Figure 1. Plexin-B2 expression and generation of cerebellum-specific Plxnb2 conditional knockout models.

(A, B, C), Plexin-B2 protein distribution in the cerebellar cortex during different stages of postnatal development. (A) Plexin-B2 immunostaining on cryostat sections immunolabeled with the Purkinje cell (PC) marker Calbindin (CaBP) and counterstained with DAPI shows that Plexin-B2 is expressed in the external granule layer (EGL). (B) Plexin-B2 immunoreactivity coincides with EdU (injected 2 hr prior to fixation to visualize proliferating cells) showing that this receptor is restricted to proliferating cerebellar granule neurons (CGNs) in the outer external granular cell layer (oEGL). It is downregulated in Tag1⁺ postmitotic CGNs in the inner EGL (iEGL). (C) High-magnification images show Plexin-B2 expression in the oEGL (stained with EdU), which regresses between P6 and P15. (D) Sagittal section of P27 cerebellum Plxnb2^-/- (full knockout) cerebellum stained with DAPI. The structure and layers of the cerebellar cortex are disorganized. Clear gaps in the internal granule layer structure can be observed (yellow asterisks), as well as patches of cells that accumulated at the cerebellar surface (arrowhead). (E) Schematic representation of the genomic Plxnb2 sequence of the conditional Plxnb2 mutant described in Deng et al., 2007. The loxP sites flanking exons 19–23 are depicted with red triangles. Plxnb2^fl/fl conditional mutant mice were crossed with En1^Cre or Wnt1-Cre mice. (F) In situ hybridization, on cerebellar sections at P4 and P7, with a probe recognizing the floxed exons of the Plxnb2 gene. Sections incubated with sense probe are devoid of signal. In cre-negative Plxnb2^fl/fl control mice, Plxnb2 mRNA is only detected in the oEGL. In both En1^Cre;Plxnb2^fl/fl and Wnt1-Cre;Plxnb2^fl/fl littermates, Plxnb2 mRNA is deleted from the oEGL. (G) Plexin-B2 immunostaining on sagittal cerebellar sections of En1^Cre;Plxnb2^fl/fl (P5) and Wnt1-Cre;Plxnb2^fl/fl (P7) animals shows the absence of Plexin-B2 protein in the EGL. Sections were also labeled with anti-CaBP antibodies and DAPI. Impaired cerebellar foliation (white arrowheads) and Purkinje cell islands (yellow asterisks) are observed in both conditional knockouts. (H) P20 sagittal cerebellar sections immunostained for CaBP and counterstained with DAPI. Both En1^Cre;Plxnb2^fl/fl and Wnt1-Cre;Plxnb2^fl/fl conditional knockouts phenocopy the cerebellar defects found in Plxnb2^-/- mutants. White arrowheads mark altered foliation, whereas yellow arrowheads in the magnified panels show surface accumulations of CGNs. Yellow asterisks indicate Purkinje cell islets. En1^Cre;Plxnb2^fl/fl mice display the Plxnb2 phenotype to a greater extent. Scale bars: (A) 1 mm. (B, C) 10 μm. (D) Low magnification 1 mm, high magnification 500 μm. (F) Low-magnification overview panels: 500 μm, high-magnification panels: 100 μm. (G) Overview panels: 500 μm, high-magnification EGL panels: 10 μm. (H) Low-magnification panels 300 μm, high-magnification panels 100 μm.