Fig. S1

Fig. S1

Epithelial Properties of the Pre-EVL Surface Layer and Imaging, Related to Figure 1

(A) Junction and polarity analysis of the surface cells. The schematic illustration shows two adjacent surface cells. Red indicates tight junctions as shown in ZO-1 staining images on the right; yellow indicates adherens junctions as shown by E-cadherin (CDH1) staining; green corresponds to the basal-lateral marker lgl as shown in (B); purple indicates aPKC (Fukazawa et al., 2010 and Krens et al., 2011). Arrows indicate focused staining signals. Scale bars: 20µm.

(B) Live imaging of embryos injected with lgl-GFP (Chalmers et al., 2005) mRNA. The GFP signal is detectable by 64-cell stage albeit being quite weak. GFP is detected on the basal lateral surface but not the apical surface. Scale bars: 10µm.

(C) Diffusion barrier function of the surface layer (pre-EVL). Embryos (128,256-Cell stages) were soaked in fluorescent dyes of different sizes and imaged for <20 min. No dye signal was detectable under the surface layer even when the exterior imaging signal is saturated. As a positive control the 10kd dye was injected into the embryo. Dye signal was found between cells (arrows) and stayed (at least 20 min). Embryos are transgenic for h2b-EGFP and mem-mCherry2.

(D) Schematic illustration of imaging timeline and protocol, including imaging set-up and spatial-temporal coverage and resolution. Embryo illustrations courtesy of Kimmel et al. (1995). See also Extended Experimental Procedures.

(E) Example time courses focusing on a lateral view and a top view, respectively. Fluorescent proteins labeling the membrane and the nucleus of cells were used. Lateral view: mem-EYFP and h2b-tomato; top view: mem-mCherry and h2b-EGFP. The lateral view images were generated using a data set partially published in Xu et al. (2012).

(F) Extended timelapse imaging data following Figure 1D. Scale bars: 20µm.

(G) Error analysis of L/R simplification in zebrafish surface cells. The image (3D maximum projection) illustrates free cell surfaces from a top view. The measurement of L in cross-sections (Figures 1D and S1F) brings an error as a difference between L1 and L2 exists.

(H) Comparison of L1/L2 and L1/R at different stages in surface cells. The L1/R difference is 13.5% ± 14.3% while L1/L2 difference is 0.5% ± 11.4%. n = 51. This indicates that using either L1 or L2 for calculating < L/R > is equivalent. The difference between L1 and L2 has a standard deviation of <11% which introduces a small amount of variability in L/R values that affects the accuracy of reported L/R distribution. See discussion in Data S1, Text 2.

(I) tg(krt4:egfp-caax) EGFP expression in differentiated EVL cells (Krens et al., 2011).

(J) Time course of tg(krt4:egfp-caax) EGFP intensity in surface cells and deep cells. n = <70 cells per time point measured. At 5.5h p = 0.08; p = 5e-6; p = 1e-14 (t tests).