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Fig. S2
Fate-mapping experiments. (A-G) Traceable wild-type endodermal (Tar* or Cas) cells were transplanted at the blastula margin of wild-type embryos and examined for taste bud cell marker expression. This experimental procedure did not noticeably affect endogenous pharyngeal taste bud development (A). At 3 dpf, grafted pharyngeal endodermal cells expressed Calb2b (B-D, n=14) or 5HT (E-G, n=10). (H-P) kaede mRNA (Ando et al., 2002) was injected into one or two cells of the marginal zone of 64-cell embryos to target endoderm-derived pharyngeal pouches (H). Embryos were left to develop until 28-30 hpf in the dark, then mounted in 1% low melting agarose. Kaede protein was photoconverted by UV light from green to red, in pharyngeal pouch cells of wild-type embryos at 28-30 hpf (I,J, p1, p2) using a 20× or 40× objective of a Zeiss Axioplan microscope with an adapted DAPI filter and pinhole to adjust the diameter of the UV light beam. We observed that red and green Kaede forms were stable after fixation. Immunohistochemistry on whole-mount embryos or vibratome sections was carried out while protecting the tissue from direct light sources and all steps were carried out in the dark to avoid bleaching. Triton was omitted during washes. This protocol was compatible with the detection of the primary rabbit anti-Calb2b antibody (Swant) or anti-5HT (Sigma) combined with a secondary anti-rabbit conjugated with Alexa Fluor 680. Red photoconverted Kaede was localized in the pharyngeal epithelial cells in the surface of pharyngeal arches (n=15) or palate (n=3) at 3 dpf and some of these cells were also immunostained for Calb2b or 5HT (K-M,N-P). Scale bars: 10 μm in A,I; 5 μm in B-H,J-P.