The T-box transcription factor Eomes/Tbr2 regulates neurogenesis in the cortical subventricular zone

Abstract

The embryonic subventricular zone (SVZ) is a critical site for generating cortical projection neurons; however, molecular mechanisms regulating neurogenesis specifically in the SVZ are largely unknown. The transcription factor Eomes/Tbr2 is transiently expressed in cortical SVZ progenitor cells. Here we demonstrate that conditional inactivation of Tbr2 during early brain development causes microcephaly and severe behavioral deficits. In Tbr2 mutants the number of SVZ progenitor cells is reduced and the differentiation of upper cortical layer neurons is disturbed. Neurogenesis in the adult dentate gyrus but not the subependymal zone is abolished. These studies establish Tbr2 as a key regulator of neurogenesis in the SVZ.

Figure 1.

CNS-specific Tbr2 inactivation causes microcephaly and behavioral abnormalities. (A) Dorsal and lateral views show a significantly reduced size of the cortex (note the more exposed tectum, arrowhead) and OB in adult mutant brains. (B) Brain, but not body, weight is significantly decreased at P1 and P60. (C) Mutants display substantial hyperactivity in the open field. (D) Hang wire exam reveals reduced grip strength. (E) Layer-specific immunostaining shows significantly reduced upper cortical layers, however normal layering is maintained (n = 3 per group). (F) The CC is thinner and smaller in the rostro-caudal dimension (red arrow, also cf. in G) and the DG lacks the lower blade (arrowhead). (G) Coronal sections show normal ventricles and absence of the anterior commissure (arrowheads). (n = animal number in bars, mean ± SEM, t-test.)

Figure 2.

Decreased proliferation in the mutant SVZ. (A) pH3 staining reveals similar numbers of mitoses on the VS but significantly reduced numbers of mitosis in nVS areas. (B) Ki67, BrdU double-staining following 30-min BrdU pulse labeling shows reduced numbers of Ki67⁺ SVZ progenitors. Cell cycle length of mutant SVZ progenitors is unchanged as indicated by similar ratios of Ki67⁺, BrdU⁺ double-positive cells over total Ki67⁺ cells. (C) Ki67, BrdU double-staining following BrdU pulse labeling 24 h earlier reveals increase rate of cell cycle exit in Tbr2 mutants, indicated by the reduced ratio of Ki67⁺, BrdU⁺ double-positive cells over total BrdU⁺ cells. (D) Activated caspase3 staining of the E15 cortex demonstrates apoptosis is unaffected.

Figure 3.

Reduced expression of SVZ markers and decreased neuronal output. (A) In situ hybridization for the VZ marker Pax6 is largely unaffected, whereas expression of the SVZ markers Svet1 and Cux2 is markedly reduced in Tbr2 mutants. (B) Immunostaining for NeuroD and Tbr1 reveals reduced numbers of cortical neurons generated in the mutant cortex. (C) Birthdating experiment of P10 cortical sections showing BrdU-labeled neurons at E16. Generation of upper cortical layer neurons is significantly ([*] P < 0.05; [***] P < 0.005) reduced in Tbr2 mutants.

Figure 4.

Tbr2 is required for differentiation and functional organization of upper cortical layer neurons. (A,B) Expression of Satb2 (A) and Brn2 (B) in upper cortical layers is reduced, whereas Brn2 in layer V is maintained (red arrows). (C,D) Expression of lower cortical layer markers Otx1 (layers V, VI) (C) and Ctip2 (layer V) (D) appears unaffected. (E) The reduced expression of upper-layer markers is not due to a general differentiation defect as indicated by normal expression of NeuN. (F) DiI tract tracing of the interhemispheric connections reveals the development of Probst bundles indicative of misrouted callosal axons (arrows). (G,H) 5HTT staining (G) and DiI tracing (H) of thalamo-cortical afferents demonstrate disturbed somatotopic organization of the Barrel cortex (arrows depict an example for the characteristic clustering of thalam-cortical afferents in layer IV).

Figure 5.

Tbr2 is required for DG development. (A) Tbr2, Ki67 double-labeling at P5 shows Tbr2 expression in progenitors of the dentate migratory stream. Most Tbr2⁺ cells are proliferating; however, a substantial fraction of proliferating cells is Tbr2⁻. (B) Overall, the number of Ki67⁺ cells in the developing mutant DG is indistinguishable from wild-type littermates. (C) Activated caspase3 staining shows substantially increased apoptosis (arrowheads) in the dentate migration stream and hilus. (D) Neurogenesis in the dentate migration stream is strongly reduced as demonstrated by NeuroD expression (arrow, hilus; arrowheads, migratory stream). (E) The adult mutant DG shows strongly reduced number of Ki67⁺ progenitors. (F) DCX staining demonstrates the absence of neurogenesis. (G) In contrast, neurogenesis in the adult SVZ is unaffected. (n = animal number in bars, mean ± SEM, t-test.)