Figure 5. Cells of Spinal Cord Character Are Induced by Combinatorial Wnt and FGF Signaling at the Late Gastrula Stage.

(A and B) Caudal (C) neural plate tissue explants (black box) were isolated from HH stage 4 embryos and embedded in collagen matrix where their rostrocaudal orientation was maintained during in vitro cultivation for 15 h, corresponding to a stage 8 embryo (A, D, F, H, and J) or for 44 h, corresponding to a stage 17 ˜30-somite embryo (B, E, G, I, and K).

(C) Schematic drawing indicating the expression pattern of Wnt (red) and Fgf genes (green) in the primitive streak and caudal ectoderm, and in the node and primitive streak, respectively. Black dotted line indicates the presumptive neural plate.

(D–K) Each row represents consecutive sections from a single explant.

(D, F, H, and J) Sox2/3 was used as a presumptive neural marker.

(D) Stage 4 C explants were DiI-labeled and cultured alone. Cells in the caudal domain of the explants, close to the DiI-labeled cells, expressed CdxB and CdxC ( n = 12 explants). White arrowhead indicates the DiI-labeled cells.

(E, G, I, and K) Sox1 was used as a general neural marker.

(E) Cells in the rostral domain of 4 C explants cultured alone expressed Krox20, whereas Hoxb4 ⁺/b8 ⁺/c9 ⁺ cells appeared in the caudal domain of the explants. A small domain of Hoxb4 ⁺/b8 ⁺/ c9 ⁻ cells, but no domain of cells expressing Hoxb4 alone, were generated ( n = 30 explants).

(F) Explants cultured in the presence of mFrz8CRD-IgG conditioned medium (300 μl/ml culture medium) generated Sox2/3 ⁺ neural cells but no CdxB or CdxC positive caudal neural cells ( n = 11 explants).

(G) Explants cultured in the presence of mFrz8CRD-IgG conditioned medium (300 μl/ml culture medium) generated Otx2 ⁺ but no, or few, caudal neural cells ( n = 12 explants).

(H) Explants cultured in the presence of SU5402 (5 μM), an inhibitor of FGF signaling, generated Sox2/3 ⁺ neural cells but no CdxB or CdxC positive caudal neural cells ( n = 8 explants).

(I) Explants cultured in the presence of SU5402 (5 μM), an inhibitor of FGF signaling, generated Otx2 ⁺ but no, or few, caudal neural cells ( n = 9 explants).

(J) Simultaneous exposure to Wnt3A (˜75 ng/ml) and FGF4 (60 ng/ml) resulted in the generation of cells that expressed CdxB and CdxC in the entire explant ( n = 17 explants). Scale bar represents 100 μm.

(K) Exposure to Wnt3A (˜75 ng/ml) and FGF4 (60 ng/ml), in combination, almost completely blocked the generation of Krox20 ⁺ rHB cells, and only Hoxb4 ⁺/b8 ⁺/c9 ⁺ spinal cord cells were generated ( n = 17 explants). Scale bar represents 100 μm.