Analysis of ciliary assembly and function in planaria

Planarians are free-living invertebrates that employ motile cilia for locomotion. Specifically, cilia that populate the ventral epithelium of the planarian body are highly conserved, with a 9+2 axoneme and a full complement of inner and outer arm dynein motors. The abundance of cilia on the planarian body, their unique accessibility, and high degree of conservation make this organism an attractive experimental model system for cilia biology. Moreover, planarians are genetically amenable and defects that compromise the function and structure of the cilia are not detrimental for their overall health, making them an ideal system for cilia gene loss-of-function studies. In this chapter, we provide information for introducing and maintaining planarians for experimental purposes in the laboratory and describe protocols for RNAi-induced gene knockdown studies. Furthermore, we elaborate on different imaging techniques used to analyze cilia physiology and structure, including live video microscopy, immunofluorescence analysis, and electron microscopy. Last, we provide assays for evaluating physical parameters of ciliary motility, including quantification of planarian gliding locomotion and measurement of ciliary beat frequency.