1 Section of Gene Function and Regulation, The Institute of Cancer Research,
Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK
2 Division of Eukaryotic Molecular Genetics, MRC National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
* Present address: Department of Developmental Neurobiology, King's College
London, Guy's Campus, London Bridge, London SE1 1UL, UK
Author for correspondence (e-mail:
Peter.Rigby{at}icr.ac.uk)
Accepted 5 March 2003
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SUMMARY |
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Key words: Hoxb4, Paraxial mesoderm, Anterior boundary, Maintenance, Transcription, RNA stability, Translation, Mouse
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INTRODUCTION |
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In flies, the establishment and maintenance of expression domains are
mechanistically distinct events. These domains are initially defined by the
regulatory cascade of gap, pair-rule and segmentation genes that also
determines the segmental structure of the embryo
(Jack and McGinnis, 1990).
However, the segmentation genes are only transiently expressed during early
embryonic development, and Hox gene domains are subsequently refined and
maintained by auto- and cross-regulatory interactions between these genes
(Miller et al., 2001
).
Moreover, genes of the Polycomb (Pc) and trithorax (trx) families are required
for the maintenance of transcriptionally silent or active states of Hox genes,
respectively (Kennison, 1995
).
The precise function of the products of Pc and trx genes has not yet been
elucidated but growing evidence indicates that they are involved in modifying
chromatin structure to maintain transcriptionally repressive or permissive
environments (Petruk et al.,
2001
; Tie et al.,
2001
).
Studies of the regulation of murine anterior Hox genes using
randomly-integrated transgenes have revealed distinct activation and
maintenance phases similar to those in Drosophila. One of the best
characterised examples is that of Hoxb1. This gene is initially
expressed in the neural tube with an anterior limit at the boundary between
rhombomeres 3 and 4. Subsequently, expression regresses and is lost from the
hindbrain, with the exception of rhombomere 4 (r4) in which high levels are
maintained. Early neural expression of Hoxb1 is controlled by
retinoic acid through a response element located 3' to the gene
(Marshall et al., 1994;
Studer et al., 1998
), whereas
maintenance of r4 expression is dependent on auto-regulation and
cross-regulation by Hoxa1, in association with the cofactor Pbx1
(Pöpperl et al., 1995
;
Studer et al., 1998
). Separate
early and late phases of neural expression have been identified for several
other Hox genes that have anterior boundaries in the hindbrain
(Gould et al., 1997
;
Gould et al., 1998
;
Maconochie et al., 1997
;
Manzanares et al., 2001
), but
little is yet known about whether equivalent phases of expression occur in
other tissues. Although many transgenic studies of more posterior Hox genes
have been performed, there are currently few examples of separate enhancers
that control early and late phases of expression. Such elements have been
identified for Hoxc8 (Bradshaw et
al., 1996
), although the molecular details of activation and
maintenance have not yet been elucidated. Interestingly, Oosterveen et al.
have recently identified a single retinoic acid response element that controls
the late phase of expression of several genes (Hoxb5, Hoxb6 and
Hoxb8) in the posterior hindbrain
(Oosterveen et al., 2003
).
Many murine homologues of Pc and trx group genes have now been identified
and shown to be involved in the regulation of Hox genes
(Gould, 1997). For example, in
mice lacking the trx group gene Mll, endogenous Hoxa7
expression is established normally but is not maintained
(Yu et al., 1998
). Conversely,
double mutation of the Pc group genes Mel18 and Bmi1 leads
to de-repression of several Hox genes in anterior regions of the embryo
(Akasaka et al., 2001
).
Interestingly, some murine Pc group genes may regulate Hox genes earlier in
development. Early activation of Hoxd11 transcription is observed in
mice lacking the Pc group gene M33, although expression is apparently
normal from 9.5 dpc onwards (Bel-Vialar et
al., 2000
). Hoxd4 and Hoxd10 are similarly
affected when the global repression of the Hoxd cluster is disrupted by
targeted genomic deletions (Kondo and
Duboule, 1999
). Thus, some Pc group genes may regulate the timing
of Hox gene activation and contribute to the generation of co-linearity.
Sequences located within a 7.4 kb genomic fragment including Hoxb4
are sufficient to recapitulate the full expression pattern of this gene in
transgenic mice (Whiting et al.,
1991). We have shown previously that the intronic enhancer (region
C) is sufficient to establish transgene expression in the paraxial mesoderm
with an anterior boundary equivalent to that of Hoxb4 but that it
cannot maintain this pattern (Gilthorpe et
al., 2002
). We have now characterised the loss of expression more
fully and show that it proceeds by a gradual regression of the anterior
boundary. We demonstrate that sequences within the Hoxb4 promoter are
necessary for continuation of the expression established by region C, and that
regulatory elements in the 3' untranslated region (UTR) of
Hoxb4 (region B) are required to maintain the correct anterior
boundary in the paraxial mesoderm throughout embryonic development. We show
that the domain of Hoxb4 expression is restricted by regulating
transcript stability in the paraxial mesoderm, and by selective translation
and/or degradation of protein in the neural tube. Furthermore, we demonstrate
that inappropriate expression of some Hoxb4 transgenes in posterior
somites is a result of escape from post-transcriptional regulation and that
this is attributable to the absence of Hoxb4 3'-UTR sequences
from transgene transcripts.
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MATERIALS AND METHODS |
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Whole-mount in situ hybridisation and immunostaining
The Hoxb4 probe was provided by R. Krumlauf (NIMR, Mill Hill). The
lacZ probe has been described elsewhere
(Teboul et al., 2002). In situ
hybridisation was performed using an InsituPro robot (Intavis,
Bergisch-Gladbach, Germany) essentially as previously described
(Summerbell et al., 2000
), but
substituting Red-Phos (Research Organics, Cleveland, Ohio) for BCIP in the
staining solution. The anti-Hoxb4 antibody was provided by A. Gould (NIMR,
Mill Hill), and immunostaining was performed as previously described
(Gould et al., 1997
). For
sectioning, embryos were embedded in 2% (w/v) agarose. 70 µm sections were
cut using a vibrotome.
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RESULTS |
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We cloned region C upstream of b4Z to produce construct Cb4Z
(Fig. 1B), and analysed
expression in transgenic mice. At 10.5 dpc, Cb4Z was expressed in more
anterior somites than CHZ, as expected
(Fig. 3C-F). However,
comparison with the distribution of the Hoxb4 protein (Fig.
3A,B)
indicated that Cb4Z did not specify the correct boundary of expression in the
somites at this stage of development. Moreover, a useful internal control
provided by staining of the dorsal root ganglia (drg) confirmed this. The
second drg (drg2) is identifiable at 10.5 dpc by its characteristic bipartite
structure and by the fact that it is the most anterior drg visible, as drg1
degenerates to form a bar-like structure
(Spörle and Schughart,
1997). The spinal nerve originating from drg2 passes through the
rostral half of the sclerotome of so7
(Spörle and Schughart,
1997
). It was clear that the spinal nerve emanating from drg2 did
not pass through a somite in which Cb4Z was expressed (Fig.
3E,F).
However, the next somite caudally was expressing lacZ, although
weakly, demonstrating that Cb4Z specifies a boundary of expression in the
paraxial mesoderm at so7/8 at this stage. This pattern was seen in all
F0 embryos in which the transgene was expressed, although
expression was often weak, not only in so8 but throughout the cervical region.
Expression in the neural tube was also more anterior with Cb4Z than CHZ
(Fig. 3C-F). The position of
the neural boundary relative to drg2 indicates that Cb4Z was expressed up to
the boundary of the spinal cord and hindbrain. Therefore, Cb4Z had maintained
the neural boundary of expression that is established by region C at 8.5
dpc.
|
Region B is required for maintenance of the somitic boundary
From these experiments it is clear that the Cb4Z construct lacks regulatory
elements required to fully recapitulate the somitic expression of
Hoxb4. All the enhancer elements necessary to recapitulate the full
expression pattern of Hoxb4 lie 3' of its transcription start
sites (Fig. 1A)
(Whiting et al., 1991). Region
A controls the proper boundary of expression in the neural tube and does not
specify any mesodermal expression. Although only lung-specific enhancer
activity has previously been ascribed to region B, constructs containing
regions C and B are able to specify the so6/7 boundary
(Whiting et al., 1991
).
Therefore, we cloned a DNA fragment consisting of regions C and B upstream of
the b4Z reporter gene to test whether region B could alter the observed
expression pattern (construct CBb4Z; Fig.
1B). At 10.5 dpc, expression of CBb4Z clearly extended one somite
more rostrally than that of Cb4Z or b4ZC (Fig.
3I,J).
Moreover, drg2 is again easily identifiable, and the nerve from drg2 was
clearly passing through the most rostral-stained somite, identifying it as
so7. This pattern was observed in all embryos expressing the CBb4Z construct,
and also in a single embryo carrying a construct in which regions C and B were
cloned 3' of the Hoxb4 promoter-lacZ reporter
(Fig. 1B; data not shown).
Interestingly, the anterior limit of neural tube expression was identical with
Cb4Z and CBb4Z (Fig.
3E,F,I,J),
which indicates that region B is involved in regulating mesodermal but not
neural expression. In addition, both constructs gave staining in the dorsal
midbrain that is attributable to ectopic activity of the Hoxb4
promoter (Whiting et al.,
1991
).
To determine whether region B can function as an independent enhancer, a construct containing only region B cloned 5' to the b4Z reporter gene was analysed in transgenic mice. Embryos were examined between 9.5 and 11.5 dpc but only random expression was observed (n=3; data not shown). This is consistent with the results of previous attempts to identify a specific enhancer function of region B in isolation (R. Krumlauf, personal communication). Therefore, we conclude that sequences within region B represent a component of the paraxial mesoderm enhancer of Hoxb4 that is functionally dependent on elements located within region C. This interaction is necessary for expression in so7 at 10.5 dpc.
Region B is required from 9.0 dpc onwards
As construct CHZ is able to establish the so6/7 boundary at 8.5 dpc, we
reasoned that Cb4Z and CBb4Z would also recapitulate this pattern and that the
single somite difference seen at 10.5 dpc must arise between these two time
points. As expected, both Cb4Z and CBb4Z were expressed with rostral limits at
so6/7 in the paraxial mesoderm, and at the hindbrain/spinal cord boundary in
the neural tube at 8.5 dpc (Fig.
4A,B).
These boundaries were maintained with both constructs until 9.0 dpc (Fig.
4C,D).
However, by 9.5 dpc the boundary in the paraxial mesoderm had shifted one
somite caudally with Cb4Z, compared with CBb4Z, although the anterior limit of
neural expression of both constructs remained identical (Fig.
4E,F).
To confirm this subtle shift in the expression boundary, transgenic embryos
previously stained for ß-galactosidase activity were treated with the
cytoplasmic stain acid fuchsin in order to visualise somites in which reporter
genes were not expressed. At 9.0 dpc there was a two-somite gap between the
limits of reporter gene expression in the paraxial mesoderm and neural tube
for both Cb4Z and CBb4Z (Fig.
4G,H).
However, at 9.5 dpc there was a clear three-somite difference between the
mesodermal and neural boundaries of Cb4Z expression
(Fig. 4I), whereas the gap
remained two somites for CBb4Z (Fig.
4J).
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Differential downregulation of reporter genes during later embryonic
development
We examined transgenic mice carrying reporter constructs CHZ, Cb4Z and
CBb4Z during later stages of development, and observed further changes in
expression patterns, notably two distinct events of downregulation. First,
expression of all three constructs in the posterior mesoderm was downregulated
between 11.0 and 12.5 dpc (Fig.
5A-I). For Cb4Z and CBb4Z this resulted in restriction of
expression to the cervical region of the embryo, although the posterior
boundary was not sharply defined for either construct (Fig.
5H,I).
By contrast, expression of CHZ was lost from the cervical somites between 8.5
and 9.5 dpc (Fig. 2) and,
therefore, downregulation in the posterior region at this stage completely
eliminated mesodermal expression of this construct
(Fig. 5G).
|
CBb4Z was also expressed in other tissues in which both Cb4Z and CHZ were
not. By 12.5 dpc, expression was visible in the follicles of the vibrissae in
the snout and in the primordia of the mammary glands
(Fig. 5I). 24 hours later,
additional expression was detected in the follicles of the tactile hairs of
the face (Fig. 5L). Moreover,
diffuse staining was seen in the skin throughout the trunk region
(Fig. 5L). This preceded the
expression in the dermal placodes of the pelage hair follicles (data not
shown), which has been reported previously for Hoxb4 transgenes
(Whiting et al., 1991).
Stabilisation of transcripts underlies inappropriate transgene
expression in posterior somites
Strong reporter gene expression in posterior somites was characteristic of
all the constructs used in this study (Fig.
3). However, this is not a domain of expression of either
Hoxb4 mRNA or protein (Fig.
6). Whole-mount in situ hybridisation detected Hoxb4
transcripts in so7 to so13 of 10.5 dpc embryos but not in more posterior
somites (Fig. 6A), and an
identical distribution of Hoxb4 protein was revealed by whole-mount
immunostaining (Fig. 6E). These
observations were confirmed by cutting sections of embryos
(Fig. 6I-L). In transverse
sections at the level of the forelimb bud, Hoxb4 transcripts were
detected in all tissue surrounding the neural tube with noticeably higher
expression in the dermomyotome (Fig.
6I). Hoxb4 protein was not detectable in the neural tube at this
axial level but was distributed in other tissues in a similar pattern to
transcripts, with relatively high dermomyotomal expression
(Fig. 6K). At posterior levels,
neither RNA nor protein were detected in somites (Fig.
6J,L).
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Translational regulation of Hoxb4 expression in the neural
tube
In contrast to the paraxial mesoderm, the distribution of transcripts and
protein was not identical in the neural tube. Hoxb4 transcripts were
detected throughout the neural tube from the rhombomere 6/7 boundary to the
posterior tip of the embryo, although staining was often noticeably weaker in
the interlimb region (Fig.
6A-C). By contrast, Hoxb4 protein was only detectable in the
posterior hindbrain and not in more posterior regions of the neural tube
(Fig. 6E-G). At the level of
the forelimb bud, Hoxb4 transcripts were distributed throughout the
neural tube, although expression was clearly stronger in the dorsal region
(Fig. 6I). By contrast, Hoxb4
protein was not detectable in the neural tube at this axial level
(Fig. 6K). At posterior levels,
transcripts were detected in the neural tube but not in the adjacent somites
(Fig. 6J), whereas protein was
not present in either of these tissues
(Fig. 6L). These differences in
the distribution of Hoxb4 transcripts and Hoxb4 protein were evident
from the earliest stages we examined (8.5 dpc; data not shown) indicating that
translational and/or post-translational regulation is a crucial mechanism in
determining the domain of Hoxb4 function in the neural tube. In addition,
strong staining was seen in the tailbud by in situ hybridisation but protein
was not detectable, which indicates that Hoxb4 is also regulated at the level
of translation in this region (Fig.
6D,H).
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DISCUSSION |
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However, it is important to note that the molecular mechanisms of
activation and maintenance are likely to be significantly different in the
neural tube and paraxial mesoderm. The activation of neural expression through
the ENE is directly controlled by retinoid signalling
(Gould et al., 1998). Region C
is sufficient to establish somitic expression of Hoxb4, and
presumably contains all the cis-acting regulatory elements required to respond
to the inductive signals that activate Hoxb4 expression in this
tissue. We have analysed region C in detail
(Gilthorpe et al., 2002
) and
have found no evidence for direct regulation of this enhancer by retinoid
signalling. The late phase of Hoxb4 expression in the neural tube is
controlled by autoregulation, and by crossregulation by other Hox proteins
(Gould et al., 1997
). As the
LNE is a Hox-responsive element, it is active in isolation from the ENE. By
contrast, region B does not function as an independent enhancer and apparently
requires interaction with region C to drive expression in so7. Although this
does not rule out the involvement of Hox proteins in maintaining somitic
expression of Hoxb4, the mechanism is clearly more complex than a
Hox-responsive element in region B that is equivalent to that in the LNE. In
addition, the r6/7 boundary can be maintained by the neural regulatory
elements on heterologous promoters (Gould
et al., 1997
; Gould et al.,
1998
; Whiting et al.,
1991
), whereas maintenance of the somitic boundary is dependent on
the Hoxb4 promoter (Gilthorpe et
al., 2002
).
Enhancer/promoter interactions in the regulation of
Hoxb4
We have previously shown that in the absence of interaction with specific
enhancers, the Hoxb4 promoter cannot recapitulate any aspect of the
proper expression pattern (Whiting et al.,
1991). However, we have now demonstrated that the Hoxb4
promoter is required to maintain the anterior boundaries of expression that
are established by the region C enhancer. To our knowledge, this represents
the first example of an active role for the promoter of a Hox gene in the
maintenance of expression. The failure of construct CHZ to maintain expression
that was initially established in the correct somitic domain is suggestive of
the involvement of the Trithorax group proteins. It is interesting to note
that in mice mutant for the trithorax gene Mll, Hoxa7
expression is established normally during late gastrulation but is completely
downregulated by 9.5 dpc (Yu et al.,
1998
). Consistent with this, we observe gradual regression of the
anterior boundaries of CHZ expression between 8.5 and 9.5 dpc. However, we do
not yet have any evidence for TrxG proteins interacting with the
Hoxb4 promoter. There may also be a requirement for the
Hoxb4 promoter in maintenance during late embryonic development, as
expression of the CHZ reporter is completely downregulated between 12.5 and
13.5 dpc; constructs containing the Hoxb4 promoter continue to
express strongly during this period.
The detailed characteristics of core promoters are important in determining
the specificity of enhancer/promoter interactions
(Smale, 2001). This is likely
to be especially pertinent to the tightly clustered Hox gene loci, where such
interactions must be correctly established and maintained in a complicated
regulatory environment; evidence from the Hoxb cluster supports this
assertion. The LNE in region A controls the late expression of Hoxb3,
as well as that of Hoxb4 (Gould
et al., 1997
). Similarly, a mesodermal enhancer located upstream
of Hoxb4 (Fig. 7B) can
activate expression through the Hoxb4 or the Hoxb5 promoters
(Sharpe et al., 1998
). By
contrast, neural- and limb-specific enhancers in the same DNA fragment
demonstrate a selective interaction with Hoxb4, and are seemingly
unable to activate transcription through the Hoxb5 promoter
(Sharpe et al., 1998
).
Furthermore, a separate neural enhancer in the Hoxb5-Hoxb4
intergenic region can drive expression through either promoter but, when
placed between them, interacts exclusively with Hoxb4, indicating
that the promoters of these two genes may compete for certain enhancers
(Sharpe et al., 1998
). The
details of the interactions between enhancers and promoters that determine
sharing, selectivity and competition in the Hoxb cluster have not yet been
elucidated but it is interesting to note the results of recent studies in
Drosophila. The presence or absence of certain components of the core
promoter (the TATA box, initiator and downstream promoter element) can define
the specificity of enhancer/promoter interactions
(Ohtsuki et al., 1998
;
Butler and Kadonaga, 2001
).
Although the promoters of the majority of mouse Hox genes are poorly
characterised, we have shown that the Hoxb4 promoter has an unusual
architecture (Gutman et al.,
1994
). It does not contain a TATA box but includes two initiators
located approximately 80 bp apart that determine the start sites of
alternative transcripts. It will be interesting to see how the specific
characteristics of this and other promoters in the Hoxb cluster contribute to
the proper spatiotemporal regulation of these genes
Regulatory organisation of PG-4 Hox genes
Murine Hox genes of paralogous group 4 (PG-4) have regulatory regions that
are organised in a broadly similar manner
(Morrison et al., 1997).
Fig. 7B summarises all the
known neural- and paraxial mesoderm-specific enhancers located close to the
Hoxa4, Hoxb4 and Hoxd4 genes, and incorporates the results
of this study. Enhancers located 3' of Hoxa4 and Hoxd4
direct somitic expression with appropriate anterior boundaries for each gene.
We have now demonstrated that sequences 3' of Hoxb4 are
similarly required for proper expression of this gene in the paraxial
mesoderm. However, region B has a restricted role in maintaining the anterior
boundary and is dependent on sequences within region C to drive somitic
expression (this study). By contrast, the 3' enhancers of both
Hoxa4 and Hoxd4 can function as regulatory regions
independently (Morrison et al.,
1997
). Interestingly, there may be some interaction between the
intron and the 5' region of Hoxa4. A transgene containing these
sequences is expressed in the paraxial mesoderm with a boundary equivalent to
that of Hoxa4 and deletion of a 2 kb fragment from the 5'
region abolishes reporter gene expression
(Behringer et al., 1993
). Using
the same parent construct, mutation of Hox binding sites in the intron
eliminates expression in the paraxial mesoderm and the posterior neural tube
(Keegan et al., 1997
). Thus,
both the upstream region and the intron would seem to be required. However,
the ability of either to function as an independent enhancer has not yet been
tested, and the relative contribution of these regions and the 3'
enhancer to the establishment and/or maintenance of somitic expression remains
unclear.
As noted above, the enhancer 5' of Hoxb4 can function
independently and can interact with the promoter of either of the neighbouring
genes (Sharpe et al., 1998).
Although this region specifies an anterior somitic boundary characteristic of
Hoxb5, this does not rule out involvement in the regulation of
Hoxb4, a hypothesis supported by comparison to Hoxd4.
Similar to Hoxb4, the 5' mesodermal enhancer of Hoxd4
directs expression with an anterior boundary caudal to that of the endogenous
gene (Zhang et al., 1997
). The
nearest gene 5' to Hoxd4 is Hoxd8, the somitic
expression of which has an anterior boundary in the lower thoracic region
(Izpisùa-Belmonte et al.,
1990
), and it is thus unlikely to be regulated by the enhancer
5' of Hoxd4. Therefore, the assumption is that this enhancer
does regulate Hoxd4 expression. Thus it seems likely that regulation
of PG-4 Hox genes involves integration of inputs from upstream and downstream
elements, and this arrangement presumably serves to determine appropriate
levels of expression. Finally, no regulatory function has yet been ascribed to
the intron of Hoxd4. Deletion of the intron from transgenes based on
the mouse or human Hoxd4 genes has no effect on the observed
expression patterns, and the intron of the human gene does not function as an
independent enhancer (Morrison et al.,
1997
; Zhang et al.,
1997
). We have previously identified a conserved block of sequence
within the introns of PG-4 genes
(Gilthorpe et al., 2002
).
Interestingly, the Hoxd4 sequences showed the least identity in these
alignments. Whether this is related to the apparent reduction in the
regulatory function of the Hoxd4 intron has not yet been
determined.
It seems that the relative inputs of 5', 3' and intron
sequences to PG-4 regulation have diverged over the course of vertebrate
evolution but that the similar regulatory organisation of these genes reflects
that of an ancestral Hox4 gene. Unfortunately, details of the regulation of
Hox genes in species other than the mouse are extremely scarce. However, we
note that the regulatory function of intronic sequences is apparently not
equivalent for the Hoxb4 genes of all vertebrate species. The intron
of the chicken gene drives expression only in posterior neural and mesodermal
tissue in transgenic mice, while the equivalent region of the pufferfish
(Fugu rubripes) gene completely lacks enhancer activity in this assay
(Morrison et al., 1995). This
is in marked contrast to the 3' neural enhancer (region A), as similar
fragments of the chicken and pufferfish genes are able to recapitulate the
r6/7 boundary in transgenic mice (Aparicio
et al., 1995
; Morrison et al.,
1995
). It is an intriguing possibility that the chicken and
pufferfish genes have retained a more robust mesodermal enhancer function in
3' regions that is characteristic of the ancestral condition, and that
the mouse gene has evolved to rely more heavily on sequences within the
intron. Further analysis of the regulatory regions of PG-4 genes from these
and other vertebrate species should provide valuable insights into the
evolution of regulatory organisation in the Hox clusters, and whether or not
this can be correlated with changes in expression boundaries.
Regulation of transcript stability in the paraxial mesoderm
In the paraxial mesoderm, the distribution of Hoxb4 transcripts is
restricted to so7-13, and protein is produced wherever transcripts are
present. By contrast, all of the constructs used in this study are expressed
in somites posterior to so13, at both the transcript and the protein level.
Comparison with other Hoxb4 transgenes
(Whiting et al., 1991),
indicates that the 3' UTR of Hoxb4 is required to destabilise
transcripts in the posterior somitic domain and thus restrict Hoxb4
expression to so7-13. Therefore, although other possibilities exist, we feel
it is likely that Hoxb4 is transcribed in all somites posterior to
the so6/7 boundary, and that post-transcriptional regulation determines the
posterior boundary and thus the definitive domain of Hoxb4
expression. Further complexity in the regulation of Hoxb4 is revealed
by the downregulation of constructs CHZ, Cb4Z and CBb4Z in the posterior
domain after 11.5 dpc. However, we do not yet know whether this late phase of
regulation involves changes in transcriptional or post-transcriptional
regulation.
Interestingly, construct CHZ uncouples the two domains of somitic expression during early development, as it maintains expression only posterior to so14 and not in the anterior definitive domain of Hoxb4 expression. We have shown that maintenance of expression in the latter domain is dependent on the Hoxb4 promoter and have attributed this to a requirement for the promoter in the maintenance of transcription. However, it is possible that sequences within this region (presumably within the 5' UTR) are necessary for the stabilisation of transcripts, and that CHZ transcripts are generated in so7-13 after 8.5 dpc but are rapidly degraded. Should this be true, it would identify contrasting roles for the 5' and 3' UTRs of Hoxb4, the former being required to stabilise transcripts and the latter to destabilise them, albeit in different domains of the paraxial mesoderm.
We have found interesting parallels between our observations on the
regulation of Hoxb4 expression and recent work on Hoxd1
(Zákány et al.,
2001). This is an unusual Hox gene in that it is not expressed in
somites but is expressed in the anterior presomitic mesoderm. This expression
occurs in pulses associated with the formation of each somite, and
Hoxd1 transcripts are rapidly excluded from somites once they have
formed. However, when a lacZ reporter was inserted into the
endogenous Hoxd1 gene, stable transcripts accumulated and were
retained in somites well after their formation. We note that in this
experiment the lacZ gene was coupled to SV40 polyA sequences, thus
removing Hoxd1 3' UTR sequences from the transcripts generated
from this locus. This correlates with our observations that Hoxb4
transgenes containing lacZ-SV40 polyA cassettes generate transcripts
that are stable in posterior somites, whereas those that contain
Hoxb4 3' UTR sequences are not. We note that many, although not
all, Hox genes have restricted domains of expression in the paraxial mesoderm,
with both anterior and posterior boundaries
(Burke et al., 1995
), and
propose the following general mechanism of Hox gene regulation in
this tissue. Specification of the anterior boundary of a given gene is
determined by the timing of transcriptional activation and is linked to the
segmentation clock that regulates somitogenesis
(Dubrelle et al., 2001
;
Zákány et al.,
2001
). Transcription then occurs in all somites posterior to this
point and the definitive posterior boundary, if any, is determined by
regulating the stability of transcripts in posterior regions through sequences
in the 3' UTR.
Translational regulation of Hoxb4 in the neural tube
We have compared the distribution of Hoxb4 transcripts and Hoxb4
protein and observed that different regulatory strategies are employed in the
neural tube and paraxial mesoderm to achieve the same end: the spatial
restriction of Hoxb4 function in the embryo. In the neural tube,
detectable levels of the protein accumulate only in an anterior subdomain of
the region in which Hoxb4 is transcribed. Although the mechanism by
which this is achieved is not yet known, we envisage two likely scenarios.
Either transcripts are selectively translated in the hindbrain and anterior
spinal cord, or Hoxb4 protein is produced throughout the neural tube and
actively and rapidly degraded in posterior regions. In Drosophila,
the homeodomain protein Bicoid (Bcd) regulates the expression of another
homeodomain protein Caudal (Cad) at the translational level. Bcd binds in a
sequence-specific manner to the 3' UTR of cad transcripts and
represses translation by interacting with proteins bound to the 5' cap
(Niessing et al., 2000;
Niessing et al., 2002
).
bcd has evolved from an ancestral PG-3 Hox gene
(Stauber et al., 1999
) and,
although no Hox proteins have yet been shown to bind RNA, it is an intriguing
possibility that translational repression of Hoxb4 is mediated by more
posteriorly expressed Hox proteins in the same manner that Bcd regulates
Cad.
Although data on the distribution of Hox proteins in the mouse embryo are
scarce, both Hoxb5 and Hoxc8 are expressed in spatially restricted domains
along the AP axis of the neural tube
(Belting et al., 1998;
Sharpe et al., 1998
;
Wall et al., 1992
). However,
for these genes the RNA is similarly localised and the regulation of
expression appears to be primarily transcriptional
(Awgulewitsch and Jacobs, 1990
;
Conlon and Rossant, 1992
).
Thus, the relative importance of transcriptional and translational control in
the neural tube may differ for individual Hox genes.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Akasaka, T., van Lohuizen, M., van der Lugt, N.,
Mizutani-Koseki, Y., Kanno, M., Taniguchi, M., Vidal, M., Alkema, M., Berns,
A. and Koseki, H. (2001). Mice doubly deficient for the
Polycomb Group genes Mel18 and Bmi1 reveal synergy and
requirement for maintenance but not activation of Hox gene expression.
Development 128,1587
-1597.
Amores, A., Force, A., Yan, Y.-L., Joly, L., Amemiya, C., Fritz,
A., Ho, R. K., Langeland, J., Prince, V., Wang, Y.-L. et al.
(1998). Zebrafish Hox clusters and vertebrate genome
evolution. Science 282,1711
-1714.
Aparicio, S., Morrison, A., Gould, A., Gilthorpe, J., Chaudhuri, C., Rigby, P., Krumlauf, R. and Brenner, S. (1995). Detecting conserved regulatory elements with the model genome of the Japanese puffer fish, Fugu rubripes. Proc. Natl. Acad. Sci. USA 92,1684 -1688.[Abstract]
Awgulewitsch, A. and Jacobs, D. (1990). Differential expression of Hox 3.1 protein in subregions of the embryonic and adult spinal cord. Development 108,411 -420.[Abstract]
Behringer, R. R., Crotty, D. A., Tennyson, V. M., Brinster, R.
L., Palmiter, R. D. and Wolgemuth, D. J. (1993). Sequences
5' of the homeobox of the Hox-1.4 gene direct tissue-specific
expression of lacZ during mouse development.
Development 117,823
-833.
Bel-Vialar, S., Coré, N., Terranova, R., Goudot, V., Boned, A. and Djabali, M. (2000). Altered retinoic acid sensitivity and temporal expression of Hox genes in Polycomb-M33-deficient mice. Dev. Biol. 224,238 -249.[CrossRef][Medline]
Belting, H.-G., Shashikant, C. S. and Ruddle, F. H. (1998). Multiple phases of expression and regulation of mouse Hoxc8 during early embryology. J. Exp. Zool. 282,196 -222.[CrossRef][Medline]
Bradshaw, M. S., Shashikant, C. S., Belting, H.-G., Bollekens,
J. A. and Ruddle, F. H. (1996). A long-range regulatory
element of Hoxc8 identified by using the pClasper vector.
Proc. Natl. Acad. Sci. USA
93,2426
-2430.
Burke, A. C., Nelson, C. E., Morgan, B. A. and Tabin, C.
(1995). Hox genes and the evolution of vertebrate axial
morphology. Development
121,333
-346.
Butler, J. E. and Kadonaga, J. T. (2001).
Enhancer-promoter specificity mediated by DPE or TATA core promoter motifs.
Genes Dev. 15,2515
-2519.
Conlon, R. A. and Rossant, J. (1992). Exogenous
retinoic acid rapidly induces anterior ectopic expression of murine
Hox-2 genes in vivo. Development
116,357
-368.
de Rosa, R., Grenier, J. K., Andreeva, T., Cook, C. E., Adoutte, A., Akam, M., Carroll, S. B. and Balavoine, G. (1999). Hox genes in brachiopods and priapulids and protostome evolution. Nature 399,772 -776.[CrossRef][Medline]
Dubrelle, J., McGrew, M. J. and Pourquié, O. (2001). FGF signalling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106,219 -232.[Medline]
Ferrier, D. E. K. and Holland, P. W. H. (2001). Ancient origin of the Hox gene cluster. Nat. Rev. Genet. 2,33 -37.[CrossRef][Medline]
Gilthorpe, J. D. and Rigby, P. W. J. (1999). Reporter genes for the study of transcriptional regulation in transgenic mouse embryos. Methods Mol. Biol. 97,159 -182.[Medline]
Gilthorpe, J., Vandromme, M., Brend, T., Gutman, A., Summerbell, D., Totty, N. and Rigby, P. W. J. (2002). Spatially specific expression of Hoxb4 is dependent on the ubiquitous transcription factor NFY. Development 129,3887 -3899.[Medline]
Gould, A. (1997). Functions of mammalian Polycomb group and trithorax group related genes. Curr. Opin. Genet. Dev. 7, 488-494.[CrossRef][Medline]
Gould, A., Morrison, A., Sproat, G., White, R. A. H. and Krumlauf, R. (1997). Positive cross-regulation and enhancer sharing: two mechanisms for specifying overlapping Hox expression patterns. Genes Dev. 11,900 -913.[Abstract]
Gould, A., Itsaki, N. and Krumlauf, R. (1998). Initiation of rhombomeric Hoxb4 expression requires induction by somites and a retinoid pathway. Neuron 21, 39-51.[Medline]
Gutman, A., Gilthorpe, J. and Rigby, P. W. J. (1994). Multiple positive and negative regulatory elements in the promoter of the mouse homeobox gene Hoxb-4. Mol. Cell. Biol. 14,8143 -8154.[Abstract]
Izpisùa-Belmonte, J. C., Dollé, P., Renucci, A., Zappavigna, V., Falkenstein, H. and Duboule, D. (1990). Primary structure and embryonic expression pattern of the mouse Hox-4.3 homeobox gene. Development 110,733 -745.[Abstract]
Jack, T. and McGinnis, W. (1990). Establishment of the Deformed expression stripe requires the combinatorial action of coordinate, gap and pair-rule proteins. EMBO J. 9,1187 -1198.[Abstract]
Keegan, L. P., Haerry, T. E., Crotty, D. A., Packer, A. I., Wolgemuth, D. J. and Gehring, W. J. (1997). A sequence conserved in vertebrate Hox gene introns functions as an enhancer regulated by posterior homeotic genes in Drosophila imaginal discs. Mech. Dev. 63,145 -157.[CrossRef][Medline]
Kennison, J. A. (1995). The Polycomb and Trithorax group proteins of Drosophila: trans-regulators of homeotic gene function. Annu. Rev. Genet. 29,289 -303.[CrossRef][Medline]
Kondo, T. and Duboule, D. (1999). Breaking colinearity in the mouse HoxD complex. Cell 97,407 -417.[Medline]
Maconochie, M. K., Nonchev, S., Studer, M., Chan, S.-K., Pöpperl, H., Sham, M. H., Mann, R. S. and Krumlauf, R. (1997). Cross-regulation in the mouse HoxB complex: the expression of Hoxb2 in rhombomere 4 is regulated by Hoxb1. Genes Dev. 11,1885 -1895.[Abstract]
Manzanares, M., Bel-Vialar, S., Ariza-McNaughton, L., Ferretti, E., Marshall, H., Maconochie, M., Blasi, F. and Krumlauf, R. (2001). Independent regulation of initiation and maintenance phases of Hoxa3 expression in the vertebrate hindbrain involve auto- and cross-regulatory mechanisms. Development 128,3595 -3607.[Medline]
Marshall, H., Studer, M., Pöpperl, H., Aparicio, S., Kuroiwa, A., Brenner, S. and Krumlauf, R. (1994). A conserved retinoic acid response element required for early expression of the homeobox gene Hoxb-1. Nature 370,567 -571.[CrossRef][Medline]
Miller, D. F. B., Rogers, B. T., Kalkbrenner, A., Hamilton, B., Holtzman, S. L. and Kaufman, T. (2001). Cross-regulation of Hox genes in the Drosophila melanogaster embryo. Mech. Dev. 102,3 -16.[CrossRef][Medline]
Morrison, A., Chaudhuri, C., Ariza-McNaughton, L., Muchamore, I., Kuroiwa, A. and Krumlauf, R. (1995). Comparative analysis of chicken Hoxb-4 regulation in transgenic mice. Mech. Dev. 53,47 -59.[CrossRef][Medline]
Morrison, A., Ariza-McNaughton, L., Gould, A., Featherstone, M.
and Krumlauf, R. (1997). HOXD4 and regulation of the
group 4 paralog genes. Development
124,3135
-3146.
Niessing, D., Driever, W., Sprenger, F., Taubert, H., Jäckle, H. and Rivera-Pomar, R. (2000). Homeodomain position 54 specifies transcriptional versus translational control by Bicoid. Mol. Cell 5,395 -401.[Medline]
Niessing, D., Blanke, S. and Jäckle, H.
(2002). Bicoid associates with the 5'-cap bound complex of
caudal mRNA and represses translation. Genes
Dev. 16,2576
-2582.
Ohtsuki, S., Levine, M. and Cai, H. N. (1998).
Different core promoters possess distinct regulatory activities in the
Drosophila embryo. Genes Dev.
12,547
-556.
Oosterveen, T., Niederreither, K., Dollé, P., Chambon,
P., Meijlink, F. and Deschamps, J. (2003). Retinoids regulate
the anterior expression boundaries of 5' Hoxb genes in
posterior hindbrain. EMBO J.
22,262
-269.
Petruk, S., Sedkov, Y., Smith, S., Tillib, S., Kraevski, V.,
Nakamura, T., Canaani, E., Croce, C. M. and Mazo, A. (2001).
Trithorax and dCBP acting in a complex to maintain expression of a homeotic
gene. Science 294,1331
-1334.
Pöpperl, H., Bienz, M., Studer, M., Chan, S.-K., Aparicio, S., Brenner, S., Mann, R. S. and Krumlauf, R. (1995). Segmental expression of Hoxb-1 is controlled by a highly conserved autoregulatory loop dependent upon exd/pbx. Cell 81,1031 -1042.[Medline]
Sharpe, J., Nonchev, S., Gould, A., Whiting, J. and Krumlauf,
R. (1998). Selectivity, sharing and competitive interactions
in the regulation of Hoxb genes. EMBO J.
17,1788
-1798.
Smale, S. T. (2001). Core promoters: active
contributors to combinatorial gene regulation. Genes
Dev. 15,2503
-2508.
Spörle, R. and Schughart, K. (1997). System to identify individual somites and their derivatives in the developing mouse embryo. Dev. Dyn. 210,216 -226.[CrossRef][Medline]
Stauber, M., Jäckle, H. and Schmidt-Ott, U.
(1999). The anterior determinant bicoid of
Drosophila is a derived Hox class 3 gene. Proc.
Natl. Acad. Sci. USA 96,3786
-3789.
Studer, M., Gavalas, A., Marshall, H., Ariza-McNaughton, L.,
Rijli, F. M., Chambon, P. and Krumlauf, R. (1998). Genetic
interactions between Hoxa1 and Hoxb1 reveal new roles in
regulation of early hindbrain patterning. Development
125,1025
-1036.
Summerbell, D., Ashby, P. R., Coutelle, O., Cox, D., Yee, S.-P.
and Rigby, P. W. J. (2000). The expression of Myf5
in the developing mouse embryo is controlled by discrete and dispersed
enhancers specific for particular populations of skeletal muscle precursors.
Development 127,3745
-3757.
Teboul, L., Hadchouel, J., Daubas, P., Summerbell, D.,
Buckingham, M. and Rigby, P. W. J. (2002). The early epaxial
enhancer is essential for the initial expression of the skeletal muscle
determination gene Myf5 but not for subsequent, multiple phases of
somitic myogenesis. Development
129,4571
-4580.
Tie, F., Furuyama, T., Prasad-Sinha, J., Jane, E. and Harte, P.
J. (2001). The Drosophila Polycomb Group proteins
ESC and E(Z) are present in a complex containing the histone-binding protein
p55 and the histone deactylase RPD3. Development
128,275
-286.
Wall, N. A., Jones, M. C., Hogan, B. L. M. and Wright, C. V. E. (1992). Expression and modification of Hox 2.1 protein in mouse embryos. Mech. Dev. 37,111 -120.[CrossRef][Medline]
Whiting, J., Marshall, H., Cook, M., Krumlauf, R., Rigby, P. W. J., Stott, D. and Allemann, R. K. (1991). Multiple spatially specific enhancers are required to reconstruct the pattern of Hox-2.6 gene expression. Genes Dev. 5,2048 -2059.[Abstract]
Yu, B. D., Hanson, R. D., Hess, J. L., Horning, S. E. and
Korsmeyer, S. J. (1998). MLL, a mammalian
trithorax-group gene, functions as a transcriptional maintenance
factor in morphogenesis. Proc. Natl. Acad. Sci. USA
95,10632
-10636.
Zákány, J., Kmita, M., Alarcon, P., de la Pompa, J.-L. and Duboule, D. (2001). Localized and transient transcription of Hox genes suggests a link between patterning and the segmentation clock. Cell 106,207 -217.[Medline]
Zhang, F., Popperl, H., Morrison, A., Kovacs, E. N., Prideaux, V., Schwarz, L., Krumlauf, R., Rossant, J. and Featherstone, M. S. (1997). Elements both 5' and 3' to the murine Hoxd4 gene establish anterior borders of expression in mesoderm and neuroectoderm. Mech. Dev. 67, 49-58.[CrossRef][Medline]