GFP
reporter gene into the 3' untranslated region of Fgf8 was
performed as previously described for the Fgf8APN
hypomorphic, conditional reporter allele
(Frank et al., 2002;
Moon and Capecchi, 2000
). The
Fgf8null allele, resulting from removal of exon 5, has
also been reported (Moon and Capecchi,
2000
). Mutant embryos are in a 75% C57Bl6, 25% SV129
background.
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TUNEL analysis and immunohistochemistry
Somite/stage-matched embryos were fixed and whole mount terminal UTP nick
end labeled (TUNEL) assays performed as described previously
(Frank et al., 2002;
Stadler et al., 2001
).
Whole-mount green fluorescent protein (GFP) detection was performed on whole embryos using a rabbit anti-GFP and FITC-conjugated anti-rabbit IgG secondary antibodies (1:1000 and 1:500, respectively, both from Molecular Probes).
For cryosectioned specimens, embryos were protected in sucrose and
gelatin-embedded. Cryosections (12 µm) were cut transversely, parallel to
the third PAA. The Ap2 transcription factor, which is expressed in
neural crest and ectoderm, was detected with a mouse monoclonal
anti-AP2
-antibody (1:25, 3B5, Developmental Studies Hybridoma Bank) and
a FITC-conjugated anti-mouse IgG secondary (1:500, Molecular Probes).
Simultaneous TUNEL was performed by adding the TMR Red in situ cell death
detection reagents (Roche) to secondary antibody incubation. Sections were
preserved in Fluoromount-G (Southern Biotechnology Associates) and analyzed by
confocal microscopy. FITC and Texas red fluorescence were recorded using a
BioRad MRC 1024 laser-scanning confocal imaging system fitted to a Leitz
Aristoplan microscope. A digital Kalman averaging filter was used to reduce
background fluorescence.
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Results |
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We previously described a conditional reporter allele,
Fgf8APN, that is hypomorphic due to the presence of a
neor gene in the 3' untranslated region of the Fgf8
locus (Frank et al., 2002;
Moon and Capecchi, 2000
)
(Fig. 1A). Mice bearing this
allele and a null allele of Fgf8 are hypomorphs (genotype
Fgf8APN/null) and die at birth with the aforementioned
complex phenotype (Frank et al.,
2002
).
For the current study, we modified the Fgf8APN allele
by removing the frt-flanked neor gene with flp-mediated
recombination in the germline of founder animals
(Dymecki, 1996)
(Fig. 1B). The resulting
Fgf8AP allele is not hypomorphic.
Fgf8AP/null compound heterozygotes survive and are
phenotypically normal (see Fig.
4A). The Fgf8AP allele was also designed as a
conditional reporter allele: exon 5 is flanked with loxP sites and coding
sequences for human alkaline phosphatase (AP) are positioned in the 3'
untranslated region of Fgf8. Cre-mediated recombination of this
allele removes exon 5 and allows expression of the AP reporter gene under
control of the Fgf8 promoter.
|
Expression of either reporter gene depends on: (1) Cre-mediated
recombination of Fgf8AP or Fgf8GFP to
generate the Fgf8APR and Fgf8GFPR null
reporter alleles (Fig. 1C) and
(2) activity of the Fgf8 locus. These reporters permit precise
determination of the temporospatial inactivation of Fgf8 in cells in
which it is expressed (Moon et al.,
2000; Moon and Capecchi,
2000
).
Domain-specific Cre-recombinase drivers for conditional ablation of
FGF8 in the pharyngeal epithelia
Deletion of exon 5 from the of Fgf8AP or
Fgf8GFP conditional alleles depends on expression and
activity of Cre-recombinase. We obtained spatial and temporal control over Cre
by inserting an IRES followed by Cre-encoding sequences into the 3'
untranslated region of two genes that are expressed in pharyngeal domains of
interest, the Ap2 and hoxa3 loci (see Materials and
methods; Fig. 2A,
Fig. 3A). As described in
detail below, to determine whether expression of these targeted Cre drivers
recapitulated the pattern of the endogenous loci, we examined the expression
of the global Cre reporter gene, Rosa26lacZ
(Soriano, 1999
). Cre-mediated
recombination of the Rosa26lacZ allele results in
ß-galactosidase production from the recombined, constituitively expressed
Rosa26 locus. Furthermore, we determined the domains of functionally
relevant recombination of Fgf8 obtained with these Cre drivers by
characterizing expression of recombined Fgf8 conditional reporter
alleles in the developing PAs.
AP2-IRESCre ablates FGF8 signaling from the PA
ectoderm
We ablated Fgf8 gene function in its PA ectodermal expression
domains from the time of PA formation with the targeted
AP2-IRESCre driver
(Fig. 2A,B). The
AP2
gene is expressed in many regions of the mouse embryo
(Mitchell et al., 1991
),
including pharyngeal NC and ectoderm
(Brewer et al., 2002
). Lineage
analyses of the AP2
-IRESCre driver in the PAs were
performed by crossing this allele into mice bearing the
Rosa26lacZ allele (genotype
AP2
IRESCre/+; Rosa26lacZ/+,
Fig. 2C). ß-Galactosidase
activity is detectable in developing PAs1 and 2, indicating that onset of Cre
activity occurred at approximately the 10 somite stage (ss, indicated in lower
right corner of each panel). Caudal ectoderm over the region that will form
PAs3-6 is also stained prior to definitive arch formation
(Fig. 2C, large red
arrowheads).
Cells that express both AP2-IRESCre and
Fgf8 were identified by staining for alkaline phosphatase (AP)
activity in an Fgf8AP/+;
AP2
IRES-Cre/+, 21 ss embryo. Analysis of
whole mount and coronal sections demonstrates that AP is expressed
specifically in the ectoderm of PAs 1-3
(Fig. 2D). This expression
pattern reflects cells of the AP2
lineage that express
Fgf8APR (generated by AP2
-IRESCre
activity), and defines the functionally relevant domains of Fgf8
inactivation.
We confirmed that AP2-IRESCre ablates Fgf8
throughout its ectodermal expression domains by comparing expression of GFP in
Fgf8GFP/+;AP2
IRES-Cre/+ embryos
versus Fgf8GFP/+;deleterCre embryos
(Fig. 2E, left and right
panels, respectively). The deleterCre transgene is active in germ
cells so Fgf8GFP is recombined in all cells of the embryo
from the earliest developmental stages
(Schwenk et al., 1995
).
Therefore, GFP expression in Fgf8GFP/+;deleterCre embryos
depends only on the activity of the Fgf8 locus. Importantly,
Fgf8GFPR expression in the PA ectoderm is the same whether
it results from the action of AP2
-IRESCre or
deleterCre (Fig. 2E).
Although Fgf8 is also expressed in the PA endoderm at this stage
(discussed below), the endodermal domain of GFP expression in the
Fgf8GFP/+;deleterCre embryo
(Fig. 2E, right panel) is
obscured by overlying ectodermal signal. Coronal sections of a 20 ss
Fgf8GFP/+; AP2
IRES-Cre/+ embryo
(Fig. 2F,G) reveal that
AP2
-IRESCre is not active in the endoderm, and that
the ectoderm of PAs 1, 2 and developing PA3 express GFP.
All of these data provide confirm that in
Fgf8;AP2-IRESCre conditional mutants
(Fgf8GFP/null; AP2
IRES-Cre/+),
FGF8 is ablated specifically in the PA ectoderm.
hoxa3-IRESCre ablates FGF8 signaling from the PA endoderm
and ectoderm
To simultaneously ablate Fgf8 gene function in both the endoderm
and ectoderm of PAs 3-6, we used the hoxa3-IRESCre driver
(Fig. 3A,B). Lineage analysis
of hoxa3-IRESCre in hoxa3IRESCre/+;
Rosa26lacZ/+ embryos shows that hoxa3-IRESCre
recapitulates the caudal to rostral progression of the endogenous
hoxa3 locus (Fig. 3C,
10-28 ss whole-mount panels). Previous reports described hoxa3
expression in endoderm and mesenchyme of PAs3 and 4
(Manley and Capecchi, 1995),
but we also found lacZ staining in the ectoderm of PAs 3-6 (see
Fig. 3C, sectioned 20 ss and 23
ss embryos). Ectodermal lacZ staining is lighter than in mesenchyme
(which may explain inability to detect this domain of hoxa3 mRNA by
in situ hybridization). Ectodermal expression was consistent in all embryos
and was also confirmed using the Fgf8GFP reporter
allele.
To evaluate functionally relevant activity of hoxa3-IRESCre in Fgf8-expressing cells, we analyzed Fgf8GFPR expression in Fgf8GFP/+; hoxa3IRESCre/+ coronally sectioned embryos (Fig. 3D) in comparison with Fgf8GFP/+;deleterCre embryos (Fig. 3E). The relative planes of ventral and dorsal coronal sections are demonstrated by the black lines labeled v and d in the 20 ss whole mount in Fig. 3C. Fgf8GFPR is clearly expressed in the endoderm and ectoderm of developing PAs 3-6, including pharyngeal pouches and clefts. At the 20 ss, ventral and dorsal sections (Fig. 3D, 20v and 20d, respectively) show Fgf8GFPR expression throughout the PA3 endoderm, including the developing third pouch (3p), and ectoderm. By the 24 ss, Fgf8GFPR expression is decreasing in the rostral endoderm of PA3, but persists in caudal endoderm and pouch (Fig. 3D, 24v and 24d, yellow arrowheads). At the 27 ss, GFP is detected throughout endoderm and ectoderm of PA4 as it forms. Remarkably, the expression pattern of GFP was the same in Fgf8GFP/+;deleterCre and Fgf8GFP/+; hoxa3IRESCre/+ embryos. Note that Fgf8 is not expressed in PA mesenchyme (Fig. 2D,F,G; Fig. 3D,E).
In concert, the data in Fig.
3 clearly show that in Fgf8GFP/null;
Hoxa3IRES-Cre/+ mutants (Fgf8;hoxa3-IRESCre mutants),
FGF8 is ablated throughout its expression domains in endoderm and ectoderm of
developing PAs 3-6. hoxa3-IRESCre activity is reproducibly present in
the endoderm from the 18-19 ss, and from the 16 ss in the ectoderm (data not
shown). Note that thymic and parathyroid epithelia arise from the third
endodermal pouch, and that `cardiac' neural crest migrates from rhombomeres
6-8 through PAs 3-6 into the OFT (Kirby et
al., 1997; Kirby and Waldo,
1990
; Kirby and Waldo,
1995
). These are the relevant domains of hoxa3-IRESCre
activity.
Phenotypes of Fgf8 domain-specific conditional mutants
Ablation of FGF8 from the PA ectoderm causes vascular defects
The consequence of specifically ablating FGF8 in the PA ectoderm was
determined by comparing Fgf8;AP2-IRESCre
ectodermal domain mutants (genotype Fgf8GFP/null or
AP/null; AP2
IRES-Cre/+) with
controls (genotypes Fgf8+/+, Fgf8AP/+,
or Fgf8+/null) and Fgf8 hypomorphs (genotype
Fgf8APN/null) at multiple developmental stages using
anatomic and molecular assays (Figs
4,
5,
6,
7).
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By contrast, only 75% of Fgf8;AP2-IRESCre
mutants survive to birth and the rest die postnatally due to lethal vascular
defects in 30% and severe craniofacial malformation in 100%. The craniofacial
defect results from complete ablation of FGF8 in PA1
(Fig. 4C). The pharyngeal
phenotype is detected as early as E9.5 as severe PA1 hypoplasia and hypoplasia
and fusion of more caudal PAs (data not shown).
The fourth PAAs form between E9.5-10.0 (25-29 ss); the right fourth PAA
forms the proximal right subclavian artery, while the left fourth PAA becomes
the aortic arch between the left common carotid and left subclavian arteries
(the segment of aorta missing in IAAB). Ninety-five percent of
Fgf8;AP2-IRESCre mutants have vascular
defects at birth resulting from abnormal formation of the fourth PAAs
(Fig. 4G,O-S;
Fig. 5; Tables
1 and
2). Thirty percent of
Fgf8;AP2
-IRESCre E18.5 conditional mutants
have the postnatally lethal vascular malformation, interrupted aortic arch
type B (IAAB, Fig. 4G,P,Q). In
addition to IAAB and subclavian artery anomalies, we observed circumflex right
aortic arch (RAA, Fig. 4O) and
RAA with right ductus arteriosus (Fig.
4E,R,S) in these mutants; defects also attributable to failed left
PAA4 formation.
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Overall, the incidence of defects attributable to abnormal fourth PAA
formation, such as IAAB, RAA or aberrant subclavian artery, is the same in
Fgf8 hypomorphs and
Fgf8;AP2-IRESCre mutants. However, the
severity of the defects at E10.5 resulting from complete ablation of FGF8 in
the PA ectoderm is much greater in
Fgf8;AP2
-IRESCre mutants when compared with
the globally deficient hypomorphs. This indicates that the pharyngeal FGF8
ectodermal domain has required function(s) during PAA4 vasculogenesis.
We have previously shown that PAA4 vasculogenesis is specifically disrupted
in Fgf8 hypomorphs: endothelial cells (ECs) are specified and
differentiated in the fourth PA as they express the VEGF receptor, Flk1 and
the cell adhesion molecule PECAM (Cleaver
and Krieg, 1999). However, the ECs fail to organize into primitive
vascular tubes (Frank et al.,
2002
). To evaluate vasculogenesis in
Fgf8;AP2
-IRESCre mutants, we examined
whether migration and specification of ECs in the fourth PA proceeds normally.
Flk1/PECAM-expressing cells were detected in hypoplastic fourth PAs of
Fgf8;AP2
-IRESCre mutants at the 25-27 ss
(during PAA4 formation) in clusters that are indistinguishable from controls,
Fgf8 hypomorphs, or hoxa3-IRESCre mutants
(discussed below, and data not shown). However, in all classes of
Fgf8 mutants at the 35-37 ss, ECs in PAA4 remain disorganized and
fail to form primitive vascular tubes, long after this vessel is patent and
pericyte recruitment is under way in controls (data not shown)
(Frank et al., 2002
). Thus,
migration and early differentiation of PAA4 ECs occurs normally in the absence
of FGF8, but subsequent vascular organization fails.
Remarkably, and unlike Fgf8 hypomorphs,
Fgf8;AP2-IRESCre mutants have normal OFT
development: that is, alignment, septation and rotation of the aortic and
pulmonary arteries are normal. In the mutant shown in
Fig. 4G, the aorta and ductus
arteriosus/pulmonary artery are normally aligned and septated, but the mutant
has the IAAB vascular defect.
Fgf8;AP2
-IRESCre mutants also have coronary
artery anomalies (Fig. 4M,N),
in isolation or associated with other vascular defects
(Table 1).
In further contrast to Fgf8 hypomorphs,
Fgf8;AP2-IRESCre mutants have normal thymic
and parathyroid glands (Fig.
4H, Table 1,
Table 3).
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Ablation of FGF8 from the PA endoderm causes glandular and aortic
valve defects
We used the hoxa3-IRESCre driver to ablate FGF8 from both the
endoderm and ectoderm of PAs 3-6 as they develop. In marked contrast to both
Fgf8 hypomorphs and
Fgf8;AP2-IRESCre mutants, all
Fgf8;hoxa3-IRESCre mutants survive to birth and most survive
beyond the neonatal period. These mutants have normal craniofacial morphology
because Fgf8 is intact anterior to PA3
(Fig. 4D). Thirty percent of
Fgf8;hoxa3-IRESCre mutants die as neonates because of the
same lethal cardiovascular malformations described in
Fgf8;AP2
-IRESCre
(Table 1 and below).
Notably, Fgf8;hoxa3-IRESCre mutants display thymic ectopy
and hypoplasia and parathyroid ectopy, hypoplasia and aplasia
(Table 3, Fig. 6A-G). Parathyroid and
thymic epithelia are derived from the anterior and posterior third pouch
endoderm, respectively. The incidence of abnormal thymic and parathyroid
development in Fgf8;hoxa3-IRESCre mutants
(Table 3) was comparable with
that of Fgf8 hypomorphs (Frank et
al., 2002). However, the severity of these defects in
hoxa3-IRESCre mutants was less than in globally deficient hypomorphs
because no hoxa3-IRESCre mutants had bilateral thymic or parathyroid
aplasia, which occurred frequently in Fgf8 hypomorphs. Fourth
pouch-derived thyroid C-cells were detected normally in
Fgf8;hoxa3-IRESCre mutants, Fgf8 hypomorphs and
controls, as assessed by anti-calcitonin immunohistochemistry (data not
shown). Notably, glandular defects are not seen in
Fgf8;AP2
-IRESCre mutants
(Fig. 4G, versus
Fig. 6A-G,
Table 3).
As FGF8 is also ablated in the ectoderm of PAS 3-6 in
Fgf8;hoxa3-IRESCre mutants, it was not surprising
to find the same PAA and coronary vascular defects in these mutants seen in
Fgf8;AP2-IRESCre mutants (see above and
Table 1). However, it is quite
remarkable that ablation of FGF8 in both the endoderm and ectoderm in
Fgf8;hoxa3-IRESCre mutants does not increase either
incidence or severity of PAA or coronary vascular defects at any developmental
stage (Tables 1,
2,
4). The same defect in vascular
tube formation in the fourth PA described in Fgf8 hypomorphs and
Fgf8;AP2
-IRESCre mutants was also detected
in Fgf8;hoxa3-IRESCre mutants. These findings
confirm that the ectodermal domain of FGF8 is specifically required for normal
PAA and coronary vascular development.
|
The glandular and valvar phenotypes of Fgf8;hoxa3-IRESCre mutants indicate that the FGF8 endodermal domain ablated by hoxa3-IRESCre has distinct functional roles in pharyngeal and aortic valve development.
Differential survival of neural crest is not the mechanism for the
distinct phenotypes of Fgf8 hypomorphic and domain-specific,
AP2a-IRESCre ectodermal and Fgf8;hoxa3-IRESCre
mutants
We previously reported that neural crest (NC) cells in developing PAs 3-6
undergo abnormal apoptosis at the 25-29 ss in Fgf8 hypomorphs
(Frank et al., 2002). As the
cardiovascular features of hypomorphs recapitulate those of NC-ablated chicks
(Kirby et al., 1985
), we
hypothesized that the high incidence of severe OFT defects in hypomorphs
(85%), resulted from abnormal NC survival in PAs 3-6 prior to entering the OFT
(Frank et al., 2002
). As we
have now demonstrated that differential ablation of FGF8 in PA ectoderm and
endoderm separates vascular from glandular and OFT defects, we questioned
whether domain-specific mutants would display unique patterns of NC apoptosis.
Therefore, we compared apoptosis in all three classes of Fgf8 mutants
by whole-mount TUNEL. We detected the same abnormal areas of apoptosis in PA3
and developing PAs 4 and 6 in Fgf8 hypomorphs and domain-specific
mutants (Fig. 7A-H, compare
white circled regions and white arrowheads in controls with yellow arrowheads
in mutants). Note that similar domains of normal (transient, stage specific)
apoptosis are present in the otocysts of all embryos, indicating appropriate
stage matching.
These observations were confirmed by assaying for apoptosis and expression
of the transcription factor Ap2 (labels NC) using double fluorescent
immunohistochemistry on serial cryosections of 25 ss embryos
(Fig. 7I-L). Minimal NC
apoptosis was detected in controls (Fig.
7, row I), whereas all three mutant classes had large abnormal
domains of apoptosis in NC migrating from rhombomeres 6-8 into the lateral
regions of PA3, and developing PAs 4 and 6 (yellow arrowheads). The hypomorph
shown had hypoplastic third PAAs (Fig.
7, row J) and large domains of abnormal apoptosis. Note the
abnormally large third PAA in
Fgf8;AP2
-IRESCre and
Fgf8;hoxa3-IRESCre mutants
(Fig. 7, rows K and L,
respectively; white asterisks mark third PAAs); consistent with abnormal
persistence and enlargement of the third PAA in mutants when the fourth PAAs
do not form (see Fig. 5C).
![]() |
Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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To determine if local FGF8 signals in the arches contribute to normal pharyngeal and cardiovascular development, and to evaluate which of the many potentially relevant Fgf8 expression domains might be responsible for different aspects of Fgf8 hypomorphic pharyngeal and cardiovascular phenotypes, we generated a system of nonhypomorphic Fgf8 conditional reporter alleles and domain-specific Crerecombinase drivers. Appropriate combinations of these alleles in murine embryos allowed us to differentially ablate FGF8 in distinct temporospatial expression domains in the developing PAs.
AP2-IRESCre unequivocally ablates FGF8 throughout
its expression domains in PA ectoderm from the time of PA formation, at least
by the 10-somite stage. By contrast, hoxa3-IRESCre is active in all
three germ layers of developing PAs 3-6 from the 16 ss in the ectoderm and the
18-19 ss in the endoderm.
These detailed expression analyses allowed us to determine the precise location and timing of overlap between Fgf8 expression and Cre expressed by the different temporospatially restricted drivers. This is crucial to discerning how ablation of FGF8 in a given domain relates to the distinct phenotypes obtained by domain-specific conditional mutagenesis and in comparison with the complex Fgf8 hypomorphic phenotype.
Domain-specific phenotypes reveal distinct, essential functions of
local FGF8 signals in cardiovascular and pharyngeal development
Fgf8 domain-specific mutants display vascular, coronary artery,
aortic valve and pharyngeal gland phenotypes due to loss of specific, local
FGF8 signals from separate expression domains in the PA ectoderm and
endoderm.
Our Fgf8 allelic series and conditional mutagenesis system reveals
a remarkable dosage sensitivity of regional vascular development to FGF8
levels in the caudal PA ectoderm. This domain of expression is required for
normal formation of the fourth (and frequently sixth) PAA (Tables
1,
2, Figs
4,
5). Although the overall
incidence of PAA4-related vascular defects (IAAB, RAA, subclavian artery
anomalies) was the same in Fgf8;AP2-IRESCre mutants
and FGF8-deficient hypomorphs, the severity of PAA defects at E10.5 was much
greater in Fgf8;AP2
-IRESCre mutants because of
complete absence of FGF8 in the PA ectoderm from the earliest stages of PA
formation. hoxa3-IRESCre mutants had (statistically) the
same incidence and severity of PAA and coronary vascular defects as
Fgf8;AP2
-IRESCre mutants, in spite of the fact that
FGF8 was ablated in both the PA endoderm and ectoderm (Tables
1,
2,
4). Ablation of FGF8 with a
Tbx1Cre transgene that is active in early endoderm and precardiac
mesoderm (but not in arch ectoderm) results in OFT and glandular defects, but
no fourth PAA-related vascular defects (J. Epstein, personal communication).
These observations indicate that FGF8 signaling from PA ectoderm is critical
for normal PAA4 development and that the vascular defects seen in all classes
of Fgf8 mutants are attributable to FGF8 deficiency in the PA
ectoderm.
By contrast, FGF8 ablation from the endoderm of PAs 3-6 in Fgf8;
hoxa3-IRESCre mutants results in thymic, parathyroid and BAV
defects. Thus the FGF8 endodermal domain makes important and distinct
contributions to development of these structures. Thyroid, parathyroid and
thymic epithelial cells are derived from pharyngeal pouch endoderm; these
glands initially have a NC component, and interactions between NC and endoderm
play an important role in early thymic and parathyroid development
(Auerbach, 1960;
Bockman and Kirby, 1984
;
Graham, 2001
;
Graham and Smith, 2001
;
LeDouarin and Jotereau, 1975
;
LeLievre and LeDouarin, 1975
).
Because FGF8 is ablated from both epithelial layers of the third pouch/cleft
of Fgf8;hoxa3-IRESCre mutants, it is possible that
glandular hypoplasia and ectopy in these mutants represents a combined effect
of absence of FGF8 in both domains. This is unlikely given that the glandular
hypoplasia and ectopy resulting from complete ablation of FGF8 in
hoxa3-IRESCre mutants is less severe when compared with the
frequent glandular aplasia seen in Fgf8 hypomorphs. It is likely that
an earlier endodermal FGF8 domain has a crucial influence on the earliest
precursors of these glands. Indeed, the aforementioned
Fgf8;Tbx1Cre mutants have thymic aplasia (J. Epstein,
personal communication).
Fgf8 mutants reveal a role for neural crest in PA and
coronary vascular development
The same regions of abnormal NC apoptosis are observed in the PAs of all
classes of Fgf8 mutants. Importantly, the only abnormal phenotypes
common to these different mutants are PAA4-derived defects and coronary
vascular anomalies. We hypothesize that signaling between NC-derived
ectomesenchyme and endothelial cells (ECs) is required for normal PA vascular
development prior to differentiation of NC into the vascular smooth muscle
cells (VSMC) and pericytes supporting the PAAs. This signaling may be
perturbed by abnormal death of NC migrating from rhombomeres 6-8 into and
through PAs 3-6 in Fgf8 mutants. Disruption of EC/ectomesenchymal
interactions may contribute to both the PAA and coronary vascular defects seen
in all classes of Fgf8 mutants in our series.
Approximately 50% of Fgf8;AP2-IRESCre and
Fgf8;hoxa3-IRESCre mutants have coronary artery (CA)
defects. Most commonly, we see a single CA arising from the right cusp of a
normal aortic valve. Coronary vessels are derived from cells in the
proepicardial organ (PEO, a hepatic-derived structure) that invade the tubular
heart and become ECs, VSMCs and pericytes of the coronary vasculature and main
CAs (Mikawa and Gourdie,
1996
). Although NC does not contribute structurally to the CAs
(Jiang et al., 2000
;
Li et al., 2002
), CA defects
have been described in NC-ablated chicks
(Hood and Rosenquist, 1992
;
Waldo et al., 1994
) and in
murine mutants of Connexin43 (Li et al.,
2002
). Cx43 is expressed in both NC and PEO cells; however, the
phenotype of Cx43-null mice suggests that the CA defects are due to abnormal
migration and/or survival of PEO-derived VSMCs in combination with perturbed
interactions between NC and PEO-derived cells in the region of the developing
CAs and aortic valve (Li et al.,
2002
). FGF8 is produced in PA ectoderm adjacent to the PEO;
deficiency or ablation of FGF8 in this domain may have similar effects.
Further investigation of the entire cardiac vasculature and expression of Cx43
and other intercellular adhesion molecules that participate in CA development,
such as
4-integrin or VCAM1 (Kwee
et al., 1995
; Yang et al.,
1996
) in Fgf8 mutants is warranted, in addition to
examination of survival, proliferation and differentiation of PEO cells. We
have not yet determined if Fgf8 or our Cre-drivers are expressed in
the proepicardial organ; if so, FGF8 autocrine or paracrine actions could play
a role in coronary vascular development.
Bicuspid aortic valve (BAV) is a mild form of OFT defect and is the most
common human congenital cardiac malformation. Semilunar (aortic and pulmonary)
valve formation requires interactions between endocardial and NC-derived
mesenchymal cells and myocardium (Ya et
al., 1998). NC progeny are present during formation of the aortic
valve leaflets at E13.5 and postnatally in the leaflets and tissues adjacent
to the CA orifices (Jiang et al.,
2000
). NC ablation or dysfunction results in severe OFT defects
and mild semilunar valve defects, such as BAV. However, existing mouse models
of abnormal semilunar valve development result from disruption of
endocardially expressed genes (de la Pompa
et al., 1998
; Lee et al.,
2000
; Ranger et al.,
1998
; Ya et al.,
1998
). BAV in Fgf8;hoxa3-IRESCre mutants is attributable
to FGF8 ablation in the endoderm since
Fgf8;AP2
-IRESCre mutants have normal aortic valves.
However, the cellular and molecular etiologies of this defect require further
investigation in the context of altered FGF8 signaling.
The OFT septum is derived largely from NC, and ablation or dysfunction of
NC results in severe OFT defects (Bockman
et al., 1989; Conway et al.,
1997a
; Conway et al.,
1997b
; Conway et al.,
1997c
; Epstein et al.,
2000
; Franz, 1989
;
Goulding et al., 1993
;
Jiang et al., 2000
;
Kirby and Waldo, 1990
).
Whole-mount TUNEL and analyses of NC apoptosis in the different classes of
Fgf8 mutants presented herein indicate that abnormal NC apoptosis at
the 25-27 ss does not cause the severe OFT defects observed in Fgf8
hypomorphs. Most NC destined for the OFT may have already traversed PAs 3-6 by
this somite stage, although OFT septation has not begun. In fact, separation
of severe OFT defects (which probably involve some manner of NC dysfunction)
from glandular and vascular defects in different classes of Fgf8
mutants, implies that FGF8 influences on NC are location and time dependent.
This raises the exciting possibility that subsets of NC, with distinct
structural fates, migrate through the PAs at different times and are supported
by distinct domains of FGF8 expression.
Outflow tract formation is not dependent on Fgf8 expression
in the PA epithelia
Our findings indicate that the frequent, severe defects in cardiac OFT
alignment, septation and growth seen in Fgf8 hypomorphs result from
altered FGF8 signaling at an earlier stage in the endoderm, or from
abnormalities resulting from decreased FGF8 in the primary and/or putative
secondary heart field. Because only one out of 33 Fgf8;hoxa3-IRESCre
mutants had a severe OFT defect and BAV, we hypothesize that onset of
hoxa3-IRESCre activity in the endoderm (at approximately the 18 ss)
may be at a relatively late stage of endoderm function during OFT development,
and that BAV is a manifestation of late endodermal FGF8 deficiency (similar to
our hypothesis regarding the glandular defects in these mutants). Expected
`wobble' in this biological system could result in occasional earlier Cre
activity in the endoderm and the rare occurrence of more severe OFT
defects.
Ablation of FGF8 in early endoderm and cardiac mesoderm (but not PA
ectoderm) with a Tbx1Cre transgene results in severe OFT defects, but
normal development of fourth PAA-derived vessels (J. Epstein, personal
communication). Although this experiment confirms the crucial roles of
distinct Fgf8 expression domains in different aspects of pharyngeal
and cardiovascular morphogenesis, the question remains whether the crucial
source of FGF8 for OFT development is derived from mesoderm or endoderm. Other
groups have reported that Tbx1 is not expressed in precardiac
mesoderm (Yamagishi et al.,
2003), which would suggest that early endoderm is the requisite
source. We are developing early endoderm-specific and precardiac mesoderm Cre
drivers to definitively address this question.
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ACKNOWLEDGMENTS |
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