Role of Ca2+ Signaling During Early Pronephric Development in Zebrafish and Xenopus Embryos

Recent studies from vertebrates have implicated a Ca2+-dependent signaling pathway in normal kidney development, because the loss of function in either of two Ca2+-related proteins is known to result in abnormal kidney development resulting in polycystic kidney disease. We have begun a study to image Ca2+ signaling during the formation of the pronephros (the functional embryonic kidney) in both intact zebrafish and Xenopus laevis embryos, as well as in Xenopus animal caps that are isolated at the blastula stage and induced to differentiate into pronephric tubules by incubation with retinoic acid (RA) and activin A (AA). By loading embryos with the Ca2+-sensitive luminescent reporter, aequorin, and using a custom-designed photon-imaging microscope to continually visualize the Ca2+ signaling patterns, we are currently generating spatial and temporal maps of the Ca2+ signals that occur during pronephric formation in these two intact model systems, as well as in animal caps. Furthermore, we have begun to explore the requirement for, and the function of, these Ca2+ transients. We loaded intact Xenopus embryos with the caged calcium chelator, diazo-2, specifically into blastomeres that are known to develop into the pronephros. When the diazo-2 is uncaged at the earliest stages of pronephros development, the tubules that form are abnormal. In addition, tubulogenesis is inhibited in animal caps (incubated with RA and AA) that are treated with BAPTA. Furthermore, when intact zebrafish embryos are incubated with the L-type calcium channel blocker nifedipine for the first 60 hours of pronephros development, tubule formation is once again inhibited; and the ducts that develop are longer and narrower than the untreated controls. Our preliminary results thus support the proposal that Ca2+ signaling plays a crucial role in kidney formation and that zebrafish and Xenopus embryos provide suitable models to image Ca2+ dynamics during organogenesis.