In situ hybridization of an antisense probe of the germline-specific gene 25M04 (putative KRAB domain zinc finger protein) reveals expression in the developing germline cells at day 14 (A, B, I) and day 20 (C, D, J) post-fertilization. Punctate staining reveals specific expression in the presumptive primordial germ cells (PGCs). Staining of PGCs is not observed in embryos that were hybridized with the sense strand probe (E–F), but some background staining is observed due to the presence of non-cellular endogenous alkaline phosphatase activity in the developing gut, pharynx, notochord and otic capsule. Panels B, D, F, and H correspond to the circumscribed regions in A, C, E, G, respectively. Panels I and J are transverse sections of the embryos shown in panels A and C. Sections have been counterstained with eosin in order to enhance contrast; arrows mark the location of PGCs positive for the 25M04 marker. This expression pattern suggests that 25M04 is involved in some aspect of PGC differentiation and/or migration. Nc = notochord, Nt = neural tube, Y = yolk.