Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
* Author for correspondence (e-mail: jacqueli{at}niob.knaw.nl)
SUMMARY
The Hox genes confer positional information to the axial and paraxial tissues as they emerge gradually from the posterior aspect of the vertebrate embryo. Hox genes are sequentially activated in time and space, in a way that reflects their organisation into clusters in the genome. Although this co-linearity of expression of the Hox genes has been conserved during evolution, it is a phenomenon that is still not understood at the molecular level. This review aims to bring together recent findings that have advanced our understanding of the regulation of the Hox genes during mouse embryonic development. In particular, we highlight the integration of these transducers of anteroposterior positional information into the genetic network that drives tissue generation and patterning during axial elongation.
Introduction
The regulation of the patterning of the trunk and tail as they develop is a function of the homeobox-containing Hox gene family, which has been evolutionarily conserved among the metazoans. The conservation of the structure and function of these genes may lie in their ability to adequately provide an identity to the anteroposterior (AP) structures during embryogenesis. An excellent example of this property is the ontogeny of the vertebral column. During this process, pairs of mesodermal blocks are established sequentially on either side of the neural tube as the vertebrate embryo develops. Although morphologically very similar, these blocks will differentiate into distinct mesodermal tissues, depending on their axial level. The identity of these blocks, or somites, is specified by their unique combinatorial expression of the Hox genes. For example, the first trunk somites to form will give rise to the most anterior prevertebrae. Their anterior (or rostral) identity is achieved through the exclusive expression of the 3' Hox genes (Fig. 1). The next somites to form acquire a more posterior (or caudal) identity through the expression of these 3' Hox genes, together with the following more 5' Hox genes. All axial and paraxial tissues between the middle of the hindbrain and the tip of the tail acquire differential and combinatorial Hox expression patterns, irrespective of whether they are segmented. Lateral plate mesoderm and spinal cord cells, for example, also express a differential combination of Hox genes depending on their ultimate axial level.
The combination of Hox genes expressed in a specific AP region has been
called its `Hox code' (Kessel and Gruss,
1991). The correspondence between the order of the Hox genes on
their chromosome and the anterior-to-posterior sequence of the structures that
express them has been called `spatial colinearity' (for reviews, see
Krumlauf, 1994
;
Kmita and Duboule, 2003
).
Furthermore, in mammals and in short germ-band insects, which, unlike
Drosophila, extend their axis progressively by adding new tissues
from their posterior end, 3' Hox genes are expressed first, whereas more
5' Hox genes are expressed later and sequentially. This latter
phenomenon has been called `temporal co-linearity' (reviewed by
Kmita and Duboule, 2003
) (see
Fig. 1). The intimate
relationship between the co-linear timing of Hox gene expression and
morphogenesis may initially have played an evolutionary constraining role in
maintaining the Hox genes in their chromosomal clusters
(Kmita and Duboule, 2003
;
Ferrier and Minguillon, 2003
).
However, an extensive analysis of the molecular mechanisms that modulate axial
Hox gene expression strongly suggests that coordinated expression is achieved
through a variety of species-dependent mechanisms. The strategy by which these
genes are expressed in a correct spatiotemporal pattern at the molecular level
appears not to matter too much, providing that the proper Hox protein
distribution is achieved (Kmita and
Duboule, 2003
).
This review focuses on recent data on Hox gene regulation that shed new
light on the integration of AP patterning into the morphogenetic programme
that drives embryogenesis. We evaluate the importance of the early
transcriptional activation of these genes in the posterior primitive streak,
as well as the role of the node region in modulating the Hox gene expression
domain during the laying down of axial and paraxial tissues. We survey recent
work on how the signalling molecules that have a crucial role in the onset of
patterning in the neural tube and mesoderm influence Hox gene transcription.
We then focus on a class of Hox regulators, the Cdx transcription factors,
that participate in tissue generation during axial extension, as well as in AP
patterning, and discuss how these processes are intimately linked. Regulatory
events that ensure the long-term memory of the transcriptional states of the
Hox genes are also discussed, together with recent findings that some
epigenetic marks that ensure the inheritance of the expression status of the
Hox genes also act as a chromatin-editing system. This system differentially
sensitises the Hox genes to transcription at early stages of development in
tissues that will only later express these genes. We also discuss other
parameters that correlate with Hox gene transcription. Finally, we consider
how the genomic area that surrounds a Hox cluster contributes to controlling
tissue-specific Hox gene expression in the limbs and, independently,
spatiotemporal expression in the trunk. [Other aspects of the regulation of
the Hox genes are discussed elsewhere
(Krumlauf, 1994;
Rijli et al., 1998
;
Trainor and Krumlauf, 2000
;
Kmita and Duboule, 2003
).]
|
In mice, temporal co-linearity of Hox gene expression is observed from the
very first transcription initiation event in the posterior primitive streak
(see Figs 1 and
2), an area that is fated to
become extra-embryonic mesoderm. The regulation of the Hox genes at this very
early stage in the posterior-most epiblast does not directly concern the
future axial and paraxial embryonic structures, as these are derived from more
anterior cells within and around the anterior streak
(Lawson et al., 1991).
However, the sequential initiation of Hox transcription will determine the
time at which the expression domains successively reach the anterior primitive
streak, or node region, from where the embryonic axis mainly extends
(Fig. 3) [Forlani et al.
(Forlani et al., 2003
) in
mice; Wacker et al. (Wacker et al.,
2004
) in Xenopus]. The precise temporal activation of the
Hox genes at the initial stages in the generation of their expression domains
is therefore crucial for establishing regional identity. For example, in the
mouse, a Hoxc8 regulatory mutation affects skeletal patterning by
causing a transient delay in the initial transcription of the Hox gene. This
mutation phenocopies many of the axial defects of the Hoxc8-null
mutant (Juan and Ruddle,
2003
).
Initial Hox transcription and the early rostral expansion of Hox expression
domains are regulated by events that are connected to the emergence and
extension of the primitive streak (Forlani
et al., 2003). Wnt signals that regulate the formation and
function of the primitive streak may modulate Hox gene expression during its
anteriorward spreading (Forlani et al.,
2003
). Fgf signalling that modulates the morphogenetic movement of
the mesoderm at the primitive streak
(Ciruna and Rossant, 2001
) may
regulate the Hox genes as well. A role for retinoic acid (RA) in initiating
Hox gene expression has also been suggested because endogenous RA has been
detected in the posterior part of early post-implantation embryos
(Hogan et al., 1992
). This
role is more difficult to ascertain, however, because at early developmental
stages, embryos with impaired RA biosynthesis exhibit rather normal initial
3' Hox gene expression domains
(Niederreither et al.,
1999
).
After expanding anteriorly in and along the primitive streak, the Hox
expression domains continue to spread and sweep through the node region (Figs
2 and
3). This region has proved to
be crucial for the generation of axial and paraxial structures
(Lawson et al., 1991;
Beddington, 1994
) (reviewed by
Joubin and Stern, 2001
), and
to constitute an `organizing' area of gene expression that is traversed by
cells that contribute to the extending axial tissues
(Joubin and Stern, 1999
).
However, cell lineage analysis in mouse embryos has shown that the Hox codes
are not fixed in the node region (Forlani
et al., 2003
), as Hox gene expression appears to be modified after
nascent mesoderm and neurectoderm have been generated there. This modulation
occurs independently in mesoderm and neurectoderm
(Forlani et al., 2003
). Below,
we discuss how a variety of regulatory influences progressively modulate Hox
gene expression in the interval between the emergence of cells from the
primitive streak and the time when they acquire their definitive Hox
identity.
Generating and patterning embryonic mesoderm
The cohorts of cells that leave the primitive streak at the neural plate
(E7.5) stage to contribute to future rostral somites express 3' Hox
genes exclusively. The next cohorts to leave the streak express these 3'
Hox genes together with more 5' Hox genes. This early Hox expression
program does not correspond to the definitive Hox codes of the axial and
paraxial descendants of these cells, which will only be fixed later on, upon
receiving additional regulatory influences. Paraxial mesoderm cells receive
patterning signals, including positional information, as they transit in the
presomitic mesoderm (PSM), after they have emerged from a zone just posterior
to the node that has been suggested to contain stem cells for the elongating
axis (see the stem cell zone shown in Figs
2 and
3)
(Nicolas et al., 1996;
Cambray and Wilson, 2002
;
Eloy-Trinquet and Nicolas,
2002
). It is in this crucially important phase that cells are
exposed to threshold values of the caudorostral gradients of Fgf
(Dubrulle et al., 2001
) and
Wnt signals (Aulehla et al.,
2003
), and to the rostrocaudally decreasing RA gradient (reviewed
by Dubrulle and Pourquié,
2004a
). These signals couple the speed of paraxial mesoderm
production during axial elongation to the rate of somite formation
(Dubrulle and Pourquié,
2004a
). The mechanism that generates the Fgf gradient was recently
elucidated (Dubrulle and Pourquié,
2004b
). Fgf gene transcription is exclusively restricted to cells
in the `stem cell zone' of the node region, and transcript levels decrease
thereafter in their descendants (which are carried away during the extension
of the axis). Fgf and Wnt proteins, key players in the maturation and
commitment of paraxial mesoderm to its segmental fate, are molecules that have
all been shown to regulate Hox genes either directly or indirectly: thus,
Wnt3a and Fgfr1 hypomorphic mutations restrict Hox gene
expression domains to more posterior positions and give rise to vertebral
anterior transformations along the AP axis
(Ikeya and Takada, 2001
;
Partanen et al., 1998
). Thus,
cells at the node do not have their definitive Hox code yet, and will acquire
it upon receiving graded signals in regions anterior to the node at later
developmental stages (Fig.
4).
|
In addition to the influence of these morphogens, Hox gene expression in
the anterior PSM is affected by mutations in genes that function in the
segmentation program itself. Loss- or gain-of-function mutations in genes of
the Notch pathway, or mutations that alter the temporal periodicity of their
expression, affect Hox gene expression in the PSM
(Zákány et al.,
2001; Cordes et al.,
2004
), and subsequently vertebral patterning
(Cordes et al., 2004
). The
effect on Hox gene expression caused by altering Wnt signalling or by mutating
genes of the oscillatory `clock mechanism' are, in fact, related, as recent
work has shown that the gradient of Wnt signals that controls PSM maturation
directly crossregulates the fluctuating expression of the segmentation genes
in the Notch pathway (Hofmann et al.,
2004
; Galceran et al.,
2004
) during somitogenesis. The very first link between the
segmentation genetic program and Hox gene expression came from the discovery
that discrete stripes of expression of the Hoxd genes exist that closely
correlate with the segmentation process caudal to the last-formed somite
(Zákány et al.,
2001
). When the Hoxd cluster was replaced with a lacZ
reporter under the control of a Hox promoter, the reporter gene also showed
bursts of reinforced gene expression, indicating that regulatory sequences
outside of the Hoxd cluster account for the dynamic expression of the genes in
the PSM, in phase with the segmentation process. These data indicate that
transient bursts of Hox gene activation occur each time cells approach the
PSM/somite transition. These bursts may provide the cells with specific PSM
`instructions', which add to the information already received by their
progenitors during early Hox gene activation in the primitive streak. This
would provide these new somites with at least a component of their AP
identity. A mutation that inactivates RBPjk (Rbpsuh - Mouse
Genome Informatics), the effector of the Notch pathway, abolishes these
stripes and leads to the downregulation of mesodermal Hox gene expression
(Zákány et al.,
2001
). Another link between the segmentation genetic program and
Hox gene expression has been highlighted in a recent study
(Cordes et al., 2004
), which
found that reduced Notch signalling caused by a dominant-negative form of the
Notch ligand Dll1 results in the anterior transformation of vertebral
identity and a posterior shift of the expression domain of several Hox genes.
In addition, the loss of the oscillatory character of lunatic fringe
(Lnfg) expression, a crucial modulator of Notch function in the PSM,
led to vertebral transformations and to a shift of the expression domain of
the same Hox genes. Rather than observing a general drop in Hox gene
expression levels in the PSM, Cordes and co-workers found that reduced Notch
signalling caused a shift in the rostral extension of the expression domains
of the Hox genes (Cordes et al.,
2004
). These two studies could appear to produce different results
because of the different ways in which the Notch pathway has been affected in
these experiments. Inactivating RBPjk, which is essentially required by the
Notch intracellular domain to transcriptionally activate its targets, totally
abolishes Notch signalling and therefore drastically affects gene expression.
Weakening the action of Dll1 or altering the cyclic expression of a modulator
of the Notch interaction with its ligands only partially affects the Notch
signalling pathway, possibly leading to a delay, rather than to a decrease, in
Hox gene expression in the PSM. In any case, although the interactions between
Hox genes and the Notch pathway at the molecular level remain unclear, these
two studies underscore the existence of a link between the acquisition of
positional identity by Hox gene expression and the activity of the genetic
cascade that drives somitogenesis.
|
In addition to patterning the paraxial mesoderm (as discussed here) and the lateral mesoderm (such as the emerging limb bud mesenchyme, which is not dealt with in detail in this review), Hox genes supply AP identity to the neurectoderm between the middle of the hindbrain and the caudal end of the embryo. We will see in the next section that the regulation of patterning in the mesoderm and neurectoderm is tightly coordinated and uses common morphogenetic signalling. And so is the control of the expression of the Hox genes in these tissues.
Regulating Hox gene expression in the neurectoderm
A distinction must be made here between the regulation of the Hox genes in
the anterior part of their neural expression domains in the forming hindbrain,
and the regulation of these genes in the posterior spinal cord, where the axis
elongates by the production of new tissue from the node region. The hindbrain
neurectoderm is generated from a small region of the epiblast that is located
anterolaterally to the node at the late streak stage
(Lawson et al., 1991;
Forlani et al., 2003
). The
anterior rhombomeres (r3 and r4) in the neurectoderm, which will form the
rostral part of the expression domain of the 3' most Hox genes, are laid
down sequentially at the neural plate (E7.5) and subsequent stages, as the
axis extends. At that time, the expression domains of the Hox genes are still
located more posteriorly (Forlani et al.,
2003
). RA is present at these stages in chick embryos at AP
positions just posterior to the forming hindbrain, where it diffuses from the
underlying mesoderm (Blentic et al.,
2003
) (Fig. 2B and
Fig. 4). It has been proposed
that this signalling molecule provides the hindbrain rhombomeres with AP
positional identity by inducing 3' to more 5' Hox genes
(Gavalas and Krumlauf, 2000
;
Dupé and Lumsden, 2001
)
in the hindbrain. Two main conditions are essential for the normal expression
of the Hox genes in the hindbrain: the distribution of the inducing signals
and the sensitivity of the promoter region of the Hox genes to these signals.
The decreasing concentration of RA diffusing from the boundary of RA
production in the mesenchyme, combined with the increasing sensitivity to RA
of 5' to 3' Hox genes, generate the unique combinations of Hox
genes expressed in r3 to r8 that define rhombomere identity
(Gould et al., 1998
;
Gavalas and Krumlauf, 2000
;
Dupé and Lumsden,
2001
). Increasing levels of retinoids are required for the
Hox-mediated specification of the identity of chick rhombomeres 3 to 8
(Dupé and Lumsden,
2001
). In the mouse, the anterior expression boundaries of
3' to 5' Hox genes in the hindbrain directly depends on endogenous
retinoids (Niederreither et al.,
2000
; Oosterveen et al.,
2003
) and on functional retinoic acid responsive elements (RAREs)
around some of the Hox genes. These RAREs are active in sequential, co-linear
time windows; this time window is earlier for the Hoxb1 RAREs than
for the Hoxb4 RARE, and for the RARE located between the
Hoxb4 and Hoxb5 (reviewed by
Gavalas and Krumlauf, 2000
;
Oosterveen et al., 2003
). The
expression of the Hox genes in the hindbrain therefore undergoes a spatially
and temporally co-linear regulation by RA. In addition to the mesoderm-derived
signals such as RA, rhombomere-specific transcription factors modulate the
expression of Hox genes in the neurectoderm itself, including the r3- and
r5-specific Krox20 (Egr2 - Mouse Genome Informatics), the r5- and r6-specific
kreisler, and Hoxb1 and Hoxb4, which act in autoregulatory
loops in r4 and r6, respectively.
|
In addition to regulating the release of cells from the stem cell zone and
their neural differentiation when they become flanked by somites, RA and Fgf
modulate a subsequent phase of neural differentiation. They are essential for
the rostrocaudal modulation of Hox gene expression during neuronal cell fate
specification in the ventral spinal cord
(Liu et al., 2001). It has
been demonstrated that graded Fgf signals from Hensen's node region and
retinoids from the cervical paraxial mesoderm both contribute to the
establishment of the rostrocaudal pattern of Hoxc gene expression in the
progenitors of chick motoneurons (Liu et
al., 2001
). At stages later than E10.5, the action of Fgf is
enhanced at posterior levels by the TGFß family member Gdf11, which
diffuses from the paraxial mesoderm and induces expression of 5' Hoxc
genes at thoracolumbar levels (Liu et al.,
2001
). These successive episodes of signalling that regulate Hox
gene expression in nascent, maturing and differentiating neurectoderm during
embryogenesis are brought together in Fig.
4. The following section focuses on the role of the Cdx
transcription factor family, which regulates the Hox genes and integrates
several posterior signalling pathways, in the genetic network that links AP
patterning to the extension of the body axis.
The Hox regulator Cdx and development of axial structures
The Cdx genes are relatives of the Hox genes. Both gene families are
believed to derive from a common ProtoHox ancestral cluster
(Pollard and Holland, 2000)
(see Box 1). The products of
the three Cdx genes directly regulate vertebrate Hox genes in mesoderm and
neurectoderm in a dose-dependent way
(Subramanian et al., 1995
;
Charité et al., 1998
;
Pownall et al., 1996
;
Isaacs et al., 1998
;
Gaunt et al., 2004
), and
modulate the morphogenesis of vertebrae. The transcriptional stimulation of
the Hox clusters by Cdx proteins occurs at Cdx-binding sites, which are often
found in clusters throughout the Hox complexes. Although recent data have
suggested that the Cdx proteins normally do not affect all Hox genes to the
same extent (van den Akker et al.,
2002
; Bel-Vialar et al.,
2002
; Houle et al.,
2003
), a complete picture of how normal Cdx inputs increase the
expression levels of the different 3' to 5' Hox genes remains to
be established. The expression of the three Cdx genes at two stages of mouse
embryogenesis is shown in Fig.
5. The data derived from the effects of Cdx mutations on
anterior-to-posterior vertebral patterning suggest that Cdx genes affect the
Hox code at `cervical' to `caudal' axial levels
(van den Akker et al., 2002
;
Houle et al., 2003
) (reviewed
by Lohnes, 2003
). Although
Cdx2 is not expressed more rostrally than the PSM, Cdx2
mutations do alter Hox gene expression and the identity of vertebrae at
cervical levels, implying that the molecular interactions between Cdx proteins
and Hox genes occur early in the PSM (van
den Akker et al., 2002
). Several other aspects of the Hox/Cdx
regulatory interaction are worth highlighting. First, the effect of combined
Cdx mutations tested so far on Hox gene expression is modest. Whether this is
due to the existence of other simultaneous regulatory pathways affecting Hox
gene expression, and/or functional redundancy between the three Cdx genes,
remains an unresolved issue. Second, it is not yet clear whether the Fgf and
Wnt morphogenetic signals are transmitted to the Hox genes via the Cdx genes
exclusively [as suggested for exogenous Fgf
(Isaacs et al., 1998
)] or
whether Hox genes also respond to these signals independently of Cdx
regulation.
|
Box 1. Cdx genes and the ancestral mechanism of axial extension Vertebrates, arthropods and short germ-band insects develop their axial
structures in strikingly similar ways, even though their somites and segmental
metameres differ substantially from each other (reviewed by
Tautz, 2004
|
A striking novel property of the Cdx transcription factors has recently
emerged. In addition to their role in transducing AP positional information,
they also play a dominant role in embryonic axial elongation, a function that
has been evolutionary conserved (Box
1). Cdx2 homozygote mutant embryos, when rescued from
their implantation defect, fail to complete the extension of their body axis
and are severely truncated posteriorly
(Chawengsaksophak et al.,
2004), while an earlier analysis of compound
Cdx1/Cdx2 mutant embryos had already revealed that these
genes have a role in axial elongation (van
den Akker et al., 2002
). The posterior body truncations of Cdx
mutants are very similar to the phenotype of loss-of-function Wnt3a
(Ikeya and Takada, 2001
) and
Fgfr1 mutants (Partanen et al.,
1998
). This finding suggests that a genetic interaction exists
between the Wnt and Fgf pathways and the Cdx transcription factors in axial
extension. Cdx2-null mutant embryos also have irregular and often
smaller somites, particularly in the posteriormost region, a feature that
possibly relates to an imbalance between mesoderm generation and the
recruitment of PSM cells into somites, as discussed in the previous section.
The Hox regulator Cdx thus plays a role in the balance between tissue
generation, mesodermal segmentation and AP patterning, clearly demonstrating
that Cdx genes belong to the constellation of genes that form an integrated
genetic network for these three processes
(Fig. 6).
|
Chromatin modifiers: prelude to Hox expression and transcriptional memory
Polycomb group (PcG) and trithorax group (trxG) proteins play an important
role in maintaining the spatially restricted silenced and active
transcriptional states of the Hox genes, respectively, in both flies and mice
(reviewed by Ringrose and Paro,
2004). Histone methylation has recently been implicated in the
long-term maintenance of gene silencing by the PcG complex
(Cao et al., 2002
;
Ringrose et al., 2004
) (see
Box 2). Another recent study
has demonstrated that the mono-ubiquitylation on lysine 9 of histone H2A
(U-H2A K9) plays an essential role in chromatin-mediated heritable gene
silencing (Wang et al., 2004
;
de Napoles et al., 2004
).
Histone lysine modification therefore plays a central role in the stability of
chromosomal states and ensures that a transcriptionally inactive, condensed
chromatin state is inherited by the progeny of a cell.
In addition to their role in the epigenetic maintenance of transcriptional
states of their target genes, PcG and trxG protein complexes probably regulate
the transcription of their targets in Drosophila
(Breiling et al., 2001;
Saurin et al., 2001
), as well
as in early mouse embryos (de Graaff et
al., 2003
). A recent study
(Milne et al., 2002
) elegantly
showed that the binding of SET domain methyl transferase activity to the
proximal promoter of human HOXC8 in cultured fibroblasts was
crucially required for transcription of the gene. The transcriptionally
repressive, mono-ubiquitylated form of histone H2A (U-H2A K9) is recruited to
the Hox promoters by the main PcG protein complex
(Wang et al., 2004
;
de Napoles et al., 2004
).
These two studies link histone K9 and K27 methylation and ubiquitylation to
Polycomb-mediated transcriptional repression, and histone K4 methylation to
active transcription.
A particularly interesting issue in the genetics of AP patterning during
embryonic development concerns the role that chromatin events play in the
early co-linear activation of the clustered Hox genes during early
embryogenesis (Duboule and Dollé,
1989; Kmita et al.,
2000
; Kmita and Duboule,
2003
). Recently, the sequential activation of clustered Hox genes
was followed in mouse embryonic stem (ES) cells
(Chambeyron and Bickmore,
2004
) and in early developing embryos
(Chambeyron et al., 2005
).
Chromatin modifications were scored across the Hoxb locus in ES cells
during RA-mediated differentiation. Acetylation at lysine 9 and dimethylation
at lysine 4 of histone H3, both marks of actively transcribed chromatin, were
increased in both Hoxb1 and Hoxb9 at an early time point,
when only Hoxb1 was expressed. These histone tags therefore are not
tightly coupled to gene transcription, but rather indicate that the genes are
in a `poised' state, ready for transcription. Another recent study of the
chromatin changes that occur during the initiation of Hox gene expression
examined the relationship between histone modification and Hoxd4
activation in Hoxd4-expressing and non-expressing embryonic tissues
(Rastegar et al., 2004
). This
study concluded that Hoxd4 acquires the marks of active chromatin at
a stage earlier than its transcriptional activation, exclusively in the
posterior embryonic territories where it will later become expressed. Again,
these histone modifications seem to confer selective transcriptional
`awareness' to the locus in the presumptive Hox expression domain.
|
Interestingly, the looping out of the clustered ß-globin genes
specifically depends on regulatory sequences around the genes
(Kosak and Groudine, 2004).
The importance of global regulatory regions for the control of clustered genes
has been recognized (Grosveld et al.,
1987
; Spitz et al.,
2003
). We will see in the next section that such global control
regions play essential roles not only in the recruitment of a set of clustered
genes to common functions but also in the differential regulation of the gene
members of a Hox cluster.
Balanced regulatory inputs from inside and outside Hox clusters
The sequentially and spatially co-linear expression of the Hox genes has to
be orchestrated in concert with morphogenesis. This harmony is realised in
part through molecular regulatory interactions that have an impact on subsets
of the Hox genes. The cluster from which most information regarding this
enigmatic regulatory process has been obtained is the Hoxd cluster. A
regulatory element on the 3' side of the Hoxd cluster has been proposed
to account for early co-linear Hoxd gene expression in the lateral plate
mesoderm of the emerging limb field along the AP axis. The existence of this
element, called the early limb control region (ELCR), has been inferred from
the effects of experimentally inverting and deleting parts of this Hox cluster
in the mouse (Zákány et al.,
2004). Expression of the Hoxd genes in the mesenchyme of the
nascent limb bud follows the same co-linearity rules as the early expression
of the Hoxd genes in axial and paraxial tissues, and the molecular regulatory
interactions occurring through the ELCR therefore must be intimately linked to
the mechanism of spatiotemporal co-linearity of expression during
embryogenesis (Zákány et
al., 2004
) (Fig.
8).
Although the integrity of the Hox clusters has been maintained in mammals,
they have not behaved through evolution as isolated islands in the genome. The
discovery of a global control region (GCR), which coordinately regulates gene
expression over large chromosomal domains
(Spitz et al., 2003),
confirmed the hypothesis (Spitz et al.,
2001
; Kmita et al.,
2002a
) that the Hoxd genes recently acquired a novel function in
limb development, in addition to their ancestral function along the main axis.
This GCR, localized about 240 kb upstream of Hoxd, contains the long-predicted
remote control element that coordinately regulates the expression of 5'
Hoxd genes in the distalmost part of the limb buds
(Spitz et al., 2003
). This
element controls the 5' Hoxd genes and two other genes, lunapark
(Lnp) and Evx2, both of which are located in the intervening
region that separates the GCR from the most 5' Hoxd gene. The GCR
harbours, in addition to the digit enhancer, a small cluster of neural
enhancers. These neural enhancers, which are conserved in mammals, drive
Lnp and Evx2 expression in patterns that differ from those
of the Hoxd genes, which are insulated from these enhancers
(Kmita et al., 2002b
) (see
Fig. 8). In the ancestral
scenario, which is still present in the genome of cartilaginous fish, the GCR
contains only the neural enhancers (Spitz
et al., 2003
). The generation of a digit enhancer within the GCR
allowed the 5' Hoxd genes to be strongly expressed in the distal-most
limb mesenchyme, where their activity probably allowed the emergence of the
digits, which have been conserved ever since.
Global control regions regulate the expression of all or groups of the clustered Hox genes, adding their effects to those of the local, Hox-proximal regulatory elements. A last potential element to add in this survey of the regulatory circuits that modulate Hox gene expression are microRNAs, which interfere with gene expression at the post-transcriptional level and have target sequences within the Hox clusters (see Box 3 for more).
|
During axial extension, cells emerging from the posterior stem cell zone do not have or receive their Hox code when leaving the node region, but the transcription of the Hox genes in these cells is regulated thereafter by multiple mechanisms. Despite recent progress, key issues remain unresolved. These include the genetic and cellular mechanism of cell generation from the stem cell zone, its relation to Hox gene expression and the control of the arrest of axial extension at later stages. The live imaging of cells released from the stem cell zone in cultured wild-type and mutant mouse embryos, coupled to the visualisation of gene expression at the cellular level, are just some approaches that promise to shed more light on these processes in the future.
Even if the molecular mechanism that underpins 3' to 5'
colinear expression of the Hox genes has so far been elusive, a corner of the
veil has been lifted. It will be exciting to discover the molecular mechanism
underlying the action of the global regulatory element that drives early
co-linear Hoxd gene expression in the emerging limb buds (ELCR)
(Zákány et al.,
2004), given that this element probably controls the early
spatiotemporal co-linearity of expression of the clustered genes along the
axis, as hypothesised in Fig.
8. In addition, the relationship between this early
spatiotemporally acting enhancer and the regulatory element presumed to
generate sequential transient bursts of Hox gene expression in the anterior
PSM (Zákány et al.,
2001
) is intriguing. Whether and how signalling by RA, Fgf and Wnt
is involved in this regulation is another puzzling issue. It will be
interesting to uncover the mechanism of action and the relationship between
these various episodes of co-linear Hox gene control during embryogenesis.
Another largely unachieved goal is the deciphering of the numerous gene interactions that involve the Hox genes in tissue generation and patterning during elongation of the axis (see Fig. 6). The emerging view suggests that the Hox genes belong to the common constellation of genes that orchestrate morphogenesis in an integrated way during embryogenesis. But much of the functional network involving Wnt, Fgf and Cdx in axial extension and patterning remains elusive. Even the issue of whether Cdx proteins affect posterior axial elongation by regulating the Hox genes remains to be addressed. The availability of many mutants and gene array technology should soon bring more order to this puzzle.
Finally, in addition to the molecular interactions between signalling effectors and the cis-acting responsive elements that lie proximal or more distal to the Hox genes, chromatin modification also prepares the genes for transcription. The physical looping out of Hox genes from their CT correlates with their co-linear expression in time and space in the embryo. It is therefore possible that chromatin events play the important role proposed long ago in setting the prerequisites for initial co-linear Hox gene expression. These events might start much earlier than the maintenance of the Hox transcription status by PcG and trxG proteins. Among the issues that remain to be resolved about these processes is whether the nuclear repositioning of the Hox genes facilitates or results from their transcriptional activation, and how gene extrusion itself is regulated at the molecular level. Exciting new discoveries in this field will surely come.
Note added in proof
Four papers have recently revealed that molecular links exist between the
generation and transmission of left-right (LR) asymmetry to body organs, and
the bilaterally symmetrical extension and patterning of the anteroposterior
axis (AP) axis. Tanaka et al. (Tanaka et
al., 2005) report that the AP patterning signals Fgf and retinoic
acid are key components of a novel mechanism that generates LR asymmetry by
unidirectionally transporting morphogens across the mouse node. Retinoic acid
signalling is subsequently needed to shield forming somites from these LR
asymmetrical cues (Vermot et al.,
2005
; Kawakami et al.,
2005
; Vermot and
Pourquié, 2005
). In the absence of retinoic acid, the
coordination between left and right somite formation is transiently disturbed,
following delayed Fgf8 front regression on one side and the desynchronization
of Notch-dependent oscillation patterns of clock gene expression (see
Hornstein and Tabin,
2005
).
ACKNOWLEDGMENTS
The authors warmly thank Guilherme Costa, Wim de Graaff and Nigel Hynes for help with the figures, and Felix Beck, Jeroen Charité, Karen Downs, Marie Kmita, Kirstie Lawson, Frits Meijlink and Aimée Zuniga for reading the manuscript. We apologize for not being able to exhaustively refer to the work of all colleagues in the field, owing to space limitations.
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