Technical Reference #1867

1867.

FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst Yojiro Yamanaka; Fredrik Lanner; and Janet Rossant, Hospital for Sick Children Research Institute, Development, 137(1867), (2010)
Link To Paper

Keywords:
ICM; Asymmetric division; Primitive endoderm; Epiblast; Nanog; Gata6; Mouse

Materials & Methods:
Time-lapse live imaging was performed on a Zeiss Axiovert inverted microscope equipped with Apotome and an environment controller. Embryos were placed on a glass-bottomed dish (MatTek) in KSOM covered with mineral oil. The objective lens used was 20 dry NA0.75. The camera used was an Axiocam MRm (1388 1044 pixels). For single eightcell tracing images were taken every 15 minutes with a 7-9 m section interval (six z-sections/time point) for 24-30 hours. The typical experimental condition was: for DIC image acquisition 2 2 binning camera gain 0 exposure time 5 milliseconds and 2.4 V halogen light volume with NA0.55 condenser setting; for RFP image acquisition Cy3 filter 2 2 binning camera gain 3 exposure time 80 milliseconds and the minimum light volume (20%) of a mercury light source with further 90% of cutoff by a neutral density filter. Twelve embryos were imaged in single experiments. After image acquisition the embryos were transferred individually to pseudo pregnant females (one embryo/single uterine horn). Embryos were recovered between embryonic day (E) 5.5 and E6.5 and the distribution of GFP expressing cells analyzed. No phototoxic effect was observed in this experimental condition as judged by a comparison of survival rates to term (control 13 pups/24 transferred; imaged 12 pups/25 transferred). To validate the accuracy of our positional live imaging tracing one researcher scored cell position using live imaging of 28 embryos which were then fixed following either the 8-16 or the 16-32 cell division. A second researcher counterstained each individual embryo with Par6 to visualize the apical membranes and independently scored cell position using the Quorum spinning disc confocal microscope with 1 m sections (see Fig. S6 in the supplementary material). Both methods agreed with each other confirming the accuracy of our experimental set-up. For PdgfraH2B-GFPmouse embryos images were taken every 15-20 minutes with 6-7 m section intervals (eight z-sections/time point) for 24-48 hours. The typical experimental condition for GFP image acquisition was: FITC filter 2 2 binning camera gain 3 exposure time 250 milliseconds and the half-light volume (50%) of a mercury light source with further 50% of cutoff by a neutral density filter.

Microscopic Technique
Time-lapse Imaging

Cell Type(s)
Mouse embryos