Determination of Germ Cells The Germ Line • Early separation of “germ line” from regular somatic cells in many but not all animals – Early specification of primordial germ cells (PGCs) – Later migration into developing somatic gonad • In gonadal environment, gametogenesis – Meiosis – Sperm/egg differentiation • Germ cell determination can either be – Autonomous – Specified by neighbors Segregation of Germ Plasm of Parascaris • Theodor Boveri (1862-1915) – Nematode of horse and pig intestines – Observed the two chromosomes through cell divisions, through gametogenesis, through development – Chromosome parts loss correlated to special cytoplasm Does a Specific Region of Cytoplasm Protect the Chromosomes from Diminution? • Centrifugation to reorient the first cleavage plane relative to the vegetal cytoplasm centrifuged: Consequences • Only in germ line is all chromosomal information retained • Concept of stem cells – Cells that give rise to one the same and one different Early Segregation of Cytoplasmic Factors Defines PGCs • Segregation of P granules in C. elegans embryo follows PGC lineage • Contain RNA binding proteins and transcription inhibitors germ cell precursor Drosophila Pole Plasm • Pole cells (=PGCs) are the first nuclei to migrate out to periphery and cellularize • Surrounded by pole plasm – mitochondria, fibrils, polar granules, polysomes, etc. – plasm similar in other organisms How do We Know Pole Plasm Specifies the Germline? • Experiments demonstrated importance of pole plasm for generating germline – Hegner (1911) removed posterior pole cytoplasm from beetle eggs -----> beetles grew up to be sterile – Mahowald (1971) prevented pole cell nuclei from reaching posterior pole plasm in Drosophila embryos -----> no germ cells made! – Mahowald (1974) pole plasm transplantation expts • Into anterior makes pole cells there (fate change) How Can We Use Genetics to Identify Components of Pole Plasm? • If mother is unable to pack all components of pole plasm into her oocytes…..? – Her progeny will be sterile – The mom will be “grandchildless” • Hunt for maternal-effect mutations that cause sterility in offspring Components of Pole Plasm • grandchildless mutant identified: – gcl (germ cell-less) – look at localization of mRNA and protein – Transcribed in nurse cells – Transported to oocyte – Transported to posterior – Translated in early cleavage – Goes to nucleus and responsible for germ cell formation Components of Pole Plasm • 1) mRNA of the gcl gene • 2) posterior determinants – also important for ant-post patterning – nanos, oskar, vasa – injection of oskar mRNA into ant of embryo -> ectopic pole cell formation – Excess oskar -> excess no. pole cells – Localizes other proteins and RNAs like nanos – vasa RNA binding protein (helicase) is well conserved and found in germ plasm of many animals Components of Pole Plasm • 3) mitochondrial ribosomal RNA (mtrRNA) – Small and large subunits of ribosome that are specifically associated with mitochondria – Exported to pole plasm and components of the granules – injection of mtrRNA into UV-treated eggs -> restores pole cell formation Components of Pole Plasm • Proteins/RNAs involved in localization and inhibition of transcription/translation • Hypothesis: – Pole cells = cells that are prevented from differentiating – Retain totipotency .