The nanos-class gene of the leech Helobdella robusta (Hro-nos) is present as a maternal transcript whose levels decay during cleavage; HRO-NOS protein is more abundant in the D quadrant cells relative to the A, B, and C quadrants; and HRO-NOS is more abundant in the ectodermal precursor cell (DNOPQ) than in its sister mesodermal precursor (DM) (Pilon and Weisblat, 1997). Here, using in situ hybridization, we show that Hro-nos mRNA is broadly distributed throughout the zygote, is concentrated in both animal and vegetal teloplasm during stage 1 and is at higher levels in DNOPQ than in DM at stage 4b. Hro-nos expression increases after stage 7, as judged by in situ hybridization, developmental RT-PCR, and western blots; this increase must therefore represent later zygotic expression. Of particular interest, during stages 9 and 10, each of 11 mid-body segments (M8-M18) has a pair of Hro-nos positive "spots" comprising of one or two large cells each. These spots later disappear in an anteroposterior progression. We find that these Hro-nos-expressing cells are of mesodermal origin, arising in a segmentally iterated manner from the M lineage, and correspond to cells previously proposed as primordial germ cells (PGCs; Bürger, 1891; Weisblat and Shankland, 1985). These results support the proposal that nanos-class genes functioned in the specification of germline cells in the ancestral bilaterian and possibly in a separate process related to embryonic polarity in the ancestral protostome.
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We thank other members of our lab for many helpful discussions and assistance in preparing this manuscript. This work was supported by NSF grant IBN 9723114 to DAW and a fellowship from the Korean Research Foundation to DK.