Live imaging of whole mouse embryos during gastrulation: migration analyses of epiblast and mesodermal cells.

Mice Histone H2B-GFP and -mCherry transgenic mice were obtained from T. Fujimori (National Institute for Basic Biology) [41] , [42] . Heterozygous embryos from intercrosses between wild-type ICR and Histone H2B-GFP ( Figure 2 , 3 , 4 , S2 ) or H2B-mCherry ( Figure 5 ) were used. All animal experiments were carried out along the guidelines and the approval of The Institutional Animal Care and Use Committee of National Institutes of Natural Sciences (Permit Number: 10A087, 10A90).

Embryo culture and imaging conditions

Mouse embryos were dissected into phenol red–free Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), preserving a portion of Reichert's membrane at the ectoplacental cone. Embryos were moved to the pocket space of the specially designed embryo holder which has been filled with medium culture from window of the cylinder ( Figure 1C ). Embryos were set into the hole of the holder and then the holder was pushed until the end of the cylinder. The embryos stayed at the position without window in order to prevent exposure of the embryo to air. The cylinder and holder was set at the stage and inserted into the culture medium in the chamber. The cylinder is elongated due to the length of cylinder holder. Embryos were cultured at 37°C with 5% CO 2 and 5% O 2 , in medium containing 40% phenol red–free DMEM, 10% FCS (Invitrogen), 50% rat serum, 100 µM Trolox (Cayman), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Gas was mixed by a GM-6000 from TOKAIHIT and blown into the air gap of the chamber. The sample holder was attached to the sample positioning system, which comprises three linear translation stages (M-111.2DG, Physik Instrumente) and one micro-rotation stage (M-116DG, Physik Instrumente). Three-dimensional image stacks were acquired every 1.5 or 3 min with a 300 ms exposure time per image and a z-spacing of 2.58 µm.

Microscopy

We used a Digital Scanned Laser Light Sheet Fluorescence Microscope (DSLM) that was modified from our previously reported implementation [8] . An argon-krypton laser (35 LTL 835–200, Melles Griot) was used as the light source. The wavelength of the laser beam (488 or 568 nm) was selected using an acousto-optical tunable filter (AA.AOTF.nC-400–650 nm-PV-TN, AA Opto-Electronic) and scanned through the sample using a two-axis high-speed scan head (VM500+, GSI Lumonics). The scanned light sheet was created with an f-theta lens (S4LFT0061/065, Sill Optics) and a low-NA objective lens (Plan-Apochromat 5×/0.16, Carl Zeiss). Fluorescence emitted from probes was detected using a water immersion lens (Achroplan 20×/0.5, Carl Zeiss) and recorded with a CCD camera (Orca AG, Hamamatsu) through a long pass filter (RazorEdge RU 488 or 568, Semrock). The image data were recorded using our custom DSLM control software, which was developed in the Microsoft. NET framework. Illumination intensity was measured at the focal point of the illumination objective lens.

Fixation and labeling

Dissected embryos were fixed and permeabilized overnight at 4°C in 4% paraformaldehyde in phosphate-buffered saline (PBS, Sigma) with 0.1% Triton X-100 (Sigma). After thorough washing, embryos were stained with Alexa Fluor 546 phalloidin (1∶100, Invitrogen) and 1 µM DRAQ5 (1∶1000, Biostatus) for one hour at room temperature. Pre-processing of image data and analysis of tracked epiblast and mesodermal cells The acquired data were cropped and aligned in time and space with custom macros in ImageJ (National Institutes of Health). For tracking nuclei in epiblast, we used a custom-made assistant program for manual tracking, which records positions indicated by the user. For mesodermal cell tracking, we used another custom-made program that automatically track nuclei after the user points the position at first time point. The tracking results were confirmed by human eye, and wrong ones were deleted. Both tools were written in C++ (code available upon request). The automated tracking is based on an image recognition algorithm pointing the center of the nuclei. To reconstruct nuclear positions as 3D computer graphics shown in Figure 5 and Movie S4 to 8, we used POV-Ray ( http://www.povray.org ). Statistical analysis was performed in Microsoft Excel. Nuclear density in Figure 4B was calculated as the number of nuclei per 100 µm curve along the apical or basal surface of epiblast. The curve was drawn 8 µm from the apical or basal tips of the nuclei. The meddle was drawn at the center of the epiblast along its apical–basal axis. A nucleus was counted when the curve crossed the apical-basal axis at a point more than 20% of the axis length from the nearest end. The distance of the nearest-neighbor nuclei shown in Figure 4C was calculated as a distance between a nucleus and nearest nucleus in a cone having bottom of same spherical radius as the nucleus and height of twice radius at the nuclear center toward apical or basal side. Nuclear radius is set as 11 µm at apical side and 16.6 µm at basal side and it increases linearly along apical–basal axis. These values were measured with ImageJ (apical n = 30, basal n = 42). Classified regions of mesoderm in Figure 5 were defined according to the following criteria: where = angle between right–left axis and proximal–distal axis, ‘lateral’ was defined as 0°≤