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First published online 13 May 2004
doi: 10.1242/dev.01165

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The Wnt/ß-catenin pathway directs neuronal differentiation of cortical neural precursor cells

¹ Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
² Department of Anatomy, Keio University School of Medicine, Tokyo 160-8582, Japan
³ Department of Molecular Neurobiology, Institute of DNA Medicine, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan
⁴ National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan

View larger version (28K): [in a new window]   Fig. 1. Ectopic expression of Wnt7a promotes neuronal differentiation in cortical NPCs. Dissociated cortical NPCs were infected with a retrovirus encoding either GFP alone (control) or both GFP and Wnt7a. The cells were then incubated for 3 days in the absence of Fgf2. (A) The percentage of TuJ1+ cells among GFP+ cells was determined. Data are the mean±s.e.m. of values from three samples. Similar results were obtained in four independent experiments. *P<0.025 versus control; t-test. (B) GFP fluorescence, TuJ1 immunofluorescence and the corresponding merged images are shown for typical fields of control and Wnt7a-expressing cells. (C) The percentage of GFAP+ cells among GFP+ cells was determined. Data are the mean±s.e.m. of values from three samples. Scale bar: 25 µm.

View larger version (39K): [in a new window]   Fig. 2. Wnt7a promotes neuronal differentiation through the ß-catenin/TCF signaling pathway. (A) NPCs were infected with a retrovirus encoding GFP alone (control) or GFP together with the ß-catenin mutants S33Y or N90 or with either wild-type (WT) or a kinase-negative mutant (KN) of MKK7-JNK1. The percentage of TuJ1+ cells among GFP+ cells was determined after culture for 2 days in the absence of Fgf2. Data are the mean±s.e.m. of values from eight samples, and similar results were obtained in three independent experiments. (B) NPCs were infected with a retrovirus encoding GFP alone (control) or GFP together with S33Y ß-catenin. The percentage of neurofilament+ cells among GFP+ cells was determined after culture for 4 days in the absence of Fgf2. Data are the mean±s.e.m. of values from eight samples, and similar results were obtained in three independent experiments. (C) GFP fluorescence, neurofilament (NF) immunofluorescence and the corresponding merged images of control cells and cells expressing S33Y ß-catenin are shown. Arrowheads indicate neurite-like protrusions induced by the expression of S33Y ß-catenin. Scale bar: 25 µm. Data are the mean±s.e.m. of values from six samples, and similar results were obtained in three independent experiments.

View larger version (12K): [in a new window]   Fig. 3. Expression of a stabilized form of ß-catenin promotes neuronal differentiation in the presence of Fgf2. (A) A model depicting the possible actions contributing to the increased neuronal population after activation of the canonical Wnt pathway. NP, neuronal progenitor; GP, glial progenitor; N, neuron; G, glia. (B) NPCs were infected with a retrovirus encoding either GFP alone (control) or both GFP and S33Y ß-catenin, and the percentage of TuJ1+ cells among GFP+ cells was determined at the indicated times after culture in the presence of Fgf2. Data are the mean±s.e.m. of values from six samples, and similar results were obtained in two independent experiments. (C) The percentage of HuC/D+ cells among GFP+ cells after culture for 2 days in the presence of Fgf2 was determined. Data are the mean±s.e.m. of values from eight samples, and similar results were obtained in three independent experiments.

View larger version (16K): [in a new window]   Fig. 4. Expression of a stabilized form of ß-catenin instructively promotes neuronal differentiation. NPCs were infected with a retrovirus encoding either GFP alone (control) or both GFP and S33Y ß-catenin at a low titer and subjected to clonal analysis. After incubation for 3 days in the presence of Fgf2, cells in each clone were stained with TuJ1 antibody and the clones were classified as containing either only TuJ1+ cells (neuron-only clone), both TuJ1+ and TuJ1- cells (neuron-containing clone), or only TuJ1- cells (precursor-only clone) (A). The cell number for each clone was determined (B). The results are representatives of three independent experiments. (C) NPCs were infected with a retrovirus encoding either GFP alone (control) or GFP together with Wnt7a, S33Y ß-catenin or wild-type (WT) ß-catenin. They were then plated at a density of 1000 cells/well in 96-well plates that had been coated with poly-HEME and incubated for 9 days in suspension culture in the presence of Fgf2, after which the number of GFP+ primary neurospheres per 1000 founding cells/well was determined. Data are the mean±s.e.m. of values from 12 samples, and are representatives of three independent experiments. *P<10-5 versus control.

View larger version (43K): [in a new window]   Fig. 5. The canonical Wnt pathway promotes NPC differentiation into neurons in the developing mouse neocortex. Mouse neuroepithelial cells were subjected to in utero electroporation at E13.5 with various constructs. Embryos were fixed 2 days after electroporation and subjected to immunostaining. (A) Immunostaining with TuJ1 (red) and antibodies to GFP (green) in cells of the same region of the dorsolateral telencephalon electroporated with a vector for GFP alone (control) or for GFP and S33Y ß-catenin. Broken lines indicate the boundary of TuJ1+ and TuJ1- areas, which coincides with the basal edge of the ventricular zone (VZ) judged by the shape of the cells. Scale bar: 100 µm. Most of the cells expressing S33Y ß-catenin migrated out of the VZ and only a small fraction of these cells (10.4%) remained in the VZ, whereas many control cells remained in the VZ (33.3%). The VZ of the cortex electroporated with S33Y ß-catenin was thinner than that of control. Similar results were obtained from three independent experiments. (B) Quantitative analysis of HuC/D expression in individual electroporated cells. Embryos were electroporated with a vector expressing either histone H2B-GFP alone or together with a vector for S33Y ß-catenin. The relative expression level (intensity/area) of the neuronal marker HuC/D within individual histone H2B-GFP+ cells was quantified by immunostaining and LSC. The percentages of cells within each range of expression are shown in the histograms. The broken lines represent the approximate expression level apparent at the border between the VZ and the intermediate zone, and the percentages of cells with expression levels less than or greater than this value are indicated (see Materials and methods). Littermates were analyzed for each experiment. (C) Immunostaining with TuJ1 (red) and antibodies to GFP (green) in cells of the same region of the dorsolateral telencephalon electroporated with vectors for histone H2B-GFP alone (control) or for histone H2B-GFP and Axin. A large number of the cells expressing Axin remained in the VZ (42.8%), compared with the number of control cells remaining (28.3%). Scale bar: 50 µm. (D,E) Quantitative analysis of HuC/D expression in individual cells electroporated with a vector for either Axin (D) or for Dkk1 (E). All data are representative of results obtained from three independent experiments.

View larger version (41K): [in a new window]   Fig. 6. ß-catenin/TCF complex directly regulates the Ngn1 promoter. (A) A schematic representation of the mouse neurogenin 1 (Ngn1) promoter (WT), and its mutant within the putative TCF binding site (-1167 to -1160) located in the Ngn1 promoter (Mut). NPCs were transfected with a vector containing the Ngn1 promoter (2.7 kb: wild type or mutant) driving luciferase expression. (B) Relative luciferase activity was measured after 13 hours of incubation. Mutation of the TCF binding site in the Ngn1 promoter reduced endogenous transcriptional activity. (C) Chromatin complex was immunoprecipitated with anti-ß-catenin (Santa Cruz Biotechnology and Sigma) or control IgG, and was subjected to PCR analysis to amplify Ngn1 genomic sequence. (D) RT-PCR analysis of Ngn1 expression. NPCs were infected with a retrovirus encoding either GFP alone (control) or both GFP and S33Y ß-catenin, and the expression level of Ngn1 mRNA was analyzed. Gapdh was used for standardization of the samples. No genomic amplification was observed from the RNA treated without reverse transcriptase (-RT).

View larger version (13K): [in a new window]   Fig. 7. Expression of a stabilized form of ß-catenin does not promote neuronal differentiation in the cortical NPCs from E10.5. (A,B) NPCs isolated from E10.5 (A) or E13.5 (B) were acutely plated on poly-D-lysine coated coverslips and infected with a retrovirus encoding either GFP alone (control) or both GFP and S33Y ß-catenin. The cells were incubated for 2 days with Fgf2, and the percentage of TuJ1+ cells among GFP+ cells was determined. *P< 0.02 and **P<10-9 versus control.