A genetic and microscopy toolkit for manipulating and monitoring regeneration in Macrostomum lignano.

Hall, R Nelson; Li, Hongquan; Chai, Chew; Vermeulen, Sidney; Bigasin, Robin R; Song, Eun Sun; Sarkar, Souradeep R; Gibson, Jesse; Prakash, Manu; Fire, Andrew Z; Wang, Bo

Hall, R Nelson; Li, Hongquan; Chai, Chew; Vermeulen, Sidney; Bigasin, Robin R; Song, Eun Sun; Sarkar, Souradeep R; Gibson, Jesse; Prakash, Manu; Fire, Andrew Z; Wang, Bo.

Affiliation

Hall RN; Department of Bioengineering, Stanford University, Stanford, CA, USA. Electronic address: nh2894@stowers.org.
Li H; Department of Electrical Engineering, Stanford University, Stanford, CA, USA.
Chai C; Department of Bioengineering, Stanford University, Stanford, CA, USA.
Vermeulen S; Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
Bigasin RR; Department of Bioengineering, Stanford University, Stanford, CA, USA.
Song ES; Department of Applied Physics, Stanford University, Stanford, CA, USA.
Sarkar SR; Department of Bioengineering, Stanford University, Stanford, CA, USA.
Gibson J; Department of Bioengineering, Stanford University, Stanford, CA, USA.
Prakash M; Department of Bioengineering, Stanford University, Stanford, CA, USA.
Fire AZ; Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
Wang B; Department of Bioengineering, Stanford University, Stanford, CA, USA. Electronic address: wangbo@stanford.edu.

Cell Rep ; 43(11): 114892, 2024 Oct 19.

Article in En | MEDLINE | ID: mdl-39427313

ABSTRACT

ABSTRACT

Live imaging of regenerative processes can reveal how animals restore their bodies after injury through a cascade of dynamic cellular events. Here, we present a comprehensive toolkit for live imaging of tissue regeneration in the flatworm Macrostomum lignano, including a high-throughput cloning pipeline, targeted cellular ablation, and advanced microscopy solutions. Using tissue-specific reporter expression, we examine how various structures regenerate. Enabled by a custom luminescence/fluorescence microscope, we overcome intense stress-induced autofluorescence to demonstrate genetic cellular ablation and reveal the limited regenerative capacity of neurons and their essential role during wound healing, contrasting muscle cells' rapid regeneration after ablation. Finally, we build an open-source tracking microscope to continuously image freely moving animals throughout the week-long process of regeneration, quantifying kinetics of wound healing, nerve cord repair, body regeneration, growth, and behavioral recovery. Our findings suggest that nerve cord reconnection is highly robust and proceeds independently of regeneration.

Key words

CP: Stem cell research; flatworms; live imaging; luminescence; nervous system; regeneration; transgenics

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