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First published online 11 September 2008
doi: 10.1242/dev.023614
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1 INSERM, U784, Laboratoire de Génétique Moléculaire du
Développement and 46 rue d'Ulm, 75230 Paris, France.
2 Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, France.
3 CNRS UMR 7622, Laboratoire de Biologie du Développement, 75252 Paris,
France.
4 Université Pierre et Marie Curie, 9 quai Saint Bernard, 75005 Paris,
France.
Author for correspondence (e-mail:
charnay{at}biologie.ens.fr)
Accepted 20 August 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Hindbrain segmentation, Pattern formation, Transcription factor
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lumsden and Krumlauf, 1996).
This subdivision presages the establishment of a stereotyped pattern of
neuronal differentiation (Clarke et al.,
1998; Lumsden and Keynes,
1989). Rhombomeres constitute units of both cell lineage
restriction (Birgbauer and Fraser,
1994; Fraser et al.,
1990) and specific gene expression
(Lumsden and Krumlauf, 1996;
Rijli et al., 1998).
Among the genes involved in hindbrain patterning, Krox20 plays a
particularly important role. It encodes a zinc finger transcription factor
(Chavrier et al., 1989;
Chavrier et al., 1988) and is
expressed in prospective and established r3 and r5 territories
(Schneider-Maunoury et al.,
1993; Wilkinson et al.,
1989). Loss- and gain-of-function experiments have shown that
Krox20 is essential for the formation of these odd-numbered segments,
and that it acts by coupling segment delimitation, specification of rhombomere
identity and cell lineage restriction
(Giudicelli et al., 2001;
Schneider-Maunoury et al.,
1997; Schneider-Maunoury et
al., 1993; Swiatek and
Gridley, 1993; Voiculescu et
al., 2001). Krox20 performs its complex function by up- or
downregulating the expression of a number of other regulatory genes
(Giudicelli et al., 2001;
Mechta-Grigoriou et al., 2000;
Seitanidou et al., 1997).
These include Hox genes, which are involved in regional and segmental
specification in the hindbrain (Lumsden
and Krumlauf, 1996; Rijli et
al., 1998). Specifically Krox20 is responsible for the direct
transcriptional activation of paralogous group (PG) 2 and 3 genes (Hoxa2,
Hoxb2 and Hoxb3) in r3 and r5, and in r5, respectively
(Giudicelli et al., 2001;
Manzanares et al., 2002;
Nonchev et al., 1996a;
Nonchev et al., 1996b;
Seitanidou et al., 1997;
Sham et al., 1993;
Vesque et al., 1996), whereas
it represses the PG 1 gene Hoxb1
(Garcia-Dominguez et al.,
2006; Giudicelli et al.,
2001).
Given the central role of Krox20 in hindbrain development,
understanding the basis of Krox20 regulation is of prime importance.
Significant progress has been made in this direction in r5, because the
transcription factors Mafb and vHnf1 (Hnf1b - Mouse Genome Informatics,
Zebrafish Information Network) have been shown to be necessary for its
expression (Hernandez et al.,
2004). In r3, however, our knowledge is more limited and somehow
confusing. Pbx and Meis proteins have been implicated in several studies.
Hence, eliminating both maternal and zygotic expression of the pbx2
and pbx4 genes in zebrafish embryos leads to a complete
transformation of the hindbrain, with no krox20 expression
(Waskiewicz et al., 2002).
Loss of Meis function in zebrafish results in phenotypes similar to a single
pbx4 mutation, with loss of the anterior domain of krox20
expression (Choe et al., 2002;
Popperl et al., 2000;
Waskiewicz et al., 2001). Pbx
and Meis usually act as co-factors for Hox proteins (for a review, see
Moens and Selleri, 2006). Pbx
proteins form complexes with Hox factors and bind bi-partite Hox/Pbx DNA
sequence motifs. Meis or Prep proteins recognize separate DNA-binding sites
but directly interact with the Hox/Pbx complexes. The involvement of Pbx and
Meis factors in Krox20 regulation therefore suggests an involvement
of Hox proteins, but other data are apparently contradictory. According to
their expression patterns, PG 2 genes could be involved in Krox20
regulation in r3. However, in the Hoxa2/Hoxb2 mouse double mutant,
Krox20 expression is not affected, indicating that PG 2 genes are not
required for normal Krox20 regulation
(Davenne et al., 1999).
Concerning PG 1 genes, they have been proposed to be expressed up to the r3/r4
presumptive rhombomere boundary (reviewed by
Lumsden and Krumlauf, 1996),
and have not been reported to overlap with the Krox20 expression
domain in r3. Nevertheless, strong evidence for their involvement in
Krox20 regulation in r3 has been obtained in Xenopus, where
Hoxd1 is expressed in the hindbrain in addition to Hoxa1 and
Hoxb1, and where knockdown of the complete PG 1 leads to loss of
Krox20 expression (McNulty et
al., 2005). In mouse and zebrafish, where only Hoxa1 and
Hoxb1 are expressed, the situation is less clear. The Hoxa1
mutation results in a patchy but caudally extended Krox20 r3 domain
(Carpenter et al., 1993;
Chisaka et al., 1992;
Dolle et al., 1993;
Lufkin et al., 1991;
Mark et al., 1993), and
combined Hoxa1 and Hoxb1 impairment further reduces
Krox20 expression in r3, but it does not prevent it
(Barrow et al., 2000;
Gavalas et al., 1998;
McClintock et al., 2002;
McNulty et al., 2005;
Rossel and Capecchi,
1999).
Altogether, the above data offer a contrasting view of Hox protein
involvement in the control of Krox20 expression in r3. To clarify
this issue, we have localized the cis-acting regulatory elements responsible
for Krox20 expression in the developing hindbrain. Three evolutionary
conserved transcriptional enhancers, designated elements A, B and C, have been
characterized (Chomette et al.,
2006). Element A is a direct auto-regulatory element, involved in
the maintenance of Krox20 expression, whereas elements B and C are
responsible for the initiation of Krox20 expression, as indicated by
their activity in a Krox20-null background
(Chomette et al., 2006).
Element B is active only in r5, whereas element C is active in the r3-r5
domain. Therefore, element C is the only initiator element in r3. In this
paper, we analyze chick element C. We establish that Hox proteins are directly
involved in Krox20 regulation and provide a comprehensive view of a
very complex regulatory network essential for hindbrain segmentation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). pCMVHoxa1, pCMVHoxb1 and pCMVHoxb9 (gifts from
R. Krumlauf, Stowers Institute, Kansas City), pCMVHoxa2 (gift from R.
Rezsohazy, Université Catholique de Louvain) and pCMVMeis2,
respectively, contain complete cDNAs of human HOXA1 (IMAGE Consortium
Clone ID 5537563), mouse Hoxb1, Hoxb9 and Hoxa2
(Matis et al., 2001) and
Meis2 (IMAGE Consortium Clone ID 4191098). For cell extract
preparation, mouse Meis2, Hoxb1 and Pbx1 (IMAGE Consortium
Clone ID 5701148) cDNAs were introduced into pAdRSVSp
(Giudicelli et al., 2003),
together with a HA epitope-coding sequence immediately before the stop
codon.
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). In ovo
electroporation was performed as described
(Giudicelli et al., 2001) at
stages HH8-HH10. Each construct or combination of constructs was tested in at
least two independent experiments, each involving eight or more embryos. The
efficiency of electroporation was controlled by co-electroporation of a GFP
reporter.
In situ hybridization, immunolabelling and X-gal staining
Whole-mount in situ hybridization was performed as described
(Giudicelli et al., 2001). The
probes for in situ hybridizations were as follows: a mouse Krox20
probe (Wilkinson et al.,
1989), a mouse Meis1 probe (gift from Sonia Garel, Ecole
Normale Supérieure), a chick Meis2 probe (gift from Nadia
Mercader, EMBL, Heidelberg), mouse Meis2, Meis3, Prep1 and
Prep2 probes synthesized from IMAGE Consortium cDNA clones 5687497,
5121146, 5721441 and 6332968, respectively. Double in situ hybridization was
performed as described (Giudicelli et al.,
2001). Alkaline phosphatase activity was revealed using the NBT/or
INT/BCIP substrates (Roche). Mouse immunolabelling was performed using rabbit
anti-Krox20 (Desmazières et al.,
2008) and rat anti-GFP (Nacalai Tesque) antibodies. For double
labelling, in situ hybridization was performed as above and rat anti-HA
antibody (Roche) was added with the anti-DIG antibody. Labelling for
β-galactosidase activity was performed as described
(Ghislain et al., 2003).
Protein extracts and band shift assays
Expression plasmids were transfected into COS-7 cells using the FuGene 6
Transfection Reagent (Roche). Cell lysates were prepared as described
(Dignam et al., 1983). Nuclear
membranes were disrupted by the addition of 0.5% Nonidet P-40, the suspension
was brought to 0.4 M NaCl and 0.2 mM EDTA. HA-tagged protein was purified from
the supernatant using the Anti-HA Affinity Matrix (Roche). Band shift
experiments were performed as described
(Chomette et al., 2006), with
the following modifications: 2 or 4 µl of Meis2 or Hoxb1/Pbx1 protein
preparations were pre-incubated on ice with 1 µg of poly(dI-dC) in 10 µl
of buffer (10 mM Tris-HCl pH 7.5, 75 mM NaCl, 1 mM EDTA, 1 mM DTT, 540 ng/ml
BSA, 12% glycerol for Meis2; 20 mM HEPES pH 7.9, 1 mM EDTA, 1 mM DTT, 2 mM
MgCl2, 6.25% Ficoll for Hoxb1/Pbx1).
Zebrafish manipulations
In situ hybridization was performed as described
(Hauptmann and Gerster, 1994),
using hoxb1a (Prince et al.,
1998) and krox20
(Oxtoby and Jowett, 1993)
probes. For cell-autonomy experiments, capped RNAs were transcribed using the
mMessage mMachine Kit (Ambion). hoxb1aMyc (gift from V. Prince,
University of Chicago) and meis1.1 RNAs were injected into one cell
at the 16- to 32-cell stage at concentrations of 60 and 75 ng/µl,
respectively. Embryos were embedded in gelatine-sucrose after whole-mount in
situ hybridization and cryosectioned. Sections (16 µm) were treated for
immunofluorescence with a primary rabbit anti-Myc antibody (Upstate
Biotechnology 06-549, 1/300) and a secondary donkey anti-rabbit antibody
(Molecular Probes, 1/400). For chromatin immunoprecipitation (ChIP) analysis,
2 nl of meis1.1 (80 ng/µl), Myc-tagged-hoxb1a (60
ng/µl) and Myc-tagged-hoxa2 (60 ng/µl, gift from V. Prince)
RNAs were injected into one cell at the four-cell stage. ChIP was carried out
as described (Havis et al.,
2006) at the 100% epiboly stage. Ten µl of the anti-Myc (9B11,
Ozyme) antibody were used per 10 µg of sonicated chromatin.
Immunoprecipitated DNA was analyzed by PCR using the following primers:
element C, 5'-gttaatgacaggaggtcg-3' and
5'-gctctctgggataaaggt-3'; and -22.4 kb sequence,
5'-tgcaaccacttgcctcac-3' and
5'-gcgctgctgttagcctcc-3'.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Two
overlapping putative Hox/Pbx sites on both strands (HP1), highly conserved
among vertebrates, and three clustered putative Meis sites (M1, M2 and M3, the
first one being also conserved) were identified within a 70 bp region
(Fig. 1; see Fig. S1 in the
supplementary material). Another highly conserved putative Hox/Pbx-binding
site (HP2) was identified in the 3' part of the enhancer. We
investigated the significance of these putative sites by gel retardation with
a 64 bp oligonucleotide carrying HP1 and the Meis sites (see Fig. S1 in the
supplementary material). This oligonucleotide was incubated with purified
proteins prepared from COS-7 cells transfected or co-transfected with
HA-tagged mouse Hoxb1, Pbx1 and Meis2 expression plasmids.
Meis2 and the Hoxb1/Pbx1 combination led to the formation of retarded bands,
which could be specifically competed out by the addition of oligonucleotides
carrying high-affinity Meis- or Hox/Pbx-binding sites, respectively
(Fig. 1A). The relatively
efficient binding of Meis2 alone might be explained by the presence of the
three sites. To establish that the binding sites were indeed those identified
in silico, we introduced mutations separately into HP1 and into the three Meis
sites (Fig. 1). Band shift
analysis demonstrated that the mutated fragments did not bind Hoxb1/Pbx1 and
Meis2, respectively (Fig. 1A).
Further experiments performed with the 896 bp fragment demonstrated that
mutations of the three Meis sites also abolished Meis2 binding (data not
shown), indicating that it does not contain any additional Meis site. We performed a similar analysis in the case of HP2. An 83 bp oligonucleotide carrying HP2 (see Fig. S1 in the supplementary material) was subjected to gel retardation in presence of Hoxb1 and Pbx1 proteins, leading to formation of specific retarded bands (Fig. 1B). Mutation of the putative Hox/Pbx site eliminated the major band, although some residual binding was maintained, the origin of which was not investigated. To establish that there was no other major Hox/Pbx-binding site within the enhancer, we repeated the band shift analysis with a larger 321 bp fragment that retained element C activity in the chick electroporation assay (data not shown) and that carries both HP1 and HP2 (see Fig. S1 in the supplementary material). Exposure of this fragment to Hoxb1 and Pbx1 led to a strong gel retardation that was dramatically reduced upon mutation of both HP1 and HP2 (Fig. 1C). The residual binding might correspond to a low-affinity binding site(s) that could not be identified by sequence analysis.
In conclusion, these data indicate that element C contains two bona fide Hox/Pbx-binding sites and several Meis sites, and therefore suggest that it could mediate aspects of the Krox20 regulation by direct binding of these factors.
Element C mediates Hox, Pbx and Meis synergy
Because element C contains binding sites for Hox/Pbx and Meis proteins, we
investigated the possibility that these factors might be able to modulate its
activity. For this purpose, a DNA construct in which element C drives a
lacZ reporter gene (cC-lacZ)
(Chomette et al., 2006) was
electroporated in the chick hindbrain together with various Hox, Meis and Pbx
expression vectors. In the first series of experiments, each expression
construct was used alone and at a concentration of 0.2 µg/µl. In the
absence of exogenous factor, element C drives reporter expression in the r3-r5
domain (Chomette et al., 2006).
Co-electroporation with the Meis2 expression vector did not
significantly affect the activity of the enhancer
(Fig. 2A,B), and
co-electroporation with the Pbx1 expression vector led to a slight
extension of the reporter expression domain
(Fig. 2C). By contrast,
co-electroporation with Hoxa1, Hoxb1, Hoxa2 or Hoxb9
expression constructs led to major extensions of the expression domain and to
increases in the level of reporter gene expression
(Fig. 2D,E; data not
shown).
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Activation by the Hox/Pbx/Meis complex requires direct binding to element C
To determine whether Krox20 is under the direct transcriptional
control of Hox, Pbx and Meis proteins, we analyzed the consequences of the
mutations preventing binding of Hox/Pbx or Meis
(Fig. 1) on enhancer activity.
This was first investigated by in ovo chick electroporation. Mutation of the
Hox/Pbx site HP1 led to complete inactivation of the enhancer
(Fig. 3A,B). By contrast, the
Meis-binding sites mutant retained its activity in r4 and r5, but lost it in
r3 (Fig. 3C). Finally, mutation
of the Hox/Pbx site HP2 largely prevented reporter expression in r3, but
preserved some activity in r4 and r5 (Fig.
3D).
The effects of the mutations were then investigated by murine transgenesis.
Whereas the wild-type enhancer led to specific lacZ reporter
expression in the r3-r5 region in more than half of the transgenic embryos
(Fig. 3E,F) (see also
Chomette et al., 2006), the
construct carrying the HP1 site mutation was completely inactive in the
hindbrain (Fig. 3E,G;
n=11). Two of these transgenic embryos showed some ectopic
lacZ expression (Fig.
3E, data not shown), indicating that in these cases the transgene
was functional and integrated in a chromatin region compatible with gene
expression. These data confirm the results obtained in the chick and strongly
suggest that direct binding of the Hox/Pbx complex to site HP1 is absolutely
required for element C activity. It should be noted, however, that the
relative activity of the enhancer in r3 and r5 compared with r4 is different
in the chick and mouse systems (compare
Fig. 3A and
Fig. 3F). This might reflect
the stage of the embryos, as the r4 activity is increased in older mouse
embryos (Chomette et al.,
2006).
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Finally, mutation of the HP2 site led to a reduction in the frequency of hindbrain-specific activity (two out of 10 embryos, as compared with five out of eight for the wild-type construct; Fig. 3E). In addition, in the positive embryos the pattern was modified: reporter expression was eliminated from r3 and significantly reduced in r4 and r5 (Fig. 3I, data not shown). Therefore, the HP2 site appears to be essential in r3, and to play a less important role in r4 and r5. Altogether, our work establishes that Krox20 is a direct transcriptional target of Hox/Pbx and Meis, and reveals different requirements for element C activity in r3, r4 and r5.
Identification of the Meis or Prep protein involved in Krox20 activation in r3
Because Meis-binding sites are necessary for the activity of
Krox20 element C in r3, we analyzed the expression patterns of the
different Meis or Prep family members in the developing hindbrain, to
determine which one(s) might be involved in Krox20 regulation. The
family includes five members in the mouse [Meis1, Meis2, Meis3, Prep1
(Pknox1 - Mouse Genome Informatics) and Prep2 (Pknox2 - Mouse Genome
Informatics)]. A detailed analysis of their expression is presented in Fig. S2
in the supplementary material. Altogether, the data indicate that during the
period corresponding to 1 to 5 somite stages (ss), which includes the stage of
initiation of Krox20 expression in the prospective r3 (1 to 3 ss),
Meis2 is the only family member expressed at the level of this
rhombomere. Therefore, Meis2 must be the member of the Meis family responsible
for the cooperation with the Hox/Pbx complex on element C, and for the
initiation of Krox20 expression in r3. Analysis of chick
Meis2 expression revealed a pattern compatible with a similar role
(data not shown). At later stages of development, the continued activity of
element C in r3 could also involve other Meis family members, in particular
Meis1.
Specificity of Krox20 activation by Hox proteins
We have shown above that the misexpression of several Hox genes in the
chick neural tube leads to general activation of a co-electroporated
lacZ reporter driven by element C. To investigate whether the
endogenous Krox20 gene would respond in a similar manner, we have
analyzed its expression by in situ hybridization. The electroporation of
Hoxa1 or Hoxb1 expression vectors led to ectopic activation
of Krox20 in r1, r2 and the midbrain-hindbrain boundary (MHB), with
rostral enlargement of the r3 domain and the presence of homogeneous patches
of Krox20-positive cells, often connected to the enlarged r3
(Fig. 4A,B). We observed no
upregulation of Krox20 in r3 and r5, nor any activation in other
regions of the neural tube (Fig.
4A,B; data not shown). Hoxa2 misexpression also led to
activation of Krox20 in a restricted domain anterior to r3
(Fig. 4C). By contrast,
Hoxb9 was unable to induce any ectopic Krox20 expression
(Fig. 4D).
Endogenous Krox20 activation by Hox PG 1 and 2 expression vectors
is consistent with the data obtained by co-electroporation, therefore
confirming the role of Hox genes in the control of Krox20 regulation.
However, this analysis also revealed differences, suggesting the existence of
additional levels of specificity. First, the activation of the endogenous gene
is strictly restricted in space, essentially to r1, r2 and the MHB. This
restriction is likely to reflect the requirement for Hox proteins to cooperate
with other factors or the involvement of repressor molecules. Second,
induction of endogenous Krox20 expression was not observed with
Hoxb9. The differences observed in the co-electroporation experiments might be
explained by the more permissive character of this assay
(Chomette et al., 2006;
Ghislain et al., 2003;
Pouilhe et al., 2007).
Krox20 and Hoxb1 domain overlap allows the direct activation of Krox20 by PG 1 proteins
Our data indicating that the initiation of Krox20 expression in r3
is under direct transcriptional control of Hox proteins raise an interesting
issue. The only Hox genes known to be expressed in r3 are the PG 2 genes
Hoxa2 and Hoxb2, but, as indicated above, they are not
required for its activation. PG 1 genes Hoxa1 and Hoxb1 are
activated earlier and independently of Krox20, but their anterior
limits of expression have not been reported to overlap with the r3
Krox20-positive domain (Barrow et
al., 2000; Lumsden and
Krumlauf, 1996). In these conditions, we are confronted with the
following alternative possibilities: either Krox20 is activated in a
cell-autonomous manner by PG 1 genes that are at least transiently
co-expressed in the same cell, or the activation relies on a non
cell-autonomous mechanism. To revisit the issue of an overlap in the
territories of expression of PG 1 genes and Krox20 at the r3-r4
boundary, we made use of a mouse knock-in line in which GFP has been
inserted into the Hoxb1 locus and recapitulates its expression
pattern (Gaufo et al., 2000).
Hoxb1GFP/+ embryos at around 5-6 ss were subjected to
double immunofluorescence analysis using antibodies directed against Krox20
and GFP. This revealed a clear overlap between the r3 Krox20
expression domain and the Hoxb1-GFP-positive domain
(Fig. 5A-C). This suggests that
at the r3-r4 boundary Krox20 is induced in cells that also express
Hoxb1 or that have recently expressed it, as the GFP protein might be
more stable than Hoxb1. To extend this conclusion to other vertebrates, we
performed double in situ hybridization for krox20 and
hoxb1a, the zebrafish homolog of mouse Hoxb1, at early
stages of r3 development in zebrafish. We found that at tailbud and 1-somite
stages, a few cells were positive for both krox20 and hoxb1a
at the level of the r3-r4 border (Fig.
5D-G, black arrowheads). In conclusion, the data obtained in the
mouse and zebrafish indicate that there is a partial and transient overlap
between the Hoxb1 and Krox20 expression domains.
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). To
avoid this complication, we turned again toward the zebrafish system, which
allows the experiment to be performed at an earlier stage in development. In
addition, the experiment can be carried out in a vhnf1-null
background (Sun and Hopkins,
2001), which ensures that krox20 activation occurs via
element C, because element B absolutely requires vHnf1 binding
(Chomette et al., 2006). We
co-injected RNAs encoding a Myc-tagged form of Hoxb1a and Meis1.1 (which
synergizes with Hoxb1a; A.S. and S.S.-M. unpublished) into one cell of 16- to
32-cell stage embryos. The embryos were collected at the 5-somite stage and
subjected to combined krox20 in situ hybridization and Myc
immunofluorescence. In injected vhnf1-/- embryos,
krox20 was ectopically activated in groups of cells within the neural
plate rostral to r3 (Fig.
6D-F). No such ectopic krox20 expression was observed
after injection of GFP RNA (52 embryos, data not shown). The large
majority of the cells expressing krox20 ectopically (417/464 cells in
eight embryos, three different injection experiments) were positive for
Hoxb1a-Myc (Fig. 6D-F). The
krox20-positive, Hoxb1a-Myc-negative cells could be, in most cases,
explained by quenching of the fluorescence by the strong in situ staining
(data not shown). Therefore, in this experiment, hoxb1a activates
krox20 in a cell-autonomous manner. The next question was whether the cell-autonomous activation of Krox20 by Hoxb1 involves a direct interaction with element C. To address this issue, we used the zebrafish system, which allows access to larger amounts of material and at earlier embryonic stages, to perform chromatin immunoprecipitation (ChIP) experiments. A conserved orthologous element C is present in the zebrafish genome and has been shown to be active in r3 and r5 (M.A.W., A.S. and S.S.-M., unpublished). Zebrafish embryos were injected into one cell at the four-cell stage with Myc-tagged hoxb1a and meis1.1 RNAs. The embryos were collected at the 100% epiboly stage, when krox20 is initially activated, and subjected to ChIP with an antibody directed against the Myc tag, followed by PCR amplification. We used two pairs of PCR primers able to amplify the core of element C, or, as a control, an unrelated sequence located within the krox20 locus. The element C sequence was specifically immunoprecipitated, whereas the control sequence was not (Fig. 6G). These data establish that Hoxb1a binds to the element C in vivo, and support a direct involvement of Hox PG 1 proteins in the regulation of Krox20 via element C.
In conclusion, altogether our data indicate that the transient overlap between the Hoxb1 and Krox20 expression domains allows Hox PG 1 proteins to directly activate Krox20.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Specificity in transcriptional activation by Hox factors
Besides unravelling the direct involvement of Hox proteins in
Krox20 regulation, our work led to further insights in their mode of
action. First, we observed differences among Hox proteins in their activation
potential regarding endogenous Krox20
(Fig. 4), suggesting that
different paralogous groups are not functionally equivalent for the activation
of Krox20. Hence, we have shown that after in ovo electroporation a
polymerized HP1 site can drive reporter expression specifically in r4, the
posterior hindbrain and the spinal cord (see Fig. S4 in the supplementary
material), a pattern very similar to that of Hoxb1. This observation
suggests that HP1 preferentially responds to Hox PG 1 proteins, consistent
with a prominent role of these proteins in the initiation of Krox20
expression. Finally, we also found that the two Hox/Pbx sites are not
equivalent, as mutation of HP1 completely abolishes the activity of the
enhancer, whereas mutated HP2 retains limited activity in r4 and r5.
The mutagenesis of the Meis-binding sites (M1-M3) revealed another
differential effect: the activity of element C was abrogated in r3, but not in
r4 and r5 (Fig. 3). Because
analysis of element C by gel retardation did not detect any additional
Meis-binding site, this suggests that the Hox/Pbx complex does not require a
Meis factor to activate the enhancer in r4 and r5, in contrast to r3. This
conclusion is consistent with analyses performed in the zebrafish that have
shown that Meis factors are absolutely required for Krox20 expression
in r3 but not in r5 (Choe et al.,
2002; Waskiewicz et al.,
2001). Variable Meis dependence has been observed among Hox
proteins (Choe and Sagerstrom,
2005; Ferretti et al.,
2000), and the different requirements of element C in r3 and r5
might reflect the involvement of different Hox proteins and/or the implication
of additional accessory factor(s). More generally, our work establishes that
element C functions according to different modes in r3 and r5. Finally, our
analysis of Meis and Prep gene expression suggests that Meis2 is a
key component of the transcriptional complex in charge of Krox20
activation in r3.
A model for the control of Krox20 expression in the developing hindbrain
Krox20 regulation appears to constitute a complex process and we
have attempted to amalgamate the observations collected in the present work
with previous data to develop a molecular model. Our consistent observations
in mouse, chick and zebrafish allow us to combine data obtained in different
vertebrate species. We will first envisage the regulation in r3
(Fig. 7). We propose that, in
contrast to what was previously thought, at around E8 in the mouse, when
Hoxa1/Hoxb1 neural domains reach their maximal rostral extensions,
their limits are located within prospective r3. This point is consistent with
recent tracing data indicating that derivatives of Hoxa1-expressing
cells are found in r3 (Makki et al., 2007), and is supported by our
observation of an overlap between Krox20 and Hoxb1
expression domains in r3. In addition, we postulate the existence of another
factor (X, unknown), whose expression domain extends caudally
(Fig. 7A,B) and will start to
overlap with the PG 1 domain around E8. This defines a transversal, narrow
stripe of cells where Krox20 is specifically activated under the
synergistic transcriptional activities of factor X, Hox PG 1, Pbx and Meis2
proteins, acting through element C. Interestingly, an essential role of
Iroquois transcription factors in the activation of krox20 in r3 has
been recently uncovered (A.S. and S.S.-M., unpublished). Factor X might
therefore be an Iroquois transcription factors or it might lie downstream to
them in the regulatory cascade. As discussed above, a complementary
involvement of Hox PG 2 proteins is also likely, although loss-of-function
analyses suggest that the major role is played by PG 1 factors. An important
feature of our hypothesis is that it provides an explanation for the
characteristic initial expression pattern of Krox20, restricted to a
very narrow stripe of cells
(Schneider-Maunoury et al.,
1993). Krox20 activation will have multiple consequences
(Fig. 7B,C). (1) It will lead
to the progressive retraction of the rostral limit of Hox PG 1 gene expression
to the future r3/r4 boundary. This is consistent with the observations that
the Hoxb1-positive domain extends within prospective r3 in a
Krox20-null mutant (Voiculescu et
al., 2001) and that ectopic Krox20 expression results in
Hoxb1 repression
(Garcia-Dominguez et al.,
2006; Giudicelli et al.,
2001). (2) Krox20 initiates several transcriptional autoregulatory
loops that are necessary for the maintenance of its own expression
(Schneider-Maunoury et al.,
1993). One of them is direct and relies on the binding of Krox20
to element A (Chomette et al.,
2006), whereas the others involve the activation of Hoxa2
and Hoxb2 (Nonchev et al.,
1996a; Sham et al.,
1993), which will replace Hox PG 1 proteins on element C. These
autoregulatory mechanisms are likely to be redundant, as the double mutation
of Hoxa2 and Hoxb2 only marginally affects the r3 domain of
Krox20 expression (Davenne et
al., 1999). (3) Expression of Krox20 also results in its
activation in neighbouring Krox20-negative cells by non-cell
autonomous autoregulation (Giudicelli et
al., 2001), a process thought to participate in the extension of
r3. The caudal extension of r3 might also rely on the progression of the front
of gene X expression. These processes will give rise to a moving
stripe of cells co-expressing Krox20 and Hoxb1 at the caudal
edge of developing r3, as we observe in mouse and zebrafish embryos
(Fig. 5). At some point (around
E8.5), these processes of extension of r3 at the expense of adjacent
rhombomeres will stop, delimiting the final extensions of r2, r3 and r4.
|
). The severe loss of Krox20 expression in r5 upon
mutation of Mafb or vHnf1
(Hernandez et al., 2004), and
the fact that these factors are likely to act only via element B (M.A.W. and
P.C., unpublished), suggests that element B is predominant. In r5, element C
functions according to a different mode than in r3: although it still requires
binding of a Hox protein, Meis factors are not necessary.
Finally, what happens in r4, where element C is active but Krox20
is not expressed? To explain this apparent contradiction, we propose that
Krox20, in addition to the positive regulatory mechanisms discussed
above, is subject to a negative regulation, which may lie downstream of the
Hox PG 1 genes and prevent Krox20 expression in r4
(Fig. 7B,C). The existence of
such a negative regulation is consistent with the inactivation of
Hoxa1, which results in an extension of the anterior domain of
Krox20 into prospective r4
(Gavalas et al., 1998;
Rossel and Capecchi, 1999),
and with the repressive activity of Nlz family members on Krox20
expression (Runko and Sagerström,
2003; Hoyle et al.,
2004).
In conclusion, a particularly interesting feature of this model resides in the initial phase of Krox20 expression in r3. We propose that a narrow band of cells is defined by the encounter of two domains extending in opposite directions. In these cells, Krox20 is very transiently activated by Hox PG 1 proteins, which disappear rapidly while Krox20 expression is maintained and propagated by different molecular mechanisms. We propose to use the term `ignition' to refer to the role of Hox PG 1 proteins in this novel type of initiation of gene expression, which may occur in other developmental processes.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/20/3369/DC1
|
ACKNOWLEDGMENTS |
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|
Footnotes |
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|
REFERENCES |
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