Defective postnatal neurogenesis and disorganization of the rostral migratory stream in absence of the Vax1 homeobox gene

Abstract

The subventricular zone (SVZ) is one of the sources of adult neural stem cells (ANSCs) in the mouse brain. Precursor cells proliferate in the SVZ and migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB), where they differentiate into granule and periglomerular cells. Few transcription factors are known to be responsible for regulating NSC proliferation, migration, and differentiation processes; even fewer have been found to be responsible for the organization of the SVZ and RMS. For this reason, we studied the ventral anterior homeobox (Vax1) gene in NSC proliferation and in SVZ organization. We found that Vax1 is strongly expressed in the SVZ and in the RMS and that, in the absence of Vax1, embryonic precursor cells proliferate 100 times more than wild-type controls, in vitro. The SVZ of Vax1(-/-) brains is hyperplastic and mostly disorganized, and the RMS is missing, causing a failure of precursor cell migration to the OBs, which as a result are severely hypoplastic. Moreover, we found that Vax1 is essential for the correct differentiation of ependyma and astrocytes. Together, these data indicate that Vax1 is a potent regulator of SVZ organization and NSC proliferation, with important consequences on postnatal neurogenesis.

Figure 1.

Vax1 expression in the SVZ and RMS. A, In situ hybridization revealing widespread Vax1 expression with in the WT telencephalic SVZ, SVZa, and RMS at P20 (sagittal cutting plane). Scale bar, 500 μm. B, In situ hybridization revealing Dlx1 expression in the SVZ, SVZa, and RMS at P20. Dlx1 labels transit amplifying cells (C-cells) and migrating neuroblasts (A-cells). Scale bar, 200 μm. LV, Lateral ventricle.

Figure 2.

Abnormal differentiation of the SVZ in Vax1^-/- mice. A, B, Hematoxylin and eosin staining of WT and Vax1^-/- mutant sagittal sections at P15. Note the expansion of the SVZ in the mutants. C, In WT brains at P15, the SVZa contains precursor cells delaminating from the SVZ and entering the RMS. D, In P15 Vax1^-/- brains, it is not possible to detect a defined SVZa. The insets show in detail the SVZa anatomy in WT (C) and Vax1 (D) mutants. E, Apoptotic cells were detected by TUNEL assay in the mutant SVZ (green signal and arrows). F, Many of the apoptotic cells shown in E, identified here by caspase-3 staining, colocalized with neuronal precursor cells (Tuj1 positive; arrows). G, Dorsal view of P15 WT (left) and KO (right) brains. Note the hypoplastic Vax1^-/- OB. LV, Lateral ventricle. Scale bars: A, B, E, 500 μm; F, 100 μm; G, H, 20 μm.

Figure 3.

Vax1^-/- OBs are severely hypoplastic, although correctly innervated. A, WT P20 OB shows well organized glomeruli (G), as revealed by TH staining. B, TH staining of the Vax1^-/- OB shows the complete absence of periglomerular cells (A, arrowheads). C, Mitral cells do not derive from migrating SVZ precursor cells and are immunoreactive for Reelin (arrowheads). D, The mitral cell layer is still present in the Vax1^-/- OB, although disorganized (see Results). E-H, DiI tracing of the olfactory nerve. The olfactory nerve is evident in P0 WT controls (E, G) and in the Vax1 mutants (F, H). All sections are in a sagittal orientation. CX, Cortex; ON, olfactory nerve. Scale bars: A, C, E-H, 500 μm; B, D, 200 μm.

Figure 4.

Vax1 mutants present an expanded SVZ, abnormally differentiated. A, B, Progenitor cells immunoreactive for the ETS transcription factor ER81 in the SVZ and in the OB of P0 WT (A) and Vax1 (B) mutants. A, Arrows indicate ER81-positive cells in the SVZ, in the RMS (dashed line), and in the outermost layers of the OB. In the absence of Vax1 (B), the SVZ appears mostly expanded, ∼10 times the size of its WT counterpart, generating an expanded zone (EZ). C, D, Sagittal sections of P0 WT (C) and Vax1^-/- (D) mutant telencephala analyzed by immunohistochemistry for the proliferation marker KI-67. In WT mice, proliferating cells are homogeneously distributed along the RMS, whereas in KOs they are located close to the ventricle (LV). The coronal section of control (E, G) and Vax1 (F, H) mutant telencephala at E15.5, analyzed by immunohistochemistry for ER81 (E, F) and ISL1/2 (G, H). CX, Cortex; cp, choroid plexus. Scale bars: A, B, 500 μm; C, D, 200 μm; E-H, 100 μm.

Figure 5.

The expanded SVZ contains long-term survival precursor cells and continuous proliferation throughout embryogenesis and postnatal life. A-A″, Birthdating experiments in WT mice. A BrdU pulse was given at E14 and subsequently revealed at P0 by immunohistochemistry in the SVZ (A′) or in the OB (A″), together with an anti-GABA antibody (green). B-B″, Same as in A-A″, but experiments were performed in Vax1^-/- mutants. C-C″, A BrdU pulse was given at P14 in WT mice and subsequently revealed by immunohistochemistry at P19 in the SVZ (C′) or in the OB (C″), together with an anti-neuronal nuclear marker (NeuN; green). D-D″, Same as in C-C″, but experiments were performed in Vax1^-/- mutants. A-D, Low-power magnification of sagittal sections stained with DAPI showing the analyzed areas (squares) in WT (A, C) and Vax1^-/- (B, D) mice. IZ, Internal zone of the mutant SVZ; EZ, external zone of the mutant SVZ. EZ, External zone; IZ, internal zone. Scale bars: A-D, 500 μm; A′-D″, 200 μm.

Figure 6.

Vax1 controls the proliferation of NSCs in vitro. A, B, Graphs indicate the growth rate of cultured NSCs isolated from the LGE of Vax1^-/- and WT mice at E15.5 (A) and P0 (B). At E15.5, in the absence of Vax1, the proliferation rate is increased of approximately two logarithmic orders of magnitude when compared with cells obtained from WT mice. At P0 (B), we could not detect any difference in proliferation between WT and Vax1^-/- cells. The total cell number is indicated on the y-axis, and days in culture are indicated on the x-axis. C, The table shows the mitotic fractions of WT and Vax1^-/- NSCs, isolated from E15.5 LGE, obtained through BrdU incorporation. Note statistically significant differences at 24 and 30 hr after plating, with Vax1^-/- cells displaying a higher rate of proliferation compared with WT NSCs (Student's t test; ^*p < 0.005). By cumulative BrdU labeling, no difference in the overall cell cycle length could be observed between WT and Vax1^-/- NSCs. D, No effect of Vax1 deletion on cell viability could be detected after 6, 24, 30, and 54 hr after plating, as determined by MTT assay. Values are expressed as absorbance at 550 nm.

Figure 7.

In the absence of Vax1, the SVZ contains undifferentiated ependymal cells. A, B, Electron microscopy images of ependymal cells of P20 wild types (A) and KOs (B). In the absence of Vax1, ependymal cells appear undifferentiated with an unusual morphology, light cytoplasms, and a reduced number of cilia (arrows indicate cilia; arrowheads indicate basal bodies). C, Quantification of multiciliated ependymal cells in the MGE and LGE of P0 Vax1^-/- brains and in the SVZ of WT controls. Note the strong reduction of multiciliated ependymal cells in the LGE of Vax1 mutant mice (the anlage of the mature SVZ) and the unvaried number of mature (uniciliated) ependyma in the mutant MGE. D, Reconstruction of a representative tract of the SVZ, highlighting the position and the characteristics of ependymal cells (gray; in case of a differentiated morphology; blue, in case of an immature morphology). Red lines indicate the nuclear membranes, and green lines indicate the cellular membranes. E, F, Immunohistochemistry for Noggin on coronal sections obtained from P15 WT (E) and Vax1^-/- (F) telencephala. Note the expression of Noggin in the ependyma (e; indicated by the arrows) of WT brains and the low levels (practically absent) of Noggin in Vax1 mutants. bb, Basal bodies; e, ependymal cell; LV, lateral ventricle. Scale bars, 100 μm.

Figure 8.

In the Vax1^-/- SVZ, astrocytes are absent or mostly immature. A-D, Immunohistochemistry using an anti-GFAP antibody, which labels astrocytes in the SVZ and in the RMS (arrows) of P15 WT (A, B) and Vax1^-/- (C, D) animals. In the absence of Vax1, astrocytes are detected in the SVZa and in the outermost area of the expanded zone (C; arrow). However, astrocytes are either absent or mainly disorganized in the RMS (D; arrows). E, F, EM images showing representative astrocytes from WT (E) and Vax1 (F) mutants. The arrowheads in E point to the readily detectable nucleoli, whereas the arrows indicate the evident circumvolutions of the nuclear membrane, typical hallmarks of mature astrocytes. Both of these features are absent or extremely reduced in the few astrocytes present in the Vax1 mutants (F). Scale bars: A, C, 500 μm; B, D, 100 μm.

Figure 9.

In Vax1^-/- mutants, neuroblasts fail to migrate to the OB. A, B, SVZ of WT and mutant Vax1^-/- mice at P0 analyzed by transmission electron microscopy. Although in wild type the SVZ contains many migrating neuroblasts, separated by clear intercellular spaces (arrows), the mutant SVZ does not present any anatomical evidence of migration. C-F, Cell migration tracing by DiI injection. In P0 WT brain (C, D), DiI-labeled cells in the SVZ can migrate to the OB after 48 hr in culture. The inset in C shows a migrating DiI-labeled cell in the OB with an elongated cell body a long leading process. No sign of migration was detected in mutant mice when a DiI crystal was placed in the internal expanded zone (EZ) (E, F). D, F, Bright-field images of C and E. Scale bars: C-F, 500 μm.

Figure 10.

Transplant experiments suggest a cell nonautonomous defect in Vax1 mutants. A, B, Low-magnification photographs showing the transplanted internal expanded zone (solid white line) from GFP-Vax1^-/- mice into a P0 WT organotypic slice culture. C, D, GFP-positive cells migrating from the transplanted internal expanded zone (A; blue square) and entering the OB after 3 d in culture. C, High-magnification of GFP-positive migrating neuroblasts in the OB (D; asterisk) with elongated cell bodies and a long leading process. E, F, Low-magnification micrographs showing the transplanted SVZ from GFP-WT mice into a P0 Vax1^-/- organotypic slice culture, showing few migrating cells from the explants. G-I, High-magnification of GFP-positive cells migrating from the transplanted SVZ. Labeled cells with elongated cell bodies and evident leading processes migrate in different directions (H, I), but no cells from the transplanted SVZ were found in the OB after 3 d in culture (J). Scale bars: A, B, E, F, 200 μm; C, G, 500 μm; D, H-J, 100 μm.