Terry Kunder Brian Landi

1. Terry Kunder Brian Landi Large-scale mutagenesis in the zebrafish: in search of genes controlling development in a vertebrate et. al. Mullins, 1994

2. So why Zebrafish? • Well suited for mutational analysis because… • Rapid development • Large brood size • External fertilization • Large embryos • Clear embryos

3. Mutagenesis Background • Mutagenic properties were tested by the use of EMS and ENU • Different toxicities observed for both mutagens • In order to assess the nature of the mutations, mutagenized fish were crossed with females carrying mutations in one or two of the four pigmentation genes • These original mutagenized fish and mutant carrying-females were subsequently mated bi-monthly for testing of the four mutant phenotypes • In the progeny, the embryo that displayed the mutation pigment would be the egg receiving the mutated sperm

4. Spermatogenesis Background • Spermatogonial cells produce spermocytes mitotically • Spermocytes undergo meiosis, which result in spermatids • Spermatids then develop into mature sperm

5. Mutagenesis of Post-Mitotic Stages of Spermatogenesis • Following the first 2 weeks, mosaics were observed in the progeny • In the paternal germline, the post-mitotic cells (i.e. spermatocytes, spermatids, or sperm) were mutated, which is why the mosaic embryos resulted • When the post-mitotic cells were mutated, only one strand of the DNA was alkylated by the mutagen, resulting in the mosaic in the progeny • After combining the egg and sperm, cellular division would produce cells that exhibited the mutation and cells that were without the mutation

6. Mutagenesis of Post-Mitotic…Cont’d • The mosaic embryos were raised and crossed with the tester strain to determine if the mutation was present in the germline cells • Results showed none of the fish contained the mutation in their germline • The researchers observed that the wild type cells were far more prevalent in the progeny than mutant pigmentation cells • To be more efficient in screening for mutations, it was important that mosaics were not present, and that full mutants were tested in the future • This led to a different experimental strategy

7. Mutagenesis of Pre-Meiotic Stages of Spermatogenesis • To create more efficient mutation screenings, the spermatogonial cells were point mutated, thus producing non-mosaic progeny • This method fixes the mutation in both strands of DNA as opposed to only one in the post-mitotic trials • The researchers knew that the mutations were only occurring in the spermatogonial cells because the progeny were displaying non-mosaic phenotypes, both wild type and mutated

8. Table 1 – Mutation Rates for ENU, EMS, and non-mutagenized fish The researchers began to assess the mutation rates of the Zebrafish The ENU treated fish showed higher mutation rates, and gave easily reproducible data 0.23% of ENU mutated 0.03% of EMS mutated 0.01% of Control mutated All embryos were viable and non-mosaic phenotypes indicating complete mutation of spermatogonial cells

9. Table 1 – Mutation… Cont’d This indicates an induction of mutations by ENU in spermatogonial stem cells of the mutagenized fish. • The mutations, though low in rate, did produce significant results • With further testing over the 8 week period, the researchers observed no experimentally distinctive changes in rate frequency in any resulting progeny • 15 weeks after treatment, progeny were still produced that contained the pigmentation mutation

10. Additional material After the pre-meiotic cells were mutated, the Zebrafish would have the ability to consistently produce progeny with the mutation because spermatogonial cells are germ line stem cells, which gives it the ability to create sperm with the mutation • After the post-mitotic cells (Spermatocytes, spermatids, or sperm) were mutated in the Zerbrafish, they would eventually fully develop into mobile sperm and be released from the male. • For this reason, these fish would initially produce progeny with the mutation, but over time they decrease and would no longer produce these mutated progeny because they would not have any mutated post-mitotic cells left available

11. References • http://www.cosmosmagazine.com/news/2475/zebrafish-could-help-humans-regrow-lost-muscle • http://www.google.com/imgres?imgurl=http://science.tjc.edu/images/reproduction/spermatogenesis.jpg&imgrefurl=http://f5biology.blogspot.com/&h=1097&w=865&sz=92&tbnid=4aqMYvP0EyeMGM:&tbnh=150&tbnw=118&prev=/images%3Fq%3Dspermatogenesis&zoom=1&q=spermatogenesis&usg=__ZA1HpohnbnOWioTQqvtg668tFaA=&sa=X&ei=HVaZTJ_CL4L7lwf_q4T4Dw&ved=0CDcQ9QEwBQ • http://www.ncbi.nlm.nih.gov/pubmed/7922324