Deadenylase depletion protects inherited mRNAs in primordial germ cells.

A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo.

Use of sea stars to study basic reproductive processes.

Echinoderms are closely related to chordates and comprise a major group of invertebrate deuterostomes. They are broadcast spawners and as such, each female accumulates millions of eggs and oocytes. These cells are readily isolated, and are often large, clear, and surrounded by accessory cells and extracellular coverings that do not prevent access to the oocyte. Sea star oocytes are stored in prophase of meiosis, and since the natural meiotic stimulus has been identified as 1-methyladenine, these cells can be induced to complete meiotic maturation as individuals, or synchronously en masse. Microinjection and culture of these cells is feasible using quantitative or repetitive methods so that hundreds of oocytes and eggs can be modified each hour. Experimentation on this organism is extensive over a rich history of reproductive and developmental biology so that new investigators can easily incorporate this organism into their repertoire of research. This review will highlight the fundamental protocols to enable a new investigator to perform an array of approaches on this organism, including oocyte isolation, microinjection, and even single cell quantitative PCR.