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Fig. 1. Whole-mount in situ hybridizations showing expression of dorsal and ventral forebrain markers in stage 23 embryos and noggin-treated explants. (A) Triple staining of cpl-1 (blue), etr-1 (purple) and XAG-1 (a light blue ring marking the cement gland), in the head. Expression of cpl-1 in the dorsal brain overlaps some etr-1 expression, though etr-1 is mainly expressed ventrally in the brain. (B) Double staining of cpl-1 (blue) and etr-1 (purple) in separate territories in noggin-treated explants. cpl-1 is expressed in several protrusions (arrowheads). (C) Double staining of XBF-1 (blue) and etr-1 (brown) in the head. XBF-1 expression is restricted to the dorsal forebrain. (D) Double staining of XBF-1, in blue, and etr-1, in purple, in noggin-treated explants. The pattern of expression of XBF-1 is identical to that of cpl-1, in B. (E) Side view of expression of XeNK-2 in the ventral neural tube. (F) XeNK-2 is not expressed in noggin-treated explants. Scale bars in A, B, and C represent 0.5 mm. D-F are the same magnification as B.
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Fig. 2. Dorsoanterior views of the overlapping expression of noggin and cpl-1 in stage 13 embryos. All embryos were processed together for double in situ hybridization; however, the brown-staining substrate was omitted from the embryos in A, and the blue substrate was omitted from the embryos in B. (A) noggin expression (blue), at the anterior edge of the neural plate, and in the prechordal plate and notochord. (B) cpl-1 expression (brown), also in the anterior neural folds. (C) An embryo double stained for noggin and cpl-1, showing overlapping expression in the anterior neural folds. (The two stains together appear bluish-brown; compare with the blue noggin- expressing notochord). (D) Higher magnification view of C. Scale bars in A and D represent 0.5 mm. B and C are the same magnification as A.
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nkx2-2 (NK2 homeobox 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
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Fig. 3. cpl-1 is induced by low doses and repressed by high doses of noggin. RT-PCR analysis of dissociated cells treated with a range of concentrations of noggin protein, as indicated, or untreated (‘control’). For comparison, non-dissociated ‘intact caps’ were treated in parallel, with medium alone (‘cont.’), or medium + 1 μg/ml noggin (‘nog’). All samples were harvested at stage 22-23. As a positive control, varying amounts of whole embryo control cDNAs were used in PCR reactions, as indicated; for a negative control, reverse transcriptase was omitted (‘−RT’). All samples were analyzed using primers specific for NCAM, cpl-1, XAG-1, epidermal keratin, EF-1α (a loading control; Krieg et al., 1989), and cardiac actin (a muscle marker; Gurdon et al., 1985).
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Fig. 5. Noggin dose-response profiles are shifted in the presence of BMP-2. RT-PCR analysis of cells dissociated at late stage 9/early stage 10 and treated with a range of concentrations of noggin protein, as indicated. Cells were then divided into two pools; one pool was untreated, and the other was additionally treated with 1 ng/ml purified BMP-2. All samples were harvested at stage 22. Controls and primers are the same as described for Fig. 3.
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Fig. 6. Continously dissociated cells express high levels of cpl-1. RT- PCR analysis of noggin dose-response in dissociated cells, treated as in Fig. 4 (N, noggin protein). After treatment, one-third of the cells were reaggregated (‘reagg.’), and the rest were kept dissociated (‘dissoc.’). All samples were cultured until stage 20. Controls and primers are the same as described for Fig. 3.
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Fig. 7. Partially localized cpl-1 expression in stage 22 cell reaggregates. Dissociated cells were untreated (A), or treated with the following noggin protein concentrations: 0.1 ng/ml (B), 1 ng/ml (C, shown at higher magnification in D), 10 ng/ml (E, shown at higher magnification in F), 100 ng/ml (G), or 1 μg/ml (H). Darkly stained protrusions are observed at 1 ng/ml noggin (C,D). Pits are observed at higher noggin concentrations (E-H, see arrowheads in F and H). Scale bars in A and D represent 0.5 mm. F is the same magnification as D; all others are the same magnification as A.
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Fig. 8. Partially localized cpl-1 expression in reaggregated cells, dissociated at stage 8 (A-C), stage 9 (D-F), or stage 10 (G-I). Cells were untreated (A,D,G), treated with 1 ng/ml noggin protein (B,E,H), or 1 μg/ml noggin (C,F,I), then cultured until stage 20. Neuralized reaggregates often appear cup-shaped (arrowhead in H). Scale bar in A represents 0.5 mm.
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Fig. 9. Regulation in five-explant recombinants, cultured until stage 22-23. (A) Recombinants were made by allowing five noggin-treated explants to heal together. If regulation occurs, one large patch of cpl-1 expression should result; otherwise, each explant should express cpl-1 independently, such that five separate patches result.
(B) cpl-1 expression in five-explant recombinants, made from explants dissected at stage 8 (a,d,g), early stage 9 (b,e,h), or late stage 9 (c,f,i). (a-c) Explants were dissected at the stages indicated, then treated and combined immediately. Stage 8 recombinants regulate to form one patch of cpl-1 expression (a), which often contains a large pit (arrowhead in a); later recombinants (b,c) exhibit multiple, fused patches of cpl-1. (d-f) Explants were dissected at the stages indicated, but aged until late stage 9 before treatment and combination. All recombinants exhibit multiple cpl-1 patches (d-f). (g-i) Explants were treated immediately after their dissection, but all were aged to late stage 9 before combination. Stage 8 recombinants regulate to form one cpl-1 patch (g), as in a. Scale bar in a represents 0.5 mm
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