Instituto Cajal CSIC, Doctor Arce 37, 28002 Madrid, Spain
* Author for correspondence (e-mail: jbarbas{at}cajal.csic.es)
Accepted 18 June 2004
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SUMMARY |
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Misexpression of LSox5 in the cephalic neural tube activated RhoB expression throughout the dorsoventral axis. Furthermore, the prolonged forced expression of LSox5 enlarged the dorsal territory in which the neural crest is generated, extended the `temporal window' of neural crest segregation, and led to an overproduction of neural crest cells in cephalic regions. In addition to HNK-1, the additional neural crest cells expressed putative upstream markers (Slug, FoxD3) indicating that a regulatory feedback mechanism may operate during neural crest generation. Thus, our data show that in addition to the SoxE genes (Sox9 and Sox10) a SoxD gene (Sox5) also participates in neural crest development and that a cooperative interaction may operate during neural crest generation, as seen during the formation of cartilage.
Key words: LSox5, Neural crest, RhoB, Peripheral glia, Chick
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Introduction |
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SoxD genes contain more exons than do other Sox genes
(Schepers et al., 2002).
Additional complexity is introduced through tightly regulated mRNA processing,
the functional significance of alternative splicing being highlighted by the
differential expression of the distinct isoforms. Indeed, a short variant of
Sox5 (SSox5) is found only in adult testis, while longer isoforms (LSox5) are
expressed in other tissues during mammalian development
(Denny et al., 1992
;
Lefebvre et al., 1998
;
Hiraoka et al., 1998
). LSox5
variants share common structural features with other group D members,
including a leucine zipper and glutamine-rich regions thought to be involved
in dimerisation (Hiraoka et al.,
1998
; Lefebvre et al.,
1998
). In the mouse, LSox5/Sox6 heterodimers cooperate with
Sox9 to activate the Col2a1 gene, which encodes type II
collagen, an extracellular matrix component that is essential for
chondrogenesis (Lefebvre et al.,
1998
). Indeed, it is in this process that LSox5 has been most
thoroughly studied. Mutations in SOX9 produce campomelic dysplasia, a
syndrome that is often associated with autosomal XY sex reversal and involves
the severe malformation of most cartilage-derived structures
(Meyer et al., 1997
).
Furthermore, mouse chimeras containing Sox9/
embryonic stem cells inactivate early cartilage markers, including the
Col2a1 gene (Bi et al.,
1999
), a phenomenon that is also observed in Sox5, Sox6
double mutant embryos (Smits et al.,
2001
). This scheme is further clarified by the severe
downregulation of LSox5 and Sox6 expression produced after
Sox9 inactivation using the Cre/LoxP system in chondrogenic cell
lineages, thus demonstrating that LSox5 and Sox6 are
genetically downstream of Sox9 in chondrocytes
(Akiyama et al., 2002
).
Interestingly, in the chick, Sox9 has been implicated in the differentiation
of the neural crest (Cheung and Briscoe, 2002) and here we characterise chick
LSox5 and show its participation in cranial neural crest development.
The neural crest is a cell population that originates at the boundary
between the neural plate and the prospective epidermis. Once specified, the
cells of the neural crest delaminate from the neural folds/neural tube by
undergoing a process of epithelium to mesenchyme transition (EMT). These cells
then migrate along characteristic pathways and differentiate into a wide
variety of derivatives upon reaching their destination, including neurons and
glia of the peripheral nervous system, pigment cells, craniofacial cartilage
and bone (LeDouarin and Kalcheim,
1999). Diffusible factors of the BMP, BMP antagonists, FGF and WNT
families, together with retinoic acid, seem to direct the first steps of
neural crest induction (Liem et al.,
1995
; Selleck et al.,
1998
; Garcia-Castro et al.,
2002
; Villanueva et al.,
2002
), although different transcription factors are thought to
interpret these extracellular signals. Indeed, the winged-helix family member
FoxD3 and Sox9 have been implicated in the induction of the neural crest
(Kos et al., 2001
;
Dottori et al., 2001
;
Cheung and Briscoe, 2003
); and
members of the Snail family of transcription factors, Snail and Slug, are
required to trigger the EMT (reviewed in
Nieto, 2002
). The neural crest
derivatives become committed at different stages of their development and some
transcription factors seem to direct this lineage commitment. At low
concentrations, Sox10 apparently maintains the multipotency of the crest cells
and at higher doses it inhibits neuronal differentiation, favouring the
generation of peripheral glia and melanoblasts
(Kim et al., 2003
;
Paratore et al., 2001
). by
contrast, FoxD3 represses melanogenesis
(Kos et al., 2001
;
Dottori et al., 2001
) and it
has been demonstrated that Sox9 is necessary for the determination of the
chondrogenic lineage in cranial neural crest cells
(Mori-Akiyama et al.,
2003
).
We have characterised chick LSox5, which shows a high degree of similarity to its mammalian counterpart and an identical HMG box. Two splice variants were found that contain specific structural motifs susceptible to post-translational modification. We show that LSox5 is expressed in premigratory cranial neural crest cells and that following delamination, LSox5 expression coincides with a characteristic pattern of crest migration. High expression levels are maintained in the crest-derived components of cranial glia, including Schwann cells and satellite glia. Misexpression of LSox5 in the midbrain and the hindbrain provoked the rapid, cell autonomous upregulation of RhoB. This misexpression of LSox5 also led to an extension of the dorsal territory and of the developmental window in which the neural crest is produced, and augmented the generation of cephalic neural crest. The less immediate effects of LSox5 include the upregulation of other neural crest markers, such as Slug, FoxD3 and Sox10. Thus, we propose that LSox5 participates in the generation of the cranial neural crest and in the subsequent differentiation of the cranial glia lineage.
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Materials and methods |
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Cloning and sequencing of chicken LSox5
Chicken LSox5 was isolated in a subtractive screen to isolate
genes involved in the early stages of brain development. A cDNA subtraction
library was generated with the `Clontech PCR select-cDNA subtraction kit'
(Clontech, catalog number K1804-1), using the anterior region of stage 8 and
stage 11 chick embryos as the `driver' and `tester' populations, respectively.
One of the clones isolated in the screen was a partial cDNA for
LSox5, and the full-length cDNAs of LSox5-I and
LSox5-II (2.3 and 2.4 kb, respectively) were obtained by PCR walking
using a stage14 lambda zap cDNA library. The nucleotide sequence of
LSox5 appears in the EMBL, GenBank and DDBJ Nucleotide Sequence
Databases under the Accession Numbers AJ626988 and AJ626989. A putative short
isoform (SSox5) results from the deletion of the first 1248 coding
nucleotides of LSox5-II. Hence, we analysed the expression of the
alternatively spliced LSox5 isoforms by RT-PCR using the flanking
oligonucleotides: sense 5'-CTCCCAGCCTTTCACCTTCCC-3'; and antisense
5'-GCTTTCTGGAGTCCCTTTTAT-3'.
In ovo electroporation
Full-length LSox5-II (772 amino acids) and SSox5 (356
amino acids) cDNAs were cloned into a pCX vector
(Niwa et al., 1991) to be used
in electroporation assays. Each plasmid (4 mg/ml) was injected between the
neural folds of stage 8 chicken embryos together with pCX-EGFP (0.5
mg/ml), and the embryos were immediately electroporated as described
previously (Itasaki et al.,
1999
) with some modifications. Electroporation was performed with
a TSS20 Intracel square pulse generator, programmed to deliver five 50
mseconds, 15 V pulses at 4 Hz, through custom-made platinum electrodes. The
eggs were sealed and allowed to develop for another 6-7, 12 or 20-24 hours, at
which point the embryos were removed and processed for in situ hybridisation
or immunohistochemistry. Control electroporation assays were performed by
injecting pCX-EGFP (4.5 mg/ml) alone.
Whole mount in-situ hybridisation
Single or double whole-mount in situ hybridisation was carried out as
described (Nieto et al.,
1996). For double hybridisation, one probe was labelled with
digoxigenin-UTP and the second with fluorescein-UTP. The probes were detected
with anti-digoxigenin or anti-fluorescein antibodies coupled to alkaline
phosphatase, and visualised with NBT-BCIP (digoxigenin) or INT-BCIP
(fluorescein; all reagents and antibodies supplied by Roche). The
LSox5 probes were prepared from the full-length LSox5-II
cDNA isoform. The fluorescein-labelled LSox5 probe was often used to
control for electroporation without masking the blue staining of the probe
under analysis. Embryos were then photographed prior to embedding in gelatin
or agarose for vibratome sectioning.
Anti-LSox5 generation and immunohistochemistry
The LSox5-II cDNA was cloned into the pRSET plasmid (Invitrogen),
from which a fusion protein was generated to immunise rabbits and obtain
antisera. Protein extracts from embryonic fibroblasts stably infected with
RCAS vectors expressing LSox5-I or LSox5-II were analysed by
western blotting to test the affinity and specificity of the sera. One serum,
32A-III, specifically recognised LSox5 in western blots and produced a pattern
of immunostaining comparable with that obtained by in situ hybridisation (not
shown). This serum was used throughout this work. For immunohystochemistry, 10
µm cryostat or 40 µm vibratome agarose sections were permeabilised with
0.5% Triton X-100 (USB), blocked with 10% FBS and incubated overnight at
4°C with the primary antibody. After washing, the cryostat sections were
incubated for 1 hour with secondary antibodies or overnight in the case of
agarose vibratome sections. The primary antibodies were used at the following
concentrations: LSox5, 1:4000; EGFP, 1:1000 (Molecular Probes); Laminin 1,
1:1000 (Sigma); Pax7, 1:1000 (DSHB)
(Ericson et al., 1996); Slug,
1:1000 (62.1E6, DSHB) (Liem et al.,
1995
); P0, 1:1000 (1E8, DSHB) (Bhattacharyya et al.,
1991); HNK-1, 1:4000 (prepared from a cell line obtained from ATCC);
Islet-1/2, 1:1000 (40.2D6, DSHB) (Ericson
et al., 1992
). Cy2- or Cy3-conjugated secondary antibodies were
used (Jackson; 1:1000 dilution). HNK-1 whole-mount immunohistochemistry was
performed as described (Nieto et al.,
1996
).
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Results |
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The two cDNA isoforms isolated in the chick encode proteins of 737 and 772 amino acids, predicted to be of 80.99 and 84.95 kDa, respectively. The amino acid sequences are highly similar to their mammalian counterparts, LSox5-II sharing 95.7% and 93% of its residues with its human and mouse orthologues, respectively. The HMG box is identical in all three species, but out of this region, the chick LSox5-II contains 113 serine/threonine residues, 10% more than its mammalian orthologues. Strikingly, the majority of the additional S/T residues are concentrated between amino acids 426-456, and within this region, four serines and one threonine are concentrated in a short nine amino acid stretch. In silico screening of EST databases, together with the analysis of the corresponding genomic entries has shown that the inclusion of this serine-rich domain (SRD) varies across species. It is constitutively incorporated in zebrafish and fugu LSox5 polypeptides, absent in human, mouse and rat, and it may be alternatively spliced in the chicken (see Fig. S2 at http://dev.biologists.org/cgi/content/full/131/18/4455/DC1 shows the DNA sequence at the intron-exon boundary of different species, providing a molecular basis for this diversity). These phylogenetic variations may reflect the evolution of mechanisms that regulate LSox5 activity which involve differential phosphorylation.
We analysed the expression of the two distinct LSox5 isoforms by RT-PCR on cDNA samples from stages 9-23, with a pair of primers flanking the inserted domain. Two bands corresponding in size to that predicted for each isoform were detected at all stages and at the same intensity. Furthermore, in western blots of whole embryo protein extracts, a serum generated against LSox5 also detected two bands of similar intensity and with apparent molecular masses slightly higher than those predicted from the primary structure (the expression of LSox5 as obtained by both RT-PCR and western blot is shown in Fig. S1 at http://dev.biologists.org/cgi/content/full/131/18/4455/DC1). Thus, we detected LSox5 in embryos at all the stages examined and no differences in expression could be detected between the two isoforms during development, either by RT-PCR or immunoblotting. Nevertheless, as these studies were performed on whole embryos, we cannot exclude the possibility that local differences do indeed exist.
LSox5 is expressed in premigratory and migratory cephalic neural crest cells
The long splice variants derived from the Sox5 gene were first
identified in the mouse. They have been shown to play a crucial role in
chondrogenesis (Lefebvre et al.,
1998) and to be expressed in the developing pancreas, as well as
in a variety of adult human tissues (Ikeda
et al., 2002
; Lioubinski et
al., 2003
). At early stages of chick development, LSox5
mRNA appeared to be distributed in the neural crest and its derivatives
(Fig. 1). The expression of
LSox5 was first detected at stage 7 in the neural folds
(Fig. 1A), closely following
the onset of the expression of other neural crest markers such as
Slug (data not shown) and FoxD3
(Kos et al., 2001
). At all the
stages analysed, LSox5 was also expressed at high levels in the
notochord (Fig. 1C).
|
To confirm whether the migratory cells that express LSox5 were
neural crest cells, we examined the expression of Slug, a marker of the
premigratory and the migratory neural crest population
(Fig. 1J) (Nieto et al., 1994;
del Barrio and Nieto, 2004
).
Within the neural tube, all cells that expressed LSox5 also expressed Slug
(Fig. 1J-L), confirming that
LSox5 is expressed in premigratory neural crest cells. Nevertheless, a
subpopulation of cells only expressed Slug, indicating that LSox5 is not
common to all premigratory crest cells. In the early stages of migration, many
cells expressed both these proteins, although as migration proceeded, LSox5
expression augmented while the expression of Slug diminished
(del Barrio and Nieto, 2004
).
The onset of Slug expression prior to that of LSox5
indicated that Slug precedes LSox5 in the genetic cascade at play during
neural crest development.
LSox5 expression is maintained in the glial lineage of the cephalic peripheral nervous system
We then compared the expression of LSox5 with that of other neural crest
markers and its distribution in neural crest derivatives. RhoB is a small
GTPase implicated in the delamination of the neural crest that lies downstream
of Slug (Liu and Jessell,
1998; del Barrio and Nieto,
2002
). RhoB mRNA is expressed in a small population of
premigratory neural crest cells and in the early migratory cells. Its
expression pattern is very similar to that of LSox5 in the
premigatory neural crest cells, suggesting that at a particular stage of
development they may be co-expressed in neural crest cells. The glycoprotein
epitope HNK1 (Tucker et al.,
1984
) is found in the majority of migratory neural crest cells (Le
Dourain and Kalcheim, 1999; del Barrio and
Nieto, 2004
). Although most migratory neural crest cells were
labelled for both HNK1 and LSox5 (Fig.
1M), an early migratory cell population contained LSox5 alone.
In the head, all the peripheral glia and most neurons in both the sensory
and autonomic ganglia are lineages derived from the neural crest (reviewed by
LeDourain and Kalcheim, 1999). The population of peripheral glia includes
satellite glia of the cranial ganglia and the Schwann cells associated with
the cranial nerves. Our immunohistochemical analysis revealed that LSox5
expression was maintained in both these glial cell types. In Schwann cell
precursors that ensheath the cranial nerves, LSox5 is co-expressed with
P0, an early marker of both myelinating and nonmyelinating cells
(Bhattacharyya et al., 1991) (Fig.
1N). In the cranial ganglia, anti-LSox5 labelled small rod-shaped
nuclei whose morphology indicated that they might correspond to satellite
glia. This population of LSox5-positive cells was clearly different from the
neuroblasts that express Lim-domain factors of the Islet class
(Fedtsova et al., 2003)
(Fig. 1O).
LSox5 misexpression in the cephalic neural tube augments the production of neural crest
To investigate whether LSox5 plays a role in the migration and/or
differentiation of the neural crest, we ectopically induced expression of the
longest LSox5-II isoform on the right-hand side of the neural tube. This was
achieved by electroporating pCX-LSox5 (together with
pCX-EGFP as a marker of transformation) into the cephalic region of
stage 8 embryos. After 20-24 hours, uniform EGFP labelling was detected on one
side of the neural tube and in the characteristic streams of migratory cranial
neural crest (Fig. 2A). A
similar distribution of ectopic LSox5 was observed by immunohistochemistry
(Fig. 2B,C). Under these
conditions, the expression of HNK1 increased on the electroporated side,
particularly in the post-otic regions, although a similar effect was also
observed at different cranial levels (Fig.
2D,E). Moreover, many of the additional HNK1-labelled cells were
located at more retarded positions along the migratory pathway, and ectopic
epithelial cells were also labelled by HNK1 throughout the electroporated side
of the neuroepithelium (Fig.
2F-H). None of these effects was observed when pCX-EGFP
alone was electroporated (not shown).
|
The induction of RhoB is an immediate response to LSox5 misexpression
Under normal conditions, the onset of Slug expression occurs
before that of LSox5. Considering that the onset of LSox5
and RhoB expression occurs within a similar timescale, and that
LSox5 is able to induce RhoB in areas where Slug
was not induced (Fig. 2O), we
examined whether LSox5 could induce RhoB within short
periods of time and whether this was independent of Slug induction.
On the transformed side of the neural tube, we could identify a number of
cells that expressed EGFP and LSox5 6-7 hours after electroporation
(Fig. 3A,B). In these embryos,
ectopic RhoB expression was also detected in the intermediate and
ventral regions of the neural tube (Fig.
3C), but no abnormal Slug expression could be observed
(not shown). Moreover, no ectopic cells expressing RhoB were observed
in the contralateral side of the tube or in control embryos electroporated
with pCX-EGFP alone (not shown). The rapid appearance of ectopic
cells expressing both LSox5 and RhoB suggested that the forced
expression of LSox5 directly induced RhoB expression in the
neural tube (Fig. 3D-F).
Interestingly, this induction was transitory in intermediate and ventral
regions of the neural tube because 12 hours after electroporation only a
fraction of the EGFP-labelled cells here expressed RhoB. Similarly,
after 20 hours, the dorsal domain of RhoB expression had expanded and
a few scattered ectopic RhoB-expressing cells were detected out of
this region.
|
|
LSox5 misexpression drives neural crest differentiation towards non-neuronal phenotypes
The persistence of LSox5 in the cephalic neural crest derived glial
lineage, and the increased expression of both Sox10 and
FoxD3 upon LSox5 misexpression, prompted us to analyse the fate of
electroporated cells. In embryos that were allowed to develop for 48 hours
after LSox5 electroporation, many EGFP-labelled cells reached the branchial
arches or were spread across the most rostral mesenchyme (not shown). Thus, it
seemed likely that these cells might be related to the non-neural crest
derivatives. In the cranial ganglia, electroporated cells were preferentially
detected at sites where Schwann cell precursors accumulate, both at the
entrance and exit of the corresponding cranial nerve
(Fig. 5). We also found
labelled cells interspersed with neuronal precursors of placodal origin at the
distal region of the trigeminal ganglion
(Fig. 5A,B), the geniculate
ganglion (Fig. 5C,D) and the
petrosal ganglion (not shown). By contrast, labelled cells were very rarely
found in the proximal region of the trigeminal ganglion where neural
crest-derived neuronal precursors accumulate. In the ciliary and superior
ganglia, the neuronal precursors are all of neural crest origin, but they did
not coincide with LSox5 overexpressing cells
(Fig. 5E-H). These data suggest
that LSox5 overexpression in neural crest cells drives the differentiation of
non-neuronal phenotypes.
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Discussion |
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Not only does the overall sequence of chick LSox5 show a high degree of
similarity to that of its mammalian orthologue but the HMG box is identical.
Nevertheless, one structural feature that distinguishes chick LSox5 derives
from the fact that 16 out of the 33 amino acid substitutions between the mouse
and the predicted chicken protein lie within residues 426-456. Strikingly,
nine of these changes imply the gain of serine or threonine residues in the
chick protein, including a specific serine rich domain (SRD). Indeed, when
scanned for putative phosphorylation sites
(Blom et al., 1999), they
mainly lie in this region of LSox5 and some of the substitutions in the chick
sequence generate new sites. This suggests that in the chicken, specific
mechanisms may exist to regulate LSox5 activity by phosphorylation. Indeed,
the regulation of Sox function by serine phosphorylation has been
demonstrated: LSox5, Sox6 and Sox9 cooperate in the transactivation of a
Col2a1 chondrocyte-specific enhancer during cartilage formation, and
the activity of Sox9 in this process is controlled by cyclic AMP-dependent
protein kinase A phosphorylation (Huang et
al., 2000
).
LSox5 expression in the neural crest
We isolated chicken LSox5 while searching for genes that are
induced or upregulated between embryonic stages 8-11, a period during which
the cephalic neural crest forms and begins to migrate. LSox5
satisfied the criteria of the screen because it is expressed in the cephalic
neural crest, both premigratory and migratory. However, we also detected
LSox5 expression in the trunk neural crest, albeit at much lower
levels (not shown). At early developmental stages, LSox5 is observed
in the neural folds, but while it is expressed in the whole of the prospective
forebrain epithelium, in more caudal regions it is restricted to the most
dorsal part of the neural tube where the neural crest form. When compared with
other neural crest markers, this expression pattern is unique to
LSox5. The two genes Slug and Sox9 are restricted
to the dorsal region of the neural plate and the onset of their expression
coincides with the generation of the neural crest. By contrast, FoxD3
is first expressed in the most rostral region of the neural folds and only
becomes restricted to the territories where the neural crest forms prior to
delamination (Kos et al., 2002). Thus, the initial expression of
FoxD3 and the persistence of LSox5 in the anterior
prosencephalon are likely to be related to developmental processes other than
the generation of the neural crest.
In the head, the onset of LSox5 expression follows the
well-established anteroposterior developmental gradient in the dorsal neural
folds/tube, as do most other neural crest markers. However, Sox9,
Slug and FoxD3 all precede LSox5, which approximately
coincides with RhoB and precedes Sox10 expression. It is
interesting to note that LSox5, as Slug, is continuously
expressed at low levels throughout rhombomere (r) 3, while other markers such
as Sox9, FoxD3, RhoB and Sox10 are never expressed in this
rhombomere (Liu and Jessell,
1998; Cheng et al.,
2000
; Kos et al., 2002) (S.P.-A., M.A.N. and J.A.B., unpublished).
Although we do not know the functional significance of this at present, the
absence of many neural crest markers may be related to the fact that the
neural crest produced in r3 undergoes massive apoptosis
(Graham et al., 1993
). Those
crest cells that survive in r3 are deviated rostrally or caudally to
contribute to the major migratory streams adjacent to r2 and r4
(Birgbauer et al., 1995
). Thus,
based on their relative expression patterns, we would locate LSox5
downstream of Sox9, Slug and FoxD3, and at the same
hierarchical level or slightly upstream of RhoB and
Sox10.
Early upregulation of RhoB caused by LSox5 misexpression
Taking into account the gene hierarchy that emerged by analysing the
endogenous expression in the neural crest, the upregulation of RhoB
appears to be an immediate consequence of LSox5 misexpression in the
neural epithelium. This upregulation is a cell-autonomous phenomenon and it
initially presents near complete penetrance, i.e. most transformed cells
become RhoB-positive regardless of their location in the dorsoventral
axis of the neural tube. With time, and in parallel with the number of
LSox5-expressing cells within the neural epithelium, the number of
RhoB-expressing cells increases dramatically at the dorsal aspect of
the neural tube, concomitant with a massive delamination of cells expressing
different neural crest markers. By contrast, the number of
RhoB-expressing cells remains low at intermediate and ventral
positions, suggesting that these environments are hostile to its induction.
RhoB has been proposed to contribute to the changes in cell shape and adhesion
that occur during the delamination of neural crest cells by regulating actin
polymerisation, the formation of focal adhesions and of stress fibres
(Liu and Jessell, 1998).
Accordingly, it should be noted that some of the cells that are induced to
express RhoB but that lie outside of the dorsal domain of neural
crest competence, remain in the neural tube and maintain an epithelial aspect.
Only a few cells located outside of, but close to, the domain of competence
for crest formation seem to be able to delaminate and enter the adjacent
mesenchyme. However, even when they are able to delaminate, these cells do not
express any neural crest markers other than the ectopic LSox5. These
results indicate that LSox5 is likely to induce RhoB expression
directly. Moreover, although this makes the cells competent to undergo changes
in cell shape, it is not compatible with them undergoing complete EMT and
fully acquiring the neural crest phenotype, unless they are located in the
crest-competent domain. We also describe the appearance of HNK1-positive cells
within the neural epithelium as a consequence of LSox5 misexpression,
a phenotype similar to that described following FoxD3 or
Sox9 misexpression (Kos et al., 2002;
Cheung and Briscoe, 2003
).
Although we have not checked if this aberrant HNK1 expression coincides with
or follows RhoB ectopic expression, it seems plausible that this
phenomenon mimics the normal development of migratory neural crest cells,
which acquire the HNK1 epitope after expressing LSox5 and
RhoB. In addition, although we do not know the molecular mechanisms
involved in this particular behaviour, they may also operate under
physiological conditions. Whereas physiological expression of Slug, Sox9 and
FoxD3 is restricted to the neural crest competent region in the dorsal neural
tube, both RhoB- and HNK1-positive cells can be found at more ventral
locations later in development (S.P.-A., M.A.N. and J.A.B., unpublished).
LSox5 misexpression increases neural crest generation
We have shown that LSox5 misexpression in the neuroepithelium
produces a dramatic increase in the generation of neural crest cells, which
express several neural crest markers, including Pax7, Slug, FoxD3, RhoB, Sox10
and HNK1. Within the head region, this effect can be detected at all levels of
the AP axis. Quantitative variation was found between embryos that might
reflect the precise timing of the electroporation and analysis, and the
efficiency of transfection. Under our standard electroporation conditions, the
extension of the `temporal window' of neural crest generation was best
visualised in the mesencephalic and rostral hindbrain regions. Other aspects
such as the increase in the number of migratory cells were more evident in the
circumpharyngeal streams. Whereas the induction of RhoB expression
can be detected soon after the appearance of LSox5, the other markers
are only induced after longer periods of time, suggesting that their induction
is indirect. Before delamination, the ectopic crest cells are located within a
crest competent domain as defined by overexpressing Slug
(del Barrio and Nieto, 2002).
The expansion of this presumptive neural crest territory is paralleled by the
extension of the region in which the basement membrane disassembles, allowing
the delamination of the neural crest cells that follow the normal migratory
pathways. In addition, the `temporal window' of neural crest generation is
also extended, as has been observed after the misexpression of other neural
crest markers, such as Noelin 1 (Barenbaum et al., 2000),
FoxD3 (Kos et al.,
2001
; Dottori et al.,
2001
), Slug (del
Barrio and Nieto, 2002
), Sox10
(Aoki et al., 2003
) and
Sox9 (Cheung and Briscoe,
2003
). As a matter of fact, many features related to the increase
of neural crest production after LSox5 overexpression are similar to
those described in the aforementioned studies. It is thought that neural crest
overproduction occurs at the expense of other CNS cell types
(Dottori et al., 2001
;
Cheung and Briscoe, 2003
).
Consequently, the overproduction of neural crest cells frequently appears to
deplete the cells from the most dorsal region of the neural tube, as evident
morphologically following LSox5 misexpression.
Regardless of the generalised phenotype of neural crest overproduction
reported here, it is of interest to place LSox5 in the genetic
cascade of neural crest development. The endogenous expression patterns
tentatively locate it downstream of Sox9, Slug and FoxD3,
implying it acts after neural crest induction and the segregation of the crest
from the other neuroepithelial cells. The results obtained after
LSox5 overexpression indicate that it lies upstream of RhoB
and Sox10, compatible with studies indicating that RhoB is
involved in the delamination process and Sox10 in the survival and
maintenance of the stem cell properties in the migratory population
(Paratore et al., 2001;
Kim et al., 2003
;
Mollaaghababa and Pavan,
2003
). However, earlier markers were also upregulated when
LSox5 was ectopically expressed in neural crest cells at more
extended times, suggesting that a feedback loop may be at work in vivo. A
precedent comes from in vitro studies of EMT induction where a balanced
cross-modulation of cell-cell adhesion molecules, cell-ECM adhesion molecules
and cytoskeletal molecules can trigger and orchestrate EMT
(Newgreen and Minichiello,
1995
; Somasiri et al.,
2001
). Moreover, in a quail neural epithelial cell system in
vitro, the pharmacological inhibition of protein kinase C immediately affects
the cytoskeleton, provoking transformation into crest-like cells
(Minichiello et al., 1999
) and
the upregulation of Slug and of Sox10 expression (D. F.
Newgreen, personal communication). In addition, a non-autonomous cell response
may be induced by extracellular molecules (membrane proteins, soluble factors
or matrix molecules). Indeed, many neural crest cells in the migratory streams
on the transfected side did not express the EGFP marker, raising the
possibility of secondary or indirect induction.
With respect to signalling molecules, previous studies have shown that
signals emanating from the non-neural ectoderm and paraxial mesoderm, such as
the BMPs, WNTs and FGFs, induce neural crest differentiation
(Liem et al., 1995;
Ikeya et al., 1997
;
LaBonne and Bronner-Fraser,
1998
). However, the overproduction of neural crest caused by the
misexpression of both FoxD3 and Sox9 has been placed
downstream of these dorsalising pathways
(Dottori et al., 2001
;
Cheung and Briscoe, 2003
). The
same seems to hold true for LSox5 misexpression. We have verified
that Bmp4, Bmp7 and Wnt1 expression remains unaltered in
treated embryos, although it was sometimes diminished as a consequence of the
depletion of neuroepithelial cells in the dorsal tube (S.P.-A., M.A.N. and
J.A.B., unpublished).
During chondrocyte differentiation, Sox9 is required for the expression of
LSox5 (Akiyama et al., 2002),
which subsequently interacts cooperatively with Sox6 and Sox9 to activate the
type II collagen gene and promote chondrogenesis
(Lefebvre et al., 1998
). We
have analysed chick Sox6 expression by in situ hybridisation,
verifying that it is not expressed in either the dorsal neural tube or in
early migratory crest (S.P.-A., M.A.N. and J.A.B., unpublished). In the dorsal
neural tube, Sox9 activates a pathway leading to neural crest induction, in
which Sox10 is later induced (Cheung and
Briscoe, 2003
). Sox10 expression is then maintained in the
peripheral glial lineage (Britsch et al.,
2001
), where it regulates the expression of P0
(Peirano et al., 2000
). Thus,
it is tempting to speculate that in neural crest development a similar
interaction between SoxE and SoxD factors to that observed during chondroblast
differentiation occurs. In this case, LSox5 would be the downstream a member
of the Sox E group, Sox9, and would interact with Sox10, another E-group
member during the premigratory stages and/or later on during the
differentiation of the glial lineage. In addition, as our misexpression
experiments suggest LSox5 may lie upstream of Sox10, the expression of which
is induced later to cooperate at stages of differentiation. Alternatively,
Sox10 expression could be independent of LSox5, but may be upregulated or
induced after forced LSox5 expression, in which case it would enter in the
pathway of neural crest generation by mimicking the role of Sox9.
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ACKNOWLEDGMENTS |
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Footnotes |
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