1 Cancer Research Labs, Queen's University, Kingston, ON K7L 3N6, Canada
2 Institut de Génétique et de Biologie Moléculaire et
Cellulaire, Collège de France, BP 163-67404 Illkirch Cedex, CU de
Strasbourg, France
* Author for correspondence (e-mail: petkovic{at}post.queensu.ca)
Accepted 16 December 2002
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SUMMARY |
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Key words: Gastrulation, Tail bud, Cytochrome P450, Homeotic transformations, Mouse mutant, Retinoids, Retinoic acid, Teratogenesis, Spina bifida, Caudal regression, Cyp26a1, RAR, Wnt3a, Brachyury, Fgf8, Hnf3b, Cdx4, Hoxd11, Tbx6, Raldh2
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INTRODUCTION |
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Although RA is undetectable during the early stages of gastrulation
(E6.5-E7.5) (Rossant et al.,
1991; Ulven et al.,
2000
), Cyp26a1 is expressed in the primitive streak and
its newly formed mesoderm (Fujii et al.,
1997
). The onset of Raldh2 expression at E7.5 complements
that of Cyp26a1, which shifts from the posterior embryo to all three
germ layers of the anterior half. At this stage, ingressing epiblast cells may
encounter a pool of RA, generated by Raldh2, in newly formed mesoderm
that surrounds the primitive streak and node region
(Niederreither et al., 1997
;
Rossant et al., 1991
), and
predominantly contributes to somite formation
(Kinder et al., 1999
). A state
of RA deficiency generated by loss of RALDH2 activity leads to severe
developmental defects that affect late-streak-derived structures, such as the
trunk somites, heart and limbs
(Niederreither et al., 1999
).
By contrast, exposure to teratogenic doses of RA at E7.5 affects development
of more anterior structures, such as the face and brain
(Iulianella and Lohnes, 1997
;
Simeone et al., 1995
). During
this stage, CYP26A1 may protect anterior structures from RA exposure, as some
Cyp26a1-null animals exhibit exencephaly
(Abu-Abed et al., 2001
).
During late gastrulation (E8.5-E10), Cyp26a1 expression shifts
posteriorly, becoming mainly restricted to the open neuropore, hindgut
endoderm and tail bud mesoderm. Thus, the domain of Cyp26a1
expression in the tail bud abuts that of Raldh2 in the trunk
mesoderm, a complementarity that persists until somitogenesis is complete
(E13). Tissues derived from the tail bud at this stage include paraxial
mesoderm of the lumbosacral region, and, to a lesser extent, lateral plate
mesoderm, notochord, posterior neural tube and hindgut
(Kinder et al., 1999
). In the
absence of CYP26A1 function, assays involving a RA-responsive lacZ
reporter transgene reveal that tail bud tissues normally devoid of RA stain
positively for RA activity (Sakai et al.,
2001
). Loss of CYP26A1 function is embryonic lethal, with mutant
embryos dying from caudal regression associated with spina bifida, imperforate
anus, agenesis of the caudal portions of the digestive and urogenital tracts,
malformed lumbosacral skeletal elements, and lack of caudal tail vertebrae
(Abu-Abed et al., 2001
;
Sakai et al., 2001
). This
phenotype indicates that CYP26A1 is also essential for maintaining
morphogenetic events during late gastrulation, probably by protecting tail bud
tissues from exposure to RA.
The caudal regression phenotype of Cyp26a1 mutants closely
resembles that of wild-type embryos treated with teratogenic doses of RA at
E8.5-E9.5 (Alles and Sulik,
1990; Kessel,
1992
; Padmanabhan,
1998
). Such RA-treated wild-type embryos show elevated levels of
apoptosis, develop ectopic neural tubes and exhibit severe downregulation of
Wnt3a expression in tail bud tissues
(Alles and Sulik, 1990
;
Iulianella et al., 1999
;
Shum et al., 1999
;
Yasuda et al., 1990
).
Interestingly, loss of WNT3A function leads to a caudal regression syndrome
similar to that observed in Cyp26a1 mutants and RA-treated wild-type
embryos (Takada et al., 1994
).
Finally, resistance to the teratogenic effects of RA has been observed in mice
that lack Rarg, which develop caudal tissues normally and show no
change in Wnt3a expression after RA treatment during late
gastrulation (Lohnes et al.,
1993
; Iulianella et al.,
1999
). Based on these observations, we were interested in testing
whether we could rescue Cyp26a1 mutants from endogenous RA
teratogenicity by disrupting the Rarg gene.
We report that loss of Rarg rescues Cyp26a1 mutants from
embryonic lethality and suppresses their caudal regression phenotype.
Furthermore, ablation of Rarg restores normal gene expression
patterns in the tail bud of
Cyp26a1-/-Rarg-/- double mutants. Our
results suggest that RA signaling mediated by RAR downregulates WNT3A
and FGF8 signaling activities, leading to defects in caudal mesoderm and
definitive endoderm formation. Using this double mutant model, not only do we
better dissect the biological role of CYP26A1 in late gastrulation and tail
bud development, but we also show that it is possible to suppress the lethal
effects of one null mutation by introducing another, a phenomenon as yet
rarely observed in mouse.
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MATERIALS AND METHODS |
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Skeletal analysis
Fetuses were skinned, eviscerated, and dehydrated in 100% ethanol.
Carcasses were stained with 0.03% Alcian Blue in 80% ethanol:20% glacial
acetic acid to detect cartilaginous elements. Specimens were then dehydrated
in 100% ethanol, cleared overnight in 1% potassium hydroxide, and stained
overnight in 0.1% Alizarin Red S in 1% potassium hydroxide. Clearing was
achieved by several changes of 20% glycerol in 1% potassium hydroxide over 1
week, followed by 50% glycerol:50% ethanol for 2-3 weeks. Specimens were
scored under a dissecting microscope and photographed with a digital
camera.
In situ hybridization analysis
Staged embryos were pooled by genotype and rehydrated in a methanol series.
Brachyury, Cdx4, Fgf8, Hnf3b, Hoxd11, Tbx6 and Wnt3a probes
were kindly provided by Drs B. Herrmann, J. Deschamps, G. Martin, S.-L. Ang,
D. Duboule, V. Papaioannou and A. McMahon, respectively. Whole-mount in situ
hybridization was performed according to Wilkinson
(Wilkinson, 1992). After
hybridization, embryos were cleared and photographed under a dissecting scope
(Leica MZ 9.5) with a digital camera.
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RESULTS |
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A Rarg-null mutant background rescues Cyp26a1 mutants from embryonic
lethality
Cyp26a1+/-Rarg+/- double
heterozygous mutant mice were intercrossed to generate compound mutants. For
simplicity, Cyp26a1 and Rarg are abbreviated A1 and
, respectively (e.g. A1-/- and
A1-/-
-/- represent
Cyp26a1-/- and
Cyp26a1-/-Rarg-/- mutants,
respectively). Embryos at E8.5-9.5, E18.5 fetuses and 10-day old newborn pups
collected from A1+/-
+/-
intercrosses were considered for analysis of Mendelian ratios; specimens were
also collected from A1+/-
-/-
females mated with A1+/-
+/-
males, in order to increase the yield of double mutant embryos and fetuses. At
E8.5-9.5, A1-/-
-/- embryos were
collected at approximately a third of the expected ratio, while other
genotypes were observed at the expected Mendelian ratios
(Table 1). At E18.5, the
A1-/-
-/- and
A1-/- genotypes were observed at two-thirds and one-third
of the expected frequencies, respectively. At 10 days post-partum,
A1-/-
-/- mutants were observed
at approx. half the expected ratio, distinguished in some cases by a shortened
and/or kinked tail (Fig. 2A-C).
No A1-/- or
A1-/-
+/- newborns were
recovered 10 days post-partum. Altogether, these results suggest that a
Rarg-null mutant background rescues approximately half of the
A1-/- mutants from lethality. As
A1-/-
-/- mutants obtained at
embryonic and fetal stages were externally indistinguishable from their
wild-type counterparts, it is likely that the
A1-/-
-/- embryos not rescued by
the Rarg-null background were resorbed before E8.5. Previously, we
have recovered Cyp26a1-/- embryos exhibiting early
lethality (Abu-Abed et al.,
2001
). We have also found that exposing
Cyp26a1-/- embryos to subteratogenic doses of RA at E7.5
increases early lethality (S. A.-A., P. D., D. M., C. W., G. M., P. C. and M.
P., unpublished), suggesting that variations in maternal RA status results in
variability in the expressivity of the Cyp26a1-/-
phenotype. Lack of RAR
may make embryos more susceptible to this
phenotype, possibly by potentiating inappropriate signaling through RAR
or RARß. We are currently attempting to characterize the cause of this
early lethality. All generated
A1-/-
-/- animals continued to
gain weight at a rate similar to that of their wild-type offspring. However,
A1-/-
-/- females were apparently
sterile, even after a 3-month exposure to fertile males. The cause of this
infertility is under investigation.
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|
The Cyp26a1-null mutant caudal regression syndrome is
rescued in a Rarg-null background
Previous analysis of Cyp26a1-null mutant embryos revealed that, in
addition to malformed lumbosacral vertebrae and lack of caudal tail vertebrae,
A1-/- mutants suffer from an open posterior neuropore
(spina bifida), as well as agenesis of the posterior urogenital and digestive
systems (Abu-Abed et al., 2001;
Sakai et al., 2001
). The most
severely affected A1-/- mutants lack the entire caudal
body and/or show various degrees of hindlimb fusions (sirenomelia). Skeletal
phenotypes for both A1-/- and
-/- null
mutants have been previously described
(Abu-Abed et al., 2001
;
Iulianella and Lohnes, 1997
;
Lohnes et al., 1993
;
Sakai et al., 2001
) (see
Table 2). Apart from the
lumbosacral truncation and lack of tail vertebrae
(Fig. 2F),
A1-/- mutants exhibit consistent abnormalities of cervical
vertebrae, most of which correspond to posterior homeotic transformations
(Table 2;
Fig. 3B). On the other hand, a
fraction of the
-/- mutants exhibit anterior transformations
of cervical and thoracic vertebrae (Table
2, and data not shown).
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|
We analyzed the vertebral patterns of
A1-/--/- double mutants at E18.5 [for a
detailed description of the wild-type vertebral patterns, see Kessel
(Kessel, 1992
)]. The
Rarg-/- background almost fully rescued
Cyp26a1-null mutants from the caudal regression syndrome. In contrast
to A1-/- mutants (Fig.
2F), A1-/-
-/- skeletons
(Fig. 2C,E) exhibited six
normal, evenly spaced lumbar, three normal sacral and 15 ossified caudal
vertebrae, whereas wild-type littermates
(Fig. 2B,D) had 18 ossified
tail vertebrae. Furthermore, A1-/-
-/-
mutants had a normal pelvic bone and correctly set hindlimbs (compare
Fig. 2B,D with 2C,E). We also
examined the effects of Rarg haploinsufficiency by analyzing two
E18.5 A1-/-
+/- mutants. Although one of
these A1-/-
+/- skeletons
(Fig. 2G) showed an
intermediate phenotype with lumbosacral vertebrae less deformed than in
A1-/- mutants and a truncated tail rudiment, the other
showed fusion and twisting of the hindlimbs, as well as malformed lumbosacral
region and no caudal tail vertebrae (data not shown). These results indicate
that the rescue of A1-/- animals by Rarg
haploinsufficiency is partial and not fully penetrant, possibly accounting for
the absence of viable A1-/-
+/- animals
in our crosses.
Suppression of other vertebral abnormalities
We also examined whether loss of RAR function may revert the
A1-/- cervical phenotype
(Table 2;
Fig. 3). The first and second
cervical vertebrae (C1 and C2) developed normally in all
A1-/-
-/- mutants examined
(Fig. 3C; n=4). By
contrast, one of the A1-/-
+/- skeletons
showed fusion between the exoccipital bone and neural arch of C1, whereas both
A1-/- skeletons exhibited this malformation (compare
Fig. 3B with 3D). In addition,
A1-/- skeletons generally showed bifidus of the neural
arch on C2 (Fig. 3B). This
latter phenotype is also observed in some of the
-/- mutants
(Lohnes et al., 1993
;
Lohnes et al., 1994
). None of
the A1-/-
-/- or
A1-/-
+/- double mutants exhibited such
malformations (Fig. 3C,D).
The A1-/--/-,
A1-/-
+/- and A1-/-
mutants showed the following posterior homeotic transformations within the
C5-T1 region: a C5 to C6 transformation, which was demonstrated by the
presence of unilateral or bilateral anterior tuberculi on C5, structures
normally characteristic of C6 (2/4
A1-/-
-/-, 1/2
A1-/-
+/-, 2/2
A1-/-); a C7 to T1 transformation, as evidenced by the
presence of ectopic ribs on C7 that were fused to either T1 or to the sternum
(4/4 A1-/-
-/-, 2/2
A1-/-
+/-, 2/2
A1-/-); and a T1 to T2 transformation, indicated by the
presence of a prominent ectopic spinous process on T1 in addition to the one
normally observed on T2 (2/4 A1-/-
-/-,
1/2 A1-/-
+/-, 2/2
A1-/-) (compare Fig.
3A-D; see Table 2
for a summary). Thus, in contrast to the rescue of the C1-C2 abnormalities, it
appears that ablation of Rarg does not rescue the posterior
transformations observed within the C5-T1 region of A1-/-
mutants. Furthermore, the A1-/-
-/- and
A1-/-
+/- compound mutants did not
display anterior homeotic transformations normally observed in the
cervicothoracic region of Rarg-/- mutants
(Iulianella and Lohnes, 1997
;
Lohnes et al., 1993
). Some
A1-/- mutants showed an L1 to T13 transformation due to
the development of unilateral or bilateral ectopic ribs on L1 (data not shown;
Table 2).
A1-/-
+/-, but not
A1-/-
-/- compound mutants, also
exhibited this anterior transformation. Finally, the tracheal rings were
ventrally fused and disrupted in all
A1-/-
-/- skeletons examined, a
malformation also observed in all Rarg-/- mutants
(Lohnes et al., 1993
)
(Table 2). Thus, not all
defects resulting from Cyp26a1 ablation are rescued by loss of
RAR
function, suggesting that other RARs may compensate for ectopic RA
signaling in the C5-T1 region.
Normal expression of genes involved in tail bud morphogenesis is
restored in E9.5 Cyp26a1/Rarg double null embryos
To characterize the molecular defects resulting from loss of CYP26A1
function, and their possible rescue by ablation of Rarg, we analyzed
genes known for their specific expression in tail bud tissues and/or whose
mutation generates developmental defects in the caudal region of the embryo.
In situ hybridization experiments were carried out at E9.5, which corresponds
to an early stage of tail bud growth in wild-type embryos, at which mild
caudal defects are seen in the A1-/- embryos Each
expression pattern was analyzed on one
A1-/--/- and two
A1-/-
+/- compound mutants, in comparison
with A1-/- and
-/- single mutants, as
well as wild-type control embryos processed in the same experimental series.
As none of the expression patterns found in
-/- mutant
embryos differed from wild-type patterns, these mutants are not discussed
further.
Wnt3a transcripts are expressed in the primitive streak, nascent
mesoderm and neuroepithelium of the developing tail bud
(Parr et al., 1993;
Roelink and Nusse, 1991
).
Wnt3a-null embryos show defects caudal to the forelimbs in that they
lack trunk somites, have a disrupted notochord and fail to form tail bud
tissues (Takada et al., 1994
).
We (Abu-Abed et al., 2001
) and
others (Sakai et al., 2001
)
have previously examined the expression of Wnt3a in
Cyp26a1-null mutants and found its expression to be downregulated
within the nascent mesoderm and neuroepithelium. By contrast, the E9.5
A1-/-
-/- embryo expressed Wnt3a
normally (data not shown). To substantiate these findings, we chose to examine
a downstream target of WNT3A signaling, the T-box gene Brachyury
(Herrmann et al., 1990
).
As reported (Abu-Abed et al.,
2001; Sakai et al.,
2001
), A1-/- embryos showed reduced
Brachyury expression in the tail bud nascent mesoderm and
neuroepithelium, whereas expression in the notochord indicated abnormal
development of this structure (compare Fig.
4A with 4D). By contrast, Brachyury expression levels in
the tail bud tissues of A1-/-
-/-
(Fig. 4B) and
A1-/-
+/-
(Fig. 4C) compound mutants were
comparable with those observed in wild-type littermates
(Fig. 4A). Interestingly, while
the A1-/-
-/- mutant displayed normal
morphology (Fig. 4B), the
A1-/-
+/- embryo was malformed caudally,
showing eversion and twisting of tail bud tissues
(Fig. 4C, inset). This
malformation was also observed, albeit more severely, in the
A1-/- mutant (Fig.
4D), indicating that restoration of Brachyury expression
may not be sufficient to achieve normal development of tail bud tissues in
compound mutants. These results confirm that down-regulation of WNT3A
signaling in A1-/- mutants affects expression of a WNT3A
downstream target.
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Fgf8-null mutants also show severe gastrulation defects and lack
several mesoderm- and endoderm-derived structures
(Sun et al., 1999).
Fgf8 is expressed in the primitive streak, dorsal half of the nascent
mesoderm, neuroepithelium and hindgut endoderm of the wild-type tail bud
(Fig. 5A)
(Crossley and Martin, 1995
).
Fgf8 expression was virtually absent from the tail bud of
A1-/- and A1-/-
+/-
embryos, except for weak expression within the nascent mesoderm
(Fig. 5B; inset, and data not
shown). Furthermore, abnormal tail bud morphology of the
A1-/- mutant suggested minimal tissue development caudal
to the posterior neuropore (PNP) in this particular embryo
(Fig. 5B, inset). By contrast,
Fgf8 expression in the A1-/-
-/-
embryo was comparable with that of the wild-type embryo (compare
Fig. 5A with 5C).
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We examined the expression of an additional hindgut endoderm marker,
Hnf3b, which is also expressed in the notochord and floor plate of
the neural tube (Fig. 5D)
(Ang et al., 1993;
Monaghan et al., 1993
;
Sasaki and Hogan, 1993
).
Hnf3b expression persisted in the notochord and floor plate of
A1-/- and A1-/-
+/-
embryos (Fig. 5E; data not
shown), although labeling indicated that both structures developed abnormally
(Fig. 5E, inset; data not
shown). In tail bud tissues caudal to the PNP, Hnf3b labeling also
indicated a reduction in the size of the hindgut pocket, whose development
appeared to stop prematurely in both A1-/- and
A1-/-
+/- mutants
(Fig. 5E; data not shown).
Abnormal patterns of expression were not detected in the
A1-/-
-/- embryo
(Fig. 5F; data not shown).
Loss of FGF signaling has been shown to disrupt expression of
caudal-related genes in Xenopus
(Northrop and Kimelman, 1994).
As Fgf8 was abnormally expressed in A1-/- and
A1-/-
+/- embryos, we examined the
expression of Cdx4, one of three known caudal-related genes
in mouse (Gamer and Wright,
1993
). Cdx4 shows a rostrocaudal gradient of expression
within tail bud tissues of E9.5 wild-type embryos, with its expression being
most intense caudally (Fig.
6A). In A1-/- mutant embryos, Cdx4
expression was downregulated in both the neuroepithelium and nascent mesoderm
of the tail bud, having an expression boundary that altogether shifted
posteriorly (compare Fig. 6A with
6B, insets); A1-/-
+/-
mutants showed a less pronounced caudal shift (data not shown). Cdx4
expression in the A1-/-
-/- embryo was
similar to that of wild-type littermates (compare
Fig. 6A with 6C).
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Pownall et al. (Pownall et al.,
1996) have demonstrated that caudal-related transcription
factors regulate the expression of Hox genes in Xenopus; similar
observations have been made in the case of Cdx1-null mice
(Subramanian et al., 1995
). We
chose to analyze the expression of Hoxd11, which together with other
5'-located Hox genes, is involved in setting up regional identities
along the lumbosacral column and posterior urogenital and digestive structures
(Favier and Dollé,
1997
). Hoxd11 is strongly expressed at E9.5 throughout
most of the tail bud tissues, its expression being stronger towards the caudal
extremity (Fig. 6D). Hoxd11 expression was downregulated in the tail bud tissues of
A1-/- embryos, especially within the hindgut, presomitic
and lateral mesoderm, and to a lesser extent in the neuroepithelium
(Fig. 6E). Moreover,
A1-/- mutants showed comparably reduced expression domains
of Cdx4 and Hoxd11 expression (compare
Fig. 6B with 6E, insets),
indicating that CDX4 may indeed regulate Hox gene expression. By contrast,
Hoxd11 expression levels in
A1-/-
-/- and
A1-/-
+/- compound mutants were
comparable with those seen in wild-type embryos
(Fig. 6F; and data not shown),
although expression was expanded along the ventral mesoderm (compare
Fig. 6D wit 6F, insets; see
Discussion).
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DISCUSSION |
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A Rarg-null background rescues most, but not all defects in
Cyp26a1-/- animals
Although Cyp26a1 and Rarg tail bud-expression patterns
overlap in a spatiotemporal manner (Fujii
et al., 1997; MacLean et al.,
2001
; Ruberte et al.,
1990
) (see Fig. 1),
CYP26A1 is critical for normal caudal development whereas RAR
is
dispensable (Abu-Abed et al.,
2001
; Lohnes et al.,
1993
; Look et al.,
1995
; Sakai et al.,
2001
). Rarg-null animals are viable, exhibiting vertebral
transformations and malformations in the cervical region, in addition to
squamous metaplasia and/or keratinization of various glandular epithelia.
Moreover, Rarg-/- embryos resist the effects of RA
toxicity on caudal development, suggesting that RAR
mediates ectopic RA
signaling in the tail bud (Lohnes et al.,
1993
). However, although caudal development proceeds normally in
Cyp26a1-/-Rarg-/- animals, ablation of
Rarg does not rescue all defects characterized in
Cyp26a1-/- mutants. In particular, the double mutants
retain several vertebral posterior transformations in the cervical region
(Fig. 2;
Table 2), thus implicating
other RARs in mediating the effects of ectopic RA after loss of CYP26A1
function.
Cyp26a1 prevents RAR-mediated disruption of WNT3A and FGF8
signaling pathways
Removal of RAR function rescues Wnt3a and Fgf8
expression, as well as normal tail bud development, in
Cyp26a1-/- mutants. Both Fgf8 and Wnt3a
genes exhibit markedly reduced expression domains in the caudal region of
Cyp26a1-/- embryos
(Fig. 4)
(Abu-Abed et al., 2001
;
Sakai et al., 2001
). These
abnormal transcript patterns could result from a combination of two events:
(1) an impaired development of the tail bud tissues that normally express
these genes; and (2) a downregulation of their expression levels (see
Fig. 5B for Fgf8) [see
Sakai et al. (Sakai et al.,
2001
) for Wnt3a]. As a result, impaired FGF8 and WNT3A
signaling is likely to affect the tail bud gastrulation movements in
Cyp26a1-/- embryos. In Wnt3a-/- and
Fgf8-/- mutants, epiblast cells appear to undergo the
gastrulation epithelial-to-mesenchymal transition, but fail to migrate
laterally or to contribute to mesoderm and endoderm formation
(Sun et al., 1999
;
Takada et al., 1994
;
Yamaguchi et al., 1999
).
Furthermore, similar to what we observed in Cyp26a1-/-
embryos, Wnt3a-/- and Fgf8-/- mutants
show decreased Brachyury (Fig.
4) and Tbx6 (data not shown) expression
(Sun et al., 1999
;
Yamaguchi et al., 1999
). The
Fgf8-/-, Brachyury-/- and
Tbx6-/- abnormal phenotypes are more severe than those
observed in Wnt3a-/- and Cyp26a1-/-
mutant embryos, with severity of the null mutant phenotypes appearing to
correspond to onset of their respective gene expression in the tail bud
(Chapman and Papaioannou,
1998
; Yamaguchi et al.,
1999
; Yoshikawa et al.,
1997
). As tail bud defects in CYP26A1 mutants are mediated by
RAR
, which only first appears in the caudal embryonic germ layers at
E8.0, they should accordingly be less severe than those observed in
Wnt3a-/- embryos. Cyp26a1-/- fetuses
indeed develop thoracic vertebrae, as well as some lumbosacral vertebral
remnants, whereas Wnt3a-/- animals show loss of all
vertebrae and tissues caudal to the forelimbs. Thus, our results show that, in
the absence of CYP26A1, RAR
downregulates WNT3A and FGF8 signaling
(Fig. 7B).
WNT3A and FGF8 are thought to affect the expression of other common
targets, such as caudal-related genes, which regulate Hox expression in
Drosophila, Xenopus and mouse
(Ikeya and Takada, 2001;
Isaacs et al., 1998
;
Lickert et al., 2000
;
Northrop and Kimelman, 1994
;
Prinos et al., 2001
). Cdx4
expression is strongest in the tail bud at a time when 5' Hox genes,
which are believed to define identity of more caudal body segments, are first
expressed (Burke et al., 1995
;
Gamer and Wright, 1993
).
Therefore, we investigated Cdx4 and Hoxd11 expression. Our
results demonstrate that loss of WNT3A and FGF8 signaling in
Cyp26a1-/- mutants coincides with downregulation of both
Cdx4 and Hoxd11. Interestingly, Hoxd11 expression
expanded ventrally in mutants lacking Rarg. A similar phenomenon was
observed in animals harboring a mutation in a 3' repressor region of the
Hoxd11 gene (Gérard et
al., 1996
). Together with our results, these observations suggest
that in the absence of RA, RAR
may repress Hoxd11 gene
activity in the ventral mesoderm by binding to its 3' repressor element,
thereby defining its rostral boundary of expression. As almost normal
Cdx4 and Hoxd11 expression is restored in
Cyp26a1-/-Rarg-/- double mutants,
ectopic RA signaling through RAR
in the tail bud appears to affect
directly and/or indirectly the expression of additional downstream targets of
WNT3A and FGF8 signaling, such as the Cdx and 5' Hox genes
(Fig. 7).
What is the function of Rarg in the tail bud?
Ablation of Rarg restored normal gene expression patterns and
essentially alleviated caudal regression in
Cyp26a1-/-Rarg-/- double mutants. What
then, besides the possible setting of the rostral boundary of expression of
Hoxd11, is the physiological function of RAR in the caudal
embryo, and why are Cyp26a1 and Rarg co-expressed in the
tail bud? Loss of RAR
function severely compromises male fertility, by
disrupting normal differentiation of glandular epithelia in the seminal
vesicles and prostate glands (Lohnes et
al., 1993
; Look et al.,
1995
). We note that
Cyp26a1-/-Rarg-/- double mutant males
and females also fail to reproduce normally (data not shown). Thus, as
Cyp26a1-/- males and females in which one Raldh2
allele has been ablated are fertile
(Niederreither et al., 2002a
),
RAR
-mediated RA-signaling is likely to perform an essential role at
some stage in the ontogeny of reproductive organs.
Reducing RA synthesis or blocking Rarg-mediated RA signaling can
rescue Cyp26a1 mutants
RALDH2 and CYP26A1, respectively, show complementary RA-synthesizing and
-catabolizing activities throughout embryonic development in several tissues
undergoing morphogenesis (Abu-Abed et al.,
2002; Fujii et al.,
1997
; MacLean et al.,
2001
; Niederreither et al.,
1997
), enzymatic activities which are conserved in the trunk and
tail bud of both mouse and chick embryos
(Berggren et al., 1999
;
Swindell et al., 1999
). Our
present data indicate that by catabolizing RA, which diffuses from neighboring
trunk somites, CYP26A1 activity prevents ectopic RAR
-mediated RA
signaling in the tail bud. Interestingly, using a lacZ-RA-reporter
transgene it has been shown that under Raldh2-haploinsufficient
conditions the extent of RA diffusion into caudal tissues is reduced, which in
turn rescues Cyp26a1-null mutant mouse fetuses from lethality
(Niederreither et al., 2002a
).
These observations suggest that below a certain threshold, RA synthesis in the
trunk does not interfere with tail bud development
(Perlmann, 2002
), even in the
presence of RAR
. In this respect we note that, although RA treatments
downregulate Raldh2 expression in the trunk
(Niederreither et al., 1997
),
Cyp26a1 gene expression is induced by RA in the tail bud and
craniofacial regions (Iulianella et al.,
1999
), indicating that feedback responses may have evolved to
regulate local RA concentrations. Moreover, we have previously shown that
RA-induced Cyp26a1 expression is mediated by RAR
(Abu-Abed et al., 1998
), and,
therefore, RAR
may be involved in regulating Cyp26a1
expression in the tail bud when endogenous RA levels exceed a certain
threshold. Thus, it is only in the absence of CYP26A1 or following
administration of excess RA, that RAR
-mediated RA signaling becomes
lethal by suppressing the expression of genes that control tail bud
morphogenesis.
Previously, several mechanisms of partial genetic suppression of lethal
null mutations have been reported in the mouse
(Kodera et al., 2002;
Litingtung and Chiang, 2000
;
Niederreither et al., 2002a
;
Tsai et al., 1998
). Our
present double mutants, in which a Rarg-null background rescues
caudal regression and neural tube defects in Cyp26a1-/-
mutants, provides a clear illustration of genetic suppression of a lethal
mutation, as the ablation of Rarg-/- allows
Cyp26a1-/- mutants to survive lethal RA levels. Finally,
it is interesting to note that in their characterization of mouse models with
neural tube defects, Juriloff and Harris
(Juriloff and Harris, 2000
)
identified several genes whose mutational phenotypes are affected by
nutritional factors. Supplementation of curly tail mice with inositol or of
splotch (Pax3) mutants with folic acid reduced the incidence of spina
bifida in both types of animals. Our previous
(Niederreither et al., 2002a
)
and present doublemutant mouse models similarly demonstrate that alterations
in RA availability have marked effects on the incidence of spina bifida, and
defects of caudal morphogenesis and neural tube formation. Whether similar
human congenital defects could result from dysregulation of mechanisms that
control the levels of RA remains to be determined.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Abu-Abed, S. S., Beckett, B. R., Chiba, H., Chithalen, J. V.,
Jones, G., Metzger, D., Chambon, P. and Petkovich, M. (1998).
Mouse P450RAI (CYP26) expression and retinoic acid-inducible retinoic acid
metabolism in F9 cells are regulated by retinoic acid receptor gamma and
retinoid X receptor alpha. J. Biol. Chem.
273,2409
-2415.
Abu-Abed, S., Dollé, P., Metzger, D., Beckett, B.,
Chambon, P. and Petkovich, M. (2001). The retinoic
acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain
patterning, vertebral identity, and development of posterior structures.
Genes Dev. 15,226
-240.
Abu-Abed, S., MacLean, G., Fraulob, V., Chambon, P., Petkovich, M. and Dollé, P. (2002). Differential expression of the retinoic acid-metabolizing enzymes CYP26A1 and CYP26B1 during murine organogenesis. Mech. Dev. 110,173 -177.[CrossRef][Medline]
Alles, A. J. and Sulik, K. K. (1990). Retinoic acid-induced spina bifida: evidence for a pathogenetic mechanism. Development 108,73 -81.[Abstract]
Ang, S. L., Wierda, A., Wong, D., Stevens, K. A., Cascio, S.,
Rossant, J. and Zaret, K. S. (1993). The formation and
maintenance of the definitive endoderm lineage in the mouse: involvement of
HNF3/forkhead proteins. Development
119,1301
-1315.
Beddington, R. S. and Robertson, E. J. (1999). Axis development and early asymmetry in mammals. Cell 96,195 -209.[Medline]
Berggren, K., McCaffery, P., Drager, U. and Forehand, C. J. (1999). Differential distribution of retinoic acid synthesis in the chicken embryo as determined by immunolocalization of the retinoic acid synthetic enzyme, RALDH-2. Dev. Biol. 210,288 -304.[CrossRef][Medline]
Burke, A. C., Nelson, C. E., Morgan, B. A. and Tabin, C.
(1995). Hox genes and the evolution of vertebrate axial
morphology. Development
121,333
-346.
Chambon, P. (1996). A decade of molecular
biology of retinoic acid receptors. FASEB J.
10,940
-954.
Chapman, D. L. and Papaioannou, V. E. (1998). Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6. Nature 391,695 -697.[CrossRef][Medline]
Crossley, P. H. and Martin, G. R. (1995). The
mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions
that direct outgrowth and patterning in the developing embryo.
Development 121,439
-451.
de Roos, K., Sonneveld, E., Compaan, B., ten Berge, D., Durston, A. J. and van der Saag, P. T. (1999). Expression of retinoic acid 4-hydroxylase (CYP26) during mouse and Xenopus laevis embryogenesis. Mech. Dev. 82,205 -211.[CrossRef][Medline]
Durston, A. J., van der Wees, J., Pijnappel, W. W., Schilthuis, J. G. and Godsave, S. F. (1997). Retinoid signalling and axial patterning during early vertebrate embryogenesis. Cell Mol. Life Sci. 53,339 -349.[Medline]
Favier, B. and Dollé, P. (1997). Developmental functions of mammalian Hox genes. Mol. Hum. Reprod. 3,115 -131.[Abstract]
Fujii, H., Sato, T., Kaneko, S., Gotoh, O., Fujii-Kuriyama, Y.,
Osawa, K., Kato, S. and Hamada, H. (1997). Metabolic
inactivation of retinoic acid by a novel P450 differentially expressed in
developing mouse embryos. EMBO J.
16,4163
-4173.
Gamer, L. W. and Wright, C. V. (1993). Murine Cdx-4 bears striking similarities to the Drosophila caudal gene in its homeodomain sequence and early expression pattern. Mech. Dev. 43,71 -81.[CrossRef][Medline]
Gérard, M., Chen, J. Y., Gronemeyer, H., Chambon, P., Duboule, D. and Zakany, J. (1996). In vivo targeted mutagenesis of a regulatory element required for positioning the Hoxd-11 and Hoxd-10 expression boundaries. Genes Dev. 10,2326 -2334.[Abstract]
Griffith, C. M. and Wiley, M. J. (1991). Effects of retinoic acid on chick tail bud development. Teratology 43,217 -224.[Medline]
Herrmann, B. G., Labeit, S., Poustka, A., King, T. R. and Lehrach, H. (1990). Cloning of the T gene required in mesoderm formation in the mouse. Nature 343,617 -622.[CrossRef][Medline]
Hollemann, T., Chen, Y., Grunz, H. and Pieler, T.
(1998). Regionalized metabolic activity establishes boundaries of
retinoic acid signalling. EMBO J.
17,7361
-7372.
Ikeya, M. and Takada, S. (2001). Wnt-3a is required for somite specification along the anteroposterior axis of the mouse embryo and for regulation of cdx-1 expression. Mech. Dev. 103,27 -33.[CrossRef][Medline]
Isaacs, H. V., Pownall, M. E. and Slack, J. M.
(1998). Regulation of Hox gene expression and posterior
development by the Xenopus caudal homologue Xcad3. EMBO
J. 17,3413
-3427.
Iulianella, A. and Lohnes, D. (1997). Contribution of retinoic acid receptor gamma to retinoid-induced craniofacial and axial defects. Dev. Dyn. 209,92 -104.[CrossRef][Medline]
Iulianella, A., Beckett, B., Petkovich, M. and Lohnes, D. (1999). A molecular basis for retinoic acid-induced axial truncation. Dev. Biol. 205, 33-48.[CrossRef][Medline]
Juriloff, D. M. and Harris, M. J. (2000). Mouse
models for neural tube closure defects. Hum. Mol.
Genet. 9,993
-1000.
Kessel, M. (1992). Respecification of vertebral identities by retinoic acid. Development 115,487 -501.[Abstract]
Kinder, S. J., Tsang, T. E., Quinlan, G. A., Hadjantonakis, A.
K., Nagy, A. and Tam, P. P. (1999). The orderly allocation of
mesodermal cells to the extraembryonic structures and the anteroposterior axis
during gastrulation of the mouse embryo. Development
126,4691
-4701.
Kodera, T., McGaha, T. L., Phelps, R., Paul, W. E. and Bona, C.
A. (2002). Disrupting the IL-4 gene rescues mice homozygous
for the tight-skin mutation from embryonic death and diminishes TGF-beta
production by fibroblasts. Proc. Natl. Acad. Sci. USA
99,3800
-3805.
Lickert, H., Domon, C., Huls, G., Wehrle, C., Duluc, I.,
Clevers, H., Meyer, B. I., Freund, J. N. and Kemler, R.
(2000). Wnt/(beta)-catenin signaling regulates the expression of
the homeobox gene Cdx1 in embryonic intestine.
Development 127,3805
-3813.
Litingtung, Y. and Chiang, C. (2000). Specification of ventral neuron types is mediated by an antagonistic interaction between Shh and Gli3. Nat. Neurosci. 3, 979-985.[CrossRef][Medline]
Lohnes, D., Kastner, P., Dierich, A., Mark, M., LeMeur, M. and Chambon, P. (1993). Function of retinoic acid receptor gamma in the mouse. Cell 73,643 -658.[Medline]
Lohnes, D., Mark, M., Mendelsohn, C., Dollé, P., Dierich,
A., Gorry, P., Gansmuller, A. and Chambon, P. (1994).
Function of the retinoic acid receptors (RARs) during development (I).
Craniofacial and skeletal abnormalities in RAR double mutants.
Development 120,2723
-2748.
Look, J., Landwehr, J., Bauer, F., Hoffmann, A. S., Bluethmann,
H. and LeMotte, P. (1995). Marked resistance of RAR
gamma-deficient mice to the toxic effects of retinoic acid. Am. J.
Physiol. 269,E91
-E98.
MacLean, G., Abu-Abed, S., Dollé, P., Tahayato, A., Chambon, P. and Petkovich, M. (2001). Cloning of a novel retinoic acid-metabolizing cytochrome P450, Cyp26B1, and comparative expression analysis with Cyp26A1 during early murine development. Mech. Dev. 107,195 -201.[CrossRef][Medline]
Mic, F. A., Molotkov, A., Fan, X., Cuenca, A. E. and Duester, G. (2000). RALDH3, a retinaldehyde dehydrogenase that generates retinoic acid, is expressed in the ventral retina, otic vesicle and olfactory pit during mouse development. Mech. Dev. 97,227 -230.[CrossRef][Medline]
Monaghan, A. P., Kaestner, K. H., Grau, E. and Schutz, G.
(1993). Postimplantation expression patterns indicate a role for
the mouse forkhead/HNF-3 alpha, beta and gamma genes in determination of the
definitive endoderm, chordamesoderm and neuroectoderm.
Development 119,567
-578.
Niederreither, K., McCaffery, P., Drager, U. C., Chambon, P. and Dollé, P. (1997). Restricted expression and retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse development. Mech. Dev. 62, 67-78.[CrossRef][Medline]
Niederreither, K., Subbarayan, V., Dollé, P. and Chambon, P. (1999). Embryonic retinoic acid synthesis is essential for early mouse post-implantation development. Nat. Genet. 21,444 -448.[CrossRef][Medline]
Niederreither, K., Abu-Abed, S., Schuhbaur, B., Petkovich, M., Chambon, P. and Dollé, P. (2002a). Genetic evidence that oxidative derivatives of retinoic acid are not involved in retinoid signaling during mouse development. Nat. Genet. 15, 84-88.
Niederreither, K., Fraulob, V., Garnier, J. M., Chambon, P. and Dollé, P. (2002b). Differential expression of retinoic acid-synthesizing (RALDH) enzymes during fetal development and organ differentiation in the mouse. Mech. Dev. 110,165 -171.[CrossRef][Medline]
Northrop, J. L. and Kimelman, D. (1994). Dorsal-ventral differences in Xcad-3 expression in response to FGF-mediated induction in Xenopus. Dev. Biol. 161,490 -503.[CrossRef][Medline]
Padmanabhan, R. (1998). Retinoic acid-induced caudal regression syndrome in the mouse fetus. Reprod. Toxicol. 12,139 -151.[CrossRef][Medline]
Parr, B. A., Shea, M. J., Vassileva, G. and McMahon, A. P.
(1993). Mouse Wnt genes exhibit discrete domains of expression in
the early embryonic CNS and limb buds. Development
119,247
-261.
Perlmann, T. (2002). Retinoid metabolism: a balancing act. Nat. Genet. 15, 1-2.
Pownall, M. E., Tucker, A. S., Slack, J. M. and Isaacs, H.
V. (1996). eFGF, Xcad3 and Hox genes form a molecular pathway
that establishes the anteroposterior axis in Xenopus.
Development 122,3881
-3892.
Prinos, P., Joseph, S., Oh, K., Meyer, B. I., Gruss, P. and Lohnes, D. (2001). Multiple pathways governing Cdx1 expression during murine development. Dev. Biol. 239,257 -269.[CrossRef][Medline]
Roelink, H. and Nusse, R. (1991). Expression of two members of the Wnt family during mouse development restricted temporal and spatial patterns in the developing neural tube. Genes Dev. 5,381 -388.[Abstract]
Rossant, J., Zirngibl, R., Cado, D., Shago, M. and Giguère, V. (1991). Expression of a retinoic acid response element-hsplacZ transgene defines specific domains of transcriptional activity during mouse embryogenesis. Genes Dev. 5,1333 -1344.[Abstract]
Ruberte, E., Dollé, P., Krust, A., Zelent, A., Morriss-Kay, G. and Chambon, P. (1990). Specific spatial and temporal distribution of retinoic acid receptor gamma transcripts during mouse embryogenesis. Development 108,213 -222.[Abstract]
Ruiz i Altaba, A. and Jessell, T. (1991). Retinoic acid modifies mesodermal patterning in early Xenopus embryos. Genes Dev. 5,175 -187.[Abstract]
Sakai, Y., Meno, C., Fujii, H., Nishino, J., Shiratori, H.,
Saijoh, Y., Rossant, J. and Hamada, H. (2001). The retinoic
acid-inactivating enzyme CYP26 is essential for establishing an uneven
distribution of retinoic acid along the anterio-posterior axis within the
mouse embryo. Genes Dev.
15,213
-225.
Sasaki, H. and Hogan, B. L. (1993).
Differential expression of multiple fork head related genes during
gastrulation and axial pattern formation in the mouse embryo.
Development 118,47
-59.
Shum, A. S., Poon, L. L., Tang, W. W., Koide, T., Chan, B. W., Leung, Y. C., Shiroishi, T. and Copp, A. J. (1999). Retinoic acid induces down-regulation of Wnt-3a, apoptosis and diversion of tail bud cells to a neural fate in the mouse embryo. Mech. Dev. 84, 17-30.[CrossRef][Medline]
Simeone, A., Avantaggiato, V., Moroni, M. C., Mavilio, F., Arra, C., Cotelli, F., Nigro, V. and Acampora, D. (1995). Retinoic acid induces stage-specific antero-posterior transformation of rostral central nervous system. Mech. Dev. 51, 83-98.[CrossRef][Medline]
Subramanian, V., Meyer, B. I. and Gruss, P. (1995). Disruption of the murine homeobox gene Cdx1 affects axial skeletal identities by altering the mesodermal expression domains of Hox genes. Cell 83,641 -653.[Medline]
Sun, X., Meyers, E. N., Lewandoski, M. and Martin, G. R.
(1999). Targeted disruption of Fgf8 causes failure of cell
migration in the gastrulating mouse embryo. Genes Dev.
13,1834
-1846.
Swindell, E. C., Thaller, C., Sockanathan, S., Petkovich, M., Jessell, T. M. and Eichele, G. (1999). Complementary domains of retinoic acid production and degradation in the early chick embryo. Dev. Biol. 216,282 -296.[CrossRef][Medline]
Takada, S., Stark, K. L., Shea, M. J., Vassileva, G., McMahon, J. A. and McMahon, A. P. (1994). Wnt-3a regulates somite and tailbud formation in the mouse embryo. Genes Dev. 8, 174-189.[Abstract]
Tsai, K. Y., Hu, Y., Macleod, K. F., Crowley, D., Yamasaki, L. and Jacks, T. (1998). Mutation of E2f-1 suppresses apoptosis and inappropriate S phase entry and extends survival of Rb-deficient mouse embryos. Mol. Cell 2,293 -304.[Medline]
Ulven, S. M., Gundersen, T. E., Weedon, M. S., Landaas, V. O., Sakhi, A. K., Fromm, S. H., Geronimo, B. A., Moskaug, J. O. and Blomhoff, R. (2000). Identification of endogenous retinoids, enzymes, binding proteins, and receptors during early postimplantation development in mouse: important role of retinal dehydrogenase type 2 in synthesis of all-transretinoic acid. Dev. Biol. 220,379 -391.[CrossRef][Medline]
Wilkinson, D. G. (1992). In Situ Hybridization: A Practical Approach. Oxford, UK: IRL Press.
Yamaguchi, T. P., Takada, S., Yoshikawa, Y., Wu, N. and McMahon,
A. P. (1999). T (Brachyury) is a direct target of Wnt3a
during paraxial mesoderm specification. Genes Dev.
13,3185
-3190.
Yasuda, Y., Konishi, H., Kihara, T. and Tanimura, T. (1990). Discontinuity of primary and secondary neural tube in spina bifida induced by retinoic acid in mice. Teratology 41,257 -274.[Medline]
Yoshikawa, Y., Fujimori, T., McMahon, A. P. and Takada, S. (1997). Evidence that absence of Wnt-3a signaling promotes neuralization instead of paraxial mesoderm development in the mouse. Dev. Biol. 183,234 -242.[CrossRef][Medline]