|
Fig. 5
Modulation of pitx2c expression affects cell division orientation during gastrulation.
(a) Schematic of the experimental design to assess the orientation of cell division in the embryo. Embryos were injected at the 1 cell stage with H2B-GFP and membrane tdTomato mRNA. Time-lapse imaging was performed from shield stage for 90 mins; quantification was performed on at least five embryos per condition with 30–60 cell divisions counted per embryo. (b) Example of a cell division oriented perpendicular to the mediolateral (ML) axis shown at two consecutive time points, t1 (upper panel) and t2 (lower panel). (c) Example of a cell division oriented parallel to the ML axis shown at two consecutive time points, t1 and t2. Yellow arrows point to the particular cell division that is measured by the white dashed lines. All images are oriented with dorsal to the right, animal pole to the top. (d–g) Rose diagrams indicating the orientation of cell division axes in wild types (d) and MZpitx2c mutants (e), and after injection of pitx2c mRNA at the 1 cell stage in wild types (f) and MZpitx2c mutants (g). The proportion of cell division angles is indicated relative to the ML axis. MZpitx2c mutants exhibit a higher proportion of cell divisions oriented less than 45° from the ML axis (e) as compared to wild-type embryos (d). Injection of 25 pg of pitx2c mRNA at the 1 cell stage randomizes cell division orientation in wild-type embryos (f), whereas it increases the proportion of cell divisions occurring perpendicular to the ML axis in MZpitx2c mutant embryos (g). Chi-square tests were performed on binned data of cell division angles.