Figure 1.
Generation of conditional Nolz-1 transgenic mice and expression of transgenes in vitro.
A: The strategy for generating nestin-Cre mediated Nolz-1eGFP transgenic mice. B–E: Expressions of the transgenes LacZ (B), eGFP (C), Nolz-1 (D) were detected in the transfected HEK293T cells. Cells co-expressing eGFP (green, C) and Nolz-1 (red, D) were indicated by the arrows. E: Merged images of C and D.
Figure 2.
Ectopic expression of transgenic Nolz-1 and eGFP in embryonic nCT brains.
A–E: In situ hybridization demonstrates ectopic expression of eGFP (A, C) and tNolz-1 (B, D) mRNAs in the cerebral cortex (CTX) of E17.5 nCT brain, but not in the control of nC brain (E). F: The Western blotting with anti-Nolz-1 antibody shows that the band of transgenic Nolz-1 protein (tNolz-1, arrowhead) was detected in the cortex of E16.5 nCT brain with size similar to that of endogenous striatal Nolz-1 protein (arrow) in the control nC and T brains. There was no detectable Nolz-1 protein in the cortex of the control nC or T brains. Asterisk indicates a non-specific band. Scale bars in B (for A, B), 200 µm, in E (for C–E), 100 µm.
Figure 3.
Ectopic expression of tNolz-1 in telencephalic neural progenitor cells of the nCT brain.
A–F: Nolz-1 and eGFP mRNA-positive cells (arrows in C and F, respectively) were present in the ventricular zone of LGE in E12.5 nCT brain (A–C, E, F), but not in the ventricular zone of the control T brain (D). Arrowheads in C and F indicate tNolz-1 and eGFP mRNA-positive cells lined up in radial lines. Ectopic Nolz-1 mRNA-positive cells (I, inset) and eGFP mRNA-positive cells (J, inset) remained in the ventricular zone of lateral ventricle (LV) of nCT brain at postnatal day 1 (P1). B, C, F, I and J show the boxed regions at high magnification in A, B, E, G and H, respectively. Scale bars in D (for C, D) and in F, 50 µm, in I (for I, J), 100 µm, in inset of I (for insets of I, J), 20 µm.
Figure 4.
Hypoplasia phenotype of the nCT brain. A:
Apparent hypoplasia of the nCT brain, particularly in the telencephalic region (arrowhead), was observed in E12.5 embryos B: Prominent hypoplasia was observed in postnatal day 1 (P1) nCT brain.
Figure 5.
Reduction of proliferative neural progenitor cells in the nCT brain.
A1-B2: BrdU pulse-labeled cells in the germinal zone were decreased in the LGE of E11.5 nCT brain (A2) compared to that in the nC brain (A1). Similar results were found in the MGE (B1, B2). C1–C2: Reduction of BrdU pulse-labeled cells in the germinal zone were also found in the LGE of E12.5 nCT brain (C2). The white double-headed arrows indicate the width of BrdU-positive germinal zone. D: Quantitative analysis of BrdU-positive cells in E12.5 forebrain regions. The graphical results represent mean ± SEM (control vs. nCT in CTX: 216±36 vs. 198±9; in LGE, 151±17 vs. 100±9; in MGE, 167±18 vs. 86±13). **, p<0.01, Student’s t test. E1–E2’: The Ki67-positive germinal zone was decreased in the nCT brain (E2) compared to the nC brain (E1). The bracketed regions in E1 and E2 are shown with DAPI counter staining at high magnification in E1’ and E2’, respectively. The white double-headed arrows indicate the width of Ki67-positive germinal zone. The arrows in E2 indicate radial lines that are devoid of Ki67-positive cells in the germinal zone of LGE and MGE. Scale bars in A1, B1, C1, E1’ for A1–2 B1–2, C1–2, E1’-2′, respectively, 100 µm; in E1 for E1–2, 150 µm.
Figure 6.
Induction of abnormal programmed cell death in the nCT brain.
A1, A2: Many TUNEL-positive apoptotic cells were found in Ki67-positive germinal zone in the LGE of E12.5 nCT brain (A2). Few TUNEL-positive apoptotic cells were found in the control T brain (A1). Note that Ki67-positive cells lining along the lateral ventricle (arrows) were significantly decreased in the nCT brain (A1, A2). B1, B2: Activated caspase-3 (AC3)-positive apoptotic cells (arrows) were detected in the germinal zone of LGE in E11.5 nCT brain (B2), but not in that of the T brain (B1). Note that the majority of AC3-positive cells were present in the region beyond the ventricular zone of nCT brain (VZ, B2). C1–C3: Scattered TUNEL-positive cells (asterisk) and TUNEL-positive cell clusters (arrows) with condensed DAPI-positive nuclei were present in E12.5 nCT LGE. Note that few TUNEL-positive cells were present in the VZ. The double-headed arrows indicate the width of VZ. Scale bars in A2 (for A1, A2), 100 µm; B1 (for B1, B2), 50 µm; C3 (for C1–C3), 20 µm.
Figure 7.
Reduction of mitotic neural progenitors in the ventricular zone of the nCT brain.
A1–C2: PH3-positive cells (arrows) were decreased in the ventricular zone (VZ) of E10.5 subpallium (A2), E11.5 (B2) and E12.5 (C2) LGE and MGE of nCT brains compared to the control littermate of nC brains (A1, B1, C1). D: Quantitative analysis shows that the numbers of PH3-positive cells in the VZ of the cortex (CTX), LGE and MGE in E12.5 nCT brains were decreased compared to the T control brains. **, p<0.01, ***; p<0.001, Student’s t test. Scale bars in A1 (for A1, A2), 50 µm; B1 (for B1, B2), 100 µm; C1 (for C1, C2), 200 µm.
Figure 8.
Promotion of cell cycle exit by tNolz-1 in the nCT telencephalon. A–I:
Double immunostaining of Ki67 (red) and BrdU (green) in E12.5 WT (A, D, G), T (B, E, H) and nCT (C, F, I) telencephalon. E12.5 brains were harvested 4 hr after pregnant mice were injected with BrdU. Many BrdU+/Ki67− cells (single arrows in C, F, I) were detected in the cortex, LGE and MGE of the nCT brain. Double arrows and arrowhead indicate BrdU?/Ki67+ cells and BrdU+/Ki67+ cells, respectively. Note that in the nCT brain (C, F, I), a few BrdU+/Ki67− cells (single arrows) appeared to line up along a radial line in the germinal zone of LGE as if they were derived from a clone of progenitors. J-R: Triple immunostaining of BrdU (green), Ki67 (red) and TuJ1 (purple). A few cells co-expressing BrdU and TuJ1 without Ki67 (single arrows) representing premature neurons were found in the germinal zone of cortex (J-L), LGE (M-O) and MGE (P-R) in the nCT brain. Double arrows indicate single Ki67-positive proliferative cells. Arrowhead indicates double BrdU− and Ki67-positive proliferative cells. Sections were counterstained with DAPI (blue). Scale bar in A (for A–I), 50 µm; in J (for J-R), 10 µm.
Table 1.
Cell cycle exit index in E12.5 brains.
Figure 9.
Ectopic and precocious TuJ1 expression in the proliferative ventricular zone of E12.5 nCT brain.
A1-D2: TuJ1 immunoreactivity was higher in the nCT forebrain (A2, B2, C2) than that in the control forebrain (A1, B1, D1). Ectopic and precocious TuJ1-positive neurons were found in the TuJ1-poor ventricular zone of the cerebral cortex (C2, arrows) and LGE (D2, arrows). The bracketed regions in D1 and D2 are shown at high magnification in the insets of D1 and D2, respectively. E: TuJ1-positive cell clusters are present in the germinal zone of the nCT brain. The bracketed region in E is shown at high magnification in F with confocal images. F, F’: TuJ1-positive cell clusters contain apoptotic nuclei with condensed DAPI staining. The arrow indicates a TuJ1-positive neuron (F) whose nucleus was presumably under the process of DNA condensation as implicated by the condensed DAPI staining (F’). The arrowhead indicates a presumed degrading cell with condensed TuJ1 immunoreactivity without apparent DAPI staining. G: Western blotting and quantification of TuJ1 protein expression in E12.5–13.5 cortex. TuJ1 expression in nCT cortex is 123±4.5% of control littermates (p<0.05). Scale bars in A1 (for A1, A2, B1, B2), 0.5 µm; C1 (for C1, C2) and in inset of D1 (for insets of D1, D2), 50 µm; D1 (for D1, D2), 100 µm; E, 40 µm; F–F’, 20 µm.
Figure 10.
Double labeling of TuJ1 and TUNEL and tNolz-1 expression in apoptotic cell clusters of the germinal zone in E12.5 nCT forebrain.
A–E : Double labeling of TuJ1 and TUNEL. A: DAPI staining. B, D: Merged images of TuJ1 (red) and TUNEL (green) in the LGE germinal zone. E: Merged images of TuJ1 (red) and DAPI (blue). B and C–E show the boxed regions at high magnification in A and B, respectively. D: TUNEL-positive signals (asterisk) were detected in some TuJ1-positive cells (arrowhead) ectopically localized in the germinal zone. The arrow indicates a single TuJ1-positive cell without TUNEL signal. F, G: tNolz-1 mRNA was detected as presumably inclusion bodies (arrows, G) in some apoptotic cell clusters in the MGE of nCT brain.
Figure 11.
Induction of abnormal apoptosis by over-expression of Nolz-1 in N18, ST14A and N2A neural cells.
A–F: Nolz-1 over-expression led to an increase of cell death in N18 cells. N18 cells were co-transfected with pNolz-1 and pEYFP constructs. Co-localization of Nolz-1 protein (red, B) and GFP (green) were found in all GFP-positive N18 cells (arrows, C and D), but not in GFP-positive cells of mock control (A, B). E: The growth curve analysis of Nolz-1-transfected N18 cells. The number of Nolz-1-transfected N18 cells was progressively decreased with increasing culture times. F: Western blotting of cleaved caspase 3 shows a 1.4-fold increase of cleaved caspase 3 in N18 cells 24 hr after transfection. G,-J: Transfection of Nolz-1 (red, H) induced active-caspase 3 (AC3, red, J) in ST14A cells 24 hr after transfection. K: Quantitative analysis of AC3-positive ST14A cells with Nolz-1 over-expression. L: Induction of cleaved caspase 3 and reduction of PCNA in Nolz-1-transfeceted N2A cells. Upper panel shows the experimental procedure. The Western blotting shows increases and decreases of cleaved caspase 3 and PCNA, respectively, in Nolz-1-transfected N2A cells M-Q: Promotion of neuronal differentiation by Nolz-1 over-expression in N2A cells. N2A cells were co-transfected with Nolz-1 and GFP. Nolz-1 over-expression induced a 15-fold increase of the number of neurite-bearing neurons (arrows, N) compared to the mock transfected group (M) 72 hr after transfection. O–Q: Nolz-1-transfected cells expressed the neuronal differentiation marker of TuJ1. R: Quantitative analysis of neurite-bearing cells with Nolz-1 over-expression. *, p<0.05; **, p<0.01; ***, p<0.001, Student’s t test. Scale bars in D (for A–D), in J (for G–J) and in Q (for O–Q), 20 µm.