Figure 4

Validation of microarray analysis. (A) Quantitative RT-PCR for individual genes with different biological roles was performed of RNA derived from GFP+ and GFP- cells from tails of 36 hpf embryos. Real time PCR ratios were determined by normalization to β-actin (equal to 1, dotted line). Only 3 genes out of the 15 tested were not significantly enriched (asterisks). (B-G) In situ hybridization of 6 genes enriched in GFP+ cells showing a primordium specific expression pattern in 36 hpf embryos. (H-O) Loss-of-function analysis on two selected genes (cd9b and f11r) enriched in GFP+ cells of 36 hpf embryos. Cldnb:gfp embryos were injected with anti-sense morpholinos (MO) against cd9b (I, L-M) and f11r (J, N-O), and compared to control (H, K). White arrows indicate the position where the primordium is at 36 hpf (H-J). Red arrowheads indicate the rosette-like structures in the primordium (K). Scale bars are 10 μm in (B-G) and (K-O).