Fig 2: Neuroanatomy of the cerebellar cortex is comparable between wildtype and Ano2-/- mice.(A) Representative immunohistochemical stainings for calbindin (green) and parvalbumin (red) on sagittal cerebellar slices of wildtype (left) and Ano2-/- mice (right). Stainings were used to compare Purkinje cell density (B) and size (C) as well as the density of molecular layer interneurons (D) between wildtype and Ano2-/- mice. DAPI (blue) was used to visualize cell nuclei. (B)–(D) No significant differences were detected in Purkinje cell density (B; wt 41.1 ± 6.4 per mm PCL, n = 61 images from N = 6 animals, Ano2-/- 38.5 ± 7.5 per mm PCL, n = 42 N = 6; Student’s t-test p = 0.0811) and diameter (C; wt 20.7 ± 3.4 µm, n = 342 cells in 61 images from N = 6 animals, Ano2-/- 21.1 ± 3.7 µm, n = 246 in 42 images N = 6; Student’s t-test p = 0.2091), as well as in the density of molecular layer interneurons (D; wt 15.9 per 105 µm3 ML IQR 13.3–20.7, n = 62 images from N = 6 animals; Ano2-/- 17.1 per 105 µm3 ML IQR 13.3–23.6, n = 45 N = 6; Wilcoxon-Mann-Whitney test p = 0.501) between wildtype and Ano2-/- mice. (E) Immunohistochemical staining for calbindin (blue), VGluT2 (green) and VGAT (red) on sagittal cerebellar slices. As expected, GABAergic synapses (red) cover the entire molecular layer, whereas climbing fiber synapses (green) only span the proximal two thirds of the molecular layer. (F) Detail of a Purkinje cell with primary and secondary dendrites (calbindin; blue) with climbing fiber synapses (VGluT2; green) and GABAergic synapses (VGAT; red). (G), (H) No significant differences were detected in density of GABAergic (G; wt 22.0 ± 6.8 per 102 µm2 ML, n = 42 images from N = 5 animals, Ano2-/- 21.8 ± 5.5 per 102 µm2 ML, n = 47 N = 5; Student’s t-test p = 0.8788) and climbing fiber synapses (H; wt 3.5 ± 1.0 per 102 µm2 ML, n = 42 images from N = 5 animals; Ano2-/- 3.2 ± 1.0 per 102 µm2 ML, n = 48 N = 5; Student’s t-test p = 0.1989) in the proximal half of the molecular layer between wildtype and Ano2-/- mice. (I) Histograms of the distances measured between adjacent climbing fiber synapses on a Purkinje cell dendrite reveal a comparable distribution of climbing fiber synapses between wildtype and Ano2-/- mice (bin size = 0.5 µm; wt n = 1698 distances in 35 images from N = 9 animals, Ano2-/- n = 1762 in 30 images N = 6). ML = molecular layer; PCL = Purkinje cell layer; GL = granule layer. Scale bars = 20 µm.

Fig 3: Dynamic changes in neuronal transcripts in Dlx2-transfected OPCs. (A) FPKM values of key neuronal transcripts in control- and Dlx2- transfected OPCs. The p-adj values for the transcripts are as follows: Gad1 1.64 × 10–19, Gad2 1.21 × 10–9, Slc32a1 0.0001761, Dlx1 1.34 × 10–23, Dlx5 0.0007, Calb2 6.80 × 10–10. * denotes p-adj < 0.05. n = 3. (B–F) The expression of Dlx1 (B), Dlx5 (C), Dlx6 (D), Dlx1as (E) and Dlx6as (F) transcripts at 2, 7, and 14 dpt obtained by qPCR. Values are for Dlx2-transfected OPCs and are normalized to control-transfected OPCs (black dotted line), and shown as fold-change over control-transfected OPCs in log10 scale. The p-values for the comparison between Dlx2-transfected cells and control-transfected cells are indicated above each dataset in parentheses. The p-values for the comparison of Dlx2-transfected cells at 2, 7, and 14 dpt are indicated above the horizontal bars. Two-way ANOVA, Sidak’s multiple comparisons test, n = 4.

Fig 4: Differentiation of OPC-derived GABAergic neurons. (A,B) Representative mCherry + cells that were recorded from in Tol2-control-transfected (A) and Tol2-Dlx2-transfected (B) OPCs at 14 dpt expressing Gad1-GFP (green) and mCherry (red). Scale bar, 30 µm. (C,D) Membrane voltage traces in response to current injection (- 50 to + 250 pA, 500 ms duration, 10 pA increments). Red indicates response to highest current injection. Recorded cells are categorized into four types, based on the electrical response to voltage clamp recordings (C), and listed in detail in table (D). (E) Co-localization of GAD67 (red) and VGAT (blue) at 14 dpt in Dlx2-transfected OPCs. Right panels show higher resolution images of the boxed region in left panel. Arrowheads indicate area where GAD67 and VGAT double positive puncta wrap around MAP2 + (green) dendrites. Scale bars, 20 µm (low resolution image) and 10 µm (higher resolution images). (F) Co-localization of inhibitory post-synaptic protein gephyrin (red) and pre-synaptic inhibitory protein VGAT (blue) at 21 dpt in Dlx2-transfected OPCs. Right panels show higher resolution images of the boxed region in left panel. Arrowheads indicate synapsetic puncta with colocalized VGAT and gephyrin. Scale bars, 20 µm (low resolution image) and 10 µm (higher resolution images). (G) Expression of inhibitory neuron subtype-specific genes at 14 dpt for control- or Dlx2-transfected OPCs analyzed by qPCR. Values are normalized to the average of control-transfected cells for each transcript. The p-values for each transcript are as follows: Sst 0.0200, Satb1 0.0247, Erbb4 0.0305. Asterisk denotes p-values < 0.05. n = 4, Student’s t-test, unpaired.