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. 2018 Aug 9;7:e39879. doi: 10.7554/eLife.39879

Figure 3. The number of iPCs diminishes with age and increases after ablation of PCs.

(A) Schematic representation of the distribution of iPCs (red) in sagittal midline sections of P1-5 cerebella (yellow, FoxP2+ and CALB1+ PCs) (B) IF analysis of iPCs (FoxP2+ and CALB1-/low, arrow) at P1.5 in No DT mice. (C) Quantification of the numbers of iPCs and PCs at P1, P5 and P30 (CALB1+: One-way ANOVA F(2.6) = 6.883, p=0.028, iPCs: Student’s t-test: p=0.0009, all cells: One-way ANOVA F(2.6) = 1.813, p=0.24, n = 3 animals/condition). Significant post hoc comparisons are shown. (D) Quantification of the numbers of iPCs at P1.5 (Two-tailed t-test, p=0.005, n = 3) and P5 (Two-tailed t-test, p=0.04, n = 3) in No DT and P1-PC-DTR mice. (E–N) Orthogonal projections of z-stack shows a EdU+ PC (CALB1+, FOXP2+) (E–I) or iPC (CALB1-/low, FOXP2+) (J–N) at 15 hr post injection (hpi) in P1-PC-DTR mice (n = 3). (O–X) Orthogonal projections of z-stack shows EdU+ and FOXP2+ cells that either express the cell cycle markers KI67 (O–S) or pH3 (T–X) at 15 hr post injection (hpi) in P1-PC-DTR mice (n = 3). Scale bars: (B) 100 μm, (E, J, O, T) 50 μm.

Figure 3.

Figure 3—figure supplement 1. iPCs are not labeled by Pcp2Cre and their numbers diminish with age.

Figure 3—figure supplement 1.

(A) IF analysis of iPCs at P1 (FOXP2+ and CALB1-/low, arrows) shows that they are not TdT+, thus they escape DT-mediated cell death. (B) Quantification of the number of iPCs shows a steady decrease in the number of cells/midline section. (One-way ANOVA F(3,9)=9.074, p=0.004, n ≥ 3 animals/condition and three sections/mouse). Significant post hoc comparisons are shown.

Figure 3—figure supplement 2. FoxP2-TdT fate mapping marks iPCs in the PCL at P1 as well as rare cells outsidethe PCL.

Figure 3—figure supplement 2.

FoxP2Flpo/+; R26FSF-TdT/+ (FoxP2-TdT; FSF = frt stop-frt) animals were analyzed at P1. (A) As predicted, all of the FOXP2+ cells in the PCL were labeled with TdT+ and some were CALB1-/low. Arrow shows a TdT+, FOXP2+ CALB1-/low cell (iPC) in the PCL. (B–D) FoxP2-TdT also marks rare PAX2+ interneurons (B), PAX6+ granule cells (C) and SOX2+ glial cells/progenitors (D), none of which reside in the PCL. These results suggest the Flpo allele is unexpectedly expressed transiently in rare embryonic progenitors of other lineages than PCs. n = 3 animals/condition were analyzed. Scale bars: 200 μm.

Figure 3—figure supplement 3. The number of FoxP2-TdT transiently marked iPCs increases 12 hr after DT injection at P1.

Figure 3—figure supplement 3.

(A–H) iPCs (TdT+, FOXP2+, CALB1-/low, arrows in the higher magnified images) are sparsely located in No DT FoxP2-TdT pups (A–F) and the number increases 12 hr after DT injection at P1 (E-H, see text, n = 3 animals/condition). Scale bars: 50 μm.

Figure 3—figure supplement 4. Microglia and glial progenitors proliferate in both No DT and DT P1-PC-DTR mice.

Figure 3—figure supplement 4.

(A–D) IF analysis of BrdU+ cells shows that (A–B) IBA1+ microglia and (C–D) SOX2+ glial progenitors proliferate 15hpi (astrocytes and NEPs). Arrows show BrdU+ IBA1+ and Sox2+ cells. n = 3 animals/condition were analyzed. Scale bars: 100 μm.

Figure 3—figure supplement 5. IF analysis of PCs at P1.5 (15h post DT injection at P1) shows that FoxP2+ cells proliferate and there are more FoxP2+ CALB1-/low cells that incorporate BrdU than FOXP2+ CALB1+ high cells.

Figure 3—figure supplement 5.

(A–-B) Analysis of co-labeling for FOXP2 (A) or CALB1 (B) with BrdU (injected 10–14 hr post DT) at 15 hpi of DT shows that more FOXP2+ CALB1-/low cells incorporate BrdU upon DT injection (lower panels) in P1-PC-DTR mice, compared to FOXP2+ CALB1+ cells. Brains of No DT mice show no PCs that incorporated BrdU (top panels). n = 3 animals/condition were analyzed. Scale bars: 100 μm.

Figure 3—figure supplement 6. IF analysis of PCs at P1.5 (15h post DT injection at P1) shows that FOXP2+ cells proliferate (Ki67+ or pH3+) and BrdU+ FOXP2+ CALB1-/low cells can be observed at P1.5 but not at P3.

Figure 3—figure supplement 6.

(A) Arrow indicates a BrdU+ iPC (CALB1-/low, FoxP2+) at 15 hr post injection (hpi) in P1-PC-DTR mice (n = 3). (B) Quantification of the number of BrdU+ cells that are also FOXP2+ cells and that are CALB1 positive or negative per midline sagittal section at P1.5 and at P3 (n = 3/ age). (C) Orthogonal projections of z-stack shows a BrdU+ and FOXP2+ cell that also expresses the cell cycle marker KI67. (D) An example of IF analysis of the nucleus of a pH3+ and FOXP2+ cell 15 hpi in P1-PC-DTR mice (n = 3). γ-tubulin staining is used to label the centrosomes. Note the subnuclear compartmentalization of FOXP2 and pH3 signals during proliferation. Scale bars: (A and C) 100 μm, (D) 5 μm.

Figure 3—figure supplement 7. Analysis of P27 and Ki67 fluorescence intensity of iPCs and CALB1+ PCs in P1 wild type mice.

Figure 3—figure supplement 7.

(A–D) P1 iPC or CALB1+ PC nuclei were defined as regions of interest and the marker fluorescence intensity and the nuclear area was measured and reported as corrected total cell fluorescence (CTCF)/nuclear area. (CTCF = Integrated Density – (Nuclear area X mean fluorescence of background readings). (A–B) iPCs show lower P27 levels compared to PCs (Students t-test, p=0.0008,>30 cells at three different section/n = 3 brains) (C–D) iPCs show higher KI67 levels compared to PCs (Students t-test, p=0.0001,>30 cells at three different section/n = 3 brains). Scale bars: 100 μm.

Figure 3—video 1. Three-dimensional projection of a z-stack from the PCL of a P1 CB showing FoxP2+ CALB1 low/- iPCs.

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DOI: 10.7554/eLife.39879.023
Arrow heads indicate iPCs (FOXP2+ and CALB1low/-) distributed alongside PCs in the PCL.