Analysis of the C. elegans Germline Stem Cell Pool

The Caenorhabditis elegans germline is an excellent model for studying the genetic and molecular regulation of stem cell self-renewal and progression of cells from a stem cell state to a differentiated state. The germline tissue is organized in an assembly line with the germline stem cell (GSC) pool at one end and differentiated gametes at the other. A simple mesenchymal niche caps the GSC pool and maintains GSCs in an undifferentiated state by signaling through the conserved Notch pathway. Notch signaling activates transcription of the key GSC regulators lst-1 and sygl-1 proteins in a gradient through the GSC pool. LST-1 and SYGL-1 proteins work with PUF RNA regulators in a self-renewal hub to maintain the GSC pool. In this chapter, we present methods for characterizing the C. elegans GSC pool and early stages of germ cell differentiation. The methods include examination of germlines in living and fixed worms, cell cycle analysis, and analysis of markers. We also discuss assays to separate mutant phenotypes that affect the stem cell vs. differentiation decision from those that affect germ cell processes more generally.