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Fig. 3 C-syne2a overexpression prevents nuclear movement and p-Smad2 nuclear translocation in LMCs, possibly through regulation of MTOC positioning and MT formation. (A,B) Nuclear positioning in LMCs in WT and C-syne2a-overexpressing embryos at 4.7 hpf. Transverse sections of WT and C-syne2a-overexpressing embryos; cell membrane and nucleus were visualized by β-cat and DAPI staining, respectively. Arrowheads indicate LMCs. (C,D) DNB and DCN in LMCs were measured in WT and C-syne2a-overexpressing embryos at 4.7 hpf. n, number of nuclei examined. ***P<0.001. (E) Angles between the center of the cell and the nucleus in LMCs relative to the medial-lateral axis were measured as shown in Fig. S1. n, number of nuclei examined. (F) Transverse sections of WT embryos; p-Smad2 and sox32 were visualized by immunohistochemical staining and FISH, respectively. The number of nuclei examined is shown bottom right. Arrowheads indicate nuclei in LMCs. Dashed lines indicate the boundary between the blastoderm and the YSL. (G,H) Transverse sections of WT and C-syne2a-overexpressing embryos; cell membrane and MTOCs were visualized by β-cat and γ-tubulin (γ-tub) staining, respectively at 4.7 hpf. The star (H) indicates an MTOC that has detached from the nucleus in C-syne2a-overexpressing embryos. (I) Percentage of embryos with attached or detached MTOCs in WT or C-syne2a-overexpressing embryos at 4.7 hpf. n, number of nuclei examined. (J-L) Transverse sections of WT, C-syne2a-overexpressing and nocodazole-treated embryos; cell membrane, MTs and nuclei were visualized by β-cat, α-tub and DAPI staining, respectively at 4.7 hpf. The number of embryos examined is shown bottom right. An asterisk (K) indicates the EVL cell. (M) Percentage of cytoplasmic area occupied by MTs in LMCs of WT, C-syne2a-overexpressing and nocodazole-treated embryos. n, number of embryos examined. Error bars indicate s.d. of 20, 23 or 21 measurements. ***P<0.001. Scale bars: 10 µm.