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Fig. 3. Ednrb wild-type neural tubes (NTs) induce tyrosinase expression in Ednrb-deficient cultures. (Top) Tissue recombination experiments. In a first step, E9.5 NT explants were isolated from (Ednrb^lacZ/+ x Ednrb^lacZ/+) matings as well as from [(Mitf ^mi–ew/Mitf ^mi–ew; Ednrb^+/+) x (Mitf ^mi–ew/Mitf ^mi–ew; Ednrb^+/+)] or [(Kitl^Sl/+; Ednrb^+/+) x (Kitl^Sl/+; Ednrb^+/+)] matings. The NTs were placed separately into individual dishes and the corresponding embryos were genotyped. On the following day, the NTs from identified Ednrb^lacZ/Ednrb^lacZ cultures were removed and replaced with NTs from the respective Ednrb^+/+ cultures. As a control, NTs from Ednrb^lacZ/Ednrb^lacZ cultures were given to other Ednrb^lacZ/Ednrb^lacZ cultures whose own NTs had been removed. The cultures were kept for 2 weeks and then stained with ß-gal and tyrosinase antibodies as described in the legend for Fig. 2 and the Materials and methods section. (Bottom panel) (A) Ednrb^lacZ/Ednrb^lacZ culture control-reconstituted with Ednrb^lacZ/Ednrb^lacZ NT. (B) Ednrb^lacZ/Ednrb^lacZ culture reconstituted with (Mitf ^mi–ew/Mitf ^mi–ew; Ednrb^+/+) NT. (C) Ednrb^lacZ/Ednrb^lacZ culture reconstituted with (Kitl^Sl/Kitl^Sl; Ednrb^+/+) NT. Ednrb^lacZ/Ednrb^lacZ cultures always generated ß-gal-positive cells. However, only (Mitf ^mi–ew/Mitf ^mi–ew; Ednrb^+/+) NTs (B), but not Ednrb^lacZ/Ednrb^lacZ NTs (A) or (Kitl^Sl/Kitl^Sl; Ednrb^+/+) NTs (C), led to the generation of tyrosinase-positive cells. Neither (Mitf ^mi–ew/Mitf ^mi–ew; Ednrb^+/+) nor (Kitl^Sl/Kitl^Sl; Ednrb^+/+) cultures carry the lacZ marker, nor are they capable of generating tyrosinase-positive cells on their own (Hou et al., 2000) (this paper), indicating that Tyr/ß-gal double-positive cells in B are derived from the Ednrb^lacZ/Ednrb^lacZ culture. Scale bar: 25 µm.

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