Gastrulation and neural tube closure defects in high dose PACRG morphant embryos. (A) Phenotypes. Note that with increasing morpholino oligonucleotide (MO) doses the anterior-posterior axis shortened, head structures were lost and the blastopore (arrowhead) failed to close. At 2 pmol/embryo (bottom) gastrulation was arrested on the injected side. (B,C) Still frames taken from timelapse movies (Additional file 5: Movie 3) at the stages indicated. (B) Uninjected control specimen. (C) Embryo injected with 2 pmol of PACRG-MO into the dorsal marginal zone at the four cell stage. Note that the dorsal lip did not form in morphant, while the uninjected ventral side gastrulated normally and in time. (D) Immunohistochemistry with an antibody against acetylated tubulin (red), demonstrating non-dynamic microtubules in the deep mesoderm of the marginal zone of the stage 11 gastrula embryo. (E,F) Rescue of neural tube closure defect (NTD) in morphants by parallel injection of a mutated PACRG-eGFP construct such that MO binding was abolished (rescue construct; see also Methods). (E) Unilateral right-sided MO injection (0.5 pmol) into the dorsal marginal zone resulted in NTD and loss of cement gland (white arrowhead) on the injected side. (F) Rescue of NTD and partial rescue of the cement gland. (G) Wild-type development upon overexpression of PACRG-eGFP mRNA. Yellow dashed line = lateral border of neural folds; white dashed line = dorsal midline; blue arrowheads = dorsal lip; green arrowheads = ventral lip; red asterisk = lack of dorsal lip formation. a = anterior; an = animal; d = dorsal; l = left; r = right; veg = vegetal.