MiniSOG2-mediated Specific Photoablation of Motor Neurons in Ascidian Embryos

Madoka K Utsumi; Taichi Akahoshi; Kotaro Oka; Kohji Hotta

doi:10.21769/BioProtoc.4537

MiniSOG2-mediated Specific Photoablation of Motor Neurons in Ascidian Embryos

Bio Protoc. 2022 Oct 20;12(20):e4537. doi: 10.21769/BioProtoc.4537.

Authors

Madoka K Utsumi¹, Taichi Akahoshi¹, Kotaro Oka^{1

2

3}, Kohji Hotta¹

Affiliations

¹ Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Kohoku, Yokohama 223-8522, Japan.
² Waseda Research Institute for Science and Engineering, Waseda University, Shinjuku, Tokyo, Japan.
³ Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung City, Taiwan.

Abstract

When understanding the neuronal function of a specific neural circuit, single-cell level photoablation of a targeted cell is one of the useful experimental approaches. This protocol describes a method to photoablate specific motor neurons via the mini singlet oxygen generator (miniSOG2), a light-oxygen-voltage (LOV)-based optogenetic tool used for ablating targeted cells in arbitrary areas. MiniSOG2 could induce the cell death pathway by generating reactive oxygen species (ROS) upon blue light illumination. Photoablation of a specific cell using the miniSOG2 was performed to show that, in Ciona intestinalis type A ( Ciona robusta) , a single pair of motor neurons, MN2/A10.64, is necessary to drive their tail muscle contraction. The membrane targeted miniSOG2 combined with neuron-specific promoter (pSP-Neurog::miniSOG2-CAAX) was electroplated into the Ciona egg and transiently expressed at specific neurons of the embryo. MN2 labeled with pSP-Neurog:mCherry-CAAX was irradiated using a 440-nm laser from the lateral side for 10 min to ablate its neural function. The behavior of the embryo before and after the irradiation was recorded with a high-speed camera. Graphical abstract.

Keywords: Ascidian; Microscopy; Motor neuron; Photoablation; Reactive oxygen species; Tunicate; miniSOG2.