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Paper text:
single and double , fluorescent and non - fluorescent , in situ hybridization and immunohistochemical stainings were performed using standard protocols . to analyze the ltbp3 loss of function phenotype
, we injected anti - sense ltbp3 morpholinos into one - cell stage wt and transgenic embryos . for genetic lineage tracing , a transgenic driver strain expressing cre recombinase in ltbp3 cells and four cre - responsive  color switching 
the driver strain was crossed individually to each of the reporter strains and their double transgenic progenies were analyzed for zsyellow protein fluorescence using confocal microscopy . to follow the migration of zebrafish shf cells , a tracking dye was injected into the zsyellow , amcyan region of tg(nkx2.5::zsyello ) ; tg(cmlc2::csy ) embryos at 24hpf
, the embryos were imaged immediately , and then again at 48 and/or 72hpf . a transgenic strain carrying a cdna encoding a constitutively active human tgf type i receptor ( caalk5 ) under control of the zebrafish heat shock promoter was generated and used to rescue the myocardial defect in ltbp3 morphant embryos .
full methods and any associated references are available in the online version of the paper at www.natre.com/nature
the four - chambered mammalian heart develops from two fields of cardiac progenitor cells ( cpcs ) distinguished by their spatiotemporal patterns of differentiation and contributions to the definitive heart [ 13 ] . 
 the first heart field differentiates earlier in lateral plate mesoderm , generates the linear heart tube and ultimately gives rise to the left ventricle . the second heart field ( shf ) differentiates later in pharyngeal mesoderm , elongates the heart tube , and gives rise to the outflow tract ( oft ) and much of the right ventricle . 
 because hearts in lower vertebrates contain a rudimentary oft but not a right ventricle [ 4 ] , the existence and function of shf - like cells in these species has remained a topic of speculation [ 410 ] . 
 here we provide direct evidence from cre / lox - mediated lineage tracing and loss of function studies in zebrafish , a lower vertebrate with a single ventricle , that latent - tgf binding protein 3 ( ltbp3 ) transcripts mark a field of cpcs with defining characteristics of the anterior shf in mammals . 
 specifically , ltbp3 + cells differentiate in pharyngeal mesoderm after formation of the heart tube , elongate the heart tube at the outflow pole , and give rise to three cardiovascular lineages in the oft and myocardium in the distal ventricle . 
 in addition to expressing ltbp3 , a protein that regulates the bioavailability of tgf ligands [ 11 ] , zebrafish shf cells co - express nkx2.5 , an evolutionarily conserved marker of cpcs in both fields [ 4 ] . 
 embryos devoid of ltbp3 lack the same cardiac structures derived from ltbp3 + cells due to compromised progenitor proliferation . 
 additionally , small - molecule inhibition of tgf signaling phenocopies the ltbp3-morphant phenotype whereas expression of a constitutively active tgf type i receptor rescues it . taken together 
 , our findings uncover a requirement for ltbp3-tgf signaling during zebrafish shf development , a process that serves to enlarge the single ventricular chamber in this species .