QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Seville, R. A. Articles by Jones, E. A. Search for Related Content PubMed PubMed Citation Articles by Seville, R. A. Articles by Jones, E. A.

Annexin IV (Xanx-4) has a functional role in the formation of pronephric tubules

Cell and Molecular Development Group, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK

View larger version (1K): [in a new window]   Fig. 1. Amino acid sequence and motif diagram of Xanx-4A. (A) Predicted amino acid sequence of Xanx-4 compared with human (72%), rat (72%), mouse (71%), cow (74%) and medaka (67%). ‘.’ indicate identical amino acids. (B) Xanx-4 motif diagram showing N-terminal PKC phosphorylation site (P), myristoylation site (m) and C-terminal core domain of four annexin repeats (shaded boxes). which contain calcium-binding sites.

View larger version (29K): [in a new window]   Fig. 2. Temporal expression profile of Xanx-4; RT-PCR analysis showing the expression pattern of Xanx-4 transcripts in Xenopus laevis unfertilised egg and embryo stages. Maternal expression is detected in the egg that is much reduced by the 32-cell stage. Zygotic expression is detected at low levels between stages 9 and 12.5, and at a significantly increased level at stage 13, which is maintained through to stage 41.

View larger version (50K): [in a new window]   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).

View larger version (51K): [in a new window]   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.

View larger version (63K): [in a new window]   Fig. 5. Northern blot analysis of RNA isolated from dissected adult organs. (A) Xanx-4 transcripts were detected at high levels in the gall bladder and intestine, and at lower levels in tadpole, lung, kidney, ovary, testis, stomach, bladder, spleen and pancreas. No transcripts were detected in neural tissues, heart, liver, muscle or skin. (B) Ethidium bromide stained agarose gel showing RNA loading for different adult tissue types prior to transfer.

View larger version (55K): [in a new window]   Fig. 6. (A) Xanx-4 MO specificity in vitro; western blot analysis showing that Xanx-4 MO interferes with in vitro translation of Myc-tagged Xanx-4 mRNA (Myc-Xanx-4) and not of Myc-tagged Xsox17ß mRNA (Myc-Xsox17ß). Lane 1: 10 ng Myc-Xanx-4 and Myc-Xsox17ß mRNA. Lane 2: 10 ng Myc-Xanx-4 and Myc-Xsox17ß mRNA and 1 µg Xanx-4 MO. Lane 3: 10 ng Myc-Xanx-4 and Myc-Xsox17ß mRNA. Lane 4: 10 ng Myc-Xanx-4 and Myc-Xsox17ß mRNA, and 5 µg Xanx-4 MO. Lane 5: 10 ng Myc-Xanx-4 and Myc-Xsox17ß mRNA. Lane 6: 10 ng Myc-Xanx-4 and Myc-Xsox17ß mRNA and 10 µg Xanx-4 MO. Lane 7: no input mRNA or MO. (B) Xanx-4 MO specificity in vivo; western blot analysis using anti-Myc antibody showing that the Xanx-4 MO interferes with in vivo translation in Xenopus embryos of Myc-tagged Xanx-4 mRNA (Myc-Xanx-4), lanes 2-4, and not of Myc-tagged Xsox17ß mRNA (Myc-Xsox17ß), lane 8. One-cell stage Xenopus embryos were injected with Myc-Xanx-4 mRNA, Myc-Xanx-4 mRNA and Xanx-4 MO, Myc-Xsox17ß mRNA, Myc-Xsox17ß mRNA and Xsox17ß MO, and cultured to stage 9, 13 and 21. Lane 1: 0.5 ng Myc-Xanx-4 mRNA. Lane 2: 0.5 ng Myc-Xanx-4 mRNA and 5 ng Xanx-4 MO. Lane 3: 0.5 ng Myc-Xanx-4 mRNA and 10 ng Xanx-4 MO. Lane 4: 0.5 ng Myc-Xanx-4 mRNA and 20 ng Xanx-4 MO. Lane 5: uninjected control. Lane 6: 0.5 ng Myc-Xsox17ß mRNA. Lane 7: 0.5 ng Myc-Xsox17ß mRNA and 10 ng Xsox17ß MO. Lane 8: 0.5 ng Myc-Xsox17ß mRNA and 10 ng Xanx-4 MO.

View larger version (49K): [in a new window]   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.

View larger version (77K): [in a new window]   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.

View larger version (19K): [in a new window]   Fig. 9. Graphical representation of data collected from cell counts derived from serial sections of pronephric tubules from uninjected control embryos and embryos injected with Xanx-4 MO and/or Xanx-4 mRNA. (A) One-cell stage embryos were injected with 10 ng control MO (control M), 10 ng Xanx-4 MO (10M), 0.5 ng Xanx-4 mRNA (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 and cell counts per pronephric tubule were obtained. The mean number of cells per tubules section in the control, control MO and Xanx-4 mRNA injected embryos were not significantly different, whereas those embryos injected with 10 ng Xanx-4 MO displayed an enlarged pronephric tubule phenotype. (B) The enlarged pronephric tubule phenotype produced by injection of Xanx-4 MO can be rescued by co-injection with Xanx-4 mRNA. One-cell stage embryos were injected with 5 ng Xanx-4 MO (5M), 5 ng Xanx-4 MO and 0.5 ng Xanx-4 mRNA (5MR), 10 ng Xanx-4 MO (10M), 10 ng Xanx-4 MO and 0.5 ng Xanx-4 mRNA (10MR), 20 ng Xanx-4 MO (20M), 20 ng Xanx-4 MO and 0.5 ng Xanx-4 mRNA (20MR), cultured to stage 40 and analysed as before. The columns represent the arithmetic mean of each sample set of counted cross sections. Error bars indicate1 standard deviation either side of the mean. MO, morpholino; MR, morpholino and mRNA rescue.

View larger version (47K): [in a new window]   Fig. 10. 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 25. Groups of five embryos were then subjected to RT-PCR using primers designed against a range of pronephric molecular markers. No effect on expression of any of the markers was observed. EF1 was used as a loading control.

View larger version (50K): [in a new window]   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).