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Fig. S1. Verification of CNC tissue in RT-qPCR samples.
RT-qPCR with different tissue marker genes were used to verify the identity of CNC tissue. CNC explants were dissected at the indicated stages. All values were normalized and calibrated to ODC expression (expression of ODC gene = 1). The bars indicate mean value with standard deviation of at least three independent experiments. CNC specific marker gene (A) twist, (B) snail and (C) slug were present in high abundance in CNC samples throughout all three stages. In the same amount of assessed RNA, the expression of twist as well as that of slug is strongly enriched in CNC sample than in whole embryo sample, whereas the expression of snail did not show this kind of enrichment. (D) The expression levels of the mesoderm marker bra in the CNC samples are barely detectable. (E) In situ hybridization (ISH) for eya1 indicates that placodal tissue is not disturbed when CNC was removed. Scale bar: 200 μm. (F) ISH for eya1 and PCNS on dissected CNC display that the explanted CNC are not contaminated with placodes. Scale bar: 100 μm. (G) ISH for AP2 on several different CNC explants. Scale bar: 200 μm. (H) Immunofluorescence staining of endogenous E-cadherin on CNC explants shown and indicated by the square in (G). CNC cells were labelled with membrane GFP (mbGFP) and explanted on a fibronectin-coated surface. DAPI staining was used to visualize cell nuclei. E-cadherin is prominently localized at cell-cell contacts. Scale bar: 20 μm. |
