Development of genetic manipulation tools in Macrostomum lignano for dissection of molecular mechanisms of regeneration

Background: The free-living marine flatworm Macrostomum lignano is a powerful model organism to study mechanisms of regeneration and stem cell regulation due to its convenient combination of biological and experimental properties, including the availability of transgenesis methods, which is unique among flatworm models. However, due to its relatively recent introduction to the research community, there are still many biological aspects of this animal model that are yet to be explored. One of such questions is the influence of the culturing temperature on Macrostomum biology. Results: Here we systematically investigated how different culturing temperatures affect the development time, reproduction rate, regeneration, heat shock response, and gene knockdown efficiency by RNA interference in M. lignano . We used marker transgenic lines of the flatworm to accurately measure the regeneration endpoint and to establish the stress response threshold for temperature shock. We found that compared to the culturing temperature of 20˚C commonly used for M. lignano , elevated temperatures of 25˚C-30˚C substantially speed-up the development, lead to faster manifestation of RNAi phenotypes, and regeneration time and increase reproduction rate without detectable negative consequences for the animal, while temperatures above 30˚C elicit a heat shock response. Conclusions: We show that altering the temperature conditions can be used to shorten the time required to establish M. lignano cultures, store important lines and optimize the microinjection procedures for transgenesis. Our findings will help to optimize the design of experiments in M. lignano and thus facilitate future research in this model organism.