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. 2013 Jul;33(13):2551-9.
doi: 10.1128/MCB.00325-13. Epub 2013 Apr 29.

Wnt7a regulates multiple steps of neurogenesis

Qiuhao Qu  1 , Guoqiang Sun, Kiyohito Murai, Peng Ye, Wendong Li, Grace Asuelime, Yuen-Ting Cheung, Yanhong Shi
Affiliations

Wnt7a regulates multiple steps of neurogenesis

Qiuhao Qu et al. Mol Cell Biol. 2013 Jul.

Abstract

Although Wnt7a has been implicated in axon guidance and synapse formation, investigations of its role in the early steps of neurogenesis have just begun. We show here that Wnt7a is essential for neural stem cell self-renewal and neural progenitor cell cycle progression in adult mouse brains. Loss of Wnt7a expression dramatically reduced the neural stem cell population and increased the rate of cell cycle exit in neural progenitors in the hippocampal dentate gyrus of adult mice. Furthermore, Wnt7a is important for neuronal differentiation and maturation. Loss of Wnt7a expression led to a substantial decrease in the number of newborn neurons in the hippocampal dentate gyrus. Wnt7a(-/-) dentate granule neurons exhibited dramatically impaired dendritic development. Moreover, Wnt7a activated β-catenin and its downstream target genes to regulate neural stem cell proliferation and differentiation. Wnt7a stimulated neural stem cell proliferation by activating the β-catenin-cyclin D1 pathway and promoted neuronal differentiation and maturation by inducing the β-catenin-neurogenin 2 pathway. Thus, Wnt7a exercised critical control over multiple steps of neurogenesis by regulating genes involved in both cell cycle control and neuronal differentiation.

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Figures

Fig 1
Fig 1
Loss of Wnt7a expression reduced neural stem/progenitor cell (collectively referred to as NSC) proliferation and self-renewal in vitro. (a) Reduced cell proliferation in NSCs isolated from Wnt7a−/− mouse brains. BrdU labeling (shown in green) of NSCs isolated from 6-week-old WT and Wnt7a−/− mouse brains. Bar, 50 μm. (b) Quantification of the percentage of BrdU-positive cells (BrdU+) out of total living cells for WT and Wnt7a−/− NSCs after treatment with BrdU for 1 h. *, P < 0.001 by Student's t test. Experimental repeats (n) = 6. (c) Staining of both WT and Wnt7a−/− NSCs with active caspase-3 (shown in green). Nuclear 4′,6-diamidino-2-phenylindole (DAPI) staining is shown in blue. Bar, 50 μm. (d) Quantification of nonapoptotic cells in WT and Wnt7a−/− NSCs. The nonapoptotic cells were negative for active caspase-3 staining. n = 3. (e) Images of representative neurospheres of WT and Wnt7a−/− NSCs from clonal analysis. Bar, 20 μm. (f) Quantification of WT and Wnt7a−/− neurosphere size. *, P < 0.005 by Student's t test. (g) Quantification of WT and Wnt7a−/− neurosphere formation rate. *, P < 0.001 by Student's t test. Error bars are standard deviations of the means for all the quantification data. n = 6 (f and g).
Fig 2
Fig 2
Reduced neural stem cell population in the hippocampal dentate gyrus (DG) of Wnt7a−/− adult mouse brains. (a) Immunostaining of the hippocampal dentate gyrus of 6-week-old WT and Wnt7a−/− mice for GFAP (green). Nuclear DAPI staining is shown in blue. (b) Quantification of GFAP+ cells per 40-μm section in the hippocampal dentate gyrus of 6-week-old WT and Wnt7a−/− mice. (c) Immunostaining for Sox2 (red) in the hippocampal dentate gyrus of 6-week-old WT and Wnt7a−/− mice. (d) Quantification of Sox2+ cells per 40-μm section in the hippocampal dentate gyrus of 6-week-old WT and Wnt7a−/− mice. Error bars are standard deviations of the means. *, P < 0.05 by Student's t test for (b and d). Bar, 100 μm (a and c). n = 3 for both WT and Wnt7a−/− mice (b and d).
Fig 3
Fig 3
The Wnt7a−/− neural progenitors exhibited lengthened cell cycles and reduced cell cycle reentry in vivo. (a) Cell cycle length analysis was performed by treating 6-week-old WT and Wnt7a−/− mice with BrdU for 30 min before brain harvest and then double staining the hippocampal dentate gyrus (DG) with BrdU (shown in green) and Ki67 (shown in red). NeuN staining (shown in blue) was included to show the structure of the dentate gyrus. Bar, 50 μm. (b) Quantification of BrdU+ Ki67+ cells in total Ki67+ cells in the DG. The labeling index of cell cycle length is defined as the percentage of BrdU+ Ki67+ cells in total Ki67+ cells. Decreased labeling index suggests lengthening cell cycles. Error bars are standard deviations of the means. *, P < 0.005 by Student's t test. n = 6 for both WT and Wnt7a−/− mice. (c) Cell cycle reentry and exit analysis was performed by treating 6-week-old WT and Wnt7a−/− mice with BrdU for 24 h before brain harvest and then double staining the hippocampal DG with BrdU (shown in green) and Ki67 (shown in red). Bar, 50 μm. (d) Quantification of BrdU+ Ki67+ cells in total BrdU+ cells in the DG. The index of cell cycle reentry is defined as the percentage of BrdU+ Ki67+/BrdU+ cells. Error bars are standard deviations of the means. *, P < 0.001 by Student's t test. n = 6 for both WT and Wnt7a−/− mice.
Fig 4
Fig 4
Loss of Wnt7a expression decreased neuronal differentiation potential and neurite complexity in vitro. (a) The Wnt7a−/− NSCs that were induced to differentiate show reduced neuronal differentiation potential, revealed by the decreased percentage of Tuj1+ cells. NSCs isolated from WT or Wnt7a−/− mouse brains were induced to differentiate for 3 days using retinoic acid (RA) and FBS. The differentiated cells were immunostained for a neuronal marker, Tuj1 (red); an astrocyte marker, GFAP (green); and an oligodendrocyte marker, O4 (light blue, in the inset). Nuclear DAPI staining is shown in dark blue. Bar, 50 μm. (b) Quantification of Tuj1+ cells in WT and Wnt7a−/− cells. *, P < 0.001 by Student's t test. (c and d) Quantification of GFAP+ cells (c) and O4+ cells (d) in WT and Wnt7a−/− cells. Error bars are standard deviations of the means for all quantifications. Experimental repeats (n) = 6 (b to d).
Fig 5
Fig 5
Loss of Wnt7a expression reduced neurogenesis and impaired dendritic arborization in dentate granule neurons in vivo. (a) Reduced numbers of BrdU+ DCX+ cells in the dentate gyrus (DG) of Wnt7a−/− brains. BrdU (green) and DCX (red) double staining in the DG of WT and Wnt7a−/− mouse brains from 6-week-old mice that were treated with BrdU for 1 week, followed by 3-week survival. Bar, 100 μm. The insets are enlarged images of the cells indicated by arrows. (b) Quantification of BrdU+ DCX+ cells in the DG of WT and Wnt7a−/− brains. The numbers represent BrdU+ DCX+ cells in the DG per 20-μm section. Error bars are standard deviations of the means. *, P < 0.001 by Student's t test. (c) Images of dendritic arborization of dentate granule neurons in age- and gender-matched WT and Wnt7a−/− mouse brains shown by DCX staining (red). Bar, 20 μm. (d) Quantification of the maximal (max) dendritic length of DCX-positive neurons in the DG of WT and Wnt7a−/− brains. Error bars are standard deviations of the means. *, P < 0.01 by Student's t test. (e) Analysis of dendritic complexity of DCX+ dentate granule neurons in WT and Wnt7a−/− brains using Sholl analysis. Errors bars are standard errors of the means. P values were determined by 2-way ANOVA. (f) Images of dendritic arborization of dentate granule neurons in age- and gender-matched WT and Wnt7a−/− mouse brains shown by Golgi staining. Bar, 50 μm. (g) Quantification of total neurite length per dentate granule neuron in the hippocampal dentate gyrus of age- and gender-matched WT and Wnt7a−/− mice. Error bars are standard deviations of the means. *, P < 0.001 by Student's t test. (h) Analysis of dendritic complexity of dentate granule neurons revealed by Golgi staining in the DG of age- and gender-matched WT and Wnt7a−/− mice using Sholl analysis. Error bars are standard errors of the means. P values were determined by 2-way ANOVA. n = 6 for both WT and Wnt7a−/− mice for all panels.
Fig 6
Fig 6
Wnt7a regulates cyclin D1 and Ngn2 expression. (a to c) RT-PCR analysis of Wnt7a (a), cyclin D1 (cyc D1) (b), and Ngn2 (c) expression in NSCs or neurons that were transduced with control RNA (siCont) or Wnt7a siRNA (siWnt). Error bars are standard deviations of the means. *, P < 0.001 by Student's t test (a to c). (d and e) ChIP analysis to measure the relative binding of β-catenin to the TCF/LEF binding site on the promoter of cyclin D1 (cycD1) (d) or Ngn2 (e) in NSCs. A diagram of the cyclin D1 and Ngn2 genes is shown, with the genomic region amplified for ChIP analysis indicated by an open box. (f and g) ChIP analysis to measure the relative binding of β-catenin to the TCF/LEF binding site on the promoter of cyclin D1 (cycD1) (f) or Ngn2 (g) when NSCs were induced to differentiate into neurons. For panels d to g, 10% cell lysates were included as the input. Both mouse IgG (IgG1) and rabbit IgG (IgG2) were included as the negative controls. The levels of the active chromatin markers AcH3 and H3K4me3 on the same promoters were measured in parallel. n = 3 for all panels. ChIP enrichment of genomic DNA for each antibody was calculated as the percentage of input in the following: 4 × 10−6 ± 1 × 10−6 for IgG1 and IgG2, 0.23 ± 0.11 (β-catenin), 0.28 ± 0.03 (AcH3), and 0.34 ± 0.04 (H3K4me3) in panel d; 0.001 ± 0.0002 (IgG1), 0.001 ± 0.0007 (IgG2), and 0.001 ± 0.0006 for all three antibodies (β-catenin, AcH3, and H3K4me3) in panel e; 1 × 10−6 to 8 × 10−6 ± 1 × 10−6 to 4 × 10−6 for IgG1, IgG2, and all three antibodies in panel f; 0.001 ± 0.0005 (IgG1), 0.001 ± 0.0001 (IgG2), 0.32 ± 0.05 (β-catenin), 0.29 ± 0.05 (AcH3), and 0.26 ± 0.04 (H3K4me3) in panel g.
Fig 7
Fig 7
Wnt7a regulates NSC proliferation and neuronal differentiation by activating β-catenin and its downstream target genes. (a) NSCs were transduced with control RNA (siCont), Wnt7a siRNA (siWnt), the combination of siWnt and the constitutively active β-catenin (siWnt + β-catenin), or the combination of siWnt and cyclin D1 (siWnt + Cyclin D1). The transduced cells were treated with BrdU for 1 h. Cell proliferation was monitored by BrdU labeling, shown in red. Nuclear DAPI staining is shown in blue. Bar, 50 μm. (b) Quantification of the percentage of BrdU+ cells in total living cells. Error bars are standard deviations of the means. *, P < 0.001 by Student's t test. n = 7. (c) NSCs were transduced with siCont, siWnt, siWnt + β-catenin, or the combination of siWnt and Ngn2 (siWnt + Ngn2) and induced to differentiate into neurons. Neuronal differentiation was monitored by immunostaining of Tuj1, shown in red. Nuclear DAPI staining is shown in blue. Bar, 50 μm. (d) Quantification of the percentage of Tuj1+ cells out of the total living cells for each treatment described in panel c. Error bars are standard deviations of the means. *, P < 0.001 by Student's t test. n = 3. (e) Images of individual neurons derived from NSCs transduced with siCont, siWnt, siWnt + β-catenin (siWnt + β-cat), or siWnt + Ngn2. Bar, 50 μm. (f) Quantification of total neurite length per neuron shown in panel e. Error bars are standard deviations of the means. *, P < 0.001 by Student's t test. n = 3. (g) Analysis of dendritic complexity of Tuj1+ neurons shown in panel e using Sholl analysis. Error bars are standard errors of the means. n = 10. P values were determined by 2-way ANOVA.
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References

    1. Gage FH. 2000. Mammalian neural stem cells. Science 287:1433–1438 - PubMed
    1. Chenn A, Walsh CA. 2002. Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science 297:365–369 - PubMed
    1. Lee SM, Tole S, Grove E, McMahon AP. 2000. A local Wnt-3a signal is required for development of the mammalian hippocampus. Development 127:457–467 - PubMed
    1. Viti J, Gulacsi A, Lillien L. 2003. Wnt regulation of progenitor maturation in the cortex depends on Shh or fibroblast growth factor 2. J. Neurosci. 23:5919–5927 - PMC - PubMed
    1. Yu HM, Liu B, Costantini F, Hsu W. 2007. Impaired neural development caused by inducible expression of Axin in transgenic mice. Mech. Dev. 124:146–156 - PMC - PubMed

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