AGEE, S.J.*; WEISBLAT, D.A.: Investigating the embryological function of maternal Hro-nos, a nanos homolog in the leech Helobdella robusta

Previous studies have shown that a nanos-class gene is expressed zygotically in germline precursors of the leech, Helobdella robusta, as in arthropods and vertebrates, suggesting that this aspect of nanos-class gene function may have been present in the ancestor of Bilateria. In addition, leech-nanos is present as an abundant maternal transcript, as in Drosophila. What is the function of this maternal transcript and is it related to the maternal function of nanos in A-P axis determination of Drosophila?
In the leech, mesodermal and ectodermal lineages are segregated during fourth cleavage, when the D macromere divides to form cells DM and DNOPQ, the precursors of segmental mesoderm and ectoderm, respectively. Hro-NOS expression peaks during this stage of development, and there is more Hro-NOS in the ectodermal precursor DNOPQ than in the mesodermal precursor DM (Pilon & Weisblat, 1997). It is also known that the ectodermal fate of DNOPQ requires the inheritance of both cortical and teloplasmic determinants (Nelson & Weisblat, 1992). Based upon these previous studies we have speculated that Hro-nos is involved in the specification of ectodermal and mesodermal precursor lineages.
To investigate this possibility, we have knocked down Hro-NOS expression by injecting anti-NOS morpholino oligonucleotides into early 1-cell embryos. Knockdown of Hro-NOS was verified by western blot and immunostaining. Phenotypes resulting from loss of Hro-NOS expression were examined by tracing ectodermal and mesodermal lineages. Anti-NOS morpholino injected embryos have abnormal ectodermal bandlets and disorganized germinal bands, consistent with the idea that Hro-NOS functions in specifying ectoderm of this segmented lophotrochozoan.