Background: The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced by means of injection of double‐stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA.Results: We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery.Conclusions: This method gives robust and reproducible results and is amenable to high‐throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems. Developmental Dynamics 242:718–730, 2013.

Planarian flatworms contain a population of adult stem cells (neoblasts) that proliferate and generate cells of all tissues during growth, regeneration and tissue homeostasis. A characteristic feature of neoblasts is the presence of chromatoid bodies, large cytoplasmic ribonucleoprotein (RNP) granules morphologically similar to structures present in the germline of many organisms. This study aims to reveal the function, and identify additional components, of planarian chromatoid bodies. We uncover the presence of symmetrical dimethylarginine (sDMA) on chromatoid body components and identify the ortholog of protein arginine methyltransferase PRMT5 as the enzyme responsible for sDMA modification in these proteins. RNA interference-mediated depletion of planarian PRMT5 results in defects in homeostasis and regeneration, reduced animal size, reduced number of neoblasts, fewer chromatoid bodies and increased levels of transposon and repetitive-element transcripts. Our results suggest that PIWI family member SMEDWI-3 is one sDMA-containing chromatoid body protein for which methylation depends on PRMT5. Additionally, we discover an RNA localized to chromatoid bodies, germinal histone H4. Our results reveal new components of chromatoid bodies and their function in planarian stem cells, and also support emerging studies indicative of sDMA function in stabilization of RNP granules and the Piwi-interacting RNA pathway.

Few animals are known to lay eggs in the absence of ovulation or copulation, as it is presumably energetically wasteful and subjected to negative selection. Characterization of Smed-boule, a member of the DAZ family of germline RNA-binding proteins, revealed that egg capsule (or capsule) production and deposition occurs independently of the presence of gametes in the planarian flatworm Schmidtea mediterranea. Reduction of Smed-boule expression by RNA-interference (RNAi) causes ablation of spermatogonial stem cells and the inability of ovarian germline stem cells to undergo oogenesis. Although animals subjected to Smed-boule RNAi lose their gametes and become sterile, they continue to lay egg capsules. Production of sterile capsules is even observed in virgin Smed-boule(RNAi) and control planarians maintained in complete isolation, demonstrating that egg production in S. mediterranea occurs independently of ovulation, fertilization, or mating. Evidence suggests that this is a conserved feature amongst Platyhelminthes, and therefore relevant to the pathology and dissemination of parasitic flatworms. These findings demonstrate that Smed-boule functions at different stages during male and female germline stem cell development, and also demonstrate that egg capsule production by planarian flatworms occurs independently of signals produced by mating or ova. Our work shows that production and deposition of egg capsules by planarian flatworms does not require fertilization, mating, ovulation, or even the existence of gametes. We also uncovered evidence for the existence of gender-specific germline stem cells in Schmidtea mediterranea, a hermaphroditic species of flatworm that develops germ cells post-embryonically. These findings surfaced from the characterization of Smed-boule, a member of the Deleted in AZoospermia gene family of RNA-binding proteins required for germline development in a broad range of animals. These findings lead to a better appreciation of the evolutionary diversity in approaches to oviparity. Additionally, discovering that egg capsule production occurs independently of germline or mating activities may carry a potential applied aspect with regards to regulating the dissemination and pathology of parasitic flatworms (such as blood flukes and tapeworms), if conserved in these organisms.