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Fig. 7. PACSIN2-MA blocks gastrulation but not fibrillogenesis or cell adhesion. Embryos were injected in both blastomeres at the two-cell stage with transcripts encoding β-galactosidase (β gal), Wt-PACSIN2 (Wt) or the mitochondria-anchored PACSIN2 (Wt-MA). (A) When control embryos reached late gastrula stage, images of a representative control embryo (Ctl) and an embryo expressing PACSIN2-MA (Wt-MA) were taken. When embryos reached early neurula stage, they were scored for their failure to close the blastopore. The graph represents the percentage of embryos failing to close their blastopore and represents the average of 3 (β gal and Wt) or 4 experiments (Wt-MA and NI). Error bars represent the standard deviation to the mean. The number of embryos analyzed was as follow: NI = 132; βgal = 76; Wt = 100; Wt-MA = 225. While both PACSIN2 and PACSIN2-MA inhibits blastopore closure, PACSIN2-MA is significantly more efficient at blocking gastrulation compared to Wt-PACSIN2 (white asterisk). (B) Immunofluorescence performed on blastocoel roofs (BCR) dissected at stage 12. The FN fibrils of the BCR were detected using a rabbit polyclonal antibody 32F (red) and the PACSIN2 protein detected with mAb 3D8 (green). PACSIN2-MA is localized in dots in the middle of the cell (arrowhead 2) or at the periphery (arrowhead 1) likely to represent mitochondria. BCR from non-injected embryos and embryos expressing PACSIN2-MA display FN fibrils (arrows). (C) Animal cap ectoderm were dissociated at stage 8 and the cells seeded on the CCBD domain of FN, in the presence (+ Activin) or absence of Activin-A (− Activin). Photographs were taken after sibling embryos initiated gastrulation movements. A magnification of the activin treated cells is also included (+ Activin 2. The control cells (NI) spread at the onset of gastrulation while cells over-expressing Wt-PACSIN2 failed to do so. The cells expressing PACSIN2-MA spread like control cells with large lamellipodia (arrow). However these cells are significantly larger, displaying multiple nuclei (red arrowhead) indicating a failure of the cytokinesis during cell division. Quantifications are described in the manuscript. (D) Transcripts encoding PACSIN2-MA were injected in 1-cell stage embryos. When embryos reached the 8-cell stage, β-galactosidase was injected in a dorsal vegetal blastomere as a lineage tracer. Control embryos only received β-galactosidase transcripts. When control embryos reached late gastrula stage, embryos were fixed, processed for β-galactosidase detection and bisected trough their dorsoentral axis. The β-galactosidase expressing cells (dorsal mesoderm) show that PACSIN2-MA perturbed the convergence/extension movement (yellow double arrows). The magnification of the ventral ectoderm (black rectangles) shows that epiboly was also perturbed (black double arrows). Yp: yolk plug; Blc: blastocoel; Ar: archenteron; D: dorsal; V: ventral. ⁎: P < 0.05. |
