PIWI proteins are expressed in the interstitial stem cells and are enriched in perinuclear granules. (A) A schematic of Hydra showing that it is composed of three cell lineages. The ectodermal (green) and endodermal (blue) epithelial cell lineages form the inside and outside of the body column. All of the epithelial cells in the body column are mitotic and maintain the lineage (9). As they divide they are displaced toward the extremities where they become postmitotic, differentiate, and eventually are sloughed from the tips of the tentacles and the foot. The interstitial cell lineage (pink) consists of the interstitial stem cells, which give rise to the differentiated nerve cells, gland cells, nematocytes (from precursor nematoblast nests), and germ cells (8). The expression of Hydra PIWI protein, Hyli and Hywi, is restricted to the body column as shown by Hyli whole-mount immunofluorescence (B and C) and Hywi and Hyli immunoblot analysis (D). (E–G) Hywi (green) and Hyli (SI Appendix, Fig. S2 D–F) are expressed in the interstitial stem cells as shown by colabeling with the C41 antibody, which labels interstitial stem cells (red) (11). Hywi (green) and Hyli (red) proteins are distributed diffusely in the cytoplasm of interstitial stem cells, and are enriched in perinuclear granules, as demonstrated by immunofluorescence (H–J) and immunoelectron microscopy (K and L). DNA is labeled with Hoechst 33342.