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Most of the human genome has now been sequenced and about 30,000 potential open reading frames have been identified, indicating that we use these 30,000 genes to functionally organize our biologic activities. However, functions of many genes are still unknown despite intensive efforts using bioinformatics as well as transgenic and knockout mice. Retrovirus-mediated gene transfer is a powerful tool that can be used to understand gene functions. We have developed a variety of retrovirus vectors and efficient packaging cell lines that have facilitated the development of efficient functional expression cloning methods. In this review, we describe retrovirus-mediated strategies used for investigation of gene functions and function-based screening strategies.

The leukocyte mono-Ig-like receptor (LMIR) belongs to a new family of paired immunoreceptors. In this study, we analyzed activating receptor LMIR4/CLM-5 as a counterpart of inhibitory receptor LMIR3/CLM-1. LMIR4 is expressed in myeloid cells, including granulocytes, macrophages, and mast cells, whereas LMIR3 is more broadly expressed. The association of LMIR4 with Fc receptor-␥ among immunoreceptor tyrosinebased activation motif-bearing molecules was indispensable for LMIR4-mediated functions of bone marrow-derived mast cells, but dispensable for its surface expression. Cross-linking of LMIR4 led to Lyn-and Syk-dependent activation of bone marrow-derived mast cells, resulting in cytokine production and degranulation, whereas that of LMIR3 did not. The triggering of LMIR4 and TLR4 synergistically caused robust cytokine production in accordance with enhanced activation of ERK, whereas the co-ligation of LMIR4 and LMIR3 dramatically abrogated cytokine production. Notably, intraperitoneal administration of lipopolysaccharide strikingly up-regulated LMIR3 and down-regulated LMIR4, whereas that of granulocyte colony-stimulating factor upregulated both LMIR3 and LMIR4 in granulocytes. Cross-linking of LMIR4 in bone marrow granulocytes also resulted in their activation, which was enhanced by lipopolysaccharide. Collectively, these results suggest that the innate immune system is at least in part regulated by the qualitative and quantitative balance of the paired receptors LMIR3 and LMIR4.The Ig-like receptors provide positive and negative regulation of immune cells upon recognition of various ligands (1-5). We identified previously leukocyte mono-Ig-like receptors (LMIRs) 2 from a cDNA library of bone marrow-derived mast cells (BMMCs). We (6) and others (7-9) demonstrated that LMIR1/MAIRI (myeloid-associated Ig-like receptor I)/CLM-8 (CMRF-35-like molecules-8) and LMIR2/MAIRII/CLM-4/ DIgR1 (dendritic cell-derived Ig-like receptor 1) are expressed mainly in myeloid cells. The human homolog of LMIR1 is CMRF-35H/IRp60 (inhibitory receptor protein of 60 kDa)/ CD300a (10 -14). The inhibitory effects of LMIR1 on mast cells and eosinophils and the activatory roles of LMIR2 in macrophages have been described recently (6,7,11). In addition to LMIRs, a variety of Ig-like paired receptors are expressed by myeloid cells (2,(15)(16)(17), but the biological significance of a paired receptor remains incompletely understood. Despite the similarity in the extracellular Ig-like domains, a striking structural difference between activating and inhibitory receptors exists in the transmembrane and cytoplasmic regions. In general, the former associate with an immunoreceptor tyrosinebased activation motif (ITAM)-or the related activating motifbearing adaptor transmembrane protein, including DAP10, DAP12, or Fc receptor-␥ (FcR␥), via a positively charged residue in the transmembrane domain, whereas the latter include an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic domain (1,5,18,19). Cells of the myeloid lineage su...

Dorsoventral patterning depends on the local concentrations of the morphogens. Twisted gastrulation (TSG) regulates the extracellular availability of a mesoderm inducer, bone morphogenetic protein 4 (BMP-4).However, TSG function in vivo is still unclear. We isolated a TSG cDNA as a secreted molecule from the mouse aorta-gonad-mesonephros region. Here we show that TSG-deficient mice were born healthy, but more than half of the neonatal pups showed severe growth retardation shortly after birth and displayed dwarfism with delayed endochondral ossification and lymphopenia, followed by death within a month. TSG-deficient thymus was atrophic, and phosphorylation of SMAD1 was augmented in the thymocytes, suggesting enhanced BMP-4 signaling in the thymus. Since BMP-4 promotes skeletogenesis and inhibits thymus development, our findings suggest that TSG acts as both a BMP-4 agonist in skeletogenesis and a BMP-4 antagonist in T-cell development. Although lymphopenia in TSG-deficient mice would partly be ascribed to systemic effects of runtiness and wasting, our findings may also provide a clue for understanding the pathogenesis of human dwarfism with combined immunodeficiency.Transforming growth factor ␤ (TGF-␤) superfamily members bone morphogenetic proteins (BMPs) are critical developmental regulators. Mutations in TGF-␤ family ligands, receptors, and signal transducers such as SMADs are associated with a number of human diseases. TSG was identified in Drosophila as one of the seven zygotic genes that govern the fate of dorsal cells in Drosophila embryos (37). TSG encodes a secreted, cysteine-rich protein that modulates the activity of the Decapentaplegic (DPP) protein, which corresponds to vertebrate BMP-4, and mutations in TSG result in defects of dorsal midline structures called amnioserosa in Drosophila (20). In searching for essential soluble factors produced from the aorta-gonad-mesonephros (AGM) region where definitive hematopoiesis arises (21), we employed the retrovirus-mediated signal sequence trap method previously developed (16), using mRNA from the AGM region of the 10.5-day-postcoitum (dpc) mouse embryo, and isolated a mouse homologue of Drosophila TSG. In 2000, Xenopus twisted gastrulation (TSG) was found to bind directly to BMP-4 to promote BMP-4 signaling by regulating the extracellular availability of BMP-4 (25). Since the dorsoventral axis is inverted between Drosophila and vertebrates and ventralizing factor BMP-4 is essential for mesoderm formation (33) and hematopoietic stem cell (HSC) survival (4), we speculated that TSG may also be involved in ventralization and mesoderm-derived organogenesis, including hematopoiesis, in mammals. Meanwhile, four groups using fine molecular analyses reported that TSG acts rather as a BMP-4 (DPP) antagonist by forming a ternary complex of TSG, BMP-4, and BMP-4 antagonist Chordin or by collaborating with a protease, Tolloid, to generate a Supersog (truncated stable form of Sog [Chordin]) in fly, fish, and frog (6,28,29,35). Thus, it is controversial whether TSG acts...