Figure 4.
MADM Reveals the Presence of Ependymal-Ependymal and Ependymal-Astrocyte Divisions at E13.5 and E14.5
(A) Experimental schematic. The Cre plasmid was electroporated in utero in MADM-11TG/GT at E13.5 or E14.5, and V-SVZ WMs were analyzed at P15–P20.
(B) Schematic representation of Cre-mediated MADM clone induction in dividing RGCs. A G2-X event results in clones of red- and green-labeled cells, and a G2-Z event generates double-labeled (yellow) and unlabeled clones of cells. Recombination occurring in G0/G1 phases of the cell cycle leads to double-labeled (yellow) cells.
(C–G) Airyscan confocal image of a P15 MADM-labeled V-SVZ whole-mount electroporated with a CRE-expressing plasmid at E14.5. The ventricular wall was stained with EGFP (green), tdTomato (red), and FOP (white) antibodies. (C) Double-labeled yellow cells issued from a G2-Z recombination event are indicated by yellow arrows. Ependymal-ependymal (D and E) and ependymal-astrocyte (F and G) clones of two sister cells are shown at high magnification (D and F) and in a 3D view (E and G). See also Videos S1 and S2.
(H) Mean percentages of all clones generated from in utero electroporation with Cre at E13.5 or E14.5 according to the number of cells per clone (n = 6 and 16 animals at E13.5 and E14.5, respectively) are represented in a histogram. Also shown are dotted curves fitting both the E13.5 and E14.5 distributions; p values were determined with the χ2 test for trend; ∗p ≤ 0.05.
(I) Average distance between cells composing the clones. Error bars represent the SEM of 29 and 44 clones at E13.5 and E14.5, respectively; p values were determined with a Mann-Whitney test; ∗∗p ≤ 0.01.
(J) Mean percentage of all clones generated from E13.5 or E14.5 containing ependymal cells only or a mixed population of ependymal cells and astrocytes (B1). Error bars represent the SEM of 29 and 44 clones at E13.5 and E14.5, respectively; p values were determined with a two-proportion Z test; ∗p ≤ 0.05.
(K) Mean percentage of ependymal and B1 cells in all clones generated from E13.5 or E14.5. Error bars represent the SEM of 117 and 134 cells at E13.5 and E14.5, respectively; p values were determined with a two-proportion Z test; ∗p ≤ 0.05.
(L) Mean percentage of E-E, E-B1, and B1-B1 cell divisions in all clones generated from E13.5 or E14.5. Error bars represent the SEM of 24 and 54 cell divisions at E13.5 and E14.5, respectively; p values were determined with a Mann-Whitney test; ∗p ≤ 0.05.
The scale bars represent 30 μm (C) and 8 μm (D–G).