An effective method for tissue-specific ablation in zebrafish is the nitroreductase (NTR)/metronidazole (MTZ) system. Expressing bacterial NTR in the presence of nitroimidazole compounds causes apoptotic cell death, which can be useful for understanding many biological processes. However, this requires tissue-specific expression of the NTR enzyme, and many tissues have yet to be targeted with transgenic lines that express NTR. We generated a transgenic zebrafish line expressing NTR in differentiated skeletal muscle. Treatment of embryos with MTZ caused muscle specific cell ablation. We demonstrate this line can be used to monitor muscle regeneration in whole embryos and in transplanted transgenic cells.
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References
1.
YuD, CaiZ, LiD, et al.Myogenic differentiation of stem cells for skeletal muscle regeneration. Stem Cells Int, 2021; 2021:8884283; doi: 10.1155/2021/8884283
2.
BeffagnaG.Zebrafish as a smart model to understand regeneration after heart injury: How fish could help humans. Front Cardiovasc Med, 2019; 6:107; doi: 10.3389/fcvm.2019.00107
3.
PipaliaTG, KothJ, RoySD, et al.Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair. Dis Model Mech, 2016; 9(6):671–684; doi: 10.1242/dmm.022251
4.
RatnayakeD, NguyenPD, RosselloFJ, et al.Macrophages provide a transient muscle stem cell niche via NAMPT secretion. Nature, 2021; 591(7849):281–287; doi: 10.1038/s41586-021-03199-7
5.
SegerC, HargraveM, WangX, et al.Analysis of Pax7 expressing myogenic cells in zebrafish muscle development, injury, and models of disease. Dev Dyn, 2011; 240(11):2440–2451; doi: 10.1002/dvdy.22745
6.
WangJ, PanakovaD, KikuchiK, et al.The regenerative capacity of zebrafish reverses cardiac failure caused by genetic cardiomyocyte depletion. Development, 2011; 138(16):3421–3430; doi: 10.1242/dev.068601
7.
CuradoS, AndersonRM, JungblutB, et al.Conditional targeted cell ablation in zebrafish: A new tool for regeneration studies. Dev Dyn, 2007; 236(4):1025–1035; doi: 10.1002/dvdy.21100
8.
PattersonLB, ParichyDM. Interactions with iridophores and the tissue environment required for patterning melanophores and xanthophores during zebrafish adult pigment stripe formation. PLoS Genet, 2013; 9(5):e1003561; doi: 10.1371/journal.pgen.1003561
9.
CuradoS, StainierDY, AndersonRM. Nitroreductase-mediated cell/tissue ablation in zebrafish: A spatially and temporally controlled ablation method with applications in developmental and regeneration studies. Nat Protoc, 2008; 3(6):948–954; doi: 10.1038/nprot.2008.58
10.
OttenC, Abdelilah-SeyfriedS.Laser-inflicted injury of zebrafish embryonic skeletal muscle. J Vis Exp, 2013(71):e4351; doi: 10.3791/4351
11.
AnlezarkGM, MeltonRG, SherwoodRF, et al.The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)—I. Purification and properties of a nitroreductase enzyme from Escherichia coli—a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT). Biochem Pharmacol, 1992; 44(12):2289–2295; doi: 10.1016/0006-2952(92)90671-5
12.
BridgewaterJA, SpringerCJ, KnoxRJ, et al.Expression of the bacterial nitroreductase enzyme in mammalian cells renders them selectively sensitive to killing by the prodrug CB1954. Eur J Cancer, 1995; 31A(13–14):2362–2370; doi: 10.1016/0959-8049(95)00436-x
13.
EdwardsDI.Nitroimidazole drugs—action and resistance mechanisms. I. Mechanisms of action. J Antimicrob Chemother, 1993; 31(1):9–20; doi: 10.1093/jac/31.1.9
14.
BergerJ, TarakciH, BergerS, et al.Loss of Tropomodulin4 in the zebrafish mutant trage causes cytoplasmic rod formation and muscle weakness reminiscent of nemaline myopathy. Dis Model Mech, 2014; 7(12):1407–1415; doi: 10.1242/dmm.017376
15.
KawakamiK.Transgenesis and gene trap methods in zebrafish by using the Tol2 transposable element. Methods Cell Biol, 2004; 77:201–222; doi: 10.1016/s0091-679x(04)77011-9
16.
GriffinK, PatientR, HolderN. Analysis of FGF function in normal and no tail zebrafish embryos reveals separate mechanisms for formation of the trunk and the tail. Development, 1995; 121(9):2983–2994; doi: 10.1242/dev.121.9.2983
17.
CoutelleO, BlagdenCS, HampsonR, et al.Hedgehog signalling is required for maintenance of myf5 and myoD expression and timely terminal differentiation in zebrafish adaxial myogenesis. Dev Biol, 2001; 236(1):136–150; doi: 10.1006/dbio.2001.0193
18.
RowRH, PeggA, KinneyBA, et al.BMP and FGF signaling interact to pattern mesoderm by controlling basic helix-loop-helix transcription factor activity. Elife, 2018; 7:e31018; doi: 10.7554/eLife.31018
19.
EllettF, PaseL, HaymanJW, et al.mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood, 2011; 117(4):e49–e56; doi: 10.1182/blood-2010-10-314120
20.
NowakKJ, WattanasirichaigoonD, GoebelHH, et al.Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy. Nat Genet, 1999; 23(2):208–212; doi: 10.1038/13837
21.
HigashijimaS, OkamotoH, UenoN, et al.High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev Biol, 1997; 192(2):289–299; doi: 10.1006/dbio.1997.8779
22.
TaborKM, BergeronSA, HorstickEJ, et al.Direct activation of the Mauthner cell by electric field pulses drives ultrarapid escape responses. J Neurophysiol, 2014; 112(4):834–844; doi: 10.1152/jn.00228.2014
23.
LecoeurH.Nuclear apoptosis detection by flow cytometry: Influence of endogenous endonucleases. Exp Cell Res, 2002; 277(1):1–14; doi: 10.1006/excr.2002.5537
24.
TuckerB, LardelliM. A rapid apoptosis assay measuring relative acridine orange fluorescence in zebrafish embryos. Zebrafish, 2007; 4(2):113–116; doi: 10.1089/zeb.2007.0508
25.
GurevichDB, NguyenPD, SiegelAL, et al.Asymmetric division of clonal muscle stem cells coordinates muscle regeneration in vivo. Science, 2016; 353(6295):aad9969; doi: 10.1126/science.aad9969
26.
KnappeS, ZammitPS, KnightRD. A population of Pax7-expressing muscle progenitor cells show differential responses to muscle injury dependent on developmental stage and injury extent. Front Aging Neurosci, 2015; 7:161; doi: 10.3389/fnagi.2015.00161
27.
LinCY, YungRF, LeeHC, et al.Myogenic regulatory factors Myf5 and Myod function distinctly during craniofacial myogenesis of zebrafish. Dev Biol, 2006; 299(2):594–608; doi: 10.1016/j.ydbio.2006.08.042
28.
YanYL, MillerCT, NissenRM, et al.A zebrafish sox9 gene required for cartilage morphogenesis. Development, 2002; 129(21):5065–5079; doi: 10.1242/dev.129.21.5065
29.
DortJ, FabreP, MolinaT, et al.Macrophages are key regulators of stem cells during skeletal muscle regeneration and diseases. Stem Cells Int, 2019; 2019:4761427; doi: 10.1155/2019/4761427
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