Molecular Regulation of Zebrafish Cardiac Maturation

Congenital heart diseases (CHDs) are the most common type of human birth defect and often feature structural abnormalities that arise during development and maturation. Many CHDs have a genetic component which provides a molecular basis for the cellular defects underlying structural malformations. During embryonic development, the vertebrate heart expands and remodels to meet the cardiovascular needs of the developing embryo in a process called cardiac maturation. In particular, the ventricular chamber matures to optimize the internal architecture for efficient conduction and contraction. Chamber maturation features formation of luminal muscular protrusions, called trabeculae, which increase myocardial mass and are often malformed in CHD. Here, zebrafish (Danio rerio) are used as an optically accessible, genetically tractable, vertebrate model to explore the conserved, molecular basis of chamber maturation Accumulating evidence indicates a critical role for cardiac contraction and the resulting fluid forces in shaping the developing heart, yet the molecular basis of this function is largely unknown. Data reported in Chapter 2 describe an essential role for cardiac contraction-responsive transcriptional changes in endocardial cells for regulating trabeculation. Cardiac contraction causes blood flow, which is likely mechanotransduced into intracellular signaling cues by endocardial primary cilia. Contraction stimulates notch1b transcription, and Notch1 activation induces expression of downstream genes ephrinb2a (efnb2a) and neuregulin-1 (nrg1) in the endocardium. Forced Notch activation rescues efnb2a and nrg1 expression in non-contractile hearts, and efnb2a is essential for trabeculation. Although ErbB2 receptor tyrosine-protein (ErbB2), an essential receptor partner in the Nrg1-ErbB2/ErbB4 signaling pathway, is necessary to stimulate trabeculation in mice and zebrafish, requirement for nrg1 has not been explored in zebrafish. In Chapter 3, CRISPR/Cas9 targeted gene editing was used to generate novel, isoform-specific mutations in nrg1. Phenotypic analysis of nrg1 mutants revealed that nrg1 is dispensable for cardiac trabeculation. However, one isoform, nrg1-III is essential for establishing the cardiac nerve plexus. Likely as a consequence of impaired cardiac innervation, nrg1 mutants have cardiac malformations and experience early mortality. In sum, this study reveals previously uncharacterized cellular and molecular relationships regulating chamber maturation.