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

Abstract

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 wholemount 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.

Heat Shock 70-kDa Protein 5 (Hspa5) Is Essential for Pronephros Formation by Mediating Retinoic Acid Signaling

“…3, DЉ-FЉ). We also examined the expression of genes involved in pronephros tubule and glomus development including smp30 (46) and nephrin (47). Expression of smp30 (tubule; 64%; 21 of 33) and nephrin (glomus; 55%; 17 of 31) was decreased on the Hspa5MO2-injected side when compared with the uninjected side (Fig.…”

Patterning the embryonic kidney: BMP signaling mediates the differentiation of the pronephric tubules and duct in Xenopus laevis

“…To further confirm the developing glomus was not affected by a reduction in the levels of BMP signaling in post-gastrula stage embryos, we also examined the transmembrane, glomal-specific genes, nephrin and neph1 (Gerth et al, 2005). In mammals, Nephrin plays a critical role in podocyte slit-diaphragm formation as well as in the development of functional mammalian glomerular filtration barriers (Huber and Benzing, 2005;Johnstone and Holzman, 2006).…”

“…Following color development, embryos were fixed for 1 hr in MEMFA at room temperature or overnight at 4°C, and either stored in 1ϫ PBS at 4°C or dehydrated in 100% methanol for longterm storage at Ϫ20°C. cRNA probes included Xenopus orthologues of the following genes: Xlim-1 (Taira et al, 1994), WT-1 (Carroll and Vize, 1996), C-ret (Durbec et al, 1996), NKCC-2, NBC1, XSGLT1K (Zhou and Vize, 2004), ClC-K (Vize, 2003), Na,K-ATPase ␤1 (Eid and Brandli, 2001), Xpax-8 ; xWT-1 (Carroll and Vize, 1996), nephrin (Gerth et al, 2005), Neph1 (NIBB clone no. XL226gex), cardiac actin (Mohun et al, 1984), and Xsmp-30 (Sato et al, 2000).…”

“…Although much has been learned regarding the development of the duct and tubular epithelia, less is known about the induction and morphogenesis of the third component of a functional nephron, the glomus (the vascularized filtration apparatus). However, despite the limited information regarding the initial induction of the glomus, similar genes, including XWT-1 and nephrin, are expressed at later stages of development in both the glomera of lower vertebrates and the glomeruli of mammals (Vize et al, 1997;Drummond et al, 1998;Gerth et al, 2005). Therefore, by examining the role of BMP signaling during the formation of the simplest and rapidly developing excretory organ (the pronephros), important information can be quickly attained and used to advance our understanding of all three types of nephric systems.…”

pdzrn3 is required for pronephros morphogenesis in Xenopus laevis

“…pax8 and hnf1b are early expressed genes, involved in the initial patterning of the marginal zone mesoderm, but also implicated in later processes of kidney morphogenesis; Na + /K + ATPase and nephrin can be used as later pronephric markers that start to be expressed since tailbud stages 25 and 26. In particular, Na + /K + ATPase encodes for a key pump enzyme, expressed at level of tubules and duct; nephrin encodes for a transmembrane protein that is expressed at level of the glomus (Eid and Brändli, 2001;Gerth et al, 2005).…”