|
Fig. 6 Modulation of cell contractility or cell-cell adhesion in the ectoderm reverses the effect of animal and lateral ectoderm on lateral mesendoderm migration (A and B) Constitutively active RhoA (CARhoA)-overexpressing animal-to-lateral (AtL) and constitutively active Mypt (CAMypt)-overexpressing lateral-to-lateral (LtL) ectoderm transplant assay. On the left, schematic representation of the experimental setup. On the right, fluorescence images of lateral mesendoderm (LME) cells and transplanted CARhoA-overexpressing animal (CARhoA AtL Ecto, red, A) or CAMypt-overexpressing lateral (CAMypt LtL Ecto, light blue, B) ectoderm (lateral view) from representative time lapses ∼70 min after the start of LME migration (∼7.35 hpf). Green, eGFP (LME cells); red, membrane RFP (CARhoA AtL Ecto); light blue, membrane RFP (CAMypt LtL Ecto). Scale bar, 100 μm. An, animal; Veg, vegetal; D, dorsal; V, ventral. See also Figure S4 and Video S10. (C) Dominant negative Cdh1 (DNCdh1)-overexpressing LtL ectoderm transplantation assay. On the left, schematic representation of the experimental setup. On the right, fluorescence images of LME cells and DNCdh1-overexpressing lateral (DNCdh1 LtL Ecto, lilac) ectoderm (lateral view) from representative time-lapse movies ∼70 min after the start of LME migration (∼7.35 hpf). Green, eGFP (LME cells); lilac, 10,000 MW Alexa Fluor 647 dextran (DNCdh1 LtL Ecto). Scale bar, 100 μm. See also Figure S4 and Video S10. (D) Migration directionality of LME cells in CARhoA AtL Ecto (red), CAMypt LtL Ecto (light blue), and DNCdh1 LtL Ecto (lilac) transplanted embryos. Number of cells: 292 from 6 CARhoA AtL Ecto (red), 162 from 4 CAMypt LtL Ecto (light blue), 325 from 4 DNCdh1 LtL Ecto (lilac). (E) Displacement (y displacement) along the animal-vegetal (AnVeg) axis of LME cells over time in transplanted embryos. Solid line represents the mean; gray ribbon displays confidence interval. Number of cells: 292 from 6 CARhoA AtL Ecto (red), 162 from 4 CAMypt LtL Ecto (light blue), 325 from 4 DNCdh1 LtL Ecto (lilac), 327 from 9 AtL Ecto (yellow), 327 from 9 LtL Ecto (blue). Statistical test on the final y displacement, Mann-Whitney test: ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. See also Figure S4. (F) Percentage of most animally migrating LME cells (for definition see Figure S3) in transplanted embryos. Red line represents the median. Number of embryos: 6 CARhoA AtL Ecto (red), 4 CAMypt LtL Ecto (light blue), 4 DNCdh1 LtL Ecto (lilac), 9 AtL Ecto (yellow), 9 LtL Ecto (blue). Statistical test, Mann-Whitney test: ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S4. In (E) and (F), datasets for AtL Ecto and LtL Ecto correspond to data shown in Figure 3.