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, Flotillin 2 knockdown is correlated with a reduction in Flotillin 1 expression

, B-Differential interference contrast images of zebrafish embryos at different stages (4-, 16-and 1000-cell stage, 75% epiboly, Prim-6 and 25

, C-The graph shows the percentage of embryos that completed a normal epiboly (white), of embryos that were strongly delayed and reached 60-75% epiboly (orange) and of embryos that were arrested/dead between 50-60% epiboly (red) at 9.5 hpf. The number of embryos analyzed for each condition (from at least three independent experiments) is

, Flotillin 1 (right panels) distribution in the dorsal part of zebrafish embryos at the indicated stages. Inserts show the entire embryos. Bar = B-The distance (B) between the DC margin and the F-actin, D-Representative confocal images of Flotillin

). Moctrl and F. Mo, MoATGFlot2) embryos. Some cells are colored to follow their movement in the layer during the recording period. Stars indicate a DC inserted in the migrating layer

, D-Trajectories of DCs in control and Flotillin MOs during 20 min

E. , DCs in control and Flotillin MOs during 20 min

F. , duration was measured in DCs of control (MoCtrl) and Flotillin MOs (MoATGFlot2) during

G. , MoCtrl) and Flotillin MO (MoATGFlot2) embryos were co-injected with GFP-CAAX

, Shown are y-z projections of images from a 15-minute time-lapse recording to visualize cell tracking during the recording period. The lines indicate cell trajectories. The graph represents the trajectories of all cells

H. , I. , and K. , Boxplots represent the quantification of the total speed (H), instantaneous speed (I), net speed (J) and persistence (K) in control (MoCtrl) and Flotillin MOs (MoATGFlot2) embryos during 30 min

, L-Schematic representation of the different speeds measured in H, I and J

. From-c-to-f:-moctrl, MoATGFlot2 (n=54 cells from n = 10 embryos) embryos from at least three independent experiments. From H to K: MoCtrl (n=41 cells Figure 3: Flotillins knock-down impairs E-cadherin and ?-catenin accumulation at cell-cell contacts of DCs A-Zebrafish embryo injected with a mRNA encoding GFP-tagged Flotillin 2 and stained using an anti-E-cadherin antibody and Hoescht (nuclei)

, Flotillin 2 and E-cadherin signals were highlighted using the «Colocalization Threshold» tool from Image J and are shown in white. Pearson's coefficient = 0.64. Bar = 10 µm

?. B-analysis-of-e-cadherin and F. -catenin, DCs at 75% epiboly in control (MoCtrl) and Flotillin MO (MoATGFlot2) embryos by confocal microscopy. Bar = 10 µm

C. and D. , Intensity profiles of E-cadherin and ?-catenin signals in control (MoCtrl)(black) and

M. O. Flotillins, MoATGFlot2)(red) embryos were measured along lines (as shown in figure B) by line scanning

E. , F. , and G. , Boxplots represent the normalized intensity values of the F-actin, E-cadherin and ?-catenin signals at cell-cell contacts (CCC) in control (MoCtrl) and Flotillins MO (MoATGFlot2) embryos

, H-Quantification of the F-actin, E-cadherin and ?-catenin signal at CCC and outside CCC (NCC)

, Shown is the CCC signal/NCC signal ratio in control (MoCtrl) and Flotillins MO (MoATGFlot2) embryos

H. Panels-e-to, For E-cadherin: n = 20 cells from n = 12 MoCtrl embryos

, For ?-catenin: n = 14 cells from n = 8 MoCtrl embryos

. I-e-cadherin, ?-catenin and actin expression assessed by immunoblotting in protein extracts from control (MoCtrl) and Flotillins MO (MoATGFlot2) embryos (50 embryos were pooled for each condition)