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Fig. 8 Centrosome positioning is uncoupled from migratory directionality in Cadherin-2 deficient GCs.
(A, B) Example for quantification of GC polarization using dorsal view confocal projections of right cerebellar lobes in WT (A) and pac-/-R (B) gata1:GFP embryos. For quantification, the polarization of GFP-expressing GCs (LWR see Figure 4B) as well as the orientation of the long axis of a GC with respect to the MHB was determined. (C) The angle between the long axis of a GC and the linear axis between MHB and URL is plotted as function of their respective LWRs. Whereas WT GCs preferentially polarize in anterior-lateral directions toward the MHB (red squares, n = 58), pac-/-R GCs display no preference in polarization, being randomly oriented in the cerebellum (blue dots, n = 66). (D, F) Method for quantification of centrosome position in GCs co-expressing the centrosome-localized centrin-td-Tomato fluorescent protein (white arrows mark centrosome, yellow dashed lines mark soma). (E) While the centrosome in more than 70% of analyzed WT GCs (red bars, n = 98) is localized toward the MHB, the centrosome in pac-/-R GCs shows no preferred orientation (blue bars, n = 120). (F) Quantification of centrosome position with respect to cell morphology. (G) In WT GCs, the centrosome preferentially locates to the leading edge, while no preferred location is found in pac-/-R GCs. (H, I) The relationship between centrosome and leading edge dynamics was quantified by simultaneously tracing these structures within the same GC (n = 3, each group), using the ImageJ manual tracking tool. (H) In WT GCs, the centrosome movements follow leading edge movements thus moving in the same direction (I, n = 13/17, see Video S12), whereas in pac-/-R GCs centrosome movements independent of the leading edge are apparent often occurring in opposite directions (I, n = 7/18 movements in same direction, see Video S13). Lat, lateral; LE, leading edge; Med, medial; MHB, midbrain-hindbrain boundary; URL, upper rhombic lip.