ABSTRACT
In this chapter, the authors discuss the techniques used when investigating the role of Two-Pore Channel 2 (TPC2) in zebrafish neuromuscular development. The role of TPC2 in the differentiation of slow muscle cells and PMNs in intact zebrafish embryos has been investigated using various approaches. The remaining injected embryos were raised to adulthood, and the founder fish were identified using the restriction endonuclease digestion assay, by excising the tip of the caudal fin as a source of DNA. The founders were then outcrossed with wild-type adults to test for the germline transmission of the tpcn2 mutation. Zebrafish embryos are reported to demonstrate sequential motile behaviours such as spontaneous coiling, and touch-evoked behaviour, during development. The effect of TPC2 knock down on the spontaneous coiling behaviour was determined by video microscopy, whereas the touch-evoked response was determined by gently touching the embryos on the head twice and recording their response.
