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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.
Figure 1. Dominant-negative dvl2 generates Wolffian ducts that are abnormally shaped, wider, and contain more cells than control ducts. Staining details for each technique are available in the Experimental Procedures section. A: Control embryo stained for proximal tubules in green (3G8) and the Wolffian duct in red (4A6). B: Lateral view of control stained for proximal tubules with ECL (green) and 4A6 (red). C: Dvl2-D2 mRNA-injected pronephros. The Wolffian duct is an abnormal serpentine shape. D: Confocal maximal projection of a control duct, stained with 4A6 (red) to highlight the pronephros and counterstained with sytox (green) for nuclei. E: Confocal maximal projection of dvl2-D2 mRNA-injected pronephros, stained as described in D. The duct is wider and has more cells than controls. F,G: Control and dvl2-D2 mRNA-injected ducts stained with 4A6 (red) and sytox (green) then viewed in optical transverse sections. The dvl2-D2 duct contains more cells, but both have an open lumen and normal epithelialization. pn, pronephric nephronproximal domain; pd, pronephric distal tubule and duct; cl, cloaca. Panels A through E show lateral views, while F and G illustrate transverse views. Scale bar = 40 microns in D,E, 20 microns in F,G.
Figure 2. Microinjection of dvl2-D2 mRNA increases the number of phospho-H3 positive cells in the pronephros. Embryos were injected with dvl2-D2 plus membrane bound green fluorescent protein (GFP) mRNAs at the eight-cell stage, grown to stage 21/22, then fixed. Fluorescent in situ hybridization (FISH) was performed in the green channel to detect the pronephros (panels A and B), tyramide enhanced immunochemistry in the red channel to detect phospho-H3 (panels C and D), and tyramide enhanced immunochemistry to detect GFP in the far red channel (shown in blue). A,C,E,G: The left column shows different channels (A,C,E) of a control sample with a merge of these presented in panel G. The right column shows a similar data set for a dvl2-D2 plus GFP mRNA-injected pronephros. F,H: Due to the sequence of development some of the phospho-H3 signal also shows up in panels E and F, but is double stained and appears as magenta in panels G and H allowing the clear distinction of phospho-H3 and GFP.
Figure 3. Inhibiting non-canonical wnt signals or promoting canonical wnt signals leads to increases in cell division, while promoting non-canonical or suppressing canonical signals results in a decrease in division rates. Controls are shown on the left, and treated embryos on the right. The pronephros is stained green by means of fluorescent in situ hybridization (FISH), dividing cells stained red by means of anti-phospho-H3, and mRNA-injected samples identified by blue green fluorescent protein (GFP) staining in all panels. Three-dimensional scanning determined which phospho-H3 positive cells were within pronephroi in each case (Table 2). A–F: Images show contralateral sides of the same embryos. G,H: Representative images with average division rates were selected as both sides of each embryo received the same treatment. Scale bars = 100 microns.
Stage 40 Xenopus laevis pronephros stained for kidney epithelia in green, and with the h3f3a antibody 1 phospho ser 10 in red to identify dividing cells
Supplemental image: does not appear in publication. GFP staining appears blue. This embryo was injected with an mRNA encoding a membrane targeted GFP. Following fluorescent in situ hybridization to detect the kidneyprimordium (green), and phospho-h3f3 antibody 1 to detect mitotic cells (magenta), the embryo was stained with invitrogen A-31852 to detect GFP derived from the injected mRNA tracer. This antibody is coupled to Alexa 647, is visualized directly, and shown in the blue channel
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