The C.elegans adult contains a renewable pool of germ cells that make up the proliferative zone (PZ), which contains mitotically dividing cells. From this pool, cells enter meiosis and differentiate, ensuring continued production of oocytes. To date, studies on the PZ have utilized mainly fixed samples. Consequently, many questions remain about the operation of the PZ: where and how large is the stem cell pool? Is there a transit-amplifying population of germ cells? How is the number and rate of cell divisions regulated during germline proliferation? To investigate these questions, I have constructed a strain containing germline-expressed histone H2B::Dendra. Dendra is a green-fluorescing protein, which, when stimulated by laser light at 405 nm, photo-converts to red fluorescence. I have worked out a protocol to individually immobilize live worms and photo-convert selected germ cell nuclei, recover the worms to plates, and image the same worms many hours later. This allows me to determine the fate of the converted cells and their daughters arising from cell division. In the worm strain used, the entire PZ contains ~200 cells. Preliminary results indicate that a pool of 40-70 cells at the distal tip of the PZ expands an average of 2.6 times in about 8 hours, filling the majority of the PZ in this timeframe. A pool of cells in the middle of the PZ expands an average of 1.6 times in the same timeframe, indicating a subset of cells in this region divide, and some do not. This entire labeled pool originally in the mid-PZ moves proximally, with ~60% of the cells already entering meiosis. Thus the mid-PZ is unlikely to contain stem cells. Finally, a pool in the proximal zone of the PZ has little to no expansion (1.1 times average), and the entire pool moves proximally and enters meiosis. This is consistent with a model in which stem cells reside in the distal pool, and divide once in about 8 hours, giving rise to both daughters that self renew to remain in the stem cell pool, and daughters that will divide one more time as they move to the middle of the PZ, after which they cease divisions and enter meiosis. I am currently refining the analysis by converting smaller pools of cells. This work establishes the parameters that govern germ cell proliferation, which in future studies can be investigated under altered conditions or in mutant situations to determine regulatory mechanisms.
In addition, I am also using this tool to analyze histone dynamics during oogenesis.