1 California Institute of Technology, Pasadena, CA 91125, USA
2 Kansas State University, Manhattan, KS 66506, USA
* Author for correspondence (e-mail: mbronner{at}caltech.edu)
Accepted 15 January 2004
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SUMMARY |
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Key words: Neural crest, Cornea, Trigeminal ganglion, Branchial arch, Quail-chick chimera
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Introduction |
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Much of what we know about neural crest derivatives comes from elegant and
now classic transplantation experiments performed in birds. Most notably,
chimeric grafts in which the neural tube of a quail embryo was transplanted in
place of a chick host neural tube were used to define the derivatives that
arose from the neural crest at various axial levels
(Weston, 1963;
Le Douarin and Teillet, 1974
)
(reviewed by Le Douarin,
1982
). In this way, it was established that cranial neural crest
cells, extending from the forebrain to the level adjacent to the third somite,
form parts of the cranial sensory ganglia
(D'Amico-Martel and Noden,
1980
; Le Lièvre and Le
Douarin, 1982
), the autonomic ciliary ganglion
(Le Douarin and Teillet, 1974
)
and the endothelium and stroma of the cornea
(Noden, 1978a
;
Johnston et al., 1979
). In
addition, they contribute to smooth muscle, connective tissue, bone and
cartilage of the face (Le Douarin,
1982
; Olsson et al.,
2001
). Furthermore, cells arising from the `cardiac' subregion of
the cranial neural crest, spanning from rhombomere 6 to the level adjacent to
the third somite (Kirby et al.,
1983
; Kirby et al.,
1985
), migrate into pharyngeal arches 3, 4 and 6. These cells
contribute to connective tissues, blood vessels and the endocardial cushion
tissue that later differentiates into the cardiac outflow tract
(Le Lièvre and Le Douarin,
1975
; Kirby et al.,
1983
). Vagal neural crest cells, arising adjacent to somites 1-7,
give rise to enteric ganglia of the gut
(Le Douarin and Teillet,
1973
). Trunk neural crest cells, arising at the level of somites
8-28, give rise to melanocytes, sensory and autonomic neurons and glia,
Schwann cells and adrenal chromaffin cells
(Weston, 1963
).
Clear differences in the potential of neural crest cells to form these
different derivatives exist along the rostrocaudal axis. This was first
demonstrated by heterotopic grafting experiments in which cranial neural crest
was grafted to trunk levels and vice versa. The results revealed that cranial
neural crest cells could contribute to all truncal derivatives but also formed
ectopic cartilage nodules after transplantation into the trunk levels
(Le Douarin and Teillet, 1974;
Le Lievre et al., 1980
). By
contrast, trunk neural crest cells contributed to some normal cranial
derivatives such as the cranial ganglia, but were unable to form cartilage of
the head (Noden, 1978a
;
Nakamura and Ayer-Le Lievre,
1982
). This suggested that the cranial neural crest has a broader
(or at least different) developmental potential than that of trunk neural
crest. However, under appropriate culture conditions, trunk neural crest cells
have been shown to acquire some properties of chondrocytes
(McGonnell and Graham,
2002
).
The fact that trunk neural crest cells can form cartilage under appropriate
conditions raises the possibility that previous techniques may not have been
sufficiently sensitive to detect all neural crest phenotypes. In the original
quail/chick transplantation experiments performed over 25 years ago, quail
cells were recognized by histological staining for heterochromatin
(Le Douarin, 1973;
Le Douarin, 1974
). This was
effective for groups of cells but not sufficiently sensitive to achieve single
cell resolution. Furthermore, differentiated cell types were assigned by
location rather than by means of cell type-specific markers that are available
today. Thus, it is possible that differentiation of some trunk neural crest
cells into appropriate cranial derivatives may have been missed. In addition,
previous experiments concentrated on skeletal derivatives; thus,
differentiation and survival of quail cells into the cornea and trigeminal
neurons after heterotopic transplantation were not examined. Given the
significance of these classic experiments to our understanding of neural crest
developmental potential along the neural axis, it seems important to revisit
the elegant grafting experiments of Le Douarin and colleagues using modern
approaches.
In order to test the relative roles of intrinsic information versus environmental influences on neural crest cell fate, we have challenged the developmental potential of cardiac and trunk neural crest cells by transplanting them into an ectopic midbrain environment. We examined their long-term ability to contribute to the cornea, trigeminal ganglia and first branchial arch cartilage and bone using a combination of cell labeling techniques and molecular markers of differentiation and positional identity. The results show that all populations contribute equally well to non-neuronal cells of the cranial ganglion and to melanocytes. Despite their ability to properly migrate along appropriate midbrain neural crest pathways, there appears to be a progressive loss of ability to contribute to the endothelium and stroma of the cornea, somatosensory neurons of the trigeminal ganglion and branchial arch cartilage with distance along the rostrocaudal axis. Expression of Hoxa2 and Hoxa3 was transiently maintained in cardiac neural crest after transplantation to the midbrain but was subsequently downregulated. This suggests that long-term maintenance of Hox gene expression cannot account for rostrocaudal differences in developmental potential of neural crest populations in this case.
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Materials and methods |
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Quail-chick grafts
Dorsal neural tube explants were ablated from the midbrain region (between
posterior mesencephalon and anterior metencephalon) of stage 9 chick hosts and
replaced with quail dorsal neural tubes of similar size from midbrain, cardiac
or trunk axial levels removed from stage 9-10 donor embryos. Eggs were sealed
with Scotch tape and chimeric embryos were reincubated for an additional 5 to
15 days. Corneas and trigeminal ganglia were analyzed from embryonic day 15
(E15) chimeras. Because bones had ossified by this stage, we chose E5-7
chimeras to look at contribution to cartilage.
Antibodies and immunostaining
E15 chimeric embryos were sacrificed and their heads collected in cold 4%
paraformaldehyde. Corneas and trigeminal ganglia were dissected out and fixed
further at room temperature for 2 hours with mild agitation then rinsed twice
in 0.1 M PBS. Trigeminal ganglia and corneas were embedded in gelatin and
cryosectioned at 10 or 12 µm. PBS containing 0.2% (w/v) bovine serum
albumin, 0.2% (v/v) Triton X-100 and 5% (v/v) heat-inactivated goat serum
(PBT) was used as antibody buffer. Quail-specific neural antibody-QN (mouse
IgG1) (Tanaka et al., 1990)
and quail-specific nuclear marker-QCPN (mouse IgG1, Developmental Studies
Hybridoma Bank [DHSB], University of Iowa) were used diluted 1:1 in PBT to
label the grafted quail cells. Rabbit anti-neuron-specific class III
ß-tubulin antibody TuJ1 (IgG2a, BABCO) was used diluted 1:500 to label
all nerves (quail and chick). TrkA rabbit IgG antibody was used diluted 1:1000
to detect neural crest-derived sensory neurons in the trigeminal ganglion.
Rabbit anti-collagen II (IgG, BABCO) was used diluted at 1:200 to detect
chondrocytes. Mouse anti-MF20 (IgG2b, DSHB) against heavy chain myosin was
used diluted 1:3 to label the ciliary muscles of the eye. HNK-1 (IgM) was used
diluted at 1:50 to label migrating neural crest cells. Fluorochrome-conjugated
goat secondary antibodies were purchased from Molecular Probes (Alexa Fluor
488 anti-mouse IgG2b, Alexa Fluor 594 anti-mouse IgG1, Alexa Fluor 594
anti-mouse IgM, and Alexa-Fluor 488 anti-rabbit IgG1 and 2a) and used diluted
1:200. Immunostained sections were counterstained with DAPI, rinsed in PBS and
mounted on slides using Perma Fluor (Immunon, Pittsburgh, PA). Fluorescent
images were captured using a Zeiss Axiocam mounted on a Zeiss Axioskop 2
microscope. Images were processed using Adobe PhotoShop (Adobe Systems).
In situ hybridization
Stage 12-18 embryos were harvested, trimmed, and fixed in 4%
paraformaldehyde. In situ hybridization was carried out as previously
described (Henrique et al.,
1995).
Microscopy and imaging
Fixed trigeminal ganglia and corneas were photographed prior to sectioning
and immunostaining using an Olympus DP10 digital camera mounted on a Zeiss
Stemi SV11 microscope. Images of all trigeminal ganglia were taken at the same
magnification. To determine the relative areas of trigeminal ganglia, images
were imported into Adobe Photoshop, digitally trimmed by erasing the sensory
root and maxillo-mandibular nerves at the point of bifurcation. The ophthalmic
branch was always cut adjacent to the spinal cord during dissection. Trimmed
images were imported into the NIH-Image program. All ganglia were traced using
the threshold option, and their trimmed areas were measured. All area
measurements were compared statistically using a Student's
t-test.
To determine the number of neurons, serial sections representing each entire ganglion were mounted on one slide and immunostained. Three representative sections with the highest numbers of neurons were chosen from each slide, and the region with the highest number of neurons was selected for imaging. The numbers of neurons from each selected region were averaged and used for statistical analysis using a Student's t-test.
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Results |
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Transplantation of dorsal neural tubes from the cardiac and trunk regions
resulted in embryos with visually similar quantities of brown/black quail
melanocytes in the head region to those seen in the control chimeras that had
received a midbrain graft (Table
1; Fig. 1B,D,F). In
some cases, part of the beak was pigmented, as expected
(Noden, 1983). Visual
assessment of the morphology of the lower beaks, where most of the
transplanted neural crest would normally migrate, did not show any size
differences in the E15 chimeras.
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Trunk neural crest cells grafted to the midbrain only contribute melanocytes to the cornea
In contrast to cardiac neural crest cells, which contributed to some
corneal derivatives, trunk crest showed little or no ability to properly
incorporate into the cornea after heterotopic transplantation. Sections
through E15 trunk neural crest chimera corneas revealed no QCPN-positive cells
in the stroma or endothelial cell layers
(Fig. 2N,O). However, a few
QCPN-positive cells were present in the limbus area, the epithelial cell layer
and ciliary muscle (Fig. 2O,P).
All the QCPN-positive cells were darkly pigmented
(Fig. 2P), indicating they had
differentiated into melanocytes, and none of the cells observed in the ciliary
muscle were MF20 positive (Fig.
2O,P, arrow). These results indicate that trunk neural crest can
migrate to the periocular region but fail to form normal corneal
derivatives.
Cardiac and trunk neural crest contribute fewer neurons to the trigeminal ganglion
Grafting of cardiac and trunk neural crest to the midbrain resulted in the
formation of trigeminal ganglia that were reduced in size and had fewer
somatosensory neurons. The normal neural crest contribution to the trigeminal
ganglion comes from the midbrain region
(D'Amico-Martel and Noden,
1983) and rostral hindbrain
(Lee et al., 2003
). These
cells migrate ventrolaterally before condensing in the dorsal region of the
forming ganglion, where they continue to undergo cell division and give rise
to neurons and supporting cells
(D'Amico-Martel and Noden,
1980
). Analysis of E15 midbrain chimera trigeminal ganglia
sections showed numerous large and small QCPN-positive nuclei
(Fig. 3A,B). All the large
cells were TUJ-1 positive (data not shown) and had the morphological
appearance of neurons. The cells with large QCPN-positive nuclei were
restricted to the proximodorsal side of the ganglion whereas the small
non-neuronal cells (which probably support cells and glia) were widely spread
throughout the ganglion (Fig.
3A). In control chimera, many of the large cells were also trkA
positive (Fig. 3C), suggesting
that they were somatosensory neurons. In contrast to control transplants,
trigeminal ganglia formed in embryos that received cardiac neural crest grafts
showed a significant reduction in the number of large QCPN-positive cells, but
the number of smaller QCPN-positive cells appeared unchanged
(Fig. 3D,E). Few of the large
cells were trkA positive (Fig.
3F). The number of large QCPN-positive neurons was even more
reduced in grafts of trunk neural crest, although the numbers of small cells
remained high (Fig. 3G,H). In
most cases, the large cells in these ganglia were trkA negative
(Fig. 3I). In some trunk neural
crest chimeras, no QCPN-positive neurons were observed in the ganglia (data
not shown), although numerous small QCPN-positive cells were present
(Table 1). These results
indicate that the ability to form trigeminal neurons decreases with the axial
level of origin, but that the ability to form support cells and/or glia is not
affected.
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The trigeminal ganglion receives a dual, and approximately equal,
contribution from the neural crest and ectodermal placode cells
(Yntema, 1944;
Hamburger, 1961
;
Lwigale, 2001
). To determine
whether the reduced number of chimeric neurons affected total ganglion size,
the relative areas of trigeminal ganglia was compared between the different
types of transplants (Fig. 4A
for midbrain controls; n=10; Fig.
4B for cardiac, n=13;
Fig. 4C for trunk,
n=10). For all chimeras, the trigeminal ganglia had the appropriate
shape, forming the ophthalmic and maxillomandibular branches. Relative areas
of cardiac chimera ganglia were not significantly different
(P=0.0798) from midbrain controls, but those of trunk chimera ganglia
were significantly smaller (P=0.0005) than the midbrain controls
(Fig. 4D).
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Fewer cardiac and trunk neural crest-derived nerves innervate the cornea
Sensory innervation of the cornea is derived from the trigeminal neural
crest (Lwigale, 2001). To
determine whether the reduced population of neurons derived from cardiac or
trunk grafts form proper functional connections and innervate their normal
peripheral targets, we immunostained chimera corneas with a quail
nerve-specific antibody (QN) and then counterstained with TuJ-1. After
midbrain neural crest transplants, numerous QN-positive neurons were present
in the corneas (Fig. 5A,B). The
pattern was similar in cardiac neural crest chimera, although fewer
QN-positive neurons were observed (Fig.
5C,D). Reflecting the large reduction in quail neurons, very few
QN-positive axons were seen in the cornea after trunk transplants
(Fig. 5E,F). In some cases no
QN-positive axons were seen in trunk chimeric corneas (data not shown). The
results show that cardiac and trunk neural crest-derived axons can make
functional connections in the cornea but their numbers decrease
progressively.
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Trunk neural crest cells do not form cartilage when grafted directly into the first branchial arch
Trunk neural crest cells have been shown to form bones and cartilage when
cultured in media that promotes bone differentiation
(McGonnell and Graham, 2002).
To determine whether trunk neural crest cells can form cartilage when placed
directly into the appropriate environment in vivo, we transplanted trunk
dorsal neural tubes directly into the region of first branchial arch of stage
12-13 host embryos (n=5). As a control, similar grafts were carried
out using midbrain dorsal neural tubes (n=2). Sections through E6-7
midbrain control chimeras showed numerous QCPN-positive cells in the tongue,
mandibular process and, in particular, in Meckel's cartilage
(Fig. 7A,B). Grafting trunk
dorsal neural tubes directly into the first branchial arch increased the
number of QCPN-positive cells in the mandibular process
(Fig. 7C) compared with the
previous experiment when they were transplanted into the midbrain dorsal
neural tube (Fig. 6H). Some of
the QCPN-positive cells formed aggregates directly adjacent to the
cartilage-forming region indicated by the collagen II antibody. However, they
never became incorporated into the cartilage-forming region or expressed
collagen II. This result suggests that trunk neural crest cells do not form
cartilage even when grafted directly in a conducive environment in vivo
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At the level from which cardiac neural crest emigrates, the hindbrain
neural tube expresses a number of Hox genes, including Hoxa2, Hoxa3, Hoxb3 and
Hox4. Of these, whole-mount in situ hybridization revealed that Hoxa2 and a3
are also expressed in migrating cardiac neural crest cells (data not shown)
(Prince and Lumsden, 1994;
Saldivar et al., 1996
). To
look at the effects of transplantation of cardiac neural folds to the midbrain
on expression of these markers of rostrocaudal identity, we analyzed embryos
between 12 to 72 hours after transplantation; previous ablation experiments
had observed that the Hox code in the branchial arches was restored by 48 to
72 post-surgery (Hunt et al.,
1995
).
At 12 hours post-transplantation, both Hoxa2 and Hoxa3 were maintained in
the caudal hindbrain neural tube after transplantation to the midbrain
(Fig. 8A,E). Migrating neural
crest cells emerging from the graft also expressed Hoxa2
(Fig. 8B,C) and a3
(Fig. 8F,G), with the latter
being more robustly expressed than the former. However, maintenance of Hox
gene expression in the neural tube and neural crest appeared to occur only
transiently after transplantation to the midbrain. After 24-72 hours
post-grafting, we observed a complete absence of expression of either Hox gene
in cardiac neural crest cells transplanted rostrally to the midbrain
(Fig. 8D,H). Furthermore,
staining was observed in the donor neural tube that became integrated into the
midbrain only at 12 hours following grafting and became down-regulated
thereafter. This contrasts with the robust expression of Hoxa2 and a3 in
migrating cardiac neural crest and branchial arches in the normal environment.
The apparent downregulation of Hoxa2 and a3 was surprising given that previous
rostral transposition of rhombomere 4 to the rostral hindbrain resulted in
maintenance of its Hox expression (Prince
and Lumsden, 1994). These results suggest that Hoxa2 and a3
transcripts are maintained from 12 hours after transplantation but
downregulated thereafter in both the cardiac neural tube and neural crest.
Thus, aspects of rostrocaudal identity are only transiently maintained after
transplantation.
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Discussion |
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Hox genes are expressed in a segmented pattern in the hindbrain, that is colinear with their sequence along the chromosome, and thought to impart rostrocaudal positional information to the neural tube. Because migrating cardiac neural crest cells express a similar Hox code to their neural tube of origin, the difference in Hox gene expression between cardiac and midbrain crest offers a possible explanation for their reduced potential to contribute to normal midbrain crest derivatives. Surprisingly, our results show that Hox genes are maintained only transiently in the caudal hindbrain neural tube and neural crest after transplantation rostrally into the midbrain region. This suggests that long-term maintenance of their Hox identity cannot explain differences in developmental potential. It remains possible, however, that the early expression of Hoxa2 and Hoxa3 in the migrating cardiac neural crest is sufficient to account for developmental differences in their ability to populate midbrain crest derivatives.
Potential to form corneal cells
Cardiac neural crest contributes minimally to the cornea and forms ectopic cell masses
Neural crest cells from the midbrain, together with those from the
posterior diencephalon region, normally migrate to the region of the eye,
where they contribute to the periocular mesenchyme
(Kontges and Lumsden, 1996;
Couly et al., 1996
). During
early development of the chick eye, ectoderm overlaying the newly formed lens
synthesizes and assembles the primary stroma
(Hays, 1980
) that serves as a
substratum for migration of neural crest cells from the diencephalon and
posterior midbrain region (Johnston et
al., 1979
; Couly et al.,
1996
). These give rise to the innermost layer of the cornea, the
endothelium (Johnston et al.,
1979
). Shortly thereafter, the presumptive cornea is invaded by a
second group of neural crest cells that migrate directly into the
extracellular matrix of the primary stroma between the epithelium and
endothelium, differentiating into corneal stromal fibroblasts, called
keratocytes (Johnston et al.,
1979
). Thus, two major cell populations of the cornea are of
neural crest origin. Finally, the growth cones of sensory nerves whose cell
bodies are located mainly in the trigeminal ganglion provide sensory
innervation to the cornea (Arvidson,
1977
; Morgan et al.,
1978
; Marfurt et al.,
1989
; Lwigale,
2001
).
Grafting of quail midbrain neural crest isotopically into chick resulted in
numerous pigmented feathers and dark rings around the corneas indicating that
some of the neural crest cells differentiated into melanocytes by E15.
Sections through the corneas revealed numerous quail cells, identified by the
QCPN antibody, that had formed stromal keratocytes and endothelial cells, as
expected (Johnston et al.,
1979). In addition, a few darkly pigmented QCPN-positive cells
were observed in the epithelium.
Both similarities and differences to these control grafts were noted after transplantation of cardiac neural crest to the midbrain. Similar to midbrain grafts, cardiac neural crest formed a dark ring around chimeric corneas and contributed to the ciliary muscle. However, ectopic and darkly pigmented masses were noted on the dorsal sides of the corneas (48%) when stage 10 cardiac neural crest was transplanted into stage 9 hosts. This indicates that grafted cardiac neural crest cells fail to mix with the host cells, and thus result in large ectopic aggregates on the dorsal side of the cornea that protrude externally. Sections through the ectopic masses revealed that they comprised pigmented and non-pigmented QCPN-positive cells. By contrast, very few QCPN-positive cells contributed to the normal portion of the corneal stroma or endothelial cell layer. These results suggest that cardiac neural crest can follow the normal migratory pathway from the midbrain region into the periocular region, but only a few of those cells can respond to the cues in this environment and contribute to the corneal cell layers or ciliary muscle.
The observed location of the large masses of transplanted cardiac neural
crest cells on the dorsal side of the cornea probably reflects the fact that
neural crest from the caudal midbrain region migrate to the dorsal side before
entering and populating the developing eye. When cardiac neural crest cells
were transplanted into more anterior regions (data not shown), similar ectopic
masses were observed protruding from the dorsal and entire posterior half of
the cornea. Except for their pigmentation and location at the dorsal side of
the cornea, these ectopic masses resemble the non-pigmented mass of tissue
overgrowing the region of the cornea in the clinical condition known as
pterygia (Coroneo et al.,
1999). Others have observed aggregates of cells in the nasal
septum when r4-r6 neural crest was transplanted into the diencephalic level
where they still maintained their Hoxa2 expression
(Couly et al., 2002
).
Overexpression of Hoxa2, or Hoxa3 and Hoxb4 in the diencephalic region has
been shown to be associated with reduced cranial cartilage, but cornea
formation was apparently normal at E7, right after its formation
(Creuzet et al., 2002
).
However, later problems in the ability to contribute to the cornea were not
examined. Our results indicate that cardiac neural crest cells do not form
normal cornea when grafted into the midbrain region, suggesting that there is
an axial difference in potential to form corneal cells between midbrain and
cardiac neural crest cells. However, our results suggest that they only
transiently maintain Hox expression in this ectopic location.
Trunk neural crest cells grafted to the midbrain only form melanocytes in the cornea
Whereas the majority of midbrain and cardiac neural crest cells migrate
into the periocular region following grafting, only a few trunk neural crest
cells were observed within the cornea. Chimeric corneas derived from trunk
grafts formed a dark ring of cells surrounding the cornea resembling those of
midbrain and cardiac chimeras. However, only a few QCPN-positive cells were
observed within the periphery of the cornea (in the limbus region adjacent to
the cornea), with little or no contribution to the stroma, endothelium,
epithelium or ciliary muscle. Furthermore, all of the QCPN-positive cells in
trunk chimeras differentiated into melanocytes, suggesting that they may lack
the ability to respond to the corneal and ciliary environments, but retain the
capacity to form melanocytes, even in an ectopic environment. Our results
suggest that trunk neural crest cells can follow the correct migration pathway
to region of the cornea and eye but fail to invade after this experimental
manipulation. Preliminary results suggest that some trunk neural crest can
populate the cornea if directly transplanted into the periocular region
(P.Y.L. and M.B.-F., unpublished). Without definitive cell markers, however,
it is unclear whether or not they differentiate properly.
Cardiac and trunk neural crest cells have limited neurogenic ability within the trigeminal ganglion
Sensory innervation of the eye, face and mouth is derived from the
trigeminal ganglion, which comprises both neural crest and ectodermal placode
cells (for a review, see Noden,
1993). Unlike placode cells, which are specified to form
neuroblasts shortly after undergoing epithelial-mesenchymal transition, neural
crest cells continue to divide after aggregating in the trigeminal ganglion
(D'Amico-Martel and Noden,
1980
) giving rise to neuroblasts, glia and other support
cells.
Consistent with previous studies, our control midbrain grafts gave rise to
numerous QCPN-positive cells in the dorsoproximal region of the trigeminal
ganglion (Lwigale, 2001),
comprising both large (neurons) and small (support) cells. The majority of the
large cells were trkA positive, suggesting that they are somatosensory
neurons. When cardiac neural crest were transplanted into the midbrain region,
significantly fewer QCPN-positive neurons were observed in the trigeminal
ganglion, but the number of QCPN-positive support cells seemed unaffected.
With trunk neural crest grafts, there was an even more significant decrease in
the percentage of trkA-positive neurons in the ganglion. For cardiac and trunk
grafts, almost the entire neural crest contribution to the ganglion was host
derived. These results suggest that there is an axial difference in potential
to generate trigeminal neurons, decreasing in a rostral to caudal order.
Previously, it was reported that the neural crest cells from caudal
rhombencephalic levels (similar to the cardiac neural crest reported in this
study) can contribute to the neuronal derivatives of the first branchial arch
(Couly et al., 1998
). However,
these authors only examined the glial cells present in the trigeminal
mandibular nerve. In agreement with their findings, our data show that the
number of non-neuronal cells (probably glia) was similar to control grafts.
However, when sections of trigeminal ganglia were carefully analyzed, reduced
numbers of neurons were observed.
Not only the percentage of neurons, but also the size of the trigeminal
ganglion at E15 were altered after heterotopic grafting. All chimeric ganglia
had a normal shape, with both major branches (ophthalmic and
maxillomandibular) morphologically similar to those formed in midbrain control
chimeric embryos. Cardiac chimera ganglia were slightly smaller but not
significantly different from controls. By contrast, the size of trigeminal
ganglia derived from trunk chimeras was significantly smaller than midbrain
controls, consistent with the result that very few trunk neural crest cells
form neurons. Noden (Noden,
1975) showed that [3H]thymidine-labeled chick trunk
neural crest cells transplanted into the midbrain region formed
morphologically normal trigeminal ganglia. Later experiments using quail-chick
chimeras (Noden, 1978b
)
revealed that although trunk neural crest cells can form neurons when
transplanted in the midbrain region, they fail to aggregate with the
ectodermal placode cells, instead forming separate ganglia. The present
results using the quail-chick technique are in agreement with Noden's initial
observation, as the ganglia in all of our grafts properly formed the major
branches. The differences between our results and Noden's later results are
likely to reside in the size of tissue grafted. In the present experiments,
chick-host neural crest cells rostral and caudal to the grafted tissue
appeared to contribute the bulk of neurons to the trigeminal ganglion
(D'Amico-Martel and Noden,
1983
; Couly and Le Douarin,
1985
; Couly and Le Douarin,
1987
) and appeared to compensate for normal ganglionic morphology
even when few or no chimeric neurons formed.
Cardiac and trunk neural crest contribute little sensory innervation to the cornea
The cornea is highly innervated by neural crest-derived neurons
(Lwigale, 2001) originating
from the ophthalmic branch of the trigeminal ganglion
(Arvidson, 1977
;
Morgan et al., 1978
;
Marfurt et al., 1989
). Because
cardiac and trunk neural crest formed some trigeminal ganglion neurons, we
analyzed chimeric corneas for the presence of quail neural crest-derived
sensory axons, using a quail nerve specific antibody-QN. Midbrain control
corneas showed numerous QN-positive nerves in the cornea. By contrast, cardiac
and trunk neural crest chimera corneas contained fewer QN-positive nerves,
decreasing as the axial level of donor tissue became more caudal. These
results indicate that the few neurons that differentiate in the trigeminal
ganglion in cardiac and trunk chimeras are viable and form normal afferent
projections to the cornea. These results contrast with those of Noden
(Noden, 1978b
), who showed
that trunk neural crest cells form ectopic ganglia that fail to make normal
afferent ophthalmic and maxillomandibular projections. As a consequence, Noden
concluded that they did not innervate their designated targets. In the present
study, our grafts resulted in normal-appearing ganglia, which may have
facilitated the proper formation of neuronal connections. In addition, the
availability of a species-specific antibody for tracking projections made it
possible to identify processes that previously may have been missed.
Cardiac but not trunk neural crest contribute to a few first branchial arch derivatives
Midbrain neural crest cells normally migrate into the branchial arch
regions, where they form specific cartilages and bones of the jaw
(Noden, 1978a;
Lumsden et al., 1991
; Konteges
and Lumsden, 1996; Couly et al.,
1996
). In the first branchial arch region, neural crest cells give
rise to cartilages and bones of the lower jaw, such as Meckel's cartilage,
quadrate, and squamosal bones (Couly et
al., 1993
; Couly et al.,
1996
; Kontges and Lumsden,
1996
). Consistent with previous grafting experiments, our isotopic
grafts into the midbrain region showed numerous QCPN-positive cells in the
mandibular process. In these chimeras, Meckel's cartilage was almost entirely
derived from quail cells.
Cardiac neural crest cells (between r6 and somite 3) normally migrate into
branchial arches 3, 4 and 6, where they contribute to blood vessels, heart and
cardiac ganglia (Bockman et al.,
1987; Kirby et al.,
1983
). When cardiac neural crest were heterotopically grafted into
the midbrain region, the lower jaws of E15 chimeras appeared normal. Sections
through E6 chimeras showed numerous cardiac neural crest cells in the
mandibular process. However, unlike midbrain controls, few such heterotopic
crest cells contributed to Meckel's cartilage, which was mostly comprised of
host cells. The few individual cardiac neural crest cells detected were
scattered throughout the cartilage. In some cases, cardiac neural crest cells
did not mix with the host cells and instead formed nodules on the ventral side
of Meckel's cartilage, similar to those observed in r1-7 rotation experiments
(Hunt et al., 1998
). Cardiac
neural crest, however, contributed significantly to the quadrate bone. By
contrast, transplantation of r4/r6 neural folds to the r1/r2 level
(Couly et al., 1998
) showed
migration into the first branchial arch, but a failure to contribute to the
cartilage and bones at that axial level. This suggests that r4/r6 neural crest
cells were unable to differentiate into derivatives of the first branchial
arch. In the present study, we grafted cardiac neural crest from a region that
normally expresses Hoxa2 and Hoxa3. Our results reveal some degree of
plasticity in this population of neural crest to form lower jaw bones.
However, the cardiac neural crest cells did not maintain Hox gene expression
in their new environment. It is possible that transient expression of Hoxa2
and Hoxa3 is sufficient to account for their reduced potential of the cardiac
neural crest to form cartilage and trigeminal neurons. However, it is clear
that the expression of Hox genes is subsequently downregulated, suggesting
that the cells are also subject to environmental regulation. This may explain
the mixed results obtained with cardiac populations such that some take on
normal midbrain fates whereas others fail to respond appropriately to their
new environment.
In contrast to cardiac crest, no differentiation of trunk neural crest
cells into cartilage was observed after heterotopic grafting, consistent with
results of the original transplantation experiments of Noden
(Noden, 1978a) and Nakamura
and Le Lievre (Nakamura and Le Lievre,
1982
). A few trunk cells migrated into the arches, and those that
did were sparsely spread throughout the entire mandibular process, which was
mostly of host origin. Some of these trunk crest cells were detected in the
region where Meckel's cartilage formed. Although the host neural folds were
extirpated prior to grafting the trunk neural folds, many host neural crest
cells, probably derived from along the adjacent neural axes, were found within
the first arch. These compensated for the loss of normal midbrain neural crest
cells, contributing to the normal lower jaws seen at E15.
Owing to the very small number of trunk neural crest cells in the
mandibular process, it is possible that they may have the ability to form
cartilage or bone if a larger population had migrated into this region. In
fact, similar grafts in axololt revealed some capacity of trunk neural crest
to from cartilage in those cases where the cells made their way to the proper
destination even when by means of a circuitous route
(Epperlein et al., 2000). To
investigate this possibility, we grafted midbrain (control) and trunk dorsal
neural tubes directly into the first branchial arch. The midbrain grafts
produced QCPN-positive neural crest cells that migrated from the graft and
populated most of the mandibular process including Meckel's cartilage. Trunk
grafts produced more neural crest cells when transplanted directly into the
first branchial arch, but unlike the midbrain control grafts, trunk neural
crest cells formed aggregates adjacent to Meckel's cartilage but not directly
contributing to it. Some of the differences between cranial and trunk neural
crest have been attributed to their migration behaviors
(Lallier et al., 1992
).
Therefore, by grafting dorsal trunk neural tubes directly into the first
branchial arch we were able to increase the number of neural crest in the
vicinity of the developing cartilage, but they failed to differentiate
appropriately. Altogether, these results suggest that it is not due to
population effect that trunk neural crest cells fail to form cartilage, but
other intrinsic reasons. Recently, it was shown that trunk neural crest can
form both bone and cartilage when cultured in appropriate media
(McGonnell and Graham, 2002
),
suggesting that trunk neural crest has skeletogenic potential in vitro. By
grafting dorsal neural tubes directly into the first branchial arch, we
challenged the trunk neural crest cells to respond to the bone-forming cues in
that environment. As they failed to form cartilage even in a conducive
environment, this suggests that, unlike the in vitro induction, the cues that
are present in vivo are not sufficient to induce trunk neural crest to form
cartilage.
Conclusion
In summary, our results show that there are axial differences between
midbrain, cardiac and trunk neural crest in potential to generate corneal
keratocytes and endothelial cells, ciliary muscle, trigeminal neurons and
first branchial arch derivatives. Although both cardiac and trunk neural crest
migrate appropriately to the periocular region, they fail to make appropriate
contributions to the cornea. Cardiac crest forms a large percentage of ectopic
masses, but only a small number localize in the cornea. Trunk neural crest
only forms melanocytes in this location. The trigeminal ganglia of chimeric
embryos are morphologically normal, but reduced in size, and contain a
significantly reduced number of somatosensory neurons, particularly for
truncal grafts. Similarly, cartilage-forming ability is reduced for cardiac
neural crest and absent for trunk neural crest after transplantation. These
results suggest a loss in neural crest capacity along the rostrocaudal axis to
form somatosensory neurons and cartilage, even after transplantation to a
permissive environment. Our results using more sensitive detection techniques
confirm the inability of trunk neural crest to form cartilaginous derivatives
in vivo, as originally described by Le Douarin and colleagues using
quail-chick chimeric transplants. It was important to readdress this issue
given the recent demonstration that trunk neural crest can form cartilage in
vitro after long-term culture in rich medium
(McGonnell and Graham, 2002).
In addition to the previously reported effect on skeletogenesis, the present
results show that transplanting neural crest from cardiac and trunk regions
into midbrain levels also affects the formation of non-skeletal crest
derivatives, such as the cornea and trigeminal neurons. Surprisingly, we show
that transplanted cardiac neural folds only transiently maintain Hox gene
expression, which is subsequently downregulated in the midbrain environment.
This contrasts with previous findings where Hox genes are maintained for long
time periods after transpositions of r4 to r2 level. Thus, long-term
maintenance of Hox gene expression is not sufficient to account for
differences in developmental potential between cardiac and midbrain neural
crest. However, initial maintenance of Hoxa2 and a3 in the population
transplanted to midbrain may be sufficient to bias the population to exhibit
reduced ability to respond to the midbrain environment.
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ACKNOWLEDGMENTS |
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