1 Howard Hughes Medical Institute, and Department of Biological Chemistry,
University of California, Los Angeles, CA 90095-1662, USA
2 Department of Orthopaedic Surgery, University of California, Los Angeles, CA
90095, USA
* Author for correspondence (e-mail: derobert{at}mednet.ucla.edu)
Accepted 21 March 2005
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SUMMARY |
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Key words: Sirenomelia, Twisted gastrulation (Twsg1), Bmp, Bmp4, Morpholino, Limb bud, TGFß, Chordin, Dorsoventral patterning, Mouse, Xenopus
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Introduction |
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Gluecksohn-Schoenheimer and Dunn published the first study on sirens in the
mouse, in these terms: `Animal with fused and more or less perfectly developed
posterior extremities like those mythical beings supposedly representing
hybrids of man and fish'
(Gluecksohn-Schoenheimer and Dunn,
1945). Until then, sirenomelia had not been described in any
mammal except humans. These mouse sirens were found in crosses between parents
carrying mutations at the T, Fused or ur loci, thus
establishing that sirenomelia can have a genetic basis. The phenotype was not
described again in the mouse until 1970
(Hoornbeek, 1970
). More
recently, two groups linked retinoic acid to sirenomelia in gene knockouts of
the retinoic acid-degrading enzyme, CYP26
(Abu-Abed et al., 2001
;
Sakai et al., 2001
). Here, we
report the appearance of the siren phenotype in Tsg/Bmp7
compound mutants.
Dorsoventral patterning in vertebrate and invertebrate embryos is
determined by a gradient of Bmp activity controlled in the extracellular space
by a network of secreted proteins. Tsg (Twsg1 Mouse Genome
Informatics) is one of these Bmp regulators and can act both to promote and
inhibit Bmp activity. When Tsg forms a stable ternary complex with Bmp and
Chordin (Chd), Bmp signaling is prevented
(Oelgeschläger et al.,
2000; Chang et al.,
2001
; Larrain et al.,
2001
; Ross et al.,
2001
; Scott et al.,
2001
). However, the presence of Tsg in the complex facilitates
cleavage of Chd by the metalloprotease Tld, enabling Bmp to signal again, and
thus promoting Bmp activity
(Oelgeschläger et al.,
2000
; Larrain et al.,
2001
; Oelgeschläger et
al., 2003a
; Oelgeschläger
et al., 2003b
). Tsg loss-of-function experiments in
several species have resulted in various phenotypes interpreted as pro-Bmp or
anti-Bmp effects. In Drosophila, Tsg mutation results in the loss of
the amnioserosa, a tissue that requires the highest levels of Bmp/Dpp activity
(Mason et al., 1994
), and
absence of Tsg also impairs Bmp/Dpp diffusion in the embryo
(Eldar et al., 2002
). In
Xenopus, microinjection of Tsg antisense morpholino oligos
(MO) has been proposed to cause phenotypes consistent with an increase in Bmp
signaling (Blitz et al., 2003
).
In zebrafish, by contrast, knockdown of Tsg dorsalizes the embryo,
indicating that Tsg has a pro-Bmp effect in development
(Little and Mullins, 2004
;
Xie and Fisher, 2005
). In the
mouse, Tsg has been shown to play a role in thymus, foregut,
craniofacial and skeletal differentiation; Tsg appears to be a Bmp antagonist
during T-cell development, yet it increases Bmp activity in skeletal growth
(Nosaka et al., 2003
;
Petryk et al., 2004
;
Zakin and De Robertis, 2004
).
In a genetic background in which Tsg-/- mutants do not
display head malformations on their own, heterozygosity for a Bmp4
null allele leads to craniofacial defects and holoprosencephaly. This genetic
interaction suggests that, in mouse, Tsg acts as a pro-Bmp4, because in the
absence of Tsg, half a dose of Bmp4 is insufficient for head development
(Zakin and De Robertis,
2004
).
Studies on Tsg have focused on interactions with Bmp4, but, as shown here,
Tsg also binds to Bmp7. In Xenopus, Tsg and Bmp7 were
knocked down using antisense MO oligos; the injected embryos displayed tail
truncations and loss of the ventral fin. In the mouse, we performed crosses
between Tsg and Bmp7 mutant lines
(Dudley et al., 1995;
Zakin and De Robertis, 2004
).
As Bmp7 mutant mice are perinatal lethal
(Dudley et al., 1995
) and
Tsg mutants are viable (Zakin and
De Robertis, 2004
), increased or new phenotypes could be readily
scored in this experimental setting. We found that
Tsg-/-;Bmp7-/- and
Tsg+/;Bmp7-/- compound mutants displayed
sirenomelia. The appearance of this rare, severe developmental phenotype
revealed that in the absence of Bmp7, two functional alleles of
Tsg are required for dorsoventral patterning of posterior mesoderm in
mammals.
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Materials and methods |
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Mouse in situ hybridization, histology and skeletal preparations
Mouse in situ hybridization on whole mounts were performed
(Henrique et al., 1995) using
the following probes: Bmp4
(Winnier et al., 1995
),
Bmp7 (Lyons et al.,
1995
), brachyury (T)
(Herrmann et al., 1990
),
Fgf8 (Crossley and Martin,
1995
), Shh (McMahon
et al., 1998
), Tsg
(Zakin and De Robertis, 2004
)
and Wnt3a (Roelink and Nusse,
1991
). Procedures for Alcian Blue and Alizarin Red skeletal
staining (Belo et al., 1998
),
and ß-gal staining of whole embryos
(Zakin and De Robertis, 2004
)
were as described.
Western blot analysis
Immunoprecipitation of pure recombinant human Bmp7, TGFß2 and Bmp4
(from R&D Systems) was performed as described
(Oelgeschläger et al.,
2000), using mouse Tsg-Flag pre-bound to anti-Flag agarose beads
(M2, Sigma). Secreted proteins for crosslinking studies were harvested from
conditioned medium from 293T cells transfected with human Tsg-HA (Tsg
containing a hemagglutinin epitope tag at the C-terminus) or mouse Tsg-Flag
(Flag epitope tag at the N-terminus), or human Bmp7. Before chemical
crosslinking, samples were dialyzed into PBS, then incubated together and
treated with DSS (disuccinimidyl suberate) as previously described
(Oelgeschläger et al.,
2000
; Larrain et al.,
2001
). An immunopurified goat antibody specific for human Bmp7
(R&D systems) was used for western blot analyses.
Tsg-MO and Bmp7-MO injections and in situ hybridization
Xenopus microinjection and in situ hybridization were performed as
described (Piccolo et al.,
1997). Tsg-MO was as previously described
(Blitz et al., 2003
). Bmp7-MO
sequence is as follows 5'-TTACTGTCAAAGCATTCATTTTGTC-3'
(underline indicates AUG start codon) and was designed using sequences
identical in the two pseudoalleles
(Heasman et al., 2000
;
Oelgeschläger et al.,
2003b
). In vitro, Bmp7-MO efficiently and specifically inhibited
translation of Xenopus Bmp7 mRNA (B.R., E.M.D.R. et al.,
unpublished). Tsg-MO (6 ng per embryo) or of Bmp7-MO (12 ng per embryo) were
injected.
Ventral marginal zone (VMZ) assay and RT-PCR
VMZ explants from stage 10 embryos were cultured in 1 x Steinberg
solution until tailbud stage 20 and processed for RT-PCR or phenotypical
analysis. The conditions and primer sequences were as described
(http://www.hhmi.ucla.edu/derobertis).
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Results |
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|
We conclude that Tsg and Bmp7 proteins are capable of establishing direct
biochemical interactions, and that the two genes share overlapping expression
domains during embryogenesis. To investigate whether genetic interactions
existed, we next crossed mice carrying mutations in the Tsg and
Bmp7 genes (Dudley et al.,
1995; Zakin and De Robertis,
2004
).
Sirenomelia and embryonic lethality in Tsg-/-;Bmp7-/- and Tsg+/;Bmp7-/- compound mutants
Tsg and Bmp7 mutant strains were mated to generate
Tsg;Bmp7 double heterozygotes.
Table 1 summarizes the results
obtained from a total of 197 neonates from
Tsg+/;Bmp7+/ intercrosses.
Interestingly, only 0.5% (n=1) of
Tsg-/-;Bmp7-/- mutants and 2.5% (n=5),
of Tsg+/;Bmp7-/- mutants were observed,
instead of the expected 6.25% and 12.5%, respectively. This suggested
embryonic lethality associated with these genotypes. Embryonic lethality
required the loss of both copies of Bmp7; for example, the number of
Tsg-/-;Bmp7+/ mutants recovered was
normal (Table 1). Phenotypes
previously known to occur when only Bmp7 is mutated (microphthalmia,
hindlimb polydactyly, sternebral defects)
(Dudley et al., 1995) were
examined, and most appeared enhanced in
Tsg-/-;Bmp7-/- and
Tsg+/;Bmp7-/- animals
(Table 1). Two of these
neonates displayed a striking sirenomelia phenotype, characterized by the
presence of a single hindlimb. Examination of embryos recovered between 9.5
dpc and 12.5 dpc confirmed that all 23 cases with clear sirenomelia had either
the Tsg+/;Bmp7-/- or
Tsg-/-;Bmp7-/- genotypes. These results
indicate that in the absence of Bmp7 there is a dose-dependent
requirement of Tsg for mouse development.
|
|
Knockdown of Tsg and Bmp7 affects ventral development in Xenopus embryos
These interactions were also observed in Xenopus loss-of-function
experiments using antisense MO oligonucleotides. Tsg-MO
(Blitz et al., 2003) and
Bmp7-MO, were injected independently and in combination, into Xenopus
embryos (Fig. 3). Injections
were performed at the site of Tsg and Bmp7 expression, in
the ventral side. Injection of Bmp7-MO at the four-cell stage produced a mild
phenotype characterized by a partial loss of the ventral fin and a
posteriorized anus (Fig.
3D,I,E,J; arrowhead in J). Similar injections of Tsg-MO produced a
phenotype characterized by a bent tail and reduced ventral fin tissue
(Fig. 3N,O). The ventral fin
tissue is an indicator of ventral mesoderm development
(Hammerschmidt and Mullins,
2002
) and in Xenopus is derived from the ventralmost
mesoderm (Tucker and Slack,
2004
). At earlier stages, Tsg-MO or Bmp7-MO caused a reduction of
the ventrally expressed gene sizzled (Fig.
3A,F,K), a marker of high Bmp signaling
(Collavin and Kirschner, 2003
;
De Robertis and Kuroda, 2004
).
The anterior marker Otx2, which is negatively regulated by Bmp, was
unaffected or slightly expanded by injection of the individual morpholinos
(Fig. 3B,G,L). When Tsg-MO and
Bmp7-MO were injected together, sizzled expression was almost
completely eliminated, indicating a loss of Bmp signaling
(Fig. 3P). Otx2
expression domain was significantly expanded at the neurula stage, also
indicating decreased Bmp signaling (Fig.
3Q). However, the head region marked by Otx2 and somitic
mesoderm marked by Myod1 appeared normal by the early tailbud stage
(Fig. 3R). When Bmp7-MO and
Tsg-MO were co-injected, the ventral fin phenotype increased markedly, causing
the development of tadpoles with a truncated tail and virtually no ventral fin
(Fig. 3S,T).
|
|
Tsg-/-;Bmp7-/- and Tsg+/;Bmp7-/- embryos lack ventral mesodermal structures
We next dissected embryos from
Tsg+/;Bmp7+/ heterozygous matings
at different stages. Most Tsg-/-;Bmp7-/- and
Tsg+/;Bmp7-/- compound mutants recovered
after 9.5 dpc were found dead. The likely cause of lethality is related to
defects in chorioallantoic fusion (discussed below). At 10.5 dpc, compound
mutant embryos appeared developmentally delayed compared with their
littermates (Fig. 5A-C). The
morphology of embryos displaying sirenomelia, in which the hindlimb buds are
fused along the ventral midline into a single bud is shown in
Fig. 5B (arrow); this fusion
can be best seen in ventral views such as the one shown in
Fig. 7D' below. We found
that 100% of Tsg-/-;Bmp7-/- (11 embryos out of
a total of 140) and 75% of Tsg+/;Bmp7-/-
(12 embryos) were abnormal. The siren phenotype was observed in 75% of
Tsg-/-;Bmp7-/- mutants and in 88% of
Tsg+/;Bmp7-/- mutants. In addition,
kinked neural tubes were often seen (data not shown). In rarer cases, 25% of
Tsg-/-;Bmp7-/- and 12% of
Tsg+/;Bmp7-/- mutants were severely
underdeveloped and displayed heart edema
(Fig. 5C).
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|
Onset of ventral defects in Tsg-/-;Bmp7-/- and Tsg+/;Bmp7-/- compound mutants
To determine the onset of this ventroposterior mesodermal phenotype,
mesodermal markers were analyzed at 8.25 dpc and 9.0 dpc
(Fig. 6). Brachyury is a
notochord and primitive streak marker required for axial elongation and
formation of posterior mesoderm (Herrmann
et al., 1990; Wilson et al.,
1995
). Its expression was not significantly changed at 8.25 dpc
(Fig. 6A,A').
Fgf8 is required for cell migration and patterning of the primitive
streak and the mesodermal cells exiting from it
(Crossley and Martin, 1995
;
Sun et al., 1999
) and for
formation of posterior mesoderm (Draper et
al., 2003
). Its expression was reduced at 8.25 dpc in mutant
embryos (Fig. 6B-C').
Expression of Wnt3a, which regulates paraxial mesoderm fates at the
expense of neuroectodermal fates
(Yoshikawa et al., 1997
), was
unchanged in the primitive streak and ectoderm (data not shown). Thus, at 8.25
dpc, mesodermal cells appear properly specified (as shown by the normal
expression of brachyury and Wnt3a), but posterior mesoderm cells may
be reduced in number, as suggested by the decrease in Fgf8
staining.
|
We found that the onset of ventral mesoderm defects could be traced prior
to the turning of the embryo and was initially characterized by a deficit in
posterior mesoderm cells expressing Fgf8. Posterior mesodermal cells
appear to be specified normally in the posterior primitive streak, but are
unable to either proliferate or survive. The lack of ventral posterior
mesoderm could also help explain the defects observed in allantoic growth and
fusion to the chorion, as the allantois increases in size by the recruitment
of cells from the posterior primitive streak
(Fujiwara et al., 2001). The
defect is not due to a failure of cells exiting the primitive streak as no
ectopic neural structures were observed in
Tsg-/-;Bmp7-/- and
Tsg+/;Bmp7-/- mutants, which would have
indicated transformation of axial mesoderm into neural fates
(Yoshikawa et al., 1997
;
Abu-Abed et al., 2001
;
Sakai et al., 2001
). We
conclude that Tsg and Bmp7 appear to be required for the
proper survival and/or proliferation of posterior ventral mesoderm cells and
their derivatives.
Development of fused hindlimb bud territory in siren mutants
We next examined the development of the hindlimb buds and posterior ventral
structures (Fig. 7). At 9.5 dpc
Bmp4 marks the ventrolateral part of the embryo in ectoderm, limb
mesenchyme and lateral plate mesoderm (arrow in
Fig. 7A)
(Ahn et al., 2001). In
Tsg-/-;Bmp7-/- embryos, Bmp4 staining
in limb bud mesoderm spans the embryo transversely (90° from the normal
orientation of the hindlimb bud), while the ventralmost mesoderm forms a
separate domain (Fig.
7A',B'). The apical ectodermal ridge marker
Fgf8 (Crossley and Martin,
1995
) is also expressed in a transverse domain in the mutant
instead of in two arches seen on each side of a wild-type embryo at 10.5 dpc
(Fig. 7C-D'). These
observations suggest that, as early as 9.5 dpc the hindlimb buds are fused in
the ventral and posterior region. Expression of Shh in hindgut
endoderm (Gofflot et al.,
1997
) was reduced in mutant embryos
(Fig. 7E-F'), which
confirms the reduction in hindgut structures observed in the histological
analysis (Fig. 5E,E' and
see Fig. S2 in the supplementary material). Expression of Shh was
normal in the notochord, but was delayed in the zone of polarizing activity
(Fig. 7E-F'). In the
tailbud, the number of cells expressing brachyury
(Gofflot et al., 1997
) at 9.5
dpc was reduced in ventral mesoderm but not in the notochord of mutant embryos
(Fig. 7G-H'). We also
note a ventral indentation continued posteriorly by a narrower tail in
Tsg-/-;Bmp7-/- embryos (arrow in
Fig. 7H'). We conclude
that in mutant embryos the hindlimb bud territory is improperly specified as
early as 9.5 dpc. The sirenomelia phenotype results from an early fusion of
the posterior hindlimb buds that correlates with a deficit of ventral
posterior mesoderm.
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Discussion |
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Genetic interactions between Tsg and members of the Bmp family
Tsg interacts biochemically with Bmp4
(Oelgeschläger et al.,
2000; Chang et al.,
2001
). In the mouse, Tsg interacts with Bmp4 in a dose-dependent
fashion; in the absence of Tsg, the loss of one copy of Bmp4
results in holoprosencephaly and branchial arch defects
(Zakin and De Robertis, 2004
).
In the present study, we found that the combined functions of Tsg and
Bmp7 are required in a different aspect of embryogenesis.
Tsg-/-;Bmp7-/- and
Tsg+/;Bmp7-/- compound mutants displayed
sirenomelia. Embryonic lethality and sirenomelia were observed only in
Bmp7-/- embryos in which one or two copies of Tsg
were also missing. Thus, the dose of Tsg is the limiting factor. Tsg was shown
to bind directly to Bmp7 (Fig.
1A) but could also interact with other Bmps, which could be
functionally redundant to Bmp7 and compensate for its loss. When one copy of
Tsg is removed in Bmp7-/- mutants, the levels of
these other Bmp signals may fall beneath the threshold required for ventral
mesoderm development. It is also possible that Bmp7 may actually modulate Tsg
function; however, this seems unlikely in view of previous biochemical work
showing that Tsg regulates the ability of Bmps to signal through Bmp receptors
(Larrain et al., 2001
;
Oelgeschläger et al.,
2003a
).
One candidate for Bmp7 compensation is Bmp4, as both
Bmp7 and Bmp4 are expressed in ventroposterior mesoderm
(Lawson et al., 1999) and
Bmp4 is required for tailbud formation in the frog
(Beck et al., 2001
). In mouse,
Bmp4;Bmp7 double heterozygotes display increased defects in the rib
cages and limbs, supporting a cooperation between Bmp4 and
Bmp7 (Katagiri et al.,
1998
). In addition, tetraploid chimeras lacking Bmp4 in
inner cell mass derivatives have impaired allantois differentiation, absence
of the vitelline artery at 8.25 dpc and kinked neural tubes
(Fujiwara et al., 2001
).
Because similar phenotypes are seen in our sirenomelic mutants, the defects
observed could be the result of a deficit in Bmp4 activity. Another candidate
for an interaction with Tsg is Bmp5. Embryonic lethality is observed in
Bmp5;Bmp7 double mutants but not in each individual mutant
(Solloway and Robertson,
1999
), and they display, among other defects, failure of
chorioallantoic fusion and kinked neural tubes
(Solloway and Robertson,
1999
), also seen in our siren mutants.
Tsg and Bmp7 are involved in posterior ventral mesoderm formation
When Tsg-MO and Bmp7-MO were co-injected into frog embryos, the loss of
ventral fin and of tail structures was observed
(Fig. 3). In Xenopus,
the ventral mesoderm has been shown to be required for ventral fin formation
(Tucker and Slack, 2004). In
zebrafish, the loss of ventral fin is associated with decreased Bmp signaling
(Bauer et al., 2001
;
Hammerschmidt and Mullins,
2002
). For example, in the mini fin mutants, increased
Bmp inhibition by Chd occurs, because Tolloid, the metalloprotease that
cleaves Chd, is mutated (Connors et al.,
1999
). Moreover, knockdown of Tsg in zebrafish results in
dorsalization consistent with a loss of Bmp signaling, and a genetic
interaction between swirl (Bmp2b) and Tsg
loss-of-function was observed; thus, Tsg functions in zebrafish to promote Bmp
signaling in dorsoventral patterning
(Little and Mullins, 2004
;
Xie and Fisher, 2005
). In
Xenopus, ventral mesodermal explants co-injected with Tsg-MO and
Bmp7-MO become dorsalized (anti-Bmp phenotype) in a dose-dependent way
(Fig. 4). Thus, as in
zebrafish, but unlike other reports (Ross
et al., 2001
; Blitz et al.,
2003
), the function of Tsg protein in the Xenopus embryo
is to promote the formation of ventral mesoderm by increasing the activity of
Bmp7 and other Bmps.
In the mouse a loss of ventral mesoderm is also observed: sirenomelia in
Tsg-/-;Bmp7-/- and
Tsg+/;Bmp7-/- compound mutants is
characterized by a reduction of Fgf8 staining at 8.25 dpc,
deficiencies of posterior lateral plate mesoderm expressing Bmp4, and
a reduction of tail bud mesoderm expressing brachyury at 8.5 dpc. In
tetraploid chimeras that lack Bmp4 in the inner cell mass, a
reduction of Fgf8 in the primitive streak has been reported
(Fujiwara et al., 2001). That
study showed that extra-embryonic Bmp4 was required for survival and
differentiation of the allantois.
Mutations in the retinoic acid degrading enzyme Cyp26 cause
sirenomelia (Abu-Abed et al.,
2001; Sakai et al.,
2001
). They differ from our
Tsg-/-;Bmp7-/- and
Tsg+/;Bmp7-/- sirens in that they also
display severely truncated tails and spina bifida. Cyp26 knockouts
showed a reduction of Wnt3a and brachyury expression, associated with
impaired mesoderm proliferation and the mesoderm adopting neural fates
(Sakai et al., 2001
). In our
mutants, we did not observe changes in the expression of Wnt3a nor
excessive neural structures, but a reduced number of cells expressing
brachyury in the tailbud mesoderm was seen. Preliminary results indicate that
cell proliferation was not significantly changed, but apoptosis was increased
in ventral posterior regions in compound mutants (L.Z. and E.M.D.R.,
unpublished). In the mouse, other work has implicated low Bmp signaling both
in decreased cell proliferation and/or increased apoptosis
(Solloway and Robertson, 1999
;
Fujiwara and Hogan, 2001
).
Although sirenomelia has variable phenotypic traits and multiple causes
(Wei and Sulik, 1996
;
Padmanabhan, 1998
), its
occurrence is always associated with defects in posterior primitive streak.
The sirenomelia phenotype observed here is consistent with a defective
differentiation of the posterior and ventral mesoderm caused by decreased Bmp
signaling.
A link between Tsg, Bmp7 and the original sirens?
Sirens were discovered in the mouse
(Gluecksohn-Schoenheimer and Dunn,
1945) among the progeny of parents carrying various combinations
of the Short-tail (T locus), anury
(t0), Fused and ur mutations. The siren
pups obtained had no tail, various degrees of reduction and fusion of elements
of the hindlimbs, abnormalities of the spine, and fusion of ribs. Even though
Tsg-/-;Bmp7-/- and
Tsg+/;Bmp7-/- sirenomelic pups do form
tails (albeit shorter), the limb bud phenotypes we observe are very similar to
those of Gluecksohn-Schoenheimer and Dunn. Could the old and new mutations be
linked in any way? We note that the T locus (including brachyury),
Fused (corresponding to Axin)
(Zeng et al., 1997
) and
Tsg are all located on chromosome 17. The us mutation
(urogenital syndrome) (Lyon and Searle,
1989
), which is phenotypically identical to the now extinct
ur (urogenital) mutant, and Bmp7 are both located on
chromosome 2. Although the respective locations of these genes on these
chromosomes are distant from each other, mutations at the T locus
correspond to important chromosomal rearrangements, often leading to
duplications and deficiencies of chromosome segments upon cell division
(Gluecksohn-Schoenheimer and Dunn,
1945
). Thus, it is conceivable, although perhaps unlikely, that
the occurrence of sirens in the initial description was associated with
disruptions of the Tsg and/or Bmp7 genes. Unfortunately,
some of the original mutations have been lost, so this is not a testable
proposition.
In subsequent work, Hoornbeek found sirenomelic neonates in crosses between
SM/J and BUA strains studied for the incidence of the `careener' phenotype
(Hoornbeek, 1970;
Schreiner and Hoornbeek,
1973
). These sirens have the same phenotype as ours (fused
hindlimbs, a tail, an abnormal umbilical artery). The genes affected in these
crosses are not known, but the carriers of the `siren' mutation
(Hoornbeek, 1970
) had tightly
twisted tails, which is of relevance because Tsg-/- or
Bmp7-/- mutants also have kinked tails
(Jena et al., 1997
;
Zakin and De Robertis,
2004
).
In conclusion, in the absence of Bmp7, two copies of Tsg are required for the proper differentiation of ventral and posterior structures. In the mouse, when Tsg and Bmp7 are mutated, the siren phenotype results from the fusion of the limb buds in the ventroposterior midline owing to a paucity of posterior ventral mesoderm. In Xenopus, knockdown of Tsg and Bmp7 results in an analogous phenotype: loss of posteroventral cell fates associated with decreased Bmp activity. These results demonstrate a common mechanism, mediated by Bmp signaling, in mouse and frog in the patterning of the dorsoventral axis.
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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/132/10/2489/DC1
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