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(2002). Briefly, host embryos were collected from a nanos3+/?��nanos3?/? cross or a ?AB cross, and donor embryos were collected from a nanos3+/?; Tg(ziwi:eGFP) X nanos3?/?; Tg(ziwi:eGFP) cross. Donor embryos were injected at the one-cell stage with 5?pl of 0.5% rhodamine-dextran in 20?mM KCl. All embryos were then incubated in fish water at either 25?��C or 28.5?��C until the sphere stage, when they were dechorionated in agarose coated dishes using 5?mg/ml pronase (Sigma) in 1X embryo media ( Westerfield, 2000). Embryos were then transferred YES1 to agarose wells in 1X embryo media using a flame-polished pipet, where 20�C50 cells from the margin of the donor blastodisc were transferred into the animal pole of the host embryo using an oil-controlled glass needle. At 24 hpf, host embryos were sorted for localization of rhodamine-dextran donor cells primarily to anterior structures. Donor embryos were genotyped at 48 hpf, and host fish were genotyped at about 40 dpf, as previously described ( Draper et al., 2007). Hosts were then sacrificed and fixed as above at 60 dpf. Low magnification images of Icotinib research buy endogenous eGFP fluorescence were collected with a Leica 16MZF stereomicroscope equipped with a Leica DFC500 digital camera prior to anti-Vasa immunohistochemistry (preformed as above). In situ hybridization was performed as above on whole fixed juvenile ovaries. Individual nanos2+ cells or sycp3+ cysts were counted in each ovary in three dimensions using a Zeiss Axioplot microscope with a 40X objective. Selleck Thiazovivin Statistical significance was determined using the Student's t-test. If oocyte production in zebrafish is a stem cell-driven process, then genes expressed in both mitotically proliferative and early meiotic germ cells should be detected during juvenile and adult stages. Mitotic and early meiotic germ cells have been identified in juvenile and adult zebrafish ovaries based on DNA morphology (Leu and Draper, 2010). To confirm the presence of germ cells that are actively entering meiosis in adult ovaries, we determined the expression patterns of homologs of the early meiotic entry genes dmc1 and sycp3, by in situ hybridization. dmc1 encodes a meiosis-specific RecA homolog that localizes to double-strand DNA breaks during recombination of homologous chromosomes during early meiosis ( Yoshida et al., 1998), and sycp3 encodes a component of the synaptonemal complex required for synapsis of homologous chromosomes during early meiotic prophase ( Lammers et al., 1994). Thus, expression of dmc1 and sycp3 can be used as markers of meiotic entry ( Mahadevaiah et al., 2001). We find that in the adult, both dmc1 and sycp3 are expressed in a subset of
