Cytokinesis in somatic cells concludes with the formation of a midbody, which is abscised to form individual daughter cells. In contrast, germ cell cytokinesis results in a permanent intercellular bridge connecting the daughter cells through a large cytoplasmic channel. During spermatogenesis, proposed roles for the intercellular bridge include germ cell communication, synchronization, and chromosome dosage compensation in haploid cells. Although several essential components of the midbody have recently been identified, essential components of the vertebrate germ cell intercellular bridge have until now not been described. Herein, we show that testis-expressed gene 14 (TEX14) is a novel protein that localizes to germ cell intercellular bridges. In the absence of TEX14, intercellular bridges are not observed by using electron microscopy and other markers. Spermatogenesis in Tex14 ؊/؊ mice progresses through the transit amplification of diploid spermatogonia and the expression of early meiotic markers but halts before the completion of the first meiotic division. Thus, TEX14 is required for intercellular bridges in vertebrate germ cells, and these studies provide evidence that the intercellular bridge is essential for spermatogenesis and fertility.cytoplasmic bridges ͉ knockout mouse ͉ male infertility ͉ male sterility ͉ ring canals

Stable intercellular bridges are a conserved feature of gametogenesis in multicellular animals observed more than 100 years ago, but their function was unknown. Many of the components necessary for this structure have been identified through the study of cytokinesis in Drosophila; however, mammalian intercellular bridges have distinct properties from those of insects. Mammalian germ cell intercellular bridges are composed of general cytokinesis components with additional germ cell -specific factors including TEX14. TEX14 is an inactive kinase essential for the maintenance of stable intercellular bridges in gametes of both sexes but whose loss specifically impairs male meiosis. TEX14 acts to impede the terminal steps of abscission by competing for essential component CEP55, blocking its interaction in nongerm cells with ALIX and TSG101. Additionally, TEX14-interacting protein RBM44, whose localization in stabile intercellular bridges is limited to pachytene and secondary spermatocytes, may participate in processes such as RNA transport but is nonessential to the maintenance of intercellular bridge stability.

Nuage are amorphous ultrastructural granules in the cytoplasm of male germ cells as divergent as Drosophila, Xenopus, and Homo sapiens. Most nuage are cytoplasmic ribonucleoprotein structures implicated in diverse RNA metabolism including the regulation of PIWI-interacting RNA (piRNA) synthesis by the PIWI family (i.e., MILI, MIWI2, and MIWI). MILI is prominent in embryonic and early post-natal germ cells in nuage also called germinal granules that are often associated with mitochondria and called intermitochondrial cement. We find that GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) co-localizes with MILI in intermitochondrial cement. Knockout of Gasz in mice results in a dramatic downregulation of MILI, and phenocopies the zygotene–pachytene spermatocyte block and male sterility defect observed in MILI null mice. In Gasz null testes, we observe increased hypomethylation and expression of retrotransposons similar to MILI null testes. We also find global shifts in the small RNAome, including down-regulation of repeat-associated, known, and novel piRNAs. These studies provide the first evidence for an essential structural role for GASZ in male fertility and epigenetic and post-transcriptional silencing of retrotransposons by stabilizing MILI in nuage.

Whereas somatic cell cytokinesis resolves with abscission of the midbody, resulting in independent daughter cells, germ cell cytokinesis concludes with the formation of a stable intercellular bridge interconnecting daughter cells in a syncytium. While many proteins essential for abscission have been discovered, until recently, no proteins essential for mammalian germ cell intercellular bridge formation have been identified. Using TEX14 as a marker for the germ cell intercellular bridge, we show that TEX14 co-localizes with the centralspindlin complex, mitotic kinesin-like protein 1 (MKLP1) and male germ cell Rac GTPase-activating protein (MgcRacGAP) and converts these midbody matrix proteins into stable intercellular bridge components. In contrast, septins (SEPT) 2, 7 and 9 are transitional proteins in the newly forming bridge. In cultured somatic cells, TEX14 can localize to the midbody in the absence of other germ cell-specific factors, suggesting that TEX14 serves to bridge the somatic cytokinesis machinery to other germ cell proteins to form a stable intercellular bridge essential for male reproduction.

The isolation of chemically induced mutations in forward genetic screens is one of the hallmarks of Drosophila genetics. However, mapping the corresponding loci and identifying the molecular lesions associated with these mutations are often difficult and labor-intensive. Two mapping methods are most often used in flies: meiotic recombination mapping with marked chromosomes and deficiency mapping. The availability of the fly genome sequence allows the establishment and usage of molecular markers. Single-nucleotide polymorphisms have therefore recently been used to map several genes. Here we show that thousands of molecularly mapped P element insertions in fly strains that are publicly available provide a powerful alternative method to singlenucleotide polymorphism mapping. We present a strategy that allows mapping of lethal mutations, as well as viable mutations with visible phenotypes, with minimal resources. The most important unknown in using recombination rates to map at high resolution is how accurately recombination data correlate with molecular maps in small intervals. We therefore surveyed distortions of recombination rates in intervals <500 kb. We document the extent of distortions between the recombination and molecular maps and describe the required steps to map with an accuracy of <50 kb. Finally, we describe a recently developed method to determine molecular lesions in 50-kb intervals by using a heteroduplex DNA mutation detection system. Our data show that this mapping approach is inexpensive, efficient, and precise, and that it significantly broadens the application of P elements in Drosophila.F orward genetic screens in Drosophila using chemical mutagens or ␥-ray irradiation have uncovered thousands of complementation groups (1, 2). However, identifying the molecular lesions that underlie the phenotypes has traditionally been labor-intensive and time-consuming. All mapping strategies depend on a detectable phenotype caused by the mutation (e.g., lethality) to establish its genetic linkage with known markers or failure to complement characterized deficiencies. One of the most common strategies is meiotic recombination mapping, and resolution and accuracy primarily depend on the availability of markers and the number of meiotic events that are scored.The publication of the fly genome (3) made it possible to generate many more precisely mapped markers on the basis of single-nucleotide polymorphisms (SNPs). SNPs are single basepair differences between homologous chromosomes. The use of SNPs as molecular markers is a two-step process. First, SNP maps have to be established or confirmed; second, SNPs have to be detected by either sequencing or restriction enzyme digestion. Several teams have recently shown that SNPs are present in sufficient numbers in the Drosophila genome to theoretically allow the mapping of every gene. They have also demonstrated the successful application of the method in a few cases (4-6). An alternative to SNPs are P elements with molecularly defined insertion sites. They are tra...