Fig. 6.

Mpp5a is necessary for apical ring formation even in the absence of cell division. All embryos are at the 28-somite stage. (A-D) Orthogonal series of horizontal (top left), transverse (top right) and sagittal (bottom) confocal planes of Pard-EGFP expression in the following embryos. (A) Wild-type sibling treated with DMSO vehicle control. Many small apical rings have formed at the tissue midline (n=3/3 embryos). (B) Wild-type sibling treated with aphidicolin to block the C-divisions. Fewer, large apical rings have formed at the tissue midline (n=4/4 embryos). (C) mpp5a^m227 mutant embryo treated with DMSO vehicle control. No apical rings have formed at the tissue midline. Pard3 is visible as spots along the midline plane (n=3/3 embryos). (D) mpp5a^m227 mutant embryo treated with aphidicolin to block C-divisions. No apical rings have formed at the tissue midline. Pard3 is visible as spots along the midline plane (n=4/4 embryos). (E) Overall body shape of wild-type siblings and mpp5a^m227 mutants with and without aphidicolin treatment. (F) Horizontal section through neural tube showing nuclear staining in wild-type embryos treated with DMSO or aphidicolin to block C-divisions. Aphidicolin-treated embryos have larger nuclei, demonstrating that S-phase division block was successful. (G) Quantification of the area of the nucleus of wild-type siblings and mpp5a^m227 mutants with and without aphidicolin treatment. Data from 100-150 nuclei were pooled from two or three embryos in each group and analysed by two-way ANOVA. Areas of aphidicolin-treated nuclei were, on average, 30 µm² bigger than those of DMSO-treated embryos (P<0.0001). There was no significant difference in the area of nuclei between wild-type and mpp5a^m227 mutant embryos (P=0.8561). Error bars denote standard deviations. (H) Quantification of the number of apical rings in wild-type siblings and mpp5a^m227 mutants with and without aphidicolin treatment. Numbers of apical rings per 1000 µm² were calculated from three or four embryos per group. No apical rings were seen in any of the mpp5a^m227 mutants. There were, on average, 40 fewer apical rings per 1000 µm² in aphidicolin-treated wild-type embryos than in DMSO-treated wild-type embryos (Student's unpaired t-test, P=0.0222). Error bars denote standard deviations.