Animals and in utero electroporation
Timed-pregnant C57BL/6JRj mice were anaesthetized by intraperitoneal (i.p.) injection of saline solution containing fentanyl (0.05 mg/kg), midazolam (5 mg/kg), and medetomidine (0.5 mg/kg). Plasmids pCAG-KDEL-sfGFP and pCAG-Sec61a-EGFP were diluted in PBS to reach a final concentration of 0.25 mg/mL and 3 mg/mL respectively and injected in the lateral ventricle of the brain of embryos from e12 to e16. Each plasmid was injected with Fast Green (2.5 mg/mL, Sigma) and electroporated using Electro Square Porator ECM830 (Harvard Apparatus) and tweezertrodes of 3 mm (Harvard Apparatus) targeting the brain cortex. Pulses were applied 5 times at 30 V for 50 ms at 950 ms intervals. Anaesthesia was reversed using i.p. injection of saline solution containing atipamezole (2.5 mg/kg) and flumazenil (0.5 mg/kg) plus buprenorphine (0.1 mg/kg). Animals were allowed to recover in a warm pad and kept under observation. The electroporated embryos were dissected as described by Shitamukai and colleagues. Briefly, 24 h after electroporation, slices of dorsal cortex were cut (approximately thickness of 300 mm) using an ophthalmic microsurgical knife (Alcon), embedded into collagen matrix (Nitta Gelatin, Cell Matrix-type A) and placed in a cell culture insert with 0.4 mm pore membrane (Millicell, 30 mm, Millipore). The insert containing the tissues was placed in a bottom glass dish containing DMEM and F12 (Sigma D6434) with supplements (FBS, HS, N2, B27 minus vitamin A, PSF, Glucose, Glutamax, FGF, EGF). The slices were cultured for 2 h (AP) or 16 h (BP) in cell culture incubator (37°C/ 5% CO2) before the FLIP experiments start.
Using a confocal laser-scanning microscope (Zeiss LSM 800, Carl Zeiss) with a water-immersion 40X objective lens (LUMPlanFl NA0.8, Olympus), heating stage and incubation chamber, brain slices were screened for cells in metaphase, apparent by spherical shape and condensed DNA. Apical mitoses were defined by location at the ventricular surface (apical progenitors, APs) and discriminated from basal mitoses, which occur at the subventricular zone (basal progenitors, BPs).
All cells were imaged in a picture frame size of 512×512 pixels and a scanning speed of 7 ms per pixel. At anaphase, a ROI was drawn in one of the poles avoiding the condensed DNA. After an initial single plane picture was taken, the ROI was bleached for approximately 3 s with the 488 nm laser line at 7% laser power (“bleaching”), 30 iterations. The alternating cycles of “bleaching” and “image acquisition” for FLIP were repeated with an interval of 12 s until cytokinesis was finished, apparent by the time point when an obvious darker line separated the two newly formed daughter cells. The laser settings were kept consistent between experiments, with laser power in the 488 nm wavelength at 4% (Sec61a-GFP) and 2.4% (KDEL-GFP) (580–750 V). A photobleaching control to determine the loss of fluorescence due to repeated imaging was carried out by measuring the loss of fluorescence in the cells that did not receive the ROI for photobleaching.
To analyze the experiments, ROIs were created in ImageJ based on the future cleavage plane in order to separate the bleached and unbleached compartments. At each frame, the mean gray value (the sum of the gray values of all the pixels in the selection divided by the number of pixels) was obtained for each compartment. The analysis was followed by normalizing fluorescence values to the first image that was taken for that compartment. After that, the normalized fluorescence value of the bleached compartment was taken at each timepoint and subtracted from the unbleached compartment. Thus, one value could be given at each time point for each cell, and all cells compared within the same graph. This analysis shows the relationship between the 2 compartments over time, such that the greater the difference of the fluorescence intensity between the two compartments (for example, a strong barrier), the larger the value. A line that remains fairly flat (for example, KDEL-GFP, LumER-GFP) would have little differences between the unbleached and bleached compartments. Cells were separated for analysis into either apical or basal progenitors. Single-cell analysis revealed different populations of cell behaviors, thus further binning was performed to characterize these specific cell populations.
To distinguish between the cells with and without a diffusion barrier, we created 3 criteria. We took the highest value of fluorescence intensity from the averaged LumER-GFP single-cell traces (specific to the type of progenitors analyzed), and then using 6 times its standard deviation, created the first criteria to discriminate between the 2 different populations of Sec61+ cells. Since the different cell population might possess distinct bleaching behavior, we establish two additional criteria (and a cell must to fit 2 out of 3 to be considering possessing a barrier). The three criteria were: I) have at least 1 time point higher than 6 times its standard deviation; II) the difference between the average of plateau (frames 10 to 25) need to be kept higher than 4 times its standard deviation; and III) the difference between the average of the peak (frames 3 to 7) and the average of the plateau cannot be higher than 30%. Using these criteria, we separate those which had a barrier, and those that did not. The single-cell traces of these 2 distinct bins were then averaged separately to demonstrate the different behaviors of these populations. Statistical analyses to determine Barrier strength were performed at each time point as following: the first image (pre-bleach) was attributed to be 100%, and all measurements after were normalized to this initial image. Then, to combine the data from all cells, the normalized fluorescence of either the unbleached or the bleached compartment at each frame is averaged together from all cells at the same frame. Graphs represent data for all the timepoint where there are at least 3 cells that still have movie frames present. Videos’ scale bar: 5 mm.
To perform statistical analysis on these curves, the traces from all cells were used to make an average non-linear fitted curve. A one-phase association was used with Y0=0, and the K and Plateau unconstrained. The numbers obtained from all of these analyses provided the barrier index (“plateau” from the best-fit values) and the standard error (“plateau” from the standard error, SE, values). This Barrier Index (barrier strength) and SE were then entered into Prism for statistical analyses.
Analyses were performed using Excel (Microsoft) or Prism (Graphpad). For comparisons of averages among three or more groups, ordinary one-way ANOVA followed by Tukey’s multiple comparison test was performed. Differences were considered significant at p <0.05.