Fig. 9. Profilin1 is specifically required for blastopore closure. (A) Schema of the injection approach to overexpress or deplete Profilin1 in or lateral to the neural folds, adapted from Wallingford and Harland (Wallingford and Harland, 2002). RNA (50 pg) encoding membrane-localized GFP injected into the lateral blastomeres at the 16-cell stage targets expression lateral of the neural folds whereas injection into the medial blastomeres targets expression to the neural folds. (B) Targeted overexpression of Profilin1 RNA (2 ng) or depletion of Profilin1 (50 ng MO) in the neural folds does not inhibit anterior neural fold closure but leads to open neural folds in the posterior, reflecting a failure of blastopore closure. This phenotype can be rescued by coexpression of N-Profilin1 (50 pg). Lateral overexpression or depletion of Profilin1 has no effect on neural fold closure. (C) Time lapse images of embryos injected with Profilin1 RNA (2 ng) or XProfilin1 MO (50 ng) showing a delay and eventual failure of blastopore closure. The XProfilin1 MO phenotype is rescued by coinjection of 50 pg N-Profilin1 RNA. (D) Depletion of XProfilin1 and XDaam1 synergistically inhibit gastrulation. Quantitation of the phenotypes observed with separate or co-injections of XProfilin1 MO and/or XDaam1 MO, Embryos with an open blastopore and significantly reduced anterior posterior (AP) axis were scored as severe embryos. Embryos with a small open blastopore or delayed blastopore closure and a shortened AP axis or bent body axis were scored as intermediate to mild. (E) A model for how Profilin1 functions in the non-canonical Wnt signaling pathway (see Discussion).