Silencing of transposable elements occurs during fetal gametogenesis in males viaArgonaute proteins, also known as PAZ Piwi domain (PPD) proteins, are members of a well-conserved family that is expressed in a variety of organisms, from fission yeasts to humans. The family can be divided into two subfamilies, Piwi and Ago, based on the primary sequence homology and expression pattern of each member. Piwi subfamily members are expressed only in germ lineage cells, whereas members of the Ago subfamily are expressed ubiquitously. The PPD proteins were initially characterized as essential molecules for stem cell selfrenewal and maintenance in Drosophila, Caenorhabditis elegans, and certain plant species (Cox et al. 1998;Moussian et al. 1998), and a member of the family is essential for stem cell function during regeneration in planaria (Reddien et al. 2005). There are three Piwi subfamily genes in the mouse genome: Miwi (mouse piwi), Miwi2,, which are termed Piwil1 (piwi-like homolog 1), Piwil4, and Piwil2, respectively, in the official nomenclature. Although they are ex-

The piwi family genes, which are defined by conserved PAZ and Piwi domains, play important roles in stem cell selfrenewal, RNA silencing, and translational regulation in various organisms. To reveal the function of the mammalian homolog of piwi, we produced and analyzed mice with targeted mutations in the Mili gene, which is one of three mouse homologs of piwi. Spermatogenesis in the MILI-null mice was blocked completely at the early prophase of the first meiosis, from the zygotene to early pachytene, and the mice were sterile. However, primordial germ cell development and female germ cell production were not disturbed. Furthermore, MILI bound to MVH, which is an essential factor during the early spermatocyte stage. The similarities in the phenotypes of the MILI-and MVH-deficient mice and in the physical binding properties of MILI and MVH indicate a functional association of these proteins in post-transcriptional regulation. These data indicate that MILI is essential for the differentiation of spermatocytes. Key words: Mili, Miwi, piwi, Mvh, Spermatogenesis SummaryMili, a mammalian member of piwi family gene, is essential for spermatogenesis

To study the mutually exclusive expression of odorant receptor (OR) genes, we generated transgenic mice that carried the murine OR gene MOR28. Expression of the transgene and the endogenous MOR28 was distinguished by using two different markers, beta-galactosidase and green fluorescent protein (GFP), respectively. Double staining of the olfactory epithelium revealed that the two genes were rarely expressed simultaneously in individual olfactory neurons. A similar exclusion was also observed between differently tagged but identical transgenes integrated into the same locus of one particular chromosome. Although allelic inactivation has been reported for the choice between the maternal and paternal alleles, this is the first demonstration of mutually exclusive activation among non-allelic OR gene members with identical coding and regulatory sequences. Such an unusual mode of gene expression, monoallelic and mutually exclusive, has previously been shown only for the antigen-receptor genes of the immune system.

The use of mutant mice plays a pivotal role in determining the function of genes, and the recently reported germ line transposition of the Sleeping Beauty (SB) transposon would provide a novel system to facilitate this approach. In this study, we characterized SB transposition in the mouse germ line and assessed its potential for generating mutant mice. Transposition sites not only were clustered within 3 Mb near the donor site but also were widely distributed outside this cluster, indicating that the SB transposon can be utilized for both region-specific and genome-wide mutagenesis. The complexity of transposition sites in the germ line was high enough for large-scale generation of mutant mice. Based on these initial results, we conducted germ line mutagenesis by using a gene trap scheme, and the use of a green fluorescent protein reporter made it possible to select for mutant mice rapidly and noninvasively. Interestingly, mice with mutations in the same gene, each with a different insertion site, were obtained by local transposition events, demonstrating the feasibility of the SB transposon system for region-specific mutagenesis. Our results indicate that the SB transposon system has unique features that complement other mutagenesis approaches.The analysis of mutant mice plays a key role in the understanding of gene functions, and the importance of this approach is expected to increase (2) with the recent availability of the mouse genome sequence (25). However, large-scale genetic screening for mice has been lagging far behind that for other model organisms, such as Drosophila melanogaster and Caenorhabditis elegans, because of the lack of a system allowing for both mutagenesis and subsequent rapid identification of the mutation. Large-scale generation of mutant mice has been conducted recently by using N-ethyl-N-nitrosourea (ENU), and a number of mutant mice with various phenotypes have been generated successfully (6, 26). The drawback of this approach is that identification of the causative point mutations is time-consuming. The embryonic stem (ES) cell-based gene trap is another effective approach (11,12,31). However, largescale generation of mutant mice, which is a prerequisite for genetic screening, is not easy because the ES cell-based methods involve labor-intensive processes such as tissue culture or embryo manipulation.Transposon-tagged mutagenesis has been used in a wide range of organisms, such as D. melanogaster (1, 32), C. elegans (13, 24), and plants (27). Although the mutation rate resulting from transposition is not as high as that in ENU mutagenesis, transposon-tagged mutagenesis has been used as an alternative genetic screening method for the following reasons. First, the genes responsible for the phenotypes can be identified rapidly by using the transposon sequence as a tag. Second, desired elements can be introduced into the transposon sequence to expand the application range of the mutant lines. This principle has been demonstrated in the P element of D. melanogaster, where various GAL4 en...

The transcriptional activity of LIM-homeodomain (LIM-HD) proteins is regulated by their interactions with various factors that bind to the LIM domain. We show that reduced expression of single-stranded DNA-binding protein 1 (Ssdp1), which encodes a co-factor of LIM domain interacting protein 1 (Ldb1), in the mouse mutant headshrinker (hsk)disrupts anterior head development by partially mimicking Lim1mutants. Although the anterior visceral endoderm and the anterior definitive endoderm, which together comprise the head organizer, were able to form normally in Ssdp1hsk/hsk mutants, development of the prechordal plate was compromised. Head development is partially initiated in Ssdp1hsk/hsk mutants, but neuroectoderm tissue anterior to the midbrain-hindbrain boundary is lost, without a concomitant increase in apoptosis. Cell proliferation is globally reduced in Ssdp1hsk/hsk mutants, and approximately half also exhibit smaller body size, similar to the phenotype observed in Lim1 and Ldb1 mutants. We also show that Ssdp1 contains an activation domain and is able to enhance transcriptional activation through a Lim1-Ldb1 complex in transfected cells, and that Ssdp1 interacts genetically with Lim1 and Ldb1 in both head development and body growth. These results suggest that Ssdp1 regulates the development of late head organizer tissues and body growth by functioning as an essential activator component of a Lim1 complex through interaction with Ldb1.