Program in Developmental Biology and Division of Basic Sciences, Fred
Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA
98109, USA
* Present address: Department of Molecular Biology, University of Texas
Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9148,
USA
Author for correspondence (e-mail:
michelle.tallquist{at}utsouthwestern.edu)
Accepted 31 October 2002
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SUMMARY |
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Key words: Chimeric analysis, Cre-loxP recombination, Neural crest, PDGF receptor, Mouse
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INTRODUCTION |
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It has been proposed that signaling through the PDGFR is required
for the development of non-neuronal neural crest of cephalic and cardiac
origin. These findings were first identified in the naturally occurring mouse
mutant patch (Ph) (Grüneberg
and Truslove, 1960
;
Morrison-Graham et al., 1992
;
Smith et al., 1991
;
Stephenson et al., 1991
),
which carries a deletion encompassing the PDGFR
gene. Ph/+
heterozygotes exhibit defects in melanocyte development and Ph/Ph
embryos exhibit recessive defects in the axial skeleton, cardiac, and cranial
neural crest. Targeted disruption of the PDGFR
locus has demonstrated
that Pdgfra and Ph mutations share nearly identical
phenotypes with the exception of the heterozygous melanocyte defect
(Soriano, 1997
), although
deficiencies in melanocyte development have been observed in chimeric animals
using null or hypomorphic PDGFR
alleles
(Klinghoffer et al.,
2002
).
More recently, several other affected tissues have been shown to require
PDGFR signaling, including kidney interstitial fibroblasts, testes
Leydig cells and intestinal villus cells (reviewed by
Betsholtz et al., 2001
).
Despite the fact that many studies have uncovered specific cellular roles for
PDGFR
, very little is known about the mechanism of action of
PDGFR
in vivo. The analysis of biological function of PDGFR
during development is complicated because many cell types express the
receptor, which results in variable phenotypes and early lethality. The
expression pattern of the PDGFR
during embryogenesis is diverse and
extensive. At E6.5, PDGFR
mRNA is expressed in visceral extra-embryonic
endoderm. After gastrulation the receptor mRNA can be seen in many areas of
mesenchyme, including the somites, limb buds and branchial arches
(Orr-Urtreger et al., 1992
;
Orr-Urtreger and Lonai, 1992
;
Schatteman et al., 1992
). Less
than one-third of the mutant embryos survive beyond E11.5 on the C57BL/6
background, making analysis of the NCC phenotypes difficult (M. D. T.,
unpublished). In the NCC population, the PDGFR
is expressed in a broad
diffuse pattern that is synonymous with regions defined for cranial and
cardiac NCCs (Orr-Urtreger et al.,
1992
; Orr-Urtreger and Lonai,
1992
; Schatteman et al.,
1992
; Takakura et al.,
1997
; Zhang et al.,
1998
). The early embryonic lethality and broad expression pattern
of the PDGFR
make elucidation of cell autonomous functions of the
receptor in NCCs very challenging.
To investigate potential cell autonomous requirements for PDGFR
signaling in cranial and cardiac NCCs, we have generated chimeric or mosaic
embryos. Traditional chimeras were generated by mixing mutant and wild-type
cells in preimplantation embryos. In these chimeras, we found a strong
selection for cells bearing the PDGFR
in several mesenchymal cell
populations, including limb bud, somite and branchial arches. To further
investigate the role of the PDGFR
, we performed tissue-specific gene
ablation using Cre/loxP recombination exclusively in the NCC lineage. We found
that loss of PDGFR
in NCCs results in craniofacial and aortic arch
defects, while many of the other phenotypes are alleviated. From these
analyses, we have determined that PDGFR
is directly required in both
cardiac and cranial NCCs, and we have further characterized the phenotypes
associated with these particular cell types.
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MATERIALS AND METHODS |
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Mice and genotyping
The targeting construct for the PDGFR floxed allele was generated
using genomic DNA that has been previously described
(Soriano, 1997
). A
loxP site was inserted into the BamHI site located upstream
of the exon encoding the signal peptide. Pgk-neo loxP was inserted at
the SmaI site. Wnt1Cre transgenic mice
(Danielian et al., 1998
) were
kindly provided by Andy McMahon (Harvard University). ROSA26R Cre reporter
mice expressing lacZ conditionally have been described previously
(Soriano, 1999
). All mice were
maintained on mixed genetic backgrounds, except for mice bearing the null
allele, which were either congenic on C57BL/6 or on 129S4.
PDGFR
fl and null alleles were genotyped using PCR primers
described for the Pdgfra-null allele
(Soriano, 1997
). The Wnt1Cre
transgene was detected by Southern blot analysis for Cre.
Flow cytometry
Flow cytometric data analysis (FACScan, Becton Dickinson, Palo Alto, CA)
was used to determine the cell-surface expression of PDGFR in branchial
arch mesenchyme. E10.5 embryos were isolated and the branchial arch region
dissected using tungsten needles. The arch tissue was dissociated using 1
mg/ml dispase (Boehringer-Mannheim) in PBS for 10 minutes at 37°C. Single
cell suspension was filtered through nylon mesh. APA5 (Research Diagnostics,
Flanders, NJ), a rat monoclonal antibody that reacts with the murine
PDGFR
, was used at a 1:200 dilution. We used goat anti Rat IgG
conjugated to phycoerythrin (Jackson Immunoresearch, West Grove, PA) as
secondary antibody.
Resin injections
Resin injections were performed using Batson's #17 Plastic Replica and
Corrosion Kit, (Polysciences; Warrington, PA). E18.5 embryos were isolated by
cesarian section. The thoracic cavity was opened and resin injected into the
left or right ventricle using a glass drawn pipette. Resin was allowed to
harden overnight at 4°C and tissue was then macerated for 1-2 hours at
50°C using Maceration Solution (Polysciences, Warrington, PA).
Intracardiac ink injection
Embryos were collected at E9.5-11.5 and India ink was injected into the
left ventricle using a finely drawn glass pipette. The embryos were then
immediately fixed in either 4% paraformaldehyde or methyl Carnoy's. Embryos
were cleared in benzyl alchol:benzyl benzoate and photographed. Vessels were
scored for presence, absence and thickness.
Immunohistochemistry and TUNEL analysis
Tissues were fixed in 4% paraformaldehyde, sectioned, deparaffinized and
rehydrated. For immunohistochemistry, endogenous peroxidase activity was
quenched using 3%H202/10% methanol in PBS at for 30
minutes. Antigens were revealed using 2N HCl at room temperature for 45
minutes followed by a 0.1% trypsin incubation at 37°C for 20 minutes.
Antibodies against smooth muscle actin (1:10000; clone 1A4, Sigma),
PECAM (1:200; clone MEC 13.3, Pharmingen) and phospho-histone H3 (1:200;
#06-570, Upstate Biotech) were incubated for 1 hour at room temperature and
detected using the appropriate secondary antibody. The HRP reaction was
performed using the Vectastain (Vector Labs) ABC and DAB kits. The TUNEL
protocol was as described by Gavrieli et al.
(Gavrieli et al., 1992
).
Briefly, nuclear proteins were stripped with 20 µg/ml proteinase K in PBS
for 10 minutes at room temperature. Sections were then washed with TdT buffer
(30mM Tris pH 7.2/140 mM cacodylic acid/1mM CoCl2). TdT reaction
was accomplished in TdT buffer with 0.3 units/µl TdT (Pharmacia) and 7.5
µM biotinylated-14-dATP (Gibco/BRL) for 1 hour at 37°C. The Vectastain
ABC and DAB kits (Vector Labs) were used to visualize the incorporated
ATP.
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RESULTS |
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Cranial and cardiac crest initiate migration at day 8 (E8); therefore, we
examined chimeric embryos at day 9 and 10 of gestation. ES cell contribution
to these chimeras ranged from 0-100%, as judged by an estimation of
ß-galactosidase-negative cell contribution to the dorsal root ganglia
(DRG), where there was no selection bias for cells of different genotypes. We
were able to obtain extensively mutant chimeras from both ES cell lines, but
chimeras with more than 50% mutant ES cell contribution were phenotypically
abnormal and often indistinguishable from Pdgfra-null embryos
(Fig. 1A-E;
Table 1). The phenotypic
abnormalities included growth retardation, dilated pericardium, surface
ectoderm blebbing, wavy neural tube and hypoplastic branchial arches. In fact,
we recovered a chimera that was 100% ES cell derived (based on PCR and
ß-galactosidase staining). The phenotype appeared identical to that
observed in Pdgfra-null embryos. Because injected ES cells contribute
primarily to epiblast derivatives
(Beddington and Robertson,
1989
), these data suggest that the phenotypes at E9.5-10.5 are due
to defects in loss of PDGR
from the embryo and/or extra-embryonic
mesoderm and not likely to be due to defects in other extra-embryonic
tissues.
|
Analysis of the chimeras in wholemount and sections revealed a striking
selection against PDGFR-positive cells in several areas of mesenchyme
(Fig. 1). These included the
limb bud progress zone, branchial arches and sclerotome
(Table 1 and data not shown).
Fig. 1F is an example of a
section through a chimera that contains a significant proportion of
Pdgfra-/- cells (70-100% in the neural tube) while much of
the mesenchyme is wild-type derived. In forelimb buds, we observed progress
zones that were almost exclusively composed of wild-type cells, while the
adjacent surface ectoderm was populated mostly by
Pdgfra-/- cells (Fig.
1F, inset). In the less mature hindlimb bud, wild-type and mutant
cells are mixed in the more proximal region, but in the distal zone there is a
concentration of wild-type cells abutting the ligand-expressing surface
ectoderm.
A similar pattern of selection was seen when we examined neural
crest-derived head and branchial arch mesenchyme. Again,
Pdgfra-/- cells contribute to the ectodermal epithelium
and the paraxial mesoderm-derived core of the branchial arches
(Fig. 1G-J) (data not shown),
but neural crest-derived ectomesenchyme is almost exclusively composed of
wild-type cells. Analysis of proliferation in the branchial arches of these
chimeras using anti-phosphohistone 3 antibody or BrdU labeling did not yield
definitive results because the number of null cells available for analysis in
the tissues of interest was limited, and the number of proliferating null
cells was negligible. Nonetheless, the strong selection against the null cells
in areas of proliferating mesenchyme of the limb and NCC-derived
ectomesenchyme suggests a cell autonomous requirement for PDGFR in
these tissues.
Conditional analysis of PDGFR in neural crest
We wanted to study further the role of PDGFR in the neural crest
lineage, but because many of the high percent chimeras possessed the
Pdgfra-/- phenotypes, we chose to analyze PDGFR
signaling in the NCCs by conditional gene ablation analysis.
Fig. 2A depicts the
construction of the mutant allele (PDGFR
fl) flanked by lox
sites (floxed). In the presence of Cre recombinase, the second and third exon
of the Pdgfra gene as well as the neo cassette will be removed,
resulting in a null allele identical to the one previously described
(Soriano, 1997
). While
homozygous mice bearing this floxed allele are viable with no overt phenotype,
the presence of the Pgk-neo loxP cassette apparently interferes with
gene expression. This result was revealed when we crossed mice bearing the
null allele to PDGFR
fl/fl. From seven litters, no
transheterozygotes were obtained at the time of weaning. These data suggest
that transheterozygotes of the Pdgfra-null allele and floxed allele
are inviable due to reduced levels of receptor expression. Because the floxed
allele is hypomorphic, we have used both the homozygous floxed mice
(PDGFR
fl/fl) and Wnt1Cre+;
PDGFR
fl/+ as littermate controls. We have observed no
craniofacial nor cardiac NCC defects in PDGFR
fl/fl embryos
or Wnt1Cre+; PDGFR
fl/+ mice.
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To investigate specifically the role of the PDGFR in NCCs, we
crossed the PDGFR
conditional allele to the Wnt1Cre mouse line. In this
line, the Cre recombinase is expressed in the dorsal neural tube as early as
embryonic day 8 (E8), and therefore in the presumptive neural crest. Several
studies have used the transgenic Wnt1Cre line in combination with the ROSA26
Cre reporter line (where ß-galactosidase is expressed upon recombination
by Cre) as a lineage marker for NCCs (Chai
et al., 2000
; Echelard et al.,
1994
; Jiang et al.,
2000
); Cre is expressed at the correct time and place to delete
the PDGFR
from the neural crest
(Fig. 2C,D). We determined the
extent of tissue-specific PDGFR
gene ablation in several ways. First,
Southern blot analysis verified that the deleted floxed allele resulted in the
expected genetic rearrangement in head mesenchyme
(Fig. 2B). Second, the
distribution of tissues with Cre recombination was investigated using the
ROSA26R Cre reporter mouse. In both wild-type and
PDGFR
fl/fl; Wnt1Cre+ embryos (also referred to as
NCC conditionals), the pattern of Cre activity follows the localization of NCC
derivatives (Fig. 2C,D). In
addition, it appears that at E9 many of the cells express
ß-galactosidase, suggesting that most cells of neural crest origin
exhibit recombination of the loxP flanked DNA. Therefore at this time
point, few if any cells have escaped Cre recombination activity and thus have
lost PDGFR
expression and this is further illustrated by the findings
from protein expression. As a final approach to examining the efficiency of
the Cre recombinase, we measured PDGFR
protein expression by flow
cytometry. Fig. 2E indicates
that as early as E10.5, PDGFR
expression in the mesenchyme of the
branchial arches has dropped below detection limits. These results indicate
that the Wnt1-driven Cre is expressed in the appropriate tissues, at the
correct time, and at sufficient levels to render NCC derivatives void of
PDGFR
protein expression.
To determine the outcome of loss of PDGFR in NCCs, we established
matings of PDGFR
fl/+;Wnt1Cre+ males to
PDGFR
fl/fl females. No viable
PDGFR
fl/fl;Wnt1Cre+ neonates were obtained from
181 offspring. In contrast to PDGFR
-/- embryos which are
resorbed several days before birth, several NCC conditional newborns were
found dead with a foreshortened snout, a sagittal median cleft, and often a
midline hemorrhage. These external defects were accompanied by a cleft palate
(Fig. 3A-B). While the extent
of the frontonasal fusion varied between NCC conditional mice, the palatal
defect remained consistent with complete failure of the shelves to fuse. This
phenotype was fully penetrant and never observed in
PDGFR
fl/fl or PDGFR
fl/+;
Wnt1Cre+ mice. The phenotype of the PDGFR
NCC conditional
and null embryos were compared at E13.5-15.5
(Fig. 3C,D).
Fig. 3D demonstrates that the
NCC conditional embryos possessed a midline facial cleft often accompanied by
a hemorrhagic bleb. Even though the midline defect was always present, it
rarely extended the length of the anterior mesencephalon as the clefts in the
null embryos do. To study the etiology of the craniofacial defects, we
examined embryos at various time points. From E8.5 to E10.5, NCC conditional
embryos were indistinguishable from littermate controls. Starting at E11.5,
NCC conditional embryos possessed a gap in the frontal nasal process
(Fig. 4E,F). At this time
point, no hemorrhaging or blebbing was evident, although by E13.5 both were
observed (Fig. 3D). External
examination of the embryos also showed that the subepidermal blebbing adjacent
to the thoracic and lumbar vertebrae and on the flanks that is observed in
Pdgfra-null embryos was not present in the NCC conditional embryos.
To determine the extent of the phenotype similarities between the NCC
conditional and null embryos, we examined histological and bone preparations.
We observed no phenotypes in the other PDGFR
-dependent tissues, such as
the kidney, lungs, intestines or bones of the axial skeleton (data not
shown).
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Craniofacial bone defects
Detailed analysis of cranial skeleton and cartilage defects in
Pdgfra-/- embryos had not been possible because most
embryos die before these structures have developed. Therefore, we examined the
NCC conditional mutants to understand the extent of the craniofacial defects
in the absence of PDGFR signaling. NCCs from the branchial arches
contribute to the bony and cartilaginous structures of the cranium in the
mouse (Trainor and Tam, 1995
),
and we analyzed these structures using Alcian Blue/Alizarin Red staining from
E17.5-P1 embryos. Grüneberg and Truslove
(Grüneberg and Truslove,
1960
) noted that the cranial cavity of Ph/+ mice was
shorter and broader than those measured in wild-type skulls. We compared the
skulls of PDGFR
fl/fl;Wnt1Cre+ to
PDGFR
fl/fl embryos at E17.5 and observed that the NCC
conditional skulls were shortened 8±1%. All the NCC conditional mutants
exhibited a cleft palate where the palatine and maxillary shelves failed to
fuse. Although the midline frontal nasal clefting was always present, the
defect ranged in severity from a full, blood-filled, cleft spanning the
anterior forebrain to the frontal nasal process to a small hemorrhage
overlying the nasal cavity. Even in the less severe examples, the nasal
capsule and the overlying frontal and nasal bones were lacking or failed to
fuse. In many embryos the vomer and nasal septum were malformed (data not
shown). Defects including rudimentary structures and incomplete ossification
were observed in many other NCC-derived bones, including the basosphenoid,
presphenoid and alisphenoid bones (Fig.
4A-D and data not shown). The hyoid bone derived from both the
second and third arches was either absent or fused to the thyroid cartilage in
all mutants examined (3/3). In one instance, only the horns of the hyoid bone
were observed (Fig. 4D). In
contrast to these defects, other cranial neural crest derivatives were very
similar to wild type. The mandible, tympanic ring and styloid process were
unperturbed in the NCC conditional embryos. Thus, the PDGFR
is required
for proper formation of some but not all cranial NCC structures.
In the mouse, cranial NCCs arise from the dorsal neural tube before it
closes and must migrate to the anterior facial primordium, where they
contribute to the frontonasal prominence and the pharyngeal arches
(Osumi-Yamashita et al., 1994;
Trainor and Tam, 1995
). It has
been proposed that PDGFR
is required for proliferation of multiple
progenitor cell types and loss of this signal results in insufficient
differentiated progeny (Betsholtz et al.,
2001
). To investigate if the cranial NCC defects observed in the
NCC conditional embryos were caused by a lack of proliferation, we performed
immunohistochemistry for phosphohistone 3 on the craniofacial mesenchyme at
several time points. Fig. 5
illustrates a typical result at E11.5. The numbers of proliferating cells in
the ectomesenchyme are the same in wild-type and mutant embryos. These data
suggest that the craniofacial defects are not caused by a global lack of
proliferation of NCC derivatives. Likewise, we observed no changes in the
number of apoptotic cells, as detected by TUNEL staining (data not shown). An
important point to note is that the wild-type and mutant arches are very
similar in size and shape, with the major exception being the lack of fusion
between the frontal nasal processes in the mutant embryo. We infer from these
observations that the majority of the NCC progeny are present within the
arches and that one cause of the craniofacial defects could be failure of
midline fusion. There are reports that pharyngeal arches can form and be
patterned in the absence of NCCs. In these studies, there were no changes in
pharyngeal arch apoptosis or proliferation, even though NCCs were absent
(Veitch et al., 1999
;
Gavalas et al., 2001
). Given
this information, loss of a subpopulation of NCCs may not be obvious using
these two techniques.
|
Cardiovascular defects in PDGFR NCC conditional embryos
Because aortic arch and thymus anomalies had been described in Ph
mutants (Morrison-Graham et al.,
1992), we examined these tissues in the PDGFR
NCC
conditional embryos. A decrease in thymus size was observed at very low
penetrance (three out of 32 E18.5 NCC conditional embryos; data not shown). To
investigate potential aortic arch defects, corrosion casts were prepared at
E18.5. In greater than 50% of the NCC conditional embryos, we identified
several arch anomalies that have been associated with defects in cardiac
crest. This number may be an underestimate of the embryos harboring defects,
because in multiple castings there was vessel rupture and incomplete filling
of the vessels. Inability to cast the vessels occurred almost exclusively in
the NCC conditional embryos. A similar loss in vessel integrity was also
observed in the Ph mutants
(Schatteman et al., 1995
).
Fig. 6 provides examples of the
range of defects observed. The most severe defect observed was persistent
truncus arteriosus (PTA; Fig.
6B), which results from failure of the conotruncus to septate.
Another common vascular anomaly that we observed was ectopic origin of the
right subclavian artery (RSA). Frequently the RSA arose from the descending
aorta (Fig. 6B,C) and in one
embryo the RSA originated at the proximal pulmonary trunk
(Fig. 6D). In total, we have
examined 23 E17.5-E18.5 NCC conditional embryos by corrosion casting. Seven of
these had incomplete filling. Ten embryos exhibited an abnormal origin of the
RSA, while three of these embryos also possessed persistent truncus
arteriosus. Six of the NCC conditional aortic arches appeared completely
normal, with the exception of a longer innominate (two out of six). Failure of
the membranous region of the ventricular septum to form is also associated
with aberrant cardiac crest development
(Kirby et al., 1983
;
Kirby and Waldo, 1990
).
Although ventricular septal defects (VSD) are difficult to detect in these
corrosion casts because of the presence of the ductus arteriosus, resin was
observed to immediately enter the right ventricle in a majority (10/16) of the
NCC conditional hearts, suggesting VSD. Histological analysis of E14.5
PDGFR
NCC conditional hearts revealed a significant VSV (four out of
four NCC conditional animals Fig.
7A,B). Near the apex of the heart a septum is present
(Fig. 7C), demonstrating a
defect in only the membranous region of the ventricular septum.
|
|
To identify the time point where the defects in the pharyngeal arches
occur, we examined the arch anatomy between E9.5-11.5 with intracardiac India
ink injections. Comparison of NCC conditional embryos and Cre-negative
littermate controls at E9.5 revealed no difference in the formation and
regression of the first and second arch arteries
(Fig. 8D), but when the third,
fourth and sixth arteries were examined at E11.5, multiple abnormalities were
present in the NCC conditional embryos. In PDGFRfl/fl;
Wnt1Cre- embryos, three arch arteries are present on each side, and
they have begun to narrow (Fig.
8A). By contrast, the PDGFR
fl/fl;
Wnt1Cre+ embryos exhibited dilated and/or reduced vessels
(Fig. 8B,C, and data not
shown). Defects were sometimes symmetrical, but often vessels on one side
appeared normal, while the other side had severe defects. Deficiencies were
observed more frequently on the right side than on the left. Although not as
dramatic as the disruptions at E11.5, defects in the fourth and sixth arch
arteries could also be identified in E10.5 embryos (data not shown). One
surprising feature of this data was the lower penetrance of aortic arch
anomalies observed in E17.5-E18.5 embryos compared with the high penetrance of
defects observed at E11.5. These data suggest that some resolution of vessel
stability may occur at later time points as has been suggested previously
(Lindsay and Baldini,
2001
).
|
NCC migration and differentiation
As described above, loss of PDGFR in NCCs does not lead to global
deficits in cell proliferation or survival. Because NCCs are a migratory
population of cells, we used fate mapping to determine the number and
distribution of NCCs as they are moving towards their destinations. For this
purpose, PDGFR
fl/fl animals were bred to mice bearing the
R26R conditional allele described above.
PDGFR
fl/fl; R26R-/- females were then mated to
PDGFRfl/+; Wnt1Cre+ males. The resulting embryos will
have the NCC lineage indelibly marked with ß-galactosidase expression
(Chai et al., 2000
;
Jiang et al., 2000
). We have
examined NCC conditional and littermate control embryos using this technique
(Fig. 9). At E9.5, NCC
migration can be observed in the cranial, pharyngeal arch and trunk regions
(Fig. 9A,B). Mutant embryos
were slightly smaller and less mature. Nonetheless, distinct tracts of NCCs
can be observed migrating through the somites
(Fig. 9B) and nerve tracks can
also be discerned. To look specifically at migration of the cardiac crest, we
examined transverse section of these embryos through the conotruncal region.
NCCs populate the cranial mesenchyme and the pharyngeal arches, as well as
migrating along the lateral walls of the aortic sac and truncus arteriosus in
both the mutant and control (Fig.
2C,D; Fig. 9C,D).
The patterns of migration of tagged NCCs appear very similar. Although a minor
reduction in density of NCCs in the pharyngeal arch regions was observed when
comparing NCC conditional embryos with littermate controls, this is likely to
be due to the maturation stage of the embryo, as more rostral areas such as
the first and second pharyngeal arch regions at E9.5 and E10.5 do not exhibit
this same reduction (Fig. 2C,D;
data not shown). We have also observed mutant and littermate embryos at E13.5
and do not see any major difference in the pattern or intensity of staining
even at this later time point (data not shown). These data indicate that
PDGFR
-negative NCCs follow the expected paths of migration, and an
initial reduction of NCCs in developing tissues is not a likely cause for the
defects that we observe in the PDGFR
NCC conditional embryos.
|
The walls of the vessels of the aortic arch are composed of endothelial
cells of mesodermal origin and VSMC of neural crest origin. To determine if
the defects observed in the arch arteries was caused by an inability of NCCs
to differentiate into VSMC, we analyzed the cellular components of the arch
arteries, using immunohistochemistry for detection of vascular endothelial
cells and VSMC markers. Cross-sections through the fourth arch artery reveal
that both endothelial and VSMC populations appear normal in mutant embryos
(Fig. 10). We also analyzed
the aortic arch regions for cell proliferation (antiphospho histone 3) and
apoptosis (TUNEL), and saw no difference between mutants and wild types in
these assays (data not shown). Taken together, these data indicate that
PDGFR signaling is required for cardiac and cranial NCC development and
demonstrate that the developmental defect is likely to be in a subset of NCCs,
rather than a global defect or in a PDGFR
-directed cellular response
that occurs at a later time point. One possibility is that PDGF regulates
cellular functions involved with remodeling of the cranial facial and aortic
arch region, such as matrix deposition.
|
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DISCUSSION |
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Chimeric analysis demonstrated a pronounced selection against null cells in
several proliferating areas of mesenchymal cell populations. In fact,
wild-type cells predominated in almost every tissue that expresses
PDGFR at high levels (Orr-Urtreger
et al., 1992
; Orr-Urtreger and
Lonai, 1992
; Reinertsen et
al., 1997
; Zhang et al.,
1998
). These results suggest a cell autonomous requirement for the
receptor. Interestingly, there was a simultaneous exclusion of wild-type cells
in regions of the head that are derived from paraxial mesoderm. Possibly there
is an early localization cue that directs PDGFR
-positive NCCs to
cranial mesenchyme, while PDGFR
non-expressing mesoderm is then
allocated to the mesodermal core. This is supported by the flow cytometry data
from PDGFR
fl/fl;Wnt1Cre+ branchial arch
mesenchyme, where only the NCC population expresses the receptor. The most
obvious selection for PDGFR
-expressing cells was in the limb buds and
branchial arches. The mutant cells in both tissues were excluded from the
distal outgrowth of the mesenchyme, although they were abundant in the
ectoderm adjacent to these regions. This pattern of selection has also been
observed in chimeras generated with Fgfr1- and Shp2-null
cells (Ciruna et al., 1997
;
Deng et al., 1997
;
Saxton et al., 2000
). These
observations suggest that rapidly proliferating mesenchyme is sensitive to
subtle changes in growth factor signaling, resulting in counter-selection of
growth factor-deficient cells.
Although no limb development defects are observed in
Pdgfra-/- embryos, a very pronounced phenotype exists in
the NCC-derived cranial bones and cartilage of the nulls. These results are in
agreement with the selection in the chimeric embryos and the phenotype in the
NCC conditional embryos. Although NCCs require PDGFR signaling, it is
unclear at what stage the signals are essential. The selection in the chimeras
occurs very early, based on the paucity of null cells in the mesenchyme of the
branchial arches, but the presence of mesenchyme in both the PDGFR
nulls and NCC conditional embryos suggests that the receptor is not absolutely
required. Therefore, we propose that PDGFR
may be important at two time
points. One may be at the juncture when NCCs are initially populating the
arches. At this stage, the PDGFR
would not be essential, but may act
synergistically with other growth factor signals to promote NCC proliferation
and/or migration. The second time point would be during the morphogenetic
processes occurring in the cranial as well as the cardiac regions beginning at
E11.5. The PDGFR
would be non-redundant with other pathways at this
time, and loss of the signals in NCCs results in the observed phenotypes.
The craniofacial defects observed in the NCC conditional embryos are
similar to those described for the Pdgfra-null mice. Because only a
few null embryos survive to E15-16, the craniofacial malformations associated
with the PDGFR have not been described in depth. Our comparison of
E13.5 embryos revealed that the cranial phenotypes in the null embryos were
often more severe with regards to the depth of the midline cleft and the
amount of hemorrhaging observed. The differences in phenotypes between the
null and the NCC conditional can be explained in several ways. The first
possibility is that the deletion of the PDGFR
locus in the NCC lineage
is incomplete. Therefore, the remaining population of receptor-positive NCCs
assuages the defects in the craniofacial mesenchyme. We find this possibility
unlikely because the flow cytometry analysis of protein expression in the
branchial arch region demonstrated near 100% efficiency of recombination as
early as E10.5. A second explanation is that there is a population of later
migrating NCCs that never express Wnt1, and therefore never lose PDGFR
expression. The final and most likely explanation is that loss of PDGF
signaling in some other cell lineage, possibly somite-derived mesoderm, could
also contribute to the severe craniofacial phenotype in the null. Mesoderm
cells mix with NCCs and form connective components such as membranes and
tendons (Kontges and Lumsden,
1996
). Therefore, this population, which is still present in the
NCC conditionals, may partially alleviate the null phenotype.
We surmised three possible scenarios for how loss of PDGF signaling results
in the observed craniofacial defects. The first could be a direct effect on
the primordia of the maxillary region, where PDGFR would be required
for some function such as proliferation, growth or migration. Loss of this
signal would lead to a reduction in the number of cranial bone progenitors.
Our experiments show that at the initial stages of NCC formation,
proliferation, migration and apoptosis are normal in the mutant embryos,
suggesting that this simple explanation does not apply to the phenotypes we
observe. A second mechanism could be that PDGFR
signals are required to
promote the differentiation of CNCC progenitors by mechanisms other than
proliferation or cell death, possibly by directing matrix deposition or tissue
remodeling. Analysis of NCC ectomesenchyme from Ph homozygotes has
demonstrated a deficit in metalloprotease 2 (MMP2)
(Robbins et al., 1999
). Loss
of this and similar proteins could affect both NCC migration and frontonasal
tissue remodeling. In the third model, PDGFR
may be important for only
a subset of cells, rather than affecting the entire cranial and cardiac NCC
population. The techniques we have used would only identify global changes in
cell number or function. Examination of distinct NCC populations using a
variety of markers is currently in progress.
It has been proposed that NCCs contribute to the frontal facial vasculature
(Etchevers et al., 2001).
Therefore, NCC-derived vascular mural cells (pericytes and VSMC) could fail to
populate the frontonasal region, resulting in a disruption of vascular flow or
potentially tissue remodeling. In this situation, the mesenchyme might not
receive the appropriate signals to mature, resulting in the multiple bone
defects that we observe. Supporting this mechanism, a hemorrhage is always
present in the frontonasal process and subsequently the nasal capsule in our
NCC conditional embryos. Although our data using a pericyte lacZ
reporter mouse line (Tidhar et al.,
2001
) indicated no defects in pericytes (data not shown), it is
possible that a unique subset of VSMC may be absent or defective. This
possibility is currently under investigation.
In addition to the defects in bone and cartilage derived from cranial NCCs,
defects in other NCC derivatives in the heart, thymus, teeth and eye have been
described in Ph and/or Pdgfra mutant embryos. The aortic
arch defects are readily observable in the NCC conditional embryos, although
the penetrance appears to be less than that reported in the Ph
embryos. The range of defects that we have observed in the NCC conditional
embryos bears a striking resemblance to abnormalities seen in chick cardiac
crest ablation experiments (Kirby et al.,
1983; Kirby and Waldo,
1990
). Although multiple deficiencies were present in the NCC
conditional embryos, the most common defects were those concentrated around
the conotruncal ridges and aorticopulmonary septum. The fourth and sixth arch
arteries contribute to the RSA, ventricular septum and trunk of the arch.
Misalignment of the right subclavian artery, ventricular septal defects and
persistent truncus arteriosus were often observed in our mutant embryos, while
defects of the common carotid and pulmonary arteries were never observed.
Therefore, we propose that the PDGFR
, rather than being essential for
all cardiac crest, may be important for morphogenesis of the fourth and sixth
arch arteries. This hypothesis is supported by the disruptions we observed in
the branchial arch arteries earlier in development. It is interesting that the
timing of observable defects in cardiac crest cells is the same as that of the
craniofacial clefting. In both situations, the NCCs have migrated to their
final destination and have adopted a mesenchymal phenotype. The common feature
for both tissue populations is that there is a significant amount of cellular
rearrangement and tissue remodeling. In the cranial region, the two frontal
nasal processes must fuse, while in the aortic arch, multiple vessels are
remodeling and/or regressing.
A multitude of mouse mutants have been described with defects in cranial
and/or cardiac neural crest derivatives. Clefting of the palate is quite
common but the median cleft extending down the forebrain as seen in the
PDGFR mutant embryos is rather rare. This defect results in a virtual
absence of the nasal capsule, agenesis of the nasal septum and cleft palate.
Similar frontal nasal clefting is seen in transcription factor knockouts
(ALX4/Cart double homozygotes) and nuclear receptor knockouts
(RAR
/
double homozygotes)
(Lohnes et al., 1994
;
Qu et al., 1999
),
respectively. How these particular signaling pathways impact one another
warrants further investigation.
The most prevalent defect observed in the aortic arch of the NCC
conditional embryos is disruption of RSA origin and failure of the conotruncal
area to septate. Both of these aortic arch anomalies can be traced back to
abnormal development of the fourth arch artery. These disruptions closely
resemble those described for haploinsufficiency of the Tbx1 transcription
factor that has been implicated in the DiGeorge syndrome phenotypes
(Jerome and Papaioannou, 2001;
Lindsay et al., 2001
;
Merscher et al., 2001
). Tbx1,
however, is expressed in the mesenchyme adjacent to the NCC component of the
aortic arch (Garg et al.,
2001
) and therefore could only interact indirectly with
PDGFR
pathway.
We have shown that although the PDGFR may play an early role in NCC
expansion, this role is nonessential. In addition, it is unlikely that the
receptor is required for general NCC migration, proliferation or survival. We
conclude that the NCC requirement for PDGFR
signaling lies in another
cellular function such as differentiation or extracellular matrix deposition,
or that PDGFR
action may be on a subset of NCC-derived cells. Loss of
the PDGFR
gene exclusively in NCC illustrates an array of defects in
both craniofacial and aortic arch development. Our results using both chimeric
and conditional analysis conclusively show that the PDGFR
is required
cell autonomously in the cranial and cardiac NCC lineages.
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ACKNOWLEDGMENTS |
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