Department of Zoology and Animal Biology, University of Geneva, Sciences
III, Quai Ernest Ansermet 30, 1211 Geneva 4, Switzerland
Present address: Molecular and Experimental Genetics, FRE2358, CNRS, Institut
de Transgénose, rue de la Férollerie, 3B, 45071, Orléans
cedex 2, France
* Author for correspondence (e-mail: denis.duboule{at}zoo.unige.ch)
Accepted 20 June 2002
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SUMMARY |
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Key words: Insulator, Gene regulation, Hox complex, Mouse
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INTRODUCTION |
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Beside this level of transcriptional regulation, many cis-acting
control sequences have been characterised by their ability to impose
particular expression patterns to nearby located genes. Various enhancer
sequences have thus been described, with distinct functional properties. For
example, in several cases, gene-specific activation was shown to result from
proximal enhancers selectively interacting with a given promoter.
Alternatively, enhancer sharing mechanisms were reported to account for the
co-expression of neighbouring genes
(Sharpe et al., 1998), a
situation favoured by the tight clustered organisation of these genes
(Bell et al., 2001
). Enhancer
sharing processes, within Hox gene clusters, were not only shown to involve
proximal enhancers, which can control the expression of neighbouring genes in
the same tissue, but also more global, distally located enhancers, which are
able to impose a particular regulation to series of contiguous genes. Examples
of such a large-scale regulation was provided by the co-expression of several
Hoxd genes in either the intestinal hernia or the developing digits
(Zakany and Duboule, 1999
;
Kmita et al., 2000a
;
Spitz et al., 2001
). In these
latter cases, co-ordinated expression of several genes at the same place was
demonstrated to be necessary to properly build up the concerned structure
(Zakany et al., 1997a
).
However, this particular regulatory strategy implies that other closely
linked gene members of the cluster, the function of which may not be relevant
in a given structure, are protected against such a global regulatory
influence, such as to prevent their mis-expression. Indeed, ectopic
transcription of Hox genes was shown be a potential source of severe
morphological and/or physiological alterations
(Knezevic et al., 1997;
McLain et al., 1992
;
Morgan et al., 1992
;
Rijli et al., 1994
;
Yokouchi et al., 1995
).
Accordingly, boundary or insulator elements must exist to restrain the action
of enhancers specifically to those relevant target genes, by isolating them
from their neighbours (Sun and Elgin,
1999
; Udvardy,
1999
). We have previously showed that a DNA segment located
between Hoxd12 and Hoxd13 could prevent both genes from
responding to a distally located intestinal hernia enhancer
(Kmita et al., 2000a
). In much
the same way, Hox clusters must themselves be isolated from external
regulatory influences to prevent enhancer sequences that are necessary for
closely located, non-Hox genes, to interfere with the precise and particular
regulation of this gene family. This requirement for a context-dependent
insulation is best exemplified by the presence of the Evx2 gene in
the immediate 5' neighbourhood of the Hoxd cluster
(D'Esposito et al., 1991
;
Bastian et al., 1992
).
Evx2 indeed displays specific expression features that are not
shared by any Hoxd genes, not even by Hoxd13, whose promoter
lies close to that of Evx2. This is best illustrated by discrete cell
types of the developing central nervous system, in both spinal cord and more
rostral parts of the brain, in various vertebrate species
(Bastian et al., 1992;
Brulfert et al., 1998
;
Dollé et al., 1994
;
Sordino et al., 1996
). In the
spinal cord, transcripts are localised in the ventrally located V0
interneurones, as well as in a population of dorsal interneurones
(Moran-Rivard et al., 2001
).
In the developing brain, Evx2 expression is detected in the
rhombencephalic isthmus area (the metencephalic-mesencephalic transition) and
extends into the superficial layer of the entire midbrain. It is also
expressed in the developing hindbrain and in part of the future cerebellum
(Dollé et al.,
1994
).
While the enhancer sequences driving Evx2 expression in the CNS
have not yet been precisely identified, experiments involving targeted genomic
rearrangements around the Evx2 locus have revealed some of their
properties. First, targeted deletions have shown that these enhancer sequences
are located at a remote position, upstream the Hoxd complex
(Kondo and Duboule, 1999).
Second, we showed that a Hoxd9/lacZ transgene was able to respond to
the Evx2 CNS-specific enhancer sequences, whenever it was relocated
upstream the Hoxd complex, 3' to Evx2
(Kondo and Duboule, 1999
).
However, the same transgene was unable to respond similarly when placed within
the complex, even when positioned immediately next to the Evx2
promoter (van der Hoeven et al.,
1996
). These results demonstrated that the Evx2 CNS
enhancers had a weak specificity for Evx2 itself, i.e. they were able
to interact with other promoters. In addition, Hoxd promoters could
respond to such regulatory controls provided they would be relocated in the
proper genomic environment, i.e. in 3' of the Evx2
transcription unit. These observations raised the question of which mechanism
could prevent Hoxd genes to respond to these CNS enhancers, in the
wild-type context. In other words, why a promoter able to respond to a given
regulatory sequence, when placed outside the cluster, was unable to do so from
within the Hoxd complex, even when localised right next to the
Evx2 promoter.
In this set of experiments, we looked for potential sequences, located between Evx2 and the Hoxd cluster, that would be able to isolate this latter cluster from the surrounding regulatory influences. We show that an evolutionary conserved DNA stretch participates in the insulation of the cluster, as revealed by novel genomic rearrangements in this locus. However, even though this sequence was sufficient to ensure proper insulation of the cluster, additional sequences, located nearby, were also found to be involved in this process. The requirement for a combined deletion in cis of these sequences in order to bypass the insulation of the cluster, raised the possibility that some functional redundancy exists between these regulatory sequences.
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MATERIALS AND METHODS |
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Recombined lines
Besides the HoxdRXII line, all mutant lines
analysed in this work were produced via trans-allelic meiotic
recombination (TAMERE) (Hérault et
al., 1998b; Kmita et al.,
2002
). Each allele was obtained in the progeny of
`trans-loxer' animals, i.e. males hemizygous for the
Sycp1-Cre transgene and trans-heterozygous for different
Hoxd alleles carrying a loxP site at given positions within
the Hoxd cluster (indicated in
Fig. 4)
(Kmita et al., 2002
). In
particular, Hoxddel(13) animals were obtained by
combining a Hoxd allele carrying a loxP site between
Evx2 and Hoxd13 (the EvDGE3
allele) (Hérault et al.,
1996
), with an allele carrying a loxP site between
Hoxd13 and Hoxd12 (HoxdRXI)
(Hérault et al.,
1998a
). Hoxddel(13-12) and
Hoxddel(13-11) animals were obtained in a similar
way, although in these latter cases, the EvDGE3
allele was combined either with HoxdRX, in which
a loxP site had been inserted between Hoxd12 and
Hoxd11 (Beckers et Duboule,
1998
), or with HoxdRIX, containing a
loxP site between Hoxd11 and Hoxd10
(Gérard et al., 1996
).
HoxdRXII-del(13) mice were obtained in the
progeny of trans-loxer, which were trans-heterozygous for
HoxdRXII and
HoxdRXI. Finally,
HoxdRXII(del(13-12) were produced trough
trans-loxer animals trans-heterozygous for both
HoxdRXII and HoxdRX
alleles. All these novel lines of mice were selected by Southern blot analysis
using tail DNA. The frequency of TAMERE was in the range of 5-10%, as reported
previously (Hérault et al.,
1998b
).
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Whole-mount in situ hybridisation (WISH) were carried out on 11.5- and
12.5-day-old foetuses, using a standard procedure and previously described
probes (Hérault et al.,
1996; Kondo et al.,
1998
).
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RESULTS AND DISCUSSION |
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These differences in regulation between Evx2 and Hoxd13
could hardly be accounted for by the specificity of enhancer/promoter
interactions, because a Hoxd9/lacZ transgene was able to respond to
these neural enhancers when placed 3' to Evx2
(Fig. 1B; RelII). This
transgene, however, behaved as a proper Hox gene when placed between
Evx2 and Hoxd13, a position at which it failed to show
expression in rostral parts of the brain and in spinal cord
(Fig. 1B). These results
indicated that the capacity of a Hox promoter to respond to Evx2 CNS
enhancers was abrogated when this promoter was positioned within the cluster,
suggesting that a potential insulating element was present between the Re10
insertion site and Evx2 (Fig.
1; red bar). Because in birds, fish and mammals Evx2 lies
at the same relative position with respect to Hoxd13
(Sordino et al., 1996), we
anticipated that a DNA sequences that would prevent the Evx2 neural
enhancers from affecting Hox gene expression may have been conserved between
these different genomes.
Comparison between Evx2 to Hoxd13 intergenic DNA
sequences, obtained from either the murine, the chick or the zebra fish loci,
revealed only two stretches of high sequence similarity localised between
Evx2 and the Re10 position (Fig.
2A,B; red bar). In the mouse genome, these two motives are located
within a 1.2 kb large fragment, starting about 1 kb upstream from the first
exon of Evx2. This region of significant sequence conservation was
referred to as region XII (RXII), following previously characterised conserved
regions within the Hoxd cluster
(Renucci et al., 1992;
Beckers and Duboule, 1998
;
Gérard et al., 1996
;
Hérault et al., 1998a
).
While sequence conservation was high between murine and avian DNAs for both
motives (67% identity over 206 nucleotides), it was less conspicuous when
compared with the fish DNA, as only short stretches of sequence identity were
scored for both motives. In this latter case, however, the core of the second
motif was clearly identified in the zebra fish locus and found at the same
relative position (Fig. 2A,B).
This unambiguously demonstrated the existence, in the zebra fish locus, of at
least one of these two blocks of homologies.
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In order to assess the function of these two conserved sequences, we
deleted them from their native genomic context by homologous recombination in
ES cells. We constructed a targeting vector containing the Evx2 to
Hoxd13 intergenic region, but in which the 1.2 kb fragment had been
deleted (Fig. 2C). After
electroporation in ES cells, clones carrying a targeted deletion of RXII were
selected and further injected into mouse blastocysts. After germline
transmission, the HoxdRXII-neo line of mice was
established. In order to prevent regulatory interferences caused by the
presence of the PGK-neomycine selection cassette,
HoxdRXII-neo animals were crossed with transgenic
mice producing the Cre recombinase (CMV-Cre mice)
(Dupe et al., 1997) to delete
the selection cassette. Therefore, the final genomic configuration of these
HoxdRXII mice was a single deletion of the 1.2 kb
fragment containing RXII (Fig.
2C), along with the presence of a loxP site. We
subsequently obtained HoxdRXII homozygous mice,
which were fully viable and fertile.
The expression of several Hoxd genes was examined at various developmental stages, in animals homozygous for the deletion of RXII, but no detectable difference was scored when compared with their wild type or heterozygous littermates. In particular, ectopic expression of Hoxd genes showing an Evx2 related CNS pattern was not observed. This result suggested that the deleted 1.2Kb DNA fragment was not able, on its own, to function as a boundary-like or insulator element, to isolate the Hoxd cluster from the upstream located Evx2 CNS enhancers. Alternatively, the apparent lack of effect of this deletion may illustrate some redundancy in this regulatory process.
Nested deficiencies of the 5' Hoxd cluster
Within the 5' part of the Hoxd cluster, several regions of
high interspecies conservation were previously identified
(Fig. 3) (RVIII to RXI). Each
individual region was assayed for potential regulatory function through
targeted deletion/mutation (Gérard
et al., 1996; Zakany et al.,
1997b
; Beckers and Doboule, 1998;
Hérault et al., 1998a
).
Although slight variations in Hoxd gene expression were occasionally
observed following these targeted modifications, none of them indicated a
potential role for these regions, by themselves, to restrict the accessibility
of Hoxd promoters to the Evx2 cis-regulatory sequences. In
order to look for their possible cooperation in the implementation of an
insulting process, we used the targeted meiotic recombination (TAMERE)
strategy (Hérault et al.,
1998b
) to generate novel genomic configurations in vivo through
Cre-mediated meiotic recombination between loxP sites carried in
trans by homologous chromosomes. In this way, we produced a set of
progressive deletions of the 5' end of the Hoxd complex,
involving one, two or three gene loci, as well as RXI, RX and RIX/RVIII,
respectively (see Kmita et al.,
2002
).
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First, we generated mice containing the SYCP-Cre transgene
(Vidal et al., 1998), along
with a Hoxd complex carrying, on one chromosome, a loxP site
positioned in the middle of the Evx2 to Hoxd13 intergenic
region. The other chromosome had a loxP site recombined either
upstream Hoxd12, between Hoxd12 and Hoxd11, or
upstream Hoxd10. During meiotic prophase, in some male germ cells,
recombination occurred between these loxP sites in trans,
leading to unequal chromosomal exchanges, thereby producing sperms carrying a
deletion of the DNA fragment located in between. In this way, mice were
produced which carried different deletions; a 12 kb large DNA fragment
covering the Hoxd13 locus (Hoxddel(13)
in (Fig. 3); a 18 kb large
fragment covering both Hoxd13 and Hoxd12 loci
(Hoxddel(13-12)), and a 23 kb large fragment
encompassing all three Hoxd13, Hoxd12 and Hoxd11 loci
(Hoxddel(13-11) in
Fig. 3). The same 5'
break point was used to engineer all three deletions, such that increasingly
large deletions concomitantly removed either one (RXI), two (RXI and RX) or
four (RXI, RX, RIX and RVIII) conserved sequences, respectively
(Fig. 3) (Kmita et al., 2002
).
Homozygous embryos were collected for each configuration and the expression
patterns of the remaining 5' Hoxd genes were examined by
whole-mount in situ hybridisation. Again, Evx2-like expression in the
CNS was not detected in any of these configurations (data not shown). This
suggested that sequences responsible, either alone or in combination, for the
insulation of the Hoxd cluster were not exclusively located within
these 23 kb large DNA fragment containing the Hoxd13 to
Hoxd11 loci, if at all present in this fragment.
Combining deletions in cis
This set of data demonstrated that none of the engineered deletions that
removed unique evolutionary conserved sequences had an effect on the
insulation of the Hoxd cluster. It also showed that larger deletions,
i.e. those that removed more than one such sequence from the cluster, were
equally ineffective in altering this particular mechanism. One remaining
possibility that could account for the insulation effect was the presence of
an element located between the Re10 site and Evx2
(Fig. 2; red bar), but outside
the 1.2 kb large fragment that contains region XII, as deletion of this
fragment had no effect. An alternative explanation is that the combined effect
of region XII and other regions included in the series of deletions described
above are responsible. We did not favour the first possibility, assuming that
such a tight mechanism, present in many vertebrate species, may likely rely
upon some sequence specificity. Therefore, we challenged the second
possibility by producing multiple deletions in cis.
We used HoxdRXII as a parental allele in targeted meiotic recombination, to engineer novel genetic configurations in which the RXII deletion was combined in cis with larger deletions (Fig. 3). This was made possible by the strategy that was used to delete region XII, which involved the positioning of a selection cassette flanked by loxP sites, within the Re10 insertion site, i.e. in the middle of the Evx2 to Hoxd13 intergenic region (Fig. 2C). Consequently, mice carrying the deletion of RXII had a loxP site at this position (Fig. 2C; HoxdRXII), as a left over of the Cre-mediated deletion of the PGKneo selection cassette. We first produced males carrying either the HoxdRXII and HoxdRXI alleles (Fig. 4B), or the HoxdRXII and HoxdRX alleles (Fig. 4C), along with the Cre. In the progeny of these trans-loxer males, we isolated both HoxdRXII-del(13) and HoxdRXII-del(13-12) animals, respectively (Fig. 4). Although a strain of HoxdRXII-del(13) homozygous mice could be established, HoxdRXII-del(13-12) animals died at birth. Homozygous embryos of both genotypes could nevertheless be collected to look at the expression of the remaining 5' Hoxd genes. We first analysed the expression of Hoxd12, Hoxd11 and Hoxd10 in the HoxdRXII-del(13) strain, i.e. mice that lack both region XII and the Hoxd13 locus. In these animals, ectopic activation of Hoxd genes was not detected within the rostral brain or in the spinal cord (not shown), as one would have anticipated from an alteration of the insulating process.
We next looked at the deletion of both RXII and the 18 kb large fragment containing the Hoxd13 and Hoxd12 loci (Fig. 5). In marked contrast to the previous configuration, a robust ectopic expression of both Hoxd11 and Hoxd10 in the anterior CNS was detected in embryos carrying these two deletions in cis. Ectopic expression of Hoxd11 and Hoxd10 was scored in anterior neural tube, in a subset of cells located dorsally (arrow), as well as in the developing hindbrain, an expression pattern clearly reminiscent of that seen for Evx2 (Fig. 5). Hoxd11 and Hoxd10 transcripts were also detected in the isthmus and in specific domains within the mesencephalon, where Evx2 is also normally detected. Although the complete Evx2 neural pattern was not entirely recapitulated by either Hoxd11 or Hoxd10, the observed gain of expression encompassed several domains that were previously defined as specific for Evx2. Ectopic expression was also observed in heterozygous embryos with a weaker staining intensity, as expected if only one copy of each gene has been activated. From these results, we concluded that the insulation of the Hoxd complex from the Evx2 regulatory influence, in a large subset of CNS cells, was achieved as a result of the presence of two DNA fragments, one of them being RXII, the other(s) lying around the Hoxd12 locus.
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The fact that the deletion of both Hoxd13 and Hoxd12 loci
did not induce expression of Hoxd11 in the Evx2 CNS domains,
indicated that RXII, which is located in the immediate neighbourhood of the
Evx2 start site, was able by itself to mediate such an insulation.
Interestingly, in Drosophila, a GAGA-dependent enhancer blocking
activity was identified within the promoter region of the orthologous gene
even-skipped (eve), and this activity was shown to prevent
5' located genes to respond to 3' located enhancers
(Ohtsuki and Levine, 1998).
Thus, in both organisms, an enhancer blocking activity was found associated
with the eve/Evx2 locus. Whether or not this observation has a
phylogenetic meaning, rather than being a mere coincidence, remains to be
established. In any case, the underlying molecular mechanisms are likely to be
distinct, as RXII does not seem to contain any GAGA-binding site.
The morphological effect of expressing Hoxd genes in the developing anterior CNS, and hence the biological relevance of this insulation, was difficult to assess as HoxdRXII;del(13-12) homozygous specimens died at birth. However, this lethality may not be directly associated to the abrogation of insulation, as neonatal death was also observed for Hoxddel(13-12) homozygous animals, i.e. animals that carried a wild-type RXII and, consequently, did not express Hoxd genes in anterior CNS. In this latter configuration, the deletion of both Hoxd13 and Hoxd12 induced the mis-expression of other Hoxd genes in a variety of embryonic structures, which may have caused lethality (data not shown). Consequently, it is as yet unclear whether such an insulator activity is required to prevent one particular gene to be expressed in developing CNS, or alternatively, if all posterior Hoxd genes would be equally detrimental when expressed there. To precisely assess the biological relevance of this insulation mechanism, specific gain of expression of 5' Hoxd genes, using conventional transgenic approaches, will be necessary.
Specificity of the insulation
The presence, at one extremity of a Hox gene cluster, of sequences with
insulating potential suggests a general requirement for isolating these
chromosomal loci from their surrounding genomic contexts. Interestingly,
various gene complexes seem to implement different mechanisms to protect
themselves from regulatory interferences
(Bell et al., 2001). For
example, the ß-globin gene complex, which shows some analogies with Hox
clusters in its functional organisation, is flanked by sequences carrying
properties of insulators (Bell et al.,
1999
; Saitoh et al.,
2000
). These latter sequences were proposed to prevent crosstalk
between ß-globin regulation, on the one hand, and unrelated regulatory
influences emanating from closely located genes, such as those encoding
odorant receptors, on the other (Bulger et
al., 1999
; Prioleau et al.,
1999
). This insulating potential was tightly associated with the
5' HS4 and the 3' HS DNAse I hypersensitive sites
(Bell et al., 1999
;
Saitoh et al., 2000
). These
sites were identified in all cell types and tissues examined, suggesting that
insulation of this gene complex is a rather generic mechanism with little cell
specificity. By contrast, the insulating activity described in this paper,
which prevents Hox genes from responding to upstream located CNS enhancers,
was ineffective in a different cellular context. Indeed, the same series of
genes was able to respond to another remote enhancer sequence, also located
upstream the cluster, which controls Hoxd gene expression in
developing digits (Spitz et al.,
2001
). This indicates that insulation of the Hoxd cluster
is tissue-specific; it is effective in CNS cells, but not in limb mesenchymal
cells (Kmita et al.,
2002
).
In the Drosophila Bithorax complex (BX-C), the gene
orthologous to mammalian 5' Hoxd genes (AbdB) is
controlled, in defined parasegments, by a series of regulatory elements
(Boulet et al., 1991;
Celniker et al., 1990
;
Sanchez-Herrero, 1991
). Such
sequences (Iab genes) are often flanked by frontabdominal elements (Fab
genes), which display insulating or boundary properties. Fab sequences are
essential for proper parasegmental identity as they prevent crosstalk between
distinct Iab (Barges et al.,
2000
; Mihaly et al.,
1997
; Zhou et al.,
1999
). Instead, in the vertebrate Hoxd complex, the
insulating activity may rather reflect a general, complex-wide protection
against anterior CNS regulation, rather than a way to implement properly a
regulatory circuitry in space and time, as is the case for
Drosophila. Therefore, it is unlikely that the mechanisms involved in
these two processes serve identical purposes. It is nonetheless possible that
RXII, as do Fab8 and the promoter targeting sequences (PTS)
identified adjacent to it (Zhou and
Levine, 1999
), contains both insulating and `enhancer positioning'
activities. Indeed, the bipartite RXII element was also shown to be involved
in the mechanism that triggers preferential interaction between the digit
enhancer and the most 5' Hoxd gene (Kmita et al., 2002a).
Therefore, the digit enhancer may have a `positioning activity', which might
help to bypass the RXII blocking activity in limbs, in a way related to the
PTS element which was shown to allow distal enhancers to overcome the
Fab8 insulation activity (Zhou
and Levine, 1999
). This capacity of the digit enhancer to overcome
the effect of RXII may not be shared by neural enhancers which, as a
consequence, would not be capable of bypassing RXII in CNS cells.
Regulatory redundancy
We show that only a combined deletion of both region XII and an 18 kb piece
of the cluster would lead to ectopic transcription of both Hoxd11 and
Hoxd10 in CNS. This observation suggests that the DNA fragment that
is able, along with RXII, to insulate the Hoxd complex lies around
the Hoxd12 transcription unit. Two DNA fragments were shown to
display significant interspecies sequence conservation within this interval;
regions XI and X (Beckers and Duboule,
1998; Hérault et al.,
1998a
). A role for RXI in insulation is unlikely as: (1) it has no
counterpart in the fish genome
(Hérault et al.,
1998a
); and (2) its deletion together with RXII in
HoxdRXII;del(13) animals had no apparent effect.
Therefore, region X appears as the best candidate element to mediate this
activity at the Hoxd12 locus. However, its inactivation in vivo,
through targeted deletion, had no detectable effect upon 5'
Hoxd gene regulation, similar to the case of RXII. This unexpected
observation was tentatively explained by the existence of redundant regulatory
processes (Beckers and Duboule,
1998
).
Regulatory redundancy is a difficult concept to accommodate with our current views of gene regulation. However, if we assume that both regions have insulating potentials, we may understand redundancy as a property associated with one particular cellular context. For example, in order to be functional in a given cell type, RXII may require factors partially specific for this cell type, to properly insulate the cluster. Likewise, in another cell type, RX may recruit a different set of factors to insulate the cluster from the influence of a different enhancer. In the case where both sets of factors would be present in CNS cells, both insulation processes would operate, hence only multiple deletions in cis would reveal this mechanism. Accordingly, the evolution and stability of either one of these two regions might have been driven separately, in different contexts, to become redundant in CNS cells. In this scheme, the question nevertheless remains as to why single deletions have no visible effect, at least in the original context wherein a given element is specifically required? Such tissues or organs might simply have been overlooked; they may, for example, involve vertebrate specific functions (rather recent evolutionary features), the alteration of which may have as yet escaped our attention.
The Hoxd cluster has been thoroughly investigated, in vivo, for
the functional relevance of evolutionary conserved DNA sequences. In its most
posterior part, i.e. between Hoxd10 and Evx2, five stretches
of non-coding sequences were found significantly conserved amongst
vertebrates. Using targeted approaches in ES cells, all five sequences were
either deleted, mutagenised and/or exchanged for an orthologous sequence
(Gérard et al., 1996;
Beckers and Duboule, 1998
;
Hérault et al., 1998a
)
(this work). Interestingly, although in some cases, slight variations in the
expression of the neighbouring genes were scored, none of these drastic
genetic modifications led to major regulatory alterations, a counter intuitive
observation that is at odd with current speculations regarding sequence
conservation outside coding sequences. The results presented in this paper may
shed some lights on this puzzling issue, as they suggest that such sequences
might relate to high order regulatory processes, rather than to gene-specific
cis-acting controls.
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ACKNOWLEDGMENTS |
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