Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA
* Author for correspondence (e-mail: frank.ruddle{at}yale.edu)
Accepted 13 June 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Enhancer, Hoxc8, Gene regulation, Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A two-step mechanism for precise temporal control of Hox gene expression in
the initiation and establishment phases has been advanced
(Gaunt and Strachan, 1996;
Kondo et al., 1998
; Kondo and
Duboule et al., 1999; Zákány
et al., 2001
). In the first step, progressive chromatin
modification of the clusters extending from 3' to 5' potentiates
gene activity. Isolation of a cis-element that mediates temporal
activation of Hox genes is consistent with this model. This element is located
outside of the 5' terminus of the Hoxd cluster. In vivo deletion of the
element prevents the premature activation of 5' Hox genes
(Kondo et al., 1998
; Kondo and
Duboule et al., 1999). Studies on the Polycomb-group (Pc-G) family further
enhances the importance of chromatin status regarding temporal regulation of
Hox expression. The Pc-G family is involved in transcriptional regulation of
Hox genes through modification of chromatin structure
(Gould, 1997
;
Gebuhr et al., 2000
;
Simon and Tamkun, 2002
). In
the Pc-G family M33 null mutants, Hoxd11 is activated
earlier while Hoxd4 is activated by retinoic acid (RA) prematurely
(Bel-Vialar et al., 2000
).
These studies suggest that in the initiation phase, a repressive regulatory
mechanism is involved in regulating the correct temporal activation of Hox
genes by modulating chromatin structure. Therefore, Hox genes must release
sequentially in a 3' to 5' direction in order to be transcribed in
the proper temporal sequence.
In the second step, the expression of Hox genes is regulated by a specific
response to transcription signals. Recent studies showed that after chromatin
opening, transcription of Hox genes are increased in part by the segmentation
clock pathway (Dubrulle et al.,
2001; Zákány et
al., 2001
). A `segmentation stripe enhancer' has been proposed on
the basis of deletion analyses and postulated to control the temporal
expression of several Hoxd genes possibly through segmentation signals
(Zákány et al.,
2001
). Other transcriptional inducers such as RA or the Caudal
(cdx) gene family of transcription factors may also be involved in
temporal regulation of Hox expression following the initial activation stage.
Several cis-regulatory elements possessing binding motifs of
candidate transcription factors have been identified. In vivo modification of
these enhancers leads to transient expression delay of Hox genes in early
mouse embryos. Although the correct expression patterns of the corresponding
Hox genes are re-established later, the adult mice display morphological
modifications (Dupé et al.,
1997
; Zákány et
al., 1997
). These data highlight the importance of exact
expression timing of Hox genes in early development and suggest that
regulation of the spatial and temporal expression of Hox genes is mediated
through diverse cis-regulatory pathways.
We consider these hypotheses in the light of our deletion of the
Hoxc8 early enhancer. The EE extends over 200 bp and is located 3 kb
upstream of the Hoxc8 promoter. The EE is highly conserved (95%) on
the basis of nucleotide sequence comparison between human and mouse. Reporter
gene analysis shows that this element is necessary and sufficient to
reconstitute the endogenous pattern of Hoxc8 expression in ectodermal
and mesodermal derivatives (Shashikant et
al., 1995). A minimum of seven putative transcription factor
binding motifs including two CDX-binding sites have been identified within the
EE by means of sequence analysis and mutational studies involving reporter
constructs in transgenic mice (Shashikant
et al., 1995
; Shashikant and
Ruddle, 1996
). In addition, recent data show that the EE may be
involved in chromatin remodeling through histone deacetylase 3
(Bayarsaihan and Ruddle, 2000
;
Tussie-Luna et al., 2002
).
Therefore, the function and structure of the EE indicate that it is not only
important for setting up the spatial expression domain of Hoxc8, but
also crucial for regulating proper Hoxc8 temporal activation.
In order to define the timing functions of the EE more precisely, we have deleted it endogenously using stem cell gene targeting technologies. Homozygous knockout animals exhibit a number of mutant phenotypes that both resemble and differ those reported for Hoxc8-coding region knockouts. Our findings show that the EE contributes to the temporal control of Hoxc8 in the context of the endogenous genome, but is not necessary for its expression, contrary to expectations based on our earlier transgene experiments. We show that the deletion of the EE delays the expression of Hoxc8 early in development at a time coincident with somitogenesis and possibly at a time when chromatin modifications are taking place within the Hox clusters. We also show that a second domain similar to the EE in both structure and function resides in the Hoxd cluster, indicating the possible existence of a distributed system regulating the temporal expression of the Hox genes.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Skeletal preparations
Skeleton of newborn mice are stained with Alcian Blue 8GX and Alizarin Red
S as previously described (van den Akker
et al., 2001). Briefly, Newborns are skinned, eviscerated and
fixed in 96% ethanol overnight followed by cartilage staining with Alcian Blue
(0.5 mg/ml Alcian Blue in 80% ethanol/20% acetic acid) overnight. Skeletons
are rinsed twice for 1 hour in 96% ethanol and cleared in 1.5% KOH for 5
hours. The bone is then stained overnight in 0.5% KOH and 0.15 mg/ml Alizarin
Red S. Stained skeletons are cleared in 0.5% KOH/20% glycerol for 3 days or
longer and stored in 20% ethanol/20% glycerol.
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed on genotyped embryos at
7.5-11.5 dpc following standard procedures. The probes used for hybridization
are mouse genomic fragments containing part of exon I of mouse Hoxb8
(211 bp), Hoxc6 (342 bp), Hoxc8 (248 bp) and Hoxc9
(291 bp). All probes are labeled with digoxigenin using standard
procedures.
DNA sequence analysis
Regions of nucleotide sequence similarity between the EE and
Hoxd11 RVIII
(Zákány et al.,
1997) are detected by importing the sequences to MacVector
(IBI-Kodak) and aligned manually. The putative transcription factor binding
motifs within the two enhancers are identified by TFSEARCH V1.3 (Yutaka
Akiyama, Kyoto University, 1995,
http://www.cbrc.jp/research/db/TFSEARCH.html)
and MatInspector V2.2
(http://transfac.gbf.de/cgi-bin/matSearch/matsearch.pl).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It should be noted, unless otherwise stated, that we refer only to the homozygous EE deleted embryos, as the heterozygous animals are in most respects identical phenotypically to wild-type embryos (Table 1). The EE homozygotes are of two types: those carrying the neo cassette and those in which the cassette has been deleted. We refer to these animals as EEneo and EElox, respectively.
|
Whole-mount in situ hybridization was performed on 7.5 to 11.5 dpc embryos in order to compare the spatiotemporal pattern of Hoxc8 expression in wild-type and EEneo embryos. Hoxc8 activity is not observed in any embryonic or extra-embryonic tissues in both wild-type and EEneo embryos at 7.5 dpc. Expression of Hoxc8 is first detected in the tail bud region, including the allantois at 8 dpc in wild-type embryos. However, expression of Hoxc8 is not detected in EEneo embryos (Fig. 2A). At 8.5 dpc, Hoxc8 is expressed in both neural tube and paraxial mesoderm in wild-type embryos. Although the strongest expression is in the posterior region of the embryos, the expression domains in both tissues are extended to more anterior regions (Fig. 2B). In EEneo embryos, expression of Hoxc8 is detected in both neural tube and paraxial mesoderm. However, the expression domains in both tissues are restricted to the caudal region and are relatively lower in intensity when compared with wild-type embryos (Fig. 2B).
|
The expression domains of Hoxc8 in the neural tube and paraxial mesoderm are well established in wild-type embryos at 10 dpc. Hoxc8 is expressed in the neural tube between somites 9 and 15 and in the paraxial mesoderm between somites 15 and 23. This expression profile is maintained until 11.5 dpc, while the expression intensity of the paraxial mesoderm in somite 22 and 23 becomes significantly weaker at 11.5 dpc (Fig. 2D-F). In EEneo embryos, the expression level of Hoxc8 within the neural tube is normal at 10 dpc. In the paraxial mesoderm, although the anterior boundary of Hoxc8 expression is established normally at somite 15, the posterior boundary of the Hoxc8 expression domain is anteriorized to somite 21 with the strongest expression between somites 16 to 19 (Fig. 2D). This failure to form a normal posterior boundary in the paraxial mesoderm continues through 11 dpc (Fig. 2E). However, expression of Hoxc8 in the paraxial mesoderm is fully recovered at 11.5 dpc. The expression intensity within somite 22 and 23 is even stronger when compared with wild-type embryos (Fig. 2F). Overall, deletion of the Hoxc8 early enhancer leads to temporal delay of Hoxc8 expression and expression domain alteration at certain developmental stages.
EElox embryos
We removed the neo cassette from EEneo ES cells to generate a EElox mouse
line using the Cre/loxP system in order to evaluate the effect of the neo
cassette on Hoxc8 expression in EE deleted embryos. Whole-mount in
situ hybridization in EElox embryos at 7.5 dpc does not show any
Hoxc8 activity (data not shown). At 8.0 dpc, while expression of
Hoxc8 is detected in the allantois in EElox embryos, the expression
domain has not spread from the allantois to more anterior regions, as in
wild-type embryos, suggesting a temporal delay of Hoxc8 expression at
an early developmental stage in EElox embryos
(Fig. 3A). However, there is no
spatial or temporal modification of the Hoxc8 expression pattern in
EElox embryos at 8.5 dpc and 9.0 dpc, showing full recovery of normal
expression (Fig. 3B,C).
|
Growth rate and behavioral phenotypes in postnatal EEneo and EElox
mice
Adult mice heterozygous and homozygous for the EE deletion (EEneo and
EElox) are obtained in the expected Mendelian ratio and are viable, healthy
and fertile. However, double homozygous crosses of our EEneo colony result in
lower fecundity when compared with wild-type or heterozygous crosses. In
addition, about 11% of the EEneo heterozygous and 16% of the EEneo homozygous
mice show stunted growth. They are smaller than their littermates at birth,
and do not attain normal weight as adults.
Approximately 12% of the heterozygous and 30% of the homozygous adult mice
of EEneo and EElox colonies show an abnormal contraction and clasping reflex
of both the fore- and hindlimbs upon tail suspension. Adult wild-type mice
extend their limbs when suspended by their tails. Although some of the
heterozygous and homozygous mice can extend their limbs when first suspended,
within a few seconds they hug their bodies with the fore- and hindlimbs. This
phenotype resembles the neurological defect observed in cyclin D1
(Sicinski et al., 1995),
Mf3 (Labosky et al.,
1997
) and Hoxb8 (van
den Akker et al., 1999
) null mice.
Skeletal phenotypes in EEneo and EElox embryos
EEneo embryos
Expression of Hoxc8 in mesodermal derivatives of the thoracic
region suggest that Hoxc8 is involved in specifying positional
identities in this region. Indeed, ablation of Hoxc8 has been shown
to induce skeletal transformations in the trunk
(Le Mouellic et al., 1992;
van den Akker et al., 2001
).
EEneo mice show a modification in temporal display of Hoxc8
expression that also results in skeletal modifications in the thoracic region
in some respects similar to those induced by the complete loss of
Hoxc8 gene expression. In the upper lumbar region, all the EEneo mice
show either a rudimentary rib or a fully developed pair of ribs on L1,
suggesting L1 is transformed anteriorly into T13
(Fig. 4A,B). Anterior
transformations in the thoracic vertebral column are also observed. In the rib
cage, about 93% of the EEneo mice develop an extra sternebra between T6 and T7
(Fig. 4D). In addition, eight
pairs of ribs attach to the sternum instead of seven in 71% of the EEneo
heterozygotes and all of the EEneo mice
(Fig. 4C,D). In about half of
the EEneo mice, the ninth ribs also attach to the sternum contralaterally
(Fig. 4D). These abnormalities
indicate minimally that T7, T8 and T9 are transformed anteriorly in EEneo
mice. In wild-type animals, T10 is called the transitional vertebra as the
dorsal process in the thoracic vertebrae are normally pointed posteriorly from
T3 to T9, and anteriorly from T11 to more caudal region
(Fig. 4E). In almost all the
EEneo mice, however, T13 becomes the transitional vertebra
(Fig. 4F), suggesting that T10,
T11 and T12 are transformed anteriorly. Furthermore, the 12th rib is
transformed anteriorly to the identity of the 11th rib in about 69% of EEneo
mice. In wild-type or heterozygous animals, the total length of the 12th rib
is about half of the length of the 11th rib, and the length of the cartilage
portion of the 12th rib is about half the length of the bony part. However, in
EEneo mice, the length of the 12th rib is almost equal to the length of the
11th rib, and the cartilage and the bony part of the 12th rib are of equal
length, similar to the appearance of the 11th rib
(Fig. 4G,H). In addition,
skeletal transformations are observed in the cervico-thoracic region in EEneo
mice with low penetrance. The anterior tuberculum (AT) that is normally
attached to the 6th cervical vertebra is attached to the 5th cervical vertebra
in 14% of the EEneo mice, and there is no AT on C6, suggesting that C5 and C6
are transformed posteriorly to the identity of C6 and C7
(Fig. 4I,J). Furthermore, 21%
of the EEneo mice developed extra ribs on C7, and these ectopic ribs are often
fused with the first rib attached to the first thoracic vertebra, suggesting a
posterior transformation of C7 into T1
(Fig. 4I,J).
|
EElox embryos
Several types of skeletal defects are observed in EElox mice. These defects
include low penetrance of posterior transformation of C7 to T1, and anterior
transformation of T7 to T6 and L1 to T13
(Table 1). The most prominent
skeletal phenotype in EElox mice is the anterior transformation of T8 to T7,
with 90% penetrance (Table
1).
Expression patterns of Hoxc6, Hoxc9, and Hoxb8 in
EEneo and EElox embryos
Several phenotypes are observed in EEneo and EElox mice that have not been
reported for Hoxc8 gene knockout mice as reported above. These
results suggest that the expression of other Hox genes paralogous to
Hoxc8 might also be modified. To investigate this possibility, we
examined mRNA expression pattern of Hoxc6 and Hoxc9, the
immediate neighboring genes flanking Hoxc8, and Hoxb8, a
Hoxc8 paralog in the Hoxb cluster. The expression pattern of
Hoxc6 in EEneo and EElox embryos at 8.5 and 9.5 dpc is unchanged with
respect to controls in both the neural tube and paraxial mesoderm
(Fig. 5A, data not shown).
However, although the expression pattern of Hoxc9 in the neural tube
is not altered, the anterior expression boundary in paraxial mesoderm is
shifted one somite posteriorly in two out of three 9.5 dpc EEneo embryos
(Fig. 5B). We do not find any
modifications of Hoxc9 expression in 9.5 dpc EElox embryos (data not
shown). The alteration of Hoxc9 expression in paraxial mesoderm in
EEneo embryos correlates with the ninth thoracic vertebra transformation
observed in EEneo mice, suggesting that the neo cassette may interfere with
the regulation of Hoxc9 transcription. We next examined the
expression pattern of Hoxb8 in 7.5, 8.5 and 9.5 dpc EEneo and EElox
embryos. No departure from baseline expression was detected
(Fig. 5C, data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has naturally been assumed that the underlying cause of these phenotypes
has been the absence of the Hoxc8 protein. However, this cannot be the
complete explanation, because in the case of the EEneo and EElox mice the
identical phenotypic modifications are found in the face of essentially normal
expression levels and limits of anterior expression of Hoxc8 in 10.0
dpc embryos. We propose that the delay of activation and forward spreading of
Hoxc8 at early developmental stages in the mutant embryos account for
the morphological aberrations in the vertebral column. The importance of
strict control of transcriptional timing of Hox gene expression has been
reported previously (Castelli-Gair and
Akam, 1995; Gérard et
al., 1997
;
Zákány et al.,
1997
). In the case of transcriptional regulation of
Hoxd11, expression of Hoxd11 for a few hours early or late
leads to anterior or posterior transformation in the lumbosacral region,
respectively. This suggests that the function of the gene was required prior
to morphogenesis (Gérard et al.,
1997
; Zákány et
al., 1997
). The correlation between the temporal delay of
Hoxc8 expression and the skeletal phenotypes in Hoxc8 early
enhancer knockout mice support this view. In EEneo embryos, the onset of
Hoxc8 expression is delayed for at least 12 hours, and the forward
spreading of the expression domain as well as the establishment of an anterior
boundary in the paraxial mesoderm is delayed by 1 day. The temporal delay of
Hoxc8 expression is associated with anterior transformations along
the trunk region with high penetrance, similar to that found in Hoxc8
gene knockout mice. EElox embryos show activation of Hoxc8 expression
at 8 dpc and their expression domain is restricted to the most posterior
region of the embryos without forward extension. However, at 8.5 dpc the
expected expression pattern is re-established. Even this slight temporal delay
is sufficient to induce anterior transformations of the seventh and the eighth
thoracic vertebrae. This indicates that the morphological identity of T7 and
T8 are determined around 8-8.5 dpc. As the chondrification of ribs and
thoracic vertebrae starts around 13 dpc
(Rugh, 1990
), our data are in
agreement with previous findings that the functional expression of Hox genes
is required 5 days before vertebra formation
(Zákány et al.,
1997
). Our results also show that even though the correct anterior
boundary of Hoxc8 is established later, it could not rescue the
aberrant regional identities induced by an Hoxc8 deficit in the
presomitic mesoderm. Taken together, our data indicate that transcriptional
activation of Hoxc8 at precise times prior to somite condensation are
crucial for establishing thoracic identities.
Our results are similar to those reported by Duboule et al. in which an
enhancer (RVIII) located between Hoxd10 and Hoxd11 was
deleted by stem cell targeting
(Zákány et al.,
1997). Similarities include (1) an enhancer of several hundred bp
highly conserved in mammals, birds, and fishes, (2) reduction in the
activation of the Hoxd genes in early embryos, (3) restoration of normal Hoxd
expression both in terms of level and anterior limits at later stages of
embryogenesis, (4) phenotypic modification in the axial skeleton similar to
those reported for Hoxd10 and Hoxd11 knockouts, and (5)
increased penetrance of expression in animals carrying a neo cassette compared
with lox animals.
The similarity in the properties of the RVIII and EE enhancers prompted us to make sequence comparisons between the two. Sequence analysis shows similarities within an upstream region of about 200 bp (Fig. 6A). There are two known protein-binding motifs (CDX and HOX) that show complete sequence identity, two Forkhead/SRY pairs that differ by only a few base pairs, congruence in serial order of the four protein-binding sites, and highly similar spacing relationships differing by only one base pair out of a total of 76 (Fig. 6A,B). This upstream region is also highly conserved for both RVIII and EE between mammals, birds and fish. The degree of expression and sequence similarity between RVIII and EE suggests the existence of a common, highly conserved and primitive mechanism of Hox gene regulation operating in different Hox clusters and paralogy groups, and possibly reflective of a broadly distributed mechanism associated with other Hox genes. We consider it likely that there is a conserved system of signals that serve to deploy the Hox genes in their proper temporal and spatial patterns. It would be sensible if signals were to activate the early expression phase of Hox gene expression. The presence of CDX elements in RVIII and EE enchancers is moreover consistent with early activation, as CDX is activated early, prior to Hox expression in a gradient fashion with highest activity in the tailbud and lowest activity cranially.
|
Comparison of EElox and EEneo mutations
It is of interest to compare the EElox and EEneo mutations in terms of
their similarities and differences as tabulated in
Table 1 and Figs
2,
3,
4,
5. First, both mutations induce
a number of identical phenocopies: C7 to T1, T7 to T6, T8 to T7 and L1 to T13.
However, the penetrance of expression is significantly greater for EEneo than
EElox in all instances and especially so for L1 to T13. In five cases,
segmental transitions are found for EEneo, but not for EElox.: C5 to C6, T9 to
T7, T12 to T10, T11 to T10 and T12 to T11. It should be noted that transitions
observed for EEneo, but not for EElox, are novel in that they do not
correspond to phenocopies for Hoxc8 null. Moreover, we show that the
EEneo mutation exerts a greater temporal delay than does EElox.
How might these similarities and differences be explained? The neo gene is
fully functional transcriptionally with necessary coding and non-coding
control elements. Thus, one explanation might be a competition between the neo
gene and neighboring gene(s) for transcription factors
(Olson et al., 1996;
van der Hoeven et al., 1996
).
We think this is unlikely for the following reasons: (1) although
Hoxc8 expression is retarded, it ultimately expresses at control
levels; and (2) some phenotypes are observed that do not correspond to
Hoxc8 null phenocopies. Although not yet proven, we speculate that
the observed phenomena can most likely be explained in terms of chromatin
configuration modifications in the Hoxc8 and surrounding genomic
domains. This interpretation argues that EElox imparts a minimal chromatin
distortion that is then corrected within a short period of time by unperturbed
chromatin elements in the immediate vicinity. We have shown previously that
the EE is highly conserved at a 95% nucleotide sequence level when compared
with representative species of the orders of mammals, indicating its critical
functionality (Shashikant et al.,
1998
). We have also shown that throughout the upstream 5'
region there are numerous elements that show lower, but still significant
levels of nucleotide sequence conservation that may also be critical
functional control elements and serve to buffer effects produced by EElox and
EEneo (Belting et al., 1998
).
Thus, in the case of EEneo, we posit that both the loss of EE and the adjacent
presence of neo distorts the chromatin configuration maximally, and this
results in the increased penetrance of Hoxc8 null phenocopies and in
addition spreads to more distant regions to allow the induction of
non-Hoxc8 null phenocopies. We believe this hypothesis is supported
by and consistent with our previous findings that the Hox complexes are devoid
of lines and sines that are reported to perturb chromatin structure and which
are highly prevalent elsewhere in the genome
(Kim et al., 2000
).
Comparisons between Hoxc8 null, EEneo, and EElox
mutations
It is of interest to compare our EE deletion mutations with Hoxc8
null mutants reported previously by others
(Fig. 7). Mid-thoracic anterior
homeotic transitions (T7 to T6 and T8 to T7) are associated with all three
mutant types, suggesting that the expression of Hoxc8 at an early
time is required for the support of normal patterning. More posterior thoracic
transformations (T9 to L1) are associated with Hoxc8 null and EEneo
deletion mutations, and only minimally so with EElox deletions. We posit that
normal patterning in this region is also dependent on Hoxc8
expression, but at a later time point. It is unlikely that Hoxc8 by itself can
mediate normal patterning of both mid- and posterior thoracic somites. Rather,
we believe, an interaction of Hoxc8 with factors specific for mid and
posterior thoracic somites is essential. This implies that the mid-thoracic
factor (protein interaction/gene activation or suppression, etc.) is available
only for interaction with Hoxc8 during an early time period, while the
postulated posterior thoracic factor is crucially available at a later
time.
|
In summary, we conclude the following: (1) normal patterning of mid and
posterior thoracic vertebrae is dependent on the early expression of
Hoxc8 at appropriate times and boundary positions; (2) there may
exist two distinct critical time points early and somewhat later that govern
normal patterning in mid- and posterior thoracic vertebrae, respectively; and
(3) expression of Hoxc8 at later time points is crucial for neural,
but less so for axial developmental, as EEneo and EElox mutations do not
seriously affect neural development, save for the limb clasping phenotype,
whereas Hoxc8 null mutations are associated with serious neural
defects (Le Mouellic et al.,
1992; Tiret et al.,
1998
).
Cis- and trans-regulation of Hoxc8
We have shown that the EE is responsible for setting up the correct
expression patterns of Hoxc8 in both initiation and maintenance
phases. In the initiation phase, the EE is required for regulating
Hoxc8 activation. Belting et al.
(Belting et al., 1998) proposed
that in the initiation phase of Hoxc8 expression (8-8.5 dpc), the EE
responds to an inductive signal emanating from the posterior tip of the embryo
in a planar fashion that drives the forward spreading of Hoxc8.
Recent studies on the coordination of segmentation clocks and Hox gene
activation shed light on the possible mechanisms of Hoxc8 regulation.
These studies suggest a two-step mechanism for precise control of Hox gene
expression in the somites. In the first step, there is an increase in Hox
cluster accessibility for transcriptional activation in presomitic cells
during gastrulation. In the second step, strong bursts of Hox expression are
activated by the segmentation clock in the presomitic mesoderm cells about to
transit to somitic cells. The activation of Hox genes in the presomitic cells
thus establishes the anterior boundaries of Hox expression domains and marks
the morphological fate of each somite
(Dubrulle et al., 2001
;
Tabin and Johnson, 2001
;
Zákány et al.,
2001
). Given the fact that the EE is important for the appropriate
temporal control of Hoxc8 activation, it is likely that it responds
to clock signals mediating the timing of Hoxc8 expression in the
presomitic mesoderm.
Alternatively, the EE may regulate Hoxc8 activation through a
FGF/CDX signaling pathway. Studies in Xenopus and chick show that
FGFs directly mediate early Hox expression, and that the effect of FGFs on Hox
regulation is mediated by CDX protein
(Pownall et al., 1996; Isaacs
et al., 1998; Bel-Vialar et al.,
2002
). As we stated in the previous discussion, the presence of
CDX binding motifs in both Hoxd11 RVIII and Hoxc8 EE
enhancers suggest CDX may direct the regulation of Hox expression via their
cis-regulatory elements. Indeed, the ability of the EE to drive a reporter
gene expression in mesoderm and neural tube is completely negated when
mutations are introduced at both CDX-binding sites within the EE.
(Shashikant and Ruddle, 1996
).
These results are consistent with the observation that in the EE deleted
embryos, the expression of Hoxc8 is delayed for a certain period of
time. Taken together, these data suggest that CDX transduces FGF signaling by
directly interacting with the EE thus regulating Hoxc8 activation and
expression.
Finally, the EE may be also important for opening and maintaining a
chromatin configuration that is active for transcription by directly binding
with chromosomal remodeling factors. In previous studies, we have identified
proteins that bind to specific sequences in the 5' region of the EE
using the yeast one hybrid methodology. One of these proteins, BEN (binding to
early enhancer) contains six helix-turn-helix domains located in separate
exons, a leucine zipper motif and a nuclear localization signal
(Bayarsaihan and Ruddle, 2000).
The structural properties of BEN are consistent with its role as a
transcription factor. BEN is a member of the TFII-I gene family. BEN and
TFII-I map side by side within the genome (human chromosome 7q 11.23) and have
undoubtedly arisen from a precursor gene by unequal crossing over and evince a
high level of functional and structural similarities. In a recent report, we
have shown that TFII-I interacts functionally with histone deacetylase 3 and
has the capability of modifying chromatin structure thus enabling access by
control factors to enhancers and promoters
(Tussie-Luna et al., 2002
). We
have also shown that BEN is dynamically regulated during early development and
is expressed from early cleavage stages through fetal development in a broad
spectrum of tissues with both cytoplasmic and nuclear localization
(Bayarsaihan et al., 2003
). The
fact that BEN and TFII-I have the capability to bind to the EE and modify its
chromatin structure lends credence to the concept that the activation of the
Hoxc8 gene may proceed through distinct stages that relate to the
previously described early activation and late maintenance phases of Hox gene
regulation (Deschamps and Wijgerde,
1993
; Deschamps et al.,
1999
).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bayarsaihan, D. and Ruddle, F. H. (2000).
Isolation and characterization of BEN, a member of the TFII-I family of
DNA-binding proteins containing distinct helix-loop-helix domains.
Proc. Natl. Acad. Sci. USA
97,7342
-7347.
Bayarsaihan, D., Bitchevaia, N., Enkhmandakh, B., Tussie-Luna, M. I., Leckman, J. F., Roy, A. and Ruddle, F. H. (2003). Expression of BEN, a member of TFII-I family of transcription factors, during mouse pre- and postimplantation development. Gene Expr. Patterns (in press).
Bel-Vialar, S., Core, 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. 15,238 -249.
Bel-Vialar, S., Itasaki, N. and Krumlauf, R. (2002). Initiating Hox gene expression: in the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups. Development 129,5103 -5115.[Medline]
Belting, H.-G., Shashikant, C. S. and Ruddle, F. H. (1998). Multiple phases of expression and regulation of mouse Hoxc8 during early embryogenesis. J. Exp. Zool. 280,196 -222.[CrossRef]
Castelli-Gair, J. and Akam, M. (1995). How the
Hox gene Ubx specifies two different segments: the significance of spatial and
temporal regulation within metamers. Development
121,2973
-2982.
Charité, J., de Graaff, W., Shen, S. and Deschamps, J. (1994). Ectopic expression of Hoxb8 causes duplication of the ZPA in the forelimb and homeotic transformation of axial structures. Cell 78,589 -601.[Medline]
Deschamps, J. and Wijgerde, M. (1993). Two phases in the establishment of Hox expression domains. Dev. Biol. 156,473 -480.[CrossRef][Medline]
Deschamps, J., van den Akker, E., Forlani, S., de Graffe, W., Oosterveen, T., Roelen, B. and Roelfsema, J. (1999). Initiation, establishment and maintenance of Hox gene expression patterns in the mouse. Int. J. Dev. Biol. 43,635 -650.[Medline]
Dubrulle, J., McGrew, M. J. and Pourquie, O. (2001). FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106,219 -232.[Medline]
Dupé, V., Davenne, M., Brocard, J., Dolle, P., Mark, M.,
Dierich, A., Chambon, P. and Rijli, F. M. (1997). In
vivo functional analysis of the Hoxa1 3' retinoic acid response
element. Development
124,399
-410.
Gaunt, S. J. (1988). Mouse homeobox gene transcripts occupy different but overlapping domains in embryonic germ layers and organs: a comparison Hox-3.1 and Hox-1.5.Development 103,135 -144.[Abstract]
Gaunt, S. J. and Strachan, L. (1996). Temporal colinearity in expression of anterior Hox genes in developing chick embryos. Dev. Dyn. 207,270 -280.[CrossRef][Medline]
Gebuhr, T. C., Bultman, S. J. and Magnuson, T. (2000). Pc-G/trx-G and the SWI/SNF connection: developmental gene regulation through chromatin remodeling. Genesis 26,189 -197.[CrossRef][Medline]
Gérard, M., Zákány, J. and Duboule, D. (1997). Interspecies exchange of a Hoxd enhancer in vivo induces premature transcription and anterior shift of the sacrum. Dev. Biol. 190,32 -40.[CrossRef][Medline]
Gilthorpe, J., Vandromme, M., Brend, T., Gutman, A., Summerbell, D., Totty, N. and Rigby, P. W. (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]
Issacs, H. V., Pownall, M. E. and Slack, J. M. W.
(1998). Regulation of Hox gene expression and posterior
development by the Xenopus caudal homologue Xcad3. EMBO
J. 17,3413
-3427.
Kieny, M., Mauger, A. and Sengel, P. (1972). Early regionalization of the somitic mesoderm as studied by the development of the axial skeleton of the chick. Dev. Biol. 28,142 -161.[Medline]
Kim, C.-B., Amemiya, C., Bailey, W., Kawasaki, K., Mezey, J.,
Miller, W., Minoshima, S., Shimizu, N., Wagner, G. and Ruddle, F.
H. (2000). Hox cluster genomics in the horn shark, Hetero
francisci. Proc. Natl. Acad. Sci. USA
97,1655
-1660.
Kondo, T., Zakany, J. and Duboule, D. (1998). Control of colinearity in AbdB genes of the mouse HoxD complex. Mol. Cell. 1,289 -300.[Medline]
Kondo, T. and Duboule, D. (1999). Breaking colinearity in the mouse HoxD complex. Cell 97,407 -417.[Medline]
Labosky, P. A., Winner, G. E., Jetton, T. L., Hargett, L., Ryan,
A. K., Rosenfeld, M. G., Parlow, A. F. and Hogan, B. L. M.
(1997). The winged helix gene, MF3, is required for
normal development of the diencephalon and midbrain, postnatal growth and the
milk-ejection reflex. Development
124,1263
-1274.
Le Mouellic, H., Condamine, H. and Brûlet, P. (1988). Pattern of transcription of the homeo gene Hox 3.1 in the mouse embryo. Genes. Dev. 2, 125-135.[Abstract]
Le Mouellic, H., Lallemand, Y. and Brûlet, P. (1992). Homeosis in the mouse induced by a null mutation in the Hox-3.1 gene. Cell 69,251 -264.[Medline]
Olson, E. H., Arnold, H.-H., Rigby, P. W. J. and Wold, B. J. (1996). Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell 85, 1-4.[Medline]
Pownall, M. E., Tucker, A. S., Slack, J. M. W. and Issacs, H.
V. (1996). eFGF, Xcad3 and Hox genes form a molecular pathway
that establishes the anteroposterior axis in Xenopus.
Development 122,3881
-3892.
Rugh, R. (1990). The Mouse: Its Reproduction and Development. Oxford: Oxford University Press.
Shashikant, C. S., Bieberich, C. J., Belting, H.-G., Wang, J. C.
H., Borbely, M. A. and Ruddle, F. H. (1995).
Regulation of Hoxc8 during mouse embryonic development:
identification and characterization of critical elements in early neural tube
expression. Development
121,4339
-4347.
Shashikant, C. S. and Ruddle, F. H. (1996).
Combinations of closely situated cis-acting elements determine
tissue-specific patterns and anterior extent of early Hoxc8 expression.
Proc. Natl. Acad. Sci. USA
93,12364
-12369.
Shashikant, C. S., Kim, C. B., Borbely, M. A., Wang, W. C. H.
and Ruddle, F. H. (1998). Comparative studies on
mammalian Hoxc8 early enhancer sequence reveal a baleen
whale-specific deletion of a cis-acting element. Proc.
Natl. Acad. Sci. USA 95,15446
-15451.
Sicinski, P., Donaher, J. L., Parker, S. B., Li, T., Fazell, A., Gardner, H., Haslam, S. Z., Bronson, R. T., Elledge, S. J. and Weinberg, R. A. (1995). Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell 82,621 -630.[Medline]
Simon, J. A. and Tamkun, J. W. (2002). Programming off and on states in chromatin: mechanisms of Polycomb and trithorax group complexes. Curr. Opin. Genet. Dev. 12,210 -218.[CrossRef][Medline]
Suemori, H., Takahashi, N. and Noguchi, S. (1995). Hoxc9 mutant mice show anterior transformation of the vertebrae and malformation of the sternum and ribs. Mech. Dev. 51,265 -273.[CrossRef][Medline]
Tabin, C. J. and Johnson, R. L. (2001). Clocks and Hox. Nature 412,780 -781.[CrossRef][Medline]
Tiret, L., Mouellic, H. L., Maury, M. and Brulet, P.
(1998). Increased apoptosis of motoneurons and altered
somatotopic maps in the brachial spinal cord of Hoxc8-deficient mice.
Development 125,279
-291.
Tussie-Luna, M. I., Bayarsaihan, D., Seto, E., Ruddle, F. H. and
Roy, A. L. (2002). Physical and functional
interactions of histone deacetylase 3 with TFII-I family proteins and
PIASxbeta. Proc. Natl. Acad. Sci. USA
99,12807
-12812.
van den Akker, E., Reijnen, M., Korving, J., Brouwer, A., Meijlink, F. and Deschamps, J. (1999). Targeted inactivation of Hoxb8 affects survival of a spinal ganglion and causes aberrant limb reflexes. Mech. Dev 89,103 -114.[CrossRef][Medline]
van den Akker, E., Fromental-Ramain, C., de Graaff, W., le
Mouellic, H., Brûlet, P., Chambon, P. and Deschamps, J.
(2001). Axial skeletal patterning in mice lacking all paralogous
group 8 Hox genes. Development
128,1911
-1921.
van der Hoven, F., Zákány, J. and Duboule, D. (1996). Gene transpositions in the HoxD complex reveal a hierarchy of regulatory controls. Cell 85,1025 -1035.[Medline]
Zákány, J., Gérard, M., Favier, B. and
Duboule, D. (1997). Deletion of a HoxD enhancer induces
transcriptional heterochrony leading to transcription of the sacrum.
EMBO J. 16,4393
-4402.
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]