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We have examined the role of FGF signalling in the development of muscle and notochord and in the expression of early mesodermal markers in Xenopus embryos. Disruption of the FGF signalling pathway by expression of a dominant negative construct of the FGF receptor (XFD) generally results in gastrulation defects that are later evident in the formation of the trunk and tail, though head structures are formed nearly normally. These defects are reflected in the loss of notochord and muscle. Even in embryos that show mild defects and gastrulate properly, muscle formation is impaired, suggesting that morphogenesis and tissue differentiation each depend on FGF. The XFD protein inhibits the expression of the immediate early gene brachyury throughout the marginal zone, including the dorsal side; it does not, however, inhibit the dorsal lip marker goosecoid, which is expressed in the first involuting mesoderm at the dorsal side that will underlie the head. The XFD protein also inhibits Xpo expression, an immediate early marker of ventral and lateralmesoderm. These results suggest that FGF is involved in the earliest events of most mesoderm induction that occur before gastrulation and that the early dorsal mesoderm is already composed of two cell populations that differ in their requirements for FGF.
Fig. 1. Translation of the FGF receptor constructs during early
development. (A) One blastomere of two-cell stage embryos were
injected with HAV0/ (H) or XFD (X) RNA. At stages 4, 8, 10.5,
14, 21 and 26, the glycosylated proteins from the injected and
uninjected (U) embryos were separated on an acrylamide gel,
transferred to a nylon membrane and incubated with an affinity
purified FGF receptor antibody. Molecular mass standards (´10-3)
are indicated on the left. (B, C) Two-cell stage embryos were
injected with HAV0/ RNA (B) and XFD RNA (C). At the mid
gastrula stage (st. 11) the embryos were fixed and the expressed
truncated FGF receptors were localized by immunohistochemistry
using an FGF receptor antibody. Scale bars, 60 um.
Fig. 2. Effects of overexpressing the control (d50, HAV0/) and
dominant negative FGF receptor (XFD) constructs on muscle and
notochord formation. (A) Embryos were injected at the two-cell stage
with either d50 (left) or XFD RNA (right). At the tailbud stage (st.
28), the embryos were fixed and subjected to immunocytochemistry
with the muscle-specific monoclonal antibody, 12/101 (brown stain).
(B, C) One blastomere at the two-cell stage was co-injected with
H A V0/and b-galactosidase RNA (B) or with XFD and b- g a l a c t o s i d a s e
RNA (C). At the early tailbud stage (st. 22) the embryos were fix e d ,
stained for b-galacosidase activity (blue stain) and then subjected to
whole-mount in situ hybridization with a muscle actin probe (black
stain). (D, E) The two dorsal blastomeres for four-cell stage embryos
were injected with HAV0/ (D) or XFD RNA (E). At the late tailbud
stage (st. 30) the embryos were fixed and subjected to whole-mount in
situ hybridization with a collagen type II probe which labels the
notochord (blue stain). Scale bars, 250 um .
Fig. 3. Effects of overexpressing the control (HAV0/) and dominant negative FGF receptor (XFD) constructs on brachyury expression. (A)
One blastomere at the two-cell stage was injected with either HAV0/ (left) or XFD (right) RNA. At the mid gastrula stage (st. 11), the
embryos were fixed and subjected to whole-mount in situ hybridization with a Xenopus brachyury (Xbra) probe (brown stain). After
staining for Xbra some embryos were then stained with the FGF receptor antibody. The embryo in B was injected with HAV0/ RNA and
the embryo in C was injected with XFD RNA. In these panels the stain for brachyury is blue while the FGF receptor protein stain is redbrown.
(D, E) The two dorsal blastomeres of four-cell stage embryos were co-injected with HAVø and b-galactosidase RNA (D) or XFD
and b-galactosidase RNA (E). At the mid gastrula stage (st. 11) the embryos were fixed and stained for b-galactosidase activity (blue
stain) and then subjected to in situ hybridization with the brachyury (Xbra) probe (brown stain). The embryos in D and E are oriented
with the dorsal side toward the top of the page. To emphasize the dorsal side, the embryos were not cleared and tilted ventrally, resulting
in the bleached appearance of the ventral side from reflected light. Scale bars, 250 um.
Fig. 4. Effects of overexpressing
the control (HAV0/) and dominant
negative FGF receptor (XFD)
constructs on Xpo and goosecoid
expression. One blastomere of the
two-cell stage embryo was injected
with either HAV0/ (A) or XFD (B)
RNA. At the mid gastrula stage (st.
11), the embryos were fixed and
subjected to whole-mount in situ
hybridization with an Xpo probe
(black stain). In A the dorsal side is
oriented toward the top of the page.
(C, D) Embryos were not injected
(C) or the two dorsal blastomeres
of four-cell stage embryos were
injected with XFD RNA (D). At
the early gastrula stage (st. 10) the
embryos were fixed and then
subjected to in situ hybridization
with the goosecoid probe (dark
purple stain). The embryos are
oriented with the dorsal side
toward the top of the page. Scale
bars, 250 um.
