Figure 6. Abnormal parallel fiber layering in Plxnb2 mutants.

(A) Coronal sections of the cerebellum of P25 mice electroporated with GFP at P7 and re-electroporated with tdTomato (tdTom) at P11. Double immunostaining for GFP and tdTomato. In control (left) the parallel fibers of CGNs that became postmitotic early (GFP⁺) are at the bottom of the molecular layer, whereas the CGNs that became postmitotic later (tdTom⁺) extend parallel fibers at the surface of the molecular layer. In En1^Cre;Plxnb2^fl/fl mutants, there is an important overlap in the molecular layer, between parallel fibers of early and late-born CGNs. The graph shows a quantification of the portion of the molecular layer that is occupied by parallel fibers of either early (GFP⁺) or late (tdTom⁺) CGNs (eg. (GFP⁺ width / ML total width) x 100%). Error bars represent SEM. The molecular layer measurements and its double-electroporated parallel fiber content was averaged from three different points per cerebellum from 7 Plxnb2^fl/fl and 4 En1^Cre;Plxnb2^fl/fl cerebella. P7 GFP ctl: 31.96 ± 2.07% vs. mut: 81.48 ± 4.53% (MWU(0) p=0.0061) and P11 tdTom ctl: 27.45 ± 2.26% vs. mut: 68.74 ± 2.75% (MWU(0) p=0.0061). (Figure 6—source data 1) (B) Coronal sections of cerebella electroporated at P7 and collected at P9 (EdU was injected 2 hr before termination). Sections were stained for GFP, CaBP, and EdU. In controls (left panel), nascent parallel fibers normally extend at the base of the iEGL, just above the tips of developing Purkinje dendritic arbors (yellow arrowheads). However, in Plxnb2 mutant (right panel) parallel fibers extend throughout the EGL and cross the Purkinje dendrites in the ML (yellow arrowheads indicate the tips of Purkinje dendrites). (C) The abnormal presence of young GFP⁺ parallel fibers deep in the molecular layer is also seen on coronal sections of cerebella electroporated at P11 and collected at P13 (Control, left panel and Plxnb2 mutant, right panel). Scale bars 50 μm.

Figure 6—figure supplement 1. Abnormal localization of parallel fiber synapses in Plxnb2 mutant.

(A) Mice were electroporated with GFP at P7 and their cerebellum collected at P9. Sagittal sections were incubated with antibodies against GFP (to label CGNs and parallel fibers), Calbindin (CaBP, Purkinje cells), and Vglut1 (parallel fiber synapses). In Controls, the density of VGlut1⁺ synapses is high along the proximal regions of Purkinje cell dendrites and low at their tips. In the molecular layer of Plxnb2 mutants, GFP⁺ fibers are disorganized, and there is a high density of Vglut1 puncta on both proximal and distal CaBP⁺ dendritic branches. Graph shows the quantification of the ratio between the fluorescent integrated density of distal and proximal Vglut1-labeling. Ctl: 0.21 ± 0.03 vs. mut 0.64 ± 0.07, MWU(0) p=0.028. Vglut1 integrated density was measured and averaged from 5 distal and 5 proximal 10 × 10 μm squares taken from different Purkinje cells per animal, from four animals for both genotypes. Error bars represent SEM (Figure 6—figure supplement 1—source data 1). (B) Sagittal sections of the cerebellum at P30, immunolabeled for Calbindin (Purkinje cells) and Vglut1 (parallel fibers/Purkinje Cell synapses). The synaptic distribution of Vglut1⁺ synapses appears similar in control and Plxnb2 mutants. Scale bars (A, B, C): 50 μm.