Abstract 2171: Nephronectin is Required for Zebrafish Heart Looping by Regulating Spatial Expression Pattern of BMP4
In recent years, it has been shown that the extracellular matrix (ECM) is not an inert scaffold but a complex and dynamic component that plays an essential role in morphogenesis, tissue maintenance, tissue repair and disease by regulating cell adhesion, cell survival, proliferation, migration, and differentiation. Yet, the function of the cardiac ECM is still poorly understood. We hypothesize that ECM components, which exhibit a transient spatial and temporal distribution during heart development, regulate important processes like heart looping. Based on a RNA expression screen we have identified nephronectin as a transiently expressed cardiac ECM gene. Nephronectin mRNA expression increases in rat heart at E11, reaches its maximum at E15 and declines thereafter. In situ hybridization experiments demonstrated that nephronectin is also in zebrafish heart transiently expressed around 44 hours post fertilization (hpf). We injected translation- as well as splicing-inhibitory morpholinos (MOs) targeting nephronectin to address whether it is essential for cardiac development. Efficient knockdown was confirmed by RTPCR. To determine changes in heart and cell morphology we utilized transgenic zebrafish (cmcl2-GFPras, cmcl2-nDsRed, flk1-GFP). We observed that in embryos injected with MOs heart development was perturbed. At 48 hpf bmp4 is usually confined to the outflow and inflow tract as well as the AV canal. In contrast, in morphants bmp4 is expressed throughout the ventricle, which is often associated with looping defects. At 52 hpf we observed in more than 80% of the morphants a cardiac looping defect. At 72 hpf morphants exhibited cardiac edema with otherwise normal overall morphology. Closer analyses revealed that endocardial cushion cells of the morphants fail to undergo morphological changes indicative for endothelial to mesenchymal transition. At the same time one can usually observe trabeculation. However, at 80 hpf in morphants we observed still monolayered myocardium with only rare signs of trabeculation. At 120 hpf the morphant heart consisted of a multilayered instead of a monolyered myocardium of enlarged cardiomyocytes. Taken together, we have identified nephronectin as a novel regulator of heart development in zebrafish.