Sp8x microscope

To analyze cardiac fusion (Fig. 1A’–E’) Tg(myl7:eGFP) embryos were fixed, manually deyolked and imaged with a Leica SP8X microscope. To analyze the anterior endoderm (Suppl. Fig. 3N–P) Tg(sox17: eGFP) embryos were fixed and imaged with an Axio Zoom V16 microscope (Zeiss).
For live imaging, Tg(myl7:eGFP) embryos were exposed to DMSO or 20μM LY at bud stage and mounted at 12 somite stage as described (84 (link)). Mounted embryos were covered with 0.1% DMSO/20μM LY in Tricaine-E3 solution and imaged using a Leica SP8 X microscope with a HC PL APO 20X/0.75 CS2 objective in a chamber heated to 28.5 °C. GFP and brightfield stacks were collected approximately every 4 min for 3 hours. After imaging, embryos were removed from the mold and incubated for 24 hrs in E3 media at 28.5 °C. Only embryos that appeared healthy 24 hours post imaging were used for analysis. The tip of the notochord was used as a reference point to correct embryo drift in the Correct 3D direct ImageJ plugin (85 ). Embryos were handled similarly for imaging protrusions, except 15 confocal slices of 1μm thickness were collected every 1.5 min with a HC PL APO 40X/1.10 CS2 objective.

Images were captured with a Leica SP8X microscope. For immunofluorescence analyses, the mouse organoids were washed with PBS for 1 hr, permeabilized with PBD0.2T (48.5 mL of PBS, 1 mL of 10% Triton, 0.5 mL of DMSO, and 0.5 g of BSA), and incubated with antibodies at 4°C overnight.
For live-cell analysis, sorted STAR⁺ and STAR^neg cells were plated on a glass-bottom 384-well plate (Corning 4581) and mounted onto a Leica SP8X microscope. The plate was held at 37°C in a microscope box and equipped with a culture chamber for humidity and 6.4% CO₂ overflow. Organoids were imaged in XYZ(T)-mode using a water 25× objective (HCX IRAPO L; numerical aperture [N.A.], 0.95) with a tunable white light laser. For higher magnification and identification of STAR-positive cells, we used a water 40× objective (HC PL APO CS2; N.A., 1.1). Post-acquisition analyses of phenotypes was performed manually using ImageJ.
For the colony-forming efficiency assays, 3,000–5,000 viable single cells were collected via FACS and plated over five wells with a density of 50–300 cells per well of a glass-bottom 384-well plate. Using bright-field and live-fluorescence imaging, single cells were monitored at day 0 and at several days of culture organoid to quantify organoid formation (colony-forming efficiency %).

To determine apical membrane eviction rates, W4 cells were transfected with Dendra-Cdc42(HVR), polarized, and imaged at 37°C in Hepes-buffered (pH = 7.4) Leibovitz’s L-15 medium (Invitrogen). Photoconversion and imaging were performed using an SP8x microscope (Leica) equipped with a temperature- and CO₂-controlled chamber using a 63× objective (HC Plan Apochromat 63×/1.40 oil) with LAS AF image acquisition software (Leica). Photoconversion of the Dendra fluorophore was performed with a pulse of 405-nm laser, and imaging was performed with a 1.5-s frame rate. To generate a fluorescent decay trace, the ratio of photoconverted (red) signal in the brush border and the total amount of red signal in the cell were determined for each time point. Moving regions of interest were drawn to correct for cell movements during imaging. Ratios were normalized and multiple traces were averaged to generate a mean fluorescence decay trace. Single exponential curve fitting was performed on the mean decay traces using MATLAB software with the general formula: f(x) = a · e^{(−b · x)} + c. A mean half-life was determined using the fitted curve. For statistical analyses, half-lives of the individual decay traces were determined and compared using an independent samples t test in SPSS with a p-value <0.05 as a cutoff for significance.