Wnt3a Promotes Hippocampal Neurogenesis by Shortening Cell Cycle Duration of Neural Progenitor Cells
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The effects of Wnt signaling on neural progenitor cells have been controversial. Activation of the canonical Wnt signaling pathway either promotes neural progenitor cell proliferation or accelerates their differentiation into postmitotic neurons. This study demonstrates that activation of the Wnt signaling pathway by itself induces neural progenitor cell proliferation but does not directly affect neuronal differentiation processes. To investigate whether Wnt signaling promotes expansion and/or differentiation of neural progenitor cells in the developing hippocampus, we prepared primary mouse hippocampal progenitors and treated them with Wnt3a in a chemically defined culture medium. Wnt3a increased the total number of cells, including the numbers of Ki67+ proliferating cells and Tuj1+ differentiated neurons. This result verified that Wnt3a promoted neural progenitor cell proliferation. Meanwhile, Wnt3a did not appear to actively enhance the neuronal differentiation process itself, because (1) the ratio of Tuj1+ cells to the total cells, and (2) the ratio of BrdU+ Tuj1+ cells to the total BrdU+ cells, were both comparable between cultures with or without Wnt3a. Indeed, Wnt3a caused no significant change in either cell survival or the proportion of symmetric and asymmetric cell divisions that directly affected neuron production. We finally demonstrated that the Wnt3a treatment simply shortened cell cycle duration of neural progenitor cells by 2.9 h. The accelerated cell cycle progression without affecting the ratio of symmetric/asymmetric cell divisions explains how Wnt signaling per se leads to the expansion of both proliferative cell population and differentiated neuronal cell population.
KeywordsWnt3a Hippocampus Cell cycle Cell proliferation Neuronal differentiation
We thank members of our laboratory for valuable discussions and technical advice. We also thank Ms. M. Teramoto for secretarial assistance. We are very grateful to Ms. Y. Saiki for technical help. This work was supported by a Grant-in-Aid for twenty-first century COE research from the Ministry of Education, Culture, Sports, Science and Technology “Cell Fate Regulation Research and Education Unit”, Grants-in-Aid #16047223 and #18053018 for Scientific Research on Priority Areas on “Elucidation of glia-neuron network mediated information processing systems” (T. K.); Grants-in-Aid for Scientific Research on Priority Areas on “Molecular Brain Science” (T. T.); CREST (T. T.); and Grants-in-Aid for Scientific Research on Priority Areas on “Self-renewal and pluripotency of the stem cells” (T. T.) from the Ministry of Education, Culture, Sports, Science and Technology. This work was also supported by Grants-in-Aid #17500255 for Scientific Research (C) from the Japan Society for the Promotion of Science (T. K.).
No significant change in the ratio of active caspase3+ cells among Tuj1+ cells between Wnt3a-treated and non-treated cultures. Neuronal cell death was assessed by double-immunostaining with anti-Tuj1 (red fluorescence) and anti-active caspase3 (rabbit IgG, BD Pharmingen, 1:1000), (green fluorescence, arrowheads) antibodies. The ratio of the number of active caspase 3+ cells to the number of Tuj1+ cells in the control culture was 4.79% (A; 16/334) and that in the Wnt3a-treated culture was 3.18% (B; 16/503). Scale bar: 10┬á┬╡m (JPEG 793 kb)
Wnt3a effects on neuronal marker expressions. There was no significant change in the percentage of NeuN+ mature neurons/total cells with and without Wnt3a (A, B, and green bars in E). Wnt3a significantly increased the percentage of GABA+ neurons (C, D, and red bars in E: P┬á=┬á0.014). Significance determined using studentΓÇÖs t tests (**P┬á<┬á0.01). Data are presented as mean┬á┬▒┬áSD (JPEG 2069┬ákb)
Expressions of neuronal sub-type markers examined by RT-PCR. A, Four-day Wnt3a treatment slightly induced interneuron marker Gad1 expression but not glutamatergic neuron marker VGluT1 expression in hippocampal cell culture. B, Four-day Wnt3a treatment induced calpactin and KA-1 but not Oct-6. Both calpactin and KA-1 are highly expressed in adult hippocampus CA3 region. PCR primers used in this figure are as follows. Gad1: GCTGGAAGGCATGGAAGGCTTTA and TGAGCCCCATCACCGTAGCA; VGluT1: ACGGTAACGCAGGTGTCCT and CCTCTCGTACTGAGCAGGA; G3PDH: GTCATCATCTCCGCCCCTTCTGC and GATGCCTGCTTCACCACCTTCTTG; calpactin: ATGCCATCCCAAATGGAGCACG and GAGGGCAATGGGATGCAAAC; KA-1: CCCATCGAGTCTGTGGATGA and TATTCTCCATTCCCAGGCCT; Oct-6: GCGAGCACTCGGACGAGGATGC and GGGGTCATGCGCTTCTCCTTCTGC (JPEG 2564 kb)
Experimental design for evaluating cell cycle kinetics (JPEG 769 kb)
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