The planarian Schmidtea mediterranea as a model for epigenetic germ cell specification: Analysis of ESTs from the hermaphroditic strain

Abstract

Freshwater planarians have prodigious regenerative abilities that enable them to form complete organisms from tiny body fragments. This plasticity is also exhibited by the planarian germ cell lineage. Unlike many model organisms in which germ cells are specified by localized determinants, planarian germ cells appear to be specified epigenetically, arising postembryonically from stem cells. The planarian Schmidtea mediterranea is well suited for investigating the mechanisms underlying epigenetic germ cell specification. Two strains of S. mediterranea exist: a hermaphroditic strain that reproduces sexually and an asexual strain that reproduces by means of transverse fission. To date, expressed sequence tags (ESTs) have been generated only from the asexual strain. To develop molecular reagents for studying epigenetic germ cell specification, we have sequenced 27,161 ESTs from two developmental stages of the hermaphroditic strain of S. mediterranea ; this collection of ESTs represents ≈10,000 unique transcripts. blast analysis of the assembled ESTs showed that 66% share similarity to sequences in public databases. We annotated the assembled ESTs using Gene Ontology terms as well as conserved protein domains and organized them in a relational database. To validate experimentally the Gene Ontology annotations, we used whole-mount in situ hybridization to examine the expression patterns of transcripts assigned to the biological process “reproduction.” Of the 53 genes in this category, 87% were expressed in the reproductive organs. In addition to its utility for studying germ cell development, this EST collection will be an important resource for annotating the planarian genome and studying this animal's amazing regenerative abilities.

nanos function is essential for development and regeneration of planarian germ cells

“…1C). nanos expression was detected much earlier during gonad development than previously examined markers of the reproductive organs (33).…”

“…10A); similar expression of Djnos was also reported in asexual D. japonica (32). This observation prompted us to test additional markers of the reproductive structures in asexual planarians (33). Of 14 genes examined that label the mature gonads in sexual planarians, 13 did not produce staining comparable with nanos in asexual worms (data not shown).…”

“…To characterize the process of germ cell formation in the planarian S. mediterranea, we identified sequences encoding the highly conserved NANOS zinc finger motif in whole-genome shotgun sequence data from the her-maphroditic strain. Using these sequences, we isolated fulllength nanos cDNAs (839 bases) from a sexual S. mediterranea cDNA library (33) [see supporting information (SI) Methods]. Northern blot analysis revealed a single Smed-nanos transcript of Ϸ0.8 kb in total RNA from sexual planarians (SI Fig.…”

“…As an additional marker for studying the appearance of germ cells in young hatchlings, we used germinal histone H4 (germinal H4) (33), a transcript that labels presumptive germ cells as well as somatic neoblasts (see below). As observed with nanos, the majority of D1 hatchlings (6 of 7) did not have obvious germinal H4 labeling outside of the neoblast population (SI Fig.…”

“…4L). germinal H4 is also expressed in ovaries and somatic neoblasts (33). In control planarians, this marker enabled us to visualize the regenerated ovaries (Fig.…”