Mitotic WNT signalling orchestrates neurogenesis in the developing neocortex (2021)
Figure 3

Figure 3. Lack of Ccny/l1 expression reduces asymmetric AP division and neurogenesis in the embryonic neocortex.

(A) IF for pan-cadherin, combined with DNA staining, to quantify asymmetric vs. symmetric division in VZ APs. Dashed white lines denote the cleavage plane, and solid lines mark the "cadherin hole". Asterisks mark the apical-most basolateral membrane flanking the "cadherin hole". Cell divisions were scored as symmetric or asymmetric when the cleavage plane bisects (left) or bypasses (right) the cadherin hole, respectively. Scale bar 2 μm.

(B) Quantification of symmetric vs. asymmetric AP division in E13.5 control and DKO neocortices, expressed as percentage of total AP divisions. Four independent embryos per genotype from three litters were analysed. A total of 110 and 99 cells were counted for controls and DKOs, respectively. Data are means ± SEM.

(C) IF for alpha-tubulin to visualize mitotic spindle and astral microtubules in E13.5 control and DKO neocortices. Central and apical-basal astral microtubules denoted by white and purple arrows, respectively. Scale bar 2 μm.

(D) Quantification of apical-basal, central and total (sum) number of astral microtubules in mitotic APs of control and DKO E13.5 neocortices. Data are means of microtubules per cell ± SEM (at least 10 cells counted per embryo)(n=3 embryos, 3 litters).

(E, F) In utero electroporation (IUE) of plasmids expressing GFP plus shControl (Co) or shCcny/l1 into the lateral ventricles of E13.5 wildtype embryos, followed by analysis at E15.5 or E17.5. (E) IF for GFP and Tbr2 in the E15.5 neocortex. (F) IF for GFP and the deep- layer neuron marker CTIP2 in the E17.5 neocortex. (E, F) White arrowheads denote double-positive cells. Insets of magnified cells (indicated by blue arrowheads) depict representative GFP + Tbr2 (E) or GFP + Ctip2 (F) co-staining. Scale bars 20 μm.

(G) Quantification of the percentage of GFP+ cells that are Tbr2+, in control and shCcny/l1 neocortices at E15.5 upon IUE at E13.5. Data are means ± SEM (n=3 embryos).

(H) Quantification of the percentage of GFP+ cells that are Ctip2+, in control and shCcny/l1 neocortices at E17.5 upon IUE at E13.5. Data are means ± SEM (n=4 embryos).

(I) IF for βIII-tubulin (Tuj1) of control (Ccny+/-; Ccnyl1-/-; shRNA Co) and mutant (Ccny+/-; Ccnyl1-/-; shRNA Ccny) NPC monolayer cultures grown in differentiation medium for seven days. Insets depict neurons with representative neurite morphology for controls and mutants. Scale bar 20 μm.

(J) Quantification of the percentage of Tuj1+ cells from (I). Data are means ± SEM (experiment performed twice in triplicates, representative experiment shown).

(K) Immunoblot analysis of protein lysates extracted from control and mutant NPCs prior to culture in differentiation medium, using an antibody against lysine 48-ubiquitin (K48-Ub) to label polyubiquitinated proteins targeted for proteasomal degradation. GAPDH, loading control. Two representative samples per genotype are shown.

Data information: Unpaired two-tailed t-test for all statistical analyses: ns, not significant; * p < 0.05, ** p < 0.01. p = 0.034 (both asterisks) (B); p = 0.0062 (D, apical-basal); p = 0.22 (D, central); p = 0.0022 (D, sum); p = 0.0030 (G); p = 0.030 (H); p = 0.0053 (J).