Overexpression of pk1 causes defective morphogenetic movements and enhances the slb phenotype. (A) Schematic of the experiment. Cells from embryos that received 5 pg pk1 RNA and rhodamin-dextran (RDx) and cells from embryos that received fluorescein-dextran (FDx) were transplanted simultaneously into the germ rings of wild-type, shield-stage hosts. (B) Lateral view of a living, tailbud-stage host embryo with dorsal to the right. Pk1-expressing cells (red) are positioned more laterally compared to wild-type cells (green). (C-E) Gain-of-function of pk1 enhances the slb phenotype. Dorsal views of a tailbud-stage embryo injected with 0.5 pg of pk1 RNA (C), a slb-/- embryo (D) and a slb-/- embryo injected with 0.5 pg pk1 RNA (E). The position and shape of prechordal plate (hgg1) in relation to the anterior edge of neural plate (dlx3) in the pk1 morphant (C) are similar to wild-type (see Fig. 2I). The prechordal plate is displaced caudally in the slb-/- embryo (D) and even further caudally in the slb-/- embryo injected with the pk1-Mo (E). (F-H) Confocal images of animal pole cells of embryos at 40% epiboly injected with 200 pg RNA encoding Dsh-GFP (F), 200 pg RNA encoding Dsh-GFP and 50 pg fz7 RNA (G) and 200 pg RNA encoding Dsh-GFP, 50 pg fz7RNA and 5 pg pk1 RNA (H). Dsh preferentially localises to vesicles (F) but relocates to the membrane in the presence of Fz7 (G). The Fz7-induced membrane localisation of Dsh is inhibited by Pk1 (H). (I) Subcellular localisation of Pk1 in animal-pole cells of 40% epiboly embryos injected with 25 pg RNA encoding Venus-Pk1 (Pk1 tagged with a modified version of EGFP). Pk1 localises in the cytoplasm. (J) Western-blot analysis of the levels of tagged-Dsh in embryos injected with 200 pg RNA encoding myc-Dsh along with 50 pg fz7 RNA in the presence or absence of 5 pg pk1 RNA. The blot was detected with either an anti-myc antibody for Dsh or an anti-β-tubulin antibody for loading control. The level of Dsh is reduced by pk1.
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