Developmental Biology 238, 303314 (2001)

doi:10.1006/dbio.2001.0407, available online at http://www.idealibrary.com on

The Role of BMP Signaling in Outgrowth and

Patterning of the Xenopus Tail Bud

Caroline W. Beck,* ,1 Malcolm Whitman, and Jonathan M. W. Slack*

*Developmental Biology Programme, Department of Biology and Biochemistry, University of
Bath, Claverton Down, Bath BA2 7AY, United Kingdom; and Department of Cell Biology,
Harvard Medical School, Boston, Massachusetts 02115

Tail bud formation in Xenopus depends on interaction between a dorsal domain (dorsal roof) expressing lunatic fringe and
Notch, and a ventral domain (posterior wall) expressing the Notch ligand Delta. Ectopic expression of an activated form of
Notch, Notch ICD, by means of an animal cap graft into the posterior neural plate, results in the formation of an ectopic
tail-like structure containing a neural tube and fin. However, somites are never formed in these tails. Here, we show that
BMP signaling is activated in the posterior wall of the tail bud and is involved in the formation of tail somites from this
region. Grafts into the posterior neural plate, in which BMP signaling is activated, will form tail-like outgrowths. Unlike
the Notch ICD tails, the BMP tails contain well-organized somites as well as neural tube and fin, with the graft contributing
to both somites and neural tube. Through a variety of epistasis-type experiments, we show that the most likely model
involves a requirement for BMP signaling upstream of Notch activation, resulting in formation of the secondary neural tube,
as well as a Notch-independent pathway leading to the formation of tail somites from the posterior wall. 2001 Academic Press
Key Words: BMP; Smad; Notch; Xhox3; evx; tail bud; somites; Xenopus.

INTRODUCTION Grafts of animal caps into the posterior neural plate

Tail bud determination in Xenopus requires a three-way
interaction between the mesodermal (M) and neural (N)
regions of the posterior neural plate, and the underlying
caudal notochord (C), which first come into contact at the
end of gastrulation (Tucker and Slack, 1995). We previously
showed that there are two types of gene involved in tail
development, those that are expressed in the region of the
future tail bud from gastrulation (early genes) and those that
are expressed from the time the tail bud begins to extend,
forming the axial tail tissues, around stage 26 (late genes)
(Beck and Slack, 1998). Notch and Delta are early-phase
genes expressed ventrally in the future posterior wall re-
gion, corresponding to the mesodermal part of the neural
plate (M). At around stage 26, expression of the late-phase
gene Lunatic fringe is detected in the dorsal tail bud region
(N), leading to activation of Notch signaling at the dorsal/
ventral boundary of the tail bud. This in turn leads to
localized Xhox3 expression in the distal tip of the tail bud as
it begins to extend (Beck and Slack, 1998, 1999).
1
To whom correspondence should be addressed. Fax: 44-1225-
826779. E-mail: c.beck@bath.ac.uk.
containing either exogenous activated Notch (Notch ICD)
or Xhox3 itself will generate ectopic tails that contain
neural tube and fin but lack somites and notochord (Beck
and Slack, 1999). Recently, a zebrafish mutant known as
mini-fin (mfn), which lacks ventral tail somites and tail fin,
was identified as a null mutation in the Tolloid gene
(Connors et al., 1999). This suggests that regulation of BMP
signaling is required, in addition to its early role in estab-
lishing the dorsalventral axis, for patterning of the tail
postgastrulation. The phenotype of mfn embryos is likely to
be attributable to a reduction in levels of BMP activity in
the ventral/posterior region. The normal expression of
BMP4 during the relevant stages is in the region immedi-
ately ventral to the future posterior wall region of the tail
bud, suggesting that BMP protein is likely to be present in
the region of tail bud outgrowth (Fainsod et al., 1994).
BMPs, with the exception of BMP1, are members of the
transforming growth factor- superfamily (Wozney et al.,
1988) with multiple roles in embryonic development
(Hogan, 1996). BMPs bind specific pairs of serine/threonine
kinase receptors as homo- or heterodimers, resulting in
transphosphorylation of the type I receptor by the type II
receptor (Heldin et al., 1997). The activated type I receptor

0012-1606/01 $35.00
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All rights of reproduction in any form reserved. 303
304
then in turn specifically phosphorylates BMP pathway-
specific Smad proteins (Smads1/5/8), enabling the forma-
tion of heterotrimeric complexes with the nonspecific co-
smad Smad4 (Heldin et al., 1997). These complexes then
translocate to the nucleus, where they are thought to
associate with companion transcription factors, such as
OAZ, to elicit a transcriptional response via binding to BMP
response elements (BREs) (Hata et al., 2000). Activation of
endogenous BMP signaling can be detected by using an
antibody that detects the phosphorylated forms of Smads1/
5/8 (Faure et al., 2000). Using this antibody, we show that
BMP signaling is activated in the M region corresponding to
the future ventral posterior wall of the tail bud and that this
activity is present from the end of gastrulation and persists
at least until the tail bud begins to emerge. This region of
the tail bud is fated to give rise to posterior somites (Gont et
al., 1993). We therefore wondered whether BMP signaling
might be involved in the formation of tail somites. Using
the same grafting test for the production of ectopic tails, we
now present evidence for such a role. Grafts containing
mRNA for BMP4, activated BMP receptor Alk3, or an
activated Smad5 mutant all produced ectopic tails, and
these contain somites in addition to neural tube and fin.
However, the tails were never found to contain notochord.
Epistasis experiments suggest a requirement for BMP sig-
naling upstream of Notch/Xhox3 in tail bud outgrowth.
The combined evidence from the results presented here
together with the previously published expression data and
mutant phenotypes strongly suggest that BMP signaling is
required for tail bud development in vertebrates.
MATERIALS AND METHODS
mRNA Synthesis
Capped synthetic mRNA was made using an SP6 megascript in
vitro transcription kit (Ambion, Austin, TX) with the addition of 5
mM cap analogue (Ambion) and reduction of GTP to 0.5 mM in the
reaction. Notch ICD, Xhox3EnR, and Xhox3VP16 in the Xenopus
expression vector pCS2 were previously described (Coffman et
al., 1993; Beck and Slack, 1999). Membrane-tethered mouse
Notch1 in CS2 (mNotchE) was a kind gift of Raphael Kopan
(Kopan et al., 1996). Mouse Smad5 WT cDNA in pMEP4 was a kind
gift of Peter ten Dijke, and the full coding sequence was amplified
by PCR primers with flanking restriction sites for BglII (5) and
EcoRV (3). This was cloned into the BamHI and StuI sites of
pCS2 to make Smad5-CS2. An inducible fusion protein version
of Smad5 was also made, by cloning the same PCR product into the
in-filled ClaI site and BamHI site of pCS2-GR, which contains
the glucocorticoid ligand-binding domain (amino acids 512777), to
create Smad5-GR CS2 (Kolm and Sive, 1995). A constitutive
mutant form, Smad5*-CS2, was also made by PCR. This lacks
amino acids 465 and 466 from the conserved SSXS domain at the
C-terminus. A dominant negative mutant form, Smad5-sbn-CS2,
was made by replacement of the 700-bp EcoRIScaI fragment of
Smad5-CS2 with a PCR fragment incorporating a single base
change, resulting in replacement of Thr 429, a conserved residue in
the L3 loop, by Ile. This reproduces the mutation responsible for
the somitabun (sbn) phenotype in zebrafish (Connors et al., 1999).
Copyright 2001 by Academic Press. All rights of reproduction in any form reserved.
Beck, Whitman, and Slack
All of the preceding products were linearized with NotI and
transcribed with SP6 to make mRNA. BMP4 mRNA was made
from BMP4 CS2 as previously described (Nishimatsu and Thom-
sen, 1998). Alk3, a constitutive form of BMPR-1 (Gln 233-Asp) was
a kind gift from Richard Harlands lab (Hsu et al., 1998). Noggin-
CSKA was a kind gift of Betsy Pownall.
Embryo Culture and DorsalVentral Phenotype
Scoring (DAI Index)
Xenopus embryos were generated and cultured as described
previously (Beck and Slack, 1999). They were staged according to
Nieuwkoop and Faber (1967). Dorsoanterior index (DAI) scores
(Kao and Elinson, 1988) were recorded at stage 40. A score of 5
indicated a wild type phenotype, with lower scores down to 0
indicating the degree of ventralization and higher scores up to 10
indicating the degree of dorsalization.
RT-PCRs
Total RNA was isolated from 10 to 12 animal caps or 5 embryos
using the method of Beck et al. (1998). Semiquantitative reverse
transcriptionpolymerase chain reaction (RT-PCR) was based on
the method of Rupp and Weintraub (1991), as modified by the
Woodland lab (see http://www.bio.warwick.ac.uk/woodland/
HRW3.htm). Primers and conditions used were: Cardiac actin, 5
GCT GAC AGA ATG CAG AAG and 3 TTG CTT GGA GGA
GTG TGT (bp 9871212, 62C annealing, 24 cycles); T3-Globin,
5 GCC TAC AAC CTG AGA GTG G and 3 CAG GCT GGT
GAG GCT GCC C (bp 328 529, 62C annealing, 24 cycles); ODC,
5 GGA GCT GCA AGT TGG AGA and 3 TCA GTT GCC AGT
GTG GTC (bp 14821541, 55C annealing, 17 cycles); Nrp-1, 5
GGG TTT CTT GGA ACA AGC and 3 ACT GTG CAG GAA
CAC AAG (bp 934 1210, 55C annealing, 24 cycles); Xhox3, 5
TTA CGC CTC ACC TGC ACA and 3 GCC AAC ATG GTG TTC
ATC (bp 10021240, 55C annealing, 24 cycles).
Luciferase Reporter Assays
A luciferase reporter driven by a promoter consisting of the BMP
responsive element of Xvent2b (275 to 152) fused to the TATA
box region (32 to 34) was kindly provided by Kris Henningfeld
(Xvent2b-lux) (Henningfeld et al., 2000). A 20-pg sample of plasmid
DNA was mixed with the appropriate mRNA and injected into
4-cell embryos. Injections were targeted into either the two dorsal
blastomeres for BMP4 (250 pg), Smad5 (2 ng), Smad5* (2 ng), or into
the two ventral blastomeres for Smad5-sbn (200 pg). Controls with
DNA alone were included for each injection set. Groups of 25
embryos for each treatment were washed and frozen in dry ice at
stage 10. Embryos were then homogenized in 500 l lysis buffer (25
mM Tris, pH 7.8; 10 mM CDTA; 10% glycerol; 1% Triton X-100;
2 mM DTT) and centrifuged at 13K rpm for 5 min, after which the
supernatant was kept. Luciferase assays were performed using 10 l
of a 1/10 dilution of protein extract with a kit for luciferase assay
(Promega, Madison, WI) according to the manufacturers instruc-
tions, and normalized to total protein (Bio-Rad protein assay;
Bio-Rad, Richmond, CA). Each sample was prepared in duplicate
and read using a MicroLumat Plus microplate luminometer LB 96V
(EG&G Berthold Electro-Optics, Salem, MA). Results show the
means and error bars represent standard error.
BMP Signaling in Tail Bud Outgrowth
Assay for Ectopic Tail Formation
Embryos were injected with synthetic mRNA into each cell at
the 4-cell stage and cultured to stage 8.5. Grafts of animal cap
tissue into the wild type host posterior neural plate at stage 13 were
as described previously (Beck and Slack, 1999). Briefly, animal cap
tissue was excised at stage 8.5 and trimmed to rectangles of 100
600 m. These were then grafted into a slit in the host neural plate,
perpendicular to the midline and 100 200 m anterior to the
blastopore. Embryos were scored for formation of ectopic tails at
stages 35/36. For inducing GR fusions, dexamethasone (Sigma, St.
Louis, MO) was added to culture media at a final concentration of
10 M from a 1000 stock solution in EtOH. For inhibition of
Notch cleavage, the protease inhibitor calpeptin (Calbiochem, San
Diego, CA) was added to a final concentration of 25 M from a 10
mM stock made up in DMSO and frozen in small aliquots.
Labeling and Histology of Grafts
Grafts were labeled with nuclear -galactosidase by injecting a
total of 50 pg of mRNA into the embryo along with the test mRNA.
X-gal staining was then used to trace the final position and
contribution of the graft (see Beck and Slack, 1999). Histology of
labeled grafts was as described previously (Beck and Slack, 1999),
except that counterstaining with picroblueblack was not used.
Marker Expression
Whole-mount in situ hybridizations for the tail bud markers
Xbra, Xhox3, and FGF8 were described previously (Christen and
Slack, 1997; Beck and Slack, 1998). The BMP4 probe used for in situ
hybridizations was also previously described (Beck and Slack,
1998). The use of 12/101 antibody (Kintner and Brockes, 1984) for
detection of tail somites was previously described (Tucker and
Slack, 1995).
Detection of Endogenous Smad 1/5/8 Activity
Embryos were prepared and stained with antibody to the type 1
receptor phosphorylated forms of Smad 1/5/8 as described in Faure
et al. (2000) with the following modifications: Embryos were stored
in MeOH after fixation, rehydrated to and then into PBS/0.3 M
sucrose/0.05% Triton X-100, then embedded in 2% low-melt
agarose (Nusieve GTG agarose; FMC BioProducts, Rockland, ME)
also containing PBS/0.3 M sucrose/0.05% Triton X-100. Embedded
embryos were subjected to sagittal bisection using a disposable
microscalpel (Feather Safety Razor Company) under PBS and re-
moved from the agarose. After washing in PBT (PBS, 2 mg/ml BSA,
0.1% Triton X-100), embryos were blocked for 2 h with PBT plus
10% normal goat serum (Jackson ImmunoResearch Laboratories,
West Grove PA) dialyzed against PBS to remove sodium azide.
Primary antibody incubation (affinity-purified anti-phospho-
Smad1/5/8) at 1:20 dilution was carried out overnight at 4C,
followed by washes of PBS for 2 h. Secondary antibody incubation
with HRP-conjugated goat anti-rabbit antibody (at 1:250 dilution)
was carried out for 1 h at room temperature and followed by washes
as above. Staining was developed with ImmunoPure Metal-
enhanced DAB Substrate Kit (Pierce, Rockford, IL), as per the
manufacturers instructions, for 510 min.
Copyright 2001 by Academic Press. All rights of reproduction in any form reserved.
305
RESULTS
BMP Signaling Is Active in the Tail Bud Region
during Development
Of the BMPs expressed in the early Xenopus embryo,
BMP4 is the best candidate for a role in tail patterning,
given its strong expression in cells immediately ventral to
the future tail bud from gastrulation (Fainsod et al., 1994).
Here, we show that BMP signaling is active in the tail bud
region itself using an antibody to the phosphorylated form
of Smads 1/5/8 (Fig. 1). This antibody has been shown to be
specific for the activated, phosphorylated forms of the BMP
pathway Smads (Faure et al., 2000). Levels of Smad 1/5/8
activation in the ventral/posterior regions correlate well
with those of BMP4 expression, shown for comparison
(Figs. 1F1I). Smad 1/5/8 phosphorylation is at its highest
levels in the deep (mesodermal) layer of the ventral blas-
topore lip at stage 12 (Fig. 1A) and this expression is
maintained in the proctodeum until tailbud stages (Figs.
1B1E). By stage 17 Smad activation is also detected in the
ventral side of the future tail bud (Fig. 1C) and this is also
maintained, at least until the tail bud begins to extend (Figs.
1D and 1E).
Constitutively Active, Dominant Negative, and
Inducible Forms of Smad5
BMP signaling utilizes the pathway specific Smads 1, 5,
and 8 (for review see Dale and Jones, 1999). In this study we
concentrated on Smad5. An activated version of mouse
Smad5, Smad5*, was created by deletion of the last two
C-terminal amino acids. This mutant lacks the second of
the phosphorylation sites in the SSXS motif. When injected
into embryos, Smad5* mRNA causes a ventralized pheno-
type (Fig. 2D), which is similar to embryos injected with
either BMP4 (Fig. 2C) or activated BMPR1 (Alk3; Fig. 2G),
although the phenotype is weaker, scoring higher on the
dorsoanterior index (Table 1).
The zebrafish mutant somitabun (sbn), which encodes a
point mutation in the L3 loop of Smad5, is known to act as
a dominant negative and to be specific to BMP signaling
(Hild et al., 1999). Since Smad5 has not yet been cloned
from Xenopus, we reproduced this mutation using the
mouse Smad5 coding region (Smad5-sbn). Expression of this
construct in early Xenopus embryos results in a dorsalized
phenotype, as expected for a dominant negative effect (Fig.
2H).
We made an inducible version of Smad5 (Smad5-GR) by
fusion of the glucocorticoid receptor ligand-binding (GR)
domain to the C-terminus of Smad5. GR fusions are inac-
tive unless released from the heat shock complex by the
addition of the steroid hormone dexamethasone at nontoxic
levels (Kolm and Sive, 1995; Hollenberg et al., 1993).
Embryos injected with Smad5-GR mRNA develop nor-
mally in the absence of inducing hormone. In the presence
of dexamethasone, they have a ventralized phenotype simi-
306 Beck, Whitman, and Slack

FIG. 1. Endogenous BMP activity in the tail bud. (AE) Embryos bisected along the anteriorposterior axis and stained using an antibody
that recognizes the activated form of Smad. Strong anti-phospho-Smad1/5/8 staining is seen in the region immediately ventral to the future
tail bud (black arrows), correlating to the expression of BMP4 mRNA. Nuclear phosphorylated BMP Smads are also present at lower levels
in the tail-forming region itself from stage 17 on. (A) stage 12, (B) stage 15, (C) stage 17, (D) stage 20, (E) stage 25. All embryos are oriented
with anterior to the left and dorsal uppermost. (FI) In situ hybridization to BMP4 to show sites of endogenous transcription. (F) BMP4
expression just anterior to the ventral and lateral lips of the blastopore (black arrow). Embryo viewed from the vegetal side with dorsal
uppermost. (GI) Expression in embryos bisected after in situ hybridization; arrow points to strong expression in the region immediately
ventral to the tail bud. Orientation is anterior to the left and dorsal uppermost. (G) stage 14, (H) stage 20, (I) stage 25. ftb, position of future
tail bud; tb, tail bud; bp, blastopore.
FIG. 2. Ventralizing activity of BMP pathway components. Embryos were injected into each of 4 cells at the 4-cell stage and analyzed at
stage 40 using the DAI index of Kao and Hopwood (1988), where a score of 5 indicates a normal embryo and a score of 0 indicates a
completely ventralized embryo. (A) Control (uninjected) embryos at stage 40 score 5 on the DAI scale (N 62). (B) Embryos injected with
2 ng Smad5 mRNA have defects in ventral fin and proctodeum. (C) Embryos injected with 250 pg of BMP4 mRNA are ventralized with an
average DAI score of 1.8 (N 44). (D) Embryos injected with 2 ng Smad5*, an activated form lacking the last two amino acids, are partially
ventralized with an average DAI score of 2.6 (N 46). (E) Embryos injected with 2 ng mRNA encoding the hormone-inducible fusion protein
Smad5-GR develop normally with an average DAI score of 4.9 (N 35). (F) Embryos injected with the same dose of Smad5-GR mRNA but
treated with 10 M dexamethasone from stage 6 are partially ventralized with an average DAI score of 2.2 (N 29). (G) Embryos injected
with 500 pg of the constitutively active BMP receptor Alk3 are extremely ventralized with an average DAI score of 1.2 (N 62). (H) Embryos
injected with 100 pg mRNA encoding dominant negative Smad5-sbn are dorsalized with an average DAI score of 6.2 (N 44).

lar to that of embryos injected with Smad5* mRNA (com- mild ventralization, seen most clearly at stages 30 32 as an
pare Figs. 2F and 2D; Table 1). By contrast, injection of WT enlargement of the proctodeum, a derivative of the ventral
Smad5 (with no C-terminal fusions) produced only a very blastopore lip (data not shown). By stage 40 the proctodeum

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BMP Signaling in Tail Bud Outgrowth 307

TABLE 1 Characterization of Constructs

Dorsoanterior Index (DAI) Scores of Injected Embryos
To further test the constitutive, dominant negative, and
mRNA ng/Embryo N DAI inducible versions of Smad5 we looked at the effects of
Controls 62 5.00
expressing these constructs in animal caps (Fig. 3). Control
Smad5 WT 2.0 39 4.94 (uninjected) animal caps formed atypical epidermis (Fig.
Smad5* 2.0 46 2.57 3A), as do animal caps injected with 2 ng WT Smad5
Alk3 2.0 14 0.14 mRNA (Fig. 3D). BMP4-injected animal caps (250 pg)
Alk3 0.5 62 1.23 formed ventral mesoderm (Fig. 3B). Ventral mesoderm was
BMP4 1.0 38 0.68 also formed by animal caps injected with 500 pg Alk3 (Fig.
BMP4 0.25 67 1.68 3C), 2 ng Smad5* (Fig. 3E), or 2 ng Smad5-GR in the
BMP4 calpeptin 0.25 25 1.16 presence (but not the absence) of dexamethasone (Figs. 3F
Smad5-GR 2.0 35 4.91
and 3G). Injection of 400 pg Smad5-sbn caused the explants
Smad5-GR dexamethasone 2.0 29 2.17
Smad5-sbn 0.1 44 6.27 to elongate as a result of the formation of axial mesoderm,
an effect that was not reversed by coinjection of BMP4 (Fig.
3H).
The type of tissue induced in these explants was con-
firmed by histology (not shown) and RT-PCR (Fig. 4A).
has been shed from the embryo in most cases, and a defect Expression of markers for ventral mesoderm (Xhox3, glo-
in the ventral fin can be seen (Fig. 2B). It is not surprising bin), axial mesoderm (cardiac actin), and neural tissue
that overexpression of WT Smad5 has a limited effect in the (Nrp-1) were compared to that of the ubiquitously expressed
absence of BMP signaling, given that ligand-dependent gene ornithine decarboxylase (ODC). As expected, BMP4
phosphorylation is required for transcriptional activity caps expressed the ventral mesoderm markers. Expression
(Heldin et al., 1997). The enhanced effect of the inducible of ventral mesoderm markers was also induced by Alk3,
form is probably attributable to the fact that the GR domain Smad5*, and Smad5-GR in the presence of dexamethasone.
itself contains some transcriptional activation activity However, globin expression was quite low in Smad5* caps.
(Webster et al., 1988). Smad5-GR caps without dexamethasone treatment ex-

FIG. 3. Effects of injecting BMP pathway components into animal caps. Animal caps were dissected at stage 8.5 and cultured in NAM/2
until control stage 30. Uninduced caps form compact wrinkled balls, those producing ventral-type mesoderm form translucent vesicles, and
those producing dorsal-type mesoderm elongate to a sausage shape. (A) Untreated animal caps do not form mesoderm. (B) Animal caps from
embryos injected with 250 pg BMP4 mRNA form ventral mesoderm. (C) Animal caps from embryos injected with 500 pg of the constitutive
BMP receptor Alk3 form ventral mesoderm. (D) Animal caps from embryos injected with 2 ng Smad5 mRNA do not form mesoderm. (E)
Animal caps from embryos injected with 2 ng of active Smad5* mRNA form ventral mesoderm. (F) Animal caps from embryos expressing
2 ng of Smad5-GR mRNA do not form mesoderm. (G) Animal caps from embryos injected with 2 ng of Smad5-GR mRNA and treated with
10 M dexamethasone form ventral mesoderm. (H) Animal caps injected with 250 pg BMP4 and 100 pg Smad5-sbn mRNA elongate, the
formation of dorsal mesoderm indicating that both endogenous and exogenous BMP4 are inhibited.

Copyright 2001 by Academic Press. All rights of reproduction in any form reserved.
308 Beck, Whitman, and Slack

FIG. 4. RT-PCR and luciferase reporter assays to demonstrate activity of Smad constructs. (A) RT-PCR to show the nature of inductions
in animal caps using markers for ventral mesoderm (Xhox3, Globin), axial mesoderm (cardiac actin), neural tissue (Nrp-1), and the loading
control gene ODC. A total of 10 12 animal caps at stage 30 were pooled for each sample and positive and negative control RNA was made
from 5 pooled WT embryos at the same stage and reverse-transcribed with (Embryo) or without reverse transcriptase enzyme (-RT).
Concentrations of injected mRNAs were as stated in Fig. 2. (B) Schematic of Xvent2b promoterluciferase reporter construct. The BMP
responsive element (BRE) is contained within the 275 to 152 portion of the promoter. The TATA box lies between 32 and 34. (CD)
Results of luciferase assays performed on protein extracts from embryos injected at the 4-cell stage and harvested at stage 10. These results
are representative of three repeat experiments and measured in duplicate to generate error bars (standard errors). (C) Dorsal injections reveal
that Smad5 (1 ng per embryo) does not stimulate luciferase activity over background, whereas Smad5* (1 ng per embryo) increases
production of luciferase threefold. BMP4 (250 pg per embryo), included as a positive control, increases luciferase production ninefold. (D)
On the ventral side, injections of reporter DNA alone produce high levels of luciferase as a result of the endogenous factors present on the
ventral side. Smad5-sbn (200 pg per embryo) reduces this activity to less than 1/3, showing that it is capable of interfering with the
endogenous BMP pathway.

pressed only a low level of Xhox3, showing minimal activ-
ity in the absence of induction, and unmodified Smad5 did
not induce any of the markers tested. Animal caps contain-
ing Smad5-sbn expressed markers for axial mesoderm and
neural tissue but not ventral mesoderm, confirming that
Smad5-sbn indeed blocks BMP4 signaling.
In addition, a series of experiments were carried out to
establish the relative ability of the constructs to activate or
repress transcription from the Xvent2B promoter, on the
dorsal or ventral sides of the embryo, respectively, using
luciferase as the reporter (Figs. 4B 4D). Smad5* was found
to elevate reporter expression on the dorsal side by a factor
of 3 (Fig. 4C), whereas Smad5-sbn was found to reduce
reporter expression on the ventral side by a factor of 3 (Fig.
4D).
Induction of Ectopic Tails
We previously described an assay for tail-forming ability
in which a piece of animal cap tissue expressing the test
gene is grafted into the posterior neural plate of a host
embryo at stage 13 (Beck and Slack, 1999). The grafts
become incorporated into the neural plate, and if they
contain an appropriate mRNA, will develop into a tail-like
structure with axial tissues formed from the graft and a fin
formed from the host. This assay was used to test the effect
of ectopic BMP signaling on secondary tail formation (Fig.
5). Grafts from animal cap tissue expressing BMP4, Alk3 or
Smad5* mRNA, or Smad5-GR mRNA in the presence of
dexamethasone form ectopic tails (Figs. 5C5F; Table 1).
Grafts containing either no mRNA (not shown), WT Smad5
mRNA (Fig. 5A), or Smad5-GR (Fig. 5B) do not produce
ectopic tails (see also Table 1). So the ability to provoke tail
formation correlates perfectly with the activity of the
constructs in whole embryo ventralization, ventral meso-
derm induction, and vent2B promoter activation. Like
ectopic tail buds induced by Notch signaling, the BMP
pathway-induced tail buds expressed characteristic molecu-
lar markers such as Xbra, FGF-8, and Xhox3 (Figs. 5G5I).
Expression of the dominant negative Smad5-sbn in animal

Copyright 2001 by Academic Press. All rights of reproduction in any form reserved.
BMP Signaling in Tail Bud Outgrowth 309

TABLE 2
Scoring of Ectopic Tail Formation by Animal Cap Grafts into the Posterior Neural Plate

mRNA ng/Embryo No. of grafts No. of ectopic tails Percentage of ectopic tails

Smad5* 2.0 70 50 71
Smad5 WT 2.0 34 0 0
Smad5-sbn 0.1 12 1 8
Smad5-GR 2.0 27 2 (v. small) 7
Smad5-GR dexamethasone 2.0 38 29 76

Alk3 0.5 32 25 78
BMP4 1.0 38 33 86

Noggin 0.05 12 0 0
Xhox3 0.4 18 13 72
Xhox3/Smad5-sbn 0.4/0.4 14 12 86
Smad5*/Xhox3VP16 2.0/0.4 15 4 (v. small) 27
Noggin/Xhox3 0.05/0.4 12 5 42

Notch ICD 0.4 36 24 67

Notch ICD/Smad5-sbn 0.4/0.4 12 7 58
Notch ICD/Noggin 0.4/0.05 12 9 75

BMP4/Smad5-sbn 0.25/0.4 12 0 0

BMP4/calpeptin 0.25/25 M 15 0 0
Notch ICD/calpeptin 0.4/25 M 11 6 54
mNotchE/calpeptin 0.1/25 M 12 0 0
mNotchE 0.1 12 7 58

cap grafts did not result in the production of any ectopic
tails (Table 1). These results show that BMP signaling can
induce the formation of ectopic tails and thereby suggest
that BMP4 could be involved in tail formation in vivo.
The secondary tails produced by the Notch pathway
contain neural tube but lack muscle and notochord (Beck
and Slack, 1999). By comparison, histological analysis of the
BMP-induced tails revealed the presence of somites as well
as neural tube and fin (Fig. 6). When the grafts were labeled
with -galactosidase mRNA, the graft tissue was seen to
produce both the somites and the neural tube of the ectopic
tail, whereas the fin and fin mesenchyme were unlabeled,
indicating that it had come from the host (Figs. 6A 6D).
More anteriorly, the graft tissue integrates into the host
dorsal axis (somites and neural tube). In Fig. 6E the somite
files of the host and ectopic tails are shown immunostained
with 12/101 antibody.
Xhox3 can block production of ectopic tails by Notch ICD
grafts as well as outgrowth of the tail bud in vivo (Beck and
Slack, 1999). The antimorphic Xhox3 construct has the
repressor domain replaced by a transactivation domain
from the VP16 protein (Xhox3VP16). Here, we show that
coexpression of Xhox3VP16 can also block the production
of ectopic tails by Smad5* (Fig. 7A). This suggests that
Xhox3 is acting downstream of BMP signaling in tail bud
outgrowth. In support of this, coexpression of either Smad5-
sbn or the BMP inhibitor Noggin with Xhox3 has no effect
on ectopic tail formation (Figs. 7B and 7C). Expression of
Xhox3 is known to be induced by BMP signaling (Dale et
al., 1992; Jones et al., 1992) and is turned on in the ectopic
tail buds produced by grafts in which BMP signaling is
activated (Fig. 5I). The production of ectopic tails by BMP4
grafts can, as expected, be blocked by the coexpression of
Smad5-sbn (Fig. 7D).

Xhox3 Acts Downstream of BMP and Notch
Signaling
Because both the Notch-Xhox3 pathway and the BMP
pathway produce outgrowth and neural tube formation in
the ectopic tails it is possible that they form part of the
same pathway. To elucidate the relationship between BMP
and Notch signaling we carried out a number of epistasis
experiments.
We previously showed that an antimorphic fusion of
BMP Signaling Acts Upstream of Notch Signaling
Grafts expressing constitutively active Notch ICD pro-
duce ectopic tails when grafted into the posterior neural
plate (Beck and Slack, 1999). Coexpression of the dominant
negative form, Smad5-sbn, or the BMP inhibitor Noggin,
does not block the formation of ectopic tails by Notch ICD
(Figs. 7E and 7F). Although these results could suggest that
BMP signaling acts upstream of Notch activation and
cleavage, the same results would be obtained if BMP acti-

Copyright 2001 by Academic Press. All rights of reproduction in any form reserved.
310 Beck, Whitman, and Slack

FIG. 5. Activation of the BMP pathway in animal cap grafts to the posterior neural plate results in the formation of ectopic tails expressing
tail bud markers. (AF) Results of grafts into the posterior neural plate. Arrows point to ectopic tails, if formed. (A) Grafts from animal caps
injected with 2 ng Smad5 mRNA do not form ectopic tails. (B) Grafts from animal caps injected with 2 ng Smad5-GR mRNA do not form
ectopic tails. (C) Ectopic tails are formed from grafts of animal caps injected with 250 pg BMP4 mRNA. (D) Ectopic tails are formed from
grafts of animal caps injected with 2 ng Smad5* mRNA. (E) Ectopic tails are formed from grafts of animal caps injected with Smad5-GR
mRNA and treated with dexamethasone from stage 6. (F) Ectopic tails are formed from grafts of animal caps injected with 500 pg Alk3
mRNA. (GI) Comparison of tissue-specific markers (dark blue) in the host tail (black arrow) and ectopic tail formed by Smad5* graft (white
arrow). (G) The tail bud marker Xbra is expressed in both host- and graft-derived tail buds. (H) FGF-8 is expressed in both host- and
graft-derived tail buds. (I) Xhox3 is expressed in both host- and graft-derived tail buds.
FIG. 6. Activation of the BMP pathway in animal cap grafts to the posterior neural plate leads to the formation of ectopic tails (white arrow)
containing somites, neural tube, and fin. (A and B) Two examples of grafts labeled with nuclear -galactosidase (blue) to show contribution of the
graft to the ectopic tail by stage 36. The fin and epidermis of the secondary tail are not labeled and are therefore induced from the host by the
graft. (C) Sagittal section through posterior of a grafted embryo at stage 36. Graft cells are labeled with nuclear -galactosidase and are found in
the somites and neural tube of the ectopic tail but not in the fin, fin mesenchyme, or epidermis. (D) Drawing of the section in C, showing the
identity of the tissues contributing to the host and graft tails. nt, host neural tube; nt, graft-derived ectopic neural tube; s, host somites; s, graft
derived ectopic tail somites; nc, notochord (host); fin, tail fin; fin, tail fin of ectopic tail (host-derived). (E) 12/101 antibody staining to show
somites (dark blue) in both host- and graft-derived tails. Black arrows denote host tails.
BMP Signaling in Tail Bud Outgrowth 311

FIG. 7. BMP signaling is required for tail bud outgrowth and acts upstream of Notch activation and cleavage. (A) Animal cap grafts
from embryos injected with 2 ng of Smad5* and 400 pg mRNA encoding the antimorphic Xhox3VP16 fusion fail to form ectopic tails
(white arrow), suggesting a requirement for Xhox3 downstream of BMP signaling. (B) Animal cap grafts from embryos injected with
400 pg Smad5-sbn and 400 pg Xhox3EnR mRNA do form ectopic tails, indicating that Xhox3 is required downstream of Smad5. (C)
Animal cap grafts from embryos injected with 400 pg Xhox3EnR mRNA and 50 pg Noggin-CSKA DNA form ectopic tails. (D) Animal
cap grafts from embryos injected with 250 pg BMP4 and 400 pg Smad5-sbn do not form ectopic tails. (E) Coinjection of 400 pg
Smad5-sbn mRNA with the same dose of Notch ICD does not block ectopic tail formation, suggesting that Notch signaling is
downstream of Smad 5 activity. (F) Coinjection of 50 pg Noggin-CSKA DNA with 400 pg Notch ICD mRNA does not block formation
of ectopic tails from the graft. (G) Calpeptin treatment of embryos grafted with Notch ICD expressing animal caps does not affect the
formation of ectopic tails. (H) Treatment of embryos grafted with animal caps expressing a membrane-tethered constitutively cleaved
form of Notch, mNotchE, with calpeptin prevents formation of ectopic tails. (I) Treatment with calpeptin also prevents the
formation of ectopic tails from grafts expressing BMP4.

vates Xhox3 without the need for Notch signaling. To
distinguish between these two possibilities we made use of
the protease inhibitor calpeptin, which has been shown to
block the intramembraneous proteolytic cleavage of Notch
normally responsible for the release of the intracellular
domain (ICD) (De Strooper et al., 1999).
Because treatment with an inhibitor may well provoke
nonspecific effects, we compared the activity of calpeptin
on tail formation by two different constitutive forms of
Notch. Notch-ICD is the normal cleavage product of Notch
activation and will activate the target genes without the
need for proteolytic processing. mNotchE is a membrane-
tethered form that is constitutively cleaved to liberate
Notch ICD. The expectation is that calpeptin should block
tail formation by grafts expressing mNotchE, which re-
quires cleavage, but not by those containing Notch-ICD,
which does not require cleavage. This prediction was borne
out (Figs. 7G and 7H), with calpeptin in both cases also
reducing the length of the host tail. We conclude that,
whatever other side effects it may have, calpeptin does
block Notch cleavage in Xenopus. The critical experiment
allowing the ordering of BMP and Notch effects is shown in
Fig. 7I. BMP4-containing grafts do not form ectopic tails in
the presence of calpeptin. BMP signaling itself is unaffected
by calpeptin, as shown by the lack of impact of incubation
with the protease inhibitor on DAI scores of BMP4-injected
embryos (see Table 1). This shows that BMP exerts its
effects on tail bud outgrowth upstream of Notch.

Copyright 2001 by Academic Press. All rights of reproduction in any form reserved.
312
FIG. 8. Model for the role of BMP signaling in tail development.
DISCUSSION
BMP4 Expression Causes Tail Bud Formation,
Outgrowth, and Patterning
In this study, we have shown that ectopic expression of
BMP4, a constitutively active BMP receptor, or an activated
form of Smad5, in the posterior neural plate of an early
Xenopus neurula results in the formation of an extra
tail-like structure. These ectopic tails express tail bud
markers appropriately and contain well-formed somites as
well as neural tube and fin. It is already known that BMP4
is expressed in the cells immediately ventral to the site of
the future tail bud (Fainsod et al., 1994) and we show here
that BMP signaling is also active in the ventral part of the
future tail bud itself, which is fated to form tail somites.
BMP4 was previously classed as a gene expressed in the late
phase, during tail bud outgrowth, because of its expression
in the tail fin (Beck and Slack, 1998). However, as shown
here, BMP signaling is active during the early phase, when
the tail bud is being determined, in the region that will give
rise to posterior somites.
BMP Signaling Is Required Upstream of Notch
Activation in Tail Bud Formation
We previously described a pathway for tail bud formation
that depends on the interaction between a ventral compart-
ment, the posterior wall, which expresses the Notch ligand
Delta, and a dorsal compartment, the dorsal roof, which
expresses both Notch itself and the signaling modulator
Lunatic Fringe. It is therefore likely that Notch activation
occurs specifically at the junction between these compart-
ments, which lies at the distal tip of the future tail. Ectopic
activation of Notch leads to formation of an ectopic tail bud
that elongates and differentiates into neural tube and fin.
The results of our epistasis experiments in the present
study suggest that BMP signaling is required upstream of
Notch activation and cleavage. Tails formed from BMP
grafts contain somites as well as neural tube; the most
likely explanation for this result is that BMP, as well as
causing activation of Notch, activates a second, Notch-
independent pathway that results in the formation of
somites from the ventral tail bud (Fig. 8).
A Role for BMPs in Vertebrate Tail Development
We have presented evidence on the appropriate expres-
sion and biological activity of BMP. To prove that it is
Copyright 2001 by Academic Press. All rights of reproduction in any form reserved.
Beck, Whitman, and Slack
involved in tail outgrowth in vivo it is also necessary to
show that removal of BMP signaling will impair tail devel-
opment. There is already considerable evidence to support
this from other vertebrate species in which experimental
genetics is possible. The zebrafish mfn gene, loss-of-
function alleles of which lack ventral tail tissues such as fin
and ventral somites, encodes the homologue of Tolloid
(Connors et al., 1999). Tolloid encodes a metalloprotease
that cleaves the BMP antagonist chordin (Piccolo et al.,
1996, 1997). Loss of Tolloid therefore leads to a decrease in
BMP signaling. Expression of the zebrafish homologue of
Xhox3, eve-1, is downregulated in the tail bud of mfn
embryos (Connors et al., 1999). Furthermore, BMP4 expres-
sion in the tail bud is much reduced in mfn mutant fish,
suggesting an autoregulatory role. Another zebrafish mu-
tant, swirl, is known to result from a point mutation in the
BMP2b gene. In these embryos, BMP4 is again downregu-
lated, leading to the formation of embryos with severe
defects in posterior and ventral structures (Kishimoto et al.,
1997). Mice that are homozygous null for BMP4 normally
die before gastrulation, although a minority of embryos
survive until later stages, exhibiting posterior truncations
(Winnier et al., 1995). Smad5 knockout mice show defects
in L-R patterning, angiogenesis, and mesenchymal defects,
but also have truncated posteriors (Chang et al., 1999, 2000;
Yang et al., 1999).
The juxtaposition of expression domains of both BMPs,
their antagonists (chordin, noggin), and agonists (Tolloid/
Xolloid) in the posterior at the time of tail bud initiation is
also indicative of a role in this process. Xolloid is expressed
in the dorsal roof and distal tip of the Xenopus tail bud,
appearing peripheral in transverse section (Goodman et al.,
1998). BMP4 is expressed in lower lateral and ventral
blastopore lips at the end of gastrulation (Fig. 1) (Fainsod et
al., 1994; Hemmati-Brivanlou and Thomsen, 1995; this
study), which go on to form the proctodeum (Gont et al.,
1993; Tucker and Slack, 1995). BMP4 protein, however, is
also likely to be present in the ventral region of the future
tail bud, as shown by the immunohistological detection of
activated Smad 1/5/8. BMP2 and BMP4 are also expressed in
the fin at later stages, during outgrowth of the tail bud-
derived tail (Beck and Slack, 1998). The BMP antagonists
chordin and noggin are expressed in the chordoneural hinge,
derived from the late dorsal blastopore lip (Smith and
Harland, 1992; Sasai et al., 1994). Expression of BMP2 and
BMP7 is ubiquitous during gastrulation (Hemmati-
Brivanlou and Thomsen, 1995; Hawley et al., 1995), provid-
ing the possibility of potent BMP heterodimer formation in
lateral and ventral regions of the posterior embryo. In
zebrafish, expression of chordino, Tolloid/minifin, and
BMP4 is in sequential anteriorposterior domains of the tail
bud (Connors et al., 1999). Expression of three novel ze-
brafish noggin genes was also previously described in the
tail bud and fin (Furthauer et al., 1999). Finally, a novel
mouse BMP, BMP11, is expressed in limb, spinal cord, and
tail bud (Gamer et al., 1999).
We consider that the evidence presented here, showing
BMP Signaling in Tail Bud Outgrowth
that Smad phosphorylation occurs in the future tail bud,
and that BMP signaling promotes tail outgrowth, together
with the properties of the relevant zebrafish and mouse
loss-of-function mutants, proves that BMP signaling is both
necessary and sufficient for tail bud outgrowth in verte-
brates.
ACKNOWLEDGMENTS
We are grateful to all those people who kindly provided reagents
for use in this work: Peter ten Dijke, Raphael Kopan, Kris Hen-
ningfeld, Marko Horb, Betsy Pownall, and Richard Harland. We
also express thanks to Andrew Ward and Marika Charalambous for
their advice on luciferase assays. This work was supported by the
Wellcome Trust, Programme Grant 43192.
REFERENCES
Beck, C. W., and Slack, J. M. (1998). Analysis of the developing
Xenopus tail bud reveals separate phases of gene expression
during determination and outgrowth. Mech. Dev. 72, 4152.
Beck, C. W., and Slack, J. M. (1999). A developmental pathway
controlling outgrowth of the Xenopus tail bud. Development
126, 16111620.
Chang, H., Huylebroeck, D., Verschueren, K., Guo, Q., Matzuk,
M. M., and Zwijsen, A. (1999). Smad5 knockout mice die at
mid-gestation due to multiple embryonic and extraembryonic
defects. Development 126, 16311642.
Chang, H., Zwijsen, A., Vogel, H., Huylebroeck, D., and Matzuk,
M. M. (2000). Smad5 is essential for left-right asymmetry in
mice. Dev. Biol. 219, 7178.
Christen, B., and Slack, J. M. (1997). FGF-8 is associated with
anteroposterior patterning and limb regeneration in Xenopus.
Dev. Biol. 192, 455 466.
Coffman, C. R., Skoglund, P., Harris, W. A., and Kintner, C. R.
(1993). Expression of an extracellular deletion of Xotch diverts
cell fate in Xenopus embryos. Cell 73, 659 671.
Connors, S. A., Trout, J., Ekker, M., and Mullins, M. C. (1999). The
role of tolloid/mini fin in dorsoventral pattern formation of the
zebrafish embryo. Development 126, 3119 3130.
Dale, L., Howes, G., Price, B. M. J., and Smith, J. C. (1992). Bone
morphogenetic protein 4: A ventralising factor in early Xenopus
development. Development 115, 573585.
Dale, L., and Jones, C. M. (1999). BMP signalling in early Xenopus
development. Bioessays 21, 751760.
De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K.,
Mumm, J. S., Schroeter, E. H., Schrijvers, V., Wolfe, M. S., Ray,
W. J., Goate, A., and Kopan, R. (1999). A presenilin-1-dependent
gamma-secretase-like protease mediates release of Notch intra-
cellular domain. Nature 398, 518 522.
Fainsod, A., Steinbeisser, H., and De Robertis, E. M. (1994). On the
function of BMP-4 in patterning the marginal zone of the
Xenopus embryo. EMBO J. 13, 50155025.
Faure, S., Lee, M. A., Keller, T., ten Dijke, P., and Whitman, M.
(2000). Endogenous patterns of TGF superfamily signaling dur-
ing early Xenopus development. Development 127, 29172931.
Furthauer, M., Thisse, B., and Thisse, C. (1999). Three different
noggin genes antagonize the activity of bone morphogenetic
proteins in the zebrafish embryo. Dev. Biol. 214, 181196.
Copyright 2001 by Academic Press. All rights of reproduction in any form reserved.
313
Gamer, L. W., Wolfman, N. M., Celeste, A. J., Hattersley, G.,
Hewick, R., and Rosen, V. (1999). A novel BMP expressed in
developing mouse limb, spinal cord, and tail bud is a potent
mesoderm inducer in Xenopus embryos. Dev. Biol. 208, 222232.
Gont, L. K., Steinbeisser, H., Blumberg, B., and De Robertis, E. M.
(1993). Tail formation as a continuation of gastrulation: The
multiple cell populations of the Xenopus tailbud derive from the
late blastopore lip. Development 119, 9911004.
Goodman, S. A., Albano, R., Wardle, F. C., Matthews, G., Tanna-
hill, D., and Dale, L. (1998). BMP1-related metalloproteinases
promote the development of ventral mesoderm in early Xenopus
embryos. Dev. Biol. 195, 144 157.
Hata, A., Seoane, J., Lagna, G., Montalvo, E., Hemmati-Brivanlou,
A., and Massague, J. (2000). OAZ uses distinct DNA- and
protein-binding zinc fingers in separate BMP-Smad and Olf
signaling pathways. Cell 100, 229 240.
Hawley, S. H. B., Wunnenberg-Stapleton, K., Hashimoto, C., Lau-
rent, M. N., Watabe, T., Blumberg, B. W., and Cho, K. W. Y.
(1995). Disruption of BMP signals in embryonic Xenopus ecto-
derm leads to direct neural induction. Genes Dev. 9, 29232935.
Heldin, C. H., Miyazono, K., and ten-Dijke, P. (1997). TGF-beta
signalling from cell membrane to nucleus through SMAD pro-
teins. Nature 390, 465 471.
Hemmati-Brivanlou, A., and Thomsen, G. H. (1995). Ventral me-
soderm patterning in Xenopus embryos: Expression patterns and
activities of BMP-2 and BMP-4. Dev. Genet. 17, 78 89.
Henningfeld, K. A., Rastegar, S., Adler, G., and Knochel, W. (2000).
Smad1 and Smad4 are components of the BMP-4 induced tran-
scription complex of the Xvent-2B promoter. J. Biol. Chem. 275,
2182721835.
Hild, M., Dick, A., Rauch, G. J., Meier, A., Bouwmeester, T.,
Haffter, P., and Hammerschmidt, M. (1999). The smad5 muta-
tion somitabun blocks BMP2b signaling during early dorsoven-
tral patterning of the zebrafish embryo. Development 126, 2149
2159.
Hogan, B. L. M. (1996). Bone morphogenetic proteins: Multifunc-
tional regulators of vertebrate development. Genes Dev. 10,
1580 1594.
Hollenberg, S. M., Cheng, P. F., and Weintraub, H. (1993). Use of a
conditional MyoD transcription factor in studies of MyoD trans-
activation and muscle determination. Proc. Natl. Acad. Sci. USA
90, 8028 8032.
Hsu, D. R., Economides, A. N., Wang, X., Eimon, P. M., and
Harland, R. M. (1998). The Xenopus dorsalizing factor Gremlin
identifies a novel family of secreted proteins that antagonize
BMP activities. Mol. Cell. 1, 673 683.
Jones, C. M., Lyons, K. M., Lapan, P. M., Wright, C. V. E., and
Hogan, B. L. M. (1992). DVR-4 (bone morphogenetic protein-4) as
a posterior-ventralising factor in Xenopus mesoderm induction.
Development 115, 639 647.
Kao, K. R., and Elinson, R. P. (1988). The entire mesodermal mantle
behaves as Spemanns organizer in dorsoanterior enhanced Xe-
nopus laevis embryos. Dev. Biol. 127, 64 77.
Kintner, C. R., and Brockes, J. P. (1984). Monoclonal antibodies
identify blastemal cells derived from dedifferentiating limb re-
generation. Nature 308, 67 69.
Kishimoto, Y., Lee, K. H., Zon, L., Hammerschmidt, M., and
Schulte-Merker, S. (1997). The molecular nature of zebrafish
swirl: BMP2 function is essential during early dorsoventral
patterning. Development 124, 4457 4466.
Kolm, P. J., and Sive, H. L. (1995). Efficient hormone-inducible
protein function in Xenopus laevis. Dev. Biol. 171, 267272.
314
Kopan, R., Schroeter, E. H., Weintraub, H., and Nye, J. S. (1996).
Signal transduction by activated mNotch: Importance of proteo-
lytic processing and its regulation by the extracellular domain.
Proc. Natl. Acad. Sci. USA 93, 16831688.
Nieuwkoop, P. D., and Faber, J. (1967). Normal Table of Xenopus
laevis (Daudin). North-Holland, Amsterdam.
Nishimatsu, S., and Thomsen, G. H. (1998). Ventral mesoderm
induction and patterning by bone morphogenetic protein het-
erodimers in Xenopus embryos. Mech. Dev. 74, 75 88.
Piccolo, S., Agius, E., Lu, B., Goodman, S., Dale, L., and De
Robertis, E. M. (1997). Cleavage of Chordin by Xolloid metallo-
protease suggests a role for proteolytic processing in the regula-
tion of Spemann organizer activity. Cell 91, 407 416.
Piccolo, S., Sasai, Y., Lu, B., and De Robertis, E. M. (1996).
Dorsal-ventral patterning in Xenopus: Inhibition of ventral sig-
nals by direct binding of chordin to BMP-4. Cell 86, 589 598.
Rupp, R. A., and Weintraub, H. (1991). Ubiquitous MyoD transcrip-
tion at the midblastula transition precedes induction-dependent
MyoD expression in presumptive mesoderm of X. laevis. Cell 65,
927937.
Copyright 2001 by Academic Press. All rights of reproduction in any form reserved.
Beck, Whitman, and Slack
Tucker, A. S., and Slack, J. M. (1995). Tail bud determination in the
vertebrate embryo. Curr. Biol. 5, 807 813.
Webster, N. J., Green, S., Jin, J. R., and Chambon, P. (1988). The
hormone-binding domains of the estrogen and glucocorticoid
receptors contain an inducible transcription activation function.
Cell 54, 199 207.
Winnier, G., Blessing, M., Labosky, P. A., and Hogan, B. L. (1995).
Bone morphogenetic protein-4 is required for mesoderm forma-
tion and patterning in the mouse. Genes Dev. 9, 21052116.
Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L. M., Whitters,
M. J., Kriz, R. W., Hewick, R. M., and Wang, E. A. (1988). Novel
regulators of bone formation: Molecular clones and activities.
Science 242, 1528 1534.
Yang, X., Castilla, L. H., Xu, X., Li, C., Gotay, J., Weinstein, M., Liu,
P. P., and Deng, C. X. (1999). Angiogenesis defects and mesen-
chymal apoptosis in mice lacking SMAD5. Development 126,
15711580.
Received for publication June 13, 2001
Accepted July 17, 2001
Published online September 13, 2001