Article Title: Comprehensive characterization of migration profiles of murine cerebral cortical neurons during development using FlashTag labeling
Figure Lengend Snippet: Characterization of cell population labeled with the FlashTag (FT) technology. A-G: 1 mM of 5- or 6-(N-Succinimidyloxycarbonyl) fluorescein 3’,6’-diacetate (CFSE) was injected into the lateral ventricles (LV) at embryonic day (E)14 ICR mice and fixed 0.5 (A), 3.5 (B), 6.5 (C), 9.5 (D) hours later. Intraperitoneal bolus injection of 5-Ethynyl-2’-deoxyuridine (EdU) was performed maternally 0.5 hours before fixation. Photomicrographs from the dorsolateral cortex were shown. In (A), FT-labeled cells positioned most apically and were often positive for phospho-histone H3 (pH3) ( Figure 1F -0.5 hour, dorsolateral: 36.1± 4.0% [mean ± standard error of means], 339 cells from 5 brains; dorsomedial: 35.2 ± 4.7%, 249 cells from 5 brains) but negative for EdU administered at the same time ( Figure 1G , -0.5 hour, dorsolateral: 0 ± 0%, 339 cells from 5 brains; dorsomedial: 0 ± 0%, 249 cells from 5 brains). The nuclei of EdU positive cells positioned basally in the VZ. 3.5 hours after FT injection, FT-labeled cells left the ventricular surface but still near it, and were no longer positive for pH3 (B) ( Figure 1F , -3.5 hours, dorsolateral: 2.8 ± 0.7%, 530 cells from 5 brains; dorsomedial: 1.1 ± 0.5%, 415 cells from 5 brains). 6.5 hours after labeling, almost no cells were adjacent to the lateral ventricle (C). 9.5 hours after labeling, most of the labeled cells were in about basal two thirds in the VZ and double labeled for EdU, suggesting that some of them reentered the S-phase (D) ( Figure 1G , -9.5 hours, dorsolateral: 15.9± 2.5%, 711 cells from 5 brains; dorsomedial: 33.4 ± 6.6 %, 546 cells from 5 brains). Schematic presentation of these experiments was shown in E. In (F), percentages of pH3+ cells out of the FT-labeled cells were shown. Magenta, pH3+ FT+/FT+ in the dorsolateral cortex. Green, pH3+ FT+/FT+ in the dorsomedial cortex. In (G), percentages of EdU+ cells out of FT-labeled cells were shown. Orange, EdU+ FT+/FT+ in the dorsolateral cortex. Blue, EdU+ FT+/FT+ in the dorsomedial cortex. H-L: EdU was administered 3 (I), 6 (J) and 9 (K) hours before FT labeling. 0.5 hours after FT labeling, brains were harvested. Schematic presentation of these experiments was shown in (H). Nuclei of the EdU labeled cells positioned more apically in brains in which EdU was administered 3.5 hours before fixation (I) compared with A, and some of the EdU labeled cells positioned at the ventricular surface to enter M phase (interkinetic nuclear migration). In brains which EdU was administered 6.5 (J) and 9.5 (K) hours before fixation, EdU-labeled cells positioned even more apically. In these mice treated with EdU 3-9 hours prior to FT, FT-labeled cells were often co-labelled with EdU (I-K) ( Figure1L , dorsolateral, -9.5 hours: 76.6 ± 2.4%, 328 cells from 5 brains; -6.5 hours: 96.1 ± 0.5 %, 304 cells from 5 brains; -3.5 hours: 81.2 ± 1.9 %, 263 cells from 5 brains. Dorsomedial, -9.5 hours: 65.1 ± 1.5%, 369 cells from 5 brains; -6.5 hours: 96.7 ± 1.2 %, 217 cells from 5 brains; -3.5 hours: 81.5 ± 1.9 %, 287 cells from 5 brains). Note that EdU and FT never co-labeled when administered simultaneously (A). In the graph in (L), percentage of EdU+ cells out of FT-labeled cells were shown. Data for -0.5 hours in (L) corresponds to those for -0.5 hours in (G). Orange, EdU+ FT+/FT+ in the dorsolateral cortex. Blue, EdU+ FT+/FT+ in the dorsomedial cortex. M-O: CytoTell Blue was injected into the LV of the E12.5 (M-N) and 15.5 (O) GAD67-GFP brains. In E15.5 dorsolateral cortex labeled at E12.5, most of the labeled cells (red) were in the deep part of the cortical plate (CP) (M, N). Vast majority of the labeled cells were negative for GFP (E12.5-15.5 dorsolateral cortex, 93.3 ± 2.5%, 1653 cells from 3 brains) (N, N1-3). In postnatal day (P)1 dorsolateral cortex labeled at E15.5 (O), most of the labeled cells were found in the superficial gray matter. Again, vast majority of the labeled cells were negative for GFP (E15.5-P1, 95.5 ± 0.5 %, 1455 cells from 5 brains) (O, O1-3). Arrowheads in (N) and (O) show rare examples of cells positive for both FT and GFP. EdU, 5-Ethynyl-2’-deoxyuridine; DAPI, 4’,6-diamidino-2-phenylindole; FT, FlashTag; pH3, phospho-histone H3. GAD-GFP, Glutamate decarboxylase 67-green fluorescent protein, VZ, ventricular zone; MAZ, multipolar cell accumulation zone; IZ, intermediate zone; SP, subplate; CP, cortical plate; PCZ, primitive cortical zone; MZ, marginal zone; LI, cortical layer I; GM, gray matter; WM, white matter. Scale bars, 20 μm (A-D, I-K), 50 μm (N, O), 200 μm (M).
Article Snippet: When nuclear staining was performed without immunohistochemistry, sections were immersed with PBS for more than 30 minutes at RT and incubated with 2.5 ng/μl of 4’,6-diamidino-2-phenylindole (DAPI; D3571; Thermo Fisher Scientific, Waltham, MA) or 0.5 μM of TO-PRO3 Iodide (T3605, Thermo Fisher Scientific) at RT for 1 hour.
Techniques: Labeling, Injection, Mouse Assay, Migration