Nephrin expression and three-dimensional morphogenesis of the Xenopus pronephric glomus

Nephrin (NHPS1) encodes a transmembrane protein of approximately 1,200 amino acids that plays a critical role in podocyte slit-diaphragm formation and the development of functional mammalian glomerular filtration barriers. In humans and mice with congenital defects in the nephrin gene, the glomerular filtration barrier is defective and protein leakage into the kidney filtrate causes a life-threatening proteinuria. This protein also plays an essential role in the formation of the stellate cells of the Drosophila Malpighian tubules. In this report, the sequence and expression of a Xenopus ortholog of nephrin is described using both conventional and novel three-dimensional (3D) visualization methodologies. Xenopus nephrin encodes a protein of 1,238 amino acids and is expressed at high levels in the forming pronephric kidney glomus, the equivalent of the mammalian glomerulus. Expression commences at stage 25 and is specific to the pronephric glomus up until at least tadpole feeding stages. Two-color fluorescent whole-mount in situ analysis of nephrin expression allowed the 3D shape of the glomus to be imaged and contrasted to the pronephric tubules throughout its morphogenesis. Confocal data processing pipelines were established to generate both volumetric and surface models of the developing pronephros, and a Web-based visualization system was used to generate dynamic and manipulable models of the forming nephric organs. This system allows simple on-line morphometric analysis of the developing pronephric components. As in fish embryos, the glomera first form laterally then migrate medially as the pronephros matures. Unlike in the zebrafish, in Xenopus, this migration stops short of complete fusion of the two glomera at the midline, but a nephrin-positive glomeral nexus does form anteriorly and links the two structures from stage 38 onward.