Figure 6. Birthdating assays demonstrate defects in laminar distribution.
(a) Experimental outline of birthdating assays: BrdU was injected at E12.5 and E16.5 and analyzed at P0 (B12.5;P0 and B16.5;P0). Total number of BrdU+ cells at P0 generated at E12.5 and E16.5 (b,j), and total number of Ctip2+ cells (layer V neurons; d,l) were not significantly different between respective controls and mutants, for both H1047R and E545K lines. (c) Distribution of BrdU+ cells in the neocortex was significantly different between control and hGFAP-cre;H1047R mutant for both early and late assays, with more cells residing in the lower cortical plate and white matter instead of mid and upper zones of the cortical plate. (e,m) Total number of layer V neurons in both H1047R and E545K mutants, born at E12.5 and at E16.5, did not significantly differ from the respective controls; but showed significant difference in their zonal distribution with Ctip2+BrdU+ cells predominating the lower cortical plate in both the mutants (f,n). Total number of Cux1+ neurons (layers II/III neurons; g,o) was significantly higher in both the mutants compared with the respective controls. The colocalization of Cux1 and BrdU was not significantly different in the H1047R mutant and control for both ages (h); but number of Cux1+ cells born at E16.5 was significantly higher in E545K mutant than in the control (p). (i,q) Zonal distribution of Cux1+ cells was significantly different between controls and mutants, with more Cux1+ cells residing at the lower portion of the P0 cortical plate. The H1047R mutant phenotype is more extreme than the E545K mutant. Data are represented as mean ± SEM. *p<0.05; **p<0.001; ***p<0.0001.