Proc Natl Acad Sci U S A
October 25, 1994;
A truncated bone morphogenetic protein receptor affects dorsal-ventral patterning in the early Xenopus embryo.
morphogenetic proteins (BMPs), which are members of the transforming growth factor beta (TGF-beta
) superfamily, have been implicated in bone
formation and the regulation of early development. To better understand the roles of BMPs in Xenopus laevis embryogenesis, we have cloned a cDNA coding for a serine/threonine kinase receptor that binds BMP-2
. To analyze its function, we attempted to block the BMP signaling pathway in Xenopus embryos by using a dominant-negative mutant of the BMP receptor. When the mutant receptor lacking the putative serine/threonine kinase domain was expressed in ventral
blastomeres of Xenopus embryos, these blastomeres were respecified to dorsal mesoderm
, eventually resulting in the formation of a secondary body axis. These findings suggest that endogenous BMP-2
are involved in the dorsal-ventral
specification in the embryo
and that ventral
fate requires induction rather than resulting from an absence of dorsal specification.
Proc Natl Acad Sci U S A
[+] show captions
FIG. 1. Amino acid sequence and homology of mTFR11. (A)
Translated amino acid sequence of the mTFR11 cDNA. The signal
peptide and transmembrane domains are indicated by a single
underline. Potential sites of N-linked glycosylation are indicated by
asterisks. The ends of the kinase domain are indicated by arrows.
Ten of the conserved cysteine residues in the extracellular domain
are in reverse print. Type I box is indicated by a double underline.
An arrowhead is under the tyrosine residue that changes to stop
codon in the dominant negative BMP receptor. (B) Homology of the
mTFR11/hALK-3 to other receptors for ligands of the TGF-(3
superfamily. Percent amino acid identity is indicated. Light-shaded
boxes, extracellular domain; black boxes, transmembrane domain;
dark-shaded boxes, type I box; hatched boxes, intracellular kinase
domain. ALK-2, -3, -5, and -6, activin receptor-like kinases 2, 3, 5,
and 6(26); xTFR11, Xenopus TFR11 (accession no. D32066); mTGFf3RI,
mouse TGF-(3 type I receptor (27); mActRI, mouse activin type
I receptor (28); mActRII, mouse activin type II receptor (13);
mTGF-fBRII, mouse TGF-f type II receptor (29);daf4, C. elegans
daf-4 gene product (30).
FIG. 2. Specific binding of iodinated BMP-4 to COS cells transfected with mTFR11. (A) 'MI-BMP4 binding to COS cells transfected with
mTFR11. Binding was performed on cell monolayers as described below and was competitively inhibited with unlabeled BMP-4. (Inset) Scatchard
analysis. o, pMV2; *, mTfr11/pMV2. (B) Specificity of mI-BMPA binding to COS cells transfected with mTFR11. Binding of 125I-BMP4 to
COS cells transfected with mTFR11 was performed with or without increasing concentrations ofunlabeled ligands. *, BMP-2; o, BMP-4;m, activin
A; *, TGF-.l1. (C) Chemical cross-linking ofiodinated BMP-4 to COS cells transfected with mTFR11/pMV2 orpMV2. COS cells were transfected
with either pMV2 (lane 1) or mTFR11/pMV2 (lanes 2-5) and incubated with 5 ng of 125I-BMP-4 per ml (all lanes) and 500 ng ofBMP-4 per ml (lane
3), 500 ng of BMP-2 per ml (lane 4), or 500 ng of activin per ml (lane 5). Molecular masses are indicated in kDa.
FIG. 3. Truncated BMP receptor inhibits BMP signaling pathway
in early Xenopus embryo. At the four-cell stage, embryos were
injected with the indicated samples into the dorsal marginal region
and allowed to develop until stage 37 or 38. (A) Distilled H20. (B)
BMP-4 mRNA, 200 pg. (C) AmnTFR11 mRNA, 200 pg. (D) BMP-4
plus AmTFR11 mRNA, 200 pg of each.
FIG. 4. Inhibition of endogenous BMP signaling pathway in the
ventral part of the embryo induces a secondary body axis.- Embryos
were injected with anti-sense &mTFR11 (A) and sense AmTFR11
(B-F) mRNA into the magnal region of the ventral two blastomeres
at the four-cell stage. Neurula embryos (A and B) show the formation
of two neural tubes in the embryo injected with sense AmTFR11
mRNA. Tadpole stage embryo (C) and D show the secondary axial
structures (n, neural tube; m, muscle); primary (10) and secondary
(20) dorsal axes are indicated. Whole-mount in situ hybridization
analysis of duplicate embryos shows induction of neural celladhesion
molecule (E) and a-actin (F) mRNA in primary (10) and
secondary (20) body axes.
FIG. 5. Effect of inhibition of BMP signaling pathway on expression
of early mesodermal markers. AmTFR11 mRNA was injected
into the marginal region of ventral blastomeres at the four-cell stage.
Uninjected control embryo (A, C, and E), and AmTFR11-injected
embryos (B, D, and F) were developed until early gastrula stage and
subjected to whole-mount in situ analysis. The probe for goosecoid
was used in A and B; the probe for Xpo was used in C and D; the
probe for Xenopus brachyury was used in E and F. Views of the
vegetal pole are shown and dorsal is up. Note that the expression of
Xpo gene disappeared in the ventral part of embryo that received
truncated BMP receptor mRNA (arrowheads) but that of brachyury
gene, normally expressed through mesoderm, was not affected.