1 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, MA 02115, USA
* Author for correspondence (e-mail: tabin{at}genetics.med.harvard.edu)
Accepted 13 June 2003
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SUMMARY |
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Key words: Cranial neural crest, Trunk neural crest, Signaling, Differentiation, Chick
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Introduction |
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These and other experiments have established a picture of the trunk neural
crest as a pluripotent population of stem-like progenitors, the fate of which
is determined by signals in the local environment. The molecular nature of the
signals regulating differentiation of the neural crest cells has been explored
mostly in the trunk, where experiments have provided a wealth of information
on how signaling molecules and transcription factors control cell
differentiation (Shah, 1996; Anderson,
1997). For example, it has been demonstrated in vivo that the
production of pigment cells depends on the WNT pathway, whereas members of the
TGFß1 superfamily have been implicated in formation of smooth muscle
cells and sensory and adrenergic neurons
(Anderson, 1997
;
Zhang et al., 1997
;
Shah et al., 1996
;
Dunn et al., 2000
).
Importantly, these in vitro results correlate well with in vivo expression
patterns, such that the in vitro defined signals are indeed produced in the
regions where the expected neural crest derivatives differentiate.
Similarly, cranial neural crest cells have been shown to receive
instructional information from surrounding tissues, such as neuroepithelium,
the head surface ectoderm, the endoderm of the foregut and the paraxial head
mesoderm (Francis-West et al.,
1998; Golding et al.,
2002
; Couly et al.,
2002
; Trainor et al.,
2002
). A number of candidate signaling molecules have been
identified that are expressed in these tissues during head development
(Wall and Hogan, 1995
;
Ferguson et al., 2000
)
(reviewed by Francis-West et al.,
1998
). Several members of the fibroblast growth factor family
(FGF1, FGF2, FGF4, FGF5 and FGF8) are expressed in the developing facial
primordia. FGF2, FGF4 and FGF8 are expressed in the epithelium covering
particular regions of the developing face
(Crossley and Martin, 1995
;
Wall and Hogan, 1995
;
Barlow and Francis-West, 1997
;
Helms et al., 1997
;
Richman et al., 1997
).
Cre-mediated inactivation of Fgf8 gene showed that FGF8 product is
required for cell survival in the first branchial arch
(Trumpp et al., 1999
). These
newborn mutant mice lack all of the structures derived from the first
branchial arch except the most distal regions. Moreover, FGF8 signaling
contributes to generation of rostrocaudal polarity in the first branchial arch
as it provides positional information to the rostral (Lhx7-
expressing) and caudal (Gsc-expressing) domains of the crest-derived
ectomesenchyme from the rostral epithelium where FGF8 is expressed
(Tucker et al., 1999
).
Members of the BMP family are also expressed in the developing head
(Roelen et al., 1997;
Francis-West et al., 1998
).
Craniofacial roles of a few members of the BMP family have been studied in
some detail (Bennet et al.,
1995
; Wall and Hogan,
1995
; Barlow and Francis-West,
1997
). For example, BMP2 and BMP4 are expressed in particular
domains of surface epithelium that are associated with mesenchyme expressing
Msx1 and Msx2 and ectopic application of beads soaked with either BMP2 or BMP4
can activate the expression of Msx1 and Msx2 and can result in the bifurcation
of certain cranial skeletal structures
(Barlow and Francis-West,
1997
). In addition, Bmp2 signaling increases cell proliferation in
the mandibular primordia whereas haplo-insufficiency of BMP4 in C57B1/6 mice
results in shorter frontal and nasal bones
(Barlow and Francis-West,
1997
).
On the cellular level, there has been a number of studies of the specific
roles of the above-mentioned signaling molecules in directing the
differentiation and proliferation of the trunk neural crest
(Nakamura and Ayer-le Lievre,
1982; Shah et al.,
1996
; Anderson,
1997
). However, there is far less information concerning the
cellular effects of these molecules on cranial neural crest. Moreover, those
in vitro studies that have been carried out with cranial neural crest are
often difficult to compare with trunk neural crest as they often used
dissimilar media or incubation conditions
(Baroffio et al., 1988
;
Shah et al., 1996
;
Anderson, 1997
;
Sarkar et al., 2001
). The
objectives of the current study were, thus, to directly compare cell
differentiation from mesencephalic (cranial) and sacral (trunk) neural crest
in response to various factors when grown under similar conditions. We focused
on the effects of FGF2/8, BMP2/4 and TGFß1. We find that cranial and
trunk crest cells differ considerably in their responsiveness to these
factors, such that the same array of differentiated cell types is produced at
different axial levels via interactions with a distinct set of differentiation
cues.
As cranial and trunk crest differ qualitatively in their responsiveness to
various signaling proteins, one would like to know the upstream cellular
factors responsible for these differences. Hox genes, a family of homeodomain
transcription factors, are known to play important roles in specifying
rostralcaudal differences in many tissues and are hence attractive candidates.
Mis-expression of Hox genes in cranial crest cells indeed suggests that these
genes are involved in setting up the crucial differences in differentiation
response between cranial and trunk crest cells. Among the differences between
trunk and cranial neural crest cells, the most well-known has been the unique
ability of cranial neural crest to produce chondrocytes. However, it has
recently been reported that trunk neural crest cells can produce chondrocytes
in long-term in vitro cultures (McGonnell
and Graham, 2002). We confirm this observation but find that this
long-term change in differentiation potential is correlated with a
downregulation of Hox genes in a subset of the neural crest cells and can,
moreover, be completely blocked by misexpression of trunk Hox genes in
vitro.
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Materials and methods |
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Concentration of all the protein cytokines was 10 ng/ml as it was found to
be optimal for chondrogenesis in previously published studies
(Sarkar et al., 2001;
Petiot et al., 2002
). At least
seven 24-well plates of cultured cells were used for each of the conditions
described unless otherwise indicated. Neural crest cells were cultured for one
week except where noted otherwise. To visualize apoptotic cells, we used 'In
Situ Cell Death Detection, POD' kit (Roche Diagnostics, GmbH) according to the
manufacturer's instructions.
Viral infections of early embryos
The RCAS(A)::CA-ß-catenin construct has been described before
(Kengaku et al., 1998). To
obtain cranial neural crest cell cultures expressing these transgene, we
infected future neural plate cells of the early stage 6+ embryos. The
following day neural crest tissue was placed in culture. Each of the
RCAS-infected cultures was tested with 3C2 antibody with subsequent
FITC-conjugated secondary antibody and DAPI stained to ensure that all cells
were infected. Only the 3C2-positive (infected) cultures were counted and
examined with cell-specific antibodies.
RT-PCR analysis of trunk neural crest cultures
Cultured cells were lysed and processed for RNA analysis using the RNeasy
kit (QIAGEN). The method for the RT-PCR analysis was previously described
(Munsterberg et al., 1995).
The primers used are: GAPDH (5'-AGTCATCCCTGAGCTGAATG and
5'-ACCATCAAGTCCACAACACG); Noelin-1 (5'-CGTGGAGAAGATGGAAAACC and
5'-GTGCCTGACCACGGGTGAGG); and Id2 (5'-GTCAGCCTCCACCACCAGCG and
5'-GGGTCCTTCTGGTACTCACG). PCR reactions were typically performed at
56°C for 30 cycles with 5% formamide, except for GAPDH, 60°C, 24
cycles, no-formamide. More details are available upon request.
Immunohistochemical procedures and in situ hybridization
The presence of the migrating crest cells in the culture was determined by
using HNK-1 or rabbit anti-p75 nerve growth factor (NGF) receptor antibody
(Chemicon International, Temecula)
(Bannerman and Pleasure, 1993;
Rao and Anderson, 1997
). p75
is believed to be an excellent marker for undifferentiated neural crest cells
(Rao and Anderson, 1997
;
Young et al., 1998
). In
addition, we occasionally used mouse antibody 20B4 (Developmental Studies
Hybridoma Bank, University of Iowa, Iowa City) to detect neural crest cells;
monoclonal anti-ColII (Chemicon International, Temecula), monoclonal
anti-ColII (Sigma) and rabbit anti-ColII (Collagen II) (Chemicon
International, Temecula) to detect chondrocytes; Cy3-conjugated anti-SMA
(smooth muscle actin) antibody (Sigma) to detect smooth muscle cells;
monoclonal anti-S100 (Sigma), rabbit anti-GFAP (glial acidic fibrillary
protein) antibody (Sigma), monoclonal Cy-3 conjugated anti-GFAP antibody
(Sigma) and mouse antibody 1E8 (Developmental Studies Hybridoma Bank,
University of Iowa, Iowa City) to detect glial cells; and rabbit
anti-neurofilament M (145 kDa) (Chemicon International, Temecula), mouse
anti-NF200 and monoclonal anti-ß-3 tubulin, clone 2G10 (Upstate
Technology, Lake Placid) to detect neuronal cells. GFAP and SMA proteins are
believed to be good markers for glial and smooth muscle cells in chicks,
respectively (Kalman et al.,
1998
; Hoya et al.,
2001
). We used biotinylated, Texas Red-, FITC-, Cy3- or
Cy5-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West
Grove and Vector Laboratories, Burlingame). All purified cytokines were
purchased from R&D Systems or were laboratory stocks (obtained from
Genetics Institute). Immunochemistry on cell cultures was performed as
described before (Bachler and Neubuser,
2001
).
In situ hybridization was performed DIG RNA probes, which were detected with antibodies conjugated with alkaline phosphatase (AP). The fluorescent double in situ hybridization on long-term TNC cultures were performed using tyramide signal amplification on HRP-conjugated anti-DIG and anti-FITC antibodies: the red Cy3 signal obtained with the TSA-Plus Fluorescence Palette System (PerkinElmer Life Sciences, Boston) and green Oregon Green 488 signal obtained with the TSA kit #9 (Molecular Probes, Eugene).
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Results |
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In the presence of both FGF2 and BMP2, the cranial neural crest cells survival (92%) was similar to that in the presence of FGF2 alone (73%) and was much higher than in the medium containing BMP2 alone (18%). A different situation was observed for the trunk neural crest cells, which survived more poorly in the FGF2+BMP2 culture (61%) when compared with cultures supplemented with BMP2.
Differentiation of cranial and trunk neural crest cells under similar
culture conditions
To examine whether the differential responses result in different cell fate
choices in trunk and cranial neural crest cultures, we used cell-type specific
antibodies and cell morphology to determine which neural crest-derived cell
types were formed. Five distinct types of differentiated cells were assayed:
pigment cells (pigment granules), chondrocytes (various anti-ColII
antibodies), smooth muscle cells (anti-smooth muscle actin (SMA), glial cells
[anti-glial acidic fibrillary protein (GAFP) and anti S100 (calcium-binding
protein)] and neuronal cells [anti-ß-3-tubulin, anti-neurofilament 145
(NF145) and anti-neurofilament 200 (NF200)]
(Fig. 3). In some cases, we
used in situ hybridization with cell-specific RNA probes to confirm the
antibody results. As used by others in previous studies
(Sarkar et al., 2001), we used
the percent of treated cultures containing a given cell type to measure
whether that cell fate is promoted by the culture conditions. Cultures were
considered to display a particular cell fate if it contained a chondrogenic
nodule or at least 10% of the cells exhibited a particular marker as
determined by antibody staining or in situ hybridization. To calculate the
statistical significance, we performed a t-test for pair-wise
comparison or ANOVA for comparison of multiple groups of samples.
|
Although, as noted above (Fig.
2), addition of FGF2 decreases the survival of trunk neural crest
by about 30%, the same differentiated cell types were found in trunk neural
crest cultures with or without FGF2, including smooth muscle cells, neuronal
cells and pigment cells with few or no chondrocytes or glial cells (Figs
3,
5). The only significant
difference in these cultures was a relative increase in percentage of pigment
cells, consistent with previous reports that FGF2 is mildly mitogenic for
melanocytes (Sieber-Blum and Zhang,
1997). Similarly, the presence or absence of FGF2 (or FGF8)
protein in the culture did not alter the differentiation of trunk neural crest
in response to BMP2, TGFß1 or canonical Wnt signaling (Figs
3,
5; not shown).
|
Dramatic differences in the response of neural crest cells derived from
different rostrocaudal levels, were seen with TGFß1. Trunk neural crest
responds to TGFß1 or FGF2+TGFß1 signaling by differentiating to
become smooth muscle cells (Figs
3,
5)
(Shah et al., 1996;
Anderson, 1997
) (data not
shown). In addition, the number of cultures undergoing gliagenesis was sharply
reduced. However, when added to the cranial neural crest culture, TGFß1
actually strongly suppressed the formation of smooth muscle cells (from 76% to
18%, Figs 3,
5). Chondrocyte cell fate was
likewise suppressed by TGFß1, and gliagenesis was also significantly
reduced. By contrast, TGFß1 promoted relatively normal rate of
melanogenesis (92% of cultures contained melanocytes) when compared with
FGF2-conditioned media alone, although very few melanocyte clusters were
observed (Fig. 3).
Additionally, in FGF2+TGFß1 cultures, the pigment cells were dispersed
and rarely formed clusters.
WNT1 signaling through the ß-catenin pathway induces trunk neural
crest cells to differentiate into pigment cells
(Dunn et al., 2000). To
compare the effect of ß-catenin signaling on trunk and cranial neural
crest, we used a replication-competent retroviral construct (RCAS) encoding a
constitutively active form of ß-catenin (CA-ß-catenin)
(Funayama et al., 1995
;
Kengaku et al., 1998
). Nearly
100% of both trunk crest and cranial neural crest cultures infected with
CA-ß catenin appeared as dense lawns of heavily pigmented cells
(Fig. 3, bottom row). These
cultures did not contain chondrocytes or neurons; however, antibodies to SMA
(recognizing smooth muscle cells) and GFAP labeled glial cells in a relatively
large proportion of the cultures (39% and 37%, respectively). About a third of
the cells positive for smooth muscle or glial markers were also pigmented
(Fig. 4). These cases might
represent either cells with mixed identities or differentiated glial and
smooth muscle cells in the process of being transformed into pigment cells
[similar process is described elsewhere
(Sherman et al., 1993
)].
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By contrast, when cultured in the presence of BMP2 or TGFß1 in addition to FGF2, there was a dramatic decrease, such that practically no cultures were scored as positive for chondrocytes (Fig. 5). A similar inhibition of chondrogenesis is seen when cells within individual cultures were counted under those culture conditions (Fig. 6). The other significant decrease in cell types under these conditions assayed by percent of cultures is in cultures populated by smooth muscle cells when treated with FGF2 and TGFß1. This culture condition also gave the lowest percentage of smooth muscle cells when counted within individual cultures (Fig. 6), although owing to the high variability between cultures, the difference from cultures treated with FGF2 alone is not statistically significant. Using the 10% cut-off for counting individual cultures (pink in Fig. 6) it is apparent that this data set is similar to that assayed in Fig. 5.
Hox genes and differentiation of cranial neural crest cells
Most of the cellular responses by cranial neural crest described above
differ dramatically from those of the trunk neural crest cells when exposed to
the same signaling factors. One significant difference along the rostrocaudal
axis is the expression of Hox genes. Indeed, regional differences between the
head and the trunk depend, in part, on the function of Hox genes
(Rijli et al., 1993;
Grammatopoulos et al., 2000
).
To examine potential roles of Hox genes in defining the regional differences
in responsiveness to various factors, we expressed two different Hox genes in
mesencephalic cranial neural crest cultures using RCAS viruses (Figs
7,
8). Hoxa2 is normally
expressed in the posterior head, a region which unlike the trunk produces
neural crest-derived cartilage although not as much as in more anterior head
regions (Sarkar et al., 2001
).
It has been shown to be an important regulator of cranial neural crest
patterning and morphology of branchial arch skeletal elements in mouse, chick
and frog (Rijli et al., 1993
;
Grammatopoulos et al., 2000
;
Pasqualetti et al., 2000
).
Hoxa2 is normally expressed in cranial neural crest cells migrating
into the second branchial arch and loss of its function leads to the homeotic
transformation of second branchial elements, into those of the first arch
(Rijli et al., 1993
).
Conversely, misexpression of Hoxa2 in the anterior head transforms
the first branchial arch into a more posterior identity
(Grammatopoulos et al., 2000
).
Hoxd10 expression is normally observed in the posterior sacral part
of the trunk with an anterior boundary at the level of somites 31-32. Neural
crest from this region does not yield any skeletal derivatives.
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We next examined the ability of Hox genes to modulate differentiation response in neural crest cell cultures. Both trunk and cranial neural crest cells give rise to a mixture of cell types when cultured in the presence of FGF2/8. However, trunk neural crest cells fail to differentiate into chondrocytes and show a more limited range of gliagenesis than CNC cells within our culture period. When mesencephalic cranial neural crest cells were infected with RCAS::Hoxa2 and treated with FGF2, the number of cultures undergoing chondrogenesis decreased from about 60% to about 40% relative to uninfected cultures (compare Figs 5, 7 and 8). None of the cranial neural crest cell cultures expressing trunk Hox gene Hoxd10 and treated with FGF2 contained chondrocytes.
This leads to a model where chondrogenesis is blocked by co-expression of anterior (e.g. Hoxa2) and posterior (e.g. Hoxd10) Hox genes, a pattern normally found in the trunk; low level of chondrogenesis occurs in the presence of only anterior Hox gene expression (e.g. Hoxa2), a pattern normally seen in metencephalic (hindbrain) crest, and high level of chondrogenesis takes place in the absence of neural crest Hox gene expression, a condition normally seen in the mesencephalon. To test this hypothesis further, we also mis-expressed Hoxd10 in primary cultures of metencephalic cranial neural crest from rhombomere 3-4 levels that normally express endogenous Hoxa2 and produce some cartilage structures. As with mesencephalic crest, we found that mis-expression of Hoxd10 inhibited chondrogenesis in metencephalic crest (19/20 cultures infected cultures; data not shown).
We also examined the differentiation of neural crest cells into other cell types in our culture conditions under the influence of Hox genes. In the presence of FGF2, mesencephalic cranial neural crest cells infected with RCAS::Hoxa2 formed large dense cultures containing pigment cells (about 80% of cultures), smooth muscle cells and glial cells, similar to uninfected cultures treated with FGF2 (Figs 5, 7, 8). By contrast, mesencephalic neural crest cultures expressing the trunk Hox gene Hoxd10 and treated with FGF2 showed a reduced number of smooth muscle cells and glial cells, although melanogenesis was not affected. This pattern of differentiation was compared with trunk neural crest cultures derived from the anterior trunk (somite 10-12 level), which expressed other Hox genes but not Hoxd10, and posterior trunk (somite 32-33 level), which expressed Hoxd10 and other Hox genes. We found that the two trunk neural crest populations differed significantly in their ability to produce smooth muscle cells, glial cells and neurons (Fig. 8). The RCAS::Hoxd10-infected cranial neural crest cells were more similar to the posterior trunk than to the anterior trunk neural crest cells, although they produced very few neurons (Fig. 8). Thus, in several key respects the differentiation pattern of cranial neural crest cells is closer to that of trunk neural crest when cells ectopically express the posteriorly expressed Hox gene Hoxd10. Thus, in several key respects the differentiation of pattern of cranial neural crest cells is closer to that of trunk neural crest when cells ectopically express the posterior Hox gene Hoxd10.
Chondrogenesis in longer-term trunk neural crest cultures
Transplantation experiments have indicated that trunk neural crest lacks
the capacity to undergo chondrogenic differentiation in vivo. Moreover, in our
in vitro culture conditions trunk neural crest cells do not produce any
chondrocytes. Nonetheless, it has recently been reported that trunk neural
crest cells, in long-term culture (2-4 weeks) can undergo chondrogenesis
(McGonnell and Graham, 2002).
This could be interpreted as reflecting an underlying potential for
chondrogenic differentiation in trunk neural crest, which is revealed only
under culture conditions that arise within the dish after several weeks.
Alternatively, the trunk neural crest cells themselves might be altered by the
long-term culture conditions such that at least a subset of these cells gain
differentiation potential that is not present in normal trunk neural crest
cells. To investigate this issue we allowed our culture to continue growing
for 2 weeks. As previously reported, these cultures of trunk neural crest
cells ultimately undergo chondrogenesis, although chondrocytes are not readily
observed until 12-14 days in culture, while chondrocytes are detected in
cranial crest culture as early as 4-5 days of culture (Figs
3,
4,
5,
6,
7,
8; data not shown).
One explanation for this could be that the neural crest loses its trunk
specificity and converts to a more cranial crest-like cell type during long
term culture. To examine this possibility, we used RT-PCR to follow the
expression of several cranial neural crest markers, which are preferentially
expressed in cranial crest in vivo. We found that Id2
(Martinsen and Bronner-Fraser,
1998) and noelin 1 (Barembaum
et al., 2000
) were strongly upregulated in trunk neural crest
cells after 2 weeks in culture to levels similar to those detected in cranial
neural crest cultures (Fig.
10A). Thus, at least in some important respects trunk neural crest
resembles cranial crest after long term culture. One possible explanation for
this could be that Hox genes, which establish differences in neural crest
along the rostrocaudal axis, are dysregulated in long-term culture.
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Discussion |
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Fig. 11 summarizes the
effect of several signaling pathways on cell differentiation in the cranial
and trunk neural crest. Note that FGF2 (and FGF8) seem to play an important
role in promoting survival, proliferation and differentiation of cranial
neural crest but not trunk neural crest cells, and that freedom from this
requirement depends, at least in part, on the expression of trunk Hox genes.
Interestingly, some factors that are inductive for a particular cell fate in
trunk neural crest cells (Anderson,
1997; Shah et al.,
1996
; Francis-West et al.,
1998
) have the opposite functions in the cranial neural crest,
e.g. BMP2/4 in neurogenesis and TGFß1 in smooth muscle formation. On the
other hand, the WNT pathway is equally potent in inducing melanogenesis in
both head and trunk crest. FGF2 and FGF8 also seem to positively regulate the
level of melanogenesis in both systems. In addition, chondrogenesis, which is
unique to cranial neural crest cells, is induced by the FGF2/8 but is
suppressed or inhibited by BMP2, TGFß1 and WNT pathways. Taken together,
the distinct modes of cell differentiation in cranial and trunk neural crest
suggest that the cranial and trunk cells possess significantly different
developmental capabilities. Interestingly, different regulatory interactions
were also recently found for the head and trunk mesoderm during the process of
myogenesis (Mootoosamy and Dietrich,
2002
) (E.T., H. Kempf, R. C. Mootoosamy, A. C. Poon, A.A., C.J.T.,
S. Dietrich and A.B.L., unpublished).
|
We did not aim in this analysis to understand the molecular mechanisms for the differences we observed. The differential survival and cell fate diversification could be due to direct regulatory control of differentiation of particular cell types, their differential induction, proliferation or death. In addition, some conditions might be supportive of survival of certain cell type progenitors but not others. All of these issues will need to be addressed in the future studies.
It is important to note that our use of combinations of different factors
revealed some interesting synergisms and antagonisms in regards to both
survival and differentiation. For example, BMP2 by itself cannot support
survival of the cranial neural crest cells in vitro, whereas FGF2 allows the
cells to survive and differentiate into chondrocytes. When FGF2 and BMP2 are
added together, the survival and proliferation are maintained but
chondrogenesis is very strongly inhibited
(Fig. 5). In a related study,
we found that a combination of FGF2/8 with sonic hedgehog has a very strong
synergistic effect on chondrogenesis both in vitro and in vivo (A.A. and
C.J.T., unpublished). These and other observations from our study are strongly
reminiscent of the previously reported differential effects of combinations of
growth factors on proliferation and differentiation of the trunk neural crest
cells. For example, the presence of stem cell factor (SCF) is required for
early trunk neural crest survival but not for the survival of melanogenic
cells. However, a combination of SCF with a neurotrophin, such as nerve growth
factor (NGF), brain-derived neurotrophic factor (BDNF) or neurotrophin 3 (NT3)
can neutralize the activity of SCF. However, a combination of SCF and NT3 can
support survival of pigment cell precursors
(Zhang et al., 1997;
Sieber-Blum, 1998
). Moreover,
this SCF+NT3 combination can be antagonized by TGFß1 signaling, which
strongly induces differentiation of the trunk neural crest cells into
sympathetic and primary sensory neurons, thus emphasizing the importance of
the concerted effects of different signaling molecules.
Our in vitro data also correlate well with the expression patterns of the
signaling molecules discussed herein. For example, the requirement for FGF2/8
to promote survival and differentiation of cranial neural crest in culture is,
moreover, consistent with the broad and persistent expression patterns of
different FGF family members (FGF1, FGF2, FGF3, FGF4, FGF5, FGF7 and FGF8) and
their receptors in the developing head
(Schneider et al., 2001;
Bachler and Neubuser, 2001
).
The in vivo functional significance of these expressions patterns has been
partially confirmed in studies where FGF8 activity was conditionally removed
from the ectoderm covering the first branchial arch using Cre/loxP technology,
resulting in loss of most of the cranial neural crest-derived structures
(Trummp et al., 1999).
Neural crest differentiation and Hox genes
Hox genes are important regulators of developmental fates, including
specification of structures formed from neural crest. Our data suggest that
Hox genes influence neural crest survival, proliferation and differentiation,
in part by controlling the differential responsiveness of neural crest cells
to various signaling pathways. Cranial neural crest cells infected with
retrovirus carrying the trunk Hox gene Hoxd10 respond to different
factors in a manner normally seen with trunk neural crest. For example,
Hoxd10 suppress chondrogenesis from the cranial neural crest cells.
Moreover, the overall survival and differentiation abilities of the
RCAS::Hoxd10-infected cranial crest cells is more similar to those of
the posterior trunk neural crest where Hoxd10 is normally expressed
than to either anterior trunk crest cells or RCAS::Hoxa2-infected
cranial crest cells. However, at least one property of trunk neural crest, the
ability to produce high numbers of neurons, was not recapitulated in cultures
of cranial neural crest cells infected with Hoxd10, suggesting that
other important regulatory factors (potentially including other Hox genes) are
involved in controlling cell diversification pathways at the trunk level. The
effect of Hoxa2 is more complicated. Hoxa2 may modulate the
extent of the response of cranial neural crest to chondrogenesis-inducing FGF
signals. This might be achieved by locally regulating the balance between
proliferative versus differentiative effects of FGF2/8
(Fig. 9). Thus, this
interpretation would take into account the fact that Hoxa2 is
required for normal patterning of the 2nd branchial arch and is expressed in
cranial neural crest cells destined to become cartilage.
Recently, it has been shown that three anterior trunk Hox genes, Hoxa2,
Hoxa3 and Hoxb4, when mis-expressed in the head are capable of
suppressing chondrogenic structures in the anterior head but differed in their
abilities to do so (Creuzet et al.,
2002). For example, Hoxa3 prevents the formation of the
first branchial arch but not the nasal septum, whereas Hoxb4
suppresses formation of nasal skeleton but allows proximal lower jaw
development. Together, these results suggest that many Hox genes are capable
of regulating differentiation of cranial crest cells, although substantial
differences exist in their exact capabilities. It might be important,
therefore, to study the effect of Hox genes on differentiation of different
crest populations within the trunk and posterior head. It might be predicted,
for example, that differentiation of cardiac crest, which originates from a
certain anteroposterior level, is also controlled by Hox genes.
It is clear that Hox expression changes the responsiveness of the cranial
cultures so that in many respects they resemble cultured trunk crest.
Conversely, we have suggested that the ability of long-term cultures of trunk
neural crest to adopt chondrogenic fates normally limited to cranial crest may
be due to the loss or downregulation of Hox expression in a subset of cells.
Although this may not reflect a physiological property of trunk neural crest,
it does suggest an underlying plasticity in Hox gene expression. The ability
of crest cells to modulate their Hox expression in foreign environments was
also seen in experiments where small pieces of dorsal rhombomeric tissue
containing crest were transferred from the rhombomere 3, 4 and 5 level to
rhombomere 2 level (Trainor and Krumlauf,
2000; Trainor and Krumlauf,
2001
).
The ability of Hox genes to alter responsiveness of different regional neural crest populations, while maintaining the extraordinary broad capacity of those cells to differentiate along diverse pathways, generates a flexibility in the way those cells can be directed in development. Thus, a mechanism is generated whereby the proper differentiation of neural crest cells can be coordinated with other tissues by taking advantage of the distinct arrays of secreted signals characterizing different regions of the embryo.
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ACKNOWLEDGMENTS |
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