Non-canonical wnt signals antagonize and canonical wnt signals promote cell proliferation in early kidney development.

Canonical and non-canonical wnt signals often have opposed roles. In this report, we use developing Xenopus embryos to demonstrate a novel anti-proliferative role for non-canonical wnt signals in the very earliest stages of kidney development. Non-canonical wnt signals were down-regulated using PDZ domain mutants of dishevelled 2 and up-regulated using wild-type vang-like 2, while canonical signals were manipulated using dominant-negative forms of lef1 or treatment with lithium. When non-canonical signals are down-regulated in the developing Xenopus pronephros, cell proliferation rates increased and when canonical signals were shutdown the opposite occurred. Treatment with lithium chloride has a powerful pro-proliferative effect on the forming nephric primordium. Together these data show that in addition to previously documented antagonisms between these distinct wnt signaling pathways, they also have opposing effects on cell division.

Multichannel wholemount fluorescent and fluorescent/chromogenic in situ hybridization in Xenopus embryos.

In situ hybridization (ISH) is widely used to study the spatial distribution of gene expression in developing embryos. It is the method of choice to analyze the normal pattern of expression of a gene and also to characterize how the expression of a gene, or a group of genes, is altered in response to experimental or genetic manipulations. The standard protocols for this technique use a chromogenic reaction that produces a purple or red precipitate in cells expressing the target gene. This technique has significant disadvantages when compared with fluorescent techniques, as it cannot detect regions of overlap and external staining masks internal staining. We present a protocol for three-channel fluorescent ISH (FISH) optimized for wholemount analysis of large vertebrate embryos. Multichannel FISH in combination with immunofluorescence or chromogenic ISH offers a suite of approaches that allow accurate mapping of overlapping gene expression patterns in two- and three-dimensions. The time required for the protocol varies depending on the number of channels sampled and ranges from 3 to 5 d plus an additional 2 d to completely wash embryos and prepare for documentation.

Collectrin/tmem27 is expressed at high levels in all segments of the developing Xenopus pronephric nephron and in the Wolffian duct.

Collectrin/tmem27 encodes a transmembrane protein that plays a critical role in amino-acid transport. Originally described as being expressed only in collecting ducts, it has subsequently also been shown to also be expressed in the S1 segment of the proximal tubule of mammalian metanephric nephrons. In this report we describe the expression of collectrin in the simple embryonic kidney of amphibians, the pronephros. Each pronephros contains a single large nephron with a proximo-distal segmentation very similar to that of mammalian metanephric nephrons. Analysis of collectrin expression in pronephroi at a variety of embryonic stages indicates that this gene is expressed at very high levels throughout the pronephric system, including proximal and distal segments and the Wolffian duct. Expression in the pronephros commences at Xenopus embryonic stage 28 which corresponds to when epithelialization begins within the pronephric mesenchyme. Like the Na+K+ATPase/atp1a1, another highly expressed pronephric marker, collectrin is also expressed in the cloaca but not in the cloacal derived posterior segment of the Wolffian duct, the rectal diverticulum. Unlike the Na+K+ATPase, which is expressed at lower levels in proximal portions of the pronephric nephron, expression of collectrin is even throughout all of the pronephric epithelia. This expression domain extends far beyond that shown to express amino-acid transporters and indicates collectrin may function in facilitating additional transport processes. Its high level of expression and broad distribution make it an excellent marker with which to examine pronephric kidney development.