LETTERS NATURE CELL BIOLOGY VOLUME 8 | NUMBER 6 | JUNE 2006 615 Plexin-A1 and its interaction with DAP12 in immune responses and bone homeostasis Noriko Takegahara 1,12 , Hyota Takamatsu 1,12 , Toshihiko Toyofuku 1,2 , Tohru Tsujimura 3 , Tatsusada Okuno 4 , Kazunori Yukawa 5 , Masayuki Mizui 1 , Midori Yamamoto 1 , Durbaka V.R. Prasad 1 , Kazuhiro Suzuki 1 , Masaru Ishii 6 , Kenta Terai 7 , Masayuki Moriya 4 , Yuji Nakatsuji 4 , Saburo Sakoda 4 , Shintaro Sato 8 , Shizuo Akira 8 , Kiyoshi Takeda 9 , Masanori Inui 10 , Toshiyuki Takai 10 , Masahito Ikawa 11 , Masaru Okabe 11 , Atsushi Kumanogoh 1,13 and Hitoshi Kikutani 1,13 Semaphorins and their receptors have diverse functions in axon guidance, organogenesis, vascularization and/or angiogenesis, oncogenesis and regulation of immune responses 1–11 . The primary receptors for semaphorins are members of the plexin family 2,12–14 . In particular, plexin-A1, together with ligand- binding neuropilins, transduces repulsive axon guidance signals for soluble class III semaphorins 15 , whereas plexin-A1 has multiple functions in chick cardiogenesis as a receptor for the transmembrane semaphorin, Sema6D, independent of neuropilins 16 . Additionally, plexin-A1 has been implicated in dendritic cell function in the immune system 17 . However, the role of plexin-A1 in vivo, and the mechanisms underlying its pleiotropic functions, remain unclear. Here, we generated plexin-A1-deficient (plexin-A1 –/– ) mice and identified its important roles, not only in immune responses, but also in bone homeostasis. Furthermore, we show that plexin-A1 associates with the triggering receptor expressed on myeloid cells-2 (Trem- 2), linking semaphorin-signalling to the immuno-receptor tyrosine-based activation motif (ITAM)-bearing adaptor protein, DAP12. These findings reveal an unexpected role for plexin- A1 and present a novel signalling mechanism for exerting the pleiotropic functions of semaphorins. To better understand the role of plexin-A1 in vivo, mice deficient in the plexin-A1 gene were generated by homologous recombination (see Supplementary Information, Fig. S1a, b), and the successful deletion of plexin-A1 was confirmed by both northern blotting and RT–PCR (see Supplementary Information, Fig. S1c, d). Mice were born with the expected Mendelian ratios from intercrosses of heterozygous mutants and the resulting plexin-A1 –/– mice were fertile. Apparent abnormalities were not observed by gross macroscopic or histological examination of the embryos (E11.5) or in the brain, kidney, lung, heart, liver and spleen of 4-week-old mice — all tissues in which plexin-A1-transcripts are expressed (see Supplementary Information, Fig. S1e, f). These obser- vations strongly suggest the existence of functional redundancy among the plexin family members during embryonic development. However, mutant mice had functional defects in the immune system, as well as morphologic abnormalities in the skeletal tissues. Therefore, the biologi- cal functions of plexin-A1 were further investigated with a focus on the immune and skeletal tissues as described below. Lymphocyte development seemed to be normal in plexin-A1 –/– mice. No differences in the expression of cell surface phenotype markers, numbers and ratios of T-cells, B-cells, macrophages and dendritic cells in the spleen and thymus were observed between wild-type and plexin-A1 –/– mice (see Supplementary Information, Fig. S1g). Plexin-A1 is highly expressed in dendritic cells 17 , and the influence of plexin-A1-deficiency on dendritic cell function was examined. FITC–dextran uptake by dendritic cells, and the appearance of fluo- rescent dendritic cells in the draining lymph nodes after skin painting with FITC, in plexin-A1 –/– mice were comparable with those seen in wild-type littermates (Fig. 1a, b). In addition, no significant differ- ences were seen in the expression levels of costimulatory molecules (including CD40, CD80, CD86 and MHC class II) between wild- type and plexin-A1 –/– dendritic cells (Fig. 1c). However, plexin-A1 –/– 1 Department of Molecular Immunology and CREST program of JST, Research Institute for Microbial Diseases, Osaka University, 3–1 Yamada-oka, Suita, Osaka 565- 0871, Japan. 2 Department of Internal Medicine and Therapeutics, Graduate School of Medicine, Osaka University, 2–2 Yamada-oka, Suita, Osaka 565–0871, Japan. 3 Department of Pathology, Hyogo College of Medicine, Hyogo 663–8501, Japan. 4 Department of Neurology, Graduate School of Medicine, Osaka University, 2–2 Yamada- oka, Suita, Osaka 565–0871, Japan. 5 Department of Physiology II, Wakayama Medical College, 811–1 Kimiidera, Wakayama, Wakayama 641–0012, Japan. 6 Department of Clinical Research, National Osaka Minami Medical Center, Kawachinagano, Osaka 586–8521, Japan. 7 Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, 3–1 Yamada-oka, Suita, Osaka 565–0871, Japan. 8 Department of Host Defence, Research Institute for Microbial Diseases, Osaka University, and ERATO, Japan Science and Technology Agency, 3–1 Yamada-oka, Suita, Osaka 565–0871, Japan. 9 Department of Molecular Genetics, Medical Institute for Bioregulation, Kyushu University, 3–1–1 Maidashi, Higashi-ku, Fukuoka 812–8582, Japan. 10 Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Siryo 4–1, Aoba-ku, Sendai 980–8575, Japan and CREST program of JST, Honcho 4–1–8, Kawaguchi, Saitama 332–0012, Japan. 11 Genome Information Research Center, Osaka University, 3–1 Yamada-oka, Suita, Osaka 565–0871, Japan. 12 These authors contributed equally to this work. 13 Correspondence should be addressed to A.K. and H.K. (e-mail: kumanogo@ragtime.biken.osaka-u.ac.jp; kikutani@ragtime.biken.osaka-u.ac.jp) Received 21 February 2006; accepted 28 April 2006; published online 21 May 2006; DOI: 10.1038/ncb1416 Nature Publishing Group ©2006