Research article

Development and disease

2489

Sirenomelia in Bmp7 and Tsg compound mutant mice: requirement
for Bmp signaling in the development of ventral posterior
mesoderm
Lise Zakin1, Bruno Reversade1, Hiroki Kuroda1, Karen M. Lyons2 and Eddy M. De Robertis1,*
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@mednet.ucla.edu)

Accepted 21 March 2005
Development 132, 2489-2499
Published by The Company of Biologists 2005
doi:10.1242/dev.01822

Development

Summary
Sirenomelia or mermaid-like phenotype is one of theprincipal human congenital malformations that can betraced back to the stage of gastrulation. Sirenomelia ischaracterized by the fusion of the two hindlimbs into asingle one. In the mouse, sirens have been observed incrosses between specific strains and as the consequence ofmutations that increase retinoic acid levels. We report thatthe loss of bone morphogenetic protein 7 (Bmp7) incombination with a half dose or complete loss of twisted
gastrulation (Tsg) causes sirenomelia in the mouse. Tsg isa Bmp- and chordin-binding protein that has multiple
effects on Bmp metabolism in the extracellular space;
Bmp7 is one of many Bmps and is shown here to bind to
Tsg. In Xenopus, co-injection of Tsg and Bmp7 morpholino

Introduction
Sirenomelia is a human developmental malformation that has
been a source of enduring interest. The abnormal fetuses
are characterized by the fusion of both legs into a single
member (Geoffroy Saint-Hilaire, 1837; Kampmeier, 1927).
Descriptions of sirenomelia in human fetuses can be traced
back to the 16th and 17th centuries, although these first reports
were still a mixture of reality and mythical imagination. In
addition to the hindlimb deformity, sirenomelic fetuses display
a single umbilical artery (instead of two) and atrophic, cystic
or absent kidneys (Feller and Sternberg, 1931). Several
mechanisms have been proposed to explain sirenomelia: (1)deficiencies in caudal mesoderm, (2) mechanical defects
resulting from lateral compression by amniotic folds and (3)trophic defects due to a deficient blood supply in the posterior
region (Padmanabhan, 1998; Sadler and Langman, 2004).
Gluecksohn-Schoenheimer and Dunn published the firststudy on sirens in the mouse, in these terms: ‘Animal withfused and more or less perfectly developed posterior
extremities like those mythical beings supposedly representing
hybrids of man and fish’ (Gluecksohn-Schoenheimer andDunn, 1945). Until then, sirenomelia had not been describedin any mammal except humans. These mouse sirens were
found in crosses between parents carrying mutations at the T,

oligonucleotides (MO) has a synergistic effect, greatly
inhibiting formation of ventral mesoderm and ventral fintissue. In the mouse, molecular marker studies indicate thatthe sirenomelia phenotype is associated with a defect in
the formation of ventroposterior mesoderm. Theseexperiments demonstrate that dorsoventral patterning of
the mouse posterior mesoderm is regulated by Bmp
signaling, as is the case in other vertebrates. Sirens result
from a fusion of the hindlimb buds caused by a defect in
the formation of ventral mesoderm.
Key words: Sirenomelia, Twisted gastrulation (Twsg1) , Bmp, Bmp4,
Morpholino, Limb bud, TGFβ, Chordin, Dorsoventral patterning,
Mouse, Xenopus

Fused or ur loci, thus establishing that sirenomelia can have agenetic basis. The phenotype was not described again in themouse until 1970 (Hoornbeek, 1970). More recently, twogroups linked retinoic acid to sirenomelia in gene knockoutsof the retinoic acid-degrading enzyme, CYP26 (Abu-Abed etal., 2001; Sakai et al., 2001). Here, we report the appearanceof 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 andChordin (Chd), Bmp signaling is prevented (Oelgeschläger etal., 2000; Chang et al., 2001; Larrain et al., 2001; Ross et al.,
2001; Scott et al., 2001). However, the presence of Tsg in thecomplex 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 invarious phenotypes interpreted as pro-Bmp or anti-Bmp
effects. In Drosophila, Tsg mutation results in the loss of theamnioserosa, a tissue that requires the highest levels of
Bmp/Dpp activity (Mason et al., 1994), and absence of Tsg

Development2490 Development 132 (10)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 theembryo, indicating that Tsg has a pro-Bmp effect indevelopment (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 headmalformations on their own, heterozygosity for a Bmp4 nullallele leads to craniofacial defects and holoprosencephaly. This
genetic interaction suggests that, in mouse, Tsg acts as a proBmp4, 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; theinjected embryos displayed tail truncations and loss of theventral 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 bereadily 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.

Materials and methods
Mouse strains and genotyping
Mice carrying the Tsg mutation (Zakin and De Robertis, 2004) werecrossed to the Bmp7 mutant strain (Dudley et al., 1995) andbackcrossed into the hybrid strain B6SJL/F1 (Jackson Laboratories).
Genotyping of pups or embryos was performed as described (Dudley
et al., 1995; Zakin and De Robertis, 2004).
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 etal., 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 β-gal staining of whole embryos (Zakin and
De Robertis, 2004) were as described.
Western blot analysis
Immunoprecipitation of pure recombinant human Bmp7, TGFβ2 andBmp4 (from R&D Systems) was performed as described(Oelgeschläger et al., 2000), using mouse Tsg-Flag pre-bound to antiFlag 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

Research article
the N-terminus), or human Bmp7. Before chemical crosslinking,samples were dialyzed into PBS, then incubated together and treatedwith 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 forwestern 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 startcodon) 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). TsgMO (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 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).

Results
Tsg and Bmp7 bind to each other and haveoverlapping expression domains
Tsg is known to interact biochemically and genetically with
Bmp4 (Oelgeschläger et al., 2000; Zakin and De Robertis,2004). To test whether Tsg could also interact directly with
Bmp7, we analyzed their biochemical interaction by coimmunoprecipitation and chemical crosslinking with DSS(Fig. 1A,A′). For co-immunoprecipitation, mouse Tsg-Flag
pre-bound to agarose beads was incubated with 15 nM humanrecombinant Bmp7, with or without the addition ofrecombinant TGFβ2 or Bmp4 (Fig. 1A). Tsg was able to pull
down Bmp7, and this binding was specific because it was notaffected by a six-fold excess of TGFβ2. Bmp4 in equimolar
amounts was able to reduce Bmp7 binding by about half,suggesting that Tsg binds both Bmps with comparable
affinities (Fig. 1A, compare lanes 2 and 4). When Tsg and
Bmp7 proteins from 293T cell culture supernatants wereincubated together and chemically crosslinked (see Materials
and methods), a bimolecular complex corresponding to ahomodimer of Tsg bound to a homodimer of Bmp7 was formed(Fig. 1A′, lane 3).
Tsg and Bmp7 expression domains overlapped in multiple
tissues during embryogenesis (Fig. 1B-D′). In particular,
histological sections showed that Tsg and Bmp7 are coexpressed in ventral mesoderm and endoderm (Fig. 1E-G). The
expression patterns of Bmp7 and Tsg have been reported
previously (Solloway and Robertson, 1999; Graf et al., 2001).
At 8.25 dpc, Tsg and Bmp7 were expressed diffusely in axial
mesoderm and lateral mesoderm, gut endoderm and allantois(Fig. 1B,B′,E). At later stages, co-expression persisted in gut
endoderm and overlapping or adjacent expression domains
were found in limb bud mesenchyme, optic and otic vesicles,
first branchial arch and posterior ventral mesoderm (Fig.
1C,C′,D,D′,F,G).
We conclude that Tsg and Bmp7 proteins are capable ofestablishing direct biochemical interactions, and that theDevelopment

Development and disease

Bmp and Tsg signaling in ventral mesoderm 2491

Fig. 1. Tsg and Bmp7 interact biochemically and are expressed in overlapping domains. (A) Bmp7 co-immunoprecipitates with Tsg. Top panel
was probed with an anti-Bmp7 antibody. Bmp7 was pulled down only in the presence of Tsg (compare lanes 1 and 2). This Tsg/Bmp7
interaction was not competed by a sixfold excess of TGFβ2 (lane 3) but was partially competed by equimolar amounts of Bmp4 (lane 4).
Middle panel provides a control for the amount of Tsg immunoprecipitated; the bottom panel serves as a loading control for antibodies on thebeads. (A′) Crosslinking of Tsg and Bmp7 proteins by DSS. A bimolecular complex of Tsg and Bmp7 is formed in lane 3. Tsg protein is HA
tagged. Top panel (crosslinked) and middle panel (no crosslinker) were probed with an anti-HA antibody; bottom panel (no crosslinker) wasprobed with anti-Bmp7 antibody. Tsg protein forms a homodimer when crosslinked (lane 1, top panel). In the presence of Bmp7 (lane 3, top
panel) a bimolecular complex (consistent with a homodimer of Tsg binding a homodimer of Bmp7) runs around 110 kDa. (B-D′) Expression of
Tsg and Bmp7 analyzed by whole-mount in situ hybridization and β-gal staining. (B,B′) Bmp7 and Tsg are co-expressed in allantois (al), axial
and lateral mesoderm (am) and gut endoderm (en) at 8.25 dpc. Tsg expression is visualized via an in-frame fusion to lacZ. (C,C′) At 9.0 dpc,
Tsg and Bmp7 are found in gut endoderm, branchial arch one (a1), eye vesicle (ev) and otic vesicle (ov). (D,D′) At 10.5 dpc, Tsg and Bmp7 areco-expressed in posterior ventral mesoderm (vm), limb bud (lb), first pharyngeal arch and basal telencephalon (bt). (E,F) Sections of 8.25 and
9.0 dpc embryos showing Tsg mRNA in endoderm, neurectoderm, head mesenchyme (hm) and ventral mesoderm (vm). (G) Section of a 9.0
dpc embryo showing expression of Bmp7 in ventral mesoderm and endoderm of the hindgut. Plane of sections are indicated in B′,C,C′. h, heart;hg, hindgut; me, mesoderm; md, mandibular component of first pharyngeal arch; mx, maxillary component of first pharyngeal arch; ne,neuroectoderm; no, node.

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 theresults 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% and12.5%, respectively. This suggested embryonic lethality

associated with these genotypes. Embryonic lethality required
the loss of both copies of Bmp7; for example, the number ofTsg–/–;Bmp7+/– mutants recovered was normal (Table 1).
Phenotypes previously known to occur when only Bmp7 ismutated (microphthalmia, hindlimb polydactyly, sternebral
defects) (Dudley et al., 1995) were examined, and mostappeared enhanced in Tsg–/–;Bmp7–/– and Tsg+/–;Bmp7–/–
animals (Table 1). Two of these neonates displayed a striking
sirenomelia phenotype, characterized by the presence of asingle 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.

2492 Development 132 (10)Research article

Table 1. Characterization of neonates obtained from Tsg+/–;Bmp7+/– intercrosses
Genotype
Tsg+/+;Bmp7+/+
Tsg+/+;Bmp7+/–
Tsg+/–;Bmp7+/–
Tsg+/–;Bmp7+/+
Tsg–/–;Bmp7+/+
Tsg–/–;Bmp7+/–
Tsg+/+;Bmp7–/–
Tsg+/–;Bmp7–/–
Tsg–/–;Bmp7–/–
Total

Neonates
18
37
55
30
12
28
11
5
1
197

Expected
percentage

Observed
percentage

Anophthalmia
microphthalmia (%)

Hindlimd
polydactyly* (%)

Sirenomelia (%)

Sternebral
defects (%)*

6.25
12.5
25
12.5
6.25
12.5
6.25
12.5
6.25

9
19
28
15
6
14
6
2.5
0.5

0
0
0
0
0
0
9 (82%)
5 (100%)1 (100%)

N/A
N/A
N/A
N/A
N/A
N/A
2 (18%)
4 (80%)
1 (100%)

0
0
0
0
0
0
0
1 (20%)
1 (100%)

N/A
N/A
N/A
N/A
N/A
N/A
5 (45%)
4 (80%)
1 (100%)

N/A, not assessed.
*Determined by skeletal staining.

Development

The sirenomelia phenotype can be seen in Fig. 2. In ventral
view, a single medial hindlimb was present in pups that hadotherwise normal forelimbs (Fig. 2A,B). Alcian blue/Alizarin
red staining of the skeletons of Tsg–/–;Bmp7–/– andTsg+/–;Bmp7–/– pups showed that the single lower limb
consisted of the fusion of two independent hindlimbs (Fig. 2CF). Most skeletal elements were duplicated, but the two limbs
were fused at the pelvis and at the heel of the foot (Fig. 2F),
and lacked the fibula. The Tsg–/–;Bmp7–/– mutant had only one
foot (designated sympus monopus) (Kampmeier, 1927) with

only five digits and a bifurcation of the first digit (Fig. 2D;
inset). The Tsg+/–;Bmp7–/– mutant had two fused feet (sympus
dipus) (Kampmeier, 1927) with nine digits, a single first digit
and a bifurcation of one of the second digits (Fig. 2F; inset).
The sirenomelia phenotype in the Tsg–/–;Bmp7–/– neonate isconsidered more severe due to the presence of only one foot.
The tail was shorter, with fewer vertebrae and delayed andreduced bone deposition with respect to the wild-type (Fig.
2C,D). As Bmps and Tsg promote chondrogenesis and bone
deposition, this is consistent with the notion that the compoundmutants have less Bmp signaling. The thoracic
region was also affected in Tsg–/–;Bmp7–/– andTsg+/–;Bmp7–/– mutants (see Fig. S1 in the
supplementary material).
In conclusion, the analysis of the skeletonsof Tsg–/–;Bmp7–/– and Tsg+/–;Bmp7–/– mutants
revealed phenotypes indicative of reduced
Bmp signaling, not observed in either mutant
alone. These results suggest that in a Bmp7null background the lack, or haploinsufficient
doses, of Tsg affects the activity of other Tsginteracting Bmps, which in vivo would be
Fig. 2. Sirenomelia in Tsg–/–;Bmp7–/– andTsg+/–;Bmp7–/– compound mutants.
(A,B) Comparison of a wild-type neonate with aTsg–/–;Bmp7–/– mutant displaying sirenomelia. The
mutant appears normal up to the level of thehindlimbs which are fused. (C-F) Alcian Blue
(cartilage) and Alizarin Red (bone) preparations ofwild-type and Tsg–/–;Bmp7–/– and Tsg+/–;Bmp7–/–
mutants. (C,D) Lateral view of the posterior regionof a wild-type and Tsg–/–;Bmp7–/– skeleton. In the
mutant, the tail is shorter and has fewer vertebrae.
The single hindlimb contains two femurs (fe), twotibias (ti), a single foot and lacks both fibulae (fi).
(E,F) Ventral views of the hindlimbs and pelvic
region of a wild-type and Tsg+/–;Bmp7–/– neonate.
The mutant hindlimb is formed by the fusion of
two hindlimbs at the level of the ischium (is); twofeet are present, fused at the level of the heel.
Insets in D and F show higher magnification viewsof the feet of Tsg–/–;Bmp7–/– and Tsg+/–;Bmp7–/–
neonates, respectively. Digits are indicated bynumbers and digit duplications by letters. il, ilium;is, ischium; p, pubic bone; ps, pubic symphysis.

Development and disease

Development

functionally redundant with Bmp7. In Bmp7–/– embryos, theseother Bmps would compensate for the loss of Bmp7; however,when Tsg function is reduced, the activity of these other Bmps
is lowered to levels incompatible with normal development.
Knockdown of Tsg and Bmp7 affects ventral
development in Xenopus embryos
These interactions were also observed in Xenopus loss-offunction experiments using antisense MO oligonucleotides.
Tsg-MO (Blitz et al., 2003) and Bmp7-MO, were injectedindependently 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 apartial 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 anindicator 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 andKuroda, 2004). The anterior marker Otx2, which is negatively
regulated by Bmp, was unaffected or slightly expanded byinjection 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 lossof Bmp signaling (Fig. 3P). Otx2 expression domain was

Bmp and Tsg signaling in ventral mesoderm 2493
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).
One of the most sensitive assays for Bmp signaling levels isprovided by the Xenopus ventral marginal zone (VMZ) explant(Fig. 4A). When such explants were prepared from embryos
injected ventrally with Bmp7-MO or Tsg-MO, they resembled
lateral marginal zone (LMZ) explants containing polarized
patches of unpigmented cells (Fig. 4B-D). When Bmp7-MO
and Tsg-MO were co-injected, the explants elongated andadopted the external appearance of dorsal marginal zone
(DMZ) explants (Fig. 4E). As shown by the RT-PCR genemarker analyses of Fig. 4F, both Tsg-MO and Bmp7-MO had
anti-Bmp effects in Xenopus. VMZ explants expressed theventral mesodermal marker β-globin (lane 4), while Tsg-MO(lane 5) or Bmp7-MO (lane 9) expressed markers present inmore lateral LMZ, explants (lane 3), such as α-actin, the neuralcrest marker slug, and the neuronal marker N-tubulin. WhenTsg and Bmp7 were knocked down simultaneously (lane 8),strong induction of somitic mesoderm (α-actin), neurons (Ntubulin), cement gland (marked by XAG) and even the dorsal
midline markers pintallavis/HNF3β and sonic hedgehog (Shh)was observed. This pattern of expression of dorsoventral
markers in double knockdowns was very similar to that ofuninjected DMZ (also known as Spemann organizer) explants

Fig. 3. Tsg and Bmp7 cooperate in ventral development in Xenopus embryos. (A,F,K,P) Control and morpholino-injected embryos at stage 12
hybridized with sizzled probe. Expression of sizzled is reduced in Bmp7-MO- or Tsg-MO-injected embryos and almost completely lost in the
Bmp7-MO/Tsg-MO-injected embryos. (B,G,L,Q) Expression of Otx2 in injected embryos at stage 13; there is an increase in Bmp7-MO/TsgMO-injected embryos. (C,H,M,R) Embryos at stage 18 hybridized with Otx2 and Myod1 probes. The expansion of Otx2 regulates and is nolonger seen at this stage. (D,E,I,J,N,O,S,T) Phenotypes of morpholino-injected embryos at (D,I,N,S) stage 39 (2 day) and (E,J,O,T) stage 42 (3day). Bmp7-MO-injected embryos have a reduced ventral fin (arrowhead); Tsg-MO-injected embryos have a bent tail and reduction of ventral
fin. Co-injection of Bmp7-MO and Tsg-MO synergize, causing a severe loss of ventral fin (arrow in S) and at later stages truncation of theentire tail region (T).

2494 Development 132 (10)Development(compare lanes 2 and 8). Importantly, we also injected several
dosages of Bmp7-MO (lanes 6-8), showing that Bmp7 and Tsg
interacted also at intermediate Bmp7 levels in Xenopus

Fig. 4. VMZ explants co-injected with Tsg-MO and Bmp7-MO arestrongly dorsalized and adopt the identity of DMZ explants.
(A) Experimental design of the DMZ, LMZ and VMZ explants.
(B) VMZs from wild-type embryos cultured until tailbud stage 20.
(C) VMZs from Bmp7-MO-injected embryos are dorsalized andresemble LMZs (inset). (D) VMZs from Tsg-MO-injected embryos
showing dorsalization (unpigmented regions). (E) VMZs from TsgMO- and Bmp7-MO-injected embryos elongate similarly to DMZ(Spemann organizer) explants (shown in the inset). (F) RT-PCR
assays of stage 26 whole embryos (WE) (lane 1), DMZs (lane 2),
LMZs (lane 3) and VMZs (lane 4) from uninjected embryos or fromembryos injected with Tsg-MO (lane 5), Bmp7-MO (lane 9), with
Tsg-MO or several doses of Bmp7-MO (lanes 6-8). VMZs fromembryos injected with both MOs express the same markers as wildtype DMZ explants (compare lanes 2 and 8), indicating strong
dorsalization and reduced Bmp signaling. VP, vegetal pole; WE,
whole embryo.

Research article
embryos. As the siren phenotype in mouse was only observed
on a Bmp7–/– background, these epistatic experiments provide
evidence that Bmp7 and Tsg interact with each other in vivo.
These results show that the wild-type function of Tsg in
Xenopus is to promote Bmp signaling, as is the case inzebrafish (Little and Mullins, 2004; Xie and Fisher, 2005).
They also show that Bmp7 and Tsg cooperate in the formationof ventral tissues, suggesting that the siren phenotype observedin mouse could be due to a loss of Bmp signaling in ventral
mesoderm.
Tsg–/–;Bmp7–/– and Tsg+/–;Bmp7–/– embryos lackventral mesodermal structures
We next dissected embryos from Tsg+/–;Bmp7+/– heterozygous
matings at different stages. Most Tsg–/–;Bmp7–/– andTsg+/–;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 thehindlimb 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 totalof 140) and 75% of Tsg+/–;Bmp7–/– (12 embryos) wereabnormal. 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 notshown). In rarer cases, 25% of Tsg–/–;Bmp7–/– and 12% of
Tsg+/–;Bmp7–/– mutants were severely underdeveloped anddisplayed heart edema (Fig. 5C).
Serial histological sections were performed on 9.5 dpc
embryos from the hindlimb bud region to the tip of the tail to
examine the anatomy of embryos with sirenomelia (Fig. 5D-G′).
At the level of the hindlimb buds in the normal embryos four
blood vessels are seen. Two dorsal aortae, which after they curveback into the tailbud, form ventrally the recurved part of the
distal aortae separating two distinct lateral coelomic cavities(Fig. 5D,E). Two thickenings of the lateral plate mesoderm markthe forming of the left and right hindlimb buds (Fig. 5E). In
mutant embryos, a single coelomic cavity was observed (Fig.
5E′, see Fig. S2 in the supplementary material), and the recurved
ventral region of the posterior aorta was absent (Fig. 5D′,E′, seeFig. S2 in the supplementary material). In compound mutants,
the posterior arterial system had a single abnormally placed
umbilical artery directly arising from the dorsal aorta (Fig. 5D′),
and a single thickening of the mesoderm was seen in the ventral
midline, marking the site of the fused hindlimb buds (Fig. 5E′).
Most defects were ventral in Tsg;Bmp7 compound mutants (Fig.
5F,F′; see Fig. S2 in the supplementary material) and, moreposteriorly, tailbud mesoderm cells were sparser (Fig. 5G,G′). In
summary, the sirenomelic phenotype is caused by a reduction ofventral mesodermal tissue that results in the fusion of thehindlimb buds and coelomic cavities. The results indicate arequirement in the mouse for Bmp signaling in ventral
mesoderm formation and/or survival.

Onset of ventral defects in Tsg–/–;Bmp7–/– andTsg+/–;Bmp7–/– compound mutants
To determine the onset of this ventroposterior mesodermalDevelopment and disease

Bmp and Tsg signaling in ventral mesoderm 2495

Development

Fig. 5. Phenotypic abnormalities in Tsg–/–;Bmp7–/– andTsg+/–;Bmp7–/– compound mutants in the mouse. (A) Wild-type
embryo 10.5 dpc. (B) Side view of a Bmp7–/–,Tsg–/– mutant
with sirenomelia; the hindlimb buds are fused into a single bud(arrow). (C) Ventral view of a severely affected Tsg+/–;Bmp7–/–
mutant embryo with heart edema and underdeveloped anterior
and posterior structures. (D-G′) Histological analyses of theposterior region of 9.5 dpc wild-type and Tsg–/–;Bmp7–/–
embryos. (D,D′) Side view of the embryos indicating the levelof sections in E-G′. The posterior arterial system is drawn
schematically and superimposed; the dorsal aorta (da) curves
ventrally in the tailbud, forming the recurved distal aorta (rda),
from which the umbilical artery (ua) originates.
(E-G′) Transverse serial sections of embryos shown in D and D′
stained with Hematoxylin and Eosin. (E,E′) Section at the levelof the right and left hindlimb buds (rhb, lhb) showing theabsence of the rda, fusion of the hindlimb buds (hb) and asingle coelomic cavity (cc) in the mutant. (F,F′) More posterior
sections showing a decreased diameter of the tail at the level ofthe aortic curvature. (G,G′) Section at the tip of the tail showing
fewer tailbud mesoderm cells and a normal neural plate in the
mutant. hg, hindgut; lhb, left hindlimb bud; n, node; nt, neuraltube; rhb, right hindlimb bud; s, somite; tbm, tail bud
mesoderm; ua, umbilical artery; va, vitelline artery.

phenotype, mesodermal markers were analyzed at 8.25
dpc and 9.0 dpc (Fig. 6). Brachyury is a notochord andprimitive streak marker required for axial elongation andformation 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 themesodermal 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 andectoderm (data not shown). Thus, at 8.25 dpc,
mesodermal cells appear properly specified (as shown by thenormal expression of brachyury and Wnt3a), but posterior
mesoderm cells may be reduced in number, as suggested by
the decrease in Fgf8 staining.
After the embryo has turned at 9.0 dpc, most mutants
displayed a small and poorly developed allantois delayed in its
fusion to the chorion (Fig. 6E′,F′). At this stage, expression ofBrachyury in tailbud mesoderm was moderately reduced in theventral mesoderm close to the tip of the tail (Fig. 6D,D′).
Normally at 9.0 dpc, Bmp4 expression is found in posterior
lateral plate mesoderm and extra-embryonic mesoderm of theallantois (Fujiwara et al., 2001). In mutant embryos, the Bmp4
expressing region closest to the tip of the tail was reduced andthe allantois failed to fuse to the chorion (Fig. 6E,E′).
We found that the onset of ventral mesoderm defects could
be traced prior to the turning of the embryo and was initiallycharacterized 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 theallantois increases in size by the recruitment of cells from theposterior primitive streak (Fujiwara et al., 2001). The defect is
not due to a failure of cells exiting the primitive streak as noectopic neural structures were observed in Tsg–/–;Bmp7–/– andTsg+/–;Bmp7–/– mutants, which would have indicatedtransformation 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 theproper 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 andposterior 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 theventralmost mesoderm forms a separate domain (Fig. 7A′,B′).
The apical ectodermal ridge marker Fgf8 (Crossley and2496 Development 132 (10)Research article
Fig. 6. Onset of ventral and posterior mesoderm
defects in Tsg–/–;Bmp7–/– and Tsg+/–;Bmp7–/–
compound mutants. (A,A′) Ventral view of posteriorprimitive streak (ps) region of wild-type and mutant
embryos stained with brachyury. The expression ofbrachyury is unchanged at 8.25 dpc in the mutant.
(B,B′) Side view of 8.25 dpc embryos before turning
hybridized with Fgf8. Staining of the primitive streak
and posterior mesoderm (pm) is fainter in the mutant.
(C,C′) Dorsal view of the posterior region of embryos
shown in B and B′, respectively. Fgf8 expression isless intense in the mutant. (D,D′) High magnification
view of the tailbud region of embryos stained withbrachyury; in the mutant staining of tailbud mesoderm
at the tip appears fainter, but notochord (n) expressionis unaffected. (E,E′) Bmp4 expression in side view of
9.0 dpc embryos (after turning). The allantois (al) andlateral plate mesoderm (lpm) are stained. (F,F′) Higher
magnification view of the tailbud region of embryos
hybridized with Bmp4. The tailbud and of the allantois
are abnormal in shape. Upon dissection, the allantois
was found not to be fused to the chorion in the mutant(chorio-allantoic fusion has normally completed bythis stage).

Development

Discussion

Martin, 1995) is also expressed in a transverse domain in the
mutant instead of in two arches seen on each side of a wildtype embryo at 10.5 dpc (Fig. 7C-D′). These observations
suggest that, as early as 9.5 dpc the hindlimb buds are fusedin the ventral and posterior region. Expression of Shh in
hindgut endoderm (Gofflot et al., 1997) was reduced inmutant 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 wasdelayed in the zone of polarizing activity (Fig. 7E-F′). In the
tailbud, the number of cells expressing brachyury (Gofflot etal., 1997) at 9.5 dpc was reduced in ventral mesoderm but notin 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 territoryis 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.

Previous work has shown that Tsg is not essential
for embryogenesis, as Tsg–/– mice are viable andfertile, but are smaller and exhibit skeletal defects(Nosaka et al., 2003; Petryk et al., 2004; Zakin and
De Robertis, 2004). However, when one copy of
Bmp4 was removed in certain genetic
backgrounds, Tsg was shown to exhibit a genetic
interaction involved in forebrain formation (Zakin
and De Robertis, 2004). As Tsg is broadly
expressed throughout the embryo and manymembers of the Bmp family exist (Hogan, 1996;
Zhao, 2003), we investigated whether genetic
interactions with Bmps were a general feature ofTsg function by analyzing Tsg;Bmp7 compoundmutants. Our results show that in the absence of
Bmp7, Tsg is required for the formation of posterior ventral
structures.
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, theloss of one copy of Bmp4 results in holoprosencephaly andbranchial arch defects (Zakin and De Robertis, 2004). In thepresent 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 thelimiting factor. Tsg was shown to bind directly to Bmp7 (Fig.
1A) but could also interact with other Bmps, which could befunctionally redundant to Bmp7 and compensate for its loss.
When one copy of Tsg is removed in Bmp7–/– mutants, thelevels of these other Bmp signals may fall beneath theDevelopment and disease

Bmp and Tsg signaling in ventral mesoderm 2497

Development

Fig. 7. Analysis of the sirenomelia phenotype.
(A,B) Side view of 9.5 dpc wild-type and mutant
embryos hybridized with Bmp4 probe. (A′,B′) Ventral
views of hindlimb bud region of embryos shown in A
and B, respectively; the tail has been excised. In the
mutant, expression of Bmp4 in the future hindlimb bud
mesoderm (hb) forms a transverse horizontal domain
instead of two lateral ones on the right (rhb) and left(lhb) hindlimb buds seen in the wild type. (C,D) Side
view of 10.5 dpc embryos hybridized with Fgf8 probe,marking the apical ectodermal ridge (aer).
(C′,D′) Ventral views of hindlimb bud region ofembryos shown in C and D, respectively. In the mutant,the aer expression domain of Fgf8 adopts a horseshoelike shape, with the two aer regions meeting posteriorly
instead of forming two lateral arches. (E,F) Side viewof 10.5 dpc embryos hybridized with sonic hedgehog(Shh) probe. (E′,F′) Ventral views of hindlimb bud
region at higher magnification. Expression of Shh inthe zone of polarizing activity (zpa) is not yet detected,
and hindgut (hg) staining is reduced in the mutant.
(G,H) Side view of 9.5 dpc embryos hybridized withbrachyury probe. (G′,H′) Higher magnification of tailregion of embryos stained for brachyury. Staining oftail bud mesoderm (tbm) is decreased ventrally in the
mutant; note the abnormal indentation (black arrow)and narrowing of the tail in the mutant. no, node; vm,
ventral mesoderm.

threshold required for ventral mesoderm
development. It is also possible that Bmp7 may
actually modulate Tsg function; however, thisseems unlikely in view of previous biochemical
work showing that Tsg regulates the ability of
Bmps to signal through Bmp receptors (Larrain etal., 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 thefrog (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 cellmass derivatives have impaired allantois differentiation,
absence of the vitelline artery at 8.25 dpc and kinked neuraltubes (Fujiwara et al., 2001). Because similar phenotypes areseen 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 isobserved in Bmp5;Bmp7 double mutants but not in eachindividual 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 wasobserved (Fig. 3). In Xenopus, the ventral mesoderm has beenshown to be required for ventral fin formation (Tucker andSlack, 2004). In zebrafish, the loss of ventral fin is associatedwith 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 inzebrafish results in dorsalization consistent with a loss of Bmp
signaling, and a genetic interaction between swirl (Bmp2b) andTsg loss-of-function was observed; thus, Tsg functions inzebrafish 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 dosedependent way (Fig. 4). Thus, as in zebrafish, but unlike otherreports (Ross et al., 2001; Blitz et al., 2003), the function ofTsg protein in the Xenopus embryo is to promote the formationof ventral mesoderm by increasing the activity of Bmp7 andother Bmps.
In the mouse a loss of ventral mesoderm is also observed:

Development2498 Development 132 (10)sirenomelia in Tsg–/–;Bmp7–/– and Tsg+/–;Bmp7–/– compoundmutants 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 showedthat extra-embryonic Bmp4 was required for survival anddifferentiation of the allantois.
Mutations in the retinoic acid degrading enzyme Cyp26 causesirenomelia (Abu-Abed et al., 2001; Sakai et al., 2001). They
differ from our Tsg–/–;Bmp7–/– and Tsg+/–;Bmp7–/– sirens in thatthey also display severely truncated tails and spina bifida. Cyp26
knockouts showed a reduction of Wnt3a and brachyury
expression, associated with impaired mesoderm proliferationand 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 ofcells 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 andmultiple causes (Wei and Sulik, 1996; Padmanabhan, 1998), its
occurrence is always associated with defects in posteriorprimitive streak. The sirenomelia phenotype observed here isconsistent with a defective differentiation of the posterior andventral mesoderm caused by decreased Bmp signaling.
A link between Tsg, Bmp7 and the original sirens?
Sirens were discovered in the mouse (GluecksohnSchoenheimer 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 elementsof the hindlimbs, abnormalities of the spine, and fusion of ribs.
Even though Tsg–/–;Bmp7–/– and Tsg+/–;Bmp7–/– sirenomelicpups do form tails (albeit shorter), the limb bud phenotypes weobserve 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 fromeach 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 isconceivable, although perhaps unlikely, that the occurrence ofsirens in the initial description was associated with disruptionsof the Tsg and/or Bmp7 genes. Unfortunately, some of theoriginal mutations have been lost, so this is not a testableproposition.
In subsequent work, Hoornbeek found sirenomelic neonatesin crosses between SM/J and BUA strains studied for the

Research article
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 notknown, 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, thesiren phenotype results from the fusion of the limb buds in theventroposterior midline owing to a paucity of posterior ventral
mesoderm. In Xenopus, knockdown of Tsg and Bmp7 results inan analogous phenotype: loss of posteroventral cell fatesassociated 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.
We thank A. Mays, D. Greenberg and D. Geissert for technicalassistance. The probes used were kind gifts from B. Hogan, A.
McMahon, G. Martin and R. Nusse. This work was supported by theNorman Sprague chair and the Howard Hughes Medical Institute, of
which L.Z. is an Associate and E.M.D.R. an Investigator.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/10/2489/DC1

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