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???displayArticle.abstract??? Bone morphogenetic proteins (BMPs) participate in the development of nearly all organs and tissues. BMP signaling is mediated by specific Smad proteins, Smad1 and/or Smad5, which undergo serine phosphorylation in response to BMP-receptor activation and are then translocated to the nucleus where they modulate transcription of target genes. We have identified a distantly related member of the Xenopus Smad family, Smad8, which lacks the C-terminal SSXS phosphorylation motif present in other Smads, and which appears to function in the BMP signaling pathway. During embryonic development, the spatial pattern of expression of Smad8 mirrors that of BMP-4. We show that an intact BMP signaling pathway is required for its expression. Overexpression of Smad8 in Xenopus embryos phenocopies the effect of blocking BMP-4 signaling, leading to induction of a secondary axis on the ventral side of intact embryos and to direct neural induction in ectodermal explants. Furthermore, Smad8 can block BMP-4-mediated induction of ventralmesoderm-specific gene expression in ectodermal explants. Overexpression of Smad8 within dorsal cells, however, causes patterning defects that are distinct from those reported in BMP-4-deficient embryos, suggesting that Smad8 may interact with additional signaling pathways. Indeed, overexpression of Smad8 blocks expression of Xbra in whole animals, and partially blocks activin signaling in animal caps. In addition, Smad8 inhibits involution of mesodermal cells during gastrulation, a phenotype that is not observed following blockade of activin or BMPs in Xenopus. Together, these results are consistent with the hypothesis that Smad8 participates in a negative feedback loop in which BMP signaling induces the expression of Smad8, which then functions to negatively modulate the amplitude or duration of signaling downstream of BMPs and, possibly, downstream of other transforming growth factor-beta (TGF-beta) family ligands.
Fig. 1. Comparison of deduced amino acid sequences of selected Smads. The complete amino acid sequences of Xenopus Smad (
XSmad) 1, 2
and 8, mouse Smad (mSmad) 6 and 7, and Drosophila Dad are shown aligned with each other. Amino acid identity between Smad8 and other
genes is indicated by black boxes while conservative substitutions are represented by gray boxes. Identical residues that are conserved among
Smads other than Smad8 are shown in red and conservative substitutions are indicated by light red shading. MH1 and MH2 domains are shown
by purple and green lines, respectively.
Fig. 2. Xenopus Smad8 transcripts are present throughout
development. Equivalent amounts of RNA isolated from
developmentally staged embryos, or from dissected animal (An) and
vegetal (Vg), or dorsal (D) and ventral (V) halves of 8-cell embryos
were analyzed for expression of Smad8 and fibroblast growth factor
receptor (FGFR, as a control for equal loading) in an RNase
protection assay.
Fig. 3. The spatial pattern of expression of Smad8 in
developing Xenopus embryos is similar to that of
BMP-4, as analyzed by whole-mount in situ
hybridization. (A,B) Vegetal views of gastrulae
hybridized with a Smad8 antisense riboprobe. Smad8
transcripts are uniformly distributed at the onset of
gastrulation (stage 10) (A) but become restricted to
cells on the ventral (V) side by the stage 11 (B).
Arrows indicate the border of expression between
ventral and dorsal (D) cells. (C) Lateral view of a
neurula (stage 15) stage embryo (anterior to the left).
Smad8 transcripts are ventrally restricted and
concentrated in cells near the anterior end.
(D-F) Lateral views of stage 22 (D), stage 25 (E) and
stage 27 (F) embryos (anterior to the left, ventral
down). During these stages, expression of Smad8
becomes increasingly restricted to the heart anlage
(closed arrowhead), to dorsal regions of the eye
(arrow), and to the brain (open arrowhead). (F) In
tailbud stage embryos, weak expression of Smad8 is
observed in the otic vesicle (white arrow), and discrete
bands of Smad8-expressing cells are observed in the anterior central nervous system (inset). (G) Lateral views of stage 30/31 embryos
hybridized with Smad8 (top) and BMP-4 (bottom) probes. Both genes are expressed in the eye, the otic vesicle (white arrow), the brain and the
heart anlage, while Smad8 (asterisk), but not BMP-4, is expressed in the olfactory placode. (H) Transverse section of a stage 27 embryo at the
level of the hindbrain. Expression of Smad8 is restricted to the dorsal part of the brain (open arrowhead). (I) Transverse sections of stage 34/35
embryos at the level of the forebrain. (Left) Expression of Smad8 in the dorsal forebrain (open arrowhead), eye (arrow) and heart (closed
arrowhead) is observed. (Right) Higher magnification view demonstrates expression throughout the heart. Embryos shown in (C-E,G) were
cleared in benzyl benzoate:benzyl alcohol (2:1).
Fig. 4. BMP signaling is required for Smad8 expression.
(A) Animal caps made to overexpress either BMP-4 or a truncated
BMP receptor (tBR) were isolated at the blastula stage and
cultured until stage 12, at which time expression of Smad8 and
FGFR was analyzed by RNase protection. Overexpression of
BMP-4 or blockade of BMP-4 signaling led to upregulation or
downregulation, respectively, of expression of Smad8 relative to
FGFR. (B) BMP-4 RNA was injected into the animal pole of 2-
cell embryos and expression of endogenous Smad8 was analyzed
by whole-mount in situ hybridization at stage 12. BMP-4 RNAinjected
embryos showed localized upregulation of Smad8
expression (arrowhead) relative to uninjected (Cont) embryos.
(C) Dorsal or ventral marginal zone cells (DMZ or VMZ) made to
overexpress BMP-4 or tBR, respectively, were isolated at stage 10
and cultured until stage 12, at which time expression of Smad8 and
EF-1a was analyzed by RT-PCR. Signal was not observed in the
absence of reverse transcriptase (RT-). Smad8 transcripts are
much more abundant in VMZ than in DMZ explants isolated from
control (Cont) embryos. Injection of BMP-4 (BMP) RNA
upregulated Smad8 expression in DMZ explants, while blockade
of BMP activity (tBR) downregulated Smad8 expression in VMZ
explants.
Fig. 5. Smad8 antagonizes BMP signaling in isolated animal caps. (A-C) Animal
caps were isolated at the blastula stage from control embryos, or from embryos
that had been injected with Smad8 RNA at the 2-cell stage, and were cultured
until sibling embryos reached stage 26. Control caps (A) differentiated into
epidermis, while Smad8-injected caps (B) formed cement glands (arrowheads).
(C) Smad8-injected, but not control explants expressed cement gland-specific
(XAG) and neural-specific (N-CAM, OtxA) genes but failed to express the dorsal
mesodermal marker a-actin (a-Act) as analyzed by RT-PCR. Signal was not
observed in the absence of reverse transcriptase (RT-). (D) Smad8 and BMP-4
RNAs were injected alone, or together, near the animal pole of 2-cell embryos
and animal caps were explanted at the blastula stage. Expression of Xwnt-8, Xbra
and EF-1a was analyzed by RT-PCR in the presence (+) and absence (-) of
reverse transcriptase (RT) when explants reached control stage 12. Co-expression
of Smad8 blocked BMP-4-mediated induction of the ventral mesodermal marker
(Xwnt-8) and pan-mesodermal marker (Xbra) in isolated animal caps.
Fig. 6. Smad8 induces a partial secondary dorsal axis when
overexpressed on the ventral side of the embryo. Tadpole stage
control embryos (A,C,E) and sibling embryos that had been injected
ventrally with Smad8 RNA at the 4-cell stage (B,D,F) are shown.
Smad8 induces the formation of a partial secondary axis (arrows,
B,D,F) that often contains a cyclopic eye (B). Immunostaining with
Tor70 antibodies reveals the presence of notochord in control
embryos (large arrowhead, C) and in the primary axis (large
arrowhead, D) but not in the secondary axis (closed arrowhead, D) of
Smad-injected embryos. In contrast, an otic vesicle is detected in
both primary (small open arrowheads, C,D) and in secondary (closed
arrowhead, D) axes. Immunostaining with a muscle-specific antibody
(E,F) reveals the presence of disorganized muscle in the secondary
axis of some Smad8-injected embryos.
Fig. 7. Ectopic expression of Smad8 in dorsal cells of
Xenopus embryos causes spina bifida and eye defects.
Smad8 RNA was injected into the dorsal marginal zone of
4-cell embryos, which were then cultured to the tadpole
stage. Embryos developed with spina bifida along with
variable eye defects (A, arrows) ranging from complete loss
of eyes (top left, dorsoanterior view, anterior to the left) to
formation of a single fused (top right, dorsoanterior view,
anterior down) or cyclopic eye (bottom right, head-on
view). (B) Immunostaining with notochord-specific (B,
dorsal view) or muscle-specific (C, dorsolateral view)
antibodies revealed that notochord and muscle were intact
in Smad8-injected embryos, but were split along either side
of the cleft caused by the defect in neural tube closure.
Fig. 8. Smad8 blocks Xbra expression in embryos and partially
antagonizes activin signaling in animal caps. (A) Smad8 or Mycepitope
tag mRNA (200 pg) was injected into one blastomere of 2-
cell embryos near the equator and the embryos were cultured until
stage 10.5. Xbra expression was analyzed by whole-mount in situ
hybridization. Arrowheads indicate loss of Xbra expression in
Smad8-injected embryos. (B) Smad7 or Smad8 mRNA (400 pg) was
injected into the animal pole of 2-cell embryos. Animal caps were
isolated at the blastula stage and cultured in the presence or absence
of human activin A (20 ng/ml) until sibling embryos reached stage
18, at which time expression of Xbra, a-actin and EF1-a was
analyzed by RT-PCR.
Fig. 9. Overexpression of Smad8 inhibits morphogenetic movements during gastrulation and causes a
dorsoanterior relocation of ventral cells. nb-gal mRNA (100 pg) was injected with (B,D,F,H,J,L) or
without (A,C,E,G,I,K) Smad8 mRNA (200 pg) into the dorsal (A,B,E,F,I,J) or ventral (C,D,G,H,K,L)
marginal zone of 4-cell embryos. (A-D) Localization of nb-gal-expressing cells (stained in light blue) in
tadpole-stage embryos. (E-H) Vegetal views of localization of nb-gal-expressing cells during gastrulation
(stage 12). Dorsal side is up. (I-L) Sagittal sections of embryos shown in E-H. Black arrowheads indicate
involution of dorsal cells, white arrowheads denote the position of the ventral lip of the blastopore. Ventral
is to the left and the animal pole is at the top in each panel.
