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Fig. 1. Neural and mesoderm induction by FGF. In situ hybridization analysis on early gastrula stage treated ectoderm cultured to the early
tailbud stage. Untreated ectoderm (B,E,H), FGF-treated ectoderm (C,F,I), and the control whole embryos (A,D,G) were examined for
expression of nrp-1 (purple) and muscle actin (M. actin, blue) (A-C); otx2 (purple) and En-2 (blue) (D-F); and Hox B9 (G-I). FGF induces both
muscle actin and nrp-1, however, expression of the neural marker is not contingent upon muscle expression. The posterior marker, Hox B9, but
not the anterior markers, otx2 or En-2, is induced by FGF. In A, the stong expression of muscle actin obscures the nrp-1 expression in the
Fig. 2. Time course of treatment with factors. (A) Ectoderm taken
from late blastula (stage 9) embryos was either treated immediately
or aged to early (stage 10.25), mid- (stage 11) or late (stage 12)
gastrula stage and treated with 50 ng/ml bFGF (F), 1 mg/ml noggin
(N), the combination (N+F), activin (A) or with an LCMR control
(L). Explants were subsequently cultured to stage 22, and harvested
for RT-PCR analysis. Expression of NCAM, En-2, Krox 20, Hox B9,
Muscle actin and Collagen Type II was analyzed. (B) Ectoderm was
treated identically as in A except, stage 12 ectoderm was not treated
since the explants were harvested at this stage to analyze expression
of the early mesoderm markers, Xbra and Goosecoid. EF-1a
expression is shown as a loading control in both panels.
Fig. 3. Noggin + FGF generates neural tissue with anterior-posterior polarity. Ectoderm from stage 10.25 embryos was treated with increasing
doses of bFGF (2 ng/ml, 10 ng/ml, 50 ng/ml) either in the absence (A-C) or presence (D-F) of 1 mg/ml noggin. Explants were cultured until
control whole embryos (G) reached the late neurula stage (stage 18). Expression of otx2 and Hox B9 were analyzed by in situ hybridization.
Otx2 (light blue) is expressed at the anterior end, and Hox B9 (purple) is expressed at the posterior end of the control embryo (G; the dark color
at the anterior end is an effect of the embryo edge, and is not Hox B9 staining). Increasing bFGF dose increases Hox B9 expression (A-C). In
the presence of noggin, increasing bFGF dose(D-F) causes the explants to elongate and to express otx2 and Hox B9 on either end of the
elongated structure. Expression of a general neural marker, nrp-1 (purple) and muscle actin (blue) was examined in whole embryos (H) and in
explants treated with 1 mg/ml noggin (I), 50 ng/ml bFGF (J), and the combination of 1 mg/ml noggin and 50 ng/ml bFGF (K). Noggin alone
induces nrp-1 expression, bFGF alone induces both muscle actin and nrp-1, and the combination of the two induces only nrp-1, no muscle actin
expression is observed.
Fig. 4. Induction of hindbrain by noggin and FGF.
Expression of Krox 20 (purple) and Muscle actin
(blue) in stage 24 control embryos (A) and in
stage 10.25 ectoderm treated with 1 mg/ml noggin
(B), 50 ng/ml FGF (C), and the mixture of noggin
and FGF (D) after culture to stage 24. Whole
embryos express Krox 20 in two stripes in the
developing hindbrain, while muscle actin is
expressed in the somites. Noggin does not induce
either gene. FGF can induce muscle actin (1/13 in
this experiment); induction of muscle is highly
stage-dependent and thus shows some variability
between experiments (compare to Fig. 1). FGF
may induce a low level of unlocalized Krox 20.
FGF + noggin explants show Krox 20 expression
localized to a stripe in the central region of the
elongated structure, and no muscle actin
Fig. 5. FGF dose response. (A) Stage 10.5 ectoderm was treated with
increasing doses of bFGF (0.08, 0.4, 2, 10, 50, 250, 1250 ng/ml) and
then cultured to the early tailbud stage (stage 20). RT-PCR analysis
of nrp-1, otx2, En-2, Krox 20, Hox B9, Muscle actin, Collagen type
II shows that when stage 10.5 ectoderm is treated with increasing
amounts of bFGF, nrp-1 expression increases. At 2 ng/ml Hox B9
and 10 ng/ml Krox 20 are induced, while En-2 or otx2 is not induced
by any dose, and the low level expression of otx2 seen in untreated
explants is repressed by 0.4 ng/ml or more of bFGF. bFGF treatment
on St 10.5 ectoderm did not induce muscle actin or collagen type II
mesoderm, and repressed XAG-1 expression. EF-1a is a loading
control. (B) When ectoderm is treated at stage 11, En-2 is induced by
a wide range of doses, with 2 ng/ml being the minimal dose.