Latent TGF-β binding protein 3 identifies a second heart field in zebrafish

Abstract

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 [1–3]. 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 [4–10]. 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-TGFβ 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 TGFβ 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 TGFβ signaling phenocopies the ltbp3-morphant phenotype whereas expression of a constitutively active TGFβ type I receptor rescues it. Taken together, our findings uncover a requirement for ltbp3-TGFβ signaling during zebrafish SHF development, a process that serves to enlarge the single ventricular chamber in this species.

Utilizing Zebrafish to Understand Second Heart Field Development

“…This technique has shown that arterial pole progenitors express both gata4 and nkx2.5 during somitogenesis, confirming that SHF progenitors originate in the ALPM [8]. Furthermore, Cre-mediated lineage tracing has confirmed that cells from the pericardial mesenchyme adjacent to the heart tube migrate into the outflow tract [9]. Taken together, these analyses show that the late-differentiating cardiomyocytes at the zebrafish arterial pole meet the criteria that define the SHF.…”

“…ltbp3 is expressed in the zebrafish SHF, and Cre-mediated lineage tracing has shown that ltbp3-expressing cells give rise to outflow tract cardiomyocytes [9]. Ltbp3-deficient embryos lack an outflow tract due to reduced SHF proliferation, a consequence of reduced TGF-β signaling [9]. This work not only illuminated Ltbp3 as a new SHF regulator but also uncovered a novel role for TGF-β signaling in SHF development.…”

“…For example, zebrafish studies may be valuable for elucidating the mechanisms that pattern the SHF into its anterior and posterior subdivisions. In addition, it will be interesting to use zebrafish to examine the factors that control differentiation of multipotent SHF cells into myocardial, endocardial, and smooth muscle lineages [9]. Zebrafish will also be valuable for exploring whether multipotent SHF cells are maintained after embryogenesis, perhaps to be deployed after injury.…”

“…The role of Ltbp3, a secreted protein that regulates TGF-β ligand availability, has been of particular interest. ltbp3 is expressed in the zebrafish SHF, and Cre-mediated lineage tracing has shown that ltbp3-expressing cells give rise to outflow tract cardiomyocytes [9]. Ltbp3-deficient embryos lack an outflow tract due to reduced SHF proliferation, a consequence of reduced TGF-β signaling [9].…”

Early frameshift alleles of zebrafish <em>tbx5a</em> that fail to develop the heartstrings phenotype

“…We injected the sgRNA complexed with Cas9 protein as solubilized RNPs [11] at sub-optimal concentration to achieve viable mosaicism (see Methods for details) in the multicolor Tg(lmo2:dsRED2;drl:EGFP;myl7:mCyan) reporter background, subsequently abbreviated as RGB. In RGB embryos, dsRED2 labels endothelial, hematopoietic, and endocardial progenitors (lmo2) in red [14], EGFP marks all lateral plate mesoderm lineages (drl) including pectoral fins in green [15], and AmCyan reveals the differentiated cardiomyocytes (myl7 ) in blue [16]; consequently, RGB enables in vivo imaging of all cardiovascular and additional LPM lineages over the first 3 days of development. From F0 outcrosses that transmitted mutant tbx5a alleles, we genotyped adult F1 zebrafish for the presence of mutated tbx5a alleles by tail clipping, PCR, sequencing, and CrispRVariants analysis [17].…”