Gerth VE et al. (2005), Nephrin expression and three-dimensional morpho...

XB-ART-1893

Dev Dyn 2005 Jul 01;2333:1131-9. doi: 10.1002/dvdy.20415.

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

Gerth VE , Zhou X , Vize PD .

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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.

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Species referenced: Xenopus laevis
Genes referenced: nphs1

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	Figure 2. Xenopus nephrin is expressed in the pronephric glomus. A: Expression of nephrin (red arrow) in a stage 26 Xenopus laevis embryo assayed by in situ hybridization. B: Enlargement of A. C: Transverse section through a late tail bud stage embryo that had been stained previously for nephrin expression by in situ hybridization. Nephrin expression is medial to the region that will form the pronephric tubules (eosin counterstain). D: Double-stained stage 31 embryo. Somites 1-6 are stained light blue (immunohistochemistry with antibody 12/101) and nephrin (in situ hybridization) is stained brown. Somites 3-5 are indicated with blue arrows, and nephrin expression with a red arrow. E: Double-stained stage 35 embryo, as in D. Two head somites have disintegrated (dashed arrows) and another (4) partially disintegrated.
	Figure 3. A-L: Xenopus nephrin expression examined by whole-mount fluorescent in situ hybridization. Anterior is to the left in all samples. A, B, and C are stage 26; D, E, and F are stage 28; G, H, and I are stage 33; J, K, and L are stage 36 embryos. A, D, G, and J are lateral views; B, E, H, and K enlargements of the panels to the left; and C, F, I, and L are dorsal views.
	Figure 5. Three-dimensional anatomy of the glomus. The Xenopus glomera were visualized by nephrin fluorescent in situ hybridization, sampled by confocal microscopy, processed in Amira to generate voltex images, then surfaced and reimaged in 3D Studio. Both voltex and surface images are shown from anterior (dorsal is up) and dorsal views (anterior is up). Stages are shown to the left, and image type and views are shown above. Scale bars, in microns, are shown in the dorsal voltex view panels.
	nphs1 (nephrin 1) expressed in Xenopus embryo via in situ hybridization, stage 31, latera; view, anterior left.
	nphs1 (nephrin 1) expressed in Xenopus embryo via in situ hybridization, stage 31, latera; view, anterior left.