Fig. S3

Fig. S3

Related to Figure 5: Pard3 localisation and BFP-ERK2 negative control.

A. Cartoon illustrating the localisation of Pard3 (green) during the maturation of neuroepithelial cells within the developing neural tube. Red dotted box shows the region imaged in figure 5D. At keel stages Pard3 broadly localises to the tissue centre before division. Midline crossing divisions (C-divisions) occur at the centre of the tissue and localise Pard3 more specifically to their cleavage planes, helping to produce a precise localisation of Pard3 at the tissue midline at rod stages. The neural tube lumen then cavitates from this midline, resulting in the opening of the neural tube. Later, NE cells undergo further rounds of division at the Pard3-labelled apical surface, resulting in the production of both NE cells and neurons.

B. En face images of the neuroepithelial apical surface labelled with anti-Pard3 antibody or Pard3-EGFP-PIF6.

C. En face images of EVL cells labelled with anti-Pard3 antibody or Pard3-EGFP-PIF6. Pard3 is present at membranes around the perimeter of cells. A small amount of fusion protein is also present in intracellular membrane inclusions.

D. Single confocal slice through the EVL of an embryo labelled with Pard3- EGFP-PIF6, PHYB-CAAX and BFP-ERK2. Signal for Pard3-EGFP-PIF6, PHYB-CAAX and BFP-ERK2 are shown in (i) and (ii) respectively. Pard3-EGFP-PIF6 is robustly recruited to the 633nm ROI, but there is no concomitant recruitment of BFP-ERK2 to this region.

E. Normalised fluorescence intensities for Pard3-EGFP-PIF6 and BFP-ERK2 from experiment shown in (D). Oneway ANOVAs with Tukey’s multiple comparison tests were carried out. Error bars denote standard error of the mean.The experiment was repeated 5 times and a significant increase in membrane intensity within the 633nm ROI when compared with membrane regions in the same cells outside the ROI was seen in 5/5 cases for EGFP, but 0/5 cases for BFP. In 2/5 cases there was also a significant increase in EGFP intensity within the cytoplasm in the 633nm ROI, but no concomitant increase in cytoplasmic BFP-ERK2.