Unit 9 Germ Plasm Germ Cells Germ Plasm Theodore Boveri germ plasm has a visibly different appearance Germ Plasm Amphibians Germ plasm contains Material associated with mitochondria mitochondrial nuage RNA helicases enzymes that alter RNA structure Gilbert 10e Fig 17 14 Germ Plasm Amphibians Gilbert 10e Fig 17 6 Germ cells in C elegans come from a distinct lineage adapted from Gilbert 10e Fig 5 20 Germ cells in C elegans come from a distinct lineage Z2 Z3 form the germ cells they are descended from posterior cells P1 P2 P3 P4 Z2 Z3 Gilbert 10e Fig 5 19 P granules localize to the posterior pole prior to first cleavage A P female pronucleus male pronucleus Wolpert et al 2e Fig 12 14 female and male pronuclei P granules AB cell P1 cell cleavage furrow P Granules C elegans Gilbert 9e Fig 5 44 PIE 1 and P granules track with germ cells in C elegans Wolpert et al 2e Fig 12 14 Germ cells P granules PIE 1 segregate to the posterior A B P granules PIE 1 is in the cytoplasm of P1 and later germ cell precursors Nance et al 2003 Development 130 5339 5350 C PIE 1 par genes are required for partitioning of germ plasm into the germ cell lineage offspring of par 3 mutant mothers have symmetric P granule distribution courtesy Ken Kemphues de novo gene has to evolve essentiality through neofunctionalization because it has no ancestral template A duplicated gene generated from an ancestral copy of its parental gene could become 13 C Joppich S Scholz G Korge A Schwendemann Chromosome Res 17 19 2009 14 H Ida et al Nucleic Acids Res 37 1423 2009 15 J L Mueller J L Page M F Wolfner Genetics 175 777 2007 Germ cells Do P granules contain the determinant Cytoplasmic Partitioning of P Granule Components Is Not Required to Specify the Germline in C elegans Christopher M Gallo Jennifer T Wang Fumio Motegi Geraldine Seydoux Asymmetric segregation of P granules during the first four divisions of the Caenorhabditis elegans embryo is a classic example of cytoplasmic partitioning of germline determinants It is thought that asymmetric partitioning of P granule components during mitosis is essential to distinguish germline from soma We have identified a mutant pptr 1 in which P granules become unstable during mitosis and P granule proteins and RNAs are distributed equally to somatic and germline blastomeres Despite symmetric partitioning of P granule components pptr 1 mutants segregate a germline that uniquely expresses P granules during postembryonic development pptr 1 mutants are fertile except at high temperatures Hence asymmetric partitioning of maternal P granules is not essential to specify germ cell fate Instead it may serve to protect the nascent germline from stress characteristic of germ cells1685 is the rhabditis elegans germ P granules are present Gallo et algeneral 2010 Science 330 presence of cytoplasmic RNA rich gran in all germ cells except mature sperm and they Tables Refere 11 Au 10 11 curso onic granu hypo ify t segre 2 3 T focal ing t fluor resul prote we a move prote Depar Medic of Me 21205 Thes To w Germ cells are transcriptionally repressed by PIE 1 pes 10 mRNA is not found in P2 pes 10 mRNA Gilbert 10e Fig 17 4 PIE 1 is required for transcriptional repression in germ cells Pole cells are the germ cell precursors in Drosophila anterior posterior pole cells Pole cells contain pole plasm the fly equivalent of germ plasm in other organisms They are the first cells to form in the embryo Gilbert 10e Fig 17 5 Pole cells in Drosophila pole plasm can be transplanted needle needle pole plasm removed from donor embryo See Gilbert 3e Fig 7 32 pole plasm injected into embryo derived from a UV irradiated egg can t make pole cells normally Pole plasm transplant can rescue the ability to make pole cells wildtype makes pole cells UV treated pole plasm transplant pole cells Conclusion Pole plasm contain a localized determinant sufficient for pole cell germ cells production Gilbert 3e Fig 7 32 UV treated no pole cells Pole plasm in Drosophila really does act as a determinant Step 1 transplant pole plasm to ANTERIOR of recipient embryo of different genotype This makes sure that the transplant didn t involve any DNA from the pink cytoplasm donor If pole cells form they should be of the second embryo s genotype Wolpert et al 2e Fig 12 13 Pole plasm in Drosophila really does act as a determinant Step 2 transplant pole cells that form to another embryo of third genotype The induced pole cells can t make it to the gonad so we transplant them into a third type of embryo to see if they function Wolpert et al 2e Fig 12 13 We are looking for cells of the second genotype in the sperm oocytes of the final flies Result functional germ cells oskar mRNA is part of a scaffold for germ plasm in Drosophila Anterior components Staufen protein oskar mRNA form scaffold Nurse cells Anterior components Modified from Gilbert 8e Fig 9 8 Testing whether oskar mRNA is sufficient to recruit germ plasm We make an engineered form of DNA the oskar gene that encodes oskar the 3 UTR of bicoid RNA bicoid mRNA is normally at the anterior so the gene if expressed in mothers will cause some oskar to be at the anterior Wolpert 2e Fig 12 15 bicoid gene 5 3 3 UTR containing bicoid localization signal oskar gene Testing whether oskar mRNA is sufficient to recruit germ plasm A anterior posterior P oskar with bicoid normal oskar 3 localization localization signal Wolpert 2e Fig 12 15 Testing whether oskar mRNA is sufficient to recruit germ plasm Pole cells at anterior Normal pole cells at posterior Conclusion oskar mRNA is sufficient to recruit pole plasm capable of forming pole cells Wolpert 2e Fig 12 15 Expression of oct4 mRNA correlates with pluripotency and ability to form germ cells in mammals Gilbert 910e Fig 17 8 Germ cells form via cell cell interactions in mammals Primordial germ cells migrating into gonad Germ cells migrate directionally in vertebrates Primordial germ cells migrate into the genital ridge gonad primordium Guidance is mediated in part by a chemokine SDF 1 PGCs sense SDF 1 via CXCR4 a receptor Gilbert 10e Fig 17 15 Adding excess SDF 1 perturbs germ cell migration in mice control SDF1 protein added Molyneaux et al 2003 Development 130 4279 86 Germ cells form from four clusters in zebrafish germ cell precursors migrate as clusters into the developing gonad a process also mediated via SDF 1 and CXCR4 Gilbert 10e Fig 17 13 SDF 1 or CXCR4 knockdown perturbs germ cell migration in zebrafish control Courtesy Erez Raz CXCR4 MO SDF1 MO