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First published online 26 March 2008
doi: 10.1242/dev.012179
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1 Department of Cell and Developmental Biology, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104-6058, USA.
2 Department of Pharmacology, Vanderbilt University School of Medicine,
Nashville, TN 37232-0494, USA.
3 Department of Cell and Developmental Biology, Vanderbilt University School of
Medicine, Nashville, TN 37232-0494, USA.
4 Center for Stem Cell Biology, Vanderbilt University School of Medicine,
Nashville, TN 37232-0494, USA.
Author for correspondence (e-mail:
trish.labosky{at}vanderbilt.edu)
Accepted 28 February 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neural crest, Foxd3, Mouse embryo, Stem cell maintenance
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Many of the cellular mechanisms crucial to the development
of an embryo are represented within this extraordinary tissue: specification
of cell type, maintenance of multipotency of progenitor cells, directed
migration of cells to their final destination and terminal differentiation of
cells. Members of the PDGF, BMP and Wnt families of secreted molecules are
important for the initial specification and subsequent maintenance of the NC
(reviewed by Steventon et al.,
2005). Many transcription factors are activated in NC in response
to these signaling cascades; these include Foxd3, Sox8, 9 and 10
(Cheung and Briscoe, 2003;
Southard-Smith et al., 1998),
Ap2 (Tcfap2a - Mouse Genome Informatics)
(Schorle et al., 1996) and
Pax3 (Epstein et al., 1991).
It is not yet clear how these factors work together to induce and then
maintain this unique cell type.
Foxd3 encodes a transcriptional repressor of the winged helix or
Forkhead family of proteins (Labosky and
Kaestner, 1998; Sutton et al.,
1996). This large family of proteins is characterized by a 100
amino acid DNA-binding domain and transcriptional activation or repression
domains (reviewed by Wijchers et al.,
2006). Foxd3 was first characterized by its expression in
embryonic stem (ES) cells and multipotent cells of the NC
(Labosky and Kaestner, 1998).
Our previous work demonstrated that Foxd3 is required early in mouse
embryogenesis; Foxd3-/- embryos fail around the time of
implantation, cells of the inner cell mass cannot be maintained in vitro, and
ES cell and trophoblast stem cell lines cannot be established
(Hanna et al., 2002;
Tompers et al., 2005). In
vivo, the pool of both embryonic and trophoblast progenitors is not maintained
without Foxd3; embryonic progenitor cells die, while trophoblast progenitors
give rise to an excess of trophoblast giant cells at the expense of the
remaining trophoblast lineage. However, the ultimate effect in both cases is
that the progenitor pool is not maintained and the cell lineage is lost.
Foxd3 is one of the earliest molecular markers of the NC lineage, it is
expressed in many organisms in premigratory and migrating NC and expression is
downregulated as the cells differentiate into most derivatives
(Dottori et al., 2001;
Labosky and Kaestner, 1998).
Ectopic expression of Foxd3 in chick neural tube and Xenopus embryos
can specify NC cell fate as measured by upregulation of HNK1 and Slug
(Dottori et al., 2001;
Kos et al., 2001;
Sasai et al., 2001). In
addition, maintained expression of Foxd3 in migrating NC interferes with
differentiation and retains NC in an undifferentiated state, evidence that
points to a potential role for Foxd3 in the control of stem cell self-renewal
(Dottori et al., 2001).
Morpholino knockdown of zebrafish foxd3 and mutations that affect
Foxd3 expression in the zebrafish NC have deleterious effects on maintenance
of the NC (Lister et al.,
2006; Montero-Balaguer et al.,
2006; Stewart et al.,
2006).
Because the deletion of Foxd3 results in embryonic lethality around the time of implantation, we employed Cre-loxP technology to delete the Foxd3 coding region specifically in NC. Here we demonstrate that without this transcriptional regulator the NC lineage fails to be maintained, resulting in catastrophic loss of most NC derivatives. Mutant mice perish at birth with a cleft face, the PNS is severely reduced and the ENS is not formed. Surprisingly, although the cardiac NC is greatly reduced early in embryogenesis, by midgestation most cardiac NC derivatives are normal, with subtle defects in a small percentage of mutant embryos. We show that the mechanism of cellular loss of most of the NC is by a failure to maintain the progenitor pool, as much of the premigratory and early migrating NC undergoes cell death. Cells that survive are able to migrate and differentiate normally but cannot completely compensate for the early loss of progenitors. Our data demonstrate that Foxd3 is required for maintenance of NC progenitor cells and, taken together with our previous work, place Foxd3 in the unique role of maintaining the undifferentiated multipotent state of three completely divergent progenitor populations of the early mammalian embryo.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ES cell electroporation and generation of mice
TL1 ES cells were electroporated as described
(Tompers and Labosky, 2004).
ES cell DNA was analyzed by Southern blot as shown in
Fig. 1A,B. Lines were screened
by PCR for the presence of the 5' loxP site. We identified three
correctly recombined cell lines out of 337 (0.9% frequency). Chimeras were
generated by blastocyst injection and offspring of chimeras were genotyped to
identify heterozygous founders. Two independent cell lines transmitted through
the germ line and because both showed the identical phenotype we restricted
our studies to one cell line. Mice were genotyped either by Southern blot or
PCR with the following primers: 5'-CGGCTTTCTTTCGGGGGAC-3' and
5'-ACATATCGCTGGCGCTGCCG-3' to give a 130 bp band for the wild-type
allele and a 220 bp band for the floxed allele.
Mouse strains
Foxd3tm2.Lby is a null mutation of Foxd3
generated previously (Hanna et al.,
2002) and is referred to as Foxd3- throughout.
Wnt1-Cre mice were a generous gift of Drs Andrew McMahon and David
Rowitch (Harvard University, Cambridge, MA). In the reporter strain
Gt(ROSA)26Sortm1Sor (referred to as ROSA26R),
Cre expression results in removal of a loxP-flanked DNA
segment that prevents expression of a lacZ gene. After Cre
activation, lacZ is detected in cells/tissues where Cre is
expressed. The transgenic line EIIaCre [Tg(EIIa-cre)C5379Lmgd, from The
Jackson Laboratories] carries a Cre transgene under control of the
adenovirus EIIa promoter that targets expression of Cre to the early
mouse embryo and acts as a ubiquitous deletor strain for our purposes. The
presence of Cre was detected with primers
5'-TGATGAGGTTCGCAAGAACC-3' and
5'-CCATGAGTGAACGAACCTGG-3', producing a band of 400 bp. All mouse
strains were maintained on an outbred mixed genetic background that was
primarily CD-1 and C57BL/6. Animals were maintained in accordance with the
rules of the University of Pennsylvania and Vanderbilt University
Institutional Animal Care and Use Committee (IACUC).
Histology and whole-mount in situ hybridization
Histology was performed using standard procedures
(Presnell and Schreibman,
1997). For whole-mount LysoTracker staining, embryos were
dissected in Hanks buffer, and incubated in 5 µM LysoTracker (Molecular
Probes) at 37°C for 1 hour. Embryos were then washed in Hanks buffer and
fixed in 4% paraformaldehyde (PFA) in PBS overnight, washed in PBS and
dehydrated in methanol before imaging with a fluorescent stereoscope. TUNEL
assay was performed in paraffin sections using the In Situ Cell Death
Detection Kit (Roche). Whole-mount immunostaining was performed as described
(Wall et al., 1992). Skeletal
preparations and 5-bromo-4-chloro-3-indolyl-β-D-galactoside
(X-Gal) staining followed standard techniques
(Nagy et al., 2003). For
histological analysis, paraffin sections (7-12 µm) were counterstained with
Eosin or Nuclear Fast Red.
The following antibodies were used: rabbit anti-Foxd3 at 1:1000
(Tompers et al., 2005); mouse
anti-PGP9.5 at 1:100 (Biogenesis); mouse anti-neurofilament (2H3 monoclonal)
at 1:1000 (Developmental Studies Hybridoma Bank); mouse anti-β III
tubulin (Tuj1; Tubb3) at 1:100 (Chemicon); rabbit anti-phosphohistone H3 at
1:200 (Upstate Biotechnology); goat anti-Fabp7 (also known as B-FABP) at 1:20
(R&D Systems); goat anti-rabbit Cy3, goat anti-mouse Cy3 and donkey
anti-goat Cy2 (Jackson ImmunoResearch Laboratories). The Vectastain ABC Kit
(Vector Laboratories) was used to detect the non-fluorescent secondary
antibodies and DAPI (1:5000, Molecular Probes) was used to highlight
nuclei.
Whole-mount in situ hybridization was performed using Costar 12-well
inserts following standard protocols
(Hanna et al., 2002). Probes
for Crabp1 (Stoner and Gudas,
1989), Dlx1 (Dolle et
al., 1992), Msx1
(Satokata and Maas, 1994),
Pdgfa (Mercola et al.,
1990), Pdgfc (Ding et
al., 2000) and Sox10
(Southard-Smith et al., 1998)
were as described previously. Digoxigenin-labeled RNA probes were prepared
using reagents from Roche Molecular Biochemicals.
Corrosion casting was performed as described
(Feiner et al., 2001) by
injecting the left ventricle of embryonic or newborn hearts first with PBS and
then with polymer containing red pigment (Polysciences #07349) using gentle
pressure. Polymer containing blue pigment was injected into the right
ventricle to monitor for possible ventricular septal defects. The casts were
cured overnight at 4°C and surrounding tissue was macerated (maceration
solution: Polysciences #07359) at 50°C for 6-8 hours.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To selectively delete Foxd3 in the NC, we used the
well-characterized Wnt1-Cre transgenic mouse line in which Cre
recombinase is expressed in migrating NC starting at 8.0-8.5 dpc
(Danielian et al., 1998). We
used reciprocal matings between Foxd3flox/flox and
Foxd3+/-; Wnt1-Cre mice to obtain
Foxd3flox/-; Wnt1-Cre (mutant) embryos. At the
four-somite stage (
8.0 dpc), Foxd3 protein was detected in migrating NC
in the head folds (Fig. 1D). By
contrast, we saw a severe reduction in Foxd3 protein in head folds of mutant
embryos, detecting only one or two cells that were weakly positive for Foxd3
protein expression (Fig. 1E,
arrow). By 9.5 dpc, no Foxd3 protein was detected in the mutant dorsal neural
tube or migratory path of NC (Fig. 1,
compare F with G). Foxd3 expression was not maintained in the
facial mesenchyme at 13.5 dpc; expression was limited to forming cranial
ganglia (Fig. 1H) and this
expression was missing in mutant embryos. In the ENS, Foxd3 protein was
expressed in the gut coils at 13.5 dpc and was missing in mutant embryos
(Fig. 1J,K). These results
demonstrate that Foxd3 is deleted specifically in the NC by
Wnt1-Cre.
Maintenance of cranial NC is dependent on Foxd3
Initially, litters were allowed to progress to term and no mutant mice were
found at weaning (21 days). However, we found one dead mutant newborn pup
(Fig. 2A) and began monitoring
litters before the mice were born (Table
1). Although the majority of 18.5 dpc littermates appeared normal
and could survive being delivered by caesarian section, the mutant mice
attempted to breathe several times but then expired.
Table 1 summarizes the number
of offspring and embryos from both
Foxd3flox/+xFoxd3+/-;
Wnt1-Cre and
Foxd3flox/floxxFoxd3+/-;
Wnt1-Cre crosses. Embryos were found in the expected ratios at all
times in development and no mutant mice survived more than a few hours.
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To monitor NC cells and their derivatives, we introduced the
ROSA26R (R26R) conditional reporter allele into the mutant
background (Soriano, 1999).
Upon expression of Cre protein, all cells and their descendants transcribe the
lacZ gene, and therefore NC cell fate can be monitored directly with
and without Foxd3 function. Note that we are comparing
Foxd3flox/-; Wnt1-Cre; R26R (mutant)
embryos with Foxd3flox/+; Wnt1-Cre; R26R
(control) embryos so that the Cre protein will be recombining two sets of loxP
sites; one to delete the Foxd3 coding region and one to delete the
stop sequence in the ROSA26 locus. β-gal-positive tissue was
observed in the frontalnasal mesenchyme in both control and mutant embryos,
indicating that NC cells contribute to these tissues in the absence of Foxd3
(Fig. 2D and see Fig. S3 in the
supplementary material).
Pharyngeal arch defects in Foxd3 mutants
At 9.5 dpc, a comparison of lineage-mapped embryos revealed that NC cells
have migrated into pharyngeal arches (PAs) 1 and 2 in both Foxd3
mutants and controls (Fig. 3A).
However, by 10.5 dpc, morphological differences in the PAs were apparent by
scanning electron microscopy (Fig.
3B). The maxillary and the mandibular prominences of PA1 were
present but reduced in size. There was a striking decrease of NC in PAs 3 and
4. Immunohistochemistry for neurofilament protein showed that all cranial
ganglia and nerves are present but smaller than normal. Some cranial nerves
were slightly misdirected (the glossopharyngeal nerve in
Fig. 3C) or had not extended as
far as in the control (the facial and vestibulocochlear nerves). Spinal nerves
along the trunk of the mutant embryos were thinner. Examining expression of
Sox10, a marker of early NC, revealed reduced expression in the
developing trigeminal, facial and vestibulocochlear ganglia and no expression
in the glossopharyngeal and vagus ganglia. Sox10 expression was also
reduced along the trunk of the embryo (Fig.
3D). Expression of other NC-specific genes in the PAs uniformly
showed reduced expression in PAs 1 and 2 (see Fig. S1 in the supplementary
material). Together, these data suggest that the initial specification of NC
occurs in Foxd3 mutants, but there is an overall failure to maintain
NC progenitors as demonstrated by a loss of PA-derived structures and a
concomitant decrease in expression of NC-specific genes.
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NC cells that migrate ventrally from the dorsal neural tube contribute to the ENS: the neurons and glia that innervate the entire gastrointestinal tract. Using the ROSA26R reporter allele, NC contribution to the ENS is easily visualized. In control midgestation embryos (14.5 dpc), the intestines were ensheathed in a plexus of NC-derived β-gal-positive cells, whereas in a Foxd3 mutant littermate the gut was completely lacking these cells (Fig. 4E,F). When the gastrointestinal tract was dissected out of 17.5 dpc embryos, the control sample was surrounded by β-gal-positive cells along the entire anterior to posterior axis, whereas the mutant sample had no NC-derived cells around the outside of the gut tube (Fig. 4G,H). Expression of PGP9.5 (Uchl1 - Mouse Genome Informatics) was readily detectable in neurons of the ENS in control embryos, whereas there were no PGP9.5-positive cells in Foxd3 mutant embryos (Fig. 4I,J). Similar results were obtained with β III tubulin antiserum used to detect neurons, and with Fabp7 antiserum used to detect glia (data not shown).
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To further investigate development of the cardiac NC, we examined
patterning of vascular NC derivatives using corrosion casting
(Fig. 5E-H). We observed normal
patterning of the aortic arch in the majority of Foxd3 mutants
(13/17, 76%) between 15.5 dpc and birth. However, in three out of 17
Foxd3 mutant embryos examined, we observed a duplication of the left
common carotid artery (Fig.
5G,G'). One mutant embryo had severe cardiac NC defects,
including type A2 persistent truncus arteriosis (PTA) in which the septation
of the outflow tract did not occur and the ductus arteriosis was absent
(Fig. 5H,H')
(Van Praagh and Van Praagh,
1965). We examined the NC lineage in both control and mutant
embryos with the ROSA26R reporter allele and saw no differences in NC
contribution to the cardiovascular system at 17.5 dpc (n=7 mutants;
see Fig. S2 in the supplementary material). Histological analysis of eight
mutant and control littermates was performed and no septal defects were
observed (see Fig. S2 in the supplementary material); however, while in the
process of performing resin casts, 1/17 (the sample shown in
Fig. 5H) displayed a
ventricular septal defect.
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Terminal differentiation of NC occurs as cells reach their final destination. We detected expression of neural and glial markers in control and mutant DRG. At 9.5 and 14.5 dpc, the neural marker β III tubulin and the glial marker Fabp7 were expressed in both control and mutant DRG (Fig. 6E-H and data not shown).
We examined cell death in control and Foxd3 mutant embryos and detected an increase in TUNEL-positive apoptotic cells in the dorsal spinal cord in the mutant, whereas there were few, if any, TUNEL-positive cells in the spinal cord of control embryos (Fig. 6I,J). Confirming this with LysoTracker Red dye, at 9.0 dpc mutant embryos showed increased apoptosis in the hindbrain, and by 10.5 dpc apoptosis was more pronounced in the posterior tail of the embryo (Fig. 6K-N). The absence of apoptosis in distal PAs of mutant embryos at 10.5 dpc (Fig. 6M) is likely to be due to the large deficit in NC cell numbers (note morphology of PAs in Fig. 6M and lineage label in Fig. 3B).
To more closely examine the curious recovery of most of the cardiac
derivates, we analyzed cell proliferation in the cardiac NC using a
combination of lineage labeling and immunohistochemistry for phosphorylated
histone H3 (pH3), a marker for cells in mitosis. Septation is complete by 13.0
dpc so we chose to examine embryos at 12.5 dpc
(Hiruma et al., 2002).
Representative sections of 12.5 dpc control and mutant embryos are shown in
Fig. 6O-R, clearly
demonstrating the paucity of NC present in the outflow tract at the most
rostral and caudal levels. The volume of the outflow tract in control and
mutant embryos was calculated as 4 mm3 and 0.7 mm3,
respectively. The proportion of pH3-positive NC cells was the same in controls
and mutants (for details, see Fig. S3 in the supplementary material). These
results suggest that although loss of Foxd3 reduces the number of cardiac NC,
these cells are still able to pattern the outflow tract and give rise to
smooth muscle cells.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Dottori et al.,
2001; Kos et al.,
2001; Sasai et al.,
2001). However, our loss-of-function analysis suggests that Foxd3
is not required for the initial specification of the NC. In
Foxd3flox/-; Wnt1-Cre mutant embryos, we do not
detect cells expressing Foxd3 at the earliest stages of NC specification
(Fig. 1D-G), yet there is still
NC present (Fig. 3). There
remains the possibility that the Wnt1-Cre transgene does not produce
Cre protein early enough and there might be a few Foxd3-expressing cells
present, but if so, our data show that they are not maintained. It is more
likely that the role of Foxd3 is in maintaining the NC, not in specifying this
cell type.
Our findings do not completely agree with those of other model systems,
highlighting differences between Xenopus, chick, zebrafish and mouse
NC. Ectopic expression of a dominant-negative FoxD3 in Xenopus
embryos suppresses NC specification as measured by the loss of NC marker
expression (Sasai et al.,
2001). Electroporation of antisense oligonucleotides specific to
Foxd3 in the chick bias the differentiation of NC towards the
melanocyte lineage (Kos et al.,
2001). There are three recent reports of mutations in the
zebrafish foxd3 locus or knockdown of foxd3 using
morpholinos (Lister et al.,
2006; Montero-Balaguer et al.,
2006; Stewart et al.,
2006). These groups reported that NC was specified normally with a
general downregulation of NC markers similar to what we have observed.
Craniofacial defects were highlighted by jaw abnormalities in the fish,
whereas defects in the mandible of our mice were minor. All three groups
demonstrated a complete loss of DRG, whereas we see smaller DRG that contain
neurons and glia. Similar to what we observe, in all cases the zebrafish ENS
was lost and increased cell death in the NC lineage was observed along with
defects in migration. Zebrafish are an ideal system to monitor changes in
different types of NC melanocytes and all three groups described
lineage-specific differences in these derivatives. For example, in the
mother superior mutation, which is a mutation somewhere outside the
coding region that affects only NC expression of foxd3, melanophores
are delayed in their development but then recover, whereas iridophores remain
greatly reduced (Montero-Balaguer et al.,
2006). Zebrafish cardiac NC contributes primarily to the
myocardial cell lineage (Li et al.,
2003). Despite this, only one group reported a slight edema in the
heart in foxd3 mutants
(Montero-Balaguer et al.,
2006).
NC patterning
We did not detect anterior-posterior patterning defects in NC of
Foxd3 mutants, although caudal NC was most severely affected. All
mutant embryos had severe facial clefting and were missing many NC-derived
facial bones. However, the mandible was present in all mutants examined. It
was shortened and thickened, similar to Dlx5-/- mice
(Acampora et al., 1999), but
its presence demonstrates that Foxd3-/- NC can
differentiate into cartilage and bone and overt cranial patterning is
unaltered. Similarly, trunk NC cells were present in reduced numbers, but
those cells migrated away from the neural tube normally and formed small DRG
that contained both neurons and glia.
By contrast, the vagal and sacral NC destined to contribute to the ENS did
not migrate to the gut. There are several possibilities for the loss of this
sub-lineage of NC. One hypothesis is that there may be expression of closely
related Fox genes in the cranial and cardiac NC, but no such compensating
family members expressed in the trunk NC caudal to somite 7. A second
hypothesis is that ENS progenitors underwent selective cell death in the
dorsal neural tube, whereas NC cells destined to populate the DRG were
maintained. There are no molecular markers to distinguish these lineages prior
to migration so we cannot test this hypothesis directly. A third possibility
is that NC cells that underwent apoptosis in the dorsal neural tube were
unspecified and the selective loss of the ENS NC is because this lineage must
migrate farther than others. Therefore, a general loss in progenitor cell
number might affect the ENS NC more severely. A fourth possibility is that
Foxd3-/- NC might not be able to enter the foregut
(Fig. 6, compare C with D). It
has been demonstrated that vagal and sacral NC, unlike the rest of the trunk
NC, possess a unique ability to enter the gut mesentery
(Serbedzija et al., 1991).
Recent studies suggest this difference might be at least partially explained
by the expression of axon guidance molecules Slit and Robo on vagal NC
(De Bellard et al., 2003). In
contrast to our results, mice lacking members of the glial cell line-derived
neurotrophic factor signaling pathway have enteric neurons in the anterior ENS
(Cacalano et al., 1998;
Pichel et al., 1996;
Sanchez et al., 1996), whereas
mutations in endothelin 3 or endothelin receptor type B cause the loss of ENS
only in the distal colon (Baynash et al.,
1994; Hosoda et al.,
1994). In Phox2b-/- embryos, there is an
initial migration of vagal NC into the proximal gut mesentery, but these
progenitors are not maintained, leaving the entire gut without innervation
(Pattyn et al., 1999). In
fact, the only other mouse model lacking the entire ENS is
Sox10Dom/Dom (Kapur,
1999; Southard-Smith et al.,
1998) and it is interesting to note that Sox10 is one of
the genes severely downregulated in Foxd3-/- mutant
embryos (Fig. 3C,
arrowheads).
One final hypothesis, not mutually exclusive to those suggested above, is that although Foxd3 appears to be equally expressed throughout the early premigratory crest, it might be differentially required in specific subpopulations of the crest. Our surprising results showing the minimal phenotype of the mutant cardiac NC supports this last hypothesis. The cardiac crest normally migrates into PAs 3, 4 and 6, eventually contributing to the cardiac outflow tract and the aorticopulmonary septum. There is a striking reduction of cardiac NC in PAs 3, 4 and 6 in Foxd3 mutant embryos compared with controls (Fig. 3A and Fig. 5A-D). However, this greatly reduced population of cardiac NC migrates into the heart field and even though there are far fewer cells (mutants contained 18% of the volume of NC of controls by lineage label), this reduced number can effectively septate the outflow tract in most cases. We did not observe alterations from the wild-type pattern of proliferation as monitored by phosphorylated histone H3 at 9.5 dpc. This suggests that the extent of migration of the cardiac NC, rather than the number of cardiac NC cells, is crucial for their ability to effectively septate the outflow tract. One of our future goals is to understand molecular differences between the cardiac NC, which can overcome the loss of Foxd3, and the rest of the NC lineage that is dependent on this transcription factor.
Conservation of Foxd3 function in progenitor cells
Development of all embryonic tissues relies on progenitor cells that are
able to self-renew and differentiate appropriately. Our initial description of
the Foxd3-/- phenotype demonstrated that this protein is
required for maintenance of epiblast cells and therefore required for
establishing ES cells (Hanna et al.,
2002). Foxd3 is similarly required in the trophoblast progenitor
lineage (Tompers et al.,
2005), a lineage completely disparate from the embryonic one
primarily affected in the null mutation. Therefore, the NC is the third
multipotent lineage that requires Foxd3 for maintenance of progenitor cells,
making an unprecedented molecular connection between these three distinct
progenitor populations. These lineages are embryonically derived and we have
yet to demonstrate that Foxd3 can play a similar role in adult-derived stem
cells. However, our results support the premise that certain self-renewal
mechanisms are likely to be conserved in very different progenitor lineages.
There are several other proteins known to regulate multiple progenitor
lineages. Bmi1 and other polycomb proteins repress genes that induce cell
death in the hematopoetic lineages, neural stem cells, NC stem cells and
cancer stem cells (reviewed by Pardal et
al., 2005). Most recently, the transcription factor Zfx has been
shown to be required for self-renewal of ES cells and maintenance of adult
hematopoietic stem cells (Galan-Caridad et
al., 2007). The proto-oncogene c-Myc has a
well-documented role in cell proliferation; recent work in Xenopus
suggests it might be involved in NC formation, whereas genetic studies in the
mouse show that a NC deletion of Myc results in a relatively mild
phenotype (Bellmeyer et al.,
2003; Wei et al.,
2007). In addition to its role in NC development, expression of
Myc along with several other `stem cell proteins' is sufficient to change the
fate of somatic cells into pluripotent stem cells
(Takahashi and Yamanaka,
2006). Finally, Sox2 is required in ES cells, TS cells and
embryonic neural lineages (Avilion et al.,
2003; Graham et al.,
2003). Sox2 is also expressed in early migrating NC, in Schwann
cells (Larysa Pevny, personal communication) and in the postnatal ENS
(Vohra et al., 2006), all
regions competent to give rise to NC stem cells. The role of Sox2 in the NC
has not been investigated, and it is tempting to speculate that there might be
a genetic interaction between Foxd3 and Sox2.
It is not clear how Foxd3 functions to maintain these progenitor pools. The
protein can function as either an activator or a repressor in different
contexts; in Xenopus mesoderm induction, FoxD3 recruits Groucho4 to
repress target genes (Steiner et al.,
2006; Yaklichkin et al.,
2007), whereas in zebrafish somite maturation, Foxd3 activates
myf5 expression directly (Lee et
al., 2006). We cannot rule out the possibility that Foxd3 might
behave similarly to Foxa proteins in modifying chromatin structure to allow
access of other co-repressors or activators to target genes
(Cirillo et al., 2002). Our
major challenges ahead are to identify Foxd3 target genes and to determine the
signaling pathways in which the protein functions.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/9/1615/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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These authors contributed equally to this work
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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