Unité 368 de l'Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France
* Author for correspondence (e-mail: charnay{at}wotan.ens.fr)
Accepted 29 November 2002
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SUMMARY |
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Key words: Krox20/Egr2, High mobility group, Sox10, Hindbrain, Cranial neural crest, Transcriptional control, Pattern formation, Segmentation, Mouse, Chick
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INTRODUCTION |
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Previous studies, involving transplantations of neural tube or neural fold,
led to the conclusion that the NC exerts an instructive role in the
specification of the regional identity of the branchial arches
(Le Douarin, 1982;
Noden, 1983
; Noden, 1988).
Furthermore, the implication of Hox genes in the AP patterning of both
rhombomeres and NC (Gendron-Maguire et
al., 1993
; Rijli et al.,
1993
; Zhang et al.,
1994
; Barrow and Capecchi,
1996
; Goddard et al.,
1996
; Studer et al.,
1996
; Gavalas et al.,
1997
; Manley and Capecchi,
1997
), together with correlated Hox codes between NC cells and
their rhombomeric region of origin (Hunt
et al., 1991a
; Hunt and
Krumlauf, 1991
), led to the elaboration of a pre-patterning model
for craniofacial development: NC cells acquire positional information (Hox
gene pattern), according to their AP level of origin in the hindbrain, which
is passively carried to peripheral tissues where it is used to instruct
ectodermic, mesodermic and endodermic tissues in co-ordinated craniofacial
development (Hunt et al.,
1991b
; Hunt et al.,
1991c
). This view has been recently tempered by several
observations, including: (i) NC plasticity in Hox gene expression
(Schilling et al., 2001
;
Trainor and Krumlauf, 2001
;
Couly et al., 2002
;
Trainor et al., 2002b
); (ii)
independent Hoxa2 gene regulation in the hindbrain and in the NC
(Prince and Lumsden, 1994
;
Maconochie et al., 1999
);
(iii) patterning roles of the isthmus, cranial mesoderm and foregut endoderm
(Trainor and Krumlauf, 2000
;
Couly et al., 2002
;
Trainor et al., 2002b
); (iv)
acquisition of AP specification by the branchial arches in the absence of NC
input (Veitch et al., 1999
;
Gavalas et al., 2001
).
Together these data suggest that the NC cells are able to respond to signals
from the environment in which they migrate and that the regional specification
of the branchial arches involves a complex integration of patterning
information from different tissues.
In spite of this new evidence, the contribution of the NC to craniofacial
development is likely to depend in part on prepatterning events that define NC
migration pathways and AP identity. We are investigating this question in the
case of the Krox20 gene. Krox20 encodes a zinc finger
transcription factor that is expressed in r3 and r5, and has been shown to
play a key role in hindbrain segmentation: it controls and co-ordinates the
formation of the r3 and r5 territories and their acquisition of odd-number
characteristics (Schneider-Maunoury et
al., 1993; Schneider-Maunoury
et al., 1997
; Giudicelli et
al., 2001
; Voiculescu et al.,
2001
). These roles are exerted through the transcriptional control
of many genes, including Hox genes and members of the Eph receptor gene family
(Sham et al., 1993
;
Vesque et al., 1993
;
Nonchev et al., 1996
;
Theil et al., 1998
;
Giudicelli et al., 2001
;
Manzanares et al., 2002
).
Together with their ephrin ligands, the Eph receptors prevent cell mingling
between adjacent rhombomeres and are involved in controlling NC migration in
separate streams (Bergemann et al.,
1995
; Gale et al.,
1996
; Smith et al.,
1997
). In the NC, Krox20 expression is mainly restricted
to cells migrating lateral to r6 towards the third branchial arch and
presumably originating from r5 in the mouse
(Schneider-Maunoury et al.,
1993
). Krox20 is rarely expressed in the NC cells
migrating rostral from r5 and has not been detected in those produced in r3
(Schneider-Maunoury et al.,
1993
). Indeed, fate-tracing analyses have not revealed any
significant contribution of Krox20-expressing cells to the r3 NC
(Voiculescu et al., 2001
). In
the r5-derived NC, Krox20 controls the transcription of several key regulatory
genes, including Hoxa2, Hoxb2, Hoxb3 and EphA4
(Sham et al., 1993
;
Nonchev et al., 1996
;
Manzanares et al., 2002
;
Theil et al., 1998
), and may
therefore play an important patterning role.
In order to understand the regulatory network controlling Krox20 in the NC, we have searched for cis-acting regulatory elements responsible for this aspect of its expression. In this paper we present the identification and characterisation of a neural crest-specific element (NCE) located 26 kb upstream of the mouse Krox20 gene that drives specific expression in the r5 stream of the NC in transgenic mice. The NCE was shown to require both Krox20 and putative high mobility group (HMG) box binding sites for proper activity. The HMG box factor Sox10, expressed specifically in the NC, was found to co-operate with Krox20 to activate the NCE and to be required for the maintenance of Krox20 expression in the r5-derived NC. This organisation suggests a mode of patterning of the r5-derived NC according to its rhombomeric origin: following the initial Krox20 expression in the premigratory NC under the control of cis-regulatory elements active in r5, Krox20 and crest-specific Sox proteins combine to activate the NCE, maintaining its expression in the delaminating NC.
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MATERIALS AND METHODS |
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DNA constructs and mutagenesis
The pEMBL2 cosmid clone 8/4 (Chavrier et
al., 1989) was used as a source of murine Krox20
extragenic sequences. Subfragments were cloned upstream of a minimal
ß-globin promoter/lacZ reporter in plasmid pBGZ40
(Yee and Rigby, 1993
), as
described previously (Ghislain et al.,
2002
). Site-directed mutagenesis of Krox20 binding sites was
performed on fragment #11 cloned in pBS using the TransformerTM
Site-Directed Mutagenesis kit (Clontech). The oligonucleotides used were:
5'-ggaggaaagcgtcggttgcaggcagg-3' (binding site 1);
5'-gcctcgaagaagtcggcgggagcctc-3' (binding site 2);
5'-ggaattcgatatctctagaatcgataccgtc-3' (selection). Deletion of the
putative HMG box binding sites was performed using the ExsiteTM PCR-based
site-directed mutagenesis kit (Stratagene) with the following
oligonucleotides: 5'-ggctggggacgggca-3' (forward);
5'-tgttctcctttccctgtctcagcag-3' (reverse). All mutated fragments
were verified by sequencing and cloned into pBGZ40. Multimerisation of
sequences containing the putative HMG box binding sites (fragment #24) was
performed on double-stranded oligonucleotides containing terminal
SpeI sites by ligation into pBS. A 7x multimer was selected and
cloned into pBGZ40.
Generation of transgenic mice and in ovo electroporation
Transgenesis and identification of transgenic embryos by PCR was performed
as described previously (Sham et al.,
1993; Ghislain et al.,
2002
). In ovo electroporation in the chick neural tube was
performed as described previously
(Giudicelli et al., 2001
).
Krox20 transactivation studies were performed by coelectroporating the
lacZ reporter constructs with a mouse Krox20 expression
plasmid, pAdRSV Krox20
(Giudicelli et al., 2001
).
In situ hybridisation, X-gal staining and sections
Whole-mount in situ hybridisation was performed as described previously
(Wilkinson et al., 1992). Alkaline phosphatase activity was revealed using the
NBT/BCIP substrate (Roche). Antisense RNA probes were prepared from a
Krox20 cDNA (Wilkinson et al.,
1989) and from Sox9 and Sox10 est clones
obtained from the MRC geneservice (IMAGE ID 4165469 and 3675437,
respectively). Mouse and chick embryos were stained for ß-galactosidase
activity in toto following fixation in 4% paraformaldehyde (PFA) in
phosphate-buffered saline (PBS) for 15 minutes at 4°C. Staining was
performed in 1 mg/ml X-gal, 5 mM K3Fe(CN)6, 5 mM
K4Fe(CN)6, 2 mM MgCl2 and 0.1% Tween-20 in
PBS at 30°C for 15 hours. For staining prior to in situ hybridization
embryos were incubated for a maximum of 4 hours in the staining solution
containing 0.02% PFA. All tissues were postfixed in 4% PFA in PBS. Embyos for
sectioning were dehydrated and embedded in paraffin wax and 10 µm sections
were prepared. Sections were stained with Nuclear Fast Red at 1 mg/ml for 10
seconds, dehydrated and mounted in Eukitt (Merck).
Isolation of the chick NCE
A pBS-HindIII sub-library of a chicken genomic BAC clone carrying
the Krox20 gene and flanking sequences
(Giudicelli et al., 2001) was
transferred to Hybond-N+ membranes and hybridised at low stringency with the
mouse NCE fragment #17, labelled with [
-32P]dATP by random
priming, according to the manufacturers instructions (Amersham Pharmacia
Biotech). A positive clone, containing a 6.4 kb insert, was digested with a
series of restriction enzymes. Following gel electrophoresis, the DNA was
transferred under alkaline conditions to Highland-N+ membrane and hybridised
at low stringency with fragment #17. This identified a 1.7 kb
ApaLI/StuI subfragment that was cloned into pBS for
sequencing and pBGZ40 for electroporation in the chick neural tube.
DNase I footprinting and band shift assays
The mouse Krox20 protein was expressed in bacteria using the pET3a system
(Novagen). Extracts were prepared from Krox20-expressing and control bacteria
as described previously (Nardelli et al.,
1992). For footprinting and band shift experiments, fragment #17,
cloned in the SpeI site of pBS, was digested with either
BamHI or XbaI, dephosphorylated, T4 polynucleotide kinase
labelled using [
-32P]ATP and digested with NotI or
SmaI, respectively. Labelled fragments were purified by
polyacrylamide gel electrophoresis. DNase I footprinting experiments were
performed as described previously (Galas
and Schmitz, 1978
) and reaction products resolved on a 6%
denaturing polyacrylamide gel. Band shift experiments have been described
previously (Nardelli et al.,
1992
).
Cell culture, transfection and ß-galactosidase assay
HeLa cells were cultured in DMEM supplemented with 10% FCS. Cells
(150,000/35 mm well) were transfected with 1 µg of DNA in duplicate using
the Fugene 6 transfection reagent (Roche). Expression plasmids encoding the
mouse Krox20 protein, pAdRSV Krox20
(Giudicelli et al., 2001) and
human Sox10 protein, pECESOX10
(Bondurand et al., 2000
) were
used. Reporter constructs were the same as those used in in vivo experiments.
The total quantity of DNA was kept constant using empty pBS vector. Following
transfection, cells were incubated for 48 hours and extracts were assayed for
ß-galactosidase activity using the chemiluminescent ß-gel reporter
gene assay (Roche). Transfections were normalised by cotransfecting a
chloramphenicol acetyltransferase (CAT) expression plasmid (pSV2CAT)
and quantitating protein using the CAT ELISA assay (Roche).
Sequence alignment
The GenBank accession numbers for human and mouse genomic contigs
containing the Krox20 gene and flanking sequence are AL133417 and
AC068424, respectively. The 1.7 kb chick sequences containing homology to the
mouse NCE have been submitted to GenBank (accession number AY117679). Sequence
alignments were performed using Dialign
(Morgenstern et al., 1996) and
identification of putative Krox20 binding sites using the rVista program (G.
G. Loots, I. Ovcharenko, L. Pachter, I. Dubchak and E. Rubin,
unpublished).
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RESULTS |
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Identification of an autoregulatory element controlling
Krox20 expression in the neural crest
In order to identify cis-acting regulatory elements controlling
Krox20 expression, we generated transgenic lines with several
constructs containing large genomic regions surrounding a Krox20
allele with a reporter lacZ gene inserted in-frame in exon 2
(Krox20/lacZ) (Ghislain et al.,
2002). This insertion is identical to the knock-in allele
(Krox20lacZ) generated previously in our lab, which was
shown to reproduce the expression profile of the endogenous gene
(Schneider-Maunoury et al.,
1993
). One of these constructs, carrying sequences between -31 kb
and +7 kb relative to the transcription start site of Krox20 (-31/+7
Krox20/lacZ), was active at the dorsal edge of the neural tube at the
level of r5 and in a stream of NC cells migrating caudally from r5, as early
as 8.5 dpc (8-10 somites) in 3/3 transgenic lines
(Fig. 2A, Fig. 3, construct #1). In the
neuroepithelium, the ß-galactosidase-expressing cells were largely
restricted to the dorsal edge consistent with the premigratory NC
(Fig. 2B). The presence of more
ventrally located ß-galactosidase-positive cells
(Fig. 2B), which are probably
of neuroepithelial identity, may be explained by a lack of commitment of some
dorsal cells to a NC fate. The expression in migratory NC cells was maintained
at later stages, the majority migrating caudal to the otic vesicle
(Fig. 2C,D). By 9.5 dpc
expression was reduced, particularly in those cells distal to their site of
origin in the third branchial arch (Fig.
2D). A small number of lacZ-positive crest cells were
also found to migrate rostal to the otic vesicle
(Fig. 2C,D). This pattern of
expression of the transgene in the r5-derived NC is identical to those
described for knock-in Krox20 alleles as well as for the endogenous
gene (Fig. 1C)
(Wilkinson et al., 1989
;
Schneider-Maunoury et al.,
1993
; Nieto et al.,
1995
; Voiculescu et al.,
2001
).
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Delimitation of the NCE
Although the genetic studies described above clearly indicate the
involvement of autoregulation in the control of the activity of the NCE, the
identification of the cis- and associated trans-acting factors is required to
determine whether the role of Krox20 is direct or indirect. As a first step,
the NCE was delimited from the original -31/+7 Krox20/lacZ transgene.
Subfragments of the -31 kb to +7 kb region containing either the
Krox20/lacZ chimera (Fig.
3, construct #2) or linked to a ß-globin minimal
promoter/lacZ reporter (Fig.
3, constructs #3-6) were tested by `transient' transgenesis at 9.5
dpc. Of the five constructs spanning this region, only one was active in the
NC, the -31 kb to -23.5 kb fragment, which reproduced the profile in 5/9
transgenic embryos (Fig. 3,
Fig. 4A, construct #6S).
Interestingly, one of the embryos, which was positive in the NC, also showed
widespread expression in r5 (Fig.
3 and data not shown). Fragment #6 was also tested by transgenesis
in antisense orientation relative to the promoter/lacZ reporter
(Fig. 3, construct #6AS). This
construct was active in 2/6 transgenic embryos in the NC, suggesting that the
NCE acts as a canonical transcriptional enhancer (data not shown).
|
In ovo electroporation in the chick neural tube offers a quick and easy
procedure to test the activity of cisregulatory elements in the
neuroepithelium (Itasaki et al.,
1999). To determine whether this approach could facilitate the
delimitation of the NCE, constructs #2-6 were electroporated into chick embryo
hindbrains at Hamburger and Hamilton (HH) stage 9 and analysed for
ß-galactosidase activity at HH stage 13-14. Similar to the results for
these constructs in mouse transgenesis, constructs #2-5 were negative whereas
construct #6 was active in the NC (Fig.
3, Fig. 4B).
Interestingly, in chick electroporation this construct was also active in r3
and r5 (Fig. 4B; see
discussion). These results support the applicability of in ovo electroporation
to delimit the NCE.
Subsequently, a series of deletions of fragment #6 were analysed for activity in the chick neural tube linked to the ß-globin/lacZ promoter/reporter (Fig. 3, constructs #7-11). These studies led to the identification of an approximately 1 kb fragment extending from -26.5 kb to -25.5 kb which, much like construct #6, was active in the NC and r3 and r5 (Fig. 3, Fig. 4C, construct #11). When tested by transgenesis in the mouse, construct #11 produced a high frequency of expression in the NC and a low frequency in r5, similar to construct #6 (Fig. 3, Fig. 4D and data not shown). The NCE was further delimited by testing external 5' and 3' deletions of the 1 kb fragment (Fig. 3, constructs #12-16). Using chick electroporation, we identified a 247 bp sequence with activity similar to the original fragment (Fig. 3, Fig. 4E, construct #17). When this fragment was tested in mouse transgenesis, it gave results similar to those of fragment #11, although the level of expression in the NC was reduced and the frequency of expression in r5 was increased (Fig. 3, Fig. 4F, construct #17). This raises the possibility that some cis-regulatory elements that contribute to the activity in the NC are located outside of fragment #17. However, as these sequences control only the level of activity in the NC and not the specificity, the characterisation of the 247 bp fragment was pursued.
As Krox20 was shown to act upstream of the NCE in the mouse
(Fig. 2C-F), we tested the
effect of Krox20 ectopic expression on the activity of this element
by co-electroporation of construct #17 with a Krox20 expression
vector (Giudicelli et al.,
2001) in the chick hindbrain. In contrast to construct #17 alone,
whose activity was limited to the endogenous Krox20 expression
domain, co-electroporation led to activation of the reporter throughout the
electroporated region (Fig. 3,
construct #17+; compare Fig. 4E with
G). This unrestricted response to Krox20 provides additional
evidence in favour of an autoregulatory mechanism controlling the activity of
the NCE.
In summary, deletion studies identified a fragment (#11) located between -26.5 kb and -25.5 kb, which reproduced the profile of the -31/+7 Krox20/lacZ transgene in the NC. Further deletions of this element resulted in the isolation of a minimal, 247 bp, Krox20-responsive NCE, which contains all of the sequences necessary for specific activity in the NC.
Isolation of a conserved chick NCE
As the mouse NCE performed comparably in the NC of both mouse and chick, we
searched for orthologous regulatory elements in the chicken genome to aid the
identification of cis-and trans-acting factors regulating Krox20 in
the NC. The putative chick NCE was isolated from a bacterial artificial
chromosome (BAC) clone containing the chicken Krox20 genomic sequence
and flanking regions (Giudicelli et al.,
2001) by hybridisation at low stringency, using fragment #17 as a
probe. A 1.7 kb, cross-hybridising subfragment was tested in chick
electroporation after linking to the ß-globin/lacZ
promoter/reporter. This construct was active in the r5 NC stream and in r3 and
r5 similar to the mouse NCE, although occasional ectopic expression in the
rostral neuroepithelium was also observed
(Fig. 4H). The nucleotide
sequences of mouse fragment #11 and of the 1.7 kb chick fragment were then
established and compared together with the human sequences that were available
in GenBank. These analyses revealed the existence of blocks of homology
corresponding approximately to mouse fragment #17 (247 bp) and consisting of
211 bp in the chick and 243 bp in man. They showed 65% and 83% sequence
identity to mouse fragment #17, respectively
(Fig. 5). Interestingly,
despite the indication that sequences flanking fragment #17 participate in NC
activity, no significant homology was detected in this region in mouse/chick
comparisons, supporting the conclusion that the essential cis-regulatory
sequences are located within fragment #17 (data not shown).
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In conclusion, together with the genetic data obtained with the Krox20 null allele, these results demonstrate the existence of a direct Krox20 autoregulation in the NC that is required for the majority of the NCE activity. The limited crest activity that remains following the elimination of the two Krox20 binding sites may be due to either residual in vivo Krox20 binding activity and/or the action of other factors.
An NCE subregion containing HMG box binding sites is involved in
crest-specific expression
In subsequent experiments, we searched for cis-acting sequences involved in
the weak crest activity observed with mutant fragment #18. We introduced a
series of internal deletions into fragment #18 and tested their effects in the
chick electroporation assay. We focused our analysis on the region of
interspecies sequence similarity since it is likely to contain the essential
regulatory elements (Fig. 5).
Two deletions, together covering the majority of this region, were first
generated (Fig. 3, constructs
#19 and #20). Whereas construct #19 behaved in a manner similar to the
original construct #18, construct #20 was completely inactive (data not
shown). The region deleted in construct #20 was further subdivided into two
deletions (Fig. 3, constructs
#21 and #22). Interestingly, both deletions were inactive in the NC (data not
shown), raising the possibility that important regulatory sequences may lie at
the junction between the two regions.
The sequences deleted in construct #20 were analysed for the presence of
putative transcription factor binding sites that were conserved between the
mouse, human and chick sequences. We identified two adjacent, conserved
sequences in a head-to-head orientation, similar to HMG box protein binding
sites (A/TA/TCAAAG, Fig. 5)
(Travis et al., 1991;
van de Wetering et al., 1991
;
Harley et al., 1994
). These
sites are located precisely at the junction between the regions deleted in
fragments #21 and #22. A mutant fragment consisting of a 15 nt deletion was
generated to eliminate the two putative HMG box binding sites
(Fig. 5). When tested in chick
electroporation this construct was inactive, suggesting that these sites
contribute to the activity of the NCE (Fig.
3, Fig. 7C,
construct #23). A 41 bp sequence encompassing these sites
(Fig. 5) was then multimerised
and tested for enhancer activity in both chick electroporation and mouse
transgenesis (Fig. 3, construct
#24). In the chick, this construct was active throughout the NC along the
electroporated region (Fig.
7D). A similar profile was observed in mouse transgenesis:
although weak, NC cells migrating from r2, r4 and r5/6 were clearly labelled
as well as some trunk crest cells (Fig.
7E).
In conclusion, the search for additional cis-acting sequences in the NCE identified an essential subregion containing conserved sequences similar to HMG box binding sites which, when multimerised, appears to exhibit enhancer activity throughout the NC, implicating the action of crest-specific HMG box factors.
Synergistic activation of the NCE by Krox20 and Sox10
Several HMG box, SRY-related Sox genes are expressed in the NC and
may therefore participate in Krox20 expression. These include two
group E Sox genes, Sox9 and Sox10
(Ng et al., 1997;
Zhao et al., 1997
;
Kuhlbrodt et al., 1998
;
Southard-Smith et al., 1998
).
Recently, Sox9 was shown to be important in the specification of NC cells in
Xenopus (Spokony et al.,
2002
). Sox10, although not required for crest formation and
migration, participates in subsequent differentiation steps
(Herbarth et al., 1998
;
Southard-Smith et al., 1998
;
Kelsh and Eisen, 2000
;
Britsch et al., 2001
;
Dutton et al., 2001
). The
possibility that Sox10 contributes to Krox20 expression in
the NC was investigated. Firstly, we analysed its expression profile by in
situ hybridisation. Sox10 expression was detected as early as 8.5 dpc
in the pre/post migratory NC, with the highest levels in r2 and r4
(Fig. 8A). However, significant
levels were also detected in dorsal r5
(Fig. 8A). Double labelling of
Krox20, by detection of ß-galactosidase activity in
Krox20+/lacZ embryos, and Sox10 revealed
a co-localisation in migrating NC, a profile that continued throughout the
stages of crest migration (Fig.
8B and data not shown).
|
In a second series of experiments we investigated the possibility that Krox20 and Sox10 co-operate in the activation of the NCE. This was performed in a transactivation assay in cultured HeLa cells, which provides a quantitative evaluation of the contribution of each factor (Fig. 9). Using the wild-type NCE construct #11 as reporter, transfection of either a Krox20 or Sox10 expression construct led to a moderate level of induction. In contrast, the combination led to a synergistic induction. In order to investigate whether the synergism between these factors requires the elements important in NCE activity, a series of NCE mutants were tested. Construct #18, mutated in the two Krox20 binding sites, was weakly responsive to Krox20, confirming that the majority of the Krox20 binding activity had been eliminated. Although the Sox10 response was not affected, the combination of Krox20 and Sox10 resulted in a considerably reduced synergistic induction when compared to the wild-type construct. This suggests that the two Krox20 binding sites are required but that additional, weaker sites, are active. These results are also consistent with our in vivo data showing that construct #18 is weakly active in the NC (Fig. 7A,B). A new mutant derivative of construct #11, carrying uniquely the 15 nt deletion of the putative HMG box binding sites (Fig. 5), was tested. While the response to Krox20 was similar to the wild-type construct, this construct responded poorly to Sox10, consistent with an interaction of this factor with these sites. The combination of Krox20 and Sox10 together resulted in a considerably weaker synergistic response than with the wild-type construct. Although the reduced activity implicates additional sites, these results suggest an important contribution of the putative HMG box binding sites. Indeed, a construct containing a 7X multimer of these sites (#24) was dramatically induced by Sox10. Finally, a construct deficient in both the Krox20 and HMG box binding sites (#23) showed only an additive response to the combination of Krox20 and Sox10, definitively establishing the importance of these sites in the synergistic response. This latter result is consistent with our in vivo data indicating that construct #23 is defective in NC activity in chick electroporations (Fig. 7C).
|
Sox10 is required for the maintenance of Krox20
expression in the migratory neural crest
To establish the involvement of Sox10 in Krox20
regulation in vivo we analysed its expression in the Sox10 mutant
line, Dom (Herbarth et al.,
1998; Southard-Smith et al.,
1998
). Comparison of wild-type (data not shown), heterozygous
(Fig. 8C) and homozygous mutant
embryos (Fig. 8D) at 9.0 dpc
revealed a strong decrease in Krox20 levels in the migrating neural
crest of the homozygous mutant, whereas the heterozygote was not affected.
This phenotype was observed throughout the stages of NC migration (data not
shown). As Sox10 mutation does not affect neural crest production and
migration (Southard-Smith et al.,
1998
; Britsch et al.,
2001
), this effect is likely due to a decrease in gene
transcription.
In conclusion our experiments indicate that Krox20 and Sox10 are co-expressed in the migrating NC, they can co-operate in the autoactivation of the NCE in vitro and Sox10 is involved in the control of Krox20 expression in vivo. This suggests that Sox10 is an essential coactivator of Krox20 autoregulation in the NC.
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DISCUSSION |
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Krox20 autoregulation
We have identified the NCE as an enhancer located upstream of
Krox20, which can drive specific expression in the r5-derived NC
stream in transgenic mice, reflecting the normal Krox20 expression
pattern in the NC. The activity of this element is largely dependent on the
integrity of two Krox20 binding sites and is abolished in a Krox20
null background. These data establish that the NCE can mediate direct,
positive autoregulation of Krox20 in the NC, raising the question as to what
extent this element contributes to Krox20 expression. Sequence
similarity searches identified elements in both man and chick that are the
orthologues of the mouse NCE (Fig.
5). The chick element exhibited an activity in the NC similar to
that of the mouse element when tested by electroporation in the chick neural
tube. These observations suggest that the NCE probably has an important
function in these cells as it has been conserved throughout the evolution of
birds and mammals. More directly, analyses performed on compound mutant
embryos, Krox20Cre/lacZ, revealed a role of
Krox20 in maintaining the level of ß-galactosidase activity and
therefore its own level in the NC (Fig.
1). Furthermore the contribution of autoregulation may be
partially masked in these studies by the stability of the ß-galactosidase
protein which is likely to be higher than that of the Krox20 protein itself.
We can conclude that autoregulation allows for prolonged Krox20
expression in the NC following the migration of these cells from the neural
tube. This may be critical for maintaining NC positional identity since
several Hox genes (Hoxa2, Hoxb2 and Hoxb3) are under
Krox20 regulation (Sham et al.,
1993; Nonchev et al.,
1996
; Manzanares et al.,
2002
). In addition to Hox genes, Krox20 controls the
expression of the receptor tyrosine kinase gene, EphA4, in the NC
(Seitanidou et al., 1997
;
Theil et al., 1998
). By
repulsion from cells expressing their Ephrin ligands, the Eph/Ephrin
signalling system may guide NC cells along their pathway (reviewed by
Holder and Klein, 1999
).
Indeed, blocking EphA4 function in Xenopus embryos causes r5-derived
NC cells, normally destined for the third arch, to migrate into the second and
fourth arches (Smith et al.,
1997
). This may partially explain why Krox20-expressing
NC, which normally migrate caudally to the otic vesicle, migrate both
rostrally and caudally in Krox20 mutant mice
(Schneider-Maunoury et al.,
1993
).
The involvement of autoregulation in the control of Krox20
expression is not specific to the NC. We have also observed this mode of
regulation in r3 and r5 (Giudicelli et
al., 2001; Voiculescu et al.,
2001
) and in the bone-forming cells (M. F., unpublished).
Autoregulation secures Krox20 expression when the factors required
for its initiation are no longer present, reflecting the importance and the
duration of the role of this factor in developmental processes. Interestingly,
distinct cis-acting elements mediate autoregulation in these tissues (this
study; M. F., unpublished; D. Chomette, unpublished). The existence of
multiple autoregulatory elements may derive from the necessity of tight
coupling of autoregulation with the presence of other tissue-specific factors
that have binding sites within each of these elements.
Sox proteins as partners for Krox20 autoregulation in the
neural crest
Analysis of the NCE carrying mutations in the two Krox20 binding sites led
to the identification of an essential subregion containing two adjacent,
head-to-head, putative HMG box binding sites
(Fig. 7C). In addition, when a
multimerised oligonucleotide containing these sites was tested in vivo,
enhancer activity was detected throughout the NC
(Fig. 7D,E). Together these
studies suggested the action of crest-specific HMG box proteins in
Krox20 autoregulation. We then provided evidence specifically
implicating members the group E Sox subfamily of HMG box proteins: (i) two
members of this group, Sox9 and Sox10, are expressed in the
pre/post migratory NC (Ng et al.,
1997; Zhao et al.,
1997
; Kuhlbrodt et al.,
1998
; Southard-Smith et al.,
1998
) and Sox10 is co-expressed with Krox20 in
r5-derived NC (Fig. 8); (ii)
Sox10 can synergise with Krox20 in NCE transactivation studies in cell culture
(Fig. 9); (iii) Krox20
levels in migrating neural crest are not maintained in Sox10 mutant
embryos (Fig. 8). Together
these data indicate that Sox10 is a crest-specific factor essential for
Krox20 autoregulation in the NC. This conclusion is consistent with
the co-operation recently observed between Sox10 and Krox20 in the activation
of the connexin 32 gene in the Schwann cell lineage
(Bondurand et al., 2001
),
suggesting that such co-operation may be a more general phenomenon.
Although our studies highlight an important role of Sox10, it may not be the only HMG box factor co-operating with Krox20 in the r5-derived NC. Sox9, which is expressed in the newly formed r5-derived neural crest (data not shown) may also be involved in an earlier step of Krox20 autoregulation. Similarly, although the mutation of either the Krox20 or HMG sites resulted in a significant decrease in the synergistic response, the response was not completely eliminated, suggesting that additional Krox20 and HMG binding sites may be involved. This is supported by sequence analyses of the NCE indicating the presence of additional putative binding sites for both factors (data not shown).
In addition to the demonstration of synergism between Krox20 and
crest-specific Sox proteins, our data suggest that these factors may also have
distinct roles in the activation of the NCE. In the mouse, the NCE is
generally active in r5-derived NC and less frequently in r5. In the
electroporated chick neural tube, the NCE is active in all territories
expressing Krox20: r3, r5 and r5-derived neural crest. Furthermore,
in the chick, ectopic expression of Krox20 leads to generalised
activation of the NCE in both the neuroepithelium and the NC. A hypothesis to
explain these data is that activation of the NCE by Krox20 may be very
dependent on the chromatin state of the NCE: in normal chromosomal chromatin
configuration, Krox20 absolutely requires co-operation with a crest-specific
factor to activate the NCE; in contrast, in `relaxed chromatin'
configurations, reached at a low frequency in mouse transgenesis, where it may
depend on the site of insertion of the transgene, and systematically after
electroporation in the chick neural tube, where the reporter plasmid is likely
to remain episomal, Krox20 can activate the NCE without the crest factor.
According to this hypothesis, the crest-specific factor might modify the local
conformation of the chromatin in order to allow activation by Krox20. The
properties of Sox proteins are consistent with such a possibility
(Wegner, 1999).
A model for Krox20 regulation in the neural crest
The present data allow us to propose a model on how Krox20
expression is set up in r5-derived NC according to its level of origin in the
hindbrain. This study has established an absolute requirement for Krox20 in
NCE activity (Fig. 2).
Consequently, the Krox20 protein must accumulate in the premigratory NC prior
to the activation of the NCE by another mechanism. This may involve the action
of regulatory elements that establish Krox20 expression in the
neuroepithelium, without discrimination between neural and NC precursors. We
propose that establishment of Krox20 expression in r5-derived NC
involves the following steps (Fig.
10). (i) Initiation of Krox20 expression in r5 by
hindbrain cis-acting initiator element(s). We have recently identified an
r3/r5-specific regulatory element, located upstream of the NCE, that
constitutes a good candidate to perform this function (D. Chomette,
unpublished). (ii) Activation of the NCE in premigratory NC cells in r5 by
co-operation between Krox20 and crest-specific Sox proteins. (iii) Maintenance
of Krox20 expression under control of the NCE in postmigratory
r5-derived NC once the initiators are downregulated. The model explains the
establishment of a registry between Krox20-expressing NC and r5 on a
cell lineage basis: sufficient amounts of Krox20 have to be inherited in
delaminating NC cells for them to initiate or maintain the autoregulatory loop
under the control of crest-specific Sox proteins. Such a mechanism ensures the
acquisition of positional information in the NC according to its rhombomeric
origin and may be involved in the control of the expression of other
patterning genes. More generally, autoregulatory mechanisms, such as those
controlling the expression of Hox genes in the hindbrain neural crest
(Pöpperl et al., 1995;
Maconochie et al., 1997
), are
likely to be important in maintaining the neural crest prepattern.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Anderson, C. B. and Meier, S. (1981). The influence of the metameric pattern in the mesoderm on migration of cranial neural crest cells in the chick embryo. Dev. Biol. 85,385 -402.[Medline]
Barrow, J. R. and Capecchi, M. R. (1996).
Targeted disruption of the Hoxb-2 locus in mice interferes with expression of
Hoxb-1 and Hoxb-4. Development
122,3817
-3828.
Bergemann, A. D., Cheng, H. J., Brambilla, R., Klein, R. and Flanagan, J. G. (1995). ELF-2, a new member of the Eph ligand family, is segmentally expressed in mouse embryos in the region of the hindbrain and newly forming somites. Mol. Cell. Biol. 15,4921 -4929.[Abstract]
Birgbauer, E. and Fraser, S. E. (1994).
Violation of cell lineage restriction compartments in the chick hindbrain.
Development 120,1347
-1356.
Bondurand, N., Pingault, V., Goerich, D. E., Lemort, N., Sock,
E., Caignec, C. L., Wegner, M. and Goossens, M. (2000).
Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg
syndrome. Hum. Mol. Genet.
9,1907
-1917.
Bondurand, N., Girard, M., Pingault, V., Lemort, N., Dubourg, O.
and Goossens, M. (2001). Human Connexin 32, a gap junction
protein altered in the X-linked form of Charcot-Marie-Tooth disease, is
directly regulated by the transcription factor SOX10. Hum. Mol.
Genet. 10,2783
-2795.
Britsch, S., Goerich, D. E., 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.
Chavrier, P., Janssen-Timmen, U., Mattei, M. G., Zerial, M., Bravo, R. and Charnay, P. (1989). Structure, chromosome location, and expression of the mouse zinc finger gene Krox-20: multiple gene products and coregulation with the proto-oncogne c-fos. Mol. Cell. Biol. 9,787 -797.[Medline]
Couly, G., Creuzet, S., Bennaceur, S., Vincent, C. and le
Douarin, N. M. (2002). Interactions between Hox-negative
cephalic neural crest cells and the foregut endoderm in patterning the facial
skeleton in the vertebrate head. Development
129,1061
-1073.
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.
Fraser, S., Keynes, R. and Lumsden, A. (1990). Segmentation in the chick embryo hindbrain is defined by cell lineage restrictions. Nature 344,431 -435.[CrossRef][Medline]
Galas, D. J. and Schmitz, A. (1978). DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 5,3157 -3170.[Abstract]
Gale, N. W., Holland, S. J., Valenzuela, D. M., Flenniken, A., Pan, L., Ryan, T. E., Henkemeyer, M., Strebhardt, K., Hirai, H., Wilkinson, D. G. et al. (1996). Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis. Neuron 17, 9-19.[Medline]
Gavalas, A., Davenne, M., Lumsden, A., Chambon, P. and Rijli, F.
M. (1997). Role of Hoxa-2 in axon pathfinding and rostral
hindbrain patterning. Development
124,3693
-3702.
Gavalas, A., Trainor, P., Ariza-McNaughton, L. and Krumlauf,
R. (2001). Synergy between Hoxa1 and Hoxb1: the relationship
between arch patterning and the generation of cranial neural crest.
Development 128,3017
-3027.
Gendron-Maguire, M., Mallo, M., Zhang, M. and Gridley, T. (1993). Hoxa2 mutant mice exhibit homeotic transformation of skeletal elements derived from cranial neural crest. Cell 75,1317 -1331.[Medline]
Ghislain, J., Desmarquet-Trin-Dinh, C., Jaegle, M., Meijer, D.,
Charnay, P. and Frain, M. (2002). Characterisation of
cis-acting sequences reveals a biphasic, axon-dependent regulation of Krox20
during Schwann cell development. Development
129,155
-166.
Giudicelli, F., Taillebourg, E., Charnay, P. and
Gilardi-Hebenstreit, P. (2001). Krox-20 patterns the
hindbrain through both cell-autonomous and non cell-autonomous mechanisms.
Genes Dev. 15,567
-580.
Goddard, J. M., Rossel, M., Manley, N. R. and Capecchi, M.
R. (1996). Mice with targeted disruption of Hoxb-1 fail to
form the motor nucleus of the VIIth nerve. Development
122,3217
-3228.
Harley, V. R., Lovell-Badge, R. and Goodfellow, P. N. (1994). Definition of a consensus DNA binding site for SRY. Nucleic Acids Res. 22,1500 -1501.[Medline]
Herbarth, B., Pingault, V., Bondurand, N., Kuhlbrodt, K.,
Hermans-Borgmeyer, I., Puliti, A., Lemort, N., Goossens, M. and Wegner, M.
(1998). Mutation of the Sry-related Sox10 gene in Dominant
megacolon, a mouse model for human Hirschsprung disease. Proc.
Natl. Acad. Sci. USA 95,5161
-5165.
Holder, N. and Klein, R. (1999). Eph receptors
and ephrins: effectors of morphogenesis. Development
126,2033
-2044.
Hunt, P. and Krumlauf, R. (1991). Deciphering the Hox code: clues to patterning branchial regions of the head. Cell 66,1075 -1078.[Medline]
Hunt, P., Gulisano, M., Cook, M., Sham, M. H., Faiella, A., Wilkinson, D., Boncinelli, E. and Krumlauf, R. (1991a). A distinct Hox code for the branchial region of the vertebrate head. Nature 353,861 -864.[CrossRef][Medline]
Hunt, P., Wilkinson, D. and Krumlauf, R. (1991b). Patterning the vertebrate head: murine Hox 2 genes mark distinct subpopulations of premigratory and migrating cranial neural crest. Development 112,43 -50.[Abstract]
Hunt, P., Whiting, J., Muchamore, I., Marshall, H. and Krumlauf, R. (1991c). Homeobox genes and models for patterning the hindbrain and branchial arches. Development Suppl. 1, 187-196.
Itasaki, N., Bel-Vialar, S. and Krumlauf, R. (1999). `Shocking' developments in chick embryology: electroporation and in ovo gene expression. Nat. Cell Biol. 1,203 -207.
Kelsh, R. N. and Eisen, J. S. (2000). The
zebrafish colourless gene regulates development of non-ectomesenchymal neural
crest derivatives. Development
127,515
-525.
Köntges, G. and Lumsden, A. (1996).
Rhombencephalic neural crest segmentation is preserved throughout craniofacial
ontogeny. Development
122,3229
-3242.
Kuhlbrodt, K., Herbarth, B., Sock, E., Hermans-Borgmeyer, I. and
Wegner, M. (1998). Sox10, a novel transcriptional modulator
in glial cells. J. Neurosci.
18,237
-250.
Le Douarin, N. M. (1982). The Neural Crest, 2nd edn. Cambridge: Cambridge University Press.
Le Douarin, N. M. and Kalcheim, C. (1999). The Neural Crest: New York: Cambridge University Press.
Le Lièvre, C. S. and le Douarin, N. M. (1975). Mesenchymal derivatives of the neural crest: analysis of chimaeric quail and chick embryos. J. Embryol. Exp. Morphol. 34,125 -154.[Medline]
Liem, K. F., Jr, Tremml, G., Roelink, H., Jessell, T. M., Granato, M., van Eeden, F. J., Schach, U., Trowe, T., Brand, M., Furutani-Seiki, M. et al. (1995). Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell 82,969 -979.[Medline]
Lumsden, A. (1990). The cellular basis of segmentation in the developing hindbrain. Trends Neurosci. 13,329 -335.[CrossRef][Medline]
Lumsden, A. and Keynes, R. (1989). Segmental patterns of neuronal development in the chick hindbrain. Nature 337,424 -428.[CrossRef][Medline]
Lumsden, A. and Krumlauf, R. (1996). Patterning
the vertebrate neuraxis. Science
274,1109
-1115.
Lumsden, A., Sprawson, N. and Graham, A. (1991). Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo. Development 113,1281 -1291.[Abstract]
Maconochie, M. K., Nonchev, S., Studer, M., Chan, S. K., Popperl, H., Sham, M. H., Mann, R. S. and Krumlauf, R. (1997). Cross-regulation in the mouse HoxB complex: the expression of Hoxb2 in rhombomere 4 is regulated by Hoxb1. Genes Dev. 11,1885 -1895.[Abstract]
Maconochie, M., Krishnamurthy, R., Nonchev, S., Meier, P.,
Manzanares, M., Mitchell, P. J. and Krumlauf, R. (1999).
Regulation of Hoxa2 in cranial neural crest cells involves members of the AP-2
family. Development 126,1483
-1494.
Manley, N. R. and Capecchi, M. R. (1997). Hox group 3 paralogous genes act synergistically in the formation of somitic and neural crest-derived structures. Dev. Biol. 192,274 -288.[CrossRef][Medline]
Manzanares, M., Nardelli, J., Gilardi-Hebenstreit, P., Marshall,
H., Giudicelli, F., Martinez-Pastor, M. T., Krumlauf, R. and Charnay, P.
(2002). Krox20 and kreisler co-operate in the transcriptional
control of segmental expression of Hoxb3 in the developing hindbrain.
EMBO J. 21,365
-376.
Morgenstern, B., Dress, A. and Werner, T.
(1996). Multiple DNA and protein sequence alignment based on
segment-to-segment comparison. Proc. Natl. Acad. Sci.
USA 93,12098
-12103.
Nardelli, J., Gibson, T. and Charnay, P. (1992). Zinc finger-DNA recognition: analysis of base specificity by site-directed mutagenesis. Nucleic Acids Res. 20,4137 -4144.[Abstract]
Ng, L. J., Wheatley, S., Muscat, G. E., Conway-Campbell, J., Bowles, J., Wright, E., Bell, D. M., Tam, P. P., Cheah, K. S. and Koopman, P. (1997). SOX9 binds DNA, activates transcription, and coexpresses with type II collagen during chondrogenesis in the mouse. Dev. Biol. 183,108 -121.[CrossRef][Medline]
Nieto, M. A., Sechrist, J., Wilkinson, D. G. and Bronner-Fraser, M. (1995). Relationship between spatially restricted Krox-20 gene expression in branchial neural crest and segmentation in the chick embryo hindbrain. EMBO J. 14,1697 -1710.[Abstract]
Noden, D. M. (1983). The role of the neural crest in patterning of avian cranial skeletal, connective, and muscle tissues. Dev. Biol. 96,144 -165.[Medline]
Noden, D. M., le Lievre, C. S. and le Douarin, N. M. (1988). Interactions and fates of avian craniofacial mesenchyme. Development 103,121 -140.[Medline]
Nonchev, S., Vesque, C., Maconochie, M., Seitanidou, T.,
Ariza-McNaughton, L., Frain, M., Marshall, H., Sham, M. H., Krumlauf, R. and
Charnay, P. (1996). Segmental expression of Hoxa-2 in the
hindbrain is directly regulated by Krox-20.
Development 122,543
-554.
Pöpperl, H., Bienz, M., Studer, M., Chan, S. K., Aparicio, S., Brenner, S., Mann, R. S. and Krumlauf, R. (1995). Segmental expression of Hoxb1 is controlled by a highly conserved autoregulatory loop dependent upon exd/pbx. Cell 81,1031 -1042.[Medline]
Prince, V. and Lumsden, A. (1994). Hoxa-2
expression in normal and transposed rhombomeres: independent regulation in the
neural tube and neural crest. Development
120,911
-923.
Rijli, F. M., Mark, M., Lakkaraju, S., Dierich, A., Dolle, P. and Chambon, P. (1993). A homeotic transformation is generated in the rostral branchial region of the head by disruption of Hoxa-2, which acts as a selector gene. Cell 75,1333 -1349.[Medline]
Schilling, T. F., Prince, V. and Ingham, P. W. (2001). Plasticity in zebrafish hox expression in the hindbrain and cranial neural crest. Dev. Biol. 231,201 -216.[CrossRef][Medline]
Schneider-Maunoury, S., Topilko, P., Seitandou, T., Levi, G., Cohen-Tannoudji, M., Pournin, S., Babinet, C. and Charnay, P. (1993). Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain. Cell 75,1199 -1214.[Medline]
Schneider-Maunoury, S., Seitanidou, T., Charnay, P. and Lumsden,
A. (1997). Segmental and neuronal architecture of the
hindbrain of Krox-20 mouse mutants. Development
124,1215
-1226.
Sechrist, J., Serbedzija, G. N., Scherson, T., Fraser, S. E. and
Bronner-Fraser, M. (1993). Segmental migration of the
hindbrain neural crest does not arise from its segmental generation.
Development 118,691
-703.
Seitanidou, T., Schneider-Maunoury, S., Desmarquet, C., Wilkinson, D. G. and Charnay, P. (1997). Krox20 is a key regulator of rhombomere-specific gene expression in the developing hindbrain. Mech. Dev. 65, 31-42.[CrossRef][Medline]
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.
Serbedzija, G. N., Bronner-Fraser, M. and Fraser, S. E.
(1992). Vital dye analysis of cranial neural crest cell migration
in the mouse embryo. Development
116,297
-307.
Sham, M. H., Vesque, C., Nonchev, S., Marshall, H., Frain, M., Gupta, R. D., Whiting, J., Wilkinson, D., Charnay, P. and Krumlauf, R. (1993). The zinc finger gene Krox20 regulates HoxB2 (Hox2.8) during hindbrain segmentation. Cell 72,183 -196.[Medline]
Smith, A., Robinson, V., Patel, K. and Wilkinson, D. G. (1997). The EphA4 and EphB1 receptor tyrosine kinases and ephrin-B2 ligand regulate targeted migration of branchial neural crest cells. Curr. Biol. 7,561 -570.[Medline]
Southard-Smith, E. M., Kos, L. and Pavan, W. J. (1998). Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model. Nat. Genet. 18, 60-64.[Medline]
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 in Xenopus.
Development 129,421
-432.
Studer, M., Lumsden, A., Ariza-McNaughton, L., Bradley, A. and Krumlauf, R. (1996). Altered segmental identity and abnormal migration of motor neurons in mice lacking Hoxb-1. Nature 384,630 -634.[CrossRef][Medline]
Swirnoff, A. H. and Milbrandt, J. (1995). DNA-binding specificity of NGFI-A and related zinc finger transcription factors. Mol. Cell. Biol. 15,2275 -2287.[Abstract]
Theil, T., Frain, M., Gilardi-Hebenstreit, P., Flenniken, A.,
Charnay, P. and Wilkinson, D. G. (1998). Segmental expression
of the EphA4 (Sek-1) receptor tyrosine kinase in the hindbrain is under direct
transcriptional control of Krox-20. Development
125,443
-452.
Trainor, P. and Krumlauf, R. (2000). Plasticity in mouse neural crest cells reveals a new patterning role for cranial mesoderm. Nat. Cell Biol. 2, 96-102.[CrossRef][Medline]
Trainor, P. A. and Krumlauf, R. (2001). Hox genes, neural crest cells and branchial arch patterning. Curr. Opin. Cell Biol. 13,698 -705.[CrossRef][Medline]
Trainor, P. A., Sobieszczuk, D., Wilkinson, D. and Krumlauf,
R. (2002a). Signalling between the hindbrain and paraxial
tissues dictates neural crest migration pathways.
Development 129,433
-442.
Trainor, P. A., Ariza-McNaughton, L. and Krumlauf, R.
(2002b). Role of the isthmus and FGFs in resolving the paradox of
neural crest plasticity and prepatterning. Science
295,1288
-1291.
Travis, A., Amsterdam, A., Belanger, C. and Grosschedl, R. (1991). LEF-1, a gene encoding a lymphoid-specific protein with an HMG domain, regulates T-cell receptor alpha enhancer function. Genes Dev. 5,880 -894.[Abstract]
van de Wetering, M., Oosterwegel, M., Dooijes, D. and Clevers, H. (1991). Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box. EMBO J. 10,123 -132.[Abstract]
Veitch, E., Begbie, J., Schilling, T. F., Smith, M. M. and Graham, A. (1999). Pharyngeal arch patterning in the absence of neural crest. Curr. Biol. 9,1481 -1484.[CrossRef][Medline]
Vesque, C., Topilko, P., Becker, N. and Charnay, P. (1993). Molecular analysis of the development of the rhombencephalon. C. R. Seances Soc. Biol. Fil. 187,364 -367.[Medline]
Voiculescu, O., Charnay, P. and Schneider-Maunoury, S. (2000). Expression pattern of a Krox-20/Cre knock-in allele in the developing hindbrain, bones, and peripheral nervous system. Genesis 26,123 -126.[CrossRef][Medline]
Voiculescu, O., Taillebourg, E., Pujades, C., Kress, C., Buart,
S., Charnay, P. and Schneider-Maunoury, S. (2001). Hindbrain
patterning: Krox20 couples segmentation and specification of regional
identity. Development
128,4967
-4978.
Wegner, M. (1999). From head to toes: the
multiple facets of Sox proteins. Nucleic Acids Res.
27,1409
-1420.
Wilkinson, D. G., Bhatt, S., Chavrier, P., Bravo, R. and Charnay, P. (1989). Segment-specific expression of a zinc-finger gene in the developing nervous system of the mouse. Nature 337,461 -464.[CrossRef][Medline]
Wilkinson, D. G. (1992). Whole-mount in situ hybridisation of vertebrate embryos. In In Situ Hybridisation: A Practical Approach (ed. D.G. Wilkinson), pp.75 -83. Oxford: IRL Press.
Wingender, E., Chen, X., Hehl, R., Karas, H., Liebich, I.,
Matys, V., Meinhardt, T., Pruss, M., Reuter, I. and Schacherer, F.
(2000). TRANSFAC: an integrated system for gene expression
regulation. Nucleic Acids Res.
28,316
-319.
Yee, S. P. and Rigby, P. W. (1993). The regulation of myogenin gene expression during the embryonic development of the mouse. Genes Dev. 7,1277 -1289.[Abstract]
Zhang, M., Kim, H. J., Marshall, H., Gendron-Maguire, M., Lucas,
D. A., Baron, A., Gudas, L. J., Gridley, T., Krumlauf, R. and Grippo, J.
F. (1994). Ectopic Hoxa-1 induces rhombomere transformation
in mouse hindbrain. Development
120,2431
-2442.
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]