Generation of Sema6B mutant mice by targeted disruption of the Sema6B gene. A, Schematic illustration of the Sema6B gene (Sema6B locus), the targeting vector, and the targeted locus. Exons are indicated by boxes. The exon 1 in the 5′ upstream is omitted in the scheme. ▿, Position of the initiation codon in the exon 2, ATG; PGK-neo-tpA, PGKneo-triple poly(A) casette; fEGFP-pA, farnesylated EGFP-poly(A) cassette. B, Southern blot analysis on the EcoRI-digested genomic DNA from targeted ES cells, using a 3′ external probe (probe in A). Note that expected sizes of DNAs, 14 kb for the wild-type (wt) allele and 8 kb for the targeted allele, are detected. C, PCR analysis using primers, P1, P2, and neo-4 in A. Expected sizes of DNAs, 1.3 kb for the wild-type allele and 0.6 kb for the mutant allele, are amplified in animals derived from Sema6B heterozygous intercrosses. D, Immunoblot analysis using goat anti-human Sema6B antibodies. A band for the Sema6B proteins is detected at 100 kDa position (arrow) in the extracts of brains from E16.5 wt but not Sema6B homozygous (−/−) mouse embryos. A band at 115 kDa position is a nonspecific binding of the antibody.

Mossy fiber projection in Sema6B mutant mice. A–J, Timm staining (A–H) and immunostaining with calbindin (I, J) of horizontal sections of the hippocampus of adult wild-type and Sema6B homozygous (Sema6B^−/−) mice. The regions corresponding to CA3c, the middle part of CA3ab, and the distal-most part of CA3ab in A and E are shown at a higher magnification in B–D and F–H, respectively. Arrowheads in J indicate mossy fibers that directly grow into the SR. DG, Dentate gyrus; DH, dentate hilus; SL, stratum lucidum; dgc, dentate granule cell; spb, suprapyramidal bundle of mossy fibers; ipb, infrapyramidal bundle of mossy fibers. These abbreviations also apply to other figures of this article. K, L, Timm staining of hippocampal slices from P5 wild-type and Sema6B−/− mice that were cultured for 5 d. Scale bars: A, E, 400 μm; I–L, 100 μm.

Expression of Sema6B and mossy fiber projection in chimeric cocultures of hippocampal slices. A, Expression of Sema6B in the hippocampus of P5 mice detected by ISH analysis. Note a strong ISH signal in pyramidal cells (py). B, Schematic diagram of the chimeric coculture. Horizontal slices of the hippocampus of P5 wild-type and Sema6B^−/− mice are divided into two pieces; a slice that contains the dentate gyrus (DG slice) and a slice that contains CA3 (CA3 slice). The DG slices and the CA3 slices were combined in various combinations and cultured for 5 d. C–F, Timm staining of the cocultures. Solid lines indicate the border of the DG slice and CA3 slice. Dotted lines indicate the boundary between SLM and SR in C–F and also SR and SP in F. Scale bars: A, 200 μm; C–F, 100 μm.

Effects of Sema6B on cultured mossy fibers. A–C, Collapse of growth cones by Sema6B. Explants of the dentate gyrus from P2 wild-type and PlxnA4 homozygous (PlxnA4^−/−) mice were cultured for 2 d and then treated with the Fc-dimerized Sema6B ectodomain Sema6Bect-Fc (100 nm) for 1 h. Mossy fibers were visualized with calbindin antibodies. Note that Sema6B induces collapse of growth cones in wild-type mossy fibers (A, arrowheads) but not PlxnA4-deficient ones (B). Incidence of collapsed growth cones was plotted against different concentration of Sema6B (C). The protein concentration for Sema6Bect-Fc was converted as a monomer. The average percentage for growth cone collapse was calculated for five littermates (>60 growth cones at each point for each mouse). D–H, Suppression of mossy fiber outgrowth by Sema6B. Explants of the dentate gyrus from P2 wild-type mice (D, F) or PlxnA4−/− mice (E, G) were cultured on monolayer sheets of 3T3 cells (D, E, 3T3) or Sema6B-expressing 3T3 cells (F, G, 6B/3T3) for 3 d and stained with calbindin antibodies. The average length of mossy fibers is given in H. The average length for the longest two mossy fibers for each dentate explant was designated as the length of mossy fibers for the given explant. The length of mossy fibers was determined for >10 explants for each mouse (from five littermates). Vertical bars indicate SEM; *p < 0.001; **p < 0.005 (Student's t test). Scale bars: A, B, 10 μm; D–G, 50 μm.

Abnormal projection of mossy fibers and mossy cell axons in Sema6A;Sema6B double knock-out mice and PlxnA4 mutant mice. A–D, Timm staining of a horizontal section of the hippocampus of an adult Sema6A^−/−;Sema6B−/− mouse. The regions corresponding to CA3c, the middle part of CA3ab, and the distal-most part of CA3ab in A are shown at a higher magnification in B–D. E, Timm staining of a horizontal section of the hippocampus of an adult PlxnA4^−/− mouse. F–I, Timm staining of horizontal sections of the dentate molecular layer of adult wild-type and mutant mice. out, Outer molecular layer; in, inner molecular layer. J–L, Calretinin immunohistochemistry of horizontal sections of the dentate gyrus of adult wild-type and mutant mice. Note that calretinin-positive mossy cell axons spread ectopically into the outer molecular layer in Sema6A;Sema6B double mutants (K, asterisks) and PlxnA4 mutants (L). Scale bars: A, E, 400 μm; F–I, 50 μm; J–L, 200 μm.

Effects of PlxnA2 on the Sema6B response. A, Effects of PlxnA2 sema domain on Sema6A-induced and Sema6B-induced growth cone collapse in mossy fibers. Recombinant proteins for Sema6A (Sema6Aect-Fc) or Sema6B (Sema6Bect-Fc) were preincubated with PlxnA2 sema domain (PlxnA2SD-Fc) or alkaline phosphatase (AP-Fc) (see Materials and Methods) and then applied to the explant cultures of the dentate gyrus from P2 mice. B, Effects of PlxnA2 on Sema6A-induced and Sema6B-induced growth cone collapse in mossy fibers. Sema6A (Sema6Aect-Fc) and Sema6B (Sema6Bect-Fc) were preabsorbed by L cells (L), PlxnA2-expressing L cells (LA2), or PlxnA4-expressing L cells (LA4) and then applied to the cultures. In A, B the average percentages in growth cone collapse were calculated for five independent experiments. C, Effects of PlxnA2 on mossy fiber outgrowth. Explants of the dentate gyrus from P2 wild-type mice were cultured on monolayer sheets of 3T3 cells, Sema6B-expressing cells (6B/3T3 cells), PlxnA2-expressing cells (A2/3T3), or cells that coexpress PlxnA2 and Sema6B (A2::6B/3T3). Data for the explant culture on 3T3 cells and 6B/3T3 cells are same as those in H. D, Effects of the PlxnA2 sema domain on mossy fiber outgrowth. Explants of the dentate gyrus were cultured on monolayer sheets of 3T3 cells, 6B/3T3 cells, or PLL-coated culture plates under the presence of PlxnA2SD-Fc (100 nm) or AP-Fc (100 nm). The average length of mossy fibers was calculated for five independent experiments. Vertical bars indicate SEM; *p < 0.001, **p < 0.01 (Student's t test).

Formation of the suprapyramidal bundle (spb) in semaphorin and plexin mutant mice. A–G, Timm staining of horizontal sections of the hippocampus of adult PlnxA2^−/−;Sema6B−/− mice (A–D), PlxnA2^−/−;Sema6B^+/− mice (E, F), and PlxnA2^−/− mice (G). The regions corresponding to CA3c, the middle part of CA3ab, and the distal-most part of CA3ab in A and E are shown at a higher magnification in B–D and F, respectively. Arrowheads in B and F indicate the positions where mossy fibers first innervated into the suprapyramidal region to form the suprapyramidal bundle. H, Thickness of the spb in adult mice. Thickness of the suprapyramidal bundle was determined within CA3ab at the position 350 μm from the CA3-CA1 boundary (A, arrow), for >15 horizontal sections of each mouse (from five mice for each line). PlxnA2 mutant mice lack the suprapyramidal bundle. Vertical bars indicate SEM; *p < 0.001, **p < 0.005, and ***p < 0.01 against wild-type mice (Student's t test). Scale bars: A, E, G, 400 μm.

Summary of functions of repellents and attractants in laminar projection of mossy fibers. A, Repulsive and attractive signals that regulate mossy fibers projection. PlxnA4 is expressed in mossy fibers and serves as a receptor for two neuronal repellents, Sema6A and Sema6B, which are expressed in CA3. PlxnA4 also mediates mossy fiber repellent(s), X, which is expressed in SLM. PlxnA2 in CA3 competitively suppresses the Sema6A-PlxnA4 interaction and thereby attenuates Sema6A-induced mossy fiber repulsion. On the other hand, PlxnA2 itself and unknown molecule(s) in CA3 (PMFA; see Discussion) function as attractants for mossy fibers. Receptors that mediate attractive signals of PlxnA2 and PMFA have not been determined (see Discussion). B, Schematic diagrams of the mossy fiber projection patterns in the suprapyramidal region of CA3ab in wild-type mice, Sema6A, Sema6B, and PlxnA2 mutant mice, and double knock-out mice for these genes. The area to which mossy fibers project is represented by the color gray. Mossy fiber repellents and attractants that have remained intact are given for each mouse line. Mossy fiber repellent, X, in SLM is omitted. Repulsive activities of Sema6A would remain intact in PlxnA2-deficent mouse lines: PlxnA2^−/−;Sema6B−/− and PlxnA2^−/− mice. On the other hand, Sema6A activities would be completely attenuated by PlxnA2 in wild-type mice and partially in Sema6B−/− mice (see Discussion). The active Sema6A in Sema6B^−/− mice is represented as ▿Sema6A. C, A model for the laminar projection of mossy fibers. The model shows that the relative strength of mossy fiber repulsion by Sema6A and Sema6B and attraction by PlxnA2 and PMFA (represented in sum by gray color) determines the areas permissive for mossy fibers to innervate into the suprapyramidal region of CA3ab. Mossy fiber repellent, X, in SLM also suppresses innervation of mossy fibers into the lamina.