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First published online 5 January 2006
doi: 10.1242/dev.02216
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1 Hubrecht laboratory, Netherlands Institute for Developmental Biology,
Uppsalalaan 8 3584 CT Utrecht, The Netherlands.
2 Faculté de Médecine Pitié - Salpêtrière, 91
Boulevard de l'Hôpital UMRS 525 INSERM/UPMC, 75634 Paris, Cedex 13,
France.
3 University of Leicester, Department of Biochemistry, University Road,
Leicester LE1 7RH, UK.
* Author for correspondence (e-mail: jacqueli{at}niob.knaw.nl)
Accepted 18 November 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cdx, Transcription factor, Mouse embryogenesis, Axial extension, AP patterning, Placental development, Allantois
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). During early development the paralogous genes Cdx1,
Cdx2 and Cdx4 are initially expressed similarly in the primitive
streak, including the base of the allantois
(Meyer and Gruss, 1993;
Gamer and Wright, 1993;
Beck et al., 1995;
Chawengsaksophak et al., 2004;
Lohnes, 2003). At somite
stages, they exhibit nested expression patterns in the posterior axial and
paraxial embryonic tissues emerging from the primitive streak and later from
the tailbud. The expression of Cdx1 extends most anteriorly and
overlaps with the expression domains of Cdx2 and Cdx4
posteriorly so that all three Cdx genes are expressed in the tailbud
(Meyer and Gruss, 1993;
Gamer and Wright, 1993;
Beck et al., 1995;
Chawengsaksophak et al., 2004;
Lohnes, 2003).
The phenotypes of Cdx1 and Cdx2 loss-of-function mutant
mice generated by homologous recombination in part reflect the fact that the
Cdx proteins are positive regulators of the Hox genes in embryonic tissues
(for a review, see Deschamps and van Nes,
2005). Cdx1-/- animals show alterations in
vertebral anteroposterior (AP) identity confined to the cervical region
(Subramanian et al., 1995).
Cdx2-/- embryos die at E3.5 because of implantation
failure (Chawengsaksophak et al.,
1997; Strumpf et al.,
2005) but Cdx2+/- embryos implant successfully
and, like Cdx1-/- homozygotes, display an anterior
homeotic shift in the axial skeleton, although more subtle and situated more
posteriorly. In addition, Cdx2+/- mice exhibit a slight
shortening of the AP axis (Chawengsaksophak
et al., 1997). Combined
Cdx1-/-/Cdx2+/- mutants have an axial
phenotype showing abnormalities greater than either mutant separately thus
reflecting functional redundancy due to gene co-expression in the presomitic
mesoderm (van den Akker et al.,
2002). The block to implantation of Cdx2-/-
embryos, that is in keeping with the strong expression of the gene in the
trophectoderm at E3.5 (Beck et al.,
1995; Chawengsaksophak et al.,
1997), can be overcome by tetraploid fusion
(Nagy et al., 1993).
Development then proceeds to E10.5 but embryos exhibit severe posterior
truncation, there being little development posterior to the forelimb buds. The
arrest of posterior tissue generation in Cdx2-/- embryos
also concerns the extra-embryonic mesoderm
(Chawengsaksophak et al.,
2004).
Progenitors of extra-embryonic mesoderm originating from the posteriormost
epiblast (Lawson et al., 1991;
Kinder et al., 1999) express
all three Cdx genes at the late primitive streak stage of development
(Meyer and Gruss, 1993;
Gamer and Wright, 1993;
Beck et al., 1995;
Davidson et al., 2003).
Cdx2 is the only Cdx gene expressed in the extra-embryonic ectoderm.
Expression is demonstrable at E3.5 in the trophectoderm, subsequently in the
extra-embryonic ectoderm of the ectoplacental cone
(Beck et al., 1995), and
eventually in the trophoblastic stem cells
(Strumpf et al., 2005).
Cdx2 is also expressed in the chorion, though it is downregulated at
the late chorionic plate stage. Expression of Cdx2 in the E9.5
placenta is strong in the spongiotrophoblasts but is absent in the adjacent
labyrinth (Beck et al., 1995).
Thus, the Cdx2-positive cells of the chorion lose expression before
they differentiate into labyrinthine syncytiotrophoblast.
We have inactivated the third mouse Cdx gene, the X-linked Cdx4, by homologous recombination in embryonic stem (ES) cells. Cdx4-/0 mutants are born healthy and are fertile. A modest contribution of Cdx4 in AP patterning, and an important role for this gene in posterior axial extension were revealed in compound Cdx1-/-/Cdx4-/0 and Cdx2+/-/Cdx4-/0 mutants. In addition, most Cdx2+/-/Cdx4-/0 compound mutant embryos die around E10.5, revealing a novel functional involvement of Cdx genes. We show here that a subset of these compound mutants is impaired in chorio-allantoic fusion, even though the allantois reaches a sufficient length to touch the chorion. The remaining majority of the Cdx2+/-/Cdx4-/0 compound mutants does undergo successful chorio-allantoic fusion, but subsequently exhibits deficient labyrinthine development, following the failure of allantoic vessels to branch and expand their network in the chorionic plate. This extends the role of the Cdx transcription factors, already known as modulators of embryonic anteroposterior patterning, to include the morphogenesis of the extra-embryonic mesoderm of the chorio-allantoic placenta, the most posterior of the structures originating from the epiblast.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genotyping
Embryos or visceral yolk sacs were genotyped by PCR using the following
primers: NEOF401 5'-GATTGCACGCAGGTTCTCC-3' and NEOR802
5'-GATGTTTCGCTTGGTGGTC-3' to detect the 401 bp Cdx4
conditional allele. Wild-type Cdx4 was genotyped with primer pair
Cdx4FOR4723 5'-TTCCCTAAAAAGACCAAAATCAAA-3' and Cdx4REV5161
5'-CCCCGGAGAACTGCCTAAC-3' (438 bp), and the Cdx4 null
allele was detected with the Cdx4FOR4723
5'-TTCCCTAAAAAGACCAAAATCAAA-3' and Cdx4REV7900
5'-AGCGCAAAACCTCACATCA-3' (400 bp) primer combination. Male
(hemizygous) Cdx4-null embryos were confirmed by the presence of the
Y-chromosome using primer pair SryFOR
5'-TTATGGTGTGGTCCCGTGGTGAG-3' and SryREV
5'-TGTGATGGCATGTGGGTTCCTGT-3' (302 bp). Cdx1 and
Cdx2 mutant embryos and mice were genotyped by PCR as described
previously (Subramanian et al.,
1995; Chawengsaksophak et al.,
1997).
Skeletal preparations
Skeletons of E15.5 embryos and newborn mice were stained according to the
following procedure: newborn mice were skinned, and both newborn mice and
E15.5 embryos were eviscerated, fixed overnight in 96% ethanol containing 1%
glacial acetic acid and stained overnight in 0.5 mg/ml Alcian Blue (Sigma)
dissolved in 80% ethanol/20% acetic acid. After rinsing twice for 1 hour in
96% ethanol, soft tissue was dissolved in 1.5% KOH for 1 and 6 hours, for
E15.5 embryos and newborns, respectively. Bone was then stained overnight in
0.5% KOH containing 0.15 mg/ml Alizarin Red (Sigma). Newborns and embryos were
destained in 0.5% KOH/20% glycerol for 1 day or longer and afterwards stored
in 20% ethanol/20% glycerol.
Histology, in situ hybridization and immunohistochemistry
Paraffin embedding and processing of the sections, including Hematoxylin
and Eosin, and PAS staining, was carried out using standard methods. Methods
for in situ hybridization on sections have been described
(Moorman et al., 2001). RNA
probes used were: Cdx1 (Meyer and
Gruss, 1993); Cdx2
(Beck et al., 1995);
Cdx4 (Gamer and Wright,
1993); Mash2
(Guillemot and al., 1994);
Hand1 (previously eHAND)
(Cserjesi et al., 1995);
Tpbp (Lescisin et al., 1998). Immunohistochemistry was performed
according to standard procedures. The following primary antibodies were used:
Pecam (BD Biosciences, 1:50), anti-cleaved caspase 3 (BD Biosciences, 1:300)
and anti-Ki67 (BD Biosciences, 1:500). Pecam signal was amplified using
Tyramid signal amplification steps (Perkin Elmer). For cleaved caspase 3 and
Ki67, Envision+ kit (DAKO) was used as a secondary reagent. Signal was
developed with 3,3' diaminobenzidine (DAB, Sigma) and sections were
counterstained with Hematoxylin.
Morphometric analysis of placental vasculature
The diameter of Pecam-stained embryonic vessels that had penetrated the
chorionic ectoderm and branched in the placental labyrinth was measured at
their minimal width on tissue sections with Leica software. Representative
sections from the central part of E9.5 placentas were analyzed in eight
wild-type controls and eight Cdx2+/-/Cdx4-/0
compound mutants. Two-tailed t-test assuming equal variance was used
to determine P for the total embryonic vessel number per placental
labyrinth and for the average embryonic vessel width.
Allantois isolation and whole-mount immunofluorescent antibody staining
E7.5 and E8.25 allantoises were dissected from 0- to 3- and 5- to 7-somite
embryos, respectively. The isolated allantois was fixed in
immunohistochemistry-zinc-fixative (BD Biosciences). Fluorescent antibody
staining was performed according to standard procedures. Briefly, allantoises
were permeabilized with 0.2% Triton X-100 in TBS, blocked for 1 hour in TNB
buffer (Amersham) at room temperature and incubated overnight at 4°C with
primary antibody against Pecam (BD Biosciences, 1:100) or Flk1 (BD
Biosciences, 1:100) in TNB buffer. After three washes with TBS,
FITC-conjugated anti-rat IgG (Jackson Immunological, 1:100) secondary antibody
in TNB was applied for 1 hour at room temperature. Allantoises were analyzed
using a Leica confocal microscope, permitting 3D compilation of individual
confocal planes.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Loss of Cdx4 function synergizes with a reduction in Cdx dosage in AP patterning, axial elongation and placentogenesis
In view of early co-expression of Cdx1, Cdx2 and Cdx4,
and the functional redundancy between Cdx1 and Cdx2 in axial
extension and AP patterning (van den Akker
et al., 2002), we set out to investigate whether additional loss
of Cdx1 and Cdx2 alleles would reveal functional cooperation
between Cdx4 and these other Cdx genes. E15.5
Cdx1-/-/Cdx4-/0 double-null mutant
embryos exhibited very subtle changes in their vertebral phenotype compared
with Cdx1-null littermates. Although 5/9 Cdx1-null embryos
carried their first rib on the 9th vertebrae (V9) unilaterally instead of on
V8 (thoracic to cervical transformation of V8, with the consequent loss of one
rib), 5/7 Cdx1/Cdx4 double null mutants did, of which 3/5 exhibited
this transformation bilaterally, manifesting a slight increase in severity and
penetrance of the Cdx1 phenotype at this V8 level. In addition, 5/7
double mutant embryos had their 8th rib pair attached to the sternum
bilaterally, while only 2/9 Cdx1-null mutants did, and 3/9
unilaterally (not shown). This represents a slight increase in the severity
and penetrance of the Cdx1-null phenotype at the V15 axial level.
Compared with E15.5 Cdx2 heterozygote mutant embryos (n=4),
which did not manifest any vertebral transformations in our genetic
background, 4/4 Cdx2+/-/Cdx4-/0 littermates had
their 8th pair of ribs attached to the sternum, manifesting an anterior
homeotic-like transformation at this V15 level. In addition, all four compound
mutant embryos had a partial or complete rib attached to V21 bilaterally, at
the position normally corresponding to the first lumbar vertebrae.
Consequently, the compound Cdx2+/-/Cdx4-/0
mutants have 14 ribs. We conclude that the Cdx4 mutation has a
specific additive effect on the Cdx1 loss of function, and on the
loss of one active Cdx2 allele, in patterning the vertebrae at
discrete thoracic levels (respectively V8 and V15, and V15 and V21).
Cdx1-, Cdx4-null embryos and Cdx1/Cdx4 double null mutants did not manifest any axial truncation (not shown). By contrast, Cdx2 was found to strongly synergize functionally with Cdx4 in posterior tissue generation during axial extension. Cdx2+/-/Cdx4-/0 compound mutant embryos exhibited truncation of embryonic structures posterior to the hindlimbs, resulting in a more anteriorly located tail bud, a phenotype much more severe than that of either mutant alone and reminiscent of, though less marked than that of Cdx2-null embryos (Fig. 2A-C).
In addition, and independently from the posterior truncations which are not
expected to lead to early embryonic lethality, most compound
Cdx2+/-/Cdx4-/0 embryos were growth retarded at
E10.5 (see Fig. 2A) and died in
utero shortly thereafter (Tables
1 and
2). The heartbeat and
circulation in the embryo, as well as circulation in the yolk sac were normal
at E9.5, but failed around E10.5. Taken together with the expression of
Cdx2 and Cdx4 in the allantoic anlage during normal
development, these findings suggested chorioallantoic placental failure. A
first analysis of the phenotype of embryos from Cdx2+/-
and Cdx4+/- intercrosses revealed that the allantois of a
small number of Cdx2+/- (three out of 54),
Cdx2+/-/Cdx4+/- (two out of 28) and
Cdx2+/-/Cdx4-/0 (seven out of 35) embryos had
not fused with the chorion at E9.5 (Fig.
3B,C; data not shown). Measurement of allantoic length around the
normal fusion stage of six to eight somites (E8.0)
(Downs and Gardner, 1995;
Downs, 2002) indicated that
the reason for the lack of fusion of the mutant allantoises with the chorion
was not a length reduction (see Fig. S1 in the supplementary material).
Histological analysis of E9.5 unfused allantoises revealed that the
chorio-adhesive mesothelium had formed, and that blood filled vessels were
present in the distal allantoic mesenchyme
(Fig. 3D). Moreover, in situ
hybridization showed that Vcam1 was expressed in the mutant E8.5
distal allantois that had failed to fuse (see Fig. S1 in the supplementary
material). The mesodermal layer of the chorion was also present in the mutant,
and expressed 
4 integrin, the VCAM1 receptor in the chorio-allantoic
fusion process. Expression of 4 integrin was still observed in the
mutant at a stage when chorio-allantoic fusion and degradation of the
chorionic mesoderm layer have occurred in the wild type (see Fig. S1 in the
supplementary material). A subset of
Cdx2+/-/Cdx4-/0 mutant allantoises is thus
unsuccessful in fusing with the chorion in spite of the fact that they reach a
sufficient length to touch it, and achieve an advanced degree of
differentiation.
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Cdx2/Cdx4 compound mutant embryos initiate but do not maintain or expand their allantoic vascular network
In order to characterize the most frequent defect causing the death of
Cdx2+/-/Cdx4-/0 embryos, we selectively
recovered compound mutant embryos that had undergone successful
chorio-allantoic fusion. At E9.5, blood vessels were present in the
Cdx2+/-/Cdx4-/0 mutant allantois, and they had
initiated penetration of the chorionic ectoderm (Fig.
4B, compare with
4A, and with enlargements in
4C and
4D). We observed some variation
in the extent of allantoic vessel penetration into the chorion, and the
mildest defects may correspond to the minority of
Cdx2+/-/Cdx4-/0 mutants that survive
(Table 2). In all E10.5 mutant
placentas that we analyzed histologically, maternal blood pools and embryonic
blood vessels were widely separated in E10.5 mutant placentas (compare
Fig. 4F,H with 4E,G), indicating an increasingly more dramatic defect with age, and showing that the
mutant phenotype is not a simple developmental delay. These findings are
sufficient to explain the high midgestation mortality as being due to
placental insufficiency.
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; Downs,
1998). Following chorio-allantoic fusion, allantoic vessels
penetrate the chorionic ectoderm and branch extensively to interdigitate
between the maternal blood sinuses (reviewed by
Cross, 2005;
Rossant and Cross, 2001). We
followed this process throughout development in mutant and wild-type placentas
using several endothelial markers. In situ hybridization of Flk1
transcripts (data not shown) and antibody staining for PECAM expression
(Fig. 5C-F) both highlighted
the limited expansion of allantoic vessels in the chorionic ectoderm in
mutants compared with controls at E9.5 and E10.5. Quantification of both the
number and the diameter of allantois-derived embryonic vessels that had
penetrated the chorionic ectoderm at E9.5 clearly documented that these
allantoic vessels branched much less extensively in Cdx mutants
(Fig. 5A,B), and almost never
came close to the maternal blood sinuses. The thin hemotrichorial membrane
(Enders, 1965) normally
developing in areas of close contact between fetal and maternal blood
circulation is almost completely absent in the mutant
(Fig. 4C,D). The defect in
establishment of an interdigitated labyrinth was more extreme at E10.5, with
maternal and embryonic blood widely separated
(Fig. 5E,F and
Fig. 4G,H).
All allantoic and placental molecular markers tested are expressed in Cdx compound mutants
Cdx2 is expressed in the placental spongiotrophoblasts but not in
the labyrinth, in both wild type and
Cdx2+/-/Cdx4-/0 mutants at E9.5
(Fig. 5G,H). Cdx4 was
not expressed in either placenta or labyrinth (data not shown).
We tested the expression of several endothelial markers [Tie1,
Tie2 (Tek - Mouse Genome Informatics), Flk1
(Kdr - Mouse Genome Informatics), Flt1, Pecam, Vegf] by
RT-PCR in the E8.25 allantois before chorio-allantoic fusion. All genes were
expressed in compound mutants and controls (see Fig. S2A in the supplementary
material). In addition we tested the expression of Angpt1, Angpt2
(previously Agpt1 and Agpt2) and Vegf, genes known to be involved in
angiogenesis during placental labyrinth development (reviewed by
Rossant and Cross, 2001) by
semi-quantitative RT-PCR at E9.25 and did not observe consistent changes in
gene expression (see Fig. S2B in the supplementary material).
The expression of trophoblast markers was assayed as well in mutants and
controls. Mash2 (Ascl2 - Mouse Genome Informatics)
(Guillemot et al., 1994) was
found to be expressed in the spongiotrophoblasts and in the differentiating
labyrinthine trophoblasts at E9.5 (Fig.
5I,J). The expression of Fzd5 expression in labyrinthine
trophoblasts and Flt1 in the spongiotrophoblast (reviewed by
Rossant and Cross, 2001) were
not changed (data not shown). Analysis of the expression of Hand1 in
the diploid trophoblasts and trophoblast giant cells
(Cserjesi et al., 1995), and
Tpbp in spongiotrophoblasts and their precursors in the ectoplacental
cone (Lescisin et al., 1998), did not reveal any change between Cdx compound
mutants and controls (Fig.
5K-N). These results confirmed the presence of all placental cell
types in E9.5 mutants and controls, and only reflected the lack of
labyrinthine cell organization in
Cdx2+/-/Cdx4-/0 mutants, compared with wild
types.
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). Although no difference could be detected between the weak
Gcm1 expression in E9.5 control and mutant placentas by in situ
hybridization (not shown), a slightly reduced expression was observed in
semi-quantitative RT-PCR analysis (see Fig. S2B in the supplementary
material). The degree of proliferation of labyrinthine endothelial cells and the possible occurrence of apoptosis were tested at several developmental stages in control and mutant embryos. Ki67 labelling did not reveal impaired proliferation (see Fig. S3 in the supplementary material), and the use of caspase 3 did not indicate increased apoptosis (see Fig. S3 in the supplementary material) at E9.5, the stage at which the allantoic vasculature initiates penetration into the chorionic trophoblast in both mutants and controls. At E10.5, apoptotic cells were more numerous in mutant than control placenta, but this difference was not restricted to the endothelial cells as it concerned chorionic trophoblasts and fetal blood cells as well, and may only reflect developmental arrest around this stage (see Fig. S3 in the supplementary material).
Early Cdx expression overlaps with Flk1 positive endothelial precursors
Cdx genes are not expressed in the labyrinth. The only place and stage
where progenitors for the allantoic vasculature are expected to express Cdx
genes is the early allantoic bud at primitive streak stages. Cdx4,
like Cdx2, is widely expressed in the early allantoic bud (E7.5),
while expression of Cdx1 is confined to the base of the allantois
(Fig. 6A-C). In order to
characterize the ontogenesis of the allantoic component of the labyrinth in
the Cdx mutants, we analyzed the distribution of Flk1-positive endothelial
precursor cells (Downs et al.,
1998; Drake and Fleming,
2000; Yamashita et al.,
2000) at an early stage. Flk1-positive cells are present in both
mutants and controls (Fig.
6D,E), in an area and at a stage (E7.5) when Cdx2 and
Cdx4 are still fully expressed in the allantois
(Fig. 6B,C). This indicates
that the precursors of allantoic endothelial cells could be affected by the
Cdx mutations.
Mutant allantoic endothelial cells often exhibit a poorer primary vessel organization
At E8.0 and E8.5, Pecam-positive endothelial cells are abundant and are
organized in a primary vessel network in both mutants and controls. Among the
mutant specimens that we examined compared with age-matched controls (nine
mutants and eight wild types), we found some variability in the extent of
maturation of this network, which may correspond to the variability in the
severity of the labyrinth defect at E9.5 described above.
Fig. 6F,G shows an example of
an E8.25 age-matched pair of allantoises shortly after chorio-allantoic fusion
and reveal a lower degree of endothelial organization into a primary vessel
network in the mutant compared with the wild type. In the (in this case)
slightly smaller mutant allantois, the endothelial cells are locally less well
organized than in the control (Fig.
6H,I). Only a minority (two out of nine) of
Cdx2+/-/Cdx4-/0 mutant allantoises isolated at
E8.25 after chorio-allantoic fusion show similar endothelial organization to
the wild types (not shown). Thus, these data reveal that mutant allantoic
endothelial cells often (seven out of nine) exhibit, to a variable extent, a
poorer degree of primary vessel organization around the time of
chorio-allantoic fusion. Although endothelial cells are abundant in both
mutants and controls, they seem to be less organized in the
Cdx2+/-/Cdx4-/0 compound mutants than in the
wild type, suggesting an incipient defect expressed later as failure to extend
and branch into the chorion between E9.5 and E10.5.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It will be interesting to find out whether Cdx genes exert their effect on
the placenta by regulating target Hox genes. This is suggested by the reported
biological activity of certain Hox genes to promote migratory behavior of
adult endothelial cells in culture
(Boudreau et al., 1997) and to
induce morphogenesis of new vascular sprouts in chick chorio-allantoic
membranes in culture (Myers et al.,
2000). Our data suggest that the regulatory function of Cdx genes
in placentogenesis is an early one, acting at the level of the progenitors of
endothelial cells in the early allantois, as Cdx genes, like Hox genes, are
downregulated in the allantois at E8.5.
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). Cdx4-null zebrafish embryos manifest a transient
deficiency in generating erythroid progenitors
(Davidson et al., 2003). The
authors suggest an early function of Cdx4 in making the posterior
lateral plate mesoderm, containing early hematopoietic progenitors, competent
to respond to genes that specify hematopoietic fate. The defect in zebrafish
Cdx4 mutants was a selective loss of blood progenitors but not of
angioblast specification, thus affecting only one of the hemangioblast
derivatives (Huber et al.,
2004; Ema et al.,
2003). Such a defect in blood development was not evident in the
Cdx compound mutant mice examined so far, but a possible slight and/or
transient delay in generating erythroid cells at any one of the stages when
blood progenitors are born (successively yolk sac blood islands, placenta, AGM
and fetal liver) (Gekas et al.,
2005; Ottersbach and Dzierzak,
2005) might not have been noticed in our experiments.
Alternatively, such a function for Cdx4 might be redundant in mice,
thus differing from the situation in zebrafish. An insufficiency may remain
hidden until the Cdx dose has been lowered below a certain threshold. The fact
that Cdx4 rescues the hematopoietic colony-forming potential in mixed
lineage leukemia (Mll) mutant ES cell-derived embryoid bodies and
enhances the frequency of hematopoietic colonies in control embryoid bodies
(Ernst et al., 2004) provides
support for this hypothesis. Vascular lineages were not affected by the Cdx4 mutation in zebrafish but the absence of an allantois in fish leaves the possibility that, after the acquisition of the chorio-allantoic placenta in mammals, the gene became specifically involved in the ontogeny of a functional allantoic vasculature. An implication of these findings is that mutations in Cdx genes might lead to pregnancy failure in humans.
Specificity versus redundancy in function among Cdx members
Among the three Cdx family members, Cdx2 is the only one that is
expressed in the early extra-embryonic ectoderm, where it ensures
establishment and maintenance of the trophectoderm lineage
(Strumpf et al., 2005),
mediating implantation of the conceptus
(Chawengsaksophak et al.,
1997). This specific expression and function only concerns
Cdx2 and may result from a trophectoderm-specific regulatory
influence selectively relating to Cdx2, without counterpart on the
other, unlinked, Cdx genes. This early Cdx2 function requires one
active allele, and is not relevant in the context of the compound mutants
analyzed in the present work.
The common Cdx functions studied here concern epiblast-derived embryonic
and extra-embryonic tissues. All three Cdx genes collaborate to pattern the
axial skeletal structures [see van den Akker et al.
(van den Akker et al., 2002)
for Cdx1/Cdx2; see this work for Cdx1/Cdx4 and
Cdx2/Cdx4, where the contribution of Cdx4 is modest but
observable in the presence of the Cdx1 and Cdx2 mutations].
The distinct AP level of the vertebral changes in the double mutants may
relate to the differences in rostral span of the expression domains of
Cdx1 and Cdx2. The three Cdx genes contribute to allow
completion of posterior axial elongation
(van den Akker et al., 2002)
(this work). Again, their contributions are unequal, as the effect of
inactivating Cdx1 or Cdx4 was visible only in combination
with a mutation in Cdx2, whereas Cdx2 heterozygocity alone
affects axial elongation (van den Akker et
al., 2002) (this work). In this case, we cannot tell whether these
differences in contribution result from sequence differences in the proteins
or from differences in the spatiotemporal regulation of their expression.
Two of the three Cdx genes (Cdx2 and Cdx4) have been
shown in this work to control the ontogenesis of the allantoic part of the
placenta, and we cannot rule out that a contribution of Cdx1 in this
function might be revealed upon further lowering Cdx dose. All three Cdx genes
are expressed in the posterior epiblast, including the most proximal area
containing the presumptive extra-embryonic mesoderm. Although loss of function
of Cdx1 or Cdx4 does not affect the extra-embryonic
derivatives by themselves, Cdx2-null mutations have previously been
shown to impair the generation of embryonic and extra-embryonic mesoderm. The
Cdx2-null allantois remains extremely short, and does not touch the chorion
(Chawengsakhophak et al., 2004). This reveals the earliest Cdx dependence of
placental ontogeny, reflected by the fact that one active Cdx2 allele
is required for outgrowth of the early allantoic bud. The allantoises in
Cdx2+/-, Cdx2+/-/Cdx4+/-
and Cdx2+/-/Cdx4-/0 mutants reach a normal
size. But these three genotypes exhibit specific subsequent defects that
compromise the ontogenesis of a proficient chorio-allantoic placenta, with a
penetrance that increases with the decrease in Cdx dose. A first defect is
manifest by the fact that some of the fully grown allantoises reach the
chorion but fail to unite with it. Several mutants in the Wnt and Fgf
signaling pathways are impaired in chorio-allantoic fusion [Wnt7b
(Parr et al., 2001); Tcf1/Lef1
(Galceran et al., 1999); Fgfr2
(Xu et al., 1998)]. This could
indicate a functional link between these signaling pathways and Cdx genes in
the chorio-allantois fusion process, in addition to the links previously
documented in the processes of embryonic axial extension and patterning
(reviewed by Lohnes, 2003;
Deschamps and van Nes, 2005).
The majority of mutant allantoises undergo chorio-allantoic fusion but exhibit
a later defect, being impaired in the establishment of a functional
endothelial network in the labyrinth. The penetrance is again dependent on the
dose of Cdx: 10% for Cdx2+/-, 20% for
Cdx2+/-/Cdx4+/- and 
100% for
Cdx2+/-/Cdx4-/0 embryos. In each of these
genotypic classes, the most frequently occurring labyrinth abnormalities are
qualitatively indistinguishable and equally severe, suggesting that
Cdx2 and Cdx4 function in an identical way, and that the
Cdx-dependence operates at the level of reaching a threshold rather than
touching different aspects of the events. A small subset of the numerous
Cdx2+/-/Cdx4-/0 embryos examined were found to
be more mildly affected than the majority of them, revealing a certain
variability in the severity of this phenotype (these exceptions probably
correspond to the occasional survivors among the embryos with this genotype).
This variability, which we expect to be proportionally present in each
genotypic class (Cdx2+/-,
Cdx2+/-/Cdx4+/- and
Cdx2+/-/Cdx4-/0) would possibly result either
from stochastic events among parameters involved in the onset of the
phenotype, or from the fact that the genetic background of embryos carrying
the new Cdx4 mutation is not homogenous.
In conclusion, our results reveal that the collaborative role of Cdx genes in posterior embryonic development extends to the stepwise establishment of a functional placental labyrinth, from allantois growth and chorio-allantoic fusion to the extension of the allantoic vascular network within the chorionic ectoderm. The data highlight a novel role for these posterior embryonic patterning genes in controlling a vital function that allows the fetus to survive through exchanges with its mother, a function presumably acquired during vertebrate evolution towards placental mammals.
The three Cdx genes exert redundant functions in mediating the generation and AP patterning of posterior embryonic structures. They might also operate redundantly in placentogenesis. In both cases, their functional contribution is quantitatively unequal, owing to either protein or regulatory differences. This suggests that the extra-embryonic mesoderm arising from the posterior part of the primitive streak is controlled by the same genetic system as the posterior axial and paraxial embryonic structures.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/3/419/DC1
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