Combinatorial Wnt, RA, and FGF Signaling Reconstruct Hox Gene Profiles Characteristic of the cHB and Spinal Cord

(A) Schematic drawing of a stage 4 chick embryo. Dotted line indicates the presumptive neural plate. Red box indicates FB explants isolated and cultured in vitro for 44 h.

(B–F) Sox1 was used as a general neural marker. Bars represent mean ± s.e.m. number of cells in Otx2⁺, Hoxb4⁺/ b8⁻/c9⁻, Hoxb4⁺/b8⁺/ c9⁻, and Hoxb4⁺/b8⁺/c9⁺ domains, respectively, as percentage of total cell number. Each row represents consecutive sections from a single explant.

(B) Control stage 4 FB explants generated Sox1⁺/Otx2⁺ but no caudal neural cells ( n = 24 explants).

(C) Stage 4 FB explants cultured in the presence of Wnt (̃150 ng/ml) and FGF (60 ng/ml) generated Hoxb4⁺/b8⁺/c9⁺ cells and only a few Krox20⁺ cells ( n = 24 explants).

(D) Cultivation in the presence of Wnt3A (̃150 ng/ml), RA (10 nM), and FGF (30 ng/ml) generated Hoxb4⁺/b8⁺/ c9⁻ cells and a few Hoxb4⁺/b8⁺/c9⁺ cells ( n = 18 explants).

(E) Cultivation in the presence of Wnt3A (̃150 ng/ml) and RA (10 nM) generated Hoxb4⁺/ b8⁻/c9⁻ cells and no, or only a few, Hoxb4⁺/b8⁺/ c9⁻ cells ( n = 28 explants).

(F) Exposure to Wnt3A (̃150 ng/ml) and RA (10 nM) in the presence of SU5402 (3 μM), an inhibitor of FGF signaling, generated Hoxb4⁺/ b8⁻/c9⁻ cells ( n = 12 explants). Scale bar represents 100 μm.