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Fig. 3. Fgf signaling is required cell autonomously in EB placodes. (A) hsp70::dn-fgfr1 donor embryos were injected with a lineage tracer (fluorescein-dextran, green). At shield stage, 25-30 donor cells were transplanted into the prospective placodal domain of wild-type hosts. Mosaic embryos were heat shocked at 10-11 hpf, collected at 24 hpf and analyzed for Pax2 protein expression (red). Panels show side view of the embryos that received either wild-type (A, top) or hsp70::dn-fgfr1 cells (A, middle and bottom). Wild-type cells readily contributed to the EB placodes (arrowheads), whereas hsp70::dn-fgfr1 cells either accumulated dorsally (arrows) or were excluded from EB placodes (A, bottom). Dotted line indicates a patch of hsp70::dn-fgfr1 cells within the EB placode that did not express Pax2. (B) In reciprocal experiments, wild-type donor embryos were injected with a lineage tracer (fluorescein-dextran, green). At shield stage, 25-30 donor cells were transplanted into the prospective placodal domain of hsp70::dn-fgfr1 hosts, mosaic embryos were heat shocked at 13.5-16 hpf, collected at 24 hpf and analyzed for Pax2 protein expression (red). Because dn-Fgfr1 protein is localized to the membrane, while fluorescein is evenly distributed throughout the cell, wild-type donor cells were easily distinguishable in the transgenic host embryos. Most of the GFP in the host is not visible (except hindbrain), because the image brightness and contrast were adjusted to visualize much brighter fluorescein-positive donor cells. Transplanted wild-type cells contribute to EB placodes in hsp70::dn-fgfr1 embryos (arrowheads, top). Scale bars: 50 µm. o, otic placode.

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