generation of transgenic lines is described in the supplementary methods . to induce recombination and subsequently genetically label cardiomyocytes , embryos / adults were treated with 4-oht followed by a wash period of at least 1 week prior to amputation . adult fish were anesthetized in 0.4%tricaine and secured ventral side up in a slotted sponge . watchmaker forceps were used to remove the surface scales and penetrate the skin , muscle , and pericardial sac . once exposed , the ventricle was gently pulled at the apex and cut with iridectomy scissors . after surgery , fish were immediately returned to system water . at the specified time points , hearts were removed and fixed in 4% paraformaldehyde overnight at 4c , washed several times in pbs , equilibrated in 30% sucrose in pbs , and frozen for cryosectioning . fish were anesthetized in 0.4%tricaine , and 0.5ml of a 2.5mg / ml solution of brdu ( in pbs ) was injected into the abdominal cavity once every 24 h for 7 d. after 14 d , hearts were removed and fixed in 4% paraformaldehyde overnight at 4c , washed several times in pbs , equilibrated in 30% sucrose in pbs , and frozen for cryosectioning . cyclapolin 9 ( sigma c6493 ) was dissolved in dmso and added to 400ml system water to a final concentration of 3m . generation of transgenic lines is described in the supplementary methods . to induce recombination and subsequently genetically label cardiomyocytes , embryos / adults were treated with 4-oht followed by a wash period of at least 1 week prior to amputation . adult fish were anesthetized in 0.4%tricaine and secured ventral side up in a slotted sponge . watchmaker forceps were used to remove the surface scales and penetrate the skin , muscle , and pericardial sac . once exposed , the ventricle was gently pulled at the apex and cut with iridectomy scissors . after surgery , fish were immediately returned to system water . at the specified time points , hearts were removed and fixed in 4% paraformaldehyde overnight at 4c , washed several times in pbs , equilibrated in 30% sucrose in pbs , and frozen for cryosectioning . fish were anesthetized in 0.4%tricaine , and 0.5ml of a 2.5mg / ml solution of brdu ( in pbs ) was injected into the abdominal cavity once every 24 h for 7 d. after 14 d , hearts were removed and fixed in 4% paraformaldehyde overnight at 4c , washed several times in pbs , equilibrated in 30% sucrose in pbs , and frozen for cryosectioning . cyclapolin 9 ( sigma c6493 ) was dissolved in dmso and added to 400ml system water to a final concentration of 3m .
although mammalian hearts show virtually no ability to regenerate , there is a growing initiative to determine whether existing cardiomyocytes or progenitor cells can be coaxed into eliciting a regenerative response . 
 in contrast to mammals , a number of non - mammalian vertebrate species are able to regenerate their hearts13 , including the zebrafish4,5 , which can fully regenerate its heart following amputation of up to 20% of the ventricle . to directly address the source of newly formed cardiomyocytes during zebrafish heart regeneration 
 , we first established a genetic strategy to lineage - trace cardiomyocytes in the adult fish , based on the cre / lox system widely used in the mouse6 . using this system , 
 we show here that regenerated heart muscle cells are derived from the proliferation of differentiated cardiomyocytes . 
 furthermore , we show that proliferating cardiomyocytes undergo limited dedifferentiation characterized by the disassembly of their sarcomeric structure , detachment from one another and expression of regulators of cell cycle progression . 
 specifically , we show that polo - like kinase1 ( plk1 ) is an essential component of cardiomyocyte proliferation during heart regeneration . 
 our data provides the first direct evidence for the source of proliferating cardiomyocytes during zebrafish heart regeneration and indicates that stem / progenitor cells are not significantly involved in this process .