Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, 701 W. 168th Street, New York, NY 10032, USA
* Author for correspondence (e-mail: vep1{at}columbia.edu)
Accepted 14 March 2003
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SUMMARY |
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Key words: Tbx4, T-box, Mouse, Allantois, Limb, Vasculogenesis
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INTRODUCTION |
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The first expression of Tbx4 in the mouse embryo is observed at
7.5 days post coitus (dpc) in the developing allantois. This expression is
maintained through at least 9.5 dpc
(Chapman et al., 1996). The
allantois is an extra-embryonic, mesodermal structure that forms a connection
between the posterior embryo and the chorion early in development. This
structure later develops into the umbilicus, and is crucial in mammals for
nutrient, waste and gas exchange between mother and embryo. Molecular pathways
involved in development of the allantois are not well characterized. However,
several targeted mutations in mice have indicated that bone morphogenetic
protein (BMP) signaling is crucial for allantois development. Embryos mutant
for Bmp4 lack an allantois
(Lawson et al., 1999
), and
chimeric embryos lacking Bmp4 specifically in the epiblast lineage
form only a small allantois that fails to fuse to the chorion
(Fujiwara et al., 2001
).
Although neither Bmp5- nor Bmp7-mutant mice have allantois
defects, the allantois of embryos doubly homozygous for both mutations fails
to undergo chorioallantoic fusion
(Solloway and Robertson,
1999
). Bmp8b mutants also have shortened allantoises, and
a reduced number of primordial germ cells
(Ying et al., 2000
).
Furthermore, mutations in the downstream effectors of BMP signaling,
Smad1 (Madh1 Mouse Genome Informatics) and
Smad5 (Madh5 Mouse Genome Informatics), produce
aberrant allantois morphology, although not as severe as that of the BMP
mutations (Chang et al., 1999
;
Lechleider et al., 2001
;
Tremblay et al., 2001
).
Several genes involved in the process of chorioallantoic fusion have been
identified. The adhesion molecule VCAM1, which is expressed in the distal tip
of the allantois, and its receptor, 4 integrin, which is expressed in
the chorion, are both required for successful chorioallantoic fusion
(Gurtner et al., 1995
;
Kwee et al., 1995
;
Yang et al., 1995
).
Additionally, several transcription factors that are required for proper
chorioallantoic fusion have been identified by targeted mutagenesis, including
the forkhead transcription factor Foxf1
(Mahlapuu et al., 2001
) and
Suppressor of Hairless homolog RBP-J
(Rbpsuh Mouse Genome Informatics)
(Oka et al., 1995
).
Connection(s) between these various signaling factors, adhesion molecules and
transcription factors have not been elucidated and previously no role in this
process has been identified for Tbx4.
During organogenesis, Tbx4 is expressed in a variety of tissues,
including the hindlimb, proctodeum, mandibular mesenchyme, lung mesenchyme,
atrium of the heart and the body wall
(Chapman et al., 1996).
Hindlimb expression of Tbx4 initiates at 9.5 dpc in the posterior
flank region known as the hindlimb field, before the hindlimb bud is formed.
Tbx5, another Tbx2 subfamily member, mirrors this expression
pattern with forelimb-specific expression starting at 8.5 dpc, when the
forelimb field is specified in the anterior flank. This reciprocal expression
of Tbx4 and Tbx5 in the hindlimb and forelimb, respectively,
is maintained throughout development
(Chapman et al., 1996
;
Gibson-Brown et al.,
1996
).
Previous work has suggested that Tbx4 plays a role in determining
hindlimb identity. Transplants of limb mesenchyme and induction of ectopic
limbs in chick show correspondence between the proportions of Tbx4
and Tbx5 expression, and the degree of forelimb versus hindlimb fate,
respectively (Gibson-Brown et al.,
1998; Isaac et al.,
1998
; Ohuchi et al.,
1998
). Ectopic expression of Tbx4 and Tbx5 in
developing chick limbs results in limb abnormalities that may represent limb
identity transformations
(Rodriguez-Esteban et al.,
1999
; Takeuchi et al.,
1999
). Supporting evidence is offered by experiments with the
homeodomain protein Ptx1 (Pitx1 Mouse Genome
Informatics), which is expressed specifically in the hindlimb
(Lanctot et al., 1997
;
Shang et al., 1997
) and which
has also been suggested to play a role in hindlimb specification. Targeted
mutagenesis of Ptx1 produces mice with shortened hindlimbs that have
characteristics of forelimbs (Lanctot et
al., 1999
; Szeto et al.,
1999
), and ectopic expression of Ptx1 in chick forelimbs
leads to both the upregulation of Tbx4, and the induction of the
hindlimb-specific Hox genes Hoxc10 and Hoxc11
(Logan and Tabin, 1999
).
Interpretation of the gain-of-function experiments with Tbx4, Tbx5
and Ptx1 in chick is confounded by the presence of endogenous
Tbx4 and Tbx5 in the experimental limbs, and by limb
abnormalities induced by overexpression of Tbx4 and Tbx5,
even in their endogenous domains
(Rodriguez-Esteban et al.,
1999; Takeuchi et al.,
1999
). We have therefore used targeted mutagenesis to investigate
the role of Tbx4 in the development of the hindlimb and other regions
of Tbx4 expression, including the allantois.
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MATERIALS AND METHODS |
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Collection of embryos
Both the Tbx4tm1Pa and the
Tbx4tm1.1Pa alleles were maintained on mixed genetic
backgrounds. Mice heterozygous for each allele were intercrossed to generate
homozygous mutant embryos. Noon on the day of a mating plug was considered 0.5
dpc. Embryos were dissected in phosphate buffered saline (PBS) with 0.2%
bovine albumin (fraction V). All embryos were scored for chorioallantoic
fusion prior to yolk sac removal. Yolk sacs were taken for PCR genotyping and
embryos were fixed in 4% paraformaldehyde in PBS at 4°C for two hours or
overnight, then dehydrated in methanol and stored.
Embryos dissected at 8.0 dpc were scored for somite number and for the extent of allantois elongation into the yolk sac cavity. Because the overall embryo size is quite variable at this point in development, allantois elongation was measured as a proportion of the distance between the posterior end of the embryo and the edge of the chorionic plate. Allantoises were scored as: `early' if they had covered two-thirds or less than this distance; `late' if they had covered more than two-thirds of the distance or if they had entered the dome formed by the chorionic plate; or `fused' if they had formed a connection to the chorion.
In situ hybridization and immunohistochemistry
Whole-mount in situ hybridization was performed according to previously
described protocols (Wilkinson and Nieto,
1993). Immunohistochemistry was performed according to standard
protocols (Davis, 1993
),
except for VCAM1 immunohistochemistry, where DMSO was omitted from the bleach,
PBSMT washes were replaced with PBSBT (4% BSA, 0.1% Triton X-100 in PBS) and
primary antibody was diluted 1:150. Embryos used for doubly phosphorylated ERK
protein (dp-ERK; EPHB2 Mouse Genome Informatics) immunostaining were
cut through the limb buds prior to bleaching to facilitate antibody
penetration. Primary antibodies used included: anti-VCAM1 (PharMingen, catalog
number 55333O), anti-phospho-Histone H3 (Upstate Biotechnology, catalog number
06570), anti-PECAM (PharMingen, catalog number 01951D) and anti-phospho-p44/42
(anti-dp-ERK; Cell Signaling, catalog number 9101). All secondary antibodies
were peroxidase-conjugated goat IgG from Jackson Immunochemicals.
PGC detection
Embryos were dissected at 8.0 dpc, scored as above and genotyped. Posterior
halves were fixed in 4% paraformaldehyde in PBS for 1 hour at 4°C, and
dehydrated in 70% ethanol for 3-6 hours. Embryos were rehydrated in NTMT, then
stained with NBT/BCIP color reaction for 3 minutes
(Wilkinson and Nieto, 1993)
and photographed.
TUNEL assay
Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)
staining was performed using a Cell Death Detection kit (Roche, catalog number
1684817). Embryos were incubated in 10µg/ml proteinase K for 8-10 minutes
depending on stage, and refixed in 4% paraformaldehyde, 0.1% glutaraldehyde
for 20 minutes at room temperature. Embryos were then incubated in TUNEL
reaction mixture at 37°C for 1 hour, blocked with 10% sheep serum in PBT
for 1 hour and incubated with the converter-POD mix (same kit) for 30 minutes
at 37°. Chromogenic reaction was developed with 0.08% NiCl2 and
250 µg/ml diaminobenzidine in PBT for approximately 30 seconds.
Histology
At 8.25 dpc and 9.5 dpc, where simultaneous histology and genotyping were
not possible, embryos were classified as mutant or normal based on the
appearance and fusion of the allantois. Three embryos were discarded from the
analysis because they were grossly retarded compared to littermates. Embryos
at 8.25 dpc and 9.5 dpc were removed intact in the uterine horns and fixed in
Bouin's fix overnight. Embryos at 10.5 dpc were dissected out of the decidua,
and the yolk sacs removed for genotyping before being similarly fixed. After
dehydration in ethanol, embryos were embedded in paraffin wax, sectioned at 8
µm thickness and stained with Hematoxylin and Eosin.
Limb bud culture
Limb buds were dissected at 10.5 dpc in DMEM supplemented with 10 mM HEPES
and 5% FCS. Explants were taken from embryos only if the embryo was alive and
had a robustly beating heart. The embryo was transected immediately rostral
and caudal to each set of limb buds (see
Fig.
4A,F). Tissue
ventral of the limb buds, including the heart and allantois, was trimmed off
and the explant was placed ventral side down on a Transwell Clear (Costar)
membrane. Explants were cultured for 4-7 days in DMEM/F-12 (Gibco BRL) with
10% FCS (Hyclone), 5x MITO+ Serum Extender (Becton Dickinson,
catalog number 355006) at 37°C in 5% CO2 in air.
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RESULTS |
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Heterozygous mice of both alleles were apparently normal and fertile. Heterozygotes were intercrossed to generate embryos homozygous for each allele. Both the Tbx4tm1Pa allele and the Cre-recombined Tbx4tm1.1Pa allele were inherited in Mendelian ratios, but all homozygous mutants dissected after 10.5 dpc were dead (Table 1). In litters that were allowed to go to term, no homozygous mutants were observed among weanlings (n=397 and n=108, for the Tbx4tm1Pa and Tbx4tm1.1Pa alleles, respectively). All analyses, including in situ hybridizations for limb marker genes, were performed on embryos homozygous for each allele. Results for each were the same, and so have been combined in this report.
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Two 10.5 dpc Tbx4-mutant embryos (representing less than 1%) have been observed in which the developing allantois had made a connection with the chorion. In both embryos the allantois was small and irregular, consisting of multiple blood-filled chambers. In each case, the allantoic attachment to the chorion was at only a single point, and had not spread over the chorionic surface. No apparent continuous vessel had formed between the embryo and the placenta, and no flow of blood could be detected.
Histological examination of Tbx4-mutant embryos
Histological examination of 8.25 dpc embryos revealed multiple
abnormalities in the allantois of Tbx4-mutant embryos. The wild-type
allantois at this stage (n=10) is a funnel shaped structure tightly
opposed to the chorion at the wide end and tapering towards the posterior end
of the embryo (Fig. 2G). The
mesenchyme near the embryo is dense and uniform, whereas near the chorion it
is loose and cavitated. In mutant embryos (n=2), the allantois was
not opposed to the chorionic plate and there was no evidence of
allantois-derived cells adherent to the chorion
(Fig. 2H), although the chorion
itself appeared normal. The mutant allantois shows dense, irregularly packed
cells in the base of the allantois, with multiple double-walled vesicles
(Fig. 2I) and numerous pyknotic
nuclei, which is indicative of dying cells.
At 9.5 dpc, the allantois of wild-type embryos (n=4) consists of moderately dense mesenchyme with a more open structure near the chorion. Blood vessels running through the allantois are visible (Fig. 2K). By contrast, the allantois of a mutant at this stage (n=1) was small and dense, and had numerous condensed cells (Fig. 2J,L). At 10.5 dpc, sections through a wild-type embryo show a developing hindlimb bud and an umbilical vessel (Fig. 2M). Sections through a Tbx4 mutant show some edema, but an otherwise normal development in embryonic tissues, including lung buds, heart, fore- and hindlimb (Fig. 2N,O). However, the allantois at this stage has not developed further, and consists of irregular mesenchyme with multiple irregularly shaped, dense condensations of cells and occasional empty vesicles (Fig. 2O).
Growth and cell proliferation in Tbx4-mutant
allantoises
Embryos dissected at 8.0 dpc were scored for allantois phenotype and somite
number (Fig. 3A). Differences
between wild-type and mutant embryos become apparent as early as the 4- to
5-somite stage, when wild-type embryos are mostly at the late allantois stage
or fused to the chorionic plate but most Tbx4-mutants are still at
the early bud stage. Heterozygous embryos at the 4- to 5-somite stage exhibit
a lag in allantois development when compared with somite-matched wild-type
controls. At the 6- to 8-somite stage, nearly all wild-type and heterozygous
embryos have undergone chorioallantoic fusion, whereas most mutant embryos are
at the early allantois stage. All wild-type and heterozygous embryos past the
8-somite stage have completed chorioallantoic fusion.
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Abnormal differentiation within the allantois in Tbx4
mutants
The differentiative capacity of the allantois in the absence of
Tbx4 was assessed by examining the expression of a number of genes
with characteristic expression patterns during normal allantois development.
Bmp4 expression, which is required for proper allantois development,
is apparent in the base of both wild-type
(Fig. 3F) and mutant
(Fig. 3G) allantoises.
Tbx2 is expressed in the wild-type allantois
(Fig. 3H) from its earliest
morphological appearance until early 9.5 dpc
(Chang et al., 1999;
Mahlapuu et al., 2001
) (L.A.N.
and V.E.P., unpublished). In Tbx4-homozygous mutants, Tbx2
expression is absent or dramatically reduced in the allantois of the 2-somite
embryo (Fig. 3I), and entirely
absent in the allantois of later embryos (data not shown).
Contrary to previous work published by this lab
(Chapman et al., 1996), we were
unable to detect Tbx5 expression in the allantois by whole-mount in
situ hybridization. No Tbx5 expression was seen in the allantois of
either mutant or wild-type embryos from 7.5 dpc to 8.5 dpc, although
expression in the heart crescent was visible from late 7.5 dpc onward as
expected (data not shown). The brachyury gene (T), which marks the
primitive streak, notochord and the base of the allantois, is expressed
normally in the Tbx4-mutant allantoises (data not shown). VCAM1, a
cell adhesion molecule required for allantoic fusion, is seen by antibody
staining in the extreme distal end of the fused allantois in the 8-somite
wild-type embryo (Fig. 3J), but
is absent from the unfused distal tip of Tbx4-mutant allantoises
(Fig. 3K).
The primordial germ cells (PGCs) form a group of alkaline
phosphotase-positive cells at the base of the allantois at 8.0 dpc, shortly
before they migrate along the dorsal mesentery to the future gonads. Their
formation is impaired in some of the BMP pathway mutations that affect
allantois development (Lawson et al.,
1999; Tremblay et al.,
2001
; Ying et al.,
2000
). In wild-type (Fig.
3L) and Tbx4-mutant (Fig.
3M) embryos, PGCs appear to be similar in number and location.
To assess vascularization in the allantois, we examined the appearance of PECAM-1 (PECAM Mouse Genome Informatics), a late marker of endothelial cell development. In wild-type embryos at the 0- to 2-somite stage, anti-PECAM-1 antibody marks small clumps of endothelial cells in the allantois (Fig. 3N). At the 3- to 4-somite stage, these PECAM-positive cells form multiple small vessels (Fig. 3O). By the 6- to 8-somite stage, these vessels have largely collected into a central vessel(s) at the base of the allantois, with unincorporated vessels still apparent distally (Fig. 3P). In Tbx4 mutants, expression of PECAM-1 initiates normally (Fig. 3L), but soon acquires a strikingly different appearance. Numerous small clumps of PECAM-positive cells appear along the length of the allantois, but these fail to elongate into vessels and no central vessel is formed (Fig. 3O,P).
In vitro culture of Tbx4-mutant hindlimbs
To investigate the role of Tbx4 in hindlimb development beyond the
time of death of Tbx4-mutant embryos, limb buds from 10.5 dpc embryos
were grown in culture. Both forelimb and hindlimb buds were dissected from
live 10.5 dpc mutant embryos and littermate controls, and explanted onto
membranes suspended on growth media (Fig.
4B,C,G,H)
to examine their developmental potential. After 3 days of growth, forelimb
buds of both normal (Fig. 4D)
and homozygous Tbx4-mutant embryos
(Fig. 4E) exhibited an increase
in the distance between the right and left distal limb margins, corresponding
to outgrowth of the limbs from the midline. Cultured limb buds also assumed
the paddle-shaped morphology of handplate stage limbs. After the same culture
period, wild-type hindlimb buds had also expanded distally and developed a
recognizable paddle-shaped handplate (Fig.
4I, n=23), but mutant hindlimb explants failed to develop
any obvious limb structures (Fig.
4J, n=11). To verify the identity of these limb
structures, in situ hybridizations were performed on cultured explants using a
Tbx4 probe from outside of the deleted region as a hindlimb marker
(Fig. 1A). The mutant
transcript was expressed normally in homozygous mutants at all stages
examined. Tbx4 was clearly expressed in the handplate paddles of
wild-type embryos (Fig. 4K),
but it was expressed only in a few scattered patches of cells, with no obvious
limb morphology, in mutant embryos (Fig.
4L).
Hindlimb bud initiation in Tbx4 mutants
To explain the absence of growth of the Tbx4-mutant hindlimbs in
culture despite the presence of a morphologically obvious hindlimb bud, we
examined the expression of genes known to be involved in the initiation and
maintenance of limb formation (Martin,
2001) prior to the development of the mutant phenotype. Limb
outgrowth is known to be maintained by a mesenchymal-ectodermal feedback loop.
Mesenchyme from the limb field signals to the overlying ectoderm through
Fgf10 to induce the apical ectodermal ridge (AER). Limb ectoderm then
signals back to the mesenchyme through several molecules, including FGFs. This
reciprocal signaling is necessary for the maintenance of many limb mesenchyme
genes, including Fgf10.
Fgf8 is an early marker of the AER, and is expressed normally in a
narrow strip along the dorsoventral margin of both wild-type and
Tbx4-mutant hindlimb buds (Fig.
5A,B), indicating
that successful initial induction of the AER has occurred. Msx1,
which lies downstream of limb ectodermal signaling
(Wang and Sassoon, 1995), is
also expressed normally in Tbx4-mutant hindlimbs
(Fig.
5C,D). The twist
gene, which is required in limb mesenchyme for the FGF-mediated feedback loop
and for proper FGF receptor expression in the limb mesenchyme
(O'Rourke et al., 2002
;
Zuniga et al., 2002
), is also
expressed normally in Tbx4 mutants
(Fig.
5E,F).
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Hindlimb patterning in Tbx4 mutants
As Tbx4 expression is specific to the hindlimb and has been
postulated to have a role in specifying hindlimb identity, we examined other
genes that have differential expression between fore- and hindlimbs. The
domain of Tbx5, which is normally expressed specifically in the
forelimb, is unaltered in Tbx4-mutant embryos
(Fig.
6A,B), indicating
that Tbx4 plays no role in maintaining this differential expression.
Likewise, the Tbx4-expression domain (visualized with a probe from
outside of the deleted region) in the hindlimb is retained in Tbx4
mutants (data not shown). Ptx1, which is co-expressed in the hindlimb
field with Tbx4, is also unaltered in the Tbx4-mutant
hindlimb field (Fig.
6C,D).
Unfortunately the death of Tbx4-mutant embryos in vivo, and the
absence of hindlimb growth in vitro, preclude the study of later fore- and
hindlimb specific genes, such as limb-specific Hoxc genes.
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Tbx2 is also normally expressed in the posterior hindlimb margin at this stage, although no function in limb development has yet been assigned to it. Tbx2 is absent from Tbx4-mutant hindlimb buds (Fig. 6K,L). This demonstrates that Tbx4 lies upstream of Tbx2 in at least two tissues, the allantois and the hindlimb.
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DISCUSSION |
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The Tbx4tm1Pa allele consists of the insertion of a lox
site and a floxed selection cassette into the introns surrounding exon 5,
which creates no disruption of the Tbx4 exon sequences or splice
sites. Nevertheless, the phenotype of this allele is indistinguishable from
the null, which indicates that these intronic insertions result in profound
gene disruption. This disruption is presumably due to the exogenous promoter
and cryptic splice sites found in the neo selection cassette, both of
which have been shown to disrupt gene function when inserted into non-coding
regions (McDevitt et al.,
1997; Meyers et al.,
1998
; Nagy et al.,
1998
). These reports have shown only partial gene disruption;
however, they involve only the neo cassette, whereas we have used a
neo-tk cassette. The addition of the tk gene results in an
additional exogenous promoter and a polyadenylation site, which probably
exacerbates interference by the neo-tk cassette. Alternatively, the
Tbx4 locus could be especially susceptible to splicing disruption, or
Tbx4 dosage sensitivity could be such that even partial disruption of
Tbx4 passes below a minimum threshold limit.
Allantois development in Tbx4 mutants
Tbx4 mutants first exhibit a defect at 8.0 dpc, when they
demonstrate a short allantois and failure of chorioallantoic fusion. At this
age, homozygous-mutant, heterozygous and wild-type embryos display
considerable overlap in phenotype (Fig.
3A). This stage marks the only appearance of a heterozygous effect
of Tbx4, resulting in a delay in chorioallantoic fusion, although all
heterozygous embryos eventually undergo chorioallantoic fusion and become
indistinguishable from their wild-type littermates. However,
homozygous-Tbx4 mutants are readily distinguished from littermates by
the failure of chorioallantoic fusion.
We have attributed the death of Tbx4-homozygous mutants at 10.5
dpc to failure of chorioallantoic fusion. In the absence of an umbilicus, the
embryo cannot exchange gasses, nutrients or waste with the maternal blood
supply. Absence of the umbilicus also alters normal blood flow patterns, most
probably accounting for the observed hemorrhage and pericardial edema. Several
other mutations that result in failure of chorioallantoic fusion produce
similar phenotypes (Gurtner et al.,
1995; Kwee et al.,
1995
; Tremblay et al.,
2001
; Yang et al.,
1995
).
Extension of the allantois from the posterior axis of the embryo to the
chorion results from a number of processes, including influx of cells from the
primitive streak, cellular proliferation and cavitation of distal tissue
(Downs and Bertler, 2000).
Embryos mutant for Tbx4 have a normal primitive streak, as evidenced
by normal expression of T, and proliferation in the allantois is also
normal (Fig.
2B,C). However,
cells near the tip of the Tbx4-mutant allantois undergo apoptosis
(Fig.
2D,E). Factors
required for cell survival in the allantois are not known. However, it is
interesting to note that Tbx2 is downregulated in
Tbx4-mutant allantoises, and previous work has implied a requirement
for Tbx2 to prevent cell cycle arrest in rapidly proliferating
tissues (Jacobs et al., 2000
).
Therefore, the role of Tbx4 in the allantois may be the maintenance
of Tbx2-mediated suppression of apoptosis.
Also, in contrast to normal development, the allantois in
Tbx4-mutant embryos shows no cavitation near the distal tip. Although
the molecular nature of this defect is unknown, we find it relevant that T-box
genes have been implicated in the regulation of adhesion molecules in several
systems. In zebrafish and Xenopus, T-box genes have been shown to
regulate paraxial protocadherin (Kim et
al., 1998; Yamamoto et al.,
1998
), and, in mice, Tbr1 is a direct activator of
reelin, an extracellular matrix glycoprotein. Mouse mutations in
T produce primitive streak cells with a substrate-dependent migration
defect (Hashimoto et al.,
1987
), and work with Fgfr1-mutant mice has revealed links
between T and cadherin regulation
(Ciruna and Rossant, 2001
).
This data suggests that the failure of allantois cavitation could be caused by
misregulation of adhesion genes in Tbx4 mutants.
Despite the various defects inhibiting allantois growth, as many as 15% of
Tbx4-mutant allantoises observed had extended into the dome of the
chorion at the 4- to 5-somite stage (Fig.
1A; L.A.N. and V.E.P., unpublished). However, chorioallantoic
fusion rarely occurs in Tbx4 mutants. Of more than 250 mutant embryos
observed, only two had established any connection between the allantois and
chorion. The lack of expression of Vcam1, a key adhesion molecule
affecting this process (Gurtner et al.,
1995; Kwee et al.,
1995
), in the Tbx4 mutants is clearly a contributing
factor. However, Vcam1 homozygous-null embryos undergo
chorioallantoic fusion in 20-50% of cases, so this defect alone is
insufficient to explain the Tbx4 phenotype. It is possible that the
combination of the lack of VCAM1 protein and the extensive apoptosis in the
tip of the allantois produces a synergistic loss of chorioallantoic fusion
potential.
In addition to the failure of chorioallantoic fusion in Tbx4 mutants, there is also a block in vascular remodeling in the allantois. Endothelial cells, evidenced by positive staining for PECAM, successfully differentiate from allantoic mesenchyme, but they remain as discreet clumps of cells and fail to remodel into primary vessels. These clumps presumably correspond to the double-layered vesicles seen histologically, and are also presumably the progenitors of the blood-filled vesicles seen morphologically in the residual allantois at 10.5 dpc. It is unclear whether the failure of endothelial cells to coalesce into vessels in Tbx4 mutants is cell autonomous, or represents a defect in signaling or adhesion characteristics in surrounding cells of the allantois.
Bmp4 has previously been identified as a key regulator of
allantois development. It is required in the extra-embryonic ectoderm for
allantois induction, and later in the allantois itself for normal development
and chorioallantoic fusion. The phenotype produced by the absence of
Bmp4 in the allantois shares many points of similarity with the
Tbx4 phenotype. Both exhibit a shortened, unfused allantois with no
apparent proliferative defect, neither express VCAM1 at the tip of the
allantois and both have defects in the vascularization of the allantois.
Previous work has suggested that T-box genes can be regulated by BMP signaling
(Koshiba-Takeuchi et al.,
2000; Smith et al.,
1991
; Yamada et al.,
2000
), and the observation that Bmp4 expression in the
allantois is unaffected by the loss of Tbx4 is consistent with an
upstream role for Bmp4 with respect to Tbx4. However, the
absence of Bmp4 also severely disrupts PGC placement and formation,
whereas this process is unaffected by a lack of Tbx4. It may be that
Tbx4 is a downstream effector of Bmp4 in the allantois,
whereas other molecules act as intermediaries for Bmp4 signaling to
the PGCs.
A model for Tbx4 function in the hindlimb
Much is known about the initiation of limb morphogenesis
(Tickle and Munsterberg,
2001). Anterior/posterior, dorsal/ventral and distal/proximal axes
are all set up early in limb bud formation. Fgf10 signaling from the
limb mesenchyme induces an apical ectodermal ridge (AER) in the overlying
ectoderm. The AER, in turn, signals back to the mesenchyme through
Fgf8 and Fgf4 to maintain Fgf10 in the region
underlying the AER known as the progress zone. Fgf10 is required to
maintain proliferation in the progress zone, which drives limb outgrowth. This
represents an FGF-mediated positive feedback loop, which is required for limb
development. A second feedback loop is set up between the AER and a region in
the posterior mesenchyme known as the zone of polarizing activity (ZPA). The
ZPA directs posterior patterning in the limb and is marked chiefly by the
expression of the secreted signaling molecule Shh. Failure of either
of these feedback loops results in the absence or dramatic reduction of limb
growth and patterning.
At early 10.5 dpc, Tbx4-mutant embryos have morphologically
obvious hindlimb buds that are similar to stage-matched wild-type controls. In
many respects, the initiation of these hindlimb buds is normal. Hindlimb
specificity in Tbx4 mutants appears unaltered, as indicated by the
presence of Ptx1 and Tbx4 expression, and the lack of
Tbx5 expression. Ventrally restricted expression of Msx2 and
correct positioning of Fgf8 expression both suggest that dorsoventral
patterning in unaffected by the absence of Tbx4
(Pizette et al., 2001).
Several genes known to be involved in limb outgrowth and/or FGF reciprocal
signaling are correctly activated, including Msx1 and the gene
encoding dp-ERK in the progress zone, Fgf8 in the presumptive AER and
Twist throughout the hindlimb mesenchyme.
However, the ablation of Tbx4 does result in some defects in
anterior/posterior patterning of the hindlimb. In normal limbs, dHand
is restricted to the posterior limb bud by the action of Gli3 in the
anterior limb bud (te Welscher et al.,
2002). dHand induces Shh in the posterior limb
bud, and Shh signaling is key for the activity of the ZPA.
Tbx4-mutant embryos die prior to the expression of Shh, but,
in mutant embryos, dHand is shifted from posterior-specific
expression to expression throughout the mutant hindlimb bud despite normal
expression of Gli3. Conversely, Tbx3, which is known to
affect posterior limb development in mouse and human
(Bamshad et al., 1997
;
Davenport et al., 2003
), is
correctly expressed in the posterior margin of the Tbx4-mutant
hindlimb. Tbx2, which is also normally expressed in the posterior
margin of the hindlimb bud, is absent in Tbx4-mutant hindlimbs. As no
function has been identified for Tbx2 in limb development, this is
notable chiefly because it shows that Tbx2 expression is ablated in
Tbx4 mutants in both of the tissues where the two genes are
co-expressed prior to the death of the mutant embryos, thus revealing a role
for a T-box gene in regulating another T-box gene.
Despite successful induction of the hindlimb bud, and of many outgrowth and
patterning genes therein, neither hindlimb outgrowth nor Fgf10
expression is maintained in Tbx4-mutant hindlimbs. Initial FGF
signaling is apparently normal in Tbx4-mutant hindlimbs, as
visualized by the induction of Fgf8 in the ectoderm and
phosphorylation of ERK in the mesenchyme. However, by 10.5 dpc, Fgf10
is absent from the distal mutant hindlimb buds and only residual flank
expression can be seen. As the absence of Fgf10 has been shown to
ablate limb formation (Min et al.,
1998; Sekine et al.,
1999
), it is unsurprising that Tbx4-mutant hindlimbs fail
to progress when grown in culture.
Similar roles of Tbx4 in the forelimb and Tbx5 in the
hindlimb have been proposed in previous studies
(Gibson-Brown et al., 1998;
Logan and Tabin, 1999
;
Ohuchi et al., 1998
;
Rodriguez-Esteban et al.,
1999
; Ruvinsky and
Gibson-Brown, 2000
; Saito et
al., 2002
; Tanaka et al.,
2002
). This study, and the recently published study of
Tbx5 in the forelimb (Agarwal et
al., 2003
), reveals that the roles of Tbx4 and
Tbx5 are subtly different. In each case, the specification of the
relevant limb field proceeds despite the lack of Tbx4 or
Tbx5. However, in the case of Tbx4 ablation, FGF signaling
is initiated but not maintained, whereas Tbx5 ablation precludes any
expression of Fgf10 or Fgf8.
By marker analysis, the Tbx4 mutation closely resembles genetic
manipulations that disrupt reciprocal FGF signaling from the AER to the limb
mesenchyme, such as mutations in Twist, an upstream regulator of
Fgfr1 in limb mesenchyme
(O'Rourke et al., 2002;
Zuniga et al., 2002
), and the
AER-specific ablation of both Fgf8 and Fgf4
(Sun et al., 2002
). All of
these disruptions exhibit successful induction of AER-specific FGFs,
disruption of some but not all anterior/posterior axis markers, normal
induction of FGF-independent limb markers, such as Msx1, and rapid
downregulation of Fgf10. In particular, mutations in Twist
result in the expansion of Shh and Hoxd11 into the anterior
of the limb bud, whereas in the absence of Fgf8 and Fgf4,
the anterior expression of Alx4 is expanded posteriorly. The anterior
expansion of Shh and Hoxd11 occurs despite the normal
expression of Gli3 in the anterior of these limbs, demonstrating the
importance of FGF signaling in the Gli3-dependent maintenance of the
anterior/posterior axis of the limb bud.
Our data shows that FGF reciprocal signaling in Tbx4-mutant
hindlimb mesenchyme is successful up to the point of ERK phosphorylation by
the FGF-activated MAPK cascade. However, we observed a failure in the
maintenance of Fgf10 and an expansion of the posterior limb domain,
as evidenced by dHand expression, both of which are consistent with a
failure in FGF signaling. This suggests that the role of Tbx4 in
early limb development is to transduce the signal of the MAPK cascade to its
final limb targets (Fig. 7), including both anterior dHand repression and Fgf10
upregulation. Aside from Gli3, the transcriptional co-factors
involved in dHand repression are not known, but it is possible that
Tbx4 is an FGF-sensitive, direct repressor of this gene;
alternatively, the derepression of dHand in Tbx4-mutant
hindlimbs could be a secondary result of disrupted FGF signaling.
Fgf10 is probably a direct target of Tbx4, as T-box binding
sites have been observed in the Fgf10 promoter and Fgf10 is
capable of T-box dependent reporter gene activation in vitro
(Agarwal et al., 2003;
Ng et al., 2002
). It seems
likely therefore that Tbx4 is a MAPK-sensitive, direct regulator of
both dHand and Fgf10.
|
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ACKNOWLEDGMENTS |
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