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Fig. 1. Isolation, characterisation and expression of Wise. (A) Outline of the screen. (B) RT-PCR analysis using Wise RNA. Wise alone (600 pg) does not induce pan-neural (NCAM) or mesodermal (myosin) markers. In the presence of noggin, increasing amounts of Wise RNA (150, 300, 600 and 1200 pg) induce progressively more posterior neural markers (en2, krox20, Hoxb9). (C) Western blot detecting HA-tagged Wise protein secreted into the medium after RNA injection into oocytes. C, control uninjected oocytes. (D,E) Recombination of noggin-expressing and Wise-expressing animal caps assayed for induction of Krox20 (D) or en2 (E). In D, the noggin RNA injected cap was marked with FIDx (to the left of the broken line) and in E the Wise cap with lacZ (to the right of the broken line). Wise induces patches of Krox20 or a ring of en2 expression (arrowheads) in a non-cell-autonomous manner in the noggin-injected cap. (F,G) In situ hybridisation of chick embryo at stage 10. Wise is expressed in the surface ectoderm (se in G) from the level of presomitic mesoderm to the posterior. Expression is also seen faintly in the head surface ectoderm. (H) RNase protection of Xenopus embryos with stages noted above each lane. Wise is first detected at an early gastrula stage (st. 10) and the expression persists into tadpole stages. ODC is a loading control. (I-N) In situ hybridisation of Xenopus embryos at indicated stages. At the neurula stages (I-L), Wise is expressed in the surface ectoderm broadly at anteroposterior levels, strongest at the edge of the neural tube and the posterior edge of the eye (arrowheads in I,J). The expression is also seen in the stomodeal-hypophyseal anlage (arrow in J, front view; arrow in L, transverse section). At the tailbud stage (M,N) the expression appears to be localised in cranial placodes, lateral line placodes and the ventral neural tube at the diencephalon level (arrowhead in N). |
