Pten deletion causes mTorc1-dependent ectopic neuroblast differentiation without causing uniform migration defects

Abstract

Neuronal precursors, generated throughout life in the subventricular zone, migrate through the rostral migratory stream to the olfactory bulb where they differentiate into interneurons. We found that the PI3K-Akt-mTorc1 pathway is selectively inactivated in migrating neuroblasts in the subventricular zone and rostral migratory stream, and activated when these cells reach the olfactory bulb. Postnatal deletion of Pten caused aberrant activation of the PI3K-Akt-mTorc1 pathway and an enlarged subventricular zone and rostral migratory stream. This expansion was caused by premature termination of migration and differentiation of neuroblasts and was rescued by inhibition of mTorc1. This phenotype is reminiscent of lamination defects caused by Pten deletion in developing brain that were previously described as defective migration. However, live imaging in acute slices showed that Pten deletion did not cause a uniform defect in the mechanics of directional neuroblast migration. Instead, a subpopulation of Pten-null neuroblasts showed minimal movement and altered morphology associated with differentiation, whereas the remainder showed unimpeded directional migration towards the olfactory bulb. Therefore, migration defects of Pten-null neurons might be secondary to ectopic differentiation.

Fig. 1.

The PI3K-mTorc1 pathway was inactive in migrating SVZ neuroblasts, but activated in OB neuroblasts. (A) Schematic brain sections indicating anatomical regions in the SVZ and OB (black boxes) from which images were captured. (B-D) IF staining of the SVZ (B,C) and the terminal RMS at the OB core (D) in coronal sections from 6-month-old (B,D) or 18-day-old (C) wild-type mice. (B) Pten (green) and Dcx (red) double IF labeling of the SVZ. Nuclei were labeled with DAPI (white). Pten was expressed in Dcx+ cells in the SVZ (overlay of Pten and Dcx, far right panel). (C,D) Mash1 (white), Dcx (red) and p-S6 (green) triple IF labeling of the SVZ (C) and OB (D) (overlay, far right panel). p-S6 was expressed in Mash1+ transit-amplifying cells but not Dcx+ neuroblasts in the SVZ (C), whereas in the OB, a substantial population of Dcx+ neuroblasts expressed p-S6 (D). The white dashed lines in the overlay mark the boundary of the SVZ in panels A and B. Scale bar: 20 μm.

Fig. 2.

Postnatal Pten deletion caused ectopic differentiated neurons in an expanded SVZ. Representative Hematoxylin & Eosin (H&E) (A,B), IHC (C-E) and IF (F) staining of the SVZ in matched coronal sections from mice injected with TM on P30-32 and analyzed more than 70 days later (n=24). A and E upper and lower panels show dorsal and ventral SVZ, respectively. (A) H&E staining showed that the Pten^cKO (cKO) SVZ was expanded compared with wild type (WT). (B) Higher magnification from WT (upper panel) and cKO (lower panel) shown in boxed areas of upper panels in A. White lines indicate the edge of the SVZ. Arrowheads indicate clusters of cells in cKO with progenitor morphology similar to those seen in wild type. (C-E) IHC staining of WT and cKO SVZ for Pten (C), NeuN (D) and Dcx (E). The expanded cKO SVZ was deficient for Pten and composed of NeuN+ and Dcx+ cells in contrast to the WT SVZ, which had only thin layers of Dcx+ cells and lacked NeuN+ cells. Lower panels in C and D show higher magnification of the white boxed areas in upper panels. In panel D, the NeuN+ cells adjacent to the WT SVZ were from surrounding striatum and the black line marks the edge of the expanded SVZ in cKO brain. (F) Mash1 (white), p-S6 (green) and Dcx (red) triple IF labeling of the expanded cKO SVZ showed a substantial overlap of p-S6+ and Dcx+ cells in the right bottom panel. Scale bars: in A, 50 μm; in B, 10 μm; in C and D upper panels, 50 μm; in C and D lower panels, 10 μm; in E, 50 μm; in F, 20 μm.

Fig. 3.

Ectopic cells in the expanded cKO SVZ expressed markers of mature interneurons of OB. (A-C) Map2 (A,B) and calretinin (C) IHC staining of the SVZ from matched coronal brain sections of wild-type (WT) or cKO mice injected with TM at P0-1 and analyzed at P18 showed strong expression of these neuronal differentiation markers in the expanded cKO SVZ that are normally expressed in the OB, but not the SVZ or RMS. B shows higher magnifications of the boxed areas in A. Black dashed lines indicate the edge of the SVZ. (D) Quantification of the number of Dcx+, NeuN+, Map2+ and calretinin+ cells in matched sections of the SVZ from mice injected with TM at P11-12 and analyzed at P31. Error bars represent s.e.m. Scale bars: in A,C, 50 μm; in B, 20 μm.

Fig. 4.

Reduced diameter of the terminal RMS and decreased granule cell density in the Pten^cKO OB. (A-C) Pten IHC staining from matched sagittal sections of the forebrain (A) or coronal section of the OB (B,C) from wild-type (WT) and cKO mice injected with TM at P0-1 and analyzed at P18 (A,B), or injected with TM at P30-32 and analyzed at 11 months of age (C). The cKO SVZ was expanded and deficient for Pten (A, arrows). The diameter of the terminal RMS in the cKO OB was diminished (B,C, arrowheads). The cell density of the GCL in the cKO OB was reduced (C). Scale bars: in A,B, 300 μm; in C, 50 μm.

Fig. 5.

Pten^cKO neuroblasts terminated tangential migration prematurely in the SVZ. Immunostaining of matched sagittal sections from wild-type (WT) and cKO mice. Cre activity was induced by TM injection at P0 and P1, BrdU was injected at P4, P5 and P6, and tissue was analyzed at P18. (A) In the OB, there were reduced BrdU+ cells in the granule cell layer of the cKO OB compared with WT. (B) In the SVZ, there was a significant accumulation of BrdU+ cells in the expanded cKO SVZ. (C) BrdU (red) and NeuN (green) double IF labeling of the SVZ from WT and cKO mice showed that a substantial number of BrdU+ cells in the expanded cKO SVZ were NeuN+, whereas there were no NeuN+ cells in the WT SVZ. Black dashed lines in B and white dashed lines in C mark the boundary of the SVZ. Scale bars: 50 μm.

Fig. 6.

mTorc1 inhibition rescued the expansion of Pten^cKO SVZ. (A-D) Representative IHC staining for Dcx (A), Pten (B), p-S6 (C) and p-Akt (D) in the SVZ of matched coronal sections from wild-type (WT) or cKO mice. Mice were treated with rapamycin or vehicle from P8 to P31, Cre activity was induced by TM injection at P11 and P12, and tissue was analyzed at P31. Rapamycin completely rescued the enlargement of the cKO SVZ (n=4 in each group). The SVZ of rapamycin-treated cKO mice remained Pten-null (B) and continued to show elevated p-Akt (D), but showed suppression of p-S6 (C), a downstream indicator of mTorc1 activity. Scale bar: 50 μm.

Fig. 7.

Ex vivo time-lapse live imaging showed that a subset of Pten^cKO neuroblasts had normal morphology, size and directional migration. (A,B) Representative static EYFP images corresponding to time-lapse migration movies from the RMS (supplementary material Movies 3, 4). A Cre expression construct was transfected by in vivo electroporation at P2 into the SVZ of WT (A) or Pten^flox/flox (cKO) (B) mice carrying the R26LSL-EYFP allele. Lower panels are higher magnification of boxed areas in upper panels. Acute brain slices were prepared at P19-20, and cells in which Cre-mediated recombination occurred were visualized by EYFP reporter expression using two-photon microscopy. There were two distinct populations of Pten^cKO (cKO) neuroblasts: cells with elongated bipolar morphology and normal directional migration (arrowheads) and cells with large rounded morphology that showed only local non-directional movement (arrows). Scale bars: upper panels, 50 μm, lower panels 20 μm. (C) Quantification of the percentage of EYFP+ cells with bipolar or non-polar morphology (n=190 for WT and n=368 for cKO). There were significantly more non-polar cells in cKO (green), than in WT (red) (P<0.0001, odds ratio=11). (D) The area of EYFP+ somata of non-polar cells was significantly greater in cKO than in WT (P<10^–12); however, the cells with bipolar morphology were of similar size in cKO and WT (P=0.66). n=22-30 for each morphology. Error bars represent s.e.m. (E) Histogram of the endpoint speed, a measure of directional migration, for bipolar migrating EYFP+ cells from WT (red) and cKO RMS (green). n=3 mice per genotype. The average endpoint speed of bipolar migrating neuroblasts in the cKO RMS (mean: 47.6 μm/hour; n=110) was not significantly different from that of WT (mean: 44.4 μm/hour; n=160) (P=0.15).