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Recent studies on Xenopus development have revealed an increasingly complex array of inductive, prepatterning, and competence signals that are necessary for proper mesoderm formation. In this study, we establish that fibroblast growth factor (FGF) signals through mitogen-activated protein kinase kinase (MAPKK) to induce mesodermal gene expression. We demonstrate that a partially activated form of MAPKK restores expression of the mesodermal genes Xcad-3 and Xbra, eliminated by the dominant-negative FGF receptor (delta FGFR). Similar to the results reported earlier with delta FGFR, expression of a dominant-negative form of MAPKK (MAPKKD) preferentially eliminates the dorsal expression of Xcad-3 and Xbra. We tested whether the regional localization of bone morphogenetic protein-4 (BMP-4) could explain why both MAPKKD and delta FGFR eliminate the dorsal and not the ventral expression of Xcad-3 and Xbra. We show that ectopic expression of BMP-4 is sufficient to maintain the dorsal expression of Xcad-3 and Xbra in embryos containing delta FGFR and that expression of a dominant-negative BMP receptor reduces the dorsal-ventral differences in delta FGFR embryos. These results indicate that regional localization of BMP-4 is responsible for the dorsal-ventral asymmetry in FGF/MAPKK-mediated mesoderm induction.
FIG. 1. Site of MAPKKmutations. Illustrated is a schematic representation of MAPKK indicating the site of the serine residue at position 222 which has beenmutated to glutamic acid forMAPKK* and to alanine for MAPKKD.
FIG. 2. Injection of RNA encoding MAPKK* expands the ventral expression patterns of Xcad-3 and Xbra in gastrula stage embryos.
Embryos at the 2- to 4-cell stage were injected in both the dorsal and ventral marginal zones with RNA encoding MAPKK* and stained
for Xcad-3 (A and B) and Xbra (A* and B*) expression at the gastrula stage by in situ hybridization. (A and A*) Uninjected embryos (B and
B*), embryos injected with 5 ng RNA encoding MAPKK*. The embryos are oriented with dorsal toward the top of the page and anterior
toward the left.
FIG. 3. RNA encoding MAPKK* injected in both the dorsal and ventral marginal zones can rescue expression of Xcad-3 and Xbra in
embryos co-injected with DFGFR. Uninjected embryos (A and A*), embryos injected with 5 ng RNA encoding MAPKK* (B and B*), embryos
injected with 1 ng RNA encoding DFGFR (C and C*), and embryos co-injected with 5 ng RNA encoding MAPKK* and 1 ng RNA encoding
DFGFR (D and D*) were fixed at the gastrula stage and stained for Xcad-3 (A–D) and Xbra (A*–D*) expression by in situ hybridization.
Vegetal view, with the dorsal side oriented toward the top of the page. Note that the ventral expansion in embryos injected with MAPKK*
is difficult to see in a vegetal view and is frequently less pronounced in embryos stained for Xbra than for Xcad-3 (see text).
FIG. 4. Injection in both the dorsal and ventral marginal zones with RNA encoding MAPKKD disrupts expression of Xcad-3 and Xbra
in a dorsal– ventral-dependent manner, an effect which is blocked by coexpression of MAPKK*. Uninjected embryos (A and A*), embryos
injected with 2 ng RNA encoding MAPKK* (B and B*), embryos injected with 5 ng RNA encoding MAPKKD (C and C*), and embryos coinjected
with 2 ng RNA encoding MAPKK* and 5 ng RNA encoding MAPKKD (D, D*) were fixed at the gastrula stage and stained for
Xcad-3 (A–D) and Xbra (A*–D*) expression by in situ hybridization. Vegetal view, with the dorsal side oriented toward the top of the
page.
FIG. 5. BMP-4 rescues the dorsal expression of Xcad-3 and Xbra eliminated in DFGFR-injected embryos when both are co-injected into
the dorsal and ventral marginal zones. Uninjected embryos (A and A*), embryos injected with 1 ng RNA encoding BMP-4 (B and B*),
embryos injected with 1 ng RNA encoding DFGFR (C and C*), and embryos co-injected with 1 ng RNA encoding BMP-4 and 1 ng RNA
encoding DFGFR (D and D*) were fixed at the gastrula stage and stained for Xcad-3 (A –D) and Xbra (A*–D*) expression by in situ
hybridization. Vegetal view, with the dorsal side oriented toward the top of the page. Note that BMP-4 caused an expansion of Xcad-3
and Xbra expression in the dorsal and ventral animal hemispheres in these embryos which are not observed in this orientation
(B and B*).
FIG. 6. RNA encoding DXBMPR injected in both the dorsal and ventral marginal zones eliminates the dorsal–ventral gradient of
sensitivity to DFGFR. Uninjected embryos (A and A*), embryos injected with 1 ng RNA encoding DXBMPR (B and B*), embryos injected
with 1 ng RNA encoding DFGFR (C and C*), and embryos co-injected with 1 ng RNA encoding DXBMPR and 1 ng RNA encoding DFGFR
(D and D*) were fixed at the gastrula stage and stained for Xcad-3 (A–D) and Xbra (A*–D*) expression by in situ hybridization. Vegetal
view, with the dorsal side oriented toward the top of the page.