Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USA
* Author for correspondence (e-mail: derobert{at}hhmi.ucla.edu)
Accepted 13 October 2003
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SUMMARY |
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Key words: Twisted gastrulation, Bmp, Tgfß, Chordin, Tolloid, Crossveinless, Holoprosencephaly, Vertebral column
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Introduction |
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Tsg has multiple biochemical activities. First, it promotes the formation
of stable ternary Bmp/Chd/Tsg complexes
(Oelgeschläger et al.,
2000; Chang et al.,
2001
; Larrain et al.,
2001
; Scott et al.,
2001
). As this ternary complex prevents binding of Bmp to its cell
surface receptors, in this aspect of its function Tsg behaves as a Bmp
antagonist. Second, the stability of these inhibitory ternary complexes is
controlled by the Tld metalloprotease, which cleaves Chd/Sog at specific sites
(Marques et al., 1997
;
Piccolo et al., 1997
). In the
presence of Tsg, Chd/Sog is a better substrate for cleavage by the Tld enzyme
(Larrain et al., 2001
;
Scott et al., 2001
;
Shimmi and O'Connor, 2003
). By
promoting Chd degradation in the presence of Tld, Tsg releases Bmp that is now
able to signal through Bmp receptors. In this second aspect of its activity,
Tsg functions to increase Bmp activity
(Piccolo et al., 1997
;
Larrain et al., 2001
;
Shimmi and O'Connor,
2003
).
The opposing functions of the Tsg protein may explain why conflicting
results have been reported in various microinjection assays. In
Xenopus, overexpression of Tsg mRNA results in Bmp-promoting
effects (Oelgeschläger et al.,
2000; Oelgeschläger et
al., 2003a
; Oelgeschläger
et al., 2003b
). The opposite observation was made in zebrafish
embryos, in which microinjection of zebrafish Tsg or Xenopus
Tsg caused dorsalization (Ross et
al., 2001
; Oelgeschläger
et al., 2003b
). These different outcomes have been attributed to
the different levels of the Tld protease in the two model embryos
(Larrain et al., 2001
). In
zebrafish, endogenous levels of the Tolloid protease are low, as indicated by
the weak phenotype of tolloid/mini-fin mutant embryos which are
viable and lack the ventral tail fin
(Connors et al., 1999
). At
these low Xolloid concentrations, microinjection of Tsg mRNA favors
the formation of inhibitory Bmp/Chd/Tsg complexes. In the Xenopus
embryo, high levels of Tld activity are present, Tsg promotes the cleavage of
Chd, and ventralization is observed. However, when the endogenous Tld protease
is inhibited by co-injection of dominant-negative Xld mRNA the
opposite result, dorsalization, is seen
(Larrain et al., 2001
). These
experiments suggest that the proteolytic cleavage of Chd constitutes the
crucial molecular switch between the anti-Bmp and Bmp-promoting activities of
Tsg.
Additional evidence on the dual activity of Tsg was provided by the study
of point mutations that dissociate Bmp binding and Chd interaction
(Oelgeschläger et al.,
2003a). Mutations in the N-terminal domain of Tsg abolish
Bmp binding. Surprisingly, these mutant Tsgs still have potent Bmp-promoting
effects in both Xenopus and zebrafish embryos
(Oelgeschläger et al.,
2003a
). There is evidence that Tsg might interact with anti-Bmp
proteins other than Chd. When Chd is mutated in zebrafish
chordino, overexpression of Tsg can further ventralize the
embryo (Oelgeschläger et al.,
2003a
). In Xenopus, embryos microinjected with antisense
Chd morpholino oligos together with Xenopus Tsg mRNA display
very severe ventralization
(Oelgeschläger et al.,
2003b
). These experiments indicate that some of the activities of
Tsg are independent of Chd. A large number of extracellular proteins contain
Bmp-binding CR modules of the type present in Chd
(Larrain et al., 2000
;
Abreu et al., 2002
;
Coffinier et al., 2002
), and
some of them interact with Tsg (C. Coffinier and E.M.D.R., unpublished). Thus,
it appears likely that Tsg interacts with other proteins in addition to Chd
and Bmps.
Although multiple activities and interactions of Tsg have been identified
in vivo and in vitro, many questions remain unanswered concerning its
physiological functions. In particular, whether Tsg is a pro-Bmp
(Oelgeschläger et al.,
2000; Oelgeschläger et
al., 2003a
; Oelgeschläger
et al., 2003b
) or is a Bmp antagonist
(Chang et al., 2001
;
Ross et al., 2001
;
Scott et al., 2001
;
Blitz et al., 2003
) in a
loss-of-function situation in mammals. We report the targeted inactivation of
the murine Tsg gene. Tsg mutant mice were viable and of
small size, and presented skeletal defects in the vertebral column. We
analyzed the interactions between the Tsg and Bmp4
genes. Tsg-/-;Bmp4+/- compound mutants
died at birth and displayed severe holoprosencephaly, eye and first branchial
arch defects. As Tsg is required for Bmp4 to function during forebrain
formation in a dose-dependent manner, we conclude that Tsg acts to promote
Bmp4 signaling during mouse head development.
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Materials and methods |
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|
In situ hybridization, histology and skeletal preparations
In situ hybridization on whole-mount or on cryostat sections was performed
as previously described (Henrique et al.,
1995). The probes used were: Bmp4
(Winnier et al., 1995
),
Dlx5 (Acampora et al.,
1999
), Fgf8 (Crossley
and Martin, 1995
), Hex
(Bedford et al., 1993
),
Shh (McMahon et al.,
1998
) and Cer1 (Belo
et al., 1997
). The Tsg probe was synthesized using the
full-length cDNA cloned in pGEMTeasy, linearized with XhoI and
transcribed with SP6 RNA polymerase. Newborns and E12.5 to E15.5 embryos were
fixed in Bouin's solution, dehydrated, cleared and embedded in paraffin wax.
Serial 7 µm sections were stained using Hematoxylin and Eosin or Mallory's
Tetrachrome method (Bachiller et al.,
2003
). Alcian Blue and Alizarin Red skeletal staining
(Belo et al., 1998
), and
Alcian Blue staining at E14.5 (Jegalian
and De Robertis, 1992
) was as described. ß-Gal staining of
whole embryos was performed by fixing the embryos in 0.2% glutaraldehyde in
PBS for 2 to 15 minutes and staining at 37°C in 1 mg/ml Xgal, 2 mM
MgCl2, 0.1 M phosphate buffer pH 7.4, 5 mM potassium ferrocyanide,
5 mM potassium ferricyanide.
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Results |
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Comparison of the expression of the Tsg mRNA and ß-galactosidase (ß-gal) activity in E6.5 to E8.5 embryos showed that the lacZ reporter gene was expressed at the same time and place as the endogenous Tsg gene (Fig. 1, compare E,E'-H,H'). Tsg expression was observed at E6.5 in the anterior visceral endoderm and forming primitive streak (Fig. 1E,E'). As gastrulation continued and mesodermal wings formed, Tsg expression was observed in the entire mesodermal layer (Fig. 1F,F',G,G'). At early headfold stage Tsg was expressed in head mesenchyme, gut endoderm and ventral neuroectoderm (Fig. 1I). At E8.5, Tsg was expressed in ventral neuroectoderm such as basal diencephalon (Fig. 1J), the endoderm of the gut, the posterior eye vesicle and the forming pharyngeal arches (Fig. 1H,H'). We conclude that the in-frame reporter gene faithfully recapitulates the endogenous Tsg expression pattern.
Tsg-/- mice are viable and have skeletal defects
To address the function of Tsg in vivo, heterozygous animals were
mated and their progeny analyzed. Viable homozygous mutants were recovered in
Mendelian proportions (e.g., in F1 crosses, of 106 neonates 25 were +/+, 57
were +/and 24 were -/-). Both male and female Tsg-/-
animals were fertile. Growth analysis of Tsg mutants showed that they
were of smaller size, weighed 10-20% less than littermates and displayed a
short tail. Eighty percent of the Tsg-/- animals presented
multiple kinks in the tail, which became more marked with age
(Fig. 2A,B). X-rays of adult
mice indicated that the caudal vertebrae of mutant mice were shorter than
normal and had lower bone density than wild-type or heterozygous animals
(Fig. 2B). In E14.5
Tsg-/- embryos, defective cartilage formation was observed
in the dorsal neural arches of most cervical and some thoracic vertebrae
(Fig. 2C,C'). This defect
persisted after birth and was characterized by the incomplete growth and
fusion (dyssymphysis) of the osseous vertebral neural arches (compare
Fig. 2D with
2D').
|
Defective chondrogenesis in the caudal region of Tsg-/- embryos
We next analyzed the pathogenesis of the vertebral defects
(Fig. 3). Formation of the
vertebral column is the result of several inductive events. First, the
notochord induces differentiation of somitic mesoderm into sclerotome. After
migrating next to the notochord, sclerotomal cells form an unsegmented
perichordal tube, which is then subdivided into alternate condensed and
uncondensed areas of mesenchyme
(Grüneberg, 1963;
Theiler, 1988
). The condensed
areas give rise to the annulus or future intervetebral disc. The annulus
separates into a fibrous outer part (annulus fibrosus) and an inner part of
prechondrogenic mesenchyme (inner annulus), forming concentric rings around
the notochord. The uncondensed areas give rise to the cartilaginous primordia
of the vertebral bodies. Finally, the notochord disappears from the developing
vertebral body (possibly extruded by the increasing pressure of the cartilage
extracellular matrix) but remains at the center of the intervertebral disc,
where it forms the nucleus pulposus
(Grüneberg, 1963
;
Theiler, 1988
;
Aszodi et al., 1998
).
|
Holoprosencephaly in Tsg-/-;Bmp4+/- mutants
We next asked whether Tsg and Bmp4 interact genetically.
Homozygous null mutants for Bmp4 die around gastrulation
(Winnier et al., 1995).
Heterozygous Bmp4 animals are viable and display craniofacial
malformations, microphthalmia, cystic kidney and preaxial polydactily
(Dunn et al., 1997
). Matings
were set up between Tsg+/-;Bmp4+/- and
Tsg+/-;Bmp4+/+ animals in B6SJL/F1
background. No additional phenotypes, compared with the ones already
described, were detected in
Tsg+/-;Bmp4+/- or
Tsg-/-;Bmp4+/+ littermates. However,
Tsg-/-;Bmp4+/- neonates displayed
severe head malformations (Fig.
4) with a 64% penetrance (seven out of 11
Tsg-/-;Bmp4+/-, n=77). In
severe cases, a single nostril, lack of mouth opening, anophtalmia and low
implantation of the external ears were observed
(Fig. 4A,A').
Histological sections of the snout region showed the absence of nasal septum,
lower mandible and tongue (Fig.
4B,B'), the latter two being derivatives of the first
branchial arch. Similar phenotypes were observed at E15.5
(Fig. 5A').
|
|
Comparison of the expression of Tsg and Bmp4
In light of the phenotypes uncovered by genetic interactions between
Tsg and Bmp4, we examined the expression of Tsg in
comparison to that of Bmp4 and Chd, specifically in the
affected structures (Fig. 6).
At E8.0 Tsg, Bmp4 and Chd are expressed in adjacent domains
in anterior regions. Tsg was diffusely expressed in ventral
neuroectoderm, head mesoderm and foregut endoderm
(Fig. 6A, Fig. 1I,J). At this stage
Bmp4 was expressed in the surface ectoderm surrounding the neural
folds and in extra-embryonic mesoderm (Fig.
6B,C) (Lawson et al.,
1999; Fujiwara et al.,
2002
). Chd was expressed in the notochord, prechordal
plate and dorsal endoderm (Fig.
6D) (Bachiller et al.,
2003
). The Tld proteases that regulate Chd activity have broad
expression patterns, which include the neuroectoderm and extra-embryonic
tissues (Scott et al., 1999
).
Tsg expression continued to be diffuse at later stages, with ventral
brain, eye, branchial arches, limb buds and ventral posterior mesoderm
expressing Tsg at higher levels
(Fig. 6E,G, Fig. 1H,H').
Bmp4 was expressed in dorsal telencephalon, eye, proximal ectoderm of
the first branchial arch, frontonasal mass, maxillary arch and limb buds,
ventral-posterior mesoderm and allantois
(Fig. 6F,H). Chd is
expressed in pharyngeal endoderm at these stages, and at low levels in the
first branchial arch (Stottmann et al.,
2001
; Bachiller et al.,
2003
). We conclude that the genetic interactions between
Bmp4 and Tsg produce phenotypes in regions in which both
genes are expressed in adjacent domains, such as the forebrain and the eye.
However, other regions in which the expression domains show strong overlap,
such as ventral mesoderm and limb buds, do not show additional phenotypes.
Presumably in these regions other Bmps compensate for the decreased level of
Bmp4. One region in which expression of Bmp4 overlaps with
Tsg and produces dose-dependent phenotypes is the first branchial
arch.
|
|
We conclude that Tsg inactivation combined with half the normal dose of Bmp4 results in the loss of Shh and Fgf8 expression in the basal telencephalon and diencephalon. Although the absence of these important signaling molecules could suffice to explain the observed holoprosencephaly, a small decrease of the forebrain territory was already observed at the neural plate stage. Formation of the AVE and ANR appeared normal in the compound mutant embryos. The holoprosencephaly phenotype in Tsg-/-;Bmp4+/- embryos arose around E8.0, concomitantly with the loss of Shh and Fgf8 expression. The results indicate that Tsg is required for the proper function of Bmp4 in the formation of the telencephalic vesicles.
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Discussion |
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Tsg is not essential for embryogenesis
Tsg has been conserved in evolution between Drosophila and the
vertebrates and is known to bind in vitro several Bmps, such as Bmp2, Bmp4 and
Bmp7, as well as the Bmp antagonist Chd
(Oelgeschläger et al.,
2000; Chang et al.,
2001
; Scott et al.,
2001
). Nosaka et al. (Nosaka
et al., 2003
) recently generated a larger deletion of the
Tsg gene that resulted in a similar phenotype to the one described
here. In addition, they reported a lethality of up to 50% of the mutants in
the first month, which we did not observe. This discrepancy may be due to a
difference in genetic backgrounds (inbred C57BL/6 versus the hybrid B6SJL/F1
background used here). Mouse Tsg had been cloned as a gene that is
differentially expressed in the thymus
(Graf et al., 2001
;
Graf et al., 2002
). Nosaka et
al. (Nosaka et al., 2003
)
identified severe deficiencies in thymocyte cell number and differentiation in
Tsg-/- mice, which were interpreted to indicate an
increase in Bmp signaling. The thymus phenotype was not analyzed here. As
discussed in the introduction, Tsg has both Bmp-promoting and anti-Bmp
properties, depending on the presence of Chd and Tld. Tsg facilitates the
formation of ternary complexes with Bmp and Chd and in this way inhibits Bmp
signaling through its cognate receptors
(De Robertis et al., 2000
)
until Chd is cleaved by Tld (Larrain et
al., 2001
). The presence of Tsg facilitates proteolytic cleavage
of Chd by Tld, promoting Bmp activity. Microinjection of antisense morpholino
oligos in zebrafish and Xenopus have provided evidence that Tsg can
inhibit Bmp signaling and that it cooperates with Chd in this function,
presumably by interfering with ternary complex formation
(Ross et al., 2001
;
Blitz et al., 2003
). Tsg
mutant proteins that are unable to bind Bmp4 can still bind to Chd and other
CR-containing proteins and have potent Bmp-promoting activity
(Oelgeschläger et al.,
2003a
). The multiple functions of Tsg made it particularly
important to analyze its function in defined genetic situations.
Despite its strong evolutionary conservation, Tsg was not required for
vertebrate embryonic development. In Drosophila, Tsg mutation results
in the loss of the amnioserosa a tissue that requires highest Bmp/Dpp activity
(Mason et al., 1994). Mutation
of Drosophila Tsg results in the lack of peak phosphorylation of the
transcription factor Mad (Ross et al.,
2001
) and in the inability of Dpp to diffuse in the embryo
(Eldar et al., 2002
). A second
Tsg gene, Drosophila Tsg2 (L. Marsh, communication to FlyBase
FBgn0000394) is affected in the crossveinless (cv) mutant.
cv causes a loss of the posterior crossveins
(Bridges, 1920
), a structure
that requires maximal Bmp signaling in the Drosophila wing. In
addition, a third Tsg homolog was identified, Drosophila
Tsg3, which only contains the C-terminal half of the protein
(Vilmos et al., 2001
).
Drosophila Tsg3 maps close to shrew, a gene also required to
achieve highest signaling levels in the Bmp/Dpp dorsoventral signaling
gradient (Vilmos et al., 2001
;
Ferguson and Anderson, 1992
).
Although the mouse genome has been sequenced, genes with small exons can
escape a BLAST search. It is conceivable that additional Tsg
homologues may exist in the mouse genome; in Xenopus a divergent
xTsg2 cDNA with biological activities similar to those of
Xenopus Tsg has been recently isolated (M. Oelgeschläger, U.
Tran and E.M.D.R., unpublished).
Tsg function in skeletal development
The loss of Tsg in mouse resulted in vertebral abnormalities. The
neural arches of the cervical and thoracic vertebrae fail to fuse at the
midline and kinked tails were observed. We examined the pathogenesis of the
vertebral defects in Tsg mutants. Instead of a single ossification
center in caudal vertebrae, Tsg-/- animals develop two
independent centers. Although in most cases the twin centers ultimately fuse,
in some vertebrae one of them fails to develop. The end result is a
wedge-shaped hemi-vertebra that leads to an abnormal angle between neighboring
vertebral articulations, causing the tail kinks
(Fig. 2G'). Tail kinks
are frequently observed in mouse skeletal mutants
(Grüneberg, 1963;
Theiler, 1988
) and can
originate from defects in sclerotome differentiation. In
Tsg-/- mice, the cartilage of the vertebral bodies was
reduced and the inner annulus, which expresses Tsg, failed to
differentiate prechondrogenic cells.
As Bmps are known to promote cartilage differentiation
(Erlebacher et al., 1995;
Hogan, 1996
;
Canalis et al., 2003
), Tsg
could be viewed as promoting Bmp activity in this tissue. Other components of
the Chd/Tsg/Bmp/Tld regulatory pathway are also expressed in the developing
skeleton (Scott et al., 1999
)
and in particular Chd is found in the outer annulus
(Coffinier et al., 2002
).
Although Bmp4 is also expressed in the outer intervertebral annulus,
no increase in the severity of the tail phenotype was found in
Tsg-/-;Bmp4+/- mutants. However, other
Bmps expressed in the tail region may have redundant functions with
Bmp4. Of particular interest is Bmp7, as Bmp7
mutant mice have kinked tails (Dudley et
al., 1995
; Jena et al.,
1997
). The analysis of the genetic interactions between Chd,
Bmp7 and Tsg in the mouse, are currently under investigation in
our laboratory. Preliminary results indicate that Tsg cooperates with
Bmp7 in the differentiation of ventral embryonic structures (L.Z., K.
Lyons and E.M.D.R., unpublished).
Tsg-/- adult mice present osteoporosis, which has been
interpreted as a loss of Bmp signaling
(Nosaka et al., 2003).
Although there is much evidence linking Bmp signals to cartilage formation, a
role for Bmps in bone differentiation is less firmly established
(Karsenty, 2000
). However,
more recent studies in transgenic mice overexpressing a dominant-negative Bmp
receptor or Noggin (Nog), in bone indicate that Bmps are required for
postnatal bone growth (Zhao et al.,
2002
; Devlin et al.,
2003
).
Tsg interacts with Bmp4
Tsg interacts genetically with Bmp4. Biochemical studies
had shown a direct interaction between Tsg and Bmps, but a genetic
demonstration that these molecules function in the same pathway in vivo was
lacking, even in Drosophila. We chose Bmp4 because of the direct
biochemical interactions between the two proteins and for the strong
Bmp4 loss-of-function phenotype in mouse. Bmp4 homozygous
mutants die at gastrulation (Winnier et
al., 1995), whereas Bmp4+/- mice present mild
haploinsufficient phenotypes with variable penetrance, making it a
particularly suitable model for uncovering potential genetic interactions
(Dunn et al., 1997
).
Tsg-/-;Bmp4+/- animals did not survive
beyond birth and presented additional phenotypes to those already found in
either Tsg-/- or Bmp4+/- animals
(Fig. 5). First, the forebrain
presented reduced telencephalic vesicles that fused into a single ventricular
cavity, the defining characteristic of holoprosencephaly
(Muenke and Beachy, 2000
).
Second, eye vesicles were formed initially but degenerated, although the
orbits and eyelids still differentiated. This phenotype suggests an
enhancement of the Bmp4+/- phenotype since microphthalmia
can result from Bmp4 haploinsufficiency
(Dunn et al., 1997
). Recently,
anophthalmia has been described in mutant mice with hypomorphic alleles of
Bmp4 (Kulessa and Hogan,
2002
) and in Cre-Foxg1;Bmp4loxP-lacZ-neo
mutants that lack Bmp4 expression in the telencephalon
(Hebert et al., 2002
;
Hebert et al., 2003
).
Bmp4 and Tsg are strongly expressed in the eye region (Figs
1 and
6). In addition, Bmp4
is essential for lens induction (Furuta
and Hogan, 1998
) and lenses were not observed in compound mutants.
Third, the lateral ganglionic eminence of the basal telencephalon did not
form. Fourth, the floor of the diencephalon was greatly thickened a site of
co-expression for Bmp4 (Furuta et
al., 1997
) and Tsg
(Fig. 1J).
Finally, the mandibular component of the first branchial arch was
hypoplastic or absent in the compound mutants. Both Bmp4 and
Tsg are expressed in the developing branchial arch
(Fig. 6). A first branchial
arch phenotype was described in
Cre-Foxg1;Bmp4loxP-lacZ-neo mutants
(Hebert et al., 2003). Of all
the phenotypes of Tsg-/-;Bmp4+/-
animals, the only one not previously reported in Bmp4 mutations was
the holoprosencephaly. It may appear surprising that Cre-Foxg1;
Bmp4loxP-lacZ-neo mutants that lack Bmp4 in the
forebrain do not have this phenotype. However, our results show that the onset
of the holoprosencephaly occurs earlier than the recombination induced by
Cre-Foxg1 mice (Hebert et al.,
2003
). It is thus likely that Bmp4 is required for the
development of the telencephalic vesicles prior to E8.5, which would not have
been revealed in the conditional Cre-Lox approach
(Hebert et al., 2003
). In
conclusion, Tsg-/-;Bmp4+/- mutants
present phenotypes similar to those observed in Bmp4 hypomorphic and
loss-of-function mutants.
Tsg, Bmp4 and holoprosencephaly (HPE)
The human Tsg gene maps close to the HPE locus 4 on
chromosome 18p11.3 (Graf et al.,
2001). However no mutations in the human Tsg gene could
be detected in familial cases of HPE at locus 4 (M. Muenke and E.M.D.R.,
unpublished observations). It has been proposed in the multi-hit hypothesis
that sporadic HPE may result from mutations in more than one gene
(Ming and Muenke, 2002
). This
is what is observed in our study, in which HPE requires mutations in two
genes, Tsg and Bmp4, to become manifest. However, we have
recently observed sporadic cases of HPE in Tsg-/- embryos
in mice that had been bred for six generations into B6SJL/F1 background.
Importantly, these Tsg-/- embryos with HPE still developed
eyes, whereas Tsg-/-;Bmp4+/- had HPE
with anophthalmia. In earlier crosses, the ones reported in this paper, we
only observed HPE in Tsg-/- embryos when one copy of
Bmp4 was removed. It is likely that a genetic modifier of unknown
nature was changed by breeding in the laboratory.
Tsg and Bmp4 are expressed in adjacent or overlapping
regions (Figs 1 and
6). The holoprosencephalic
phenotype of the Tsg-/-;Bmp4+/-
mutants is not of early onset, for the anterior visceral endoderm and the
anterior neural ridge were normally formed. At E8.5 the expression of two
crucial signaling factors, Fgf8 and Shh, was defective in
Tsg-/-;Bmp4+/- embryos and this can
explain the phenotypes observed. Shh is required for the growth of
the ventral forebrain and when mutated causes a more severe HPE than the one
described here since it includes cyclopia
(Chiang et al., 1996;
Ishibashi and McMahon, 2002
).
In addition, Shh-/- mice fail to express Fgf8 in
the ventral forebrain (Ohkubo et al.,
2002
). Fgf8 null embryos die at gastrulation
(Sun et al., 1999
), but the
study of Fgf8 hypomorphic alleles has demonstrated its requirement
for forebrain formation (Meyers et al.,
1998
; Garel et al.,
2003
) and first branchial arch development
(Trumpp et al., 1999
;
Abu-Issa et al., 2002
).
Multiple experiments in mouse and chick embryos have shown that signals
from Bmps, Shh and Fgf8 regulate the growth of the telencephalon through
proliferation and cell death (Furuta et
al., 1997; Rubenstein et al.,
1998
; Anderson et al.,
2002
; Ohkubo et al.,
2002
; Storm et al.,
2003
). For example, the implantation of Bmp4-coated beads causes
HPE (Furuta et al., 1997
;
Ohkubo et al., 2002
). Ectopic
expression of Bmp4 and Bmp5 in the chick forebrain causes
cyclopia and HPE (Golden et al.,
1999
). Chd;Nog double mutants, which should have
increased Bmp signaling, also show HPE
(Bachiller et al., 2000
;
Anderson et al., 2002
).
However, ectopic expression of the Bmp antagonist Nog also inhibits
telencephalic growth (Ohkubo et al.,
2002
). Therefore, it appears that the growth of the forebrain
requires a fine balance of Bmp, Fgf8 and Shh signaling and that both excessive
and insufficient signaling can result in similar phenotypes
(Ohkubo et al., 2002
).
Is the HPE in Tsg-/-;Bmp4+/- embryos indicative of a pro-Bmp4 or an anti-Bmp4 effect? This question can be answered by a simple genetic argument. In the absence of Tsg, two copies of Bmp4 are compatible with normal head development. However, when in addition one copy of Bmp4 is removed, development of the ventral forebrain and first branchial arch are impaired. We conclude from these dose-dependent genetic interactions that during head development Tsg is required to promote Bmp4 signaling.
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ACKNOWLEDGMENTS |
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