FIGURE 5.

ASXL3 knockdown disrupts head formation. (A) Stage 37 tadpole stage embryos were examined for axial defect phenotypes. Control embryos (CE – four left panels) were injected with 12 ng ASXL3-MO at the one-cell stage (four right panels) The ASXL3 morphant embryos are squat, less elongated, with poor trunk and head development. In the CE group 100% of the embryos developed normally development (n = 41) versus the ASXL3-MO group in which 100% of the embryos had a disrupted axial phenotype (n = 41). Four representative embryos are shown for each group. (B) Embryos were microinjected with ASXL3-MO (3 or 6 ng) into one-blastomere at the two-cell stage, so only half the embryo is perturbed. At tailbud stage 22, in situ hybridization was performed to the twist gene; twist is a marker for craniofacial neural crest cells (top panel control embryo/CE, 100% normal; n = 30). In middle and bottom panel, left side is injected and right side is normal. Twist expression is inhibited and (asymmetric, 60%; n = 40) in the left panel (ASXL3-MO), but its expression is normal on the uninjected side of the embryo in the right panel. (C) Embryos were injected with ASXL3-MO (12 ng) at the one-cell stage. Embryos were stained for cartilage formation at tadpole stages. Control embryos (90%, n = 27; panel) normal head cartilage staining (upper panel, left: head shot; right: dissected head cartilage). All morphant embryos (100%, n = 14) had reduced and abnormal head cartilage staining (lower panel, left: head shot; right: dissected head cartilage). (D,E) In situ hybridization was performed with a Xenopus laevis ASXL3 probe to neurula (D; dorsal view, anterior-posterior is top-bottom) and tadpole stage 35 (E) embryos.