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Fig. 7. Phenotypical analysis of ADAM19 KD at tailbud stage. (A) Whole-mount in situ hybridization of tailbud stage embryos (stages 22–24). Embryos were injected in one blastomere of the two-cell stage with 10 ng of MO19 or CMO. mRNA encoding β-galactosidase (β-Gal) was co-injected as a tracer. The embryos were processed by whole mount in situ hybridization with the markers myosin light chain (MLC), N-tubulin, NRP1, Sox2 and ADAM11. The injected side, as detected by β-Gal activity (light blue), is on the left side for all embryos. The percentages of embryos displaying these phenotypes are indicated and represent the average of three independent experiments. The black arrowheads point to the area of decreased gene expression. For Twist the embryo heads are presented as a side view. The injected side of each embryo is to the left. Decrease in twist expression is visible (arrowhead) in embryos injected with MO19. (B) Quantitative real-time PCR was performed on embryos injected at the one-cell stage with either the MO19 alone or with the ADAM19 rescue mRNA (MO19 + A19R) and allowed to develop until neurula (stage 17) and tailbud stage (stage 24). Alternatively, the MO19 was injected in 2 dorsal-animal blastomeres at the 8-cell stage (MO19 Ectoderm) to target dorsal ectoderm but not mesoderm derivatives. All genes were compared to non-injected control embryos. The relative abundance of MLC, N-tubulin, Sox2 and ADAM11 gene expression was measured by the 2(-δCT) method and normalized to α-tubulin. Results are presented as the log 2 of the fold change. Error bar correspond to the standard error. Asterisks represent a P < 0.05 using a standard t test assuming unequal variance. |
