Developmental Neurobiology, National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK
* Author for correspondence (e-mail: james.briscoe{at}nimr.mrc.ac.uk)
Accepted 18 August 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Sox9, Neural crest, SoxE group transcription factors, Chick
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The signalling events that instruct neural crest development have received
much attention. Inductive interactions between the epidermal ectoderm and
neural plate are required for induction and several candidate signals have
been proposed to mediate this event. Most consideration has been given to the
BMP and Wnt groups of secreted factors. Members of both families of molecules
are expressed in the relevant tissues at appropriate times during development
and there is evidence that each family of proteins is necessary and sufficient
to induce neural crest (Dickinson et al.,
1995; Selleck and
Bronner-Fraser, 1995
; Liem et
al., 1995
; Liem et al.,
1997
; Ikeya et al.,
1997
; Selleck et al.,
1998
; Garcìa-Castro et
al., 2002
). Although these studies suggest apparently
contradictory models, it is possible that both sets of signals are involved in
neural crest induction, perhaps at different stages of development or in
different capacities (Aybar and Mayor,
2002
). Additional signals such as fibroblast-derived growth
factors (FGFs) and retinoic acid have also been implicated in the induction
and differentiation of neural crest and their role also remains to be
clarified (Mayor et al., 1997
;
Villanueva et al., 2002
).
Within prospective neural crest cells, the transcriptional programme that
is initiated in response to the inductive signal is not clearly defined. The
dorsal neural tube expresses several transcription factors in response to
neural-crest-inducing signals. Examples include Pax3, Msx1-3 and Zic1-3.
Although these proteins have been implicated in neural crest induction, they
are also expressed in neural progenitors that generate dorsal interneurons and
so are unlikely to be involved solely in neural crest induction
(Epstein et al., 1991;
Liem et al., 1995
;
Houzelstein et al., 1997
;
Nakata et al., 1997
;
Nakata et al., 1998
). Several
transcription factors have been identified that are restricted to developing
neural crest. These include AP2, Id2, FoxD3 and Slug, but the epistatic
relationships between these proteins and the relative contribution of each to
neural crest specification remain unclear. Loss of AP2 results in defects in
neural crest development (Schorle et al.,
1996
; Zhang et al.,
1996
) and the forced expression of AP2 in frog embryos is
sufficient to induce neural crest differentiation
(Luo et al., 2003
). However,
AP2 is initially expressed throughout the ectoderm, suggesting that other
factors must be involved in restricting neural crest induction to the
appropriate region (Luo et al.,
2002
). Id2, a basic helix-loop-helix transcription factor, is
sufficient to induce neural crest characteristics in the chick neural tube
(Martinsen and Bronner-Fraser,
1998
), but Id2 is expressed only in cranial regions and mice
lacking Id2 have no reported neural crest defects
(Yokotak et al., 1999
). The
zinc-finger transcription factor Slug and the forkhead class transcription
factor FoxD3 are the strongest candidates for general neural-crest-specifying
factors. Both are expressed transiently in neural crest cells prior to
delamination (Nieto et al.,
1994
; Dottori et al.,
2001
; Kos et al.,
2001
). However, the absence of Slug in mouse does not affect
delamination (Jiang et al.,
1998
) and the effect of ectopic expression of Slug is limited to
increasing the amount of neural crest specification in cranial regions of the
neural tube (del Barrio and Nieto,
2002
). Moreover, although the forced expression of FoxD3 induces
some aspects of neural crest differentiation in the ventral neural tube, it is
not sufficient to induce cells that exhibit all the characteristics of neural
crest (Dottori et al., 2001
;
Kos et al., 2001
). Together,
these studies raise the possibility that other transcription factors are
involved in the specification of prospective neural crest cells.
Members of the Sox gene family of high-mobility-group (HMG) domain
containing transcription factors are candidates for playing a role in neural
crest specification. Sox proteins are involved in several processes during
embryogenesis. Based on the amino acid sequence of the HMG domain, Sox
proteins can be divided into ten sub groups
(Bowles et al., 2000). Subgroup
E consists of three members (Sox8, Sox9 and Sox10) that are expressed in
several developing tissues, including the neural crest. Mice lacking Sox8
develop to adulthood without severe defects
(Sock et al., 2001
). By
contrast, loss of function analyses have identified roles for Sox9 and Sox10
in neural crest development. In frog embryos, morpholino-mediated depletion of
Sox9 results in loss of neural crest progenitors
(Spokony et al., 2002
). It is
not clear whether this reflects a requirement for Sox9 in neural-crest cells
or a role for Sox9 in controlling neural-crest-inducing signals, and it
remains to be established whether Sox9 is sufficient to initiate neural crest
development. Loss of function studies indicate that Sox10 has a role in later
aspects of neural crest development. In mice and zebrafish lacking Sox10, the
early specification of neural crest is unaffected but the later
differentiation of peripheral glial cells and melanocytes is disrupted
(Britsch et al., 2001
;
Dutton et al., 2001
).
Moreover, recent studies (Kim et al.,
2003
; Paratore et al.,
2002
) have proposed a role for Sox10 in maintaining the
multipotency of neural crest stem cells as well as directing differentiating
cells to non-neuronal fates. Together, these studies have focused attention on
SoxE genes in neural crest development, but the role these genes play in the
early events in neural crest differentiation remain to be resolved. Because
functional redundancy between SoxE family members might limit the phenotypes
observed in the loss of function analyses, we have taken a gain of function
approach to examine the role these genes play in neural crest induction.
We demonstrate that SoxE genes are expressed in premigratory neural crest and that Sox9 is an early marker of prospective neural crest, preceding markers of migratory neural crest. Forced expression of Sox9 or other group-E Sox genes in the neural tube induces ectopic neural crest differentiation at the expense of central nervous system (CNS) neuronal generation. Strikingly, although Sox9 induces many neural crest markers along the entire dorsal-ventral axis of the neural tube, efficient emigration of ectopic neural crest is restricted to the most dorsal regions. Ventral to this, delaminating cells are observed only infrequently. This supports a model in which the induction and delamination of neural crest are independent events. Consistent with this, RhoB, which has been implicated in promoting delamination, is not induced by Sox9, raising the possibility that delamination is initiated by the upregulation of a subset of factors including RhoB. In the periphery, SoxE-transfected neural crest cells migrate along typical neural crest routes and display characteristics of glial and melanocyte neural crest derivatives but are excluded from neuronal lineages, indicating that continued expression of SoxE genes biases differentiation to certain neural crest lineages. Together, our findings indicate that SoxE genes act at two stages of neural crest differentiation first as cell intrinsic determinants of neural crest, initiating neural crest development and segregating this lineage from the neuroepithelium, and subsequently directing differentiation decisions in the periphery, biasing neural crest cells to glial cell and melanocyte lineages and away from neuronal fates.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chick in ovo electroporation and BrdU labelling
Chick Sox9 cDNA (Kamachi et
al., 1999), chick Sox8 cDNA
(Cheng et al., 2001
), chick
Sox10 cDNA (Cheng et al.,
2000
) and Wnt3a (a gift from N. Itasaki) were inserted
upstream of an internal ribosomal entry site (IRES) and nuclear localization
sequence (nls) tagged GFP in pCAGGS expression vector
(Niwa et al., 1991
).
Sox2 cDNA was cloned into the pCS2+ expression vector (a gift from E.
Remboutsika). Chick embryos were electroporated with DNA at 2.5 µg
µl1, for co-transfections, pCAGGS-IRES-nls-GFP was used
at 500 ng µl1. Briefly, plasmid DNA was injected into the
lumen of HH stage 10-11 neural tubes, electrodes placed either side of the
neural tube and electroporation carried out using a BTX electroporator
delivering five 50 millisecond pulses of 30 V
(Briscoe et al., 2001
).
Transfected embryos were allowed to develop to the specified stages then
dissected, fixed and processed for immunohistochemistry. Transfection of
pCAGGS-IRES-nls-GFP alone does not affect expression of neural markers or
neural crest markers (data not shown). For BrdU labelling, 100 µl of 200 ng
µl1 BrdU (Roche Biochemicals) was applied on top of the
transfected embryos in ovo 1 hour before harvesting.
Neural plate explants
For explant culture of electroporated neural tissue, HH stage 10 embryos
were electroporated with pCAGGS-Sox9-IRES-nls-GFP or pCAGGS-IRES-nls-GFP as a
control and incubated in ovo for 1-2 hours before isolating the neural
explants (Yamada et al., 1993;
Briscoe et al., 2001
). Neural
explants were cultured in collagen matrix (Vitrogen) with F12 medium
containing penicillin/streptomycin and Mito+ Serum Extender
(Collaborative Biomedical Products) for 48 hours before assaying GFP and HNK-1
expression (Liem et al.,
1995
). For transplantations, electroporated [i] (intermediate
neural tube) regions were placed between the neural tube and somite at the
forelimb level of HH stage 12-14 chick embryos. Transplanted embryos were
incubated for 72 hours before processing for immunohistochemistry.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Sox9 induces neural crest differentiation in neural cells
The expression profile of Sox9 raised the possibility that Sox9 is
involved in neural crest specification. To test this idea, Sox9 was
ectopically expressed in the neural tube of stage 10-11 chick embryos by in
ovo electroporation. A bicistronic vector was used that encoded Sox9 and
nuclear targeted GFP (a marker used to identify transfected cells), resulting
in the unilateral mosaic expression of Sox9 in the neural tube
(Briscoe et al., 2001). Our
analysis focuses on trunk regions of the neural tube between the forelimbs and
hindlimbs. We first examined the expression of the migratory-neural-crest
marker HNK-1 (Bronner-Fraser,
1986
). Embryos transfected with a control GFP vector did not
induce ectopic HNK-1 at any of the time points examined (data not shown). By
contrast, extensive, robust ectopic induction of HNK-1 production was detected
in neural progenitor cells of embryos electroporated with Sox9 12-48
hours after electroporation (Fig.
2D-I and data not shown, in 30/30 embryos). The activity of Sox9
appeared to be cell autonomous only transfected cells induced HNK-1
and adjacent untransfected cells lacked HNK-1 expression
(Fig. 2F,I). Moreover,
transfected Sox9 also induced other markers of neural crest including
Slug, Cad6B and Cad7 (see
Fig. 4). Transfection of
vectors directing expression of Sox8 and Sox10 also induced HNK-1 expression
with similar kinetics (data not shown, 15/15 embryos). Conversely, the
transfection of a Sox gene from a different subgroup (Sox2, a group B
Sox gene) did not induce ectopic HNK-1 (data not shown, 6/6 embryos).
Together, these data suggest that Sox9 and other group E Sox genes
induce neural crest differentiation in cells normally expected to generate CNS
neurons.
|
|
The finding that Sox9 expression induced migration of cells in vitro prompted us to examine in ovo electroporated embryos in more detail. 24-48 hours post-transfection (hpt), a marked increase in the number of HNK-1+ neural crest cells was observed migrating away from the transfected side of the neural tube (Fig. 2J,K and data not shown, n=6). These cells appeared to originate dorsally (Fig. 2G-I). Consequently, by 24 hpt, the most dorsal regions of the neural tubes were frequently depleted of cells compared with the untransfected side of the embryo (Fig. 2G-I). Consistent with this, there was an increase in basement membrane disassembly on the transfected side of embryos (Fig. 2L, data not shown). Ventral to this, there were Sox9-transfected cells expressing HNK-1 that migrated laterally through the pial surface (Fig. 2I,L,M). It was, however, noticeable that the delamination of Sox9-expressing cells in these intermediate and ventral regions of the neural tube was limited to a minority of the transfected population (Fig. 2L,M). Most Sox9-expressing cells at these dorsal-ventral positions remained within the neural tube and displayed the pseudostratified epithelial morphology characteristic of the neural tube despite expressing HNK-1 (Fig. 2D-I,M). Together, these data indicate that Sox9 can induce the differentiation of neural crest cells and, in some circumstances, these cells emigrate from the neural tube. However, cells only delaminate efficiently from dorsal regions of the neural tube. More ventrally, most cells remain within the neuroepithelium, suggesting that the initiation of neural crest delamination is constrained to the dorsal neural tube.
Sox9 induction of neural crest does not require BMP or Wnt
signals
The ability of Sox9 to induce markers and behaviour of neural
crest cells prompted us to examine the pathway of neural crest induction. The
secreted factors BMP4, BMP7, Wnt1 and Wnt3a are expressed in dorsal regions of
the neural tube and have been implicated in inducing neural crest
differentiation (Liem et al.,
1995; Ikeya et al.,
1997
; LaBonne and
Bronner-Fraser, 1998
;
Garcìa-Castro et al.,
2002
). Forced expression of Sox9 did not induce
expression of BMP4 (Fig.
3A-C, n=6), BMP7
(Fig. 3D-F, n=6) or
Wnt1 (Fig. 3G-I, n=6) at any time point examined. Indeed, in transfected embryos,
endogenous expression of these signalling molecules was downregulated in the
roof plate by 12 hpt (Fig.
3B,E,H); this is likely to be due to the increased delamination
and consequent loss of roof plate cells from this region. Furthermore,
transfection of a construct directing the expression of the BMP antagonist
Noggin together with Sox9 did not inhibit induction of ectopic neural crest
(M.C. and J.B., unpublished). Moreover, Sox9 did not induce expression of
Pax3, Pax7 or other genes characteristic of generic dorsal neural
tube identity that have previously been characterized as responding to
dorsalizing signals (data not shown) (Liem
et al., 1995
; Liem et al.,
1997
).
|
Sox9 induces a subset of early neural crest markers
We next examined factors that have been implicated in the cell autonomous
neural crest differentiation programme. Premigratory crest cells express
Slug, FoxD3 and Cad6B
(Nieto et al., 1994;
Luo et al., 2002
;
Kos et al., 2001
;
Nakagawa and Takeichi, 1998
),
whereas migratory crest cells express Sox10, Cadherin7
(Cad7) and HNK-1 (Cheng
et al., 2000
; Nakagawa and
Takeichi, 1998
; Bronner-Fraser,
1986
). We first focused on intermediate and ventral regions of the
neural tube, where Sox9 did not initiate efficient delamination.
Expression of Sox9 in these regions induced the expression of neural
crest markers. Similar to the endogenous expression profile, ectopic
Slug and Cad6B (Fig.
4A-F, n=16) were transient, induced at 6 hpt and
downregulated by 12 hpt (Fig.
4B,E, n=11). The induction of migratory neural crest
markers was slower but sustained. Robust Cad7 expression was not seen
until 12 hpt (Fig. 4H,
n=8) and ectopic expression of Sox10 was first evident 12-24
hpt (Fig. 4N,O; 10/12 embryos).
FoxD3, which is expressed in both premigratory and migrating neural
crest, was induced only at later times, 12-24 hpt
(Fig. 4K,L; 9/12 embryos),
raising the possibility that the early and late phases of FoxD3
expression are independently controlled.
We next turned our attention to the dorsal region of the neural tube where ectopic Sox9 induces a marked increase in the numbers of neural crest cells delaminating. In this domain, as ventrally, expression of premigratory neural crest markers Slug and Cad6B (Fig. 4A,D) was detected at 6 hpt. By 12 hpt, a decrease in the expression of these markers was evident (Fig. 4B,E). At later time points, 12-24 hpt, the migratory markers Cad7, FoxD3 and Sox10 (Fig. 4H,I,K,L,N,O) were observed in or adjacent to the dorsal neural tube. The rapid downregulation of premigratory markers on the transfected side of the neural tube contrasts with untransfected regions, where expression of premigratory markers continues in the dorsal midline at these stages (Fig. 4B,E). Taken together, these data suggest that Sox9 is sufficient to induce expression of neural crest markers in the neural tube. The inhibition at 12 hpt of premigratory markers in dorsal regions suggests that expression of Sox9 accelerates the premigratory to migratory transition of prospective neural crest cells, synchronizing the differentiation of all prospective neural crest cells. By 12 hpt, therefore, all cells in the dorsal neural tube have transited through the premigratory stage and matured to more differentiated stages, leaving none to continue expressing premigratory markers.
In contrast to the expression of the neural crest markers examined above,
Sox9 failed to induce RhoB at any time point examined
(Fig. 4P-R, n=18).
RhoB is expressed in premigratory neural crest and has been
implicated in promoting the delamination of neural crest cells
(Liu and Jessell, 1998).
Although the expression of RhoB was downregulated by 12 hpt in the
dorsal domain of progenitors (Fig.
4Q) in a similar manner to the other premigratory makers examined
(Fig. 4B,E), ectopic
RhoB expression was never observed at ventral or intermediate
positions (Fig. 4P-R). This
indicates that Sox9 is not sufficient to induce RhoB. The
lack of RhoB induction together with the finding that Cad7
(Fig. 4H,I) and HNK-1
(Fig. 2D-I), which are normally
restricted to migratory neural crest cells
(Nakagawa and Takeichi, 1998
;
Bronner-Fraser, 1986
), are
expressed within the neural tube in cells with a pseudostratified epithelial
morphology support the idea that, although Sox9 initiates the
transcriptional programme of neural crest development, it is not sufficient to
promote the delamination of neural crest cells. The efficient emigration of
neural crest cells is constrained to dorsal regions of the neural tube and the
expression of RhoB appears to define the region that contains cells
competent to delaminate.
Sox9 suppresses the normal differentiation programme of
neural progenitor cells
The induction of neural crest differentiation in Sox9-transfected
neural progenitor cells led us to examine whether this was at the expense of
the normal differentiation programme of neural cells. To address this, we
assayed the expression of progenitor and neuronal subtype markers in the
neural tube of Sox9-electroporated embryos. Ectopic Sox9
repressed the expression of the neural progenitor markers Pax7
(Fig. 5A-C, n=7),
Pax6 (Fig. 5D-F,
n=6), Nkx6 (Fig.
5G-I, n=6), Irx3, Olig2 and Nkx2.2
(data not shown) (Briscoe et al.,
2000; Novitch et al.,
2001
). The repressive activity of Sox9 was cell
autonomous only transfected cells demonstrated a change in expression
profile of the progenitor makers. Consistent with these data, Sox9
blocked the generation of classes of spinal neurons including Lbx1
(Fig. 5J-L, n=8) and
Pax2 (Fig. 5M-O,
n=6) expressing interneurons
(Müller et al., 2002
;
Burrill et al., 1997
) and
MNR2+/HB9+ motor neurons
(Fig. 5P-R, n=6)
(Tanabe et al., 1998
). These
neurons are each generated at different dorsoventral positions and, within
each domain, cells that did not express Sox9 generated neuronal
subtypes in a position-appropriate manner, indicating that the repression of
neuronal generation is also cell autonomous. We conclude that the expression
of Sox9 leads to cell autonomous suppression in neural progenitor
identity and neuronal differentiation.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sox9 and induction of neural crest development
Forced expression of Sox9 in the neural tube initiates a programme
of neural crest development. Consistent with these data, loss of function
analyses in Xenopus suggest that Sox9 is required for neural crest
development (Spokony et al.,
2002). Whether Sox9 is necessary for the specification of neural
crest in other species remains to be determined. The overlapping production of
the related proteins Sox8 and Sox10, and the demonstration that each SoxE gene
is sufficient to induce neural crest differentiation suggests functional
redundancy between SoxE genes that might partially or fully compensate for the
loss of an individual family member. Thus, it is possible that only limited
neural crest defects will be seen in Sox9 loss-of-function
mutants.
Sox9 is expressed in many cell types in addition to the neural
crest (Zhao et al., 1997) and
appears to play a role in the development of many tissues
(da Silva et al., 1996
;
Bell et al., 1997
). In each
tissue, Sox9 is proposed to carry out a distinct biological function and to
regulate a different subset of genes. Moreover, although Sox9
expression is restricted to prospective neural crest regions of the early
neural tube, it is subsequently expressed more broadly in neural progenitors,
in which it appears to have a role in CNS glial development
(Claus Stolt et al., 2003
). It
seems likely that this reflects changing competence of neural progenitors over
time early neural progenitors respond to Sox9 by neural crest
induction, whereas later progenitors have lost their ability to do this. The
transcriptional regulation by Sox genes usually requires DNA-binding
cofactors, hypothesized to provide target specificity, that differ between
tissues (Kamachi et al.,
2000
). In the case of Sox9, different cofactors have been
identified in chondrocytes (Lefebvre et
al., 1998
) and genital ridge cells
(de Santa Barbara et al.,
1998
). It is therefore possible that a partner necessary for
neural crest induction is expressed in neural progenitors; the identity of
this putative cofactor remains to be determined, but our data suggest that it
is expressed throughout early neural progenitors but is subsequently
downregulated so that later expression of Sox9 no longer promotes
neural crest induction.
Pathway of neural crest induction by Sox9
Neural crest induction has been divided into several sequential steps.
Initially, prospective neural crest cells are segregated from dorsal neural
progenitors by an inductive signal. Subsequently, these cells delaminate from
the neural tube and begin their migration into the periphery
(Le Douarin and Kalcheim,
1999). Our data indicate a role for SoxE genes at two distinct
steps in neural crest development. In the first step, Sox9 acts cell
autonomously downstream of the initial inductive event to induce a range of
neural crest properties including the expression of HNK-1, Slug, Cad6B,
Cad7, FoxD3 and Sox10.
The temporal sequence of gene induction in Sox9-transfected cells
is largely consistent with the temporal order of gene expression in endogenous
neural crest. Sox9 rapidly and transiently induces factors characteristic of
premigratory neural crest such as Slug and Cad6B
(Nieto et al., 1994;
Nakagawa and Takeichi, 1998
).
Markers of migratory neural crest, such as Sox10, HNK-1 and
Cad7 (Cheng et al.,
2000
; Bronner-Fraser,
1986
; Nakagawa and Takeichi,
1998
) are induced more slowly and maintained at 24 hpt. These data
are consistent with the idea that Sox9 expression is an early
response to neural crest induction signal and initiates the neural crest
differentiation programme. One exception is FoxD3, the endogenous
expression of which occurs in premigratory neural crest and migratory neural
crest (Dottori et al., 2001
;
Kos et al., 2001
). However,
ectopic Sox9 does not induce FoxD3 until 24 hpt, the time at which
migratory neural crest markers are induced. This raises the possibility that
FoxD3 expression in neural crest cells can be divided into two phases
an early Sox9-independent phase and a later phase in which the neural
crest differentiation programme initiated by Sox9 induces FoxD3
expression. Given the evidence that forced expression of FoxD3 is
also sufficient to induce some aspects of neural crest differentiation
(Dottori et al., 2001
;
Kos et al., 2001
), it will be
interesting to determine the epistatic relationship between Sox9 and
FoxD3.
In embryos transfected with Sox9, endogenous premigratory neural
crest markers are downregulated 12 hpt earlier than would normally be
expected. There is also a marked increase in the number of migratory neural
crest cells originating from this region. These data are consistent with a
model of neural crest development that involves the continuous recruitment of
premigratory neural crest cells from adjacent neuroepithelium
(Bronner-Fraser and Fraser,
1988; Bronner-Fraser and
Fraser, 1989
; Selleck and
Bronner-Fraser, 1995
). In Sox9-transfected embryos, the
transcriptional programme of premigratory neural crest is initiated in all
Sox9-expressing cells simultaneously, accordingly all cells in the
dorsal region of the neural tube commence neural crest differentiation
synchronously. Thus, these cells pass through the premigratory stage by 12 hpt
with the consequence that the pool of cells that could be progressively
recruited to replenish the premigratory neural crest population is
exhausted.
Coupling neural crest induction and delamination
In Sox9-transfected embryos, the depletion of cells from the
neural tube and the marked increase in the number of delaminating neural crest
cells are only evident in dorsal regions. In intermediate and ventral neural
tube regions, only a small proportion of Sox9-expressing cells
delaminate. Consistent with this, expression of HNK-1 and
Cad7 [markers normally restricted to migratory neural crest cells
(Bronner-Fraser, 1986; Nakagawa
and Takeichi, 1988)] is found within the neural tube of transfected embryos in
cells with a pseudostratified epithelial organization. These data suggest that
cells in ventral and intermediate regions of the neural tube are not competent
to delaminate efficiently in response to neural crest induction. It is
possible that emigration is restricted to the dorsal region by cell-intrinsic
or -extrinsic signals constraining delamination in intermediate or ventral
regions. Alternatively, it is possible that a signal is required dorsally to
induce delamination directly. In support of this second idea, Sela-Donenfeld
and Kalcheim have provided evidence that roof-plate-resident BMPs are
necessary to promote the emigration of premigratory neural crest cells
(Sela-Donenfield and Kalcheim,
1999
). Thus, the dorsal restriction of delamination of
Sox9-induced neural crest might reflect the range of effective BMP
signalling that is sufficient to promote delamination.
RhoB has been identified as a cell intrinsic determinant of neural crest
delamination (Liu and Jessell,
1998). Ectopic expression of RhoB is not observed in
Sox9-transfected embryos raising the possibility that the lack of
RhoB accounts for the low frequency of delamination in intermediate and
ventral regions of the neural tube. Moreover, the dorsal region (in which
forced expression of Sox9 does result in robust increased neural
crest migration) encompasses the region of endogenous RhoB
expression. The upregulation of RhoB therefore appears to demarcate a
region of cells competent to delaminate and the expression of RhoB or
factors with a similar expression profile might provide the molecular
mechanism that triggers neural crest delamination. Thus, the coordinated
induction of Sox9 and RhoB in dorsal regions of the neural
tube might act to couple the sequential steps of neural crest induction and
delamination during neural crest development.
Although ectopic delamination of Sox9-expressing cells in the
intermediate and ventral neural tube is inefficient, it can still be observed,
albeit at low frequency. Our findings suggest that Sox9 might induce
delamination in a RhoB-independent manner. However, we cannot rule out the
possibilities that RhoB induction occurs at a low level in our experiments or
that other members of the Rho family partially substitute for the lack of RhoB
(Liu and Jessell, 1998).
Dottori et al. (Dottori et al.,
2001
) have suggested that ectopic expression of FoxD3
induces neural crest delamination in a RhoB-independent manner but, in these
experiments, similar considerations also need to be taken into account;
emigration of neural crest was relatively inefficient and HNK-1
expression was prominent in the neural tube.
SoxE-expressing neural crest acquires properties of glial cells and
melanocytes but not neurons
Trunk neural crest cells adopt one of a range of potential fates and these
can be distinguished by the migration pathway, morphology and gene expression
profile of the cell (Le Douarin and
Kalcheim, 1999). Our data suggest that Sox9 is expressed
by the progenitors of all neural crest derivatives and
Sox9-expressing cells migrate along the routes of normal neural crest
migration, and are subsequently to be found residing in sympathetic and dorsal
root ganglions, peripheral nerves and underneath the ectoderm. The expression
of a SoxE gene in these migrating neural crest cells, however, biases the
differentiation pathways taken by these cells. Sox9-positive cells
are excluded from neuronal cell types, suggesting that the expression of
Sox9 is incompatible with neuronal differentiation.
Sox9-expressing cells do, however, acquire the characteristics of
glial cells, as shown by the presence of Sox9-expressing cells along
the peripheral nerve and the identification of Sox9+ cells
that co-express P0. Sox9-expressing cells were also observed entering
the dorsal lateral migration pathway, underlying the ectoderm. This pathway is
characteristic of melanocytes (Reedy et
al., 1998
). In the chick, neural crest cells typically migrate
along this route only at later stages of development, and these cells are
restricted to generating melanocytes
(Reedy et al., 1998
). The
finding of increased numbers of Sox9-expressing cells migrating via
this pathway in transfected embryos raises the possibility that Sox9
induces the precocious development of melanocytes.
The restriction of Sox9-expressing cells to a subset of neural
crest derivatives might indicate that Sox9 induces crest progenitors with
restricted differentiation potential. However, most of the lineage analysis
(Bronner-Fraser and Fraser,
1988; Bronner-Fraser and
Fraser, 1989
) and in vitro culture studies
(Stemple and Anderson, 1992
)
suggest that the fate of neural crest cells is not restricted prior to their
emergence from the neural tube. The Wnt signalling pathway has been shown to
influence the differentiation of melanocytes
(Ikeya et al., 1997
;
Hari et al., 2002
). The
induction of Wnt3a by Sox9 might therefore direct a proportion of
Sox9-expressing neural crest cells to this lineage. Alternatively,
this activity of Sox9 might reflect a later role for SoxE group genes
in the periphery. Sox10 has been implicated in regulating glial and
pigment cell differentiation (Britsch et
al., 2001
; Dutton et al.,
2001
). We demonstrate that Sox9 induces Sox10
expression in ectopic neural crest 12-24 hpt, so it is possible that
upregulation of Sox10 results in the induction of particular fates in
the periphery at the time cells differentiate. Alternatively, functional
equivalency between SoxE genes might account for the ability of Sox9
to direct neural crest cells towards non-neuronal fates. In this view,
Sox9 takes over the role of Sox10 and directly controls the
fate decisions. In conclusion, our study indicates that Sox9 plays an
important role in the developmental programme of neural crest cells, initially
inducing neural crest differentiation and then biasing the differentiation of
migrating neural crest cells to non-neuronal cells types.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aybar, M. J. and Mayor, R. (2002). Early induction of neural crest cells: lessons learned from frog, fish and chick. Curr. Opin. Genet. Dev. 12,452 -458.[CrossRef][Medline]
Bell, D. M., Leung, K. K., Wheatley, S. C., Ng, L. I., Zhou, S., Ling, K. W., Sham, M. H., Koopman, P., Tam, P. P. and Cheah, K. S. (1997). SOX9 directly regulates the type-II collagen gene. Nat. Genet. 16,174 -178.[Medline]
Bell, K. M., Western, P. S. and Sinclair, A. H. (2000). Sox8 expression during chick embryogenesis. Mech. Dev. 94,257 -260.[CrossRef][Medline]
Bhattachyaryya, A., Frank, E., Ratner, N. and Brackenbury, R. (1991). P0 is an early marker of the Schwann cell lineage in chickens. Neuron 7,831 -844.[Medline]
Bowles, J., Schepers, G. and Koopman, P. (2000). Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. Dev. Biol. 227,239 -255.[CrossRef][Medline]
Briscoe, J., Pierani, A., Jessell, T. M. and Ericson, J. (2000). A homeodomain code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101,435 -445.[Medline]
Briscoe, J., Chen, Y., Jessell, T. M. and Struhl, G. (2001). A Hedgehog-insensitive form of patched provides evidence for direct long-range patterning activity of Sonic hedgehog in the neural tube. Mol. Cell 7,1279 -1291.[CrossRef][Medline]
Britsch, S., E., Goerich, D., Riethmacher, D., Peirano, R. I.,
Rossner, M., Nave, K.-A., Birchmeier, C. and Wegner, M.
(2001). The transcription factor Sox10 is a key regulator of
peripheral glial development. Genes Dev.
15, 66-78.
Bronner-Fraser, M. (1986). Analysis of the early stages of trunk neural crest migration in avian embryos using monoclonal antibody HNK-1. Dev. Biol. 115, 44-55.[Medline]
Bronner-Fraser, M. and Fraser, S. (1988). Cell lineage analysis shows multipotentiality of some avian neural crest cells. Nature 335,161 -164.[CrossRef][Medline]
Bronner-Fraser, M. and Fraser, S. (1989). Developmental potential of avian trunk neural crest cells in situ. Neuron 3,755 -766.[Medline]
Burrill, J. D., Moran, L., Goulding, M. D. and Saueressig,
H. (1997). PAX2 is expressed in multiple spinal cord
interneurons, including a population of EN1+ interneurons that require PAX6
for their development. Development
124,4493
-4503.
Cheng, Y.-C., Cheung, M., Abu-Elmagd, M. M., Orme, A. and Scotting, P. J. (2000). Chick Sox10, a transcription factor expressed in both early neural crest cells and central nervous system. Brain Res. Dev. Brain Res. 121,233 -241.[Medline]
Cheng, Y.-C., Lee, C. J., Badge, R. M., Orme, A. T. and Scotting, P. J. (2001). Sox8 gene expression identifies immature glial cells in developing cerebellum and cerebellar tumours. Brain Res. Mol. Brain Res. 92,193 -200.[Medline]
Claus Stolt, C., Lommes, P., Sock, E., Chaboissier,
M.-C., Schedl, A. and Wegner, M. (2003). The Sox9
transcription factor determines glial fate choice in the developing spinal
cord. Genes Dev. 17,1677
-1689.
da Silva, S. M., Hacker, A., Harley, V., Goodfellow, P., Swain, A. and Lovell-Badge, R. (1996). Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds. Nat. Genet. 14,62 -68.[Medline]
Davis, C. A., Holmyard, D. P., Millen, K. J. and Joyer, A. L. (1991). Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum. Development 111,287 -298.[Abstract]
de Santa Barbara, P., Bonneaud, N., Boizet, B., Desclozeaux, M.,
Moniot, B., Sudbeck, P., Scherer, G., Poulat, F. and Berta, P.
(1998). Direct interaction of SRY-related protein SOX9 and
steroidogenic factor 1 regulates transcription of the human anti-Mullerian
hormone gene. Mol. Cell. Biol.
18,6653
-6665.
del Barrio, M. G. and Nieto, M. A. (2002).
Overexpression of Snail family members highlights their ability to promote
chick neural crest formation. Development
129,1583
-1593.
Dickinson, M. E., Selleck, M. A., McMahon, A. P. and
Bronner-Fraser, M. (1995). Dorsalization of the neural tube
by the non-neural ectoderm. Development
121,2099
-2106.
Dottori, M., Gross, M. K., Labosky, P. and Goulding, M.
(2001). The winged-helix transcription factor Foxd3 suppresses
interneuron differentiation and promotes neural crest cell fate.
Development 128,4127
-4138.
Dutton, K. A., Pauliny, A., Lopes, S. S., Elworthy, S., Carney,
T. J., Rauch, J., Geisler, R., Haffter, P. and Kelsh, R. N.
(2001). Zebrafish colourless encodes Sox10 and specifies
non-ectomesenchymal neural crest fates. Development
128,4113
-4125.
Epstein, D. J., Vekemans, M. and Gruss, P. (1991). Splotch (Sp2H), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homedomain of Pax3. Cell 67,767 -774.[Medline]
Garcìa-Castro, M. I., Marcelle, C. and Bronner-Fraser,
M. (2002). Ectodermal Wnt function as a neural crest inducer.
Science 297,848
-851.
George-Weinstein, M., Foster, R. F., Gerhardt, J. V. and
Kaufman, S. J. (1993). In vitro and in vivo expression of
7 integrin and desmin define the primary and secondary myogenic
lineages. Dev. Biol.
156,209
-229.[CrossRef][Medline]
Hamburger, H. and Hamilton, H. L. (1953). A series of normal stages in the development of the chick embryo. J. Morphol. 88,49 -92.
Hari, L., Brault, V., Kléber, M., Lee, H.-Y., Ille, F.,
Leimeroth, R., Paratore, C., Suter, U., Kemler, R. and Sommer, L.
(2002). Lineage-specific requirements of ß-catenin in neural
crest development. J. Cell Biol.
159,867
-880.
Hollyday, M., McMahon, J. A. and McMahon, A. P. (1995). Wnt expression patterns in chick embryo nervous system. Mech. Dev. 52,9 -25.[CrossRef][Medline]
Houzelstein, D., Cohen, A., Buckingham, M. E. and Robert, B. (1997). Insertional mutation of the mouse Msx1 homebox gene by an nlacZ reporter gene. Mech. Dev. 65,123 -133.[CrossRef][Medline]
Ikeya, M., Lee, S. M., Johnson, J. E., McMahon, A. P. and Takada, S. (1997). Wnt signaling required for expansion of neural crest and CNS progenitors. Nature 389,966 -970.[CrossRef][Medline]
Jiang, R., Lan, Y., Norton, C. R., Sundberg, J. P. and Gridley, T. (1998). The Slug gene is not essential for mesoderm or neural crest development in mice. Dev. Biol. 198,277 -285.[CrossRef][Medline]
Kamachi, Y., Cheah, K. S. E. and Kondoh, H.
(1999). Mechanism of regulatory target selection by the SOX
high-mobility-group domain proteins as revealed by comparison of SOX1/2/3 and
SOX9. Mol. Cell. Biol.
19,107
-120.
Kamachi, Y., Uchikawa, M. and Kondoh, H. (2000). Pairing SOX off with partners in the regulation of embryonic development. Trends Genet. 16,182 -187.[CrossRef][Medline]
Kim, J., Lo, L., Dormand, E. and Anderson, D. J. (2003). SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells. Neuron 38, 17-31.[Medline]
Knecht, A. K. and Bronner-Fraser, M. (2002). Induction of the neural crest: a multigene process. Nat. Rev. Genet. 3,453 -461.[Medline]
Kos, R., Reedy, M. V., Johnson, R. L. and Erickson, C. A.
(2001). The winged-helix transcription factor FoxD3 is important
for establishing the neural crest lineage and repressing melanogenesis in
avian embryos. Development
128,1467
-1479.
Kuhlbrodt, K., Herbarth, B., Sock, F., Hermans-Borgmeyer, I.
and Wegner, M. (1998). Sox10, a novel transcriptional
modulator in glial cells. J. Neurosci.
18,237
-250.
LaBonne, C. and Bronner-Fraser, M. (1998).
Neural crest induction in Xenopus: evidence for a two signal model.
Development 125,2403
-2414.
Le Douarin, N. M. and Kalcheim, C. (1999). The Neural Crest. Cambridge, UK: Cambridge University Press.
Lefebvre, V., Li, P. and de Crombrugghe, B.
(1998). A new long form of Sox5 (L-Sox5), Sox6 and Sox9 are
coexpressed in chondrogenesis and cooperatively activate the type II collagen
gene. EMBO J. 17,5718
-5733.
Liem, K. F. J., Tremml, G., Roelink, H. and Jessell, T. M. (1995). Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell 82,969 -979.[Medline]
Liem, K. F., Tremml, G. and Jessell, T. M. (1997). A role for the roof plate and its resident TGFß-related proteins in neuronal patterning in the dorsal spinal cord. Cell 91,127 -138.[CrossRef][Medline]
Liu, J. and Jessell, T. M. (1998). A role for
RhoB in the delamination of neural crest cells from the dorsal neural plate.
Development 125,5055
-5057.
Luo, T., Matsuo-Takasaki, M., Thomas, M. L., Weeks, D. L. and Sargent, T. D. (2002). Transcription factor AP-2 is an essential and direct regulator of epidermal development in Xenopus.Dev. Biol. 245,136 -144.[CrossRef][Medline]
Luo, T., Lee, Y.-H., Saint-Jeannet, J.-P. and Sargent, T. D.
(2003). Induction of neural crest in Xenopus by
transcription factor AP2. Proc. Natl. Acad. Sci.
USA 100,532
-537.
Martinsen, B. J. and Bronner-Fraser, M. (1998).
Neural crest specification regulated by the helix-loop-helix repressor Id2.
Science 281,988
-991.
Mayor, R., Guerrero, N. and Martinez, C. (1997). Role of FGF and Noggin in neural crest induction. Dev. Biol. 189,1 -12.[CrossRef][Medline]
Megason, S. G. and McMahon, A. P. (2002). A
mitogen gradient of dorsal midline Wnts organizes growth in the CNS.
Development 129,2087
-2098.
Müller, T., Brohmann, H., Pierani, A., Heppenstall, P. A., Lewin, G. R., Jessell, T. M. and Birchmeier, C. (2002). The homeodomain factor Lbx1 distinguishes two major programs of neuronal differentiation in the dorsal spinal cord. Neuron 34,551 -562.[Medline]
Nakagawa, S. and Takeichi, M. (1998). Neural
crest emigration from the neural tube depends on regulated cadherin
expression. Development
125,2963
-2971.
Nakata, K., Nagai, T., Aruga, J. and Mikoshiba, K.
(1997). Xenopus Zic3, a primary regulator both in neural
and neural crest development. Proc. Natl. Acad. Sci.
USA 94,11980
-11985.
Nakata, K., Nagai, T., Aruga, J. and Mikoshiba, K. (1998). Xenopus Zic family and its role in neural and neural crest development. Mech. Dev. 75, 43-51.[CrossRef][Medline]
Nieto, M. A. (2001). The early steps of neural crest development. Mech. Dev. 105, 27-35.[CrossRef][Medline]
Nieto, M. A., Sargent, M. G., Wilkinson, D. G. and Cooke, J. (1994). Control of cell behaviour during development by Slug, a zinc finger gene. Science 264,835 -839.[Medline]
Niwa, H., Yamamura, K. and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 15,193 -199.
Novitch, B., Chen, A. I. and Jessell, T. M. (2001). Coordination regulation of motor neuron subtype identity and pan-neural properties by the bHLH repressor Olig2. Neuron 396,749 -753.
Paratore, C., Eichenberger, C., Suter, U. and Sommer, L.
(2002). Sox10 haploinsufficiency affects maintenance of
progenitor cells in a mouse model of Hirschsprung disease. Hum.
Mol. Genet. 11,3075
-3085.
Persson, M., Stamataki, D., Welscher, P. te. andersson, E., Böse, J., Rüther, U., Ericson, J. and Briscoe, J. (2001). Dorsal-ventral patterning of the spinal cord requires Gli3 transcriptional repressor activity. Genes Dev. 16,2865 -2878.[CrossRef]
Reedy, M. V., Faraco, C. D. and Erickson, C. A. (1998). The delayed entry of thoracic neural crest cells into the dorsolateral path is a consequence of the late emigration of melanogenic neural crest cells from the neural tube. Dev. Biol. 200,234 -246.[CrossRef][Medline]
Schaeren-Wiemers, N. and Gerfin-Moser, A. (1993). A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: In situ hybridization using digoxigenin-labeled cRNA probes. Histochemistry 100,431 -440.[Medline]
Schorle, H., Meier, P., Buchert, M., Jaenisch, R. and Mitchell, P. J. (1996). Transcription factor AP-2 essential for cranial closure and craniofacial development. Nature 381,235 -238.[CrossRef][Medline]
Sela-Donenfield, D. and Kalcheim, C. (1999).
Regulation of the onset of neural crest migration by coordinated activity of
BMP4 and Noggin in the dorsal neural tube. Development
126,4749
-4762.
Selleck, M. A. and Bronner-Fraser, M. (1995).
Origins of the avian neural crest: the role of neural plate-epidermal
interactions. Development
121,525
-538.
Selleck, M. A., Garcìa-Castro, M, I., Artinger, K. B. and
Bronner-Fraser, M. (1998). Effects of Shh and Noggin
on neural crest formation demonstrate that BMP is required in the neural tube
but not ectoderm. Development
125,4919
-4930.
Sock, E., Schmidt, K., Hermanns-Borgmeyer, I., Röse, M, R.
and Wegner, M. (2001). Idiopathic weight reduction in
mice deficient in the high-mobility-group transcription factor Sox8.
Mol. Cell. Biol. 21,6951
-6959.
Spokony, R. F., Aoki, Y., Saint-Germain, N., Magner-Fink, E. and Saint-Jeannet, J.-P. (2002). The transcription factor Sox9 is required for cranial neural crest development. Development 129,421 -432.[Medline]
Stemple, D. L. and Anderson, D. J. (1992). Isolation of a stem cell for neurons and glia derived from the mammalian neural crest. Cell 71,973 -985.[Medline]
Tanabe, Y., William, C. and Jessell, T. M. (1998). Specification of motor neuron identity by the MNR2 homeodomain protein. Cell 95, 67-80.[Medline]
Vallstedt, A., Muhr, J., Pattyn, A., Pierani, A., Mendelsohn, M., Sander, M., Jessell, T. M. and Ericson, J. (2001). Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneurons specification. Neuron 31,743 -755.[Medline]
Villanueva, S., Glavic, A., Ruiz, P. and Mayor, R. (2002). Posterization by FGF, Wnt, and retinoic acid is required for neural crest induction. Dev. Biol. 241,289 -301.[CrossRef][Medline]
Yamada, T., Pfaff, S. L., Edlund, T. and Jessell, T. M. (1993). Control of cell pattern in the neural tube: motor neuron induction by diffusible factors from notochord and floor plate. Cell 73,673 -686.[Medline]
Yokotak, Y., Mansouri, A., Mori, S., Sugawara, S., Adachi, S., Nishikawa, S. and Gruss P. (1999). Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2. Nature 397,702 -706.[CrossRef][Medline]
Zhang, J., Hagopian-Donaldson S., Serbedzija G., Elsemore, J., Plehn-Dujowich, D., McMahon, A. P., Flavell, R. A. and Williams, T. (1996). Neural tube, skeletal and body wall defects in mice lacking transcription factor AP-2. Nature 381,238 -241.[CrossRef][Medline]
Zhao, Q., Eberspaecher, H., Lefebvre, V. and de Crombrugghe, B. (1997). Parallel expression of Sox9 and Col2a1 in cells undergoing chondrogenesis. Dev. Dyn. 209,377 -386.[CrossRef][Medline]