1 Department of Anatomy, Faculty of Medicine, The Chinese University of Hong
Kong, Hong Kong, China
2 Neural Development Unit, Institute of Child Health, University College London,
London WC1N 1EH, UK
* Author for correspondence (e-mail: wy-chan{at}cuhk.edu.hk)
Accepted 24 March 2004
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SUMMARY |
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Key words: Cardiac neural crest, Migration, splotch, Labelling, Transplantation, Embryo culture, WGA-Au, DiI, Grafting, Outflow tract, Persistent truncus arteriosus
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Introduction |
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In mammals, a role for the cardiac neural crest during cardiovascular
development has been established through studies of several mouse mutants with
congenital heart defects that affect the outflow tract and great vessels
(Esptein, 1996; Conway et al.,
1997; Yanagisawa et al.,
1998
; Brannan et al.,
1994
; Lo et al.,
1999
; Winnier et al.,
1999
). Moreover, neural crest defects have been implicated in the
pathogenesis of some forms of human congenital heart disease including
DiGeorge/velocardiofacial syndrome (Lipson
et al., 1991
; Driscoll,
1994
) and retinoic acid embryopathy
(Rothman et al., 1995
).
The majority of information on neural crest migration has been derived from
chick embryos using quail-to-chick chimaeras, in situ cell labelling,
immunohistochemistry and ablation of the premigratory neural crest
(Le Douarin and Kalcheim,
1999). In mammals, the lack of morphological features or specific
markers that allow neural crest cells to be identified throughout the majority
of their developmental time course has hampered the investigation of their
migration and differentiative fate. Exogenous cell labelling in rodents has
provided information on the migratory pathways of the neural crest to the
heart (Fukiishi and Morriss-Kay,
1992
; Osumi-Yamashita et al.,
1996
). Moreover, several molecular markers, both intrinsic
(Conway et al., 1997
) and
transgenic (Lo et al., 1997
;
Liu et al., 1994
;
Means and Gudas, 1997
;
Serbedzija and McMahon, 1997
),
have also been used to trace the initial migratory population of the cardiac
neural crest. Definitive evidence for a contribution of the mammalian neural
crest to cardiovascular development was provided by fate-mapping studies in
the mouse using a transgenic system based on Cre/loxP recombination
(Jiang et al., 2000
). The
conclusion from these studies is that the mammalian cardiac neural crest
arises from the occipital region of the neural tube and populates the outflow
tract similarly to avians.
Despite these several previous studies, several unanswered questions remain
concerning the mammalian cardiac neural crest. First, the specific
rostrocaudal level of origin of the cardiac neural crest lineage has not been
determined. Second, there has been no detailed spatio-temporal study of the
route taken by the mammalian neural crest en route to the heart. Third,
considerable controversy surrounds the issue of the cell-autonomy or
non-cell-autonomy of the neural crest defects in the splotch mutant
mouse, a much-studied mammalian genetic model of abnormal cardiac neural crest
development (Auerbach, 1954;
Franz, 1989
;
Epstein, 1996
). In the present
study, we have investigated these three questions using an experimental design
involving exogenous labelling of mouse cardiac neural crest, followed by whole
embryo culture in order to trace neural crest cell migration in the intact
embryo. We used two cell labels, wheat germ agglutinin gold conjugates
(WGA-Au) (Smits-van Prooije et al.,
1986
; Chan and Tam,
1988
; Trainor and Tam,
1995
) and DiI, to label the premigratory neural crest by direct
microinjection or by orthotopic transplantation of a fragment of labelled
neural crest tissue into an unlabelled embryo. In addition, we followed neural
crest migration in the splotch (Sp2H) mouse
mutant and performed reciprocal neural crest transplants between embryos of
different genotypes to determine the degree of cell autonomy of the
splotch neural crest migration defect.
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Materials and methods |
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Mouse strains and embryo isolation
Random bred ICR (Institute of Cancer Research, Harlan, Oxfordshire, UK)
mice were used for studies of normal neural crest migration. Random bred
splotch (Sp2H) mice, which harbour an intragenic
deletion of the Pax3 gene, were genotyped as described previously
(Epstein et al., 1991). Both
mouse strains were kept under a 12-hour light:12-hour dark cycle at the
Laboratory Animal Services Centre of The Chinese University of Hong Kong. Noon
on the day of finding a copulation plug was designated E0.5 assuming that
copulation occurred around midnight. Pregnant mice were sacrificed by cervical
dislocation at E8.0 to E8.5, and embryos at various somite stages were
dissected from the decidua in PB1 medium
(Whittingham and Wales, 1969
).
The visceral yolk sac and ectoplacental cone were kept intact while Reichert's
membrane and its adherent parietal endodermal cells were removed
(Chan and Tam, 1988
;
Chan and Lee, 1992
).
In situ focal labelling
DiI or WGA-Au solution was directly deposited in the vicinity of the neural
crest by microinjection using a Leitz micromanipulator on a Wild
stereomicroscope (Chan and Tam,
1988; Chan et al.,
2003
). Injection pipettes were prepared from glass micropipettes
of internal diameter 0.85 mm (GC100T-15, Clark Electromedical Instruments,
Kent, UK) using a vertical pipette puller (KOPF Instruments, Model 720,
Tujunga, CA, USA) to give an internal diameter of approximately 10-15 µm.
The tip of the injection pipette was heat-polished with a microforge
(Narishige Scientific, MF-79, Setagaya-ku, Tokyo, Japan). The deep red WGA-Au
solution or the slightly reddish-yellow DiI solution was slowly dispensed from
the injection pipette, under the control of an oil pump (DeFonbrune,
CIT-ALCATEL, Paris, France), onto the dorsal margins of the neuroepithelium at
different axial levels of the hindbrain
(Fig. 1A) after the injection
pipette pierced through the visceral yolk sac and the amnion. At the level of
somites 4 and 5, where the neural tube was already closed, the injection
pipette punctured the surface epithelium to label the dorsal neural tube. Care
was taken not to spill the labelling solution onto the surface epithelium, so
as to avoid labelling the epibranchial placodes, which are known to contribute
migrating cells within the cranial mesenchyme
(Lumsden et al., 1991
;
Le Douarin and Kalcheim,
1999
). The injection site was checked carefully after injection.
If the labelled neural crest region was found to extend over a rostrocaudal
distance of more than one somite length, or if the labelling site did not
correspond to the intended region, the embryo was discarded from further
analysis.
|
Embryo culture
Embryos after labelling or grafting were cultured in groups of five in 50
ml serum bottles containing 5 ml of heat-inactivated (56°C for 30
minutes), immediately centrifuged pure rat serum. The cultures were kept in a
roller system at 37°C for a maximum of 48 hours
(Chan and Tam, 1988;
Chan and Lee, 1992
). Assessment
of mean somite number, mean crown-rump length and a series of morphological
features revealed no significant differences between E8.5 non-mutant ICR
embryos developing in culture for 48 hours and E8.5 control embryos developing
entirely in vivo (data not shown). Moreover, focal labelling and orthotopic
grafting had no significant effect on the success of embryonic development in
culture. E8.5 embryos from splotch litters, whether focally labelled,
grafted or unoperated, grew and developed in culture with no significant
differences from ICR embryos (data not shown).
Processing of cultured embryos
Embryos that had been labelled with WGA-Au or grafted with a
WGA-Au-labelled fragment were dissected free of extraembryonic membranes,
rinsed in PBS, fixed in Carnoy's fixative for 45 minutes and processed for
paraffin wax histology. Serial transverse sections of 6 µm thickness were
prepared through the hindbrain region and stained with silver
(Chan and Lee, 1992). Embryos
labelled with DiI were rinsed in PBS, fixed in 4% paraformaldehyde for 18 to
20 hours and dehydrated in a graded series (10-30%) of sucrose solutions
before embedding in OCT compound (Tissue-TEK). Serial transverse sections were
cut at 16 µm on a cryostat (Shandon, Cheshire, UK) and examined under a
Zeiss epifluorescence microscope equipped with a rhodamine filter.
Cell counting
Following WGA-Au focal injection, labelled neural crest cells with silver
granules in their cytoplasm were counted only in sections in which more than
90% of the cells of the dorsal part of the neural tube were silver
granule-laden. This ensured that only fully labelled embryos were analysed in
detail. Cell counts were performed at 400x magnification on a Zeiss
Axioplan2 microscope linked to a Spot-Cooled Color Digital Camera (Diagnostic
Instruments, Sterling Heights, MI, USA). Images were captured with a MetaMorph
Imaging System (Universal Imaging Corporation, Downington, PA, USA). The
number of labelled cells at each time point was computed from at least five
embryos. The regions where cell counting was performed were defined as: (1)
trigeminal and facio-acoustic ganglia: discrete aggregates of cells in the
cranial mesenchyme; (2) second pharyngeal arches: all labelled cells beneath
the pharyngeal arch epithelium; (3) medial neural crest migratory pathway:
labelled cells within the mesenchymal region between neural tube and somite
extending dorsoventrally from the dorsal edge of the somite to its ventral
tip; (4) peri-aortic mesenchyme: the circular area surrounding the dorsal
aorta with a width equal to the radius of the vessel; (5) mesenchymal region
lateral to the pharynx: all cells between the dorsoventral extent of the
pharynx and the surface epithelium.
Statistical analysis
Morphological scores were compared by Chi-squared test whereas somite
numbers, crown-rump lengths and cell numbers were compared by unrelated
Student's two-tailed t-test. The level of significance was set at
P<0.05.
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Results |
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When ProRhB was labelled (Fig. 2A), a stream of labelled cells was observed to traverse the cranial mesenchyme to the second pharyngeal arch (Fig. 2B). No labelled cells were found in the cardiac outflow tract (Fig. 3) or any other cardiac structures. When the labelling site was ProRhC, labelled cells were distributed along a subepithelial pathway to the third pharyngeal arches (Fig. 2C,D). This pathway extended dorsoventrally from the neural crest to the vicinity of the developing pharynx (Fig. 2D). A similar distribution of labelled cells was found when a DiI-labelled neural crest fragment was grafted orthotopically to ProRhC (Fig. 2E,F). Of the 41 embryos labelled or grafted at the level of ProRhC, only three (7.3%) showed labelled cells in the cardiac outflow tract (Fig. 3), indicating that the neural crest from ProRhC is not a major source of cardiac neural crest.
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At subsequent stages, further ventral migration through the pharyngeal arches appeared to slow down, whereas the third to sixth pharyngeal arches were undergoing expansion. Translocation of labelled cells became visible ventral to the pharynx at the 21-somite stage (n=22; Fig. 5H). Labelled neural crest cells continued to arise from the neural tube, at the level of ProRhC to S4, until the 23-somite stage (n=25), at which time the basal aspect of the dorsal neuroepithelium resumed a smooth contour and emigrating cells were no longer observed (Fig. 5I). At 26 somites (n=7), some of the most ventrally migrating neural crest cells reached the proximal end of the cardiac outflow tract (Fig. 6A), and by 32 somites (n=5) a few neural crest cells could be seen within the mesenchyme of the outflow tract (Fig. 6B).
|
Cardiac neural crest migration in splotch embryos at the level of
S2 was compared with non-cardiac neural crest migration from the levels of
ProRhA and ProRhB. The spatio-temporal distribution of labelled neural crest
from ProRhA and ProRhB of wild type (+/+), heterozygous
(Sp2H/+) and homozygous
(Sp2H/Sp2H) splotch embryos was found
to be very similar to that observed in non-mutant ICR embryos (data not
shown). At 9, 12-13, 16-17 and 20-21 somites, the numbers of labelled cells in
the trigeminal ganglion, facio-acoustic ganglion and second pharyngeal arches
were similar in ICR and in all three splotch genotypes
(Fig. 7A-C). Hence, the
Pax3 mutation in splotch does not appear to have a major
effect on the migration of neural crest cells at the level of the rostral
hindbrain, consistent with previous findings
(Serbedzija and McMahon,
1997).
|
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At 16 to 17 somites, many labelled cells had migrated to the region lateral to the developing pharynx of +/+ (n=9) and Sp2H/+ (n=12) embryos (Fig. 8G,H), whereas in Sp2H/Sp2H (n=11) embryos, significantly fewer labelled cells were present and these had only reached the peri-aortic mesenchyme (Fig. 7E, Fig. 8I). Labelled neural crest was not present in the mesenchyme lateral to the pharynx of Sp2H/Sp2H embryos at this stage (Fig. 7F, Fig. 8I). By 20 to 21 somites, labelled cells in both +/+ (n=5) and Sp2H/+ (n=18) embryos had translocated ventral to the pharynx (Fig. 8J,K), whereas Sp2H/Sp2H embryos (n=8) exhibited fewer labelled cells and these were no farther ventral than the pharynx (Fig. 7F, Fig. 8L).
In conclusion, up to the 21-somite stage, significantly fewer labelled cells were observed in the medial pathway and peri-aortic mesenchyme of Sp2H/Sp2H embryos, compared with +/+ and Sp2H/+ littermates, whereas splotch homozygotes showed no labelled neural crest ventral to the pharynx (Fig. 7D-F).
Interactions between the splotch neural crest and its migratory environment
To determine whether the retarded neural crest migration in
splotch embryos is cell-autonomous or regulated by the migratory
environment, we performed orthotopic grafting of WGA-Au-labelled neural crest
fragments at the 5- to 6-somite stage between embryos of different
splotch genotypes (Table
1). All grafts were performed without knowledge of the genotype
combination which was revealed only retrospectively. A total of 135
splotch embryos were grafted, of which 103 (76%) had the graft
successfully placed in the neural crest region, and were subsequently
cultured. After 24 hours of culture, 12 embryos (12%) had failed to undergo
axial rotation and were discarded. Of the 25
Sp2H/Sp2H recipient embryos, 16 (64%) exhibited
an open cranial neural tube suggesting that grafting had not normalised
cranial neural tube closure.
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Discussion |
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Axial level of origin of the mouse cardiac neural crest
In birds, the region of the neural crest contributing cells to the
developing heart arises from the axial level between the midotic placode and
the caudal limit of somite 3 (Kirby and
Waldo, 1990; Kirby and Waldo,
1995
; Miyagawa-Tomita et al.,
1990
). The neural crest from rhombomeres 6 to 8, which extend down
as far as somite 4, has also been shown to contribute to the wall of the
pharyngeal arch arteries and the aorto-pulmonary septum
(Couly et al., 1998
). In
mammals, however, the rostrocaudal extent of the cardiac neural crest along
the neural axis has not been clearly delineated. DiI labelling of the
premigratory neural crest in rat embryos showed that cells from the level of
the first to fourth occipital somites migrate to the cardiac outflow tract.
Colonisation of cardiac structures by labelled cells was not observed when the
neural crest was labelled just rostral to the first somite or just caudal to
the fourth somite (Fukiishi and
Morriss-Kay, 1992
). The discrepancy in the apparent rostral extent
of the cardiac neural crest in the chick and rat has been attributed to
anatomical differences between the species: the region between the post-otic
and pre-somitic hindbrain is longer in mammals than birds, whereas an
additional pair of occipital somites is found in birds compared with mammals
(Fukiishi and Morriss-Kay,
1992
).
More recently, transgenic fate-mapping using Cre-loxP technology has
provided information on the cardiac neural crest of the mouse. Cells
expressing the lacZ reporter, expressed because of activation of the
Wnt1 promoter in the premigratory neural crest, were detected in the
developing pharyngeal arch arteries and the cardiac outflow tract with
similar, although not identical, distribution patterns to those found in avian
hearts (Jiang et al., 2000).
These findings have confirmed the existence of a cardiac neural crest lineage
in mammals (Lo et al., 1997
;
Yamauchi et al., 1999
;
Jiang et al., 2000
;
Li et al., 2000
). However,
because the transgenes are expressed in the neural crest at all axial levels
(Jiang et al., 2000
;
Li et al., 1999
;
Lo et al., 1997
;
Pietri et al., 2003
) or
expressed only at relatively advanced stages when the neural crest cells have
already come close to the heart (Tremblay
et al., 1995
), it is difficult from these studies to ascertain the
axial levels of origin of the cardiac neural crest.
In the present study, we performed a rostrocaudal, region-by-region
analysis of neural crest migratory fate, and found that the mouse cardiac
neural crest arises from a length of neural tube extending from the post-otic
hindbrain to the caudal limit of somite 4. This observation agrees with
previous findings in the chick. We demonstrated, moreover, that the neural
tube at the level of somite 2 is quantitatively the most prolific source of
cardiac neural crest. Cells arising from ProRhC (the post-otic region rostral
to the first somite) (Osumi-Yamashita et
al., 1996), and from the neural tube at the level of somites 3 and
4, contribute fewer cells to the developing outflow tract. This finding
suggests that the mouse neural crest has rather specific migratory fates,
depending on its precise axial level of origin, and that there is unlikely to
be much mixing between neural crest populations emanating from adjacent levels
of the neural tube. This is in contrast to the chick where the pharyngeal
arches 3 to 6 receive neural crest cells from a region of neural tube up to
three somites in length (Shigetani et al.,
1995
).
Migratory pathways taken by cardiac neural crest in the mouse
Our study shows that the mouse cardiac neural crest emigrates from the
neural tube between the 7- and 23-somite stages. In a similar way to the
cranial neural crest (Le Douarin and
Kalcheim, 1999; Chan and Tam,
1988
), cardiac neural crest cells derived from the post-otic,
pre-somitic ProRhC level were restricted to the dorsolateral pathway beneath
the surface epithelium and lateral to the cardinal vein. In contrast, neural
crest emanating from the levels of somites 1 to 4 exhibited a much more
complex migration pattern, following dorsolateral, medial and intersomitic
pathways, as previously described for the avian cardiac neural crest and for
the trunk neural crest of both birds and mammals (for a review, see
Le Douarin and Kalcheim,
1999
). In birds, the early migrating cardiac crest occupies the
dorsolateral pathway prior to colonisation of the medial pathways
(Tucker et al., 1986
;
Kuratani and Kirby, 1991
;
Reedy et al., 1998
), whereas
we found mouse cardiac neural crest cells migrating simultaneously along all
three pathways, similar to that described for early mouse trunk neural crest
migration (Serbedzija et al.,
1990
). Spatially, the chick and mouse pathways also differ with
the chick cardiac neural crest traversing the dorsolateral pathway at the
levels from the somites 1 to somite 4 as in the mouse, but occupying the
medial and intersomitic pathways only in regions caudal to somite 2
(Kuratani and Kirby 1991
).
Moreover, the chick medial pathway can be subdivided into ventrolateral
(sclerotomal) and ventromedial (adjacent to the neural tube) components,
whereas this subdivision was not distinct in the mouse.
The chick neural crest, migrating on the dorsolateral pathway, temporarily
ceases its migration and forms a distinct longitudinal mass of cells within
the lateral body wall, dorsolateral to the dorsal edge of the pericardium
(Kuratani and Kirby, 1991;
Kuratani and Kirby, 1992
).
This `circumpharyngeal crest' subsequently populates the expanding pharyngeal
arches. In the mouse, we did not observe the precocious arrival of neural
crest prior to the lateral expansion of the caudal pharyngeal arches, and no
distinct mass of `circumpharyngeal crest' cells was observed within the
lateral body wall.
Migration of cardiac neural crest in the splotch mutant mouse
Splotch is a well-recognized genetic model of neural crest and
neural tube defects (Auerbach,
1954). Homozygous loss of Pax3 function in
splotch mice (Epstein et al.,
1991
; Epstein et al.,
1993
; Goulding et al.,
1993
; Vogan et al.,
1993
) produces a phenotype closely resembling that of chick
embryos following cardiac neural crest ablation. This has led to the idea that
cardiac neural crest development is compromised in splotch
(Epstein, 1996
;
Conway et al., 1997
).
Several lines of evidence support defective migration of neural crest cells
from the neural tube to the heart in splotch embryos. Use of a
Wnt-1::lacZ transgenic reporter revealed a severe reduction in the
number of neural crest cells emigrating from both vagal and rostral trunk
levels of the splotch neural tube
(Serbedzija and McMahon,
1997). Moreover, a reduced number of migrating splotch
neural crest cells has been deduced indirectly from the observation of
decreased expression of various neural crest markers and transgenes
(Conway et al., 1997
;
Conway et al., 2000
;
Tremblay et al., 1995
;
Serbedzija and McMahon, 1997
;
Epstein et al., 2000
).
Alternatively, the Cx43-lacZ transgene is reported to be expressed
similarly in the cardiac neural crest of wild type and splotch
embryos. This has prompted the conclusion that Pax3 is not essential
for early cardiac neural crest migration but, rather, is important for
fine-tuning the migratory behaviour of the cardiac neural crest cells upon
their arrival in the heart (Esptein et al., 2000). Our findings do not support
this latter conclusion, but instead confirm a reduction in the cardiac neural
crest population of splotch embryos during the migratory phase
towards the heart. We detected a delay by 3 somite stages (i.e. approximately
6 hours of development) in the onset of cardiac neural crest emigration from
the splotch homozygous neural tube. This early, transient delay in
the onset of neural crest emigration can explain why splotch embryos
at more advanced stages have been reported to exhibit normal neural crest
emigration (Conway et al.,
1997
; Conway et al.,
2000
). Moreover, our direct labelling of the premigratory neural
crest shows that although mutant neural crest cells are capable of migrating
along normal pathways, the number of the cells en route is significantly
reduced in splotch homozygotes, with a discernible reduction also in
heterozygotes. Hence, our studies support the suggestion that a decrease in
the number of migrating neural crest cells is the primary defect that
ultimately leads to a lack of cardiac neural crest-derived cells in the
outflow tract of splotch embryos
(Conway et al., 2000
).
We observed a reduced number of migrating neural crest cells only in the
cardiac neural crest population of splotch embryos. Neural crest
cells arising rostral to the otic vesicle appeared in normal numbers, with
normal colonisation of cranial ganglia. Hence, there is an increase in the
severity of the neural crest defect along the rostrocaudal axis of
splotch embryos, consistent with previous findings
(Auerbach, 1954;
Serbedzija and McMahon,
1997
).
Cell autonomy versus non-cell autonomy in splotch cardiac neural crest defects
An issue of some controversy is whether the defect of cardiac neural crest
migration in splotch embryos is autonomous to the neural crest cell
lineage, or whether it reflects an influence of the mutant migratory
environment (non-cell autonomous). Transgenic reconstitution of Pax3
expression solely in the dorsal neural tube (including the neural crest
lineage) was reported to rescue neural tube closure, cardiac septation and
dorsal root ganglion formation in splotch embryos
(Li et al., 1999). This
finding has been taken as strong evidence for a cell autonomous role of
Pax3 in the neural crest, although the study did not reveal whether
the rescuing transgene was expressed in early cranial or paraxial mesoderm,
tissues that provide the immediate migratory environment for the emigrating
neural crest. Several other lines of evidence support a different view of the
splotch neural crest defect. Neural crest cells derived from the
caudal trunk level (Serbedzija and
McMahon, 1997
) or vagal level
(Conway et al., 2000
) of
splotch embryos can follow normal migratory pathways after
transplantation to chick embryos, suggesting that the migratory environment
plays a crucial role in regulating neural crest migration. Similarly, chimaera
studies, in which wild type and Pax3-deficient cells co-exist in the
same embryo (Mansouri et al.,
2001
), have supported a non-cell autonomous mechanism for the
splotch neural crest defect. Abnormalities of extracellular matrix
composition in early splotch embryos are consistent with an adverse
effect of the migratory environment on the neural crest
(Henderson et al., 1997
).
Our results offer a possible resolution of this controversy. We transplanted labelled premigratory neural crest cells between splotch embryos of different genotypes and found that the defects of cardiac neural crest migration occurred only when both donor and recipient embryos were of mutant genotype. Having either a wild-type neural crest lineage, or a wild-type migratory environment, was sufficient to rescue neural crest migration. Alternatively, even a splotch heterozygous neural crest lineage performed poorly in a homozygous mutant environment. Interestingly, the reverse was less true. Homozygous splotch neural crest cells migrated more effectively in a heterozygous environment. We conclude that the splotch mutant effect is mediated both through the neural crest cell lineage and the migratory environment, but that the effect of the environment may be more potent.
There is much evidence to support a critical role for reciprocal
interactions between neural crest and environment in regulating migration (for
a review, see Le Dourain and Kalcheim,
1999). The molecular composition of the neural crest cell surface,
and of the migratory pathways, are both highly heterogeneous as well as
temporally dynamic (Le Douarin and
Kalcheim, 1999
; Garcia-Castro
and Bronner-Fraser, 1999
;
Christiansen et al., 2000
;
Maschhoff and Baldwin et al., 2000; Perris
and Perissinotto, 2000
). Different neural crest sub-populations,
often varying in developmental potential, respond differently to a given
migratory cue and continuously modify their fates during migration
(Weston, 1991
;
Vaglia and Hall, 1999
;
White et al., 2001
).
Conversely, migrating neural crest cells also actively participate in shaping
the molecular composition of their own migratory routes. It has been suggested
that Pax3 expression may be required to regulate cell surface
properties (Mansouri et al.,
2001
), both on neural crest and its migratory environment, perhaps
via downstream regulation of Msx2
(Kwang et al., 2002
). It
remains to be determined precisely how this molecular pathway may enhance
neural crest migration, and how its genetic disruption leads to defects of the
cardiac neural crest.
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ACKNOWLEDGMENTS |
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