RESUMO
Tissue development and regeneration are dynamic processes involving complex cell migration and cell-cell interactions. We have developed a protocol for complementary time-lapse and three-dimensional (3D) imaging of tissue for developmental and regeneration studies which we apply here to the zebrafish cardiac vasculature. 3D imaging of fixed specimens is used to first define the subject at high resolution then live imaging captures how it changes dynamically. Hearts from adult and juvenile zebrafish are extracted and cleaned in preparation for the different imaging modalities. For whole-mount 3D confocal imaging, single or multiple hearts with native fluorescence or immuno-labeling are prepared for stabilization or clearing, and then imaged. For live imaging, hearts are placed in a prefabricated fluidic device and set on a temperature-controlled microscope for culture and imaging over several days. This protocol allows complete visualization of morphogenic processes in a 3D context and provides the ability to follow cell behaviors to complement in vivo and fixed tissue studies. This culture and imaging protocol can be applied to different cell and tissue types. Here, we have used it to observe zebrafish coronary vasculature and the migration of coronary endothelial cells during heart regeneration.
Assuntos
Células Endoteliais , Peixe-Zebra , Animais , Células Endoteliais/metabolismo , Coração/diagnóstico por imagem , Imageamento Tridimensional/métodosRESUMO
Heart disease remains the single largest cause of death in developed countries, and novel therapeutic interventions are desperately needed to alleviate this growing burden. The cardiac lymphatic system is the long-overlooked counterpart of the coronary blood vasculature, but its important roles in homeostasis and disease are becoming increasingly apparent. Recently, the cardiac lymphatic vasculature in zebrafish has been described and its role in supporting the potent regenerative response of zebrafish heart tissue investigated. In this review, we discuss these findings in the wider context of lymphatic development, evolution and the promise of this system to open new therapeutic avenues to treat myocardial infarction and other cardiopathologies.
RESUMO
The cardiac lymphatic vascular system and its potentially critical functions in heart patients have been largely underappreciated, in part due to a lack of experimentally accessible systems. We here demonstrate that cardiac lymphatic vessels develop in young adult zebrafish, using coronary arteries to guide their expansion down the ventricle. Mechanistically, we show that in cxcr4a mutants with defective coronary artery development, cardiac lymphatic vessels fail to expand onto the ventricle. In regenerating adult zebrafish hearts the lymphatic vasculature undergoes extensive lymphangiogenesis in response to a cryoinjury. A significant defect in reducing the scar size after cryoinjury is observed in zebrafish with impaired Vegfc/Vegfr3 signaling that fail to develop intact cardiac lymphatic vessels. These results suggest that the cardiac lymphatic system can influence the regenerative potential of the myocardium.