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Involvement of Ras/Raf/AP-1 in BMP-4 signaling during Xenopus embryonic development.
Xu RH, Dong Z, Maeno M, Kim J, Suzuki A, Ueno N, Sredni D, Colburn NH, Kung HF.
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Previously, we elucidated the role of bone morphogenetic protein 4 (BMP-4) in the dorsal-ventral patterning of the Xenopus embryo by using a dominant negative mutant of the BMP-4 receptor (DN-BR). The present paper describes the involvement of Ras, Raf, and activator protein 1 (AP-1) in BMP-4 signaling during Xenopus embryonic development. The AP-1 activity was determined by injecting an AP-1-dependent luciferase reporter gene into two-cell-stage Xenopus embryos and measuring the luciferase activity at various developmental stages. We found that injection of BMP-4 mRNA increased AP-1 activity, whereas injection of DN-BR mRNA inhibited AP-1 activity. Similar inhibitory effects were seen with injection of mRNAs encoding dominant negative mutants of c-Ha-Ras, c-Raf, or c-Jun. These results suggest that the endogenous AP-1 activity is regulated by BMP-4/Ras/Raf/Jun signals. We next investigated the effects of Ras/Raf/AP-1 signals on the biological functions of BMP-4. DN-BR-induced dorsalization of the embryo, revealed by the formation of a secondary body axis or dorsalization of the ventral mesoderm explant analyzed by histological and molecular criteria, was significantly reversed by coinjection of [Val12]Ha-Ras, c-Raf, or c-Jun mRNA. Furthermore, the BMP-4-stimulated erythroid differentiation in the ventral mesoderm was substantially inhibited by coinjection with the dominant negative c-Ha-Ras, c-Raf, or c-Jun mutant. Our results suggest the involvement of Ras/Raf/AP-1 in the BMP-4 signaling pathway.
FIG. 1. Effects on AP-1 activity by BMP-4 signals in the Xenopus
embryo. (AP-1)4-Luc plasmid DNA was injected into the two blastomeres
of two-cell-stage embryos at 50 pg per embryo with mRNAs
encoding BMP-4 (1 ng), DN-BR (1 ng), DN-BR (1 ng) plus c-Jun (2
ng), or ,B-galactosidase (3 ng) as a control. After injection, five embryos
per group were pooled at various developmental stages and homogenized
to prepare a cell extract. AP-1-dependent luciferase activity in
the extract was measured and expressed as integrated light units (ILU)
(25). Data are averages summarized from 5 separate experiments. *,
P < 0.05; **, P < 0.01 compared to control according to Student's t
test.
FIG. 2. Morphological and histological analysis of ventral mesoderm explant combined with animal pole tissue. Morphology (A, C, and E) and
histology (B, D, and F) were analyzed on the explants from embryos injected with 1 ng of mRNA encoding 13-galactosidase (A and B), DN-BR
alone (C and D), or DN-BR coinjected with 2 ng of [Vall2]Ha-Ras mRNA (E and F). These synthetic mRNAs were 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. Explants were cultured
for 3 days and then photographed. Some explants were fixed and sectioned for hemotoxylin/eosin staining. Injection of DN-BR mRNA induced
a dorsal-like phenotype including an eye-like capsule (e) and a cement gland (cg) (C) and notochord (nc), muscle (m), and neural tissue (n) (D).
Coinjection of DN-BR mRNA with [Val12]Ha-Ras mRNA reversed these phenotypic changes, showing a typical ventral phenotype without an
eye-like capsule or a cement gland (E); histologically, these explants contained blood cells (b) without the appearance of muscle, neural tissue, and
notochord (F). The reversed phenotypes were similar to those of the control ,B-galactosidase mRNA-injected explants (A and B). [Bar = 156 ,um
(A, C, E) or 50 ,uM (B, D, F).]
FIG. 3. Effects of [Va112]Ha-Ras, c-Raf, and c-Jun on DN-BR
inhibition of erythroid differentiation as measured by Ta-globin
expression. One nanogram of DN-BR mRNA was injected alone or
with 2 ng of WT-BR [Valt2]Ha-Ras, c-Raf, or c-Jun mRNA into the
animal pole area at the two-cell stage. Three nanograms of 13-galactosidase
mRNA was injected as a control. The resulting animal pole
tissue with ventral mesoderm at stage 10+ was excised and cultured as
described in Fig. 2. Five explants per group were harvested and
processed by Western blot analysis with monoclonal antibody L5.41,
which recognizes larval Ta-globin (28).
FIG. 4. BMP-4-stimulated erythroid differentiation is inhibited by
DN-Ras, DN-Raf, and DN-Jun. One nanogram of BMP-4 mRNAwas
injected alone or with 2 ng of DN-Ras, DN-Raf, or DN-Jun mRNA
into the animal pole area at the two-cell stage. Three nanograms of
,B-galactosidase mRNA was injected as a control. The resulting animal
pole tissue at stage 7 was excised and sandwiched with ventral
mesoderm at stage 10+. The explants were cultured and analyzed for
Ta-globin expression as described in Fig. 3.
FIG. 5. Proposed model for BMP-4 signaling pathway. , Stimulation
or activation; - - -, inhibition.
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