Maier et al. Supplementary material Semaphorin 4C and 4G are Plexin-B2 ligands required in cerebellar development 2 Supplementary tables 4 Supplementary figures 1 Supplementary Table 1. Exencephaly and postnatal survival of Sema4C mutants (on C57BL/6 background) Genotype Expected frequency Observed frequency Exencephaly Embryonic stages (n=47)* Wild type 25% 25% (12/47) 0% Sema4c+/- 50% 45% (21/47) 0% Sema4c-/- 25% 30% (14/47) 36% (5/14) Postnatal animals (n=417)* Wild type 25% 27% (113/417) Sema4c+/- 50% 61% (253/417) Sema4c-/- 25% 12% (51/417) * Offspring from Sema4c+/- x +/- matings. Embryonic stages scored at E15-E18, postnatal animals scored at P21. 2 Supplementary Table 2. Cerebellar phenotypes of Sema4C and Sema4G mutants (on CD-1 outbred background) Ectopic Fusion of Gap in Normal Gap in IGL granule cells lobules IGL of cerebellum of lobule II in molecular VIII/IX* lobule X Genotype n layer weak strong Wild type 17 100% 0% 0% 0% 0% 0% Sema4c+/- 11 70% 10% 0% 10% 0% 10% Sema4c-/- 10 20% 70% 0% 40% 0% 40% Sema4c+/-; Sema4g+/- 16 55% 18% 0% 18% 0% 18% Sema4c+/-; Sema4g-/- 13 40% 20% 0% 20% 0% 40% Sema4c-/-; Sema4g+/- 10 20% 30% 0% 50% 0% 60% Sema4c-/-; Sema4g-/- 11 0% 55% 0% 55% 55% 82% *Fusions of lobules VIII and IX were scored as “weak” when ectopic granule cells formed a band of cells at the fusion line, and as “strong” when a continuous bridge of granule cells connected the IGL of the two lobules. 3 Supplementary figure legends Maier et al. Suppl. Fig. 1 Expression Sema4 and Plexin-B genes in specific cerebellar cell types P10 cerebellar sections stained by in situ hybridization for Sema4 and Plexin-B gene expression (purple stain) and co-labeled by immunohistochemical markers (brown stain). Antibodies against S100 and glial fibrillary astrocytic protein (GFAP) were used for labeling of Bergmann glia and astroglia, against Pax6 for granule cells, against Calbindin for Purkinje cells, and against Olig2 for oligodendrocytes. BG, Bergmann glia; AG, astroglia; EGL, external granule cell layer; IGL, Internal granule cell layer; PC, Purkinje cells; OG, oligodendroglia. Scale bar: 25 m. Suppl. Fig 2 Plexin-B2 EUCOMM mutation The Plexin-B2 mutant allele generated by the European Conditional Mouse Mutagenesis program (EUCOMM). Official allele symbol: Plxnb2tm1(EUCOMM)Wtsi; abbreviated here as Plxnb2EUC1a. In contrast to the previously reported Plexin-B2 mutation (Friedel et al., 2007), the Plxnb2EUC1a is viable as homozygous mutant on C57BL/6 background. This discrepancy could be either due to slight differences in the genetic background (the EUCOMM mutations is in pure isogenic C57BL/6, while the Friedel et al., 2007 mutation was generated in 129P2 ES cells and then backcrossed as congenic line to C57BL/6N), or to the fact that the EUCOMM mutation contains still small amounts of wildtype protein (see Western blot below), which may result in a hypomorphic mutation. The EUCOMM allele can be further modified by Flp and Cre recombinases to generate conditional or deletion alleles, respectively (see www.eucomm.org). (A) The EUCOMM targeted trapping vector, which is flanked by two 5' and 3' homology arms was inserted by homologous recombination into the intron between exon 6 and 7. The critical exons 7, 8, and 9 are flanked by loxP sites. This cassette acts like a gene trap insertion and disrupts Plexin-B2 function and reports its expression. FRT, target site for FLP recombinase; loxP, target site for Cre recombinase; SA, splice acceptor; TM, transmembrane domain; gal, -galactosidase; T2A, self cleavage peptide; pA, polyadenylation sequence. 4 (B) Western blot analysis of lysates from P5 cerebella with anti-Plexin-B2 antibody directed against a peptide downstream of the targeting site shows reduction of Plexin-B2 protein in Plxnb2EUC1a-/- mutants to less than 5% of wild type levels (80 kDa band). Detection of ß-actin served as loading control. (C) Plxnb2EUC1a-/- mutants in co-isogenic C57BL/6N background show pigmentation defects at distal fore and hind limbs, and at the ventral midline. (D) Sagittal cerebellum sections of Plxnb2EUC1a+/- and -/- animals stained for -galactosidase activity. Plxnb2EUC1a+/- animals appear phenotypically normal. Severe cerebellar defects are found in Plxnb2EUC1a-/- animals, largely identical to defects described in a previous loss-of- function mutation (Friedel et al., 2007). Lobules I-III and VI-VII are fused and the cerebellar lamination is profoundly disturbed. Lobule VIII and IX are fused and also lobule X is disrupted. Ectopic clusters of granule cells are found at the subpial surface. Scale bar in (D): 400 m. Suppl. Fig 3 Targeting of the Sema4G gene (A) For construction of the targeting vector, a 350 bp fragment containing the first exon with the initiation codon and the second exon was replaced with a neo resistance cassette. A Herpes simplex virus thymidine kinase (HSV-TK) gene was attached downstream of the 3’ homology arm for selection against random integration. (B) Nissl staining of Sema4G mutant section reveals no cerebellar phenotype. Scale bar in (B): 400 m. Suppl. Fig 4 Sema4B mutants show normal cerebellar morphology (A) Insertion of the secretory gene trap vector pGT1TMpfs in the line RST 235 into the Sema4B gene (Leighton et al., 2001). Official allele name (Mouse Genome Informatics Database, The Jackson Laboratory): Sema4bGt(RST235)Byg. (B) Adult cerebellum of a homozygous Sema4B mutant stained for -galactosidase activity of the trap reporter, which revealed Sema4B gene expression in Bergmann glia and astroglia. No cerebellar phenotype was detectable. Scale bar in (B): 400 m. 5 Maier et al. Suppl. Fig. 1 Sema4a Sema4b Sema4c Sema4d Sema4g PC BG BG BG BG GFAP GFAP GFAP GFAP Calbindin EGL IGL OG AG S100 Pax6 Olig2 Plxnb1 Plxnb2 Plxnb3 EGL BG OG IGL GFAP Pax6 Olig2 Maier et al. Suppl. Fig. 2 A B wt PB2 PB2 C +/- -/- Plexin-B2 80 kDa ß-actin 43 kDa Plxnb2EUC1a -/- D Plxnb2EUC1a +/- Plxnb2EUC1a -/- P30 P30 ß-gal ß-gal Maier et al. Suppl. Fig. 3 A B Sema4g-/- Ad Nissl Maier et al. Suppl. Fig. 4 A B RST235 RST235 Sema4b secretory trap Sema4b -/- insertion P90 .