Fig. 2. Analysis of Fgf response element (FRE) of Xcad3. (A) Experimental design for the embryonic cell culture assay used in B. Ectodermal tissues were isolated from stage 10 gastrula embryos. The dissociated cells were inoculated into microculture wells at 200 cells/well. After completion of reaggregation by brief centrifugation, cells were cultured in the presence of increasing concentrations of bFgf until control embryos reached stage 23. The transcriptional levels of two position-specific neural markers were analyzed by RT-PCR (Kengaku and Okamoto, 1995). (B) High-dose-dependent activation by Fgf of endogenous Xcad3. Autoradiographs are shown of RT-PCR products of the transcripts from En2, an anterior neural marker gene and Xcad3, both of which were co-amplified with EF1 transcript, an internal standard (upper panels). Each RT-PCR product was quantified by a laser image analyzer and values for En2 () and Xcad3 () transcripts normalized to EF1 transcript are presented as percentages of the respective maximum value and plotted against bFgf dose (graph). (C) Experimental design for the embryonic cell culture assay used in D. Xcad3/LUC reporter and pRL-CMV plasmids were coinjected into four animal blastomeres of eight-cell stage embryos. When they reached stage 10, ectodermal tissues were isolated and processed as in A. To compare directly Fgf dose-dependence of Xcad3/LUC reporter with that of endogenous Xcad3, two parallel sets of cultures were prepared; one was assayed for luciferase activity, while the other was assayed for transcriptional levels of endogenous Xcad3. (D) Comparison of the Fgf dose-dependence profiles for a Xcad3/LUC reporter and endogenous Xcad3. Eight-cell stage embryos were injected with a Xcad3/LUC reporter depicted below the graph and an internal standard plasmid pRL-CMV and processed as described in C. Normalized Xcad3/LUC reporter activities are presented as percentages of the maximum value and plotted against bFgf dose (). Transcript levels of endogenous Xcad3 was assayed and plotted as in B (). (E,F) Presence of FRE in the intron 1. Chimeric constructs injected are indicated below each graph: they contained either Xcad3 intron1 (E) or SV40 enhancer sequence (F). Reporter activities of these constructs were analyzed as in D and presented in arbitrary units. Note that inclusion of intron1 sequence in reporter constructs is essential for dose-dependent response to Fgf.

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