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Vertebrate kidney organogenesis is characterised by the successive formation of the pronephros, the mesonephros and the metanephros. The pronephros is the first to form and is the functional embryonic kidney of lower vertebrates; although it is vestigial in higher vertebrates, it is a necessary precursor for the other kidney types. The Xenopus pronephros is a simple paired organ; each nephron consists of a single large glomus, one set of tubules and a single duct. The simple organisation of the pronephros and the amenability of Xenopus laevis embryos to manipulation make the Xenopus pronephros an attractive system in which to study organogenesis. It has been shown that pronephric tubules can be induced to form in presumptive ectodermal tissue by treatment with RA and activin. We have used this system in a subtractive hybridisation screen that resulted in the cloning of Xenopus laevis annexin IV (Xanx-4). Xanx-4 transcripts are specifically located to the developing pronephric tubules, and the protein to the luminal surface of these tubules. Temporal expression shows zygotic transcription is upregulated at the time of pronephric tubule specification and persists throughout pronephric development. The temporal and spatial expression pattern of Xanx-4 suggests it may have a role in pronephric tubule development. Overexpression of Xanx-4 yields no apparent phenotype, but Xanx-4 depletion, using morpholinos, produces a shortened, enlarged tubule phenotype. The phenotype observed can be rescued by co-injection of Xanx-4 mRNA. Although the function of annexins is not yet clear, studies have suggested a role for annexins in a number of cellular processes. Annexin IV has been shown to have an inhibitory role in the regulation of epithelial calcium-activated chloride ion conductance. The enlarged pronephric tubule phenotype observed may be attributed to incorrect modulation of exocytosis, membrane plasticity or ion channels and/or water homeostasis. In this study, we demonstrate an in vivo role for annexin IV in the development of the pronephric tubules in Xenopus laevis.
Fig. 3. Xanx-4 mRNA is localised to the pronephric tubules. (A)Whole-mount (stage 36) and (B) section (stage 42) in situ hybridisation of an Xanx-4 DIG-labelled antisense RNA probe. Expression of Xanx-4 is restricted to the pronephric tubules. The control sense probe showed no staining pattern (data not shown).
Fig. 4. Xanx-4 protein is localised to the luminal surface of the pronephric tubules. Whole-mount (A) and section (B) stage 40 embryos stained with anti-Anx-4 antibody show that Xanx-4 protein is specifically localised to the pronephric tubules and in section to the apical surface of the pronephric tubule epithelium.
Fig. 7. Whole-mount antibody staining of stage 40 Xanx-4 MO-injected embryos identifies a tubule phenotype. One-cell stage Xenopus embryos were injected with 10 ng of Xanx-4 MO, cultured to stage 40 and subjected to whole-mount antibody staining with pronephric tubule specific antibody 3G8. (A) Normal uninjected control embryo. (B) Embryo injected with Xanx-4 MO shows shortened, enlarged tubule phenotype.
Fig. 11. Analysis of the effects of Xanx-4 depletion or overexpression on the expression of pronephric marker genes by in situ hybridisation. Embryos were injected at the one-cell stage with 0.5 ng Xanx-4 mRNA or 10 ng Xanx-4 MO and cultured to stage 27. The embryos were then subjected to in situ hybridisation using specific probes prepared from pronephric molecular markers Xlim-1 (A,D,G), XPax-8 (B,E,H) and xWT1 (C,F,I). No effect on expression of XPax-8 or Xlim-1 was observed; however, a reduced field of xWT1 expression was observed in the embryos injected with Xanx-4 mRNA (compare C with F).
Fig. 8. Cryostat transverse sections of stage 40 Xenopus pronephroi stained with tubule-specific antibody 3G8 and counterstained with Hoechst. One-cell stage embryos were injected with 10 ng Xanx-4 MO, 10 ng Xanx-4 MO and 0.5 ng Xanx-4 mRNA, cultured to stage 40 and subjected to whole-mount antibody 3G8 staining. The embryos were acrylamide embedded, cryostat sectioned at 12 μm and lifted on to subbed slides. The slides were counterstained with Hoechst, inspected under light and u.v. illumination. (A) A schematic representation of B showing pronephric tubule sections illustrating true transverse sections, which were scored (filled) and partial longitudinal sections which were not counted (unfilled). (C,D) Normal uninjected control embryo. (E,F) Embryo injected with 10 ng Xanx-4 MO showing enlarged pronephric tubule phenotype.
(G,H) embryo injected with 0.5 ng Xanx-4 mRNA and 10 ng Xanx-4 MO showing partial rescue of pronephric tubule phenotype. C,E,G are viewed under UV illumination to identify Hoechst nuclei staining. B,D,F,H are viewed under partial white light and u.v. illumination to identify 3G8 pronephric tubule staining and Hoechst nuclei staining.
Fig. 4. Xanx-4 protein is localised to the luminal surface of the pronephric tubules. Whole-mount (A) and section (B) stage 40 embryos stained with anti-Anx-4 antibody show that Xanx-4 protein is specifically localised to the pronephric tubules and in section to the apical surface of the pronephric tubule epithelium.
