Highlights d New assay to monitor DNA break repair with sister chromatid or same DNA molecule d Sister chromatid or same DNA molecule used late in prophase I as repair partner d Sequencing of noncrossover conversion tracts of homologindependent repair events d XPF-1 promotes late homolog-independent chromatid repair events

Arranged in a spatial-temporal gradient for germ cell development, the adult germline of Caenorhabditis elegans is an excellent system for understanding the generation, differentiation, function, and maintenance of germ cells. Imaging whole C. elegans germlines along the distal-proximal axis enables powerful cytological analyses of germ cell nuclei as they progress from the pre-meiotic tip through all the stages of meiotic prophase I. To enable high-content image analysis of whole C. elegans gonads, we developed a custom algorithm and pipelines to function with image processing software that enables: 1) quantification of cytological features at single nucleus resolution from immunofluorescence images; and, 2) assessment of these individual nuclei based on their position within the germline. We demonstrate the capability of our quantitative image analysis approach by analyzing multiple cytological features of meiotic nuclei in whole C. elegans germlines. First, we quantify double strand DNA breaks (DSBs) per nucleus by analyzing DNA-associated foci of the recombinase RAD-51 at single-nucleus resolution in the context of whole germline progression. Second, we quantify the DSBs that are licensed for crossover repair by analyzing foci of MSH-5 and COSA-1 when they associate with the synaptonemal complex during meiotic prophase progression. Finally, we quantify P-granule composition across the whole germline by analyzing the colocalization of PGL-1 and ZNFX-1 foci. Our image analysis pipeline is an adaptable and useful method for researchers spanning multiple fields utilizing the C. elegans germline as a model system.

Arranged in a spatial-temporal gradient for germ cell development, the adult germline of Caenorhabditis elegans is an excellent system for understanding the generation, differentiation, function, and maintenance of germ cells. Imaging whole C. elegans germlines along the distal-proximal axis enables powerful cytological analyses of germ cell nuclei as they progress from the pre-meiotic tip through all the stages of meiotic prophase I. To enable high-throughput image analysis of whole C. elegans gonads, we developed a custom algorithm and pipelines to function with image processing software that enables: 1) quantification of cytological features at single nucleus resolution from immunofluorescence images; and, 2) assessment of these individual nuclei based on their position within the germline. We demonstrate the capability of our quantitative image analysis approach by analyzing multiple cytological features of meiotic nuclei in whole C. elegans germlines. First, we quantify double strand DNA breaks (DSBs) per nucleus by analyzing DNA-associated foci of the recombinase RAD-51 at the single-nucleus resolution in the context of whole germline progression. Second, we quantify the DSBs that are licensed for crossover repair by analyzing foci of MSH-5 and COSA-1 when they associate with the synaptonemal complex during meiotic prophase progression. Finally, we quantify P-granule composition across the whole germline by analyzing the colocalization of PGL-1 and ZNFX-1 foci. Our image analysis pipeline is an adaptable and useful method for researchers spanning multiple fields utilizing the C. elegans germline as a model system.

Conservation over three mammalian genera—the mouse, rat, and human—has been found for a subset of the transcripts whose level differs between the adenoma and normal epithelium of the colon. Pde4b is one of the triply conserved transcripts whose level is enhanced both in the colonic adenoma and in the normal colonic epithelium, especially adjacent to adenomas. It encodes the phosphodiesterase PDE4B, specific for cAMP. Loss of PDE4B function in the ApcMin/+ mouse leads to a significant increase in the number of colonic adenomas. Similarly, Pde4b-deficient ApcMin/+ mice are hypersensitive to treatment by the inflammatory agent DSS, becoming moribund soon after treatment. These observations imply that the PDE4B function protects against ApcMin-induced adenomagenesis and inflammatory lethality. The paradoxical enhancement of the Pde4b transcript in the adenoma versus this inferred protective function of PDE4B can be rationalized by a feedback model in which PDE4B is first activated by early oncogenic stress involving cAMP and then, as reported for frank human colon cancer, inactivated by epigenetic silencing.

The preservation of genome integrity during sperm and egg development is vital for reproductive success. During meiosis, the tumor suppressor BRCA1/BRC-1 and structural maintenance of chromosomes 5/6 (SMC-5/6) complex genetically interact to promote high fidelity DNA double strand break (DSB) repair, but the specific DSB repair outcomes these proteins regulate remain unknown. Here we show that BRCA1/BRC-1 and the SMC-5/6 complex limit intersister crossover recombination as well as error-prone repair pathways during meiotic prophase I. Using genetic and cytological methods to monitor repair of DSBs with different repair partners in Caenorhabditis elegans, we demonstrate that both BRC-1 and SMC-5/6 repress intersister crossover recombination events, with meiotic cells becoming more dependent upon these proteins to repair DSBs in late meiotic prophase I. Sequencing of conversion tracts from homolog-independent DSB repair events indicates that BRC-1 regulates intersister/intrachromatid noncrossover conversion tract length. Moreover, we find that BRC-1 also specifically inhibits error prone repair of DSBs induced at mid-pachytene. Finally, we reveal that functional BRC-1 enhances DSB repair defects in smc-5 mutants by repressing theta-mediated end joining (TMEJ). Taken together, our study illuminates the coordinate interplay of BRC-1 and SMC-5/6 to regulate DSB repair outcomes in the germline.