The role of vasculature and blood circulation in zebrafish swimbladder development

Background: Recently we have performed a detailed analysis of early development of zebrafish swimbladder, a homologous organ of tetrapod lung; however, the events of swimbladder development are still poorly characterized. Many studies have implicated the role of vascular system in development of many organs in vertebrates. As the swimbladder is lined with an intricate network of blood capillaries, it is of interest to investigate the role of the vascular system during early development of swimbladder. Results: To investigate the role of endothelial cells (ECs) and blood circulation during development of the swimbladder, phenotypes of swimbladder were analysed at three different stages (similar to 2, 3 and 5 dpf [day postfertilization]) in cloche (clo) mutant and Tnnt2 morphants, in the background of transgenic lines Et(krt4: EGFP)(sq33-2) and Et(krt4: EGFP)(sqet3) which express EGFP in the swimbladder epithelium and outer mesothelium respectively. Analyses of the three tissue layers of the swimbladder were performed using molecular markers hb9, fgf10a, acta2, and anxa5 to distinguish epithelium, mesenchyme, and outer mesothelium. We showed that the budding stage was independent of ECs and blood flow, while early epithelial growth, mesenchymal organization and its differentiation into smooth muscle, as well as outer mesothelial organization, were dependent on ECs. Blood circulation contributed to later stage of epithelial growth, smooth muscle differentiation, and organization of the outer mesothelium. Inflation of the swimbladder was also affected as a result of absence of ECs and blood flow. Conclusion: Our data demonstrated that the vascular system, though not essential in swimbladder budding, plays an important role in the development of the swimbladder starting from the early growth stage, including mesenchyme organization and smooth muscle differentiation, and outer mesothelial organization, which in turn may be essential for the function of the swimbladder as reflected in its eventual inflation.