Tube and lumen formation are essential steps in forming a functional vasculature. Despite their significance, our understanding of these processes remains limited, especially at the cellular and molecular levels. In this study, we analyze mechanisms of angioblast coalescence in the zebrafish embryonic midline and subsequent vascular tube formation. To facilitate these studies, we generated a transgenic line where EGFP expression is controlled by the zebrafish flk1 promoter. We find that angioblasts migrate as individual cells to form a vascular cord at the midline. This transient structure is stabilized by endothelial cell-cell junctions, and subsequently undergoes lumen formation to form a fully patent vessel. Downregulating the VEGF signaling pathway, while affecting the number of angioblasts, does not appear to affect their migratory behavior. Our studies also indicate that the endoderm, a tissue previously implicated in vascular development, provides a substratum for endothelial cell migration and is involved in regulating the timing of this process, but that it is not essential for the direction of migration. In addition, the endothelial cells in endodermless embryos form properly lumenized vessels, contrary to what has been previously reported in Xenopus and avian embryos. These studies provide the tools and a cellular framework for the investigation of mutations affecting vasculogenesis in zebrafish.

Defects in cardiac valve morphogenesis and septation of the heart chambers constitute some of the most common human congenital abnormalities. Some of these defects originate from errors in atrioventricular (AV) endocardial cushion development. Although this process is being extensively studied in mouse and chick, the zebrafish system presents several advantages over these models, including the ability to carry out forward genetic screens and study vertebrate gene function at the single cell level. In this paper, we analyze the cellular and subcellular architecture of the zebrafish heart during stages of AV cushion and valve development and gain an unprecedented level of resolution into this process. We find that endocardial cells in the AV canal differentiate morphologically before the onset of epithelial to mesenchymal transformation, thereby defining a previously unappreciated step during AV valve formation. We use a combination of novel transgenic lines and fluorescent immunohistochemistry to analyze further the role of various genetic (Notch and Calcineurin signaling) and epigenetic (heart function)pathways in this process. In addition, from a large-scale forward genetic screen we identified 55 mutants, defining 48 different genes, that exhibit defects in discrete stages of AV cushion development. This collection of mutants provides a unique set of tools to further our understanding of the genetic basis of cell behavior and differentiation during AV valve development.

RNA splicing is a major contributor to total transcriptome complexity; however, the functional role and regulation of splicing in heart failure remain poorly understood. Here, we used a total transcriptome profiling and bioinformatic analysis approach and identified a muscle-specific isoform of an RNA splicing regulator, RBFox1 (also known as A2BP1), as a prominent regulator of alternative RNA splicing during heart failure. Evaluation of developing murine and zebrafish hearts revealed that RBFox1 is induced during postnatal cardiac maturation. However, we found that RBFox1 is markedly diminished in failing human and mouse hearts. In a mouse model, RBFox1 deficiency in the heart promoted pressure overload–induced heart failure. We determined that RBFox1 is a potent regulator of RNA splicing and is required for a conserved splicing process of transcription factor MEF2 family members that yields different MEF2 isoforms with differential effects on cardiac hypertrophic gene expression. Finally, induction of RBFox1 expression in murine pressure overload models substantially attenuated cardiac hypertrophy and pathological manifestations. Together, this study identifies regulation of RNA splicing by RBFox1 as an important player in transcriptome reprogramming during heart failure that influence pathogenesis of the disease.