Figure 3. MEK1 is required for Ventx2 clearance and asymmetric distribution during cell division.

(A,B) Four-cell embryos were injected in each cell with 50 pg GFP-CAAX, 50 pg Ventx2-Myc, 50 pg 2SAVentx2-Myc RNAs, and 25 ng Mk-MO, as indicated. Embryos were fixed at blastula stage 9, or gastrula stage 11, cryosectioned and processed for anti-Myc (red), and anti-GFP (green) immunostaining, and DNA was stained with DAPI (blue). Graphs show the percentage of Myc positive nuclei (DAPI positive) over the total number of injected cells (GFP positive) from four independent experiments. (B) 3D reconstruction of confocal slices of mitotic Myc positive nuclei labeled by DAPI from stage nine sectioned embryos. Sister mitotic chromosomes are referred to as α (more intense Myc staining), and β (less intense Myc staining). The A-V arrows indicate the animal-vegetal axis. Note the asymmetric cortical Ventx2-Myc signal in the MEK1 morphant cell. (C). The graph shows the ratios of Myc signal intensity betweenα and β sister nuclei.

DOI: http://dx.doi.org/10.7554/eLife.21526.009

Figure 3—figure supplement 1. Ventx2 degradation and asymmetric distribution require MEK1 activity.

(A) In silico analysis of phosphorylation sites in the Ventx2 protein and prediction of kinases involved, with Kinasephos2 software. (B) Schematic representation of the Ventx2 protein. HD indicates the homeodomain (blue box), and the PEST destruction motif is highlighted in red. Note that Serine 140, which is required for Ventx2 degradation, is a predicted target of MAPK. (C) 50 pg Ventx2-Myc RNA was injected into both blastomeres at the two-cell stage. 50 pg GFP-Myc-RNA was co-injected as an internal loading control. Embryos were allowed to develop until the indicated stages, and exogenous Ventx2 was detected by anti-Myc immunostaining on Western blot. The graph shows the ratios of Ventx2-Myc over a-tub signals measured from the Western blot. (D) Four-cell embryos were injected in each cell with 50 pg GFP-CAAX, 50 pg Ventx2-Myc, 50 pg 2SAVentx2-Myc RNAs and with 25 ng Mk-MO as indicated. Embryos were fixed at blastula stage 9, cryosectioned and processed for anti-Myc (red), anti-GFP (green) and anti-g-tubulin (centrosomes, white) immunostaining, and DNA was stained with DAPI (blue). Panels represent compiled confocal slices to visualize entire mitotic nuclei.

DOI: http://dx.doi.org/10.7554/eLife.21526.011