Lamb TM and Harland RM (1995), Fibroblast growth factor is a direct neural ind...

XB-ART-19068

Development 1995 Nov 01;12111:3627-36.

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Fibroblast growth factor is a direct neural inducer, which combined with noggin generates anterior-posterior neural pattern.

Lamb TM , Harland RM .

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Neural tissue in developing Xenopus embryos is induced by signals from the dorsal mesoderm. Induction of anterior neural tissue could be mediated by noggin, a secreted polypeptide found in dorsal mesoderm. We show that bFGF, a known mesoderm inducer of blastula staged ectoderm, induces neural tissue from gastrula stage ectoderm. The type of neural tissue induced by bFGF from stage 10.25 ectoderm is posterior, as marked by Hox B9 expression. When bFGF and noggin are combined on early gastrula stage ectoderm, a more complete neural pattern is generated and no mesodermal tissue is detected. Explants treated with noggin and bFGF elongate and display distinct anterior and posterior ends marked by otx2 and Hox B9 expression, respectively. Furthermore, treatment of early gastrula ectoderm with noggin and bFGF results in the induction of En-2, a marker of the midbrain-hindbrain junction and Krox 20, a marker of the third and fifth rhombomeres of the hindbrain. Neither of these genes is induced by noggin alone or bFGF alone at this stage, suggesting a synergy in anterior-posterior neural patterning. The response of later gastrula (stage 11-12) ectoderm to bFGF changes so that Krox 20 and En-2 are induced by bFGF alone, while induction of more posterior tissue marked by Hox B9 is eliminated. The dose of bFGF affects the amount of neural tissue induced, but has little effect on the anterior-posterior character, rather the age of the ectoderm treated is the determinant of the response. Thus, an FGF signal may account for posterior neural induction, and anterior-posterior neural patterning could be partly explained by the actions of noggin and FGF, together with the changing response of the ectoderm to these factors.

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Species referenced: Xenopus
Genes referenced: actc1 ag1 col2a1 egr2 en2 fgf2 gsc hoxb9 ncam1 nog nrp1 otx2 tbxt

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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 spinal cord.
	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 expression.
	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.