Foxg1 haploinsufficiency reduces the population of cortical intermediate progenitor cells: effect of increased p21 expression

Abstract

Foxg1 is a transcription factor that is critical for forebrain development. Foxg1(+/Cre) mice were used to test the hypotheses 1) that the subventricular zone (SZ) generates supragranular neurons, 2) that Foxg1-regulated activities define the output from the SZ, and 3) that Foxg1 is involved in the suppression of p21-initiated cell-cycle exit. Foxg1(+/Cre) mice have thinner neocortices than wild-type controls, specifically in the supragranular layers, as detected by Brn2 immunostaining. Cell proliferation in the ventricular zone (VZ) and SZ was examined to investigate the reduction in upper layer neurons. The number of cycling VZ cells was similar in Foxg1(+/+) and Foxg1(+/Cre) brains. Interestingly, cell proliferation in the SZ and intermediate progenitor cell (IPC) production (noted by Tbr2-immunostaining) was reduced in Foxg1(+/Cre) brains. These decreases coincided with increased expression of the cell-cycle inhibitor p21 in the VZ and SZ. Furthermore, colocalization of p21 with markers of cell proliferation and IPCs indicated that p21 was temporally expressed to influence the proliferative fate of IPCs. Thus, the present data are consistent with the above hypotheses, particularly, that during corticogenesis, Foxg1-regulated activities enable the expansion of the IPC population likely through suppression of p21-dependent cell-cycle exit.

Figure 1.

Expression of Foxg1. (A) A representative gel of PCR products from amplification of genomic DNA from Foxg1^+/+, Foxg1^+/Cre, and Foxg1^Cre/Cre mice. The 186- and 220-bp bands result from amplification of the Foxg1 wild-type and null allele, respectively. Foxg1 immunoblots showed decreased Foxg1 protein in the Foxg1^+/Cre cortex and the absence of Foxg1 protein in the Foxg1^Cre/Cre. (B) Immunohistochemical preparations show differential expression of Foxg1 in forebrains of 15.5-day-old wild-type and heterozygous mice. Scale bars are 500 μm.

Figure 2.

Cortical growth defects in the Foxg1^+/Cre fetal brain. The full depth of the cerebral wall is shown for Foxg1^+/+ and Foxg1^+/Cre fetuses on E13.5, E15.5, and E17.5. Samples were immunostained for Tuj1 expression. Disparity in thickness of the cortical plate (CP)was evident on E15.5 and E17.5 (see Table 1). IZ, intermediate zone; MZ, marginal zone; PP, preplate; SZ, subventricular zone; VZ, ventricular zone. Scale bars are 100 μm (G13.5) and 200 μm (E15.5 and E17.5).

Figure 3.

Brn2 and Tbr1 expression in cortex of Foxg1^+/+ and Foxg1^+/Cre mice on postnatal day 6. The organization of cerebral cortex in the Foxg1^+/+ and Foxg1^+/Cre mice is depicted in the cresyl violet–stained sections (left). Brn2 and Tbr1 (middle and right, respectively) were expressed in separate compartments of cortex. Brn2 expression in superficial cortex was reduced in Foxg1^+/Cre mice, whereas Tbr1 expression was similar in the deep laminae of both genotypes of mice (see Table 2). Scale bars are 100 μm.

Figure 4.

Cell proliferation in Foxg1 haploinsufficient mice. (A) Pregnant dams were injected with IdU and BrdU 1.5 h apart and fetuses were collected 30 min later. Representative images of IdU/BrdU/Ki-67 triple labeling from brains of Foxg1^+/+ and Foxg1^+/Cre mice on E13.5, E15.5, and E17.5 shows the distributions of BrdU−/IdU+/Ki-67+ (yellow arrows) close to the ventricular surface and BrdU+/IdU+/Ki-67+ (white arrows) in the outer third of the VZ and intermingled in the SZ. Note that the depth of the VZ was similar between the 2 genotypes at all ages, whereas the SZ was clearly reduced in the Foxg1^+/Cre fetuses. Scale bars are 100 μm. (B) Total cell-cycle length (T_C) (left) and the length of S-phase (T_S) (right) were determined for Foxg1^+/+ and Foxg1^+/Cre mice on E13.5, E15.5, and E17.5. * denotes a significant difference relative to the wild type at the same age, # indicates a significant difference from the mean T_C on E13.5 (within genotype), and @ denotes significant differences relative to the expression on E15.5 (within genotype). (C) The illustration describes the experimental paradigm for determining the cell-cycle kinetics (see Quantitative Analyses, Materials and Methods) and the color scheme to immunolabeling in the micrographs. (D) The total number of proliferating (Ki-67+ cells) cells in a 62 500 μm² field in the VZ (left) and SZ (right) is shown. Separate analyses for Foxg1^+/+ and Foxg1^+/Cre mice were performed. Notations as for (B).

Figure 5.

Production of IPCs in Foxg1^+/+ and Foxg1^+/Cre fetuses. (A) Sections of the cerebral walls of Foxg1^+/+ and Foxg1^+/Cre fetuses on E13.5 (top) and E16.5 (bottom) were immunolabeled with an antibody to Tbr2, an IPC cell marker. Scale bars are 100 μm. (B) Pregnant dams were injected with BrdU on E12.5 (top) or E15.5 (bottom) and their fetuses collected 16 (E13.5) or 24 h later (E16.5), respectively. Sections were processed for BrdU (red) and Tbr2 (green) immunolabeling. Yellow cells coexpressed BrdU and Tbr2. Scale bars are 100 μm. (C) The 2 graphs depict the total number of Tbr2+ cells in a defined portion of the cerebral wall of Foxg1^+/+ and Foxg1^+/Cre mice at E13.5 and E16.5 (left) and an estimation of Tbr2-cell production as determined by the number of Tbr2+/BrdU+ cells divided by the total BrdU+ population (right). Asterisks identify statistically significant (P < 0.05) differences between genotypes.

Figure 6.

p21 expression among Tbr2+ cells. (A) p21+ cells were evident in the VZ and SZ (green arrows) of brains of Foxg1^+/+ (left) and Foxg1^+/Cre (right) fetuses on E13.5 (top) and E16.5 (bottom). In both genotypes, p21+/Tbr2+ cells (yellow arrows) were present. Scale bars are 100 μm. (B) Quantification of p21+ cell number in Foxg1^+/+ and Foxg1^+/Cre at E13.5 and E16.5 reveals a temporal increase in p21+ cells in both genotypes, though significantly more so in the Foxg1^+/Cre cortex. Asterisks identify statistically significant differences between genotypes (P < 0.05).

Figure 7.

Expression of Tbr2 and p21 in proliferating and postproliferative cells. (Top) Sections of brains from 16.5-day-old Foxg1^+/+ (left) and Foxg1^+/Cre (right) fetuses were immunolabeled with antibodies to Tbr2 (blue), p21 (green), and PCNA (red), a marker of proliferating cells. Tbr2+/p21+/PCNA+ cells are indicated by white arrows, Tbr2−/p21+/PCNA+ cells are indicated by yellow arrows, and green arrows indicate Tbr2−/p21+/PCNA− cells. (Bottom) Most p21+ cells, both Tbr2- (green arrow) and Tbr2+ (blue arrows) did not coexpress p27, though examples of p21–p27 coimmunoreactivity were present (white and yellow arrows). Scale bars are 100 μm.