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First published online 22 February 2006
doi: 10.1242/dev.02289
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1 INSERM, U784, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex
05, France.
2 UMR CNRS 7622, Université Pierre et Marie Curie, 9 quai Saint-Bernard,
75252 Paris Cedex 05, France.
* Author for correspondence (e-mail: charnay{at}biologie.ens.fr)
Accepted 18 January 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Hindbrain segmentation, Pattern formation, Transcriptional enhancers, vHnf1, Mouse, Chick
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)
(reviewed by Lumsden and Keynes,
1989; Lumsden and Krumlauf,
1996; Wingate and Lumsden,
1996). The rhombomeres behave as compartments, constituting units
of cell lineage restriction (Fraser et
al., 1990; Birgbauer and
Fraser, 1994) and domains of specific gene expression (reviewed by
Lumsden and Krumlauf, 1996;
Rijli et al., 1998). This
subdivision presages the metameric pattern of neuronal specification in the
hindbrain (Lumsden and Keynes,
1989; Clarke et al.,
1998), underlies the pathways of neural crest cell migration into
the branchial arches and participates in their patterning
(Lumsden et al., 1991;
Serbedzija et al., 1992;
Birgbauer et al., 1995;
Kulesa and Fraser, 2000;
Trainor and Krumlauf, 2000;
Trainer et al., 2002; Ghislain et al.,
2003), thus playing an essential role in craniofacial
morphogenesis.
Numerous genes have been implicated at different levels of the segmentation
process, including the initial formation of segmental territories
(Lufkin et al., 1991;
Chisaka et al., 1992;
Schneider-Maunoury et al.,
1993; Frohman et al.,
1993; McKay et al.,
1994; Barrow et al.,
2000; Waskiewicz et al.,
2001; Deflorian et al.,
2004; Choe and Sagerstrom,
2004; McNulty et al.,
2005), the specification of their AP identities
(Rijli et al., 1993;
Studer et al., 1996;
Seitanidou et al., 1997;
Rossel and Capecchi, 1999;
Bell et al., 1999) and their
stabilisation by restriction of cell intermingling between adjacent
rhombomeres (reviewed by Pasini and
Wilkinson, 2002), and development of specific cell populations at
boundaries (Cheng et al.,
2004; Amoyel et al.,
2005). Segment formation and specification are highly intricate
processes in the hindbrain, with several genes participating in both aspects
(Gavalas et al., 1998;
Rossel and Capecchi, 1999;
Voiculescu et al., 2001).
Krox20, which encodes a zinc-finger transcription factor
(Chavrier et al., 1988), plays
a key role in coupling segmentation and specification of rhombomere identity
(Voiculescu et al., 2001).
Krox20 is one of the earliest genes expressed in a segmental pattern
in the developing hindbrain, specifically in odd-numbered rhombomeres r3 and
r5 (Wilkinson et al., 1989;
Schneider-Maunoury et al.,
1993). Krox20 mutation leads to loss of r3 and r5
(Schneider-Maunoury et al.,
1993; Swiatek et al.,
1993), owing to mis-specification of these territories, which
acquire r2 and r4, and r6 identities, respectively
(Voiculescu et al., 2001).
Krox20 has been shown to exert its function by up- and downregulating the
expression of numerous genes, including Hox genes of the paralogous groups 1
to 3, such as Hoxb1, Hoxa2, Hoxb2 and Hoxb3, which are also
involved in the specification of segmental AP identity
(Sham et al., 1993;
Nonchev et al., 1996a;
Nonchev et al., 1996b;
Vesque et al., 1996;
Seitanidou et al., 1997;
Giudicelli et al., 2001;
Manzanares et al., 2002) (M.
Garcia-Dominguez, P. Gilardi and P.C., unpublished), and the EphA4 receptor
gene, which is involved in the segregation of cells between odd- and
even-numbered rhombomeres (Theil et al.,
1998). Krox20 has also been shown to positively regulate its own
expression, both cell-autonomously and non cell-autonomously, and these
processes are thought to play an essential role in the extension and
stabilisation of r3 and r5 territories
(Schneider-Maunoury et al.,
1993; Giudicelli et al.,
2001).
Given the central role of Krox20 in hindbrain development, the
efforts invested to elucidate the molecular mechanisms controlling this
process have led to the identification of putative upstream Krox20 regulators.
Like the other segmentation genes in the hindbrain, Krox20 appears to
be under the control of several signalling pathways involved in long-range
patterning, including the Wnt (Nordstrom
et al., 2002), retinoic acid (RA) (reviewed by
Gavalas and Krumlauf, 2000;
Dupe and Lumsden, 2001;
Niederreither et al., 2003)
and FGF (in r5) (Marin and Charnay,
2000; Maves et al.,
2002; Walshe et al.,
2002) cascades. It is likely that a large part of the effects of
these signalling pathways are indirect and relayed by a series of
transcription factors involved in hindbrain segmentation, including paralogous
group 1 Hox gene products and their associated factors
(Chisaka and Capecchi, 1991;
Lufkin et al., 1991;
Carpenter et al., 1993;
Dolle et al., 1993;
Mark et al., 1993;
Helmbacher et al., 1998;
Rossel et al., 1999; Barrow et al.,
2000; Choe et al.,
2002; Waskiewicz et al.,
2001; Waskiewicz et al.,
2002; McNulty et al.,
2005), vHNF1 (Sun and Hopkins,
2001; Wiellette and Sive,
2003; Choe and Sagerstrom,
2004; Hernandez et al.,
2004) and MafB (Frohman et
al., 1993; Cordes and Barsh,
1994; McKay et al.,
1994; Moens et al.,
1996; Manzanares et al.,
1999).
Despite the knowledge of these various genetic connections, no clear
picture of Krox20 regulation has emerged. This is probably due, on
the one hand, to the complexity of the network, which involves multiple
feedback loops as well as regulators playing variable functions at different
times and places; and, on the other hand, to our ignorance of the direct
interactions existing between network members. In order to understand the
molecular mechanisms controlling Krox20 expression and to identify
its direct regulators, we have initiated an analysis of its cis-acting
sequences. This strategy has previously allowed us to unravel the molecular
basis of Krox20 regulation in the neural crest, developing Schwann
cells and bone-forming cells (Ghislain et
al., 2002; Ghislain et al.,
2003; Ghislain and Charnay,
2006) (M.F. and P.C., unpublished). In the present paper, we have
screened over 200 kb surrounding the chicken Krox20 locus and have
identified the essential regulatory information controlling Krox20
expression in the hindbrain. Three cis-acting elements were characterized,
designated A, B and C, which are conserved between birds and mammals. These
elements, located far upstream of the gene play different, although
overlapping roles: B and C are involved in the initiation of Krox20
expression in r5 and r3/r5, respectively, whereas element A is involved in the
maintenance of Krox20 expression in these rhombomeres. We demonstrate
that vHNF1 binds to element B and constitutes a direct transcriptional
activator of Krox20 in r5 and that the maintenance function is
achieved by direct autoregulation, with Krox20 binding to multiple sites
within element A. Considering the central position occupied by Krox20
in the complex gene network governing hindbrain segmentation, the present work
constitutes an important step towards its elucidation.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) were
used as a source of chicken Krox20 extragenic sequences. BAC121 was
digested with SalI to obtain sub-fragments of 42 kb (3) and 65 kb (4)
(Fig. 1A). Fragment 3 was
inserted in cosmid pTCF using the Gigapack III packaging extract (Stratagene)
and used to obtain SalI-XbaI fragment 6,
NotI-BamHI fragment 7, BamHI-BamHI
fragment 8, BamHI-SalI fragment 9
(Fig. 2A). The 30 kb fragment 5
was cloned by PCR using primers indicated in Table S1 (see supplementary
material) and digested with BamHI to obtain fragments 10 and 11.
Fragment 10 was digested with AflII to obtain fragments 12 and 13.
Fragment 12 was digested with NcoI to obtain fragments 14 and 15
(Fig. 2A). Chicken elements A
(XcmI-HindIII), 12.1 (StuI-ScaI), B
(EcoRI-StyI), 12.3 (BstYI-BstYI) and C
(BglII-ClaI) were derived from fragments 7, 12 and 14,
respectively. Mouse elements A, B and C were cloned by PCR using primers
indicated in Table S1 in the supplementary material. Fragments 6-9, 11, 12, 14
and 15, and chicken and mouse conserved elements were cloned into pBGZ40
(Yee and Rigby, 1993) upstream
of the minimal ß-globin promoter/lacZ reporter.
Mutagenesis of the Krox20-binding sites in element cA was performed using the
Transformer Site-Directed Mutagenesis Kit (Clontech) and mutagenesis of the
vHnf1-binding site in element cB was performed using the ExSite PCR-Based
Site-Directed Mutagenesis Kit (Stratagene).
Mouse lines, generation of transgenic mice, genotyping and in ovo electroporation
The Krox20cre allele
(Voiculescu et al., 2000) and
the transgenic lines cA-lacZ, cB-lacZ and cC-lacZ generated
in this work were maintained in a mixed C57B16/DBA2 background. Fragment
purification and transgenesis were performed as described previously
(Sham et al., 1993;
Ghislain et al., 2002). BAC
constructs 1 and 2 were injected as supercoiled plasmids and transgenic
embryos were identified by PCR with BAC vector specific primers (see Table S1
in the supplementary material). Transgenic embryos for fragment 4 were
identified by PCR using primers specific for the chick sequence (see Table
S1). Transgenic embryos for fragments #3, #5, #10 and #13 were obtained by
co-injection of equimolar amounts of the respective fragment and of the
Krox20/lacZ reporter construct
(Ghislain et al., 2002). PCR
using primers specific for the chick sequence (see Table S1) and
Krox20/lacZ allele
(Schneider-Maunoury et al.,
1993) was performed to identify the fragments and reporter,
respectively, in transgenic embryos. Transgenic embryos for chick elements A,
B and C constructs were identified using the primers indicated in Table S1.
Transgenic embryos for the other constructs were identified with primers
specific for lacZ (Ghislain et
al., 2002). In ovo electroporation in the chick neural tube was
performed as previously described
(Giudicelli et al., 2001) at
stages HH8-HH10. Each construct was tested in at least two independent
experiments, each involving eight or more embryos. Co-electroporation
experiments with pAdRSVKrox20 were carried out as described
(Giudicelli et al., 2001).
In situ hybridization and X-gal staining
Whole-mount in situ hybridization was performed as previously described
(Giudicelli et al., 2001;
Wilkinson et al., 2002), using
a chicken Krox20 probe
(Giudicelli et al., 2001), or
a mouse Hoxb1 probe (Wilkinson et
al., 1989). Alkaline phosphatase activity was revealed using the
NBT/BCIP substrate (Roche). Simple lacZ labelling and double
labelling for ß-galactosidase activity and Hoxb1 mRNA of mouse
and chick embryos were performed as described previously
(Ghislain et al., 2003).
Protein extracts 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). The human HNF1ß/vHNF1, isoform A protein was
prepared from human embryonic kidney HEK 293 cells as described
(Cereghini et al., 1992;
Barbacci et al., 2004). To
prepare the probes, clones of element A in pBS carrying wild-type or mutant
Krox20-binding sites were digested with HindIII and XhoI,
and clones of element B in pGEM5 carrying the wild-type or mutant
vHnf1-binding sites were digested with SphI and SpeI. All
fragments were dephosphorylated and labelled using T4 polynucleotide kinase
and [
-32P]-ATP. Labelled fragments were purified using
Microspin S-200 HR Columns (Amersham) and used in band shift experiments as
previously described (Nardelli et al.,
1992; Cereghini et al.,
1992).
Nucleotide sequence analyses
Identification of mouse sequences homologous to chicken fragments #7, 8, 12
and 14 and analysis of Krox20 locus in the chick, mouse and other
species were performed using the Sanger Institute (ENSEMBL Project) website.
Sequence alignments were performed using the mVista software
(Frazer et al., 2004) and
identification of putative Krox20-binding sites using the rVista software
(Loots et al., 2002).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Ghislain et al.,
2003). Although these studies identified several cis-acting
elements involved in the control of various aspects of the Krox20
expression pattern, including its regulation in the r5-derived neural crest
(Ghislain et al., 2003), they
did not reveal transcriptional enhancers responsible for Krox20
expression in r3 and r5, suggesting that such elements were located outside of
the tested region. This led us to modify our approach to be able to reach
regulatory elements potentially located much farther from the gene: we
searched for such regulatory elements within the chicken genome, which is
about threefold more compact than the mouse one, and used BAC clones to scan
larger regions. This strategy was based on the hypothesis that Krox20
cis-acting regulatory sequences would be conserved between the two
species, which was supported by the conservation of the expression patterns
(Irving et al., 1996;
Giudicelli et al., 2001). We
isolated two chicken BAC clones (BAC 121 and 27), which together cover the
region between –100 kb and +107 kb of the transcription start site
(Fig. 1A)
(Giudicelli et al., 2001).
These clones were introduced by transgenesis into the mouse genome and the
expression pattern of the chick Krox20 gene was analysed by in situ
hybridization with a chick specific probe in F0 embryos at embryonic day 8.5
(E8.5). About half of the transgenic embryos obtained with BAC 121
(n=9), covering region –100 kb to +53 kb
(Fig. 1A, construct #1),
strongly expressed chick Krox20 in the r3-r5 region, with a lower
level extending into groups of cells in posterior r2 and anterior r6
(Fig. 1B). By contrast, no
transgenic embryo injected with BAC 27 (n=5), covering region
–28 kb to +107 kb (Fig.
1A, construct #2), showed any expression of the gene in the
rhombomeres. This suggests that important element(s) for Krox20
expression in the hindbrain are located between positions –100 kb and
–28 kb.
This possibility was investigated by testing three sub-fragments of BAC 121
(Fig. 1A). Fragment 3 contains
the most upstream sequence and is 42 kb long
(Fig. 1A). To evaluate its
cis-acting activity, it was co-injected with a Krox20/lacZ reporter
construct consisting of a 11.5 kb mouse genomic fragment containing
Krox20 with an in-frame insertion of lacZ in exon 2
(Ghislain et al., 2002). This
latter construct is not active in the hindbrain, but responds to
transcriptional enhancers, and it leads to synthesis of a chimeric protein
with ß-galactosidase activity
(Ghislain et al., 2002;
Ghislain et al., 2003).
Transgenic embryos co-injected with fragment #3 and the Krox20/lacZ
reporter expressed ß-galactosidase specifically in r3 and r5
(Fig. 1A,C). Fragment 4 covers
the following 65 kb of chick sequence, including the Krox20 gene
(Fig. 1A). The analysis of its
cis-acting activity in transgenic embryos was therefore performed by in situ
hybridization with the chick probe and revealed a pattern of expression
identical to that obtained with the entire BAC 121 in all transgenic embryos
(Fig. 1A,D). Fragment 5
contains a 30 kb fragment covering the region of fragment 4 that is not
present in BAC 27 (Fig. 1A). In
the transgenic embryos co-injected with fragment #5 and the
Krox20/lacZ reporter, strong ß-galactosidase activity was
detected in r3 and r5, and lower levels in r4 and in a few cells of the caudal
part of r2 and the rostral part of r6 (Fig.
1A,E; data not shown). These data indicate that the original
activity observed with BAC 121 is likely to correspond to multiple cis-acting
elements present in fragments 3 and 5.
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) and transgenesis in mouse embryos. Two fragments, 7 and
8, were found to drive specific reporter expression in r3 and r5 by in ovo
electroporation (Fig. 2A-C),
whereas the others were negative (Fig.
2A and data not shown). When the different constructs were tested
by mouse transgenesis, construct 7 was active in r3 and r5, consistent with
the electroporation data (Fig.
1A,F), whereas construct 8 did not lead to any reporter expression
in the hindbrain (Fig. 2A,G),
in contrast to the results of electroporation. Fragment 10, because of its
large size (20 kb), could be analysed only by transgenesis after co-injection
with the Krox20/lacZ reporter construct. It led to strong expression
in the r3-r5 region in transgenic embryos
(Fig. 2A and data not shown).
Construct 11, which presented no activity when tested by electroporation, was
active in r3 and r5 in only one transgenic embryo (n=10,
Fig. 2A). As it was an isolated
case, the analysis of this fragment was not pursued. Fragment 10 was then divided in two pieces (constructs 12 and 13) and subsequently fragment 13 also in two pieces (fragments 14 and 15), which were tested in both systems with the exception of fragment 13, which was tested only by transgenesis because of its large size (15 kb). In transgenic embryos, fragments 13 and 14 presented an activity similar to that of fragment 10 (Fig. 2A,I; data not shown). By electroporation, fragment 14 led to strong reporter expression in r3 and r5, with lower levels in r4 (Fig. 2A,E). Fragment 12 was found to drive specific reporter expression in r5 in both chick and mouse systems (Fig. 2A,D,H). Fragment 15 was not active in transgenic embryos nor in the electroporation assay.
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Functional conservation of three regulatory elements between birds and mammals
The nucleotide sequences of the three chicken fragments active in
transgenic experiments (7, 12, 14) were established and compared with the
mouse genome, to identify sequences conserved during evolution that might
correspond to functional cis-acting elements. The divergence of about 300
million years between the two species suggests that conserved non-coding
sequences are likely to have a functional role
(Duret and Bucher, 1997).
Sequences with significant homology to mouse genomic sequences were identified
within each fragment (Fig. 3A).
Fragment 7 contained one conserved block of sequence of 410 bp, designated
element A (see Fig. S1 in the supplementary material). Fragment 12 contained
three conserved blocks: 12.1 (550 bp), element B (480 bp) and 12.3 (200 pb)
(see Fig. S1; data not shown). Finally, fragment 14 contained one
well-conserved block of 220 bp and a proximal weakly conserved block of 100 bp
that, together, correspond to element C (see Fig. S1).
The five chicken conserved elements (cA, c12.1, cB, c12.3 and cC) were then inserted upstream of the ß-globin promoter-lacZ reporter construct to determine whether they carry the cis-regulatory activities observed with the entire fragments. The constructs were tested both by in ovo electroporation and mouse transgenesis. Elements A and C recapitulated the patterns obtained with fragments 7 and 14, respectively (Fig. 3A-C,E; data not shown). Among the three conserved sequences present in fragment 12, only element B led to specific expression in r5 like the entire fragment (Fig. 3A,B,D and data not shown). The two other elements were negative in the hindbrain (Fig. 3A,B; data not shown). Finally, to establish whether sequence homology was indeed reflecting functional conservation, we cloned the mouse sequences homologous to elements A, B and C (mA, mB and mC) upstream of the ß-globin promoter-lacZ reporter and tested them by in ovo electroporation. These elements led to ß-galactosidase expression patterns very similar to their chick counterparts (Fig. 3F-H and data not shown; compare with Fig. 2B,D,E). In conclusion, using a phylogenetic footprinting approach, we have precisely identified three Krox20 cis-regulatory elements that show overlapping hindbrain activities and are functionally conserved between birds and mammals.
Elements B and C are involved in initiation of Krox20 expression and element A in autoregulation
As the different Krox20 cis-acting elements lead to overlapping
patterns of expression, we wondered whether they might have redundant
functions. To address this issue, we performed a time-course analysis of their
activities and investigated whether they require the Krox20 protein for their
function. Indeed it is known that Krox20 can activate its own expression
(Giudicelli et al., 2001) and
the analysis of mutants homozygous for a null Krox20 allele
(Krox20/lacZ) shows a loss of lacZ expression, rapid in r3
and more gradual in r5 (Schneider-Maunoury
et al., 1993). To perform these studies, we generated mouse
transgenic lines with the chick A, B and C elements driving the ß-globin
promoter-lacZ reporter. To assess the role of Krox20 on these
elements, the transgenes were transferred into a Krox20-null
background (Krox20Cre/Cre)
(Voiculescu et al., 2000).
Element A was shown to be active from at least the six-somite stage (ss) in r3
and r5 in wild-type embryos, and the expression was specifically maintained in
these rhombomeres until at least E9.5 (Fig.
4B-D). In Krox20-null embryos at 6 ss, a stage when
endogenous Krox20 gene expression can still be detected in
mis-specified r3 (r3*) and r5 (r5*) territories
(Voiculescu et al., 2001),
element A showed no activity (Fig.
4A). This establishes that element A requires the Krox20 protein
to drive reporter expression and therefore that it constitutes an
autoregulatory element. This conclusion is fully consistent with the pattern
generated by element A in wild-type hindbrain, which faithfully reflects the
presence of the Krox20 protein (E. Taillebourg, unpublished).
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Element C was activated around the 2 ss in r3 and the 5-6 ss in r5 in
wild-type embryos (Fig. 4J,K),
reflecting the normal pattern of Krox20 hindbrain expression
(Schneider-Maunoury et al.,
1993; Wilkinson et al.,
1989). This early activation also occurred in Krox20-null
embryos (Fig. 4I),
demonstrating that the activity of element C is independent of the Krox20
protein at these stages. Around the 8 ss, element C led to strong expression
in r3 and r5 and in r5-derived neural crest, with an additional patchy
expression in r4, where it overlapped with the Hoxb1-positive domain
(Fig. 4L,P). At the 13 ss, the
expression in r4 became homogenous and by E9.5 was throughout the r3 to r5
region (Fig. 4N,O).
ß-Galactosidase activity persisted in r5 until at least E11.5 (data not
shown). In Krox20-null embryos at the 13 ss, expression was also
observed in r4 and r5* (Fig.
4M; note that at this stage endogenous Krox20 expression
in r3* is lost)
(Schneider-Maunoury et al.,
1993). Altogether, these data indicate that element C is able to
drive reporter expression in r3 and r5 with a time-course very similar to the
endogenous Krox20 gene, both in wild type and Krox20 null
backgrounds. This latter point indicates that this element does not require
the Krox20 protein for its activity and therefore can function as an initiator
element for Krox20 expression. However, in contrast to the endogenous
gene, it appears to be increasingly active in r4 from the 8 ss.
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vHNF1 is a direct transcriptional activator of Krox20 in r5
The knowledge of Krox20 cis-regulatory sequence opens the way to
the search for its direct transcriptional regulators. As a first step, we
investigated the possibility that vHNF1, which has been implicated in
Krox20 regulation on the basis of loss- and gain-of-function
experiments, may constitute such a factor
(Sun and Hopkins, 2001;
Wiellette and Sive, 2003;
Choe and Sagerstrom, 2004;
Hernandez et al., 2004). We
searched for sequences close to the vHNF1 consensus binding site within
elements A, B and C (Tronche et al.,
1997). Only one putative site was identified, within element B, in
a region completely conserved between mouse and chick
(Fig. 5A). In band shifts
experiments using element B as a probe, cellular extracts containing vHNF1
produced a slow migrating complex that was absent with control extracts
(Fig. 5B). Furthermore the
retarded complex could be supershifted with an antibody directed against
vHNF1, establishing the presence of vHNF1. Finally, a mutation of a
palindromic sequence, which is known to abolish vHNF1 binding, was introduced
into the putative vHNF1 binding site of element B
(Fig. 5A)
(Cereghini et al., 1988). This
prevented the formation of a complex with vHNF1. Altogether, these experiments
establish that element B contains a unique bona fide vHNF1-binding site.
To determine whether the vHNF1-binding site was playing a role in the enhancer activity of element B, we compared wild-type and mutated versions driving the lacZ reporter in the chick electroporation system. Whereas the wild-type enhancer led to specific lacZ expression in r5 (Fig. 5C, see also Fig. 3D), the mutated enhancer was completely inactive (Fig. 5D). These data strongly suggest that the initiator activity of element B in r5 requires vHNF1 binding and therefore that Krox20 constitutes a direct transcriptional target of vHNF1.
Direct autoregulation driven by element A
As we have shown that element A requires the Krox20 protein for its
activity, we wondered whether this element might be activated by ectopic
Krox20 and whether Krox20 was acting on this element by direct binding. To
address these issues, we first co-electroporated the element A-reporter
plasmid with a Krox20 expression construct in the chick neural tube.
Whereas in the absence of ectopic Krox20 the reporter expression is limited to
r3 and r5 (Fig. 6C), its
presence leads to reporter expression along the entire electroporated area,
including the whole hindbrain (Fig.
6D). This indicates that element A is able to respond to exogenous
Krox20.
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;
Wingender et al., 2000), seven
sites were identified (Fig.
6A). To investigate the function of these sites, each was mutated
by the introduction of a single substitution at the central position of the
site, a mutation that has previously been shown to eliminate Krox20-binding
activity both in vitro and in vivo
(Nardelli et al., 1992;
Sham et al., 1993). As
expected, bandshift experiments confirmed that the mutated element A had
totally lost Krox20 binding (Fig.
6E). Furthermore, in in ovo electroporation experiments, the
reporter construct driven by the mutated element A was completely inactive in
r3 and r5 (Fig. 6F) and
unresponsive to ectopic Krox20 introduced by co-electroporation
(Fig. 6G). These data
demonstrate that element A constitutes a direct transcriptional target of
Krox20 and is therefore part of an autoregulatory loop presumably involved in
the maintenance of Krox20 expression.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Oxtoby and Jowett, 1993). In
the mouse they are located on chromosome 10, in a gene desert of 290 kb
between Krox20 and Nrbf2, the first gene identified upstream
of Krox20. Their distance from the promoter (in the order of 200 kb
in mouse and man) is unusual among mammalian enhancer elements. Each hindbrain Krox20 cis-acting element presents specific properties. Element A is active in r3 and r5. It cannot function in the absence of Krox20 and responds to ectopic Krox20 protein. It contains Krox20-binding sites that are absolutely required for its activity. Therefore this element appears as the cis-acting component for a direct, positive autoregulatory loop (see below). By contrast, elements B and C can work independently of Krox20 and its downstream genes and do not respond to Krox20 ectopic expression (data not shown). The activity of element B is restricted to r5, whereas that of element C, initially restricted to r3 and r5, extends to r4 as well at later stages of development. These properties suggest that these two elements may be responsible for the initiation of Krox20 expression in the hindbrain (see below).
In addition to elements A, B and C, our analyses revealed another
cis-acting sequence, located on an 8 kb DNA fragment (construct 8), which was
active in r3 and r5 in chick electroporation experiments but not in transgenic
mouse embryos (Fig. 2A,C,G). We
have compared the sequence of this fragment to the mouse genome and found a
200 bp island conserved in sequence and location (data not shown). The 200 bp
sequence carries r3/r5 enhancer activity in the chick electroporation assay
and this enhancer is strictly dependent on Krox20 binding (data not shown).
Our interpretation of these data is that construct 8 carries another direct
autoregulatory element, which normally does not function in the hindbrain
during segmentation stages, but at another stage and probably in another
tissue. Upon electroporation, the constrains normally exerted on this element
might be relaxed, so that Krox20, which is necessary for its activity, might
become sufficient. We have previously observed that another Krox20
autoregulatory element, the activity of which is normally restricted to
r5-derived neural crest, behaves in a very similar manner upon electroporation
(Ghislain et al., 2003). It
will therefore be interesting to determine which aspect of Krox20
expression is normally governed by the novel autoregulatory element present in
fragment 8.
Cell type-specific autoregulatory elements may be required for stringent regulation
Strikingly, our analysis of the Krox20 cis-regulatory landscape
revealed the existence of multiple direct autoregulatory elements with cell
type-specific activities. Besides element A and the sequence present in
fragment 8, we have so far identified two other such enhancers: the NCE that
is required for the maintenance of Krox20 expression in r5-derived
neural crest cells (Fig. 7)
(Ghislain et al., 2003) and a
bone forming cell-specific element that is responsible for the persistence of
Krox20 expression in this latter cell type (M.F. and P.C.,
unpublished). This raises the question of the existence of multiple cell
type-specific autoregulatory elements versus a unique, global one, as is the
case during Drosophila embryo segmentation for driving the stripe
expression pattern of several pair-rule genes
(Dearolf et al., 1989;
Han et al., 1998;
Small et al., 1996;
Andrioli et al., 2002;
Riddihough and Ish-Horowicz,
1991). We propose that cell type-specific autoregulatory elements
allow for a much more precise regulation of the gene in each situation, as
they are not only dependent on Krox20, but also on other transcription factors
that bring in additional specificity (as does Sox10 in the case of the NCE)
(Ghislain et al., 2003).
Specific autoregulatory elements work in association with a particular
initiator element(s) and together establish a highly regulated cell
type-specific expression (Fig.
7), which may be required in the case of Krox20 because of its key
role in several developmental processes
(Schneider-Maunoury et al.,
1993; Topilko et al.,
1994; Levi et al.,
1996) (S. Garel, P. Topilko and P.C., unpublished).
Molecular mechanisms of Krox20 regulation in the hindbrain and neural crest
The properties of the elements identified in this study allows us to
propose a molecular model for Krox20 regulation in the hindbrain and
neural crest (Fig. 7). The
characteristics of elements B and C suggest that they are responsible for the
initiation of Krox20 expression in r3 and r5 (initiator elements). In
such a case, why is there an apparent redundancy in r5, where both elements
are active? It is possible that elements B and C are indeed largely redundant,
providing higher security for the system. Alternatively, the two elements
might work in a synergistic manner, funnelling information from two separate
pathways to reach a level of Krox20 sufficient for starting the autoregulatory
loop (see below).
The isolation of elements B and C opens the way to the identification of
the transcription factors that are directly acting on them. As a first step in
this direction, we have shown that a vHNF1-binding site within element B is
absolutely required for its r5-specific enhancer activity. Although vHNF1
involvement in Krox20 regulation was already known, a direct
transcriptional role was not expected: on the basis of the relative
vHnf1 and Krox20 expression patterns and partial rescue of a
vHnf1 mutation by val expression in zebrafish, it was
proposed that vHNF1 regulates Krox20 only indirectly, via val/MafB
and other unknown factors (Wiellette and
Sive, 2003). Our data, which support a direct involvement of vHNF1
(Fig. 7), force a revision of
this hypothesis, at least in mouse and chick.
An intriguing observation with element C is that its activity, although initially restricted to r3 and r5, progressively extends into r4, a territory where Krox20 is never expressed. The same is true for the larger fragments from which element C is derived, including BAC 121, although in the latter case the chicken Krox20 gene is present in the construct and this might affect its activity. The simplest hypothesis for explaining such behaviour is that the regulation of Krox20 involves a repression mechanism in r4 and that in our transgenic analyses this repression does not occur properly, possibly because a necessary cis-acting element is absent from our constructs.
Once elements B and C have established a threshold level of Krox20 in r3
and r5 cells, the autoregulatory element A is likely to take the relay and
maintain or amplify this level by a direct, positive feedback loop
(Fig. 7). It therefore appears
as a maintenance element. There is solid physiological evidence in favour of
the importance of such a process: mouse Krox20 knockout does not
affect its initial activation, but leads to rapid downregulation of its
expression, without loss of the cells
(Schneider-Maunoury et al.,
1993; Voiculescu et al.,
2001). Furthermore, ectopic expression of Krox20 in the
hindbrain leads to activation of the endogenous gene
(Giudicelli et al., 2001). As
discussed above, like the other Krox20 autoregulatory elements,
element A is likely to be cell type-specific and therefore rely on additional
factor(s) that provide this specificity. The availability of the nucleotide
sequence of this element will provide a means to identify this factor. In
addition, such a factor might be responsible for switching off the positive
feedback loop when Krox20 expression is downregulated in the
hindbrain.
As shown previously, the regulation of Krox20 in r3 and r5 also
involves a non cell-autonomous autoregulatory mechanism, which may be involved
in the extension of odd-numbered territories
(Giudicelli et al., 2001). As
elements B and C are not trans-activated by Krox20, they are not likely to be
implicated in this process. By contrast, this could be the case for element A.
However, as element A activity is abrogated by mutation of its Krox20-binding
sites, this would imply that non cell-autonomous activation of Krox20
requires the Krox20 protein in the receiving cell. It has been recently shown
that some homeodomain transcription factors can be transferred from cell to
cell (for a review, see Prochiantz and
Joliot, 2003). It will therefore be extremely interesting to
investigate whether this could also be the case for Krox20.
Finally, elements A, B and C are also likely to provide the threshold level
of Krox20 protein necessary, in conjunction with the crest-specific factor,
Sox10, to initiate another positive feedback loop, involving the NCE, at work
in the r5-derived neural crest (Fig.
7) (Ghislain et al.,
2003). Together, these elements explain the expression of
Krox20 in the r5-derived neural crest and will provide a detailed
molecular understanding of its patterning.
|
ACKNOWLEDGMENTS |
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|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/7/1253/DC1
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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