Wnt3a promotes hippocampal neurogenesis by shortening cell cycle duration of neural progenitor cells

Abstract

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.

Fig. 1

Increases in both the total number of cells and the number of neurons following Wnt3a treatment. a, b Hippocampal progenitor cells were cultured with or without 50 ng/ml Wnt3a for 4 days. Anti-Tuj1(green fluorescence) and anti-GFAP (red fluorescence) antibodies were used for double-immunostaining. Cells were counterstained with Hoechst 33258. a A control culture and b a Wnt3a-treated culture. c The total number of cells per cm² in Wnt3a-treated cultures was increased by 1.4-fold compared with that in control cultures (Wnt3a 90 ± 10.4, control 60.8 ± 7.9). Significance determined using student’s t tests (P < 0.005). Data are presented as mean ± SD. Wnt3a also increased the number of Tuj1⁺ neurons by 1.4-fold (Wnt3a 62.6 ± 7.3, control 43.3 ± 7: P < 0.005). There was no significant difference in the ratio of Tuj1⁺ cells to the total number of cells between Wnt3a-treated and control cultures. In Wnt3a-treated cultures, the proportion of Tuj1⁺ cells among all cells was 68.9%, whereas that in control cultures was 69.2%. Wnt3a did not affect the number of GFAP⁺ cells. At least five independent cultures were tested, and the number of cells in four different fields in each well was counted. Data are presented as mean ± SD. Five independent cultures were tested and cells in three different fields in each well were counted. Scale bar 10 μm. (Color figure online)

Fig. 2

Increase in the number of phosphorylated histone H3-positive proliferating cells induced by Wnt3a. a, b Hippocampal cells treated with or without Wnt3a for 4 days were immunostained for phosphorylated histone H3 (PH3; green nuclei, arrowheads), a marker for late G2- and M-phase progression. Cells were counterstained with Hoechst 33258. c Wnt3a increased the number of PH3⁺ cells per cm² by 1.38-fold, indicating that Wnt3a-induced cell proliferation (Wnt3a 46.41 ± 3.92, control 33.75 ± 3.08). Data are presented as mean ± SD. Significance determined using student’s t tests (***P < 0.005). At least five independent cultures were tested and cells in four different fields in each well were counted. Scale bar 10 μm. (Color figure online)

Fig. 3

Only a limited number of resting cells entered cell division following Wnt3a stimulation. Proliferating cells in standard N2/DMEM/F12 medium were labeled with BrdU for the first 24 h. The cells were then cultured with or without Wnt3a for a further 24 h. The cells were fixed and double-immunostained with anti-BrdU (a) and anti-Ki67 (b) antibodies. BrdU⁻/Ki67⁺ cells were supposed to represent cells that started to proliferate in response to Wnt3a treatment (arrows a–c). Such cells were infrequently observed in both Wnt3a-treated and non-treated cultures (less than 0.5%; see also Table 1). Scale bar 5 μm. (Color figure online)

Fig. 4

Wnt3a increased the number of Tuj1-positive neurons differentiated from progenitors. Proliferating progenitor cells were pulse-labeled with BrdU for the first 4 h of culture, and incubated with or without Wnt3a for 4 days (a control culture, b Wnt3a-treated culture). Cells were immunostained with anti-BrdU (red fluorescence) and anti-Tuj1 (green fluorescence) antibodies. Wnt3a increased the total number of BrdU⁺ cells in this paradigm, suggesting that Wnt3a promoted neural progenitor cell proliferation (red columns in c, Wnt3a 34.2 ± 4.5, control 21.9 ± 3.9: P < 0.005). Wnt3a also increased the number of Tuj1⁺/BrdU⁺ cells, which were neurons differentiated from BrdU-incorporating progenitor cells (arrowheads a, b, green columns c. Wnt3a 24.8 ± 3.1, control 14.5 ± 5.6: P < 0.005). However, there was no significant difference in the percentage of Tuj1⁺/BrdU⁺ neurons among BrdU⁺ cells between Wnt3a-treated and control cultures. Four independent cultures were tested and cells in three different fields in each well were counted. Significance determined using student’s t tests (***P < 0.005). Data are presented as mean ± SD. Scale bar 10 μm. (Color figure online)

Fig. 5

No difference in the rate of asymmetric cell division between Wnt3a-treated and control cultures. E15 hippocampal cells were exposed to GFP-retrovirus for the first 4 h of culture, and then cultured with or without 50 ng/ml Wnt3a in N2/DMEM/F12 for 32 h. Most clones consisted of two GFP⁺ cells and were analyzed by immunostaining with anti-GFP (green fluorescence) and anti-Tuj1 (red fluorescence) antibodies. N Tuj1⁺/GFP⁺ neurons. P Tuj1⁻/GFP⁺ undifferentiated cells. [N; N] neuron and neuron division in b, [P; P] progenitor and progenitor cell division in c, [N; P] neuron and progenitor cell division in d. a The number of [P; P] clones was slightly increased but the neuron containing clones was not statistically changed ([N; N] Wnt3a 29 ± 4.15, control 27 ± 5.93: not significant. [P; P] Wnt3a 47.5 ± 4.42, control 40.83 ± 3.76: P < 0.05. [N; P] Wnt3a 11 ± 3.22, control 14.5 ± 2.88: not significant). Six independent cultures were tested and clones in two well each were counted. Data are presented as mean ± SD. Scale bar 2.5 μm. (Color figure online)

Fig. 6

Shortening of cell cycle length by Wnt3a. Hippocampal progenitors cells were first pulse-labeled with GFP-virus for 1 h of the 4-h preincubation (a blue arrow a), and cultured for 36 h with or without Wnt3a. The cells were then pulse-labeled by BrdU at various time points, as indicated by red arrows in a. The cells were immunostained with anti-GFP (b) and anti-BrdU (c) antibodies (merge in d). GFP⁺/BrdU⁺ cells represented cells that re-entered into the second cell cycle after virus integration (arrowheads b–d). The largest number of GFP⁺/BrdU⁺ cells was observed in the Wnt3a-treated culture, when the cells were exposed to BrdU from 12 to 14 h after virus infection. The largest number of GFP⁺/BrdU⁺ cells was observed in the control culture, when the cells were exposed to BrdU from 14 to 16 h after virus infection. Scale bar 5 μm. Significance determined using student’s t tests (*P < 0.05, **P < 0.01). Data are presented as mean ± SD (n = 3/group). (Color figure online)