a– d) Whole-mount in situ hybridization analysis of ERK7 expression. Magnified images show regions outlined with white dashed lines. ( a) The embryos at stage 12 (vegetal view), stage 16 (dorsal view), stage 25 and stage 33/34. Black arrows, cloaca; white arrowheads, ear vesicle; black arrowheads, nephrostomes. ( b) The dorsal explant (left panel) and sagittal section (right panel) of the stage 16 embryo. The GRP is outlined by the black dotted line or indicated by the open arrowhead. a, anterior; l, left; p, posterior; r, right. ( c) An embryo at stage 13 (lateral view). ( d) Whole-mount in situ hybridization of ERK7 followed by immunostaining with anti-acetylated α-tubulin (Ac.-tub., green). ( e, f) The efficiency of ERK7 MO. ( e) The indicated sets of MO (10 ng per cell) and mRNA (500 pg per cell) were injected into the two blastomeres at the two-cell stage, and the embryos were harvested at stage 10.5. EGFP mRNA (100 pg per cell) was used as an injection control. ( f) Each MO (10 ng per cell) was injected into the animal region of four blastomeres at the four-cell stage. Injected embryos were harvested at stage 33/34. Endogenous ERK7 protein was concentrated by immunoprecipitation from embryo lysates. ( g, h) Knockdown experiments of ERK7. Control MO (20 ng per cell) or ERK7 MO (10 or 20 ng per cell) was injected into the dorsal ( g) or ventral ( h) marginal region of two blastomeres at the four-cell stage. The injected embryos were fixed and observed at stage 33/34. The phenotypes were classified into three groups (severe, mild or normal) according to the extent of the defects in head morphology ( g) or body length ( h). Scale bars, 300 μm.