A conserved regulatory program drives emergence of the lateral plate mesoderm

Cardiovascular cell lineages emerge with kidney, smooth muscle, and limb skeleton progenitors from the lateral plate mesoderm (LPM). How the LPM emerges during development and how it has evolved to form key lineages of the vertebrate body plan remain unknown. Here, we captured LPM formation by transgenic in toto imaging and lineage tracing using the first pan-LPM enhancer element from the zebrafish gene draculin (drl). drl LPM enhancer-based reporters are specifically active in LPM-corresponding territories of several chordate species, uncovering a universal LPM-specific gene program. Distinct from other mesoderm, we identified EomesA, FoxH1, and MixL1 with BMP/Nodal-controlled Smad activity as minimally required factors to drive drl-marked LPM formation. Altogether, our work provides a developmental and mechanistic framework for LPM emergence and the in vitro differentiation of cardiovascular cell types. Our findings suggest that the LPM may represent an ancient cell fate domain that predates ancestral vertebrates

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self-renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self-renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP-Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple-repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal

Next-generation plasmids for transgenesis in zebrafish and beyond

Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible, modular system. Here, we established several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene-regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2, and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Lastly, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker active prior to hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish, and other models

Next-generation plasmids for transgenesis in zebrafish and beyond

Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible, modular system. Here, we established several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene-regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse betaglobin minimal promoter coupled to several fluorophores, CreERT2, and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein Cerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Lastly, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker active prior to hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish, and other models

A conserved regulatory program drives emergence of the lateral plate mesoderm

Cardiovascular cell lineages emerge with kidney, smooth muscle, and limb skeleton progenitors from the lateral plate mesoderm (LPM). How the LPM emerges during development and how it has evolved to form key lineages of the vertebrate body plan remain unknown. Here, we captured LPM formation by transgenic in toto imaging and lineage tracing using the first pan-LPM enhancer element from the zebrafish gene draculin (drl). drl LPM enhancer-based reporters are specifically active in LPM-corresponding territories of several chordate species, uncovering a universal LPM-specific gene program. Distinct from other mesoderm, we identified EomesA, FoxH1, and MixL1 with BMP/Nodal-controlled Smad activity as minimally required factors to drive drl-marked LPM formation. Altogether, our work provides a developmental and mechanistic framework for LPM emergence and the in vitro differentiation of cardiovascular cell types. Our findings suggest that the LPM may represent an ancient cell fate domain that predates ancestral vertebrates

Early frameshift alleles of zebrafish tbx5a that fail to develop the heartstrings phenotype

Tbx5 is a key transcription factor for vertebrate heart and forelimb development that causes Holt-Oram syndrome when mutated in humans. The classic zebrafish mutant for tbx5a named heartstrings (hst) features recessive absence of pectoral fins and a spectrum of heart defects, most-prominently featuring the name-giving stretched heart tube. The mutation of the hst allele is a stop codon that is predicted to result in a truncated Tbx5a protein that might feature residual activity. Here, using CRISPR-Cas9 mutagenesis, we generated zebrafish strains for two new tbx5a frameshift alleles in the first coding exon: tbx5a c.21_25del and tbx5a c.22_31del, abbreviated as tbx5aΔ5 and tbx5aΔ10. Homozygous and trans-heterozygous combinations of these new tbx5a alleles cause fully penetrant loss of pectoral fins and heart defects including changes in cardiac marker expression akin to hst mutants. Nonetheless, distinct from hst mutants, homozygous and trans-heterozygous combinations of these tbx5a frameshift mutants do not readily manifest the stretched hst heart phenotype. Our observation points out the importance and value of comparing phenotypes from different classes of mutant alleles per gene

Continuous addition of progenitors forms the cardiac ventricle in zebrafish

The vertebrate heart develops from several progenitor lineages. After early-differentiating first heart field (FHF) progenitors form the linear heart tube, late-differentiating second heart field (SHF) progenitors extend the atrium and ventricle, and form inflow and outflow tracts (IFT/OFT). However, the position and migration of late-differentiating progenitors during heart formation remains unclear. Here, we track zebrafish heart development using transgenics based on the cardiopharyngeal gene tbx1. Live imaging uncovers a tbx1 reporter-expressing cell sheath that continuously disseminates from the lateral plate mesoderm towards the forming heart tube. High-speed imaging and optogenetic lineage tracing corroborates that the zebrafish ventricle forms through continuous addition from the undifferentiated progenitor sheath followed by late-phase accrual of the bulbus arteriosus (BA). FGF inhibition during sheath migration reduces ventricle size and abolishes BA formation, refining the window of FGF action during OFT formation. Our findings consolidate previous end-point analyses and establish zebrafish ventricle formation as a continuous process

Persistent Ventricle Partitioning in the Adult Zebrafish Heart

The vertebrate heart integrates cells from the early-differentiating first heart field (FHF) and the later-differentiating second heart field (SHF), both emerging from the lateral plate mesoderm. In mammals, this process forms the basis for the development of the left and right ventricle chambers and subsequent chamber septation. The single ventricle-forming zebrafish heart also integrates FHF and SHF lineages during embryogenesis, yet the contributions of these two myocardial lineages to the adult zebrafish heart remain incompletely understood. Here, we characterize the myocardial labeling of FHF descendants in both the developing and adult zebrafish ventricle. Expanding previous findings, late gastrulation-stage labeling using drl-driven CreERT2 recombinase with a myocardium-specific, myl7-controlled, loxP reporter results in the predominant labeling of FHF-derived outer curvature and the right side of the embryonic ventricle. Raised to adulthood, such lineage-labeled hearts retain broad areas of FHF cardiomyocytes in a region of the ventricle that is positioned at the opposite side to the atrium and encompasses the apex. Our data add to the increasing evidence for a persisting cell-based compartmentalization of the adult zebrafish ventricle even in the absence of any physical boundary

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo

Tbx5a lineage tracing shows cardiomyocyte plasticity during zebrafish heart regeneration

During development, mesodermal progenitors from the first heart field (FHF) form a primitive cardiac tube, to which progenitors from the second heart field (SHF) are added. The contribution of FHF and SHF progenitors to the adult zebrafish heart has not been studied to date. Here we find, using genetic tbx5a lineage tracing tools, that the ventricular myocardium in the adult zebrafish is mainly derived from tbx5a+ cells, with a small contribution from tbx5a− SHF progenitors. Notably, ablation of ventricular tbx5a+-derived cardiomyocytes in the embryo is compensated by expansion of SHF-derived cells. In the adult, tbx5a expression is restricted to the trabeculae and excluded from the outer cortical layer. tbx5a-lineage tracing revealed that trabecular cardiomyocytes can switch their fate and differentiate into cortical myocardium during adult heart regeneration. We conclude that a high degree of cardiomyocyte cell fate plasticity contributes to efficient regeneration