HSCI Retreat 2020 Abstract 12

Zebrafish Chemical Compound Screen Uncovers Inducers of Skeletal Muscle Engraftment Across Species

Sahar Tavakoli,*1,2 Isaac Adatto,1,2 Sara Ashrafi Kakhki,1 Victoria S Chan,1,2 Haleh Fotowat,3 Eric Gähwiler,1,4 Margot E Manning,1,2 Kathleen A Messemer,1 Apoorva Rangan,1 Song Yang,2 Amy J Wagers,#1,5,6 and Leonard I Zon#1,2,7–9 
1 Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Stem Cell Institute, Cambridge, MA, USA
2 Stem Cell Program and Division of Hematology/Oncology, Boston, MA, USA
3 Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA    
4 Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland
5 Paul F. Glenn Center for the Biology of Aging, Harvard Medical School, Boston, MA, USA 
6 Joslin Diabetes Center, Boston, MA, USA 
7 Howard Hughes Medical Institute, Boston, MA, USA 
8 Children's Hospital and Dana Farber Cancer Institute, Boston, MA, USA
9 Harvard Medical School, Boston, MA, USA

These authors contributed equally to this work
* Presenting and corresponding author: sahartavakoli@fas.harvard.edu

Submitted: Jun 11, 2020; Published online: Jul 27, 2020



Genetic muscle disorders compromise muscle function through degeneration of muscle fibers, increased inflammation and impaired muscle regeneration. Transplantation of genetically normal muscle progenitors could be an approach to rescue muscle wasting and boost repair; however, this approach has shown limited utility thus far due to the typically poor engraftment efficiency of cultured progenitors. To define regulators of muscle engraftment that could be targeted to improve transplantation outcomes, we developed a novel cross-species screening platform, employing zebrafish and mouse, to discover chemical compounds that promote muscle progenitor engraftment in vivo. Muscle cells derived from zebrafish blastomeres were treated for 4 hours with biomolecules and transplanted into the flanks of adult zebrafish (n=15/ biomolecule). We focused our screening on a well-annotated library of 230 lipids, since lipids are known to enhance cell migration and regulate the homeostasis and regenerative function of adult tissue stem cells. Using limit-dilution assays, potential "hits" from our primary screen were identified and re-evaluated in replicate transplantation experiments. We discovered two lipids that promote zebrafish muscle progenitor cell engraftment in vivo: lysophosphatidic acid (LPA) and niflumic acid (NFA). Using bioluminescence imaging, we further ascertained that both NFA and LPA enhance muscle stem cell (satellite cell) engraftment in mouse as well (mean BLI radiance ± SEM- NFA: 27.2E+6 ± 6.8 E+6 p/s; LPA: 25.6 E+6 ±4.4 E+6 p/s; vehicle-treated cells: 8.2 E+6 ± 1.4 E+6 p/s; n = 15, 1-way ANOVA, p ? 0.05), indicating conservation of the pro-myogenic activities of these compounds across vertebrate species. Studies in sapje-like (dystrophin mutant) fish transplanted with NFA-treated or LPA-treated cells showed higher engraftment efficiency, significantly better swimming performance and greater ability to swim against a water current, compared to fish engrafted with vehicle-treated control cells. Mechanistically, the pro-myogenic activities of LPA and NFA appear to be associated with increased cytoplasmic Ca2+ and down-regulation of muscle development genes. RNA sequencing analysis also revealed upregulation of myoblast fusion regulating genes, including myomaker (Tmem8c) and Ccl8, in LPA-treated satellite cells. In summary, successful application of this cross-species approach has uncovered evolutionarily conserved pathways regulating muscle regeneration, suggesting new potential opportunities for treating muscle disease by enhancing myogenic contributions of transplanted muscle progenitors.