NFIA Controls Telencephalic Progenitor Cell Differentiation through Repression of the Notch Effector Hes1

Delayed development of ventricular zone progenitors in Nfia mutants. A, B, Coronal paraffin sections of E16 wild-type and Nfia^−/− brains stained with hematoxylin. The ventricular zone was wider in the mutant (arrow in B). C, Quantification of ventricular zone width at E14, E16, and E18 revealed that the ventricular zone was significantly wider in the Nfia mutants compared to controls at both E16 and E18. D, E, PH-H3 immunohistochemistry on coronal paraffin sections of E16 brains. F, Quantification of the number of PH-H3-positive cells revealed significantly more mitotic cells in the mutant ventricular zone at both E16 and E18. G, H, Immunohistochemistry against Pax6 on coronal brain sections at E16. K, Cell counts demonstrated that there were significantly more Pax6-positive cells in the ventricular zone of Nfia mutants (arrow in H) compared to controls. No difference in the number of Tbr2-positive cells within the hippocampus was detected at E16. I, J, Tbr2 immunohistochemistry at E18 revealed higher expression of Tbr2 expression in Nfia^−/− hippocampi in late gestation. L, M, Expression of nestin in wild-type (L) and Nfia^−/− (M) brains at E18. N, qPCR on wild-type and Nfia^−/− hippocampal tissue revealed a significant increase in both nestin and Tbr2 mRNA levels in the mutant at E18. *p < 0.05, **p < 0.001, Student's t test. Scale bar (in M): A–H, 250 μm; I–M, 300 μm.

Neuronal development is delayed in the hippocampus of Nfia^−/− mice. A–F, Expression of Tbr1 in coronal sections of wild-type (A, C, E) and Nfia^−/− (B, D, F) brains. At E13, expression of Tbr1 was seen in the Cajal–Retzius cells at the pial surface of the wild type and mutant (arrows in A, B). At E14 in the wild type, a broad band of ventricular zone-derived Tbr1-positive neurons was observed within the ammonic neuroepithelium (arrowhead in C). In the mutant, however, these cells were not present at E14 (D), and were not detected until E16 (arrowhead in F). G–L, Birth dating of proliferating cells in the hippocampus. At E14, a pulse of BrdU was used to label cells in S-phase. Wild-type and Nfia^−/− hippocampi were analyzed 2 d later at E16. In the wild type, little colocalization of BrdU (green) and Tbr1 (red) was evident (G–I). J–L, In Nfia^−/− hippocampi, however, there were significantly more cells in which BrdU and Tbr1 were colocalized (L, arrowheads). Scale bar (in L): A, B, 80 μm; C, D, 100 μm; E, F, 250 μm; G–L, 50 μm.

Delay in the generation of subfield markers and dentate granule neurons. A–F, In situ hybridization against the hippocampal subfield markers SCIP (A, B) and KA1 (C–F). Expression of SCIP was markedly reduced in the mutant at E18 (open arrowhead in B), whereas expression of KA1 in the mutant was reduced at E16, but appeared comparable to the control at E18. G, qPCR on wild-type and Nfia^−/− tissue confirmed these findings, with SCIP mRNA levels at E18 and KA1 mRNA levels at E16 significantly reduced in Nfia^−/− mice compared to controls. H–K, Expression of Prox1 in coronal sections of wild-type (H, J) and Nfia^−/− (I, K) brains. At E16 in the wild type (H), Prox1-positive dentate granule neurons were observed in the dentate neuroepithelium (arrow in H) and within the incipient dentate gyrus (arrowhead in H). In the mutant, however, expression was only observed in the dentate neuroepithelium (arrow in I). At E18, expression of Prox1 in the mutant had recovered (K), but the dentate granule neurons did not form the distinctive chevron shape of the dentate gyrus that was seen in the control (arrowhead in J). L, qPCR on wild-type and Nfia^−/− tissue demonstrated an early delay in the generation of dentate granule neurons, as levels of Prox1 mRNA in the mutant were significantly reduced at E16, but not E18. *p < 0.05, Student's t test. Scale bar (in K): A, B, E, F, J, K, 300 μm; C, D, H, I, 250 μm.

Glial development is curtailed in the telencephalon of Nfia^−/− mice. Expression of GLAST (A–F) and GFAP (G, H) in wild-type and Nfia^−/− hippocampi. At E14, expression of GLAST in the wild type was detected in the ammonic neuroepithelium (arrow in A) and the fimbrial glioepithelium (arrowhead in A), whereas expression in the mutant was only observed in the fimbrial glioepithelium (arrowhead in B). At both E16 and E18, expression of GLAST in the ammonic epithelium of the mutant became apparent (D, F), but GLAST mRNA levels in the mutant were lower than in the control at both these ages (I). Expression of GFAP within the ammonic epithelium (arrows in G, H) and fimbrial glioepithelium (arrowheads in G, H) was markedly reduced in Nfia^−/− hippocampi. This finding was confirmed by qPCR, which demonstrated a significant decrease in Gfap mRNA in the mutant hippocampus at both E16 and E18 (J). At E18, expression of GFAP in the wild type was observed in the dorsal telencephalon (K, arrows) and the septum (arrowhead in K). In the mutant, GFAP expression in the cortex and septum was dramatically reduced, with low levels of GFAP observed only at the cortical midline (arrow in L). *p < 0.05, Student's t test. Scale bar (in L): A, B, 100 μm; C, D, 250 μm; E–H, 300 μm; K, L, 400 μm.

Upregulation of the Notch signaling pathway in Nfia mutants. A–R, In situ hybridization on E16 wild-type and Nfia^−/− brains. Expression of the Notch effectors Hes1 (A–D) and Hes5 (E–H) in the ventricular zone of the hippocampus and neocortex was markedly higher in the mutant compared to littermate controls (arrows in B, D, F, H). Dll1 (I–L) was also expressed at higher levels in the hippocampus (arrow in J) and cingulate cortex (arrow in L) of Nfia mutants. The proneural gene Ngn2 (M–P) was also expressed at a higher level in the hippocampus and neocortex of Nfia mutants (arrows in N, P). Expression of Mash1 was comparable between wild-type and mutant hippocampi (Q, R). S, qPCR validation of the microarray results using independent biological replicates from E16 wild-type and Nfia^−/− hippocampi. Genes in the top section were significantly upregulated, genes in the middle section were unchanged, and genes in the bottom section were significantly downregulated in Nfia^−/− tissue. Statistical significance for upregulated and downregulated genes was determined using a Student's t test with a significance level of p < 0.05. Scale bar (in R): A, B, E, F, I, J, M, N, Q, R, 250 μm; C, D, G, H, K, L, O, P, 400 μm.