The heart is the first functional organ to form during vertebrate development. Congenital heart defects are the most common type of human birth defect, many originating as anomalies in early heart development. The zebrafish model provides an accessible vertebrate system to study early heart morphogenesis and to gain new insights into the mechanisms of congenital disease. Although composed of only two chambers compared with the four-chambered mammalian heart, the zebrafish heart integrates the core processes and cellular lineages central to cardiac development across vertebrates. The rapid, translucent development of zebrafish is amenable to in vivo imaging and genetic lineage tracing techniques, providing versatile tools to study heart field migration and myocardial progenitor addition and differentiation. Combining transgenic reporters with rapid genome engineering via CRISPR-Cas9 allows for functional testing of candidate genes associated with congenital heart defects and the discovery of molecular causes leading to observed phenotypes. Here, we summarize key insights gained through zebrafish studies into the early patterning of uncommitted lateral plate mesoderm into cardiac progenitors and their regulation. We review the central genetic mechanisms, available tools, and approaches for modeling congenital heart anomalies in the zebrafish as a representative vertebrate model.

Rationale: cMyBP-C (cardiac myosin-binding protein-C) is a critical regulator of heart contraction, but the mechanisms by which cMyBP-C affects actin and myosin are only partly understood. A primary obstacle is that cMyBP-C localization on thick filaments may be a key factor defining its interactions, but most in vitro studies cannot duplicate the unique spatial arrangement of cMyBP-C within the sarcomere. Objective: The goal of this study was to validate a novel hybrid genetic/protein engineering approach for rapid manipulation of cMyBP-C in sarcomeres in situ. Methods and Results: We designed a novel cut and paste approach for removal and replacement of cMyBP-C N′-terminal domains (C0–C7) in detergent-permeabilized cardiomyocytes from gene-edited Spy-C mice. Spy-C mice express a TEVp (tobacco etch virus protease) cleavage site and a SpyTag (st) between cMyBP-C domains C7 and C8. A cut is achieved using TEVp which cleaves cMyBP-C to create a soluble N′-terminal γ C0C7 (endogenous [genetically encoded] N′-terminal domains C0 to C7 of cardiac myosin binding protein-C) fragment and an insoluble C′-terminal SpyTag-C8-C10 fragment that remains associated with thick filaments. Paste of new recombinant ( r )C0C7 domains is achieved by a covalent bond formed between SpyCatcher (-sc; encoded at the C′-termini of recombinant proteins) and SpyTag. Results show that loss of γ C0C7 reduced myofilament Ca ²⁺ sensitivity and increased cross-bridge cycling ( k _tr ) at submaximal [Ca ²⁺ ]. Acute loss of γ C0C7 also induced auto-oscillatory contractions at submaximal [Ca ²⁺ ]. Ligation of r C0C7 (exogenous [recombinant] N′-terminal domains C0 to C7 of cardiac myosin binding protein-C)-sc returned pCa ₅₀ and k _tr to control values and abolished oscillations, but phosphorylated (p)- r C0C7-sc did not completely rescue these effects. Conclusions: We describe a robust new approach for acute removal and replacement of cMyBP-C in situ. The method revealed a novel role for cMyBP-C N′-terminal domains to damp sarcomere-driven contractile waves (so-called spontaneous oscillatory contractions). Because phosphorylated (p)- r C0C7-sc was less effective at damping contractile oscillations, results suggest that spontaneous oscillatory contractions may contribute to enhanced contractility in response to inotropic stimuli.

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 establish 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. Finally, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker that is active before 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.

The development of paired appendages was a key innovation during evolution and facilitated the aquatic to terrestrial transition of vertebrates. Largely derived from the lateral plate mesoderm (LPM), one hypothesis for the evolution of paired fins invokes derivation from unpaired median fins via a pair of lateral fin folds located between pectoral and pelvic fin territories¹. Whilst unpaired and paired fins exhibit similar structural and molecular characteristics, no definitive evidence exists for paired lateral fin folds in larvae or adults of any extant or extinct species. As unpaired fin core components are regarded as exclusively derived from paraxial mesoderm, any transition presumes both co-option of a fin developmental programme to the LPM and bilateral duplication². Here, we identify that the larval zebrafish unpaired pre-anal fin fold (PAFF) is derived from the LPM and thus may represent a developmental intermediate between median and paired fins. We trace the contribution of LPM to the PAFF in both cyclostomes and gnathostomes, supporting the notion that this is an ancient trait of vertebrates. Finally, we observe that the PAFF can be bifurcated by increasing bone morphogenetic protein signalling, generating LPM-derived paired fin folds. Our work provides evidence that lateral fin folds may have existed as embryonic anlage for elaboration to paired fins.

The heart is formed from diverse cell lineages that assemble into a functional unit, including the pericardium, a mesothelial sac that supports movement, homeostasis, and immune responses. However, its developmental origins remain unresolved. Here, we find the pericardium forms within the lateral plate mesoderm from dedicated mesothelial progenitors that are distinct from the classic heart field. Imaging of transgenic zebrafish reporters documents lateral plate mesoderm cells that emerge lateral of the heart field among a continuous mesothelial progenitor band. Single-cell transcriptomics and trajectories of hand2-expressing lateral plate mesoderm reveal distinct populations of mesothelial precursors, including pericardial precursors. Their mesothelial gene expression signature is conserved in mammals and carries over to post-natal development. Light sheet imaging and machine learning-supported cell tracking documents the migration of pericardial precursors from the edge of the heart field to form the pericardial cavity. Genetic perturbations reveal this process occurs independently of heart formation, with canonical Wnt/β-catenin signaling modulating pericardial cell number and tissue rigidity. We connect the pathological expression of secreted Wnt antagonists of the SFRP family found in pediatric dilated cardiomyopathy to increased pericardial stiffness in neonatal rats. Altogether, our data integrate pericardium formation as an independent process into heart morphogenesis.