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The biological effects of endogenous bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta family, on embryonic development of Xenopus laevis were investigated by using a functionally defective mutant of the BMP-4 receptor (delta mTFR11), which blocks the BMP signaling pathway. Injection of delta mTFR11 RNA into either the animal pole area or ventral marginal cells at the two-cell stage induced a dorsal phenotype in the explant of ventral mesoderm with animal poletissue from stage 10+ embryo, even though the normal fate of this explant is a "mesenchymal ball" containing blood cells. These explants with the dorsal phenotype contained muscle, neural tissue, eye capsule, and cement gland. Northern blot analysis showed an increase of cardiac alpha-actin mRNA and a decrease of T alpha-globin mRNA expression, providing further evidence of a conversion from ventral to dorsal phenotype. Although injection of delta mTFR11 RNA did not induce mesoderm in an animal cap culture, the same tissue injected with delta mTFR11 RNA can alter the differentiation fate of uninjected ventral mesodermal explant from ventral to dorsal type, suggesting specific interaction of animal poletissue and prospective ventral mesoderm in vivo.
FIG. 1. Morphological observation of the 3-day explants of
ventral mesoderm (VM) with animal pole (AP) tissue injected with
H20 (A) or AmTFR11 RNA (B-D). In A-C, H20 (A) or 5 ng of
A&mTFR11 RNA (B and C) was injected into the animal pole area at
the two-cell stage, and the resulting animal pole tissue at stage 10+
was excised together with ventral mesoderm (B) or combined with
ventral mesoderm from a different embryo (C). In D, 5 ng of
AmTFR11 RNA was injected into ventral mesoderm, and the resulting
ventral mesoderm at stage 10+ was excised together with animal
pole tissue. Injection of AmTFR11 RNA induced a tight structure,
similar to a typical dorsal mesoderm explant. Note the obvious
eye-like capsule and cement gland that were found in the explants
injected with AmTFR11 RNA into the animal pole area (B and C).
FIG. 2. Histological observation of the 3-day explants of ventralmesoderm with animal pole tissue after injection of AmTFR11 RNA (A-C)
or H20 (D). Five nanograms of AmTFR11 RNA was injected into the animal pole area (A and B) at the two-cell stage or into the ventral marginal
zone (C) at the four-cell stage, and the resulting tissue at stage 10+ was excised with ventralmesoderm (A and B) or with animal pole tissue
(C), respectively. Neural tissues (n) and muscle (m) were differentiated in the explants injected with AmTFR11 (A-C). However, notochord
(nc) was only seen in the explants injected with AmTFR11 RNA into ventralmesoderm (C). In contrast, a typical ventralmesoderm structure
containing blood cells (b) was developed in the explants injected with H20 (D, control). A and B were different sections of the embryos. (E and
F) Sections of explant in a lineage-tracing experiment using P-galactosidase as a marker. One-half nanogram of capped RNA coding
P-galactosidase was injected into the ventral marginal zone at the four-cell stage, and the resulting ventralmesoderm at stage 10+ was combined
with animal pole tissue that was previously injected with 5 ng of AmTFR11 RNA (E) or was combined with animal pole tissue without injection
of AmTFR11 (F). Explants were cultured for 3 days, fixed, processed for 5-bromo-4chloro-3-indolyl ,3-D-galactoside staining, and sectioned.
Note that ventralmesoderm-derived cells, which express ,-galactosidase, differentiated to muscletissue by the injection with AmTFR11 RNA
into the animal pole area (E). In the control without injection of AmTFR11 RNA, these cells differentiated to blood cells and mesenchymal cells
(F). (Bars in A and E = 50 uan; same magnification in A-D and E and F, respectively.)
FIG. 3. Morphology (A) and Northern blot analysis (B) of the
explants injected with increasing doses of AmTFR11 RNA. Expression
of cardiac a-actin, Ta-globin, and EF-la mRNAs was determined
by Northern blot analysis. The embryo was injected with
AmTFR11 RNA into the animal pole area at the two-cell stage, and
the resulting tissue at stage 10+ was excised together with ventral
mesoderm and cultured for 3 days. Various concentrations of
AmTFR11 were used: sample 1, H20 injection; sample 2, 0.04 ng of
RNA; sample 3, 0.2 ng of RNA; sample 4, 1 ng of RNA; sample 5,
5 ng of RNA; sample 6, dorsal mesoderm and animal pole tissue
cultured as a control.
FIG. 4. Effect of activin and AmTFR11 RNA injection on the
expression of cardiac a-actin and Ta-globin mRNA expression.
Northern blot analysis was performed to quantitate the expression of
Ta globin, cardiac a-actin, and EF-la mRNAs. In lanes 1-4, RNA
was injected into the animal pole area at the two-cell stage, and the
resulting tissue at stage 10+ was excised together with ventral
mesoderm and cultured for 3 days. Lane 1, no injection; lane 2, 5 ng
of AmTFR11 RNA; lane 3, 0.5 ng of activin (8A RNA; lane 4, dorsal
mesoderm with animal pole tissue cultured as a control. In lanes 5-8,
RNA was injected into the animal pole area at the two-cell stage, and
the animal cap at stage 7 was cultured for 3 days. Lane 5, no injection;
lane 6,0.5 ng of activin PA RNA; lane 7,5 ng ofAmTFR11 RNA; lane
8, 5 ng ofXenopus BMP-4 RNA. Dorsal mesoderm with animal pole
tissue (lane 9) or ventral mesoderm with animal pole tissue (lane 10)
was cultured for 3 days as a control. Note that activin and AmTFR11
RNA can dorsalize the explants of ventral mesoderm with animal
pole tissue (lanes 2 and 3), but only activin can induce mesoderm in
the animal cap explant (lane 6).
FIG. 5. Hypothetical roles of BMP on the ventralzing and
dorsalizing activities. In the normal embryo without AmTFR11 RNA
injection (A), endogenous BMP signaling stimulates and enhances
ventralizing activity (VA) and ventralizes (V) the adjacent prospective
ventral mesoderm (VM). At the same time, the dorsalizing
activity (DA) is inhibited by BMP signal(s). In the embryo injected
with AmTFR11 RNA (B), the disruption of BMP signaling in the
animal pole region (AP) relieves the inhibition of dorsalizing activity
of BMP and turns on the dorsalizing activity and dorsalizes (D) the
adjacent prospective ventral mesoderm. Plus represents stimulatory
activity and minus represents inhibitory activity. A solid line indicates
that the signals are transmitted, and a dotted line indicates that
the signals are inhibited or abrogated.
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