Figure 6.
Figure 6. Ncad expression is reduced in cells lacking Sip1. (A) Quantitative PCR analysis of neural tubes and migratory crest cells dissected from HH9 embryos that were electroporated on the left side with ContMO and on the right side with Sip1MO at HH4. Ncad expression was compared with GAPDH expression. Error bars are standard deviation between samples and P < 0.046 (n = 4). The data shown here are one representative experiment with expression data from four embryos, and this was repeated four times. Asterisk indicates statistical significance. (B) Western blot analysis of Ncad protein expression after the injection of 1 mM ContMO and 1 mM Sip1MO showing that Ncad protein expression is decreased after Sip1MO electroporation. β-Tubulin was used as a loading control. The data shown here are representative of one experiment out of two repeats. In this experiment, protein was pooled from 8–10 chicken whole embryos. (C) Whole-mount in situ hybridization for Ncad expression in embryos lacking Sip1 on the right side shows a decrease in Ncad. (D) Confocal image of a section from an embryo, electroporated with ContMO on the left side and Sip1MO (green) on the right side, showing decrease in Ncad protein (red) expression (Z = 8 µm). (E and F) Whole-mount immunohistochemistry using an antibody for Snail2 of an embryo electroporated with (E) Sip1MO on the right side or (F) Sip1MO + Ncad-GFP DNA on the right side. Snail2 expression is reduced in the Sip1MO-injected cells, and this effect is rescued by co-electroporation of Sip1MO + Ncad-GFP cells. (e′) A section from the embryo in E showing loss of Snail2 protein. (e′′) Overlay of Snail2 and Sip1MO. (f′) A section from the embryo in F at the level indicated by dashed white line shows that although Snail2 appears reduced in whole mount, Snail2 expression and numbers of migratory NC cells are partially rescued. (G) Whole-mount immunohistochemistry using an antibody to Ecad on an embryo electroporated with Sip1MO + Ncad-GFP DNA on the right side shows that although Ecad protein is still increased on the injected side, it is at a lower level in the cells injected with high levels of Ncad-GFP and Sip1MO (indicated by dashed circle and white arrow) than with Sip1MO alone. Bars: (C and D) 30 µm; (E and F) 200 µm; (e′ and f′) 100 µm; and (G and H) 50 µm. The whole-mount and section data shown here are from a single representative experiment out of at least three repeats.

Ncad expression is reduced in cells lacking Sip1. (A) Quantitative PCR analysis of neural tubes and migratory crest cells dissected from HH9 embryos that were electroporated on the left side with ContMO and on the right side with Sip1MO at HH4. Ncad expression was compared with GAPDH expression. Error bars are standard deviation between samples and P < 0.046 (n = 4). The data shown here are one representative experiment with expression data from four embryos, and this was repeated four times. Asterisk indicates statistical significance. (B) Western blot analysis of Ncad protein expression after the injection of 1 mM ContMO and 1 mM Sip1MO showing that Ncad protein expression is decreased after Sip1MO electroporation. β-Tubulin was used as a loading control. The data shown here are representative of one experiment out of two repeats. In this experiment, protein was pooled from 8–10 chicken whole embryos. (C) Whole-mount in situ hybridization for Ncad expression in embryos lacking Sip1 on the right side shows a decrease in Ncad. (D) Confocal image of a section from an embryo, electroporated with ContMO on the left side and Sip1MO (green) on the right side, showing decrease in Ncad protein (red) expression (Z = 8 µm). (E and F) Whole-mount immunohistochemistry using an antibody for Snail2 of an embryo electroporated with (E) Sip1MO on the right side or (F) Sip1MO + Ncad-GFP DNA on the right side. Snail2 expression is reduced in the Sip1MO-injected cells, and this effect is rescued by co-electroporation of Sip1MO + Ncad-GFP cells. (e′) A section from the embryo in E showing loss of Snail2 protein. (e′′) Overlay of Snail2 and Sip1MO. (f′) A section from the embryo in F at the level indicated by dashed white line shows that although Snail2 appears reduced in whole mount, Snail2 expression and numbers of migratory NC cells are partially rescued. (G) Whole-mount immunohistochemistry using an antibody to Ecad on an embryo electroporated with Sip1MO + Ncad-GFP DNA on the right side shows that although Ecad protein is still increased on the injected side, it is at a lower level in the cells injected with high levels of Ncad-GFP and Sip1MO (indicated by dashed circle and white arrow) than with Sip1MO alone. Bars: (C and D) 30 µm; (E and F) 200 µm; (e′ and f′) 100 µm; and (G and H) 50 µm. The whole-mount and section data shown here are from a single representative experiment out of at least three repeats.

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