1 Division of Developmental Neurobiology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Stowers Institute for Medical Research, 1000 East 50th Street,
Kansas City, Missouri 64110, USA
* Present address: Centre de biologie du développement, UMR 5547 CNRS;
118 route de Narbonne 31062 Toulouse cedex 4, France
Author for correspondence (e-mail:
rek{at}stowers-institute.org)
Accepted 14 August 2002
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SUMMARY |
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Key words: Hox genes, FGF signaling, Retinoids, AP patterning, Neural development, Gene regulation, Chick development, caudal, Cdx, Electroporation
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INTRODUCTION |
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Understanding how the expression of homeotic genes is established and
maintained is of critical importance, since experiments in many species have
shown that shifts in expression boundaries can lead to transformations and
alterations of segmental identity (reviewed by
Maconochie et al., 1996;
McGinnis and Krumlauf, 1992
;
Moens and Prince, 2002
;
Trainor et al., 2000
).
Regulatory analyses in transgenic and targeted mice have proved to be a useful
tool in characterizing some of the upstream regulatory components of the Hox
network, through the identification of local cis-acting enhancers in
Hox loci. Using reporter genes, it has been possible to reconstruct patterns
of expression for many of the 3' members of the Hox complexes that
appear to be identical to their endogenous counterparts. With respect to the
nervous system and the hindbrain in particular, the combined action of several
components is required to set the precise location of anterior Hox expression
boundaries (reviewed by Trainor et al.,
2000
). A common mechanistic theme used by several of the 3'
Hox genes involves the early activation of expression through the transient
action of factors like Kreisler, Krox20 or retinoid receptors, followed by the
maintenance of these domains through auto- and cross-regulatory interactions
mediated by the Hox genes themselves
(Manzanares et al., 2001
). For
example, Hoxb1 and Hoxb4 are directly activated in the CNS
by transiently acting retinoid-dependent enhancers, which in turn sets their
later segment-restricted domains of expression through triggering of separate
auto/cross-regulatory elements (Gavalas et
al., 2001
; Gould et al.,
1998
; Marshall et al.,
1994
; Morrison et al.,
1997
; Morrison et al.,
1995
; Pöpperl et al.,
1995
; Studer et al.,
1998
; Studer et al.,
1996
; Whiting et al.,
1991
). However, to date it has been very difficult to reconstruct
the proper patterns and anterior boundaries of expression for more 5'
genes in Hox clusters by using local regulatory regions in transgenic
approaches. No regulatory elements of the retinoid, kreisler, Krox20
or auto-/para-/cross-regulatory type have been identified from the 5'
genes. One reason for this is that the 5' genes might depend upon
regulatory mechanisms that involve long-range interactions and the sharing of
distal control regions, as suggested for HoxD genes in the limbs or
HoxB genes in the CNS and mesoderm
(Gould et al., 1997
;
Sharpe et al., 1998
;
van der Hoeven et al., 1996
).
Alternatively these differences might be due to the fact that they require
many different components or arise as a consequence of the temporal
differences in expression of 3' versus more 5' Hox genes. Hence
cis-mechanisms and signaling pathways regulating 3' versus
5' Hox genes may be very different and this could be correlated with
differences in patterning the head versus the trunk.
In the process of neural induction, cells first take on an anterior
character and then under the influence of posteriorizing signals adopt
progressively more posterior fates (Slack
and Tannahill, 1992). Relatively little is known about the precise
signaling pathways and the balance between them that establishes Hox
expression and AP patterning during development or the cis regions
that integrate this information. There is emerging evidence that the compound
auto- and cross-regulatory Hox-responsive elements are also part of the
mechanisms that serve to integrate some of the diverse signaling inputs that
modulate Hox expression in later stages
(Affolter and Mann, 2001
;
Grieder et al., 1997
;
Ryoo et al., 1999
;
Saleh et al., 2000
). Retinoid,
FGF and WNT signaling have all been experimentally linked with early
posteriorizing activity. Considerable evidence has shown that in vivo,
retinoic acid (RA) is an overall mediator or modulator of Hox expression
(Gavalas, 2002
;
Gavalas and Krumlauf, 2000
;
Marshall et al., 1996
). Excess
RA causes a transformation of neural and mesodermal segments toward a
posterior identity, accompanied by an anterior shift in Hox gene expression
boundaries (Conlon, 1995
;
Conlon and Rossant, 1992
;
Kessel and Gruss, 1991
;
Marshall et al., 1992
;
Morrison et al., 1997
;
Simeone et al., 1995
). The
response of Hox genes to exogenous RA in embryos varies in a concentration and
stage-dependent manner that correlates with the position of genes in a
cluster. Inversely, retinoid deficient diets or blocking the retinoic acid
pathway result in anteriorization of rhombomeres and many other AP patterning
defects (Dupé et al.,
1997
; Dupé et al.,
1999
; Gale et al.,
1999
; Kolm et al.,
1997
; Niederreither et al.,
1999
; Niederreither et al.,
2000
; White et al.,
2000
; White et al.,
1998
). Functional retinoic acid
response elements (RARE) have been identified in several
Hox gene regulatory regions (Dupé
et al., 1997
; Gould et al.,
1998
; Huang et al.,
1998
; Langston et al.,
1997
; Langston and Gudas,
1992
; Manzanares et al.,
2000
; Marshall et al.,
1994
; Packer et al.,
1998
; Studer et al.,
1998
; Studer et al.,
1994
; Zhang et al.,
2000
). Furthermore, signals from mesoderm play important roles in
patterning neural tissue and retinoids have been shown to be associated with
several of these signaling events (Ensini
et al., 1998
; Gould et al.,
1998
; Itasaki et al.,
1996
; Liu et al.,
2001
; Sockanathan and Jessell,
1998
).
Recently, many links between the FGF pathway and regulation of Hox genes
have begun to be revealed. In the chick embryo FGF signalling plays a critical
role in primary and secondary neural induction and the node is an important
source of FGF signals that influence the potential of neural tissue
(Mathis et al., 2001;
Storey et al., 1998
;
Streit et al., 2000
;
Wilson et al., 2001
;
Wilson et al., 2000
). In FGFRI
hypomorphic mutants, the expression of Hoxd4 is shifted posteriorly
by one somite and the expression domain of Hoxb9 is shifted
posteriorly in the lateral mesoderm
(Partanen et al., 1998
), thus
suggesting a rule for FGF pathways in the AP patterning of the mesoderm. The
strongest evidence for the involvement of the FGF pathway for Hox gene
induction in neural tissues comes from the Xenopus
(Lamb and Harland, 1995
;
Pownall et al., 1996
). FGFs in
the presence of a BMP antagonist will induce posterior neural markers
(Lamb and Harland, 1995
).
Pownall and collaborators showed that neural tissues cultured in sandwich with
e-FGF-soaked beads express posterior Hox genes and that a dominant negative
form of this receptor (XFD) impairs the early expression of the same genes
(Pownall et al., 1996
).
Moreover, the same study showed that the vertebrate homologues of
Drosophila caudal gene, Cdx genes, are the intermediaries of
this FGF-mediated Hox induction (Isaacs et
al., 1998
; Pownall et al.,
1996
). In the mouse, Cdx genes have been shown to induce
global changes in Hox expression, as illustrated by Cdx1 null mutant
embryos, which show severe homeotic transformations accompanied by a change in
several Hox gene boundaries in the mesoderm
(Subramanian et al., 1995
).
Furthermore, a DNA motif able to bind CDX protein in vitro has been isolated
in the regulatory regions of Hoxc8, Hoxb8 and Hoxa7 that are
believed to be important for their regulation
(Charité et al., 1998
;
Subramanian et al., 1995
;
Taylor et al., 1997
). This
suggests a possible mechanism for the initiation of 5' Hox genes in
chick and mouse through FGF- and CDX-dependent pathways. However Cdx
genes are not exclusively involved in mediating FGF signals. RARE and LEF/TCF
binding motifs have been found in a regulatory region of the Cdx1
gene (Houle et al., 2000
;
Prinos et al., 2001
), and
strong genetic synergy between Cdx1, Wnt3a and retinoid receptors has
been shown in mesodermal patterning (Allan
et al., 2001
; Prinos et al.,
2001
). These results indicate that WNT and RA signaling play
important roles in the early activation of Cdx1 expression. Taken
together these studies suggest that Cdx genes provide a mechanism by
which RA, WNT and FGF signaling may be differentially balanced and integrated,
which could be important for distinct regulation of 5' and 3' Hox
genes.
Thus, signaling and control mechanisms involved in regulation of 5' Hox genes in the spinal cord are still poorly understood in comparison to regulation of 3' Hox genes in the hindbrain. It is believed that RA acts in a graded manner to activate nearly all Hox genes, either directly or indirectly. The relative insensitivity of posterior 5' Hox genes to RA could reflect a lack in their inherent ability to respond to retinoids or may be due to temporal windows of competence in the response itself. The precise contribution of Cdx genes to the regulation of posterior genes versus anterior genes in the neural tube and what signaling pathways they respond to in this process are not understood.
In this study we have systematically examined the contribution of RA, FGF and CDX pathways in the regulation of HoxB genes in the chick neural tube. We first compared the regulation of Hoxb4 and Hoxb9 in detail and then extended the analysis to the other members of the complex. In these early stages of neural development, our results surprisingly identify two distinct groups of HoxB genes based on their reciprocal abilities to respond to RA or FGF signals. This suggests that at these stages they are not regulated in a progressive colinear manner by a graded balance between FGF and RA signaling. Together these results illustrate the importance of understanding regulatory events that modulate Cdx expression to integrate the response of Hox genes to signaling pathways that establish their spatial domains of expression.
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MATERIALS AND METHODS |
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In vitro culture of embryos
For in vitro culture, fertilized chick eggs were incubated to stages 5-7 at
37°C. Embryos were dissected out carefully in L-15 medium, with the
blastoderm remaining intact. The sheet of blastoderm was folded along the body
axis with the ventral surface inside, sealed in a sandwich shape and
transferred to a plastic tube containing DMEM +10% FCS. Tubes were filled with
5% CO2 and incubated on a roller culture at 37°C for 6 hours or
overnight. FGF recombinant proteins (R&D systems) were applied at 200 or
400 ng/ml, Sugen 5402 (Calbiochem) at 25 µM and all-trans-retinoic acid
(Sigma) at 0.7 µM.
DNA constructs and electroporation of DNA
The Xenopus dnRAR1 was a gift from Nancy Papalopulu
(Blumberg et al., 1997
) and we
have previously shown that this construct blocks reporter genes under the
control of retinoid response elements from Hoxb4 and Hoxd4
when electroporated into chick embryos
(Itasaki et al., 1999
;
Gould et al., 1998
). The e-FGF
full-length cDNA was a gift from E. Amaya
(Amaya et al., 1991
). The
Xenopus Xcad3, XcadVP16 and XcadEnR constructs have been
examined and compared in Xenopus by Issacs, Pownall and Slack
(Isaacs et al., 1998
;
Pownall et al., 1996
) who
generously provided these reagents. In Xenopus XcadVP16 will
phenocopy Xcad3 activity in neural assays, but it will also mimic
effects of other Xcad proteins in other contexts
(Isaacs et al., 1998
;
Pownall et al., 1996
). Hence,
it is a useful reagent for activating general CDX targets and not just those
of a particular Cdx gene, such as Xcad3. In our hands
Xcad3 worked poorly in chick, while the XcadVP16 was a
robust activator. The XcadEnR construct replaces the activation
domains of Xcad3 and XcadVP16 with a repressor domain from engrailed,
converting the construct from an activator to a repressor of CDX targets.
Different DNA constructs were electroporated unilaterally or bilaterally as described previously (Itasaki et al., 2000). Briefly, 1-5 µg/µl DNA was injected in the neural tube using a glass pipette. Since this is a closed tube DNA remains confined to neural tissue. Then, electrodes were positioned on opposite sides of the neural tube. For unilateral electroporations, five pulses of 50 mseconds at 20 volts were applied to allow the entry of the DNA into one side of the neural tube (DNA is negatively charged so only moves to the positive pole). For bilateral electroporations the position of the electrodes was reversed and the electroporation repeated.
In situ hybridization and probes
Whole-mount in situ hybridization was performed with digoxigenin-labeled
probes as described previously (Henrique
et al., 1995). All the following probes were hybridized at
70°C overnight. Hoxb1 cDNA (2.0 kb)
(Maden et al., 1991
)
Hoxb4 cDNA (1.2 kb) (Yokouchi et
al., 1991
); Hoxb6 cDNA (300 bp)
(Wedden et al., 1989
),
Hoxb7 cDNA (1.6 kb) (Yokouchi et
al., 1991
); Hoxb8 cDNA, 850 bp, a gift from K. Olberg;
cdxA cDNA (2.488 kb) (Frumkin et
al., 1993
); cdxB cDNA (1.082 kb)
(Morales et al., 1996
).
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RESULTS |
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Hoxb9 is first detected in the caudal neural plate at HH stage 7/8 (Fig. 1F). As the embryo grows caudally, the expression becomes broader and stronger in the neural folds (Fig. 1G,H). At HH stage 10 the anterior limit of Hoxb9 in the neural tube is at the level of the prospective 9th somite (Fig. 1H) and stays fixed at the same limit until HH stage 11 (Fig. 1I and not shown). At this stage, it is also expressed in the posterior mesoderm. The boundary of Hoxb9 expression then begins to regress caudally as the embryo continues to elongate, eventually reaching the level of the 20th somite at HH stage 17 (Fig. 1J). These patterns distinguish three phases for the Hoxb9 establishment in the neural tube: an initiation phase, taking place at HH stage 8; a phase of expansion until HH stage 9; and a phase of posterior regression from HH stage 11 onwards. This profile is dynamic and quite different from the one observed for Hoxb4, which is expressed more anteriorly and for which the expression in the neural tube spreads anteriorly rather than regressing posteriorly during elongation of the embryo. These observations suggest a very different pattern of regulation for these two genes during development.
Differential response of Hoxb4 and Hoxb9 in the CNS
to retinoids and somitic mesoderm
Little is known concerning the regulation of Hoxb9 and we wanted
to investigate if signaling pathways that influence Hoxb4 also act on
Hoxb9. In previous studies, we used in ovo electroporation of a
dominant negative form of the retinoic receptor 1
(dnRAR
1) and tissue grafting to show that retinoid signaling
and somitic mesoderm are essential for the early neural expression of
Hoxb4 (Gould et al.,
1998
; Itasaki et al.,
1996
) (see also Fig.
2C,D). Here, we evaluated the role of the retinoic acid pathway
and somitic mesoderm in the induction of Hoxb9 in the neuroectoderm.
First, HH stage 7-12 embryos were incubated in vitro with 0.7 µM
RA overnight prior to analysis of Hoxb4 and Hoxb9 expression
by in situ hybridization. RA treatment leads to the anteriorization of
Hoxb4 expression in the neural tube
(Fig. 2B) whereas it has no
effect on Hoxb9 expression (Fig.
2F). This illustrates a difference in ability of the genes to
response to ectopic RA. To test if RA signaling is essential for normal
Hoxb9 regulation, we electroporated the dnRAR
1
construct unilaterally into the left side of the neural tube of different
staged embryos and compared the expression of Hoxb9 and
Hoxb4 after 24 hours of in ovo incubation. As expected from our
previous work (Gould et al.,
1998
) expression of dnRAR
1 in the electroporated
side blocks the activation of Hoxb4 if compared to the non
electroporated control side (Fig.
2A,C). In contrast, the expression of Hoxb9 is not
affected by the presence of dnRAR
1
(Fig. 2G). Hence unlike
Hoxb4, Hoxb9 appears not to require RA signaling for its normal
expression and lacks the ability to respond to exogenous RA treatment
throughout all of the early stages we tested.
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Posterior somites are able to reprogram Hoxb4 expression in the
rhombomeres when grafted in the preotic region
(Fig. 2D) and this inducing
capacity is increased for more posterior somites, suggesting a graded signal
with a stronger influence in the posterior part of the embryo
(Gould et al., 1998;
Itasaki et al., 1996
). To
assess the effect of somites in inducing Hoxb9 in the CNS, we grafted
posterior somites (s23 to s25) from a stage 25 donor embryo into a more
anterior region in a stage 10 host embryo, positioning them just anterior to
the normal AP boundary of Hoxb9 in the neural tube at the level of
somites 7-9. As shown in Fig.
2H, the presence of the graft does not modify Hoxb9
expression in the spinal cord after 24 or 48 hours of incubation. These
results demonstrate differences in the ability of Hoxb9 and
Hoxb4 to respond to posterior inducing signals such as RA and somitic
mesoderm.
Hoxb4 and Hoxb9 display different sensitivities to
FGF treatment
We next investigated the potential role of the FGF pathway in
Hoxb4 and Hoxb9 induction in the avian neural tube. HH stage
7-9 embryos were cultured overnight in vitro in the presence of 200
ng/ml of FGF2 (Fig. 3) or FGF4
(not shown) recombinant proteins. Analyses by in situ hybridization showed
that Hoxb9 expression is shifted anteriorly in the neural tube up to
the level of the otic vesicle (Fig.
3E). This shift is specific to the neural tube, as the somitic
boundary is not affected. However, this effect could be mediated through a
cascade of events initiated in the mesoderm. To exclude this possibility, we
over-expressed the Xenopus homologue of FGF4, e-FGF, specifically in
the neural tube, by in ovo electroporation of a DNA construct. In this case,
Hoxb9 expression is also induced and extends anteriorly in the
electroporated side of the neural tube
(Fig. 3F). This confirms that
the FGF effect can be mediated or initiated specifically in the neural tube.
For comparison, we also examined the sensitivity of Hoxb4 to FGF2,
FGF4 and e-FGF treatments under the same experimental conditions. The
Hoxb4 expression domain remains unaffected by both in vitro
FGF treatment (Fig. 3B) and
e-FGF electroporation (Fig.
3C). We also tested different FGF members to see if this effect on
Hoxb9 was specific to FGF2, FGF4 or e-FGF and if the insensitivity of
Hoxb4 was due to the use of these particular FGF ligands. Using FGF8
or FGF10 there was no shift in Hoxb9 or Hoxb4 expression
(not shown), suggesting that this effect on Hoxb9 specifically
involves signaling through FGF2 and/or FGF4 and their receptors and that the
insensitivity of Hoxb4 to FGF treatment is a general property of its
responsiveness.
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Cdx genes are targets of FGF signaling
Cdx genes appear to play key roles in the response to axial
signaling. It has been shown in Xenopus that Cdx genes are
the mediators of FGF signaling to initiate Hox gene expression
(Pownall et al., 1996). To
assess if the Cdx genes could be the targets of FGF signaling in the
avian embryo, we treated HH stage 5 to 7 embryos with FGF2/4 in vitro under
conditions that induce Hoxb9 and monitored expression of
cdxA and cdxB. Both gene expression domains were
anteriorized in the neural tube upon FGF treatment
(Fig. 4B,D). Moreover, the
change in cdxA expression was detectable after 6 hours, showing that
Cdx genes are early targets of FGF signaling. This induction is
temporally dynamic, as HH stage 7 embryos treated overnight showed no
persistent cdxA expression as observed for untreated embryos (not
shown). This shows that FGF has the ability to induce Cdx expression
in early stages but this effect is stage dependent and expanded Cdx
expression in neural tissue is not maintained in the later stages.
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When we compared the kinetics of the response to FGF between Hoxb9
and cdx genes, it appeared that cdxA and cdxB
respond earlier/faster than Hoxb9. This observation raised the
possibility that CDX proteins could act downstream of FGF signaling to
activate the avian Hoxb9 gene. To assess this possibility, we
utilized a construct (XcadVP16) encoding a fusion protein between the
Xenopus caudal 3 (Xcad3) and the VP16 activation domain.
Xcad3 is a homologue of the avian cdxB gene and
XcadVP16 has been shown in Xenopus to strongly transactivate
targets in a manner similar to Xcad3
(Isaacs et al., 1998;
Pownall et al., 1996
), and
also acts as a general activator of CDX target genes in other contexts. Using
in ovo electroporation, we over-expressed XcadVP16 unilaterally in
the left side of the neural tube and assayed for its effects on Hoxb9
expression after a further 20 hours of in ovo incubation. Compared with the
control side or non-electroporated embryos, we detected an upregulation of
Hoxb9 anterior to its normal domains of expression in patchy groups
of cells only in the electroporated side
(Fig. 5B,E). This demonstrates
that increasing CDX activity by ectopic expression of XcadVP16 is
sufficient to induce neural expression of Hoxb9.
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To confirm that the FGF pathway is acting though cdx genes to
induce Hoxb9 expression, we overexpressed a dominant negative form of
Xcad3 (XcadEnR) by electroporation in one side of the neural
tube and cultured the whole embryos overnight in the presence of FGF2. This
construct encodes a fusion protein between Xcad3 and the transcriptional
repression domain of Engrailed and has been shown in Xenopus to act
as a dominant negative form of Xcad3 and CDX activity
(Isaacs et al., 1998;
Pownall et al., 1996
). The
expression of this construct in the dorsal/left side of the neural tube
impairs the upregulation of Hoxb9 dependent upon FGF2 treatment
(Fig. 5C,F). We noted that
endogenous Hoxb9 expression was not affected using XcadEnR,
however this expression is initiated at earlier stages. In attempts to block
this endogenous domain by performing electroporations at earlier stages (HH
5-7), the survival rate of embryos following the manipulations is poor and
precludes analysis. Hence activation (XcadVP16) and inhibition
(XcadEnR) of CDX activity have reciprocal effects upon Hoxb9
expression. These results strongly suggest that FGF2-induced ectopic
expression of Hoxb9 in the neural tube is cdx dependent.
Sensitivity to RA or FGF defines two groups of Hoxb genes
related to their position in the cluster
The above results suggest that posterior versus anterior or midcomplex
HoxB genes respond in distinct manners to different signaling
pathways. This `opposed' or reciprocal sensitivity is rather unexpected
because Hox genes are clustered and believed to be coordinately regulated
along the complex in a sequential manner, which is the basis of temporal and
spatial colinearity. There could be a progressive shift in the balance between
RA and FGF regulation across the complex or specific groups of genes may
exclusively be able to respond to one or both of these pathways in the neural
tube to regulate Hox expression. Therefore, we examined the level of
sensitivity of Hox expression to RA and FGF signals across the HoxB
complex. Fig. 6 shows in situ
hybridization of chick embryos with several HoxB genes in control,
RA-treated and FGF2-treated embryos. We found that Hoxb1, Hoxb3 and
Hoxb5 react as Hoxb4 and are sensitive to retinoic acid
treatment and insensitive to FGF2 treatments
(Fig. 6). In chick,
Hoxb2 is not normally expressed at significant levels in the neural
tube, unlike in the mouse (Vesque et al.,
1996). The other genes we tested, Hoxb6, Hoxb7 and
Hoxb8 behave as Hoxb9 and are rapidly anteriorized upon FGF2
treatment (Fig. 6). They are
also refractory to retinoic acid treatment
(Fig. 6) and their expression
is unaffected by the presence of the dnRARa1 construct (not shown).
These results divide the HoxB complex into two groups of genes based
on their differential sensitivity to RA or FGF at these stages. Surprisingly
despite varying concentrations, timing and stage of analysis none of the genes
simultaneously showed sensitivity to both RA and FGF treatments. This suggests
that the change in the regulation or shift in responsiveness is not
progressive along the complex, but undergoes a distinct switch. The position
of the shift in RA or FGF responsiveness of Hox genes may be time-dependent.
Hence, the specific Hox genes in each of these complementary groups may vary
in later stages of development or other tissues. In some contexts Hox genes
may simultaneously respond to both signals.
|
The role of cdx in altering the Hox response to FGF and
RA
In evaluating the potential basis for this sharp change in response, it is
striking to note that the anterior limits of expression of the HoxB
genes (Hoxb6-Hoxb9) before and after FGF treatment never pass through
the hindbrain/spinal cord boundary (Fig.
6). Our experiments with Hoxb9 and XcadEnR
showed that CDX activity is important in mediating the response to FGF and we
noted that the normal and FGF-induced domains of cdxA and
cdxB expression similarly never extend anteriorly into the hindbrain
(Figs 4,
5). The Hox genes not
responsive to FGF (Hoxb1-Hoxb5) have normal limits of expression in
the hindbrain, where Cdx is not expressed. Hence, there is a good
correlation between Hox genes that are expressed with anterior limits in the
spinal cord, responsiveness to FGF and domains of Cdx expression. The
apparent insensitivity of 3' HoxB genes to FGF treatment may
not arise through lack of ligands or receptors, but could instead reflect a
loss in the competence of cells in the preotic region to activate Hox genes
upon exposure to FGF. It is possible that restriction or absence of
Cdx expression in more anterior regions could be a limiting factor
that modulates the competence to FGF response. This is consistent with our
results using the XcadEnR construct to block CDX activity, and shows
that CDX-dependent activity can act downstream of FGF signaling to modulate
5' Hox genes in chick neural tube.
In order to investigate these potential links between cdx
expression and competence, we examined the effects of elevating CDX activity
upon Hoxb4 expression. Experiments in Xenopus have
demonstrated that a fusion between Xcad3 and the VP16 activation domain
(XcadVP16) will phenocopy Xcad3 activity in neural assays,
and also mimic effects of other Xcad proteins in other contexts
(Isaacs et al., 1998;
Pownall et al., 1996
). Hence,
it is a useful reagent for increasing CDX activity and activating general CDX
targets. As shown in Fig. 7A,
electroporation of the activated XcadVP16 fusion construct into the left side
of the neural tube leads to an anterior induction and extension of the
Hoxb4 expression domain into the hindbrain and midbrain territories.
This shows that Hoxb4 is capable of being induced by CDX activity,
and suggests that mechanisms limiting CDX expression to more posterior regions
of the neural tube prevent posterior Hox genes from responding to FGF
signaling.
|
Cdx genes are not only involved in mediating FGF signals as RARE
and LEF/TCF regulatory motifs have been found in the Cdx1 gene
(Houle et al., 2000;
Prinos et al., 2001
), and
there is genetic synergy between Cdx1, Wnt3a and retinoid receptors
in mesodermal patterning (Allan et al.,
2001
; Prinos et al.,
2001
). Therefore we wanted to exclude the possibility that the
induction of Hoxb4 expression by the activated XcadVP16
construct reflected a role for Cdx genes in mediating a retinoid
response of Hoxb4. Towards this end we electroporated embryos with a
XcadEnR vector, incubated them in RA overnight and assayed for
Hoxb4 expression. The pattern of RA-induced anteriorization of
Hoxb4 expression we previously observed in the hindbrain
(Fig. 2B) was unaltered by the
presence of XcadEnR (data not shown). This shows that in the context
of this experiment, the response of Hoxb4 to RA is not going through
a CDX-dependent pathway in the hindbrain.
XcadVP16 can activate Hoxb9 in the hindbrain in
association with FGF
While we observed that FGF did not induce an anterior shift of
Hoxb6-Hoxb9 that extended into the hindbrain, the results above open
the possibility that all HoxB genes can potentially be activated by
FGF in the hindbrain if CDX activity is provided. We first tested the
potential of XacdVP16 alone to activate Hoxb9 in the preotic
region and found that, unlike Hoxb4
(Fig. 7A) it had no effect (not
shown). However, we tested the combined effect of FGF treatment and
XcadVP16 expression to see if we could bypass the restriction of
induction in the hindbrain. HH stage 8+ embryos were electroporated with
XcadVP16 in the left side of the neural tube and simultaneously
treated in culture with FGF overnight. Under these conditions, the neural
domain of Hoxb9 now extends to the most anterior rhombomeres in the
hindbrain, as compared to the non-electroporated but FGF-treated control side
on the right (Fig. 7B). This
result suggests that the expression of Hoxb9 in the hindbrain
requires not only the presence of CDX activity, but other events controlled by
FGF. As Cdx genes have been shown to possess autoregulatory feedback
loops that maintain their expression following early activation
(Prinos et al., 2001), it is
possible that the addition of FGF is required to reinforce XcadVP16
activity and stimulate such a feedback circuit to induce Hoxb9.
Together these results illustrate the importance of regulatory events that
modulate CDX expression to integrate the response to signaling pathways and
control the ability and spatial extent of the Hox response.
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DISCUSSION |
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Differences in the dynamic nature of Hoxb4 versus
Hoxb9 expression
In the neural tube, the Hoxb4 and Hoxb9 expression
domains are established following a very different sequences of events.
Typical of most other 3' Hox genes
(Maconochie et al., 1996),
Hoxb4 expression is first initiated in the posterior part of the
neural tube and this domain spreads forward over time, eventually reaching a
distinct anterior boundary that is maintained in later stages
(Fig. 1). This progressive
process does not reflect the output or response of a single control region but
is mediated by the combined activities of a series of neural regulatory
regions (Gould et al., 1998
;
Gould et al., 1997
;
Sharpe et al., 1998
;
Whiting et al., 1991
). In
contrast, Hoxb9 is activated directly at an axial level that
constitutes its most anterior limit of expression and then its neural AP
boundary regresses caudally during the later stages of development
(Fig. 1). This posterior
regression suggests that the factors activating Hoxb9 are continually
changing their spatial distribution or activity and indicates the absence of
mechanisms that maintain a sharp and distinct fixed boundary. This pattern for
Hoxb9 is also consistent with the posterior shift in Cdx
expression in the developing chick neural tube.
Activation of early Hox expression by FGF is mediated via CDX
activity
We demonstrated that FGF treatment leads to an anteriorization of the
expression domain of several HoxB (Hoxb6-Hoxb9) genes in the
chick neural tube. In addition, we showed that cdx gene activity is
required to transduce this FGF signal by using a dominant negative form of
Xcad3 (XcadEnR) (Fig.
5). Furthermore ectopic expression of XcadVP16 can induce
3' HoxB genes and in combination with FGF induce 5'
HoxB members in the hindbrain. These results are consistent with the
Hox expression data obtained in Xenopus neural tissue following
modulation of CDX activity (Isaacs et al.,
1998; Pownall et al.,
1996
) and also with ectopic expression of chick cdxB in
cardiac tissue, which induces a posterior program of Hox expression
(Ehrman and Yutzey, 2001
).
Such a relationship was also suggested by the presence of CDX recognition
boxes in the vicinity of mouse Hox regulatory regions
(Charité et al., 1998
;
Subramanian et al., 1995
;
Taylor et al., 1997
). However,
null mutations of Cdx1 in mouse lead to the mis-regulation of
anterior Hox genes only in the mesoderm, not in neural tissue
(Subramanian et al., 1995
).
This could be due to a difference in the function of Cdx genes
between mouse and chick. FGFR1 mutants display changes in Hox expression
exclusively in the mesoderm and expression of Cdx genes is not
affected (Partanen et al.,
1998
). This is a hypomorphic allele but could also reflect the
fact that the FGF effect we observed is not mediated thought FGFR1. When we
tested different FGF ligands, only FGF4 and FGF2 (not FGF8 or FGF10) had an
effect on Hox expression. These two members of the FGF family can use FGFR1,
FGFR3 or FGFR4 to transduce their signal
(Szebenyi and Fallon, 1999
).
All three receptors are present in the chick neural tube
(Walshe and Mason, 2000
) and
it is thus possible that FGFR3 or FGFR4 are preferentially used or can
compensate for FGFR1 in the neural context of our experiments.
Sensitivity to early RA or FGF treatment in the neural tube defines
two distinct groups in the HoxB complex
When we compared the RA and FGF sensitivity for all of the HoxB
genes in early chick embryos, we did not find any genes for which the anterior
boundary was anteriorized or induced by both treatments at the stages
examined. Rather, the responsiveness of members of the HoxB complex
to the two signaling pathways seemed to be mutually exclusive during the
stages examined. The sharp reciprocal transition from RA to FGF responsiveness
in moving from the 3' (Hoxb1 to Hoxb5) to the 5'
(Hoxb6-Hoxb9) Hox genes is surprising
(Fig. 6). In mouse the 3'
Hox genes do not respond uniformly to RA treatment, as there is a progressive
temporal shift in their competence or ability to respond to RA during
gastrulation, such that successively more 5' genes respond in later time
windows (Bel-Vialar et al.,
2000; Conlon, 1995
;
Conlon and Rossant, 1992
;
Marshall et al., 1992
;
Morrison et al., 1997
). Hence,
it had been suggested that the most posterior 5' Hox genes might also be
progressively sensitive to RA in later stages at the end of or after
gastrulation. While our experiments demonstrated a clear drop-off in RA
responsiveness, they focused on the early stages of expression, during the
phase when initial Hox patterns are being established. Therefore it is
possible there may be further changes or shifts in the RA sensitivity of
5' Hox genes at later stages. However, later RA responsiveness might be
complicated because it occurs during a maintenance or a refinement phase of
Hox expression, as has been proposed for retinoic acid function in mouse
mesoderm and skeletal structures (Kessel,
1992
; Kessel and Gruss,
1991
).
HoxB genes and the competence to respond to the FGF
signaling pathway
Interestingly, upon FGF treatment the expression of the FGF responsive
5' Hox genes reach the same anterior level just posterior to the otic
vesicle, which corresponds to the limit between the hindbrain and the spinal
cord. Hence, the inability of the 3' Hox genes to respond to early FGF
signaling may be directly or indirectly related to a lack of competence of the
hindbrain itself in response to FGF treatment. Like the sharp transition in RA
response, this too is surprising, as in later stages, FGF is expressed in the
region and FGF treatment of the chick neural tube leads to ectopic expression
Krox20 and kreisler in the hindbrain while inhibition of FGF
signaling downregulates their hindbrain domains
(Marin and Charnay, 2000).
Hoxa2 expression is also modulated in the anterior chick hindbrain in
response to FGF8 signals generated at the mid/hindbrain isthmus
(Irving and Mason, 2000
;
Trainor et al., 2002
). Hence,
FGFs are expressed in the hindbrain region and the hindbrain is capable of
responding to FGF signaling in some contexts or stages.
Our results show that the FGF responsiveness of 5' Hox genes is
dependent upon CDX activity and that the response of Cdx expression
is itself limited to the spinal cord region. This raises the possibility that
the lack of competence in FGF response of 3' Hox genes in the hindbrain
is a consequence of the restriction in Cdx activation in the
hindbrain following FGF treatment. Supporting this idea we showed that
Hoxb4 is expanded in the hindbrain upon XcadVP16 ectopic
expression and that the combination of Cdx gene activity and FGF are
able to induce Hoxb9 in the hindbrain. These findings are consistent
with ectopic expression experiments using adenoviral vectors in chick cardiac
tissues, where anterior expression of cdxB is capable of inducing
posterior genetic programs, such as expression of Hoxa6, Hoxc6 and
Hoxc8 (Ehrman and Yutzey,
2001). We also found that the dominant negative XcadEnR
construct does not antagonize the RA-induced expansion of Hoxb4
expression in the hindbrain, indicating that the RA response is not being
directed through CDX activity at this stage.
These results suggest that in early stages all Hox genes are competent to
respond to the FGF signal if CDX proteins are present. During gastrulation,
Cdx expression domains are very dynamic and move posteriorly during
regression of the node (Frumkin et al.,
1993; Marom et al.,
1997
; Morales et al.,
1996
). Since the node is a source of FGF
(Mathis et al., 2001
), it is
possible that the neural plate in the pre-otic region is transiently exposed
to FGF signals during regression of the node. This early FGF input together
with transient Cdx expression could be important in some aspect of
activating expression of anterior Hox genes. The progressive posterior
regression of Cdx expression in the chick might account for a sliding
scale or morphogenetic gradient that sets different AP identities
(Frumkin et al., 1993
). Our
experiments are consistent with this view and we propose that over time, as
each Hox gene gets activated, it sees a more posterior domain of Cdx
expression and consequently has a more posterior limit of expression. This
might help to explain the posterior shift in Hoxb9 expression we
detected by in situ. analysis. As there are multiple cdx products
differentially expressed over time, specificity of recognition in any
individual member could add a further degree of complexity in regulating of
Hox expression or integrating separate signaling pathways.
FGF and models for the accessibility state of Hox loci
Models that attempt to explain the colinear properties of Hox complexes
frequently incorporate global regulation of graded or differential
accessibility of a complex, with variations in the availability of upstream
factors needed to activate local control elements
(Bel et al., 1998;
Kmita et al., 2000
;
Sharpe et al., 1998
). The fact
that even the 5' posterior gene Hoxb9 can be activated by CDX
activity in the hindbrain, implies that at the stage we did our experiments,
all members of the HoxB complex might be accessible in the CNS.
This situation with respect to Hox expression and FGF signaling could be explained in several ways. The first would be that Hox loci are all equally accessible to FGF signaling along the entire AP axis at the time of our experiments and differential modulation is dependent upon variations in the transcription factors (CDX) needed to potentiate respective Hox expression. In this model the anterior boundaries of the different Hox genes in the CNS would not reflect a graded accessibility state of their complex, but instead would be set by FGF though modulation of the timing and the extent of the Cdx expression domain along the AP axis.
Another model to explain the overall accessibility state is that applying
FGF provokes the opening of the Hox complexes in a more anterior position than
normal. This renders them more accessible to transcription factors (CDX
proteins) along the entire AP axis, inducing anterior shifts in expression. It
has been proposed that for a defined AP position, Hox complexes become
progressively more accessible over time
(Gaunt, 2000). In this model,
all Hox loci become accessible along the entire axis at a certain time only
after an FGF-dependent internal clock controlling the relative accessibility
of each gene had fully opened a complex
(Fig. 8A). There is evidence of
a segmental clock in the mesoderm and it has been recently suggested that the
activation of Hox genes is in phase with the segmentation clock and that FGF
is involved in the regulation of the rhythm of this clock
(Dubrulle et al., 2001
;
Zakany et al., 2001
). However
to date, such a clock has not been described for the neural tube and it is
possible that these two tissues use different strategies to define AP values.
Alternatively events or a clock patterning mesoderm may indirectly regulate
events in the neural tube through tissue interactions.
|
Our results argue in favor of a model that includes the dynamic nature of
factors like CDX and modulation of accessibility of the complex. It is clear
that Cdx expression in the chick neural tube is dynamic and dependent
upon FGF signaling and that FGF-mediated stimulation of Hox genes requires
CDX. This highlights the importance of the regulatory interactions between FGF
and Cdx genes, in accord with both models. However, while robust
upregulation of Hoxb4 in the hindbrain can be mediated
Xcad3VP16 alone, induction of Hoxb9 is more patchy or
limited and does not extend as far anteriorly in the hindbrain
(Fig. 7). This suggests a
difference in the competence or accessibility state of Hoxb9 versus
Hoxb4 in the hindbrain. When we overexpressed XcadVP16 in
combination with FGF treatment, Hoxb9 was also strongly upregulated
in the hindbrain, whereas neither of these treatments alone was sufficient to
induce Hoxb9 expression in this domain. This suggests that in
addition to the input from CDX, FGF treatment has in some way rendered the
Hoxb9 locus more accessible, thus allowing the activation of
Hoxb9 transcription. In this combined model
(Fig. 8), FGF signaling would
have a dual role in modulating the accessibility of the Hox complex along the
axis and in activating the expression of Cdx. However, we cannot
exclude the possibility that the combination of ectopic Cdx and FGF
treatment is more effective at inducing Hoxb9, as a result of the
action or induction of other factors/co-factors required to transduce the
CDX-mediated signal and trans-activate the 5' Hox genes in the
hindbrain. It has been suggested that the retinoic acid pathway could also be
involved in the accessibility state of the complexes
(Bel-Vialar et al., 2000;
Kmita et al., 2000
) and
retinoid nuclear receptors are known to be part of complexes containing HAT
and HDAC chromatin remodeling enzymes
(Featherstone, 2002
).
Therefore any progressive opening of chromatin and accessibility of Hox
complexes could be controlled by a balance between the influence of retinoid
and FGF signaling pathways.
Finally, recent results show that in the neural plate cells adopt
successively different mature fates as they move or are forced out of the node
region toward more anterior regions
(Mathis et al., 2001). These
same authors also present data indicating that FGF signals in a node stem zone
are used to maintain a consistent pool of immature neural precursors during
elongation of the tube. This data opens the alternative possibility that FGF
acts as a caudalizing factor for the neural tube because it prolongs the
window of time during which cells are exposed to additional caudalizing
factors, FGFs, WNTs or retinoids
(Vasiliauskas and Stern,
2001
). Our results lend strength to the idea that cdx
genes appear to integrate signaling from multiple signaling pathways and it is
tempting to suggest that CDX is a pivotal general caudalizing factor.
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ACKNOWLEDGMENTS |
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