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The TGF-beta superfamily of growth factors is known to transmit signals to the nucleus mainly through the Smads, intracellular signaling components that are highly conserved from nematodes to humans. The signaling activity of the Smads is regulated by their ligand-stimulated phosphorylation through Ser/Thr kinase receptors. Here, to examine the in vivo role of BMP, we investigated the spatio-temporal activation of BMP-regulated signals during Xenopus development, using a polyclonal antibody that specifically recognizes the phosphorylated form of BMP-regulated Smads. BMP signaling was observed uniformly in embryos as early as stage 7, but was restricted to the ventral side of the embryo at the late blastula stage, supporting the proposed role of BMP4 as a ventralizing factor in Xenopus embryos. In addition, localized staining was detected in several developing organs, consistent with the predicted function of BMP family members in organogenesis.
Fig. 1 Specificity of PS1 antibody. Specificity
of PS1 antibody was analysed by Western
blotting and immunohistochemistry. A Embryos
were injected with mRNAs at the 2-
cell stage into the animal pole, and the animal
caps at stage 8.5 were analyzed by Western
blotting with PS1 antibody. Uninjected
embryos (Un), embryos injected with 50pg of
BMP4 mRNA (BMP4), co-injected with
50pg of BMP4 and 500pg of DN-BMPR IA
(BMP4 Ï DN-BMPR IA). Blots were reprobed
with anti-actin antibody as internal
control. B, C Embryos were stained with unimmunized
control serum (B) or PS1 antibody
(C) at stage 9. DâJ Embryos were injected
with mRNAs at the 2-cell stage into the
animal pole, and stained with PS1 antibody
at stage 8.5. The animal hemisphere of control
(uninjected) embryo (D), injected with
50pg of BMP4 mRNA (E), 50 pg of BMP2
mRNA (F), 200 pg of BMP7 mRNA (G), 50
pg of Activin b B mRNA (H), 500 pg of
DN-BMPR IA (I), and coinjected with 50 pg
of BMP4 mRNA and 500 pg of DN-BMPR
IA (J).
Fig. 2 PS1 staining pattern in blastula and
gastrula. Xenopus blastula- and gastrula-stage
embryos were stained with PS1 antibody. A,
B Animal (A) and lateral (B) view of wholemount-
stained and cleared embryos at stage
7. C Horizontally sectioned surface through
the equator at stage 8.5. DâH Sectioned embryos
along D-V axis at stage 9 (D), stage 10
(F), stage 11 (G) and stage 12.5 (H). E Horizontally
sectioned stage-9 embryo through
the equatorial region. Arrowhead in F indicates
dorsal blastopore lip. Arrows in G, H
indicate graded PS1 staining. (D, dorsal side;
V, ventral side; St., stage)
Fig. 3 PS1 staining pattern in neurula. Xenopus
neurula-stage embryos were stained
with PS1 antibody. A, B Anterior (A) and
posterior (B) view of whole-mount- stained
stage-14 embryos. Arrows indicate intensive
staining at the presumptive cement gland region
(A) and around the ventral blastopore
(B). C Anterior view of whole-mountstained
stage-17 embryo. Arrow indicates intensive
staining at the presumptive cement
gland region. Arrowheads indicate staining
at the eye primordia. D Transverse section
of the eye primordia at stage 18. FâG Transverse
sections of stage-13 (E), stage-15 (F),
and stage-20 (G) embryos. Arrowheads in F
indicate PS1 positive sensorial layer beneath
neural fold. Arrowheads in G indicate PS1
positive cells in dorsal part of forming neural
tube and neural crest. (no, notochord; St.,
stage)
Fig. 4 PS1 staining pattern in tail-bud-stage
embryos. Xenopus tail-bud-stage embryos
were stained with PS1 antibody. AâC Wholemount-
stained embryos at stage 25 (A) and
at stage 35 (B, C). D Transverse section
through the head region (indicated by d in A)
at stage 25. Arrowheads indicate the PS1
positive dorsal region of optic vesicle. E
Transverse section through the pharynx anlage
(indicated by e in A) at stage 25. F
Transverse section through the spinal cord at
stage 25. G Transverse section through the
visceral arches (indicated by g in B) at stage
35. H-J Transverse sections through the heart
region (H), the eye (I), and the otic vesicle
(J) of stage-35 embryos. (cg, cement grand;
ec, endocardial cell layer; fg, frontal grand;
lp, loof plate; lv, lens vesicle; no, notocord;
ov, optic vesicle; ph, pharynx anlage; va, visceral
arches; vm, ventral mesoderm; St.,
stage)
