Figure 5. Plxnb2 mutant CGNs display aberrant proliferative and tangential stages.

(A) Schematic representation of the in vivo cerebellum electroporation protocol. See Materials and methods for details. (B) Cerebellar sections of electroporated brains at 1 day, 2 days, 1 week and 3 weeks after electroporation at P7, to illustrate the different stages of CGN development. Sections were immunostaining for GFP, Calbindin (CaBP), to label Purkinje cells. EdU was injected 2 hr prior to fixation, to label proliferating CGNs in the oEGL. One day after electroporation, GFP⁺ CGNs are still proliferating or became postmitotic and initiated tangential migration. Two days after electroporation GFP⁺ CGNs start to migrate radially toward the IGL. One week after electroporation all GFP⁺ CGNs reached the IGL, where they start growing dendrites. After 3 weeks, GFP⁺ cells have their characteristic morphology with 3–4 claw-shaped dendrites. (C) Immunohistochemistry of coronal sections of cerebellum 1 day post-electroporation. GFP shows the electroporated CGNs and EdU, which was injected 2 hr before fixation, labels proliferating CGNs. Both the distribution and the morphology of migrating Plxnb2 mutant GFP⁺ CGNs are altered. (D) The graph shows aberrant process length of tangentially migrating CGNs in En1^Cre;Plxnb2^fl/fl pups. Error bars represent SEM. Bipolar leading process (longest process): ctl 70.86 ± 3.94 μm vs. mut 52.12 ± 2.92 μm, MWU(955) p=0.0002. Bipolar trailing process: ctl 31.1 ± 2.68 μm vs. mut 23.1 ± 1.84 μm, MWU(1117) p=0.0057. Unipolar leading process: ctl: 52.71 ± 9.32 μm vs. mut 45.07 ± 3.02 μm. MWU(416) p=0.75 (not significant). Forty-four wildtype bipolar cells and 73 mutant bipolar cells, and 16 wildtype unipolar and 66 mutant unipolar cells of 6 wildtype and 4 Plxnb2 mutant animals were quantified. (Figure 5—source data 1) (E) Quantification of the % of EdU⁺ and GFP⁺ GCNs. In Plxnb2 mutants, many bipolar GFP⁺ GCNs are also EdU⁺, unlike in controls (see Figure 5—figure supplement 1). By contrast the % EdU⁺/GFP⁺ GCN precursors is similar in Plxnb2^fl/fl controls and En1^Cre;Plxnb2^fl/fl mutants. A total of 447 ctl and 297 mutant precursors, and 451 ctl and 533 mutant bipolar CGNs were counted, from 6 wildtype and 4 Plxnb2 mutant animals. Error bars represent SEM. Precursors: ctl 53.91 ± 3.01% vs. mut 42.97 ± 9.51%, MWU(8) p=0.48 (not significant). Bipolar cells: ctl 6.82 ± 1.17% vs. mut 27.53 ± 4.86%, MWU(0) p=0.0095 (Figure 5—source data 1). (F) P8 coronal sections of the cerebellum, 1 day post-electroporation. Mitotic CGNs in the EGL are immunostained with anti-H3P antibodies. At this stage, GFP⁺ cells are either in a precursor state (outlined and marked P) or display a clear bipolar morphology (outlined and marked B) and express Tag1, a marker of tangentially migrating CGNs. In controls, only CGN precursor cells express H3P, whereas in Plxnb2 mutants, H3P is found in precursors but also in some Tag1⁺ bipolar CGNs. (G) Coronal sections of the cerebellum 2 days post-electroporation. GFP immunostaining labels the electroporated CGNs, and EdU (injected 2 hr before fixation) stains proliferating CGNs. Calbindin (CaBP) labels Purkinje cells. GFP⁺ cells were counted and grouped in radial, tangential and precursor cell stages based on their morphology. In controls, most CGNs have reached radial stage 2 days after electroporation. By contrast, many GFP⁺ CGNs are still in the tangential phase in Plxnb2 mutants. Radial CGNs are not labeled by EdU. Graph shows that in Plxnb2 mutants, more cells are in the radial stage (ctl 50 ± 2.77% vs. mut 38.28 ± 2.37%, MWU(1) p=0.0159) and less cells in the tangential stage (ctl 34 ± 1.33% vs. mut 47.85 ± 2.37%, MWU(0) p=0.0079). There is no significant difference in cells still in the precursor stage (ctl 16 ± 1.98% vs. mut 13.9 ± 1.71%. MWU(10) p=0.65). Error bars represent SEM. 899 ctl and 744 mutant CGNs were counted, from five animals per genotype (Figure 5—source data 1). (H) Sagittal sections of the cerebellum more than 3 weeks after electroporation with GFP. Electroporated CGNs are stained with GFP, Purkinje cells with Calbindin (CaBP) and sections were counterstained with DAPI. Three different types of defects are seen in Plxnb2 mutants: (I) Parallel fibers that usually occupy a thin part within the molecular layer (Ia) disperse through the entire molecular layer in the mutant (Ib); (II) Whereas the white matter of control cerebella is devoid of parallel fibers (IIa), some mutant CGNs send their axons into the cerebellar white matter (IIb); and (III) ectopic patches of CGNs accumulate at the cerebellar. Ectopic CGNs still acquire their characteristic morphology. Scale bars: (B, C, E): 50 μm; D: 10 μm; (F) overview panels: 500 μm, high-magnification panels: 20 μm.

Figure 5—figure supplement 1. Identity of electroporated cells and quantification of morphological features.

(A) Immunohistochemistry of coronal sections of cerebellum 2 days post-electroporation. GFP shows the electroporated cells and Pax6 marks pre- and post-mitotic CGNs. Graph shows that 99.6 ± 0.21% of all GFP⁺ cells are also Pax6⁺. Error bars represent SEM. 198 ctl and 382 mutant GFP⁺ cells were counted and pooled from four animals. (Figure 5—figure supplement 1—source data 1) (B) Graph shows quantification of cell body measurements of GFP⁺ CGNs 1-day-post-electroporation as shown in Figure 5C. Cells can be classified as precursors, of CGNs with unipolar or bipolar morphologies. No difference is seen between controls and Plxnb2 mutants. Width/length ratio of 74 ctl and 159 mut CGNs from three different pups per genotype were calculated. Bipolar cells: ctl 0.47 ± 0.02 vs. mut 0.45 ± 0.02, MWU(1400) p=0.25; Unipolar cells: ctl 0.42 ± 0.03 vs. mut 0.47 ± 0.02, MWU(382) p=0.43; Precursors: ctl 0.62 ± 0.03 vs. mut 0.57 ± 0.03, MWU(509) p=0.26. Error bars represent SEM. (Figure 5—figure supplement 1—source data 1) (C) EGL of cerebella electroporated 24 hr before isolation at P8 were equally divided in two bins. Six control and four mutant brains were analyzed. All GFP⁺ cells were counted and quantified as multipolar or bipolar. CGN-appearance per bin was then analyzed as a percentage of total CGNs in that morphology. Percentage of all multipolar cells residing in bin 1 ctl 87.5 ± 3.19% vs. mut 70.41 ± 1.2% MWU(0) p=0.0095; Percentage of all bipolar cells residing in bin 2 ctl 90.22 ± 2.64% vs. mut 52.24 ± 2.34% MWU(0) p=0.0095; Bipolar in ML ctl 5.19 ± 2.84% vs. 27.89 ± 4.17% MWU(0) p=0.0095. Error bars represent SEM. (D) High magnification images of Immunohistochemical staining of En1^Cre;Plxnb2^fl/fl P8 brains showing bipolar GFP⁺-CGNs that bear both proliferation markers EdU (injected 2 hr before brain isolation) and H3P. (E) Representative examples of Plxnb2^fl/fl and En1^Cre;Plxnb2^fl/fl CGNs 2 days post-electroporation. Axons and leading processes are pointed out. Graphs show axon and leading process lengths, and ratio of cell body width and length. Error bars represent SEM. No significant difference is found in process length (leading process: ctl 28.11 ± 0.72 μm vs. mut 30.86 ± 1.67 μm, MWU(17024) p=0.27; axon length: ctl 60.07 ± 2.5 μm vs. mut 64.98 ± 3.0 μm, MWU(17380) p=0.44). Plxnb2 mutant CGNs in their radial phase appear slightly rounder (width/length ratio ctl 0,47 ± 0.01 vs. mut 0.55 ± 0.01, MWU(13379) p<0.0001). A total of 199 ctl and 183 mut CGNs were analyzed from at least three different pups per genotype. (Figure 5—figure supplement 1—source data 1) (F) Representative examples of Plxnb2^fl/fl and En1^Cre;Plxnb2^fl/fl CGNs, 2 and 3 weeks after electroporation. (G) Whisker plots show quantification results from cells as in (F). In P20 En1^Cre;Plxnb2^fl/fl mutants, more CGNs have acquired their characteristic mature morphology, with 3–4 claw-shaped dendrites. 198 ctl and 251 mutant CGNs at P20, and 204 ctl and 213 mutant CGNs at P30 were analyzed from at least three different pups from each age and genotype. No significant difference was found in cell body measurements at P20 and P30, and the number of dendrites was also similar at P30. However, at P20 a higher portion of mutant CGNs, electroporated at P7 with GFP, already pruned their dendrites to the amount of 4 (MWU(21308) p<0.0001) (Figure 5—figure supplement 1—source data 1). Scale bar (A, C, D): 10 μm.

Figure 5—figure supplement 2. Misplaced and misprojecting CGNs keep their identity.

(A) Immunohistochemistry of a sagittal section from an En1^Cre;Plxnb2^fl/fl cerebellum, more than 3 weeks after electroporation with GFP. GFP labels electroporated CGNs, Calbindin (CaBP) stains Purkinje cells, MOG stains myelin, and sections were counterstained with DAPI. (B) High magnification of the framed region in (A) showing that CGN axons misprojecting in the white matter are not myelinated, similar to parallel fibers in control cerebella. Scale bars: (A): 500 μm; (B): 5 μm.