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First published online 25 June 2008
doi: 10.1242/dev.015909
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Department of Development and Genetics, The Babraham Institute, Babraham, Cambridge CB22 3AT, UK.
* Author for correspondence (stephen.gaunt{at}bbsrc.ac.uk)
Accepted 6 June 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cdx, Overexpression, Homeotic transformation, Hox, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The establishment of vertebrate Hox gene expression
patterns along the developing AP axis is poorly understood. Two plausible
models are the instructional (morphogen) gradient model and the timing model
(Gaunt, 2000). In the gradient
model, Hox expression boundaries establish at threshold concentrations of a
Hox gene activator. Suggested activators are retinoic acid, Fgf, Wnt and Cdx
proteins (Deschamps and van Nes,
2005). The timing model, by contrast, proposes that patterns are
generated by the sequential activation of Hox genes within the growing
primitive streak/tailbud region
(Dollé et al., 1989).
Consistent with this model, Hox genes have been shown to be activated
sequentially, 3' to 5', along their clusters (temporal
co-linearity) (Izpisúa-Belmonte et al., 1991).
The Cdx proteins are regulators of Hox genes within both neurectoderm and
mesoderm (Bel-Vialar et al.,
2002; Charité et al.,
1998; Ehrman and Yutzey,
2001; Epstein et al.,
1997; Isaacs et al.,
1998). Knockout of Cdx genes may result in posterior shifts of
both Hox gene expression and vertebral morphologies
(Chawengsaksophak et al., 1997;
Subramanian et al., 1995;
van den Akker et al., 2002;
van Nes et al., 2006).
Posterior shifts of vertebral types are commonly described as
posterior-to-anterior homeotic transformations
(van den Akker et al., 2002).
The Cdx proteins are distributed as posterior-to-anterior concentration
gradients along the developing embryo (Beck
et al., 1995; Gamer and
Wright, 1993; Meyer and Gruss,
1993), and evidence from Cdx/lacZ reporter mice has shown
that these gradients may form by decay of Cdx protein in cells once they leave
the region of intense Cdx gene expression within the primitive streak/tailbud
(Gaunt et al., 2003;
Gaunt et al., 2005).
An important issue is whether the Cdx protein gradients function as
morphogen gradients for the specification of Hox gene expression boundaries. A
proposal here is that the expression boundary of a Hox gene may move forward
in the gradient until its enhancer elements bind a minimal threshold level of
Cdx protein. In this scenario, it is the dose of Cdx protein in a cell that
can determine whether or not it will express a given Hox gene. Struhl et al.
(Struhl et al., 1989)
identified criteria to test for a morphogen gradient in Drosophila,
and these can also be applied to the mouse. Thus, two experimental predictions
are that a mouse Hox expression boundary might be shifted forwards: (1) by
increase in the number of Cdx-binding elements within the Hox gene enhancer,
so making the gene more sensitive to the endogenous Cdx protein gradient; and
(2) by increase in the dose of Cdx product within the normal tailbud domain,
so shifting the Cdx protein decay gradient forward along the embryo. The first
of these two predictions has already been examined
(Charité et al., 1998;
Gaunt et al., 2004).
Multimerization of the Hoxb8 or Hoxa7 enhancer elements
results in forward shift in the expression boundaries of Hox/lacZ
reporters in transgenic mice. This effect depends upon intact Cdx-binding
motifs within the additional enhancer elements. These studies do not, however,
provide clear-cut evidence for the Cdx morphogen gradient model as enhancer
multimerization also causes earlier activation of the transgene
(Gaunt et al., 2004), making
the main findings also explicable in terms of the timing model.
In the present paper we adopt the second approach described above. We
describe lines of mice (OE1, OE2 and OE4) overexpressing Cdx1, Cdx2
and Cdx4 transgenes under the control of their own promoter/enhancer
elements (Gaunt et al., 2003;
Gaunt et al., 2005). These OE
transgenes are transcribed in early embryos within normal Cdx expression
domains where they generate elevated levels of Cdx proteins. For
Cdx2, we show that raised levels of Cdx protein in tailbuds result in
a forward extension of the protein gradient along the embryonic axis. When
crossed with a Hoxa7/lacZ reporter line, OE1, OE2 and OE4 lines show
anterior shifts in Hox/lacZ expression boundaries without, at least
for OE1 and OE4, any accompanying change in the initial timing of
Hoxa7/lacZ activation. This is consistent with a role for Cdx protein
morphogen gradients in the positioning of Hox expression boundaries. The new
transgenic lines display homeotic, mainly anterior-to-posterior,
transformations in vertebral types, and also forelimb (OE1) and tail (OE2)
abnormalities.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Gaunt et al., 2005). The 450
bp fragment of lacZ/SV40 DNA remaining in the 3' untranslated
regions (UTRs) of the OE constructs provides a polyA signal and permits
detection by PCR and in situ hybridization. Constructs were cut from
Bluescript vectors using KpnI plus SpeI (Cdx1),
XhoI plus SpeI (Cdx2) or KpnI plus
NotI (Cdx4).
Transgenic embryo production was as described
(Gaunt et al., 2003),
injecting DNA into embryos of F1 (CBAxC57Bl6) mice. Transgenic lines were
maintained as heterozygotes by crossing with normal F1 mice. Embryos were
taken to be at 0.5 days of development at midday on the day of the copulation
plug. Genotyping of embryos by PCR was carried out on DNA prepared from
extra-embryonic membranes (for embryos over 8.5 days) or from the embryos
themselves after lacZ staining and photography (for embryos up to 8.5
days).
Cdx protein detection in embryos
For western blotting, tissue lysates were prepared from pooled
stage-matched embryo samples. These were run on 10% SDS/PAGE gels and then
blotted on to Immobilon-P (Millipore) membranes. Membrane blocking and
antibody incubations were as described in Abcam protocols. Primary antibodies
were anti-Cdx1 (ab24000, Abcam), anti-Cdx2 (CDX2-88, BioGenex), anti-Cdx4
(ARP32765, Aviva Systems) and anti-GAPDH (ab9484, Abcam). Secondary antibodies
were HRP-conjugated anti-mouse or anti-rabbit IgG (Abcam), and detection was
by luminescence using Millipore Immobilon HRP substrate. The fold-levels of
Cdx overexpression for each OE line were estimated by densitometry scans using
the Scion Image programme.
For immunohistochemistry, embryos were fixed for 2 hours in 80% methanol and 20% DMSO (v/v), and then washed/stored in methanol prior to embedding in paraffin wax. Sections (10 µm) were de-waxed in xylene and rehydrated prior to staining with anti-Cdx2 monoclonal antibody and a mouse-on-mouse HRP detection kit (Vector labs). Sections were counterstained with 0.5% Neutral Red.
Skeleton preparations, lacZ staining and in situ hybridization
Most skeleton preparations were made upon pups within the first 2 days of
life (van den Akker et al.,
2001). The Hoxa7/lacZ reporter mouse line and the
staining method to detect lacZ activity were as described
(Gaunt et al., 2004). This
mouse line carries a chick Hoxa7/lacZ transgene containing at least
one functional and evolutionarily-conserved Cdx-binding motif. In situ
hybridization was carried out as reported previously
(Gaunt et al., 1999).
Transgene expression was detected using a 450 bp lacZ/SV40 probe
(Gaunt et al., 2003). The
Mox1 probe was prepared from IMAGE clone 3984366
(Lennon et al., 1996).
Hoxb4 and Hoxa10 probes were as described by Gaunt et al.
(Gaunt et al., 1989;
Gaunt et al., 1999).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three independent OE2 founder mice showed kinked tails. Only two of these allowed production of independent lines for analysis in this study. Offspring of both show kinked tails with terminal scabs in some but not all transgenic pups (Fig. 2C). The most severely affected newborns also have shortened tails with multiple protuberances, haemorrhages and scabs at the tip (Fig. 2D). The third OE2 founder mouse sires only non-transgenic live pups. All his transgenic offspring die at 8.5 to 11 days gestation displaying large clubbed tailbuds (Fig. 1G). We find a similar pattern of embryonic death and club-tailing in a smaller proportion of the transgenic embryos produced by the OE2 transgenic lines.
Independently derived OE4 founder mice were screened for defects in the vertebral patterning of their offspring. Two lines, producing similar defects, were examined in this study.
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). Protein levels for Cdx1 decline earlier than for Cdx2 and
Cdx4. All three proteins form doublets on SDS gels, suggestive of
post-translational modification (Gross et
al., 2005).
Fig. 3B shows a time-course
study with the OE2 line 1. Transgenic embryos show clear reduction in total
Cdx2 protein by 28-somite stage, which is similar to that seen with normal
embryos (Fig. 3A). The earlier
fall in Cdx1 protein and later fall in Cdx4
(Fig. 3A) are also likely to be
mimicked in, respectively, the OE1 and OE4 transgenic embryos as similar
time-courses have already been described for mRNA expression from the
endogenous promoter sequences used in our constructs
(Gaunt et al., 2003;
Gaunt et al., 2005).
The above studies showed the importance of using embryos at the same developmental stage when comparing transgenic and non-transgenic embryos sired by OE studs. We used 15- to 16-somite stages (OE2 and OE4) and 10- to 12-somite stages (OE1). For each mouse line, the levels of the corresponding Cdx proteins are seen to be higher in the transgenic embryos (Fig. 3C). For Cdx2, the study included embryos sired by the OE2 founder whose transgenic progeny always die at 8.5 to 11 days gestation. These embryos show much higher levels of Cdx2 protein than those produced by the two OE2 lines. We therefore distinguish between OE2 peak-expressers and OE2 lines in this and subsequent experiments.
The OE transgenes include a 450 bp non-translated fragment that contains
SV40 polyA and terminal lacZ sequences. Riboprobes to this
fragment allow detection of transgene expression by in situ hybridization
(Fig. 3D). OE1, OE2 and OE4
transgenes are mainly confined in their expression to the primitive
streak/tailbud region of 8.5 to 8.7 day embryos, with mRNA boundaries in
paraxial mesoderm located some distance posterior to the presomitic/somitic
boundary (not shown, but as previously found for endogenous Cdx1,
Cdx2 and Cdx4 genes) (Gaunt
et al., 2005).
Forward shift in the Cdx2 protein gradient along the axis of OE2 embryos
Of the three anti-Cdx antibodies examined in this study, the anti-Cdx2
antibody provided the highest signal-to-background ratio when tested in
immunohistochemistry. This antibody allowed clear visualization of the Cdx2
protein gradient within sections of normal 8.7 day embryos
(Fig. 4A-C). The distribution
is similar to that described earlier for Cdx2/β-galactosidase protein
expression in reporter mice (Gaunt et al.,
2005). Posterior neural and mesoderm tissues are intensely
labelled and the level of labelling declines on proceeding forward along the
embryo. Clear posterior-to-anterior gradients are seen within the newly formed
somites (Fig. 4C) and
developing neural tube (Fig.
4B).
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)] in OE2 (Fig.
4D) but only to SIV in wild type
(Fig. 4C).
Axial skeletal defects in OE1, OE2 and OE4 newborn mice
Table 1 summarizes the
defects observed. Most of the defects may be classified as
anterior-to-posterior transformations, symbolized P
A in
Table 1 and van den Akker et
al. (van den Akker et al.,
2002). However, some of the more caudal defects are
posterior-to-anterior (P
A) transformations. For each OE type we examined
skeletal defects in newborns of the two independent transgenic lines. As the
range of defects observed was apparently the same for lines 1 and 2, the data
are pooled in Table 1.
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In the thoracic region, the nuchal ligament normally extends from v9 (Fig. 5H) to the skull. The thoracic attachment may be shifted forward in OE1, OE2 and OE4 mice (Fig. 5A,C,E,F,G). The caudal-most vertebra with rib attached to the sternum is normally v14 (Fig. 6F). This vertebral phenotype is shifted forward in some OE1 (Fig. 6C), OE2 and OE4 mice. The caudal-most vertebra bearing ribs is normally v20 (Fig. 6B,F). This vertebral phenotype may be shifted anteriorly in OE1 (Fig. 6A,C), OE2 (Fig. 7A,B) and OE4 mice. All of these thoracic vertebral defects are anterior-to-posterior transformations. However, some OE1 and OE4 mice show posterior-to-anterior transformations in the thoracic region. Thus, some OE1 mice show posterior shift in the thoracic attachment of the nuchal ligament (not shown), and some OE1 and OE4 mice show posterior shifts in both the caudal-most vertebra with rib attached to sternum, and in the caudal-most vertebra bearing ribs (Fig. 6D). Rib fusions and mis-alignments of ribs on the sternum are common in all OE mice (Fig. 6E).
In the lumbar region there is normally some homeotic variation. Thus, wild-type mice may show either five or six lumbar vertebrae, so that the first vertebra contributing to the sacrum may be either v26 or v27 (Fig. 6B). Vertebra 27 of normal animals may sometimes show a lumbar phenotype on one side and sacral on the other. In some OE1 mice the first vertebra with a sacral phenotype is shifted forward, in an anterior-to-posterior transformation, up to the level of v24 (Fig. 6A). However, some OE4 mice show a posterior shift in the first sacral vertebra (posterior-to-anterior transformation; not shown). Like wild-type, OE1 and OE4 mice show five or six lumbar vertebrae. OE2 mice may show seven lumbar vertebrae (Fig. 7A,B) and mis-alignments of lumbar vertebrae (Fig. 7C).
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OE1, OE2 and OE4 effects upon expression of a Hoxa7/lacZ reporter
Heterozygous OE1 line 1, OE2 peak-expresser or OE4 line 1 stud males were
crossed with homozygous Hoxa7/lacZ females, and the resultant embryos
examined by β-galactosidase protein staining
(Fig. 8). Each embryo was
genotyped by PCR, allowing OE1-, OE2- or OE4-positive embryos to be compared
directly for β-galactosidase distribution with their OE1-, OE2- or
OE4-negative littermates.
At 10.5 days, the anterior limits of β-galactosidase protein activity
are readily assessed relative to the boundaries of the forelimb, which
normally extends from prevertebra (v) 4 to 9. In OE non-transgenic embryos
(Fig. 8A)
(Gaunt et al., 2004) the
boundaries are located four somites posterior to the forelimb in paraxial and
lateral plate mesoderms (at the level of v13) and at a level just behind the
anterior border of the forelimb in spinal ganglia (sg) (at the level of sg5).
β-galactosidase activity boundaries in OE1-, OE2- and OE4-positive
embryos are shifted anteriorly relative to OE-negative littermates, and these
forward shifts occur in spinal ganglia, prevertebrae and lateral plate
mesoderm (Fig. 8B-D). The
shifts are greatest in OE1 embryos: up to the level of sg2, and up to the
levels of v2 in lateral plate mesoderm and v11 in paraxial mesoderm
(Fig. 8B).
The Hoxa7/lacZ transgene commences expression in wild-type embryos
at headfold stage (Fig. 8E)
(Gaunt et al., 2004). To test
for an effect of OE1, OE2 and OE4 upon the initial timing of
Hoxa7/lacZ activation, OE transgenic embryos sired by the same studs
as used in Fig. 8B-D were
examined at pre-, early- and later-headfold stages (8 to 8.25 days of
gestation) (Fig. 8E). OE1 and
OE4 transgenic embryos show no difference from wild-type in the time of
initial Hox/lacZ activation: all commence expression at the later
headfold stage. By contrast, OE2-positive embryos commence expression earlier,
at the pre-headfold stage.
OE1 and OE4 effects upon endogenous Hox gene expressions
We examined expressions of Hoxb4 and Hoxa10 by in situ
hybridization upon sections of 12.5 day OE1 and OE4 embryos (see Fig. S1 in
the supplementary material). Expressions of these Hox genes mark,
respectively, the second cervical and first lumbar vertebrae
(Gaunt et al., 1989;
Gaunt et al., 1999). OE1, but
not OE4, embryos showed forward shift of Hoxb4 to the level of the
first cervical vertebra. Hoxa10 expression is apparently normal in
both OE1 (not shown) and OE4 embryos.
Forelimb and tailbud defects in OE mice
The forelimb defects in OE1 (lines 1 and 2) mice most commonly appear as
in-turning of the feet with reduction in the number of digits, from one to
four (Fig. 2A). Seventy-nine
percent (n=16) of OE1 transgenic offspring showed forelimb defects
after staining of skeletons (see Fig. S2 in supplementary material). OE1
hindlimbs are normal. There is no consistent defect in forelimb skeletons.
Loss of the radius is common, apparently causing in-turning of the foot. The
humerus or humerus plus radius and ulna may be absent. It is likely that
absence of skeletal elements in the forelimbs of OE1 mice can be explained by
the small size of the forelimb buds seen at 10.5 days gestation and caused by
their restriction posteriorly (Fig.
2F). The posterior and anterior limits of the limbud are normally
specified by the position of Hox expression boundaries within the flank
mesoderm (Cohn et al., 1997).
For OE1, Hoxa7/β-galactosidase protein expression is dramatically shifted
forward in flank mesoderm (Fig.
8B). It therefore seems likely that forward shifts in endogenous
Hox expression are responsible for shifting anteriorly the posterior limits of
OE1 limbuds. Occasional forelimbs in OE1, OE2 and OE4 mice show increased
numbers of digits, with otherwise normal limb skeletons (see Fig. S2D in
supplementary material). We considered that these might result from larger
limbuds, caused by forward shift in Hox boundaries that specify their anterior
limits. However, we did not detect larger limbuds in either OE1, OE2 or OE4
embryos.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Charité et al., 1998;
Ehrman and Yutzey, 2001) or
ubiquitous (Epstein et al.,
1997; Isaacs et al.,
1998) activation of Cdx product anterior to the normal domains of
Cdx expression will induce more anterior expression of Hox genes. These
findings include some of the earliest evidence that Cdx genes are upstream
regulators of Hox genes. An important difference in our work, however, is that
we now show that elevation of Cdx gene transcription within the normal spatial
domain is also able to anterioriorize Hox expression, with accompanying
homeotic transformations.
Our findings are complementary to Cdx gene knockout analyses
(Chawengsaksophak et al., 1997;
Subramanian et al., 1995;
van den Akker et al., 2002;
van Nes et al., 2006) in which
the effects of reduced Cdx expression are analysed. The knockout studies have
not, however, examined for shifts in Cdx protein gradients along the embryo,
or provided any evidence for changes in the timing of Hox gene activation. The
Cdx knockout studies presented so far cannot, therefore, shed light on the
relative merits of gradient- or temporal co-linearity-based mechanisms for the
positioning of Hox expression boundaries.
OE1, OE2 and OE4 homeotic defects extend to different anterior limits
Increased doses of Cdx proteins result in homeotic transformations in
vertebral types. These transformations are usually most anterior for OE1
(skull/v1 level), more posterior for OE2 (v1/v2 level) and most posterior for
OE4 (v7 level). It is unlikely that these differences simply reflect different
levels of overexpression within individual lines of transgenic mice, as (1)
for overexpressers of each Cdx gene, the same results were obtained for two
independently derived lines; and (2) OE1 line 1 (4-fold normal Cdx1 levels)
shows more anterior defects than does OE4 line 1 (30-fold normal Cdx4). We
consider it more likely that the different positions of defects in OE mice
reflect the different anterior limits of expression normally shown by the
various Cdx genes (Gaunt et al.,
2005).
Nature of OE1, OE2 and OE4 homeotic transformations
Apart from a spatial difference, the nature of the homeotic transformations
are apparently similar for all three OE lines, supporting earlier conclusions
that there is normally overlap in function between the three Cdx proteins
(van den Akker et al., 2002;
van Nes et al., 2006). The
effects of increased Cdx protein dose on homeotic shifts are largely opposite
to those of reduced Cdx gene product, as examined in Cdx knockouts
(Subramanian et al., 1995;
van den Akker et al., 2002).
However, in contrast to the clear homeotic transformations found in OE4
embryos, Cdx4 mutants display transformations only when combined with
mutations in Cdx1 or Cdx2
(van Nes et al., 2006).
Many of the obvious homeotic effects in OE mice are located in the neck and
anterior thoracic vertebrae and may be classified as anterior-to-posterior
transformations. For example, all three transgenic lines show forward shift of
thoracic-type vertebrae, with attached ribs, into the region that is normally
cervical. The reasons for cervical vertebral fusions and splits are less
apparent and we suggest the following. Fusion between v1 and the skull is
common in OE1 mice. v1 forms normally from the posterior half of somite 5 (s5)
and the anterior half of s6, and the occipital condyles of the skull form from
anterior s5 (Couly et al.,
1993). If the level of Cdx protein is elevated in anterior s5 then
we suggest that this tissue acquires v1 characteristics and therefore fails to
split from v1, so producing a skull/v1 fusion. Splits within v1 are also seen
in OE1 mice. If the level of Cdx protein is elevated in anterior s6 then we
suggest that this tissue acquires v2 characteristics and fails to fuse
properly to s5, so producing a split. Both of these fusions and splits can
therefore be explained as anterior-to-posterior transformations, and the
mechanisms we describe may also explain vertebral (and rib) fusions and splits
observed at other levels of the vertebral column. Anterior-to-posterior
transformations are also seen in posterior thoracic and lumbosacral regions.
For example, the most caudal thoracic vertebrae with ribs attached to sternum
and the most caudal vertebrae bearing ribs are shifted forward in some OE1,
OE2 and OE4 mice, and the first sacral vertebra is shifted forward in some OE1
mice.
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The extent of the transformations is highly variable between individual
OE1, OE2 or OE4 mice. Similarly there is much variability between the
vertebral patterning of individual Cdx knockout mice
(Subramanian et al., 1995;
van den Akker et al., 2002)
and of mice with defects in Cdx1 gene regulation
(Pilon et al., 2007). In
striking contrast, however, the vertebral formula and patterning of normal
mice is highly conserved. We suggest that overlapping and interacting patterns
of developmental gene expression in embryos may, over evolutionary time, have
reached a state of mutual compatibility and equilibrium. When embryos are
tipped out of this equilibrium by, in our case, overexpression of Cdx genes,
the end result is wide variability between individuals in the exact nature and
extent of the defects.
Retinoic acid administered to pregnant mice generates phencopies in their
offspring of the vertebral transformations that we now find in OE mice. Thus,
retinoic acid given to mothers at 7.3 days pregnancy results in
anterior-to-posterior transformations, while administration at 8.5 days also
produces some posterior-to-anterior transformations in the posterior thoracic
and lumbosacral regions (Kessel and Gruss,
1991). It is known that Cdx1 is upregulated by retinoic
acid (Houle et al., 2000;
Houle et al., 2003). Our
results therefore indicate that effects of retinoic acid on vertebral
patterning could, at least in part, be mediated by its effect on
Cdx1. Transpositions in vertebral morphologies have also been
reported in Wnt3a and Fgfr1 mutant mice
(Ikeya and Takada, 2001;
Partanen et al., 1998). As Wnt3a and Fgf are expressed in
the primitive streak/tailbud (Deschamps and
van Nes, 2005), and both Wnt3a
(Pilon et al., 2006) and Fgf
(data from chick) (Bel-Vialar et al.,
2002) proteins are known activators of Cdx1 and
Cdx4, it is likely that at least some of their effects on vertebral
patterning could also be mediated by Cdx protein levels
(Lohnes, 2003). Pilon et al.
(Pilon et al., 2007) have
recently shown that normal Cdx1 expression in embryos depends upon
both Wnt and retinoic acid stimulation.
The defects in the tail vertebrae of OE2 mice seem unlikely to be homeotic
transformations. Vertebral mis-alignments and tail splits in OE2 mice might
instead be due to our observed effects of Cdx2 overexpression on
abnormal growth and segmentation in the tailbud mesoderm. Cdx2
knockout in mice (van den Akker et al.,
2002) and Cad knock-down in insects
(Shinmyo et al., 2005) both
result in premature axial termination. Our results suggest that this effect in
mice may, as in insects, be due to impaired growth in the tailbud.
Cdx proteins as regulators of Hox expression
When crossed with Hoxa7/lacZ reporter mice all three OE lines
produce forward shifts in Hoxa7/lacZ expression in 10.5 day embryos.
We also find a forward shift in the expression of endogenous Hoxb4 in
OE1 embryos, but not OE4. These shifts in Hox expression boundaries are
opposite in direction to those reported in Cdx knockout mice
(Subramanian et al., 1995;
van den Akker et al., 2002)
and in mice defective in Cdx1 regulation
(Houle et al., 2003;
Pilon et al., 2007). We
propose that dose of Cdx protein normally plays a key role in the positioning
of Hox gene expressions. Differential dose of Cdx protein along the axis is
provided by the additive effects of the three Cdx proteins, as each has a
different anterior boundary of expression. In addition, differential dose in
gradients is provided by the decay of Cdx proteins in cells left behind by the
regressing tailbud (Gaunt et al.,
2003).
We next consider whether Cdx effects are mediated in embryos by morphogen
gradient or timing mechanisms, reviewed as models 1 and 2 by Gaunt
(Gaunt, 2000). A Cdx morphogen
gradient model predicts (1) that Hox boundary positions are responsive to dose
of Cdx proteins, (2) that Cdx proteins are expressed in gradients along the
head-tail axis, and (3) that upregulation of Cdx protein should shift forward
the position of the gradient, thereby generating a forward shift in Hox
expression boundaries. These predictions are consistent with the observations
described in this paper. In terms of the timing model, it is less obvious why
increased dose of Cdx protein should shift Hox expression boundaries forwards.
Such a shift would presumably be achieved by an earlier initial activation of
Hox expression. We did detect earlier onset of Hoxa7/lacZ expression
in OE2 embryos sired by the peak-expresser line, but not in either OE1 or OE4
embryos. We earlier reported precocious expression of Hoxa7/lacZ
constructs that contain multiple Cdx-binding sites
(Gaunt et al., 2004).
Together, these findings lead us to conclude that dose of Cdx protein binding
to a Hox enhancer may indeed influence its time of initial expression. A
similar conclusion has been reached in Xenopus
(Isaacs et al., 1998).
However, OE1 line 1 embryos show no precocious expression of
Hoxa7/lacZ yet display greater forward shifts in their later
expression than OE2 embryos sired by the peak-expresser stud. This lack of
clear correlation between the time of Hoxa7/lacZ first expression and
the final position of its expression boundary leads us to conclude that Hox
expression boundaries cannot simply be set by the time of their initial
expression (temporal co-linearity model).
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; Forlani et al.,
2003). The model of Fig.
9 can accommodate these observations. It assumes that temporal
co-linearity and Cdx gradients operate together in the establishment of Hox
boundaries. The proposal is that Cdx protein concentration can regulate the
rate and extent of sequential Hox gene activation (temporal co-linearity). A
forward shift in the Cdx protein concentration gradient of OE mice would
result, in this model, in a forward shift in Hox gene expression boundaries.
For both OE1 and OE4, embryos from lines showing modest (four- to fivefold)
increase in Cdx protein levels show the same magnitude of homeotic vertebral
shifts as do embryos of higher expresser lines. This appears inconsistent with
a simple morphogen gradient model but may be explained if temporally co-linear
Hox gene activation becomes the rate-limiting step. In the proposal of
Fig. 9 the co-linear activation
of Hox genes in the tailbud can alone generate approximately correct patterns
of Hox gene expression but then these later become refined or focused along
Cdx protein gradients. Refinement of Hox boundaries initially generated by
temporal co-linearity in the tailbud affords Cdx gradients an important yet
possibly subtle contribution, and could explain why some authors have
concluded that the effects of Cdx gene knockout on anterior boundaries of Hox
expression are modest (Deschamps and van
Nes, 2005) or even not detected
(Tabariès et al.,
2005).
Cdx overexpresser mice display homeotic shifts most clearly over anterior
parts of the vertebral column. Other morphogens might regulate Hox boundaries
more posteriorly. Gdf11 encodes one possible candidate
(McPherron et al., 1999) as
knockout mice show posterior-to-anterior transformations in the anterior
lumbar region. As for Cdx, Gdf11 product apparently exerts a dose-dependent
effect upon vertebral shifts.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/15/2511/DC1
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ACKNOWLEDGMENTS |
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