Fig 2: 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 En1Cre;Plxnb2fl/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 Plxnb2fl/fl controls and En1Cre;Plxnb2fl/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—source data 1.CGN morphology in vivo and colocalization with proliferation markers.

Fig 3: Aberrant proliferation of CGN precursors in Plxnb2 mutant explants.(A) EGL explants from P4-P5 cerebella at DIV1. Two hr prior to fixation, 10 µM EdU was added to the culture medium. Cultures were stained for EdU, H3P, and DAPI. (B) The number of DAPI+ nuclei/migrating cells around DIV1 explants, is significantly decreased in Plxnb2 mutants (485.79 ± 34.77 cells) compared to controls (748.89 ± 53.54 cells; MWU(290.5) p=0.0001). Error bars represent SEM. 36 ctl and 34 mut explants were analyzed from three different experiments. (Figure 9—source data 1) (C) At DIV1, there is no significant difference in the total amount of EdU+ cells (that incorporated EdU in the last 2 hr) per explant. Ctl 75.19 ± 11.28 vs. mut 72.03 ± 7.85 cells (MWU(596.5) p=0.86. Error bars represent SEM. Thirty-six ctl and 34 mut explants were analyzed from three different experimental replicates (Figure 9—source data 1). (D) Likewise, the portion of EdU+ cells also positive for H3P (an M-phase marker) at the moment of fixation) is similar in controls (13.77 ± 3.80%) and mutants (14.10 ± 0.93%, MWU(477) p=0.11). Error bars represent SEM. Thirty-six ctl and 34 mut explants were analyzed from three different experimental repeats (Figure 9—source data 1). (E) EGL explants from P4-P5 cerebella electroporated with GFP ex vivo. Ten µM EdU was added to the medium after 6 hr of culture. Explants were fixed at DIV1 or DIV2 and EdU incorporation was quantified in multipolar and bipolar GFP+ cells. (F) Quantification of the proportion of multipolar and bipolar GFP+ CGNs that have taken up EdU in the past 18 hr at DIV1 (EdU administered from 6 to 24 hr after plating). Multipolar ctl: 9.394 ± 1.35% vs. mut 13.75 ± 2.31%, MWU(595) p=0.10, not significant; bipolar ctl: 0.41 ± 0.27% vs. mut 0.47 ± 0.27%, MWU(740) p=0.78, not significant. Error bars represent SEM. 2814 ctl and 890 mut GFP+ CGNs were counted from 47 ctl and 33 mut explants from three experimental replicates (Figure 9—source data 1). (G) Quantification of the proportion of multipolar and bipolar GFP+ CGNs that have taken up EdU in the past 42 hr at DIV2 (EdU administered from 6 to 48 hr after plating). Multipolar ctl: 69.81 ± 4.50% vs. mut 77.03 ± 6.55%, MWU(189) p=0.25, not significant. Bipolar ctl: 7.76 ± 1.00% vs. mut 23.3 ± 4.20%, MWU(113) p=0.0001. Error bars represent SEM. 2284 ctl and 617 mut GFP+ cells were counted from 32 ctl and 20 mut explants from three experimental repeats (Figure 9—source data 1). Scale bars 100 µm, high magnifications 10 µm.Figure 9—source data 1.EGL explants: in vitro proliferation.

Fig 4: Developmental time course of cerebellar Plxnb2 phenotype.The time course of cerebellar foliation and lamination during early postnatal cerebellar development is delayed in Plxnb2 conditional knockout. (A) Pax6 immunostaining labels both pre- and postmitotic CGNs in the developing cerebellum, and Calbindin (CaBP) labels Purkinje cells. In controls, many cerebellar fissures have formed at P0, and deepen further at P2 and P4, whereas the cerebellum of En1Cre;Plxnb2fl/fl mutant displays a very shallow primary fissure (prf) at P0 and aberrant fissure development over time. Furthermore, ectopic Purkinje cell islets (arrowheads) are observed in Plxnb2 mutant internal granule layer. (B) 3D-Light sheet microscope imaging of TO-PRO-3 stained and iDISCO+ cleared (see Materials and methods) P4 cerebellum illustrating the foliation delay in Plxnb2 conditional KO. Right panels are optical sections (coronal or horizontal) through 3D-reconstructed images. Plxnb2 mutants develop aberrant shallow fissures and additional folia in different orientations. Abbreviations: pfr: primary fissure, ppf: prepyramidal fissure, sf: secondary fissure, pcn: precentral fissure, pcuf: preculminate fissure, pfl: posterolateral fissure. Scale bars: overview panels (A, B): 500 µm, magnifications in (A): 100 µm.

Fig 5: 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 En1Cre;Plxnb2fl/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 Plxnb2fl/fl and 4 En1Cre;Plxnb2fl/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—source data 1.Parallel fiber distribution.