Figure 7. Plxnb2 CGNs recapitulate in vitro the developmental defects found in vivo.

(A) EGL explants from P4-P5 wildtype cerebella, fixed after 1, 2, 3, and 5 days in vitro (DIV). Immunocytochemistry for βIII-tubulin and DAPI shows that cells migrate away from the explant and extend long neurites. After DIV2, cells start accumulating in clusters around the original explant. (B) EGL explants from P4-P5 Plxnb2^fl/fl and En1^Cre;Plxnb2^fl/fl cerebella at DIV1. Cultures were stained for DAPI and βIII-tubulin. Plxnb2 mutant explants show DAPI⁺ nuclei closer to the explant and different neurite outgrowth. (C) DAPI⁺ nuclei around the explant were counted and their migration was assessed using a Sholl-analysis. Graph shows that less cells migrate from En1^Cre;Plxnb2^fl/fl explants and that they stay closer to the explant. Multiple t-test with the Holm-Sidak method were applied to the mean intersections of DAPI⁺ nuclei with the Sholl circles. p<0.0001. A total of 36 controls and 34 mutant explants were analyzed from three different experiments. Error bars represent SEM. (Figure 7—source data 1) (D) EGL explants from cerebella electroporated ex vivo with GFP and fixed at DIV1 and DIV2. Immunocytochemistry for GFP and DAPI shows the morphology of migrating cells. GFP⁺ CGNs have either a multipolar (m) or a bipolar (b) shape. After DIV2, almost all GFP⁺ cells have a bipolar morphology, with their trailing process attached (b–a) or not (b–na) to the explant. (E) Quantification of the proportion of GFP⁺ CGNs with multipolar or bipolar morphologies. Data is expressed as percentage from total number of GFP⁺ cells per explant ± SEM. DIV1 multipolar: ctl 48.88 ± 2.65% vs. mut 65.42 ± 2.37%, MWU(353) p<0.0001. DIV1 bipolar: ctl 51.12 ± 2.65% vs. mut 34.58 ± 2.37%, MWU(353) p<0.0001. DIV2 multipolar ctl 11.36 ± 1.33% vs. mut 17.58 ± 3.02%, MWU(226) p=0.13, not significant. DIV2 bipolar ctl 88.67 ± 1.37% vs. mut 80.31 ± 3.07%, MWU(189) p=0.03. All GFP⁺ cells (amounts between brackets) were counted from 46 ctl (2728) and 33 mut (835) explants (DIV1) and 32 ctl (2284) and 19 mut (617) explants (DIV2) from at least three different experimental repeats. (Figure 7—source data 1) (F) Quantification (Box-plots) of leading and trailing process length of bipolar GFP⁺ CGNs at DIV1. Leading ctl 106 ± 3.9 μm vs. mut 67.6 ± 5.62 μm, MWU(11147) p<0.0001; trailing ctl 79.5 ± 3.4 μm vs. mut 40.9 ± 2.76 μm, MWU(68833) p<0.0001. A total of 385 ctl and 93 mut cells were analyzed from 29 ctl and 13 mut explants from three experimental repeats (Figure 7—source data 1). (G) Proportion of bipolar GFP⁺ CGNs attached to the explant. DIV1 attached: ctl 52.19 ± 2.49% vs. mut 50.55 ± 4.27%, (MWU(709.5)) p=0.63, not significant. DIV2 attached: ctl 53.63 ± 2.69% vs. mut 34.92 ± 2.31%, MWU(36.5) p<0.0001. All GFP⁺ cells (amounts between brackets) were counted from 46 ctl (2728) and 33 mut (835) explants (DIV1) and 32 ctl (2284) and 19 mut (617) explants (DIV2) from at least three different experimental repeats (Figure 7—source data 1). Scale bars: overviews 500 μm (A, B, D); magnifications in (D): 50 μm.

Figure 7—figure supplement 1. Migrating cells have a CGN identity.

(A) Immunocytochemistry of DIV2 EGL explants for Pax6 (pre- and postmitotic CGNs) with DAPI counterstaining. Cells were counted in 400 × 400 pixel squares close to the explant. Almost all migrating cells are Pax6⁺ in controls (Pax6⁺/DAPI⁺ ctl 98.78 ± 0.28%) and Plxnb2 mutants (98.64 ± 0.43%, MWU(68) p=0.83, not significant). In total 2412 and 2361 DAPI⁺ cells were counted from 19 ctl and 18 mut explants respectively from three different experiments at DIV1 and DIV2 (Figure 7—figure supplement 1—source data 1). (B) Immunocytochemistry of EGL explants at DIV2 for GFAP (glia) with DAPI staining. Explants contain glia but their cell bodies do not exit the explant. Scale bars: low magnifications 100 μm, high magnifications 10 μm.

Figure 7—figure supplement 2. Electroporated cells migrating away from EGL explants have CGN identity.

(A) EGL explants from P4-P5 cerebella electroporated ex vivo with GFP just before culture. Immunocytochemistry for GFP and Pax6 with DAPI staining shows that in both controls and Plxnb2 mutants (almost) all GFP⁺ cells are also Pax6⁺ (white arrows; Pax6⁺/GFP⁺ ctl 98.77 ± 0.70% vs. mut 98.61 ± 0.64%, MWU(28.5) p=0.72, not significant). Error bars represent SEM. Cells were counted from 8 DIV1 explants from both genotypes (667 ctl and 348 mut cells) (Figure 7—figure supplement 2—source data 1). (B) Ex vivo GFP-electroporated EGL explants at DIV2 immunostained for GFP and Sema6A (tangentially migrating CGNs) and counterstained with DAPI. High magnifications show that GFP⁺ cells co-express Sema6a. (C) Ex vivo GFP electroporated EGL explants at DIV2 immunostained for GFP and GFAP and counterstained with DAPI. High magnifications show that GFP⁺ cells are not positive for glial markers. Scale bars: (A): 50 μm; (B, C): overview panels 100 μm, high magnifications 10 μm.