(C) Projection of pachytene nuclei labeled by FISH with 5S rDNA probe and stained with DAPI. REC-8 complexes provide stable cohesion. Surprisingly, a WAPL-1-impartial mechanism removes cohesin before metaphase I. Our studies provide insight into how meiosis-specific cohesin complexes are regulated to ensure formation of euploid gametes. DOI: http://dx.doi.org/10.7554/eLife.10851.001 germ line, which contains a complete time course of meiotic prophase, we demonstrate that WAPL-1 antagonizes cohesin binding from the onset of meiosis, and show that cohesin complexes containing the COH-3/4 kleisins are specifically targeted by WAPL-1. By antagonizing the binding of COH-3/4 complexes to axial elements, WAPL-1 acts as a regulator of meiotic chromosome structure and SCC. Moreover, we also show that SCC is usually modulated by WAPL-1 and recombination during the chromosome Corticotropin Releasing Factor, bovine remodeling process that starts at the end of pachytene, and report that a WAPL-1-impartial mechanism removes cohesin during the oocyte maturation process preceding metaphase I. Results WAPL-1 is required for fertility In order to investigate the role of WAPL-1 during meiotic prophase, we used a deletion allele, homolog (Physique 1A). Western blot analysis on whole-worm protein extracts showed that this WAPL-1 protein is usually absent in mutants, confirming that this deletion is usually a null allele of (Physique 1B). Homozygous mutants (referred from now on as mutants) are viable, but display a reduction in brood size and high levels of embryonic lethality (Physique 1C). In addition to these reproductive defects, mutants also displayed somatic defects, as demonstrated by the high incidence of larval arrest amongst Corticotropin Releasing Factor, bovine the hatched embryos (Physique 1C) and by the Corticotropin Releasing Factor, bovine presence of an egg laying defect in adult worms. In order to prevent the accumulation of somatic defects, all the analysis presented here was performed in homozygous worms derived from heterozygous mothers. Open in a separate window Physique 1. WAPL-1 localizes to germ line nuclei and promotes viability.(A) Structure of the gene, red bar indicates the region deleted in the allele. (B) Western blot demonstrates that is a null allele and that a protein of the expected size is present in worms carrying a transgene. (C) mutants display reduced fertility and larval lethality, numbers in parenthesis indicate total?number of embryos analysed per genotype . (D) Projections of whole-mounted germ lines stained with DAPI, the different stages of meiotic prophase are noted above the WT germ line, with transition zone made up of nuclei Corticotropin Releasing Factor, bovine in leptotene and zygotene. Note that overall germ line business in mutants is similar to WT. (E) Projections of diakinesis oocytes stained with DAPI, six bivalents are present in both WT and mutants. (F) Whole-mounted germ line from a transgenic worm homozygous for the deletion and for a single copy transgene stained with DAPI and anti-GFP antibodies. Note that the intensity of GFP::WAPL-1 decreases in transition zone and peaks again during late pachytene. (G) Insets from germ line shown in F showing GFP::WAPL-1 staining in transition zone and pachytene nuclei, note that GFP::WAPL-1 intensity is very high in transition zone nuclei that do not display chromosome clustering (arrowheads). Physique 1figure supplement 1 shows quantification of GFP::WAPL-1 intensities along the germ line. Scale bars in E and G = 5 m. DOI: http://dx.doi.org/10.7554/eLife.10851.003 Figure 1figure supplement 1. Open in a separate windows Quantification of GFP::WAPL-1 intensity.Y axis depicts average nuclear intensity of GFP signal per nucleus (calculated from maximum intensity projections made from non-deconvolved Z-stacks acquired with the same settings), X axis indicates stages at which intensity was measured, and error bars indicate standard deviation. Note the drop in GFP::WAPL-1 fluorescence intensity between pre-leptotene and leptotene nuclei. Differences between pre-leptotene and leptotene nuclei, and between leptotene and late pachytene nuclei are significant (mutant germ lines appears largely normal, with clearly defined mitotic and meiotic compartments in which the different stages of meiotic prophase can be easily identified (Physique 1D). In fact, observation of diakinesis oocytes (the last stage of meiotic prophase) showed that both wild type and mutant oocytes displayed 6 DAPI-stained bodies, demonstrating that WAPL-1 is not required for chiasma formation (Physique 1E). Nonetheless, the reduced fertility of Mouse monoclonal to RFP Tag mutants suggested that WAPL-1 may play important functions in the germ line. Thus, we investigated the staining pattern of WAPL-1 during meiosis by creating transgenic worms homozygous for the deletion and for a single-copy insertion of a transgene that expresses a GFP::WAPL-1 fusion protein using the 5 and 3 UTRs from the Corticotropin Releasing Factor, bovine locus. Expression of this transgene largely rescued the fertility defects of mutants (Figure 1C), and western blot analysis confirmed the presence of a band.