Figure 6. PCP signaling controls cell pivot by maintaining the local concentration of N-Cadherin.

(a–d) Immunofluorescence with α-N-Cadherin (Ncad) antibody (green) and DAPI (red) in the frozen sections of wild-type tissues demonstrated that Ncad was present both on the membrane and in the cytoplasm, particularly enriched in cell-cell junctions (a). These patterns appeared to be unchanged in Fzd7-ΔPDB (b) or Fzd7 expressing cells (c). However, junctional Ncad in Fzd7 positive tissues was reduced (c). This was confirmed by normalizing junctional Ncad intensity to total Ncad intensity in each pair of sister cells (d) (n = 10, 10, eight for wild-type, Fzd7-ΔPDB, and Fzd7 tissues, respectively). See also Figure 6—source data 1. (e–g) In the chick metacarpals expressing Ncad-GFP (green) via RCAS infection, live imaging revealed that the fusion protein was enriched in the interface of sister cells in wild-type tissues (e) (n = 6) and tissues coexpressing Fzd7-ΔPDB (n = 6) over time (f). In contrast, in the presence of Fzd7, Ncad was initially concentrated in the junctions but then diminished as the cytoplasmic bridge between sister cells disconnected (g) (n = 7). (h) Quantifying Ncad-GFP expression along the post-cleavage furrow. GFP intensity was measured every one hour after cell division (T = 1 hr was the time when junctional Ncad-GFP was started to be observed) and normalized by dividing the maximal intensity during the time course (F/Fmax). Changes of cumulative F/Fmax showed that Ncad-GFP signal in Fzd7 expressing cells dropped about 50% 5 hr after cytokinesis. See also Figure 6—source data 2. (i–l) The normal function of Ncad is required for maintaining sister cell contact. Live imaging was performed on H2B-GFP (green) and mCherry (red) positive tissues cultured in the medium with α-Ncad antibody (1:10 dilution) (i), demonstrating the disengagement of sister cells (j) (n = 5). Similar cell behaviors were observed in the tissues expressing H2B-GFP (green) and a dominant-negative mutant of Ncad fused to mCherry through T2A sequence (red) (k, l) (n = 5). The signal intensity of the images in i was adjusted from the corresponding movie to present nuclei morphologies more clearly. In both cases, no complete pivoting was observed in cell doublets (n = 15 and 20 for α-Ncad antibody and dnNcad-T2A-mCherry, respectively). See also Figure 6—source data 3, Figure 6—source data 4. (m) Schematic diagram to show functional blocking of Ncad causes sister cells to separate. (n) Schematic diagram to summarize the roles of PCP signaling and junctional Ncad in regulating single column formation: (1) the absolute level PCP signaling is essential for oriented cell division, (2) PCP signaling reduces Ncad enrichment at the post-cleavage furrow, (3) normal junctional Ncad function is required for cell association and pivot. Scale bars: 4 μm. ** denotes p<0.01; * denotes p<0.05 (Wilcoxon Rank-Sum Test).

Figure 6—figure supplement 1. Ncad is enriched in the post-cleavage furrow of dividing cells.

(a) Chick humerus was sectioned and stained with both α-Ncad antibody and phalloidin Alexa-647. One representative image was presented to show the localization of Ncad (a1) and contractile ring (a2). (b–d) Enlarged images of sister cells in (a). In the cleavage furrow highlighted by actin-rich contractile ring (b1), Ncad was not strongly expressed (b2), whereas this protein was concentrated between the cells with weak junctional phalloidin signal (c, d). Polygons (yellow) were drawn along the border of strong phalloidin (b1) or Ncad (c2, d2) to select the regions of interest (ROI) for fluorescence intensity analysis. (e) Pearson correlation coefficients between of Ncad and phalloidin signal in the ROI were calculated to evaluate colocalization of Ncad and cleavage furrow. In the graph, the black column represented the ROI with high Ncad signal in which the colocalization analysis was performed (n = 23); the light grey column was vise versa (n = 20). In either case, the colocalization efficiency was below 0.5 (1 represents perfect correlation, 0 represents no correlation, −1 represent perfect anti-correlation). Taken together, it shows this protein is not enriched in the cleavage furrow, but likely in the post-cleavage furrow instead. See also Figure 6—figure supplement 1—source data 1. Scale bars: 7 μm.

Figure 6—figure supplement 2. Perturbing PCP activity does not affect Ncad transcription.

Double DNA in situ hybridization against Fzd7 (green) and Ncad (red) and was performed in the frozen cartilage sections sparsely expressing either Fzd7 (a) (n = 25) or Fzd7-ΔPDB (b) (n = 23) via low-titer RCAS infection. In both cases, the expression level of Ncad mRNA appeared to be normal when compared with the uninfected chondrocytes within the same context. Scale bars: 12 μm.

Figure 6—figure supplement 3. Ncad-GFP intensity analysis confirms sister cell association.

Based on the live imaging of Ncad-GFP expressing cells (Figure 6e–g), polyline kymograph analysis seen in Figure 2b was applied to quantify GFP intensity across sister cells (a). T = 1 hr was the time when Ncad-GFP was started to be observed between sister cells. The F/Fmax curve in wild-type (n = 6) (b), Fzd7-ΔPDB (n = 6) (c) and Fzd7 (n = 7) (d) positive tissues was presented. Before cell division (T = 0 hr), Ncad distribution was generally uniform in the cells, as demonstrated by the flat profile of the blue lines. Shortly after division (T = 5 hr), Ncad-GFP was enriched in the junction, as suggested by the bell-shaped profile of the red and green lines. Later on (T = 10 hr), while junctional enrichment of Ncad was still maintained in wild-type (b) and Fzd7-ΔPDB (c) expressing cartilage, it vanished in Fzd7 expressing tissues (d). The dual-small-peak profile of the purple line confirms the premature separation of sister cells. See also Figure 6—figure supplement 3—source data 1, Figure 6—figure supplement 3—source data 2, Figure 6—figure supplement 3—source data 3.