Tcf3 Represses Wnt–β-Catenin Signaling and Maintains Neural Stem Cell Population during Neocortical Development

<div><p>During mouse neocortical development, the Wnt–β-catenin signaling pathway plays essential roles in various phenomena including neuronal differentiation and proliferation of neural precursor cells (NPCs). Production of the appropriate number of neurons without depletion of the NPC population requires precise regulation of the balance between differentiation and maintenance of NPCs. However, the mechanism that suppresses Wnt signaling to prevent premature neuronal differentiation of NPCs is poorly understood. We now show that the HMG box transcription factor Tcf3 (also known as Tcf7l1) contributes to this mechanism. Tcf3 is highly expressed in undifferentiated NPCs in the mouse neocortex, and its expression is reduced in intermediate neuronal progenitors (INPs) committed to the neuronal fate. We found Tcf3 to be a repressor of Wnt signaling in neocortical NPCs in a reporter gene assay. Tcf3 bound to the promoter of the proneural bHLH gene <i>Neurogenin1 (Neurog1)</i> and repressed its expression. Consistent with this, Tcf3 repressed neuronal differentiation and increased the self-renewal activity of NPCs. We also found that Wnt signal stimulation reduces the level of Tcf3, and increases those of Tcf1 (also known as Tcf7) and Lef1, positive mediators of Wnt signaling, in NPCs. Together, these results suggest that Tcf3 antagonizes Wnt signaling in NPCs, thereby maintaining their undifferentiated state in the neocortex and that Wnt signaling promotes the transition from Tcf3-mediated repression to Tcf1/Lef1-mediated enhancement of Wnt signaling, constituting a positive feedback loop that facilitates neuronal differentiation.</p></div

Tcf3 increases neurosphere-forming activity and represses neuronal differentiation of NPCs.

<p><b><i>A</i></b>, E11.5 NPCs were plated at 1.0×10⁵ cells/cm² with FGF2 and infected with a retrovirus encoding GFP alone (control), both GFP and Tcf3 (Tcf3), both GFP and ΔN-Tcf3 (ΔN-Tcf3) or both GFP and Tcf1 (Tcf1) and incubated for 1 d with FGF2. The cells were trypsinized and replated at 0.66×10⁵ cells/cm² cells and incubated for 2 d with FGF2 (undifferentiated condition), and then treated with Wnt3a or control for 24 h in the presence of FGF2 and cultured for another 2 d without FGF2 to induce differentiation. The percentage of βIII-tubulin⁺ cells among GFP⁺ cells was determined by immunostaining. <b><i>B</i></b>, E11.5 NPCs were plated at 1.0×10⁵ cells/cm² with FGF2 and infected with control or Tcf3. The cells were incubated for 3 d with FGF2 (undifferentiated condition) and for another 2 d without FGF2 to induce differentiation. The percentage of βIII-tubulin⁺ cells among GFP⁺ cells was determined by immunostaining. <b><i>C</i></b>, E11.5 NPCs were cultured for 1 d with FGF2 and infected with a retrovirus encoding GFP and Luc-shRNA (control), both GFP and Tcf3-shRNA #1 (Tcf3 shRNA #1), or GFP and Tcf3-shRNA #2 (Tcf3 shRNA #2). Then the cells were incubated for 3 d with FGF2 and for another 2 d without FGF2 to induce differentiation. The percentage of βIII-tubulin⁺ cells among GFP⁺ cells was determined by immunostaining. <b><i>D</i></b>, E11.5 NPCs were infected with a retrovirus encoding control, Tcf3, ΔN-Tcf3 or Tcf1 and incubated in suspension culture for 4 d with FGF2 (primary sphere). Cells were plated in suspension at a low density and incubated for 7 d in the presence of FGF2 (secondary sphere). Data represent the number of formed cell aggregates (neurospheres). <b><i>A–D</i></b>, Data represents mean ± SEM (<b><i>A–C</i></b>) and mean ± SD (<b><i>D</i></b>).</p

Tcf3 directly represses transcription of Neurog1 and N-myc.

<p><b><i>A</i></b>, Chromatin complex was immunoprecipitated from E11.5 neocortical lysates with anti-Tcf3. The immunoprecipitates were subjected to qPCR analysis. <b><i>B,C</i></b>, NPCs were infected with a retrovirus encoding control, Tcf3 shRNA #1 or Tcf3 shRNA #2 and incubated with FGF2 for 3 d. Cells were cultured for another 6 h in the presence (undifferentiated condition) or absence (differentiated condition) of FGF2. The mRNA levels of Tcf3 (<b><i>B</i></b>) and Neurog1 (<b><i>C</i></b>) were determined by qPCR analysis. Data obtained in differentiated condition are shown in <b><i>B,C</i></b>. Similar results were obtained in undifferentiated condition (not shown). <b><i>D</i></b>, E11.5 NPCs were infected with a retrovirus encoding control or Tcf3 and incubated with FGF2 for 3 d. The level of Neurog1 mRNA was determined by qPCR analysis. <b><i>E,F</i></b>, NPCs were infected with a retrovirus encoding control, Tcf3 shRNA #1 (<b><i>E</i></b>), Tcf3 shRNA #2 (<b><i>E</i></b>) or Tcf3 (<b><i>F</i></b>) as <b><i>C,D</i></b>. Then the cells were incubated in the presence of FGF2 for 3 d. The level of N-myc mRNA was determined by qPCR analysis. <b><i>B–F</i></b>, Data are normalized with GAPDH mRNA (arbitrary unit). <b><i>A–F</i></b>, Data represents mean ± SEM.</p

Wnt signaling decreases the expression of Tcf3 and increases that of Tcf1 and Lef1 containing β-catenin binding domain.

<p><b><i>A–B</i></b>, E11.5 NPCs were incubated in the presence of FGF2 with control, recombinant Wnt3a (<b><i>A</i></b>) or CHIR99021 (<b><i>B</i></b>) for 0, 3 or 6 h. The mRNA levels of Tcf3, Tcf4, Tcf1 (1st-2nd exon shown in <b><i>E</i></b>) and Lef1 (1st-2nd exon shown in <b><i>E</i></b>) were determined by qPCR analysis. Data are normalized with GAPDH mRNA (arbitrary unit) and represent the mean of three independent samples ± SD. <b><i>C</i></b>, E11.5 NPCs were incubated in the presence of FGF2 with control or recombinant Wnt3a for 24 h. Cell lysates were subjected to Western blotting analysis with antibodies to Tcf3 and GAPDH. <b><i>D</i></b>, E11.5 NPCs were incubated in the presence of FGF2 with control or CHIR99021 for 0, 3 or 6 h. The mRNA levels of Tcf3-l and Tcf3-s were determined by qPCR analysis as <b><i>B</i></b>. <b><i>E–F’</i></b>, E11.5 NPCs were incubated in the presence of FGF2 with control or recombinant Wnt3a for 4 h. Poly-A selected RNA was subjected to Northern blotting analysis with Tcf1 or Lef1 domain specific probes shown in <b><i>E</i></b>. <b><i>F,F’</i></b>, Markers represent 28S (upper) and 18S (lower) ribosomal RNA. Ethidium bromide (EtBr) staining is shown as loading control (lower panel).</p

PD-L1/PD-L2-expressing B-1 cells inhibit alloreactive T cells in mice

<div><p>B cells constitute a complex system of antigen-presenting cells (APCs) and exist as distinct subsets that differ in their lineage affiliation, surface molecule expression, and biological function, thus potentially regulating the immune response. In this study, we investigated the immune-regulatory roles of murine B cell subsets as regulatory APCs targeting alloreactive T cells. Either splenic B cells, peritoneal cavity (PerC) B cells, or non-B cells from Balb/c mice were intravenously injected into B6 mice. Serum levels of anti-Balb/c antibodies in the recipients of PerC B cells were significantly lower than those in the recipients of splenic B cells and PerC non-B cells, as determined over a 4-week period after the injection. Mixed-lymphocyte reaction (MLR) assays using splenocytes from the B6 mice at 2 weeks after the injection revealed the significantly reduced anti-Balb/c T cell-responses in the recipients of PerC B cells, as compared to those in the recipients of splenic B cells or untreated control mice. Since PerC B cells contained MHC class II⁺ CD80⁺ CD86⁺ PD-L1⁺ PD-L2⁺ cells among the CD5⁺ B-1a cell subset, PerC B cells from Balb/c mice were pre-incubated with anti-PD-L1/PD-L2 mAbs prior to injection. This treatment abrogated their immune-regulatory effects on anti-Balb/c T cells in the MLR assays. In addition, the inoculation with Balb/c PerC B cells significantly prolonged the survival of subsequently grafted Balb/c hearts in B6 mouse recipients, whereas that with SPL B cells did not. These findings indicate that the PerC B cells, including PD-L1/PD-L2 B-1a cells, may suppress T cells responding to allostimulation, and thus may be optimal for donor lymphocyte injection.</p></div

Tcf3 is expressed in Pax6⁺ NPCs in the developing neocortex.

<p><b><i>A,A’</i></b><i>,</i> In situ hybridization of coronal sections of the E11.5 (<b><i>A</i></b>) and E14.5 (<b><i>A’</i></b>) mouse cortex for the Tcf3 mRNA. <b><i>B–H”</i></b>, Coronal sections of E14.5 neocortex immunostained as indicated. VZ, ventricular zone; SVZ, subventricular zone; IMZ, intermediate zone; CP, cortical plate (<b><i>B</i></b>). Higher magnification of VZ in <b><i>C–G</i></b> are shown in <b><i>C’–G’</i></b>. Scale bars, 100 µm (<b><i>A,A’,B</i></b>) and 20 µm (<b><i>C–G’</i></b>). The percentages of Tcf3⁺Pax6⁻ cells, Tcf3⁺Pax6⁺ cells and Tcf3⁻Pax6⁺ cells among either Tcf3⁺ or Pax6⁺ cells in the VZ were determined by immunostaining (<b><i>H</i></b><i>,</i> below). The percentages of Tcf3⁺Tbr2⁻ cells, Tcf3⁺Tbr2⁺ cells and Tcf3⁻Tbr2⁺ cells and those of Tcf3⁺Neurog1⁻ cells, Tcf3⁺Neurog1⁺ cells and Tcf3⁻Neurog1⁺ cells are shown in <b><i>H’,H”</i></b>. Venn diagram of these percentages (<b><i>H-H”</i></b>, above). <b><i>I</i></b>, dissociated cells from E14.5 neocortices of Nestin-d4-Venus transgenic mouse were sorted into Nestin-d4-Venus -, Nestin-d4-Venus +, Nestin-d4-Venus ++, and Nestin-d4-Venus +++ fractions by using FACS (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094408#pone.0094408.s001" target="_blank">Fig. S1</a>). The mRNA level of Tcf3 in each fractions was determined by qPCR analysis. Data represents mean ± SEM (<b><i>H-H”, I</i></b>).</p

T-cell allo-response after the intravenous injection of allogeneic B cells, as evaluated by performing CFSE-MLR assay.

<p>SPL or PerC B cells, or PerC non-B cells from Balb/c mice were isolated by magnetic sorting with CD19 microbeads, and were injected into B6 mice through the tail vein. T-cell alloreactivity was determined by performing CFSE-MLR assay 2 weeks after the adoptive transfer. The CFSE-MLR assay was performed using B6 mice injected with Balb/c SPL B cells (n = 6), Balb/c PerC B cells (n = 6), and Balb/c PerC non-B cells (n = 6); control B6 mice were not injected with any cells (n = 7), as responders. SIs and PFs were calculated as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178765#pone.0178765.ref022" target="_blank">22</a>]. (A) CFSE-labeled CD4⁺ (upper row) and CD8⁺ (lower row) T cell division in the MLR assay was quantified by performing FCM analysis. Representative plots and images are shown. CFSE-labeled splenocytes from B6 mice were used as responders, and irradiated (30 Gy) splenocytes from Balb/c mice were used as stimulators. (B) The SIs and PFs of splenocytes from B6 mice in the CFSE-MLR are shown. *<i>p</i> < 0.05. Data are presented as mean ± SEM, and are representative of two independent experiments with six to seven mice per group.</p

CFSE-MLR assay after the intravenous injection of PerC B cells pre-incubated with anti-PD-L1 and/or anti-PD-L2 mAbs.

<p>PerC B cells from Balb/c mice were isolated, pre-incubated with anti-PD-L1 and/or anti-PD-L2 mAb for 30 min, and injected to B6 mice through the tail vein. Isotype-matched control IgGs were used instead of anti-PD-L1 or anti-PD-L2 mAb. Rat IgG2b was used instead of anti-PD-L1 mAb, and Rat IgG2a was used instead of anti-PD-L2 mAb. Mice in the control group did not receive adoptive transfer of cells or mAbs. T-cell alloreactivity was determined by performing the CFSE-MLR assay 2 weeks after the intravenous injection. Upper row shows the mean SIs of CD4⁺ and CD8⁺ T cells, and lower row shows the mean PFs of CD4⁺ and CD8⁺ T cells; *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, and ****<i>p</i> < 0.00001. Data are presented as mean ± SEM, and are representative of two independent experiments with four to five mice per group.</p

Allo-Ab assay after intravenous injection of allogeneic B cells.

<p>B cells of SPL or PerC, or non-B cells of PerC isolated from Balb/c mice were injected into B6 mice trough the tail vein. To evaluate allo-Ab production, sera of recipient B6 mice were obtained every week after the adoptive transfer, and were incubated together with splenocytes from Balb/c mice as target cells. Mice in the control group received no adoptive transfer of cells. (A) Allo antibody response after the intravenous injection. Anti-Balb/c IgM or IgG was evaluated by FCM assay, and MFI was calculated (error bars, SEM). CD19⁺ B cells of the Balb/c splenocytes were excluded from the analyses, because they included surface IgM or IgG expressing cells. (B) Subclasses of anti-Balb/c IgGs produced after the intravenous injection. Anti-IgG1, anti-IgG2a/2b, or anti-IgG3 was used to detect the production of allo-IgG subclasses and evaluated by FCM assay. Data are presented as MFI ± SEM; *<i>p</i> < 0.05, and **<i>p</i> < 0.01 (mice receiving PerC B cells vs mice receiving SPL B cells); †<i>p</i> < 0.05, ††, and <i>p</i> < 0.01, and ††† <i>p</i> < 0.001 (mice receiving PerC B cells vs mice receiving PerC non-B cells). Data are representative of two independent experiments, with six mice per group.</p

Phenotypic analysis of each B cell subset isolated from Balb/c mice.

<p>Naïve B cells from the SPL, PerC, and liver were stained with various combinations of mAbs directed against IgM, CD19, CD21, CD11b, CD5, PD-L1, PD-L2, FasL, TRAIL, CD80, CD86, and MHC class II, and were analyzed by performing FCM. Then, these cells were divided into the following subsets: CD21^intIgM^int follicular (FO) B cells, CD21^highIgM^high marginal zone (MZ) B cells, and CD21^-/lowIgM^high B-0 cells from the SPL, and CD11b⁺CD5⁺ B-1a cells, CD11b⁺CD5^- B-1b, and CD11b^-CD5^- B-2 cells from the PerC and liver (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178765#pone.0178765.s002" target="_blank">S2 Fig</a>). (A) Histograms of PD-L1, PD-L2, FasL, TRAIL, CD80, CD86, and MHC class II on each B cell subset by performing FCM analysis. (B) Percentage (mean ± SEM) expression of each membrane marker expressed on each B cell subset. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, and ****<i>p</i> < 0.00001(Student’s <i>t</i>-test). Data are representative of three experiments with three mice per group.</p