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In the vertebrate embryo, development of the excretory system is characterized by the successive formation of three distinct kidneys: the pronephros, mesonephros, and metanephros. While tubulogenesis in the metanephric kidney is critically dependent on the signaling molecule Wnt-4, it is unknown whether Wnt signaling is equally required for the formation of renal epithelia in the other embryonic kidney forms. We therefore investigated the expression of Wnt genes during the pronephric kidney development in Xenopus. Wnt4 was found to be associated with developing pronephric tubules, but was absent from the pronephric duct. Onset of pronephric Wnt-4 expression coincided with mesenchyme-to-epithelium transformation. To investigate Wnt-4 gene function, we performed gain- and loss-of-function experiments. Misexpression of Wnt4 in the intermediate and lateralmesoderm caused abnormal morphogenesis of the pronephric tubules, but was not sufficient to initiate ectopic tubule formation. We used a morpholino antisense oligonucleotide-based gene knockdown strategy to disrupt Wnt-4 gene function. Xenopus embryos injected with antisense Wnt-4 morpholinos developed normally, but marker gene and morphological analysis revealed a complete absence of pronephric tubules. Pronephric duct development was largely unaffected, indicating that ductogenesis may occur normally in the absence of pronephric tubules. Our results show that, as in the metanephric kidney, Wnt-4 is critically required for tubulogenesis in the pronephric kidney, indicating that a common, evolutionary conserved gene regulatory network may control tubulogenesis in different vertebrate excretory organs.
FIG. 1. Wnt-4 is expressed in the pronephric anlage and pronephric tubules. Wnt-4 transcripts were detected by whole-mount in situ hybridization. (A, F) Lateral views of Xenopus embryos with anterior to the left. (A) Wnt-4 expression in the pronephric anlage (arrowhead) of a stage 19 embryo. Expression is also present in the brain (b) and neural tube (nt). (B) At stage 24, Wnt-4 expression is prominently detected in the pronephric tubule anlage (arrowhead) and the ventralblood islands (bi). (C) At stage 32, Wnt-4 expression was associated with the pronephric tubules. Inset shows an enlargement of the pronephros with the three nephrostomes (arrowheads). Other regions with Wnt-4 expression include visceral arches (va), the neural tube, and discrete sections of the mid- and hindbrain (mb, hb). (D, E) Sections of stage 24 (D) and 32 (E) embryos hybridized in whole mount with Wnt-4 antisense probes. Embryos were embedded in agarose and transverse sections (30 ﰂm) were cut at the level of the developing pronephric kidney. Sections are oriented with dorsal to the top. (D) Wnt-4 expression was detected in the pronephric anlage (pa) and neural tube (nt). The inset shows an enlargement of Wnt-4 expression in the somatic layer (so) of the intermediate mesoderm. The splanchnic layer (sp) and endoderm (ed) are devoid of any expression. (E) Wnt-4 expression is associated with the developing pronephric tubules (pt) and the floor plate (fp). The inset shows a close-up view of Wnt-4 expression in the pronephric tubules. (F) A stage 32 embryo stained for Pax-2 expression to visualize the pronephric kidney with tubules (arrowheads) and duct (pd) is shown for comparative purposes. Scale bars: 600 ﰂm (A, B, C, F); 200 ﰂm (inset in C); 140 ﰂm (D, E); 40 ﰂm (insets in D, E).
FIG. 2. Overexpression of Wnt-4 affects morphogenesis of pronephric tubules. Single V2 blastomeres of eight-cell-stage embryos were coinjected with 0.25 ng of Wnt-4 mRNA and the lineage tracer nuclear -galactosidase (nucgal, 0.1 ng). Embryos were fixed at stage 33/34, processed for gal activity, and hybridized with a LIM-1 antisense probe. (A) Control side of a Wnt-4-injected embryo. (B) Enlargement of the area bordered in (A). Note the characteristic expression of LIM-1 in the nephrostomal sections of the pronephric tubules (arrowheads)
FIG. 3. Inhibition of Xenopus Wnt-4 translation in vitro by Wnt-4 antisense morpholinos. Plasmids (50 ng) encoding Wnt-4, HAWnt-4, and Wnt-1 were used as templates in cell-free coupled transcriptionranslation reactions. Several morpholinos [Wnt4-MO; sense control, Wnt4(sc)-MO; four-mispaired control, Wnt4(mp)-MO; and a standard control, Cont-MO] were tested for inhibition of translation. Cell-free transcriptionranslation reactions were performed in the presence of [35S]methionine and analyzed by SDSAGE/autoradiography. (A) Dose-response analysis of inhibition of Wnt-4 translation by Wnt4-MO. The amount of radiolabeled protein produced was quantified by using a phosphorimager. The control sample, which was not treated with Wnt4-MO, was set at 100%. Significant reduction of protein synthesis occurred with 1 and 3 ��M Wnt4-MO. (B) Sequence-specific inhibition of Wnt-4 translation by Wnt4-MO. Morpholinos (1 ��M) were used to inhibit translation of Wnt-4. Only Wnt4-MO was effective in blocking Wnt4 translation. (C, D) In vitro translation of HAWnt4 (an HA-tagged Wnt-4 variant lacking the complementary target sequence for Wnt4-MO) and Xenopus Wnt-1 was essentially unperturbed in the presence of Wnt4-MO. Morpholinos were used at 1 uM.
FIG. 4. In vivo suppression of the Wnt-4 overexpression phenotype by Wnt-4 antisense morpholinos. Both blastomeres at the two-cell stage were injected with either 0.25 ng Wnt-4 mRNA alone or Wnt-4 mRNA and Wnt4-MO (0.25 ng each). RNA encoding cytoplasmic -galactosidase mRNA (cytgal) was coinjected as a lineage tracer. Embryos were fixed at stage 33/34, processed for gal activity, and classified according to their phenotype. (A) Injection of Wnt-4 mRNA frequently causes a shortened body-axis phenotype. In the experiment shown, 31% (n 25) of the embryos were normal or mildly affected. Moderate phenotypes were observed in 64% (n 52) and severe truncations were found with 5% (n 4) of the embryos. Examples of embryos with phenotypes ranging from mild to severe are shown. (B) Coinjection of Wnt4-MO rescues Wnt-4-injected embryos. Out of the injected embryos (n 41), 85% developed with no discernable phenotype. Examples of rescued embryos are shown. A mild shortened body-axis phenotype was observed with 15% of the embryos. Scale bars: 1.1 mm (A); 1 mm (B)
FIG. 5. Down-regulation of pronephric tubule-specific gene expression caused by injection of Wnt4-MO. Coinjections of 5 ng morpholinos [Wnt4-MO: B, F, J, N; or Wnt4(mp)-MO: D, H, L, P] and the lineage tracer cytBgal (0.1 ng) were performed into single V2 blastomeres at the eight-cell stage. Injected embryos were fixed at stages 29/30 (A) or 33/34 (E) and processed for Beta-gal activity. Expression of various marker genes was visualized by whole-mount in situ hybridization. Embryos with control and injected sides, respectively, are shown accompanied by enlargements of the pronephric region (a). (A) Injection of Wnt4-MO resulted in specific down-regulation of nephrostomal Wnt-4 expression (arrowheads), whereas injection of Wnt4(mp)-MO did not affect Wnt-4 expression. (E) Absence of discernable tubular Pax-2 expression (arrowheads) was observed in Wnt4-MO, but not in Wnt4(mp)-MO injected embryos. Expression of Pax-2 in the pronephric duct (pd) was largely unaffected by injection of either MO. (I) Pronephric expression of CLC-K is essentially normal in MO-injected embryos. Reduced coiling of the anterior duct portion (arrowhead), however, is observed with Wnt4-MO-injected embryos. (M) Injection of Wnt4-MO, but not Wnt4(mp)-MO, abolishes SGLT-1L expression (arrowhead). Scale bars: 400 um (A); 120 um (a).
Fig. 6. Coinjection of HAWnt-4 rescues SGLT-1L expression in Wnt4-MO-treated Xenopus embryos. Single V2 blastomeres of eight-cell stage embryos were injected with 5 ng Wnt4-MO alone (A) or in conjunction with 0.25 ng HAWnt-4 mRNA (B, C). Cytoplasmic beta-galactosidase mRNA (cytBgal; 0.1 ng) was coinjected as a lineage tracer. Tailbud embryos (stage 33/34) were processed for Beta-gal activity and hybridized with antisense SGLT-1L probes. (A) Injection of 5 ng Wnt4-MO abolishes pronephric expression of SGLT-1L. The enlargement of the pronephric tubule region shows the presence of Beta-gal-expressing cells (pale blue), but absence of SGLT-1L transcripts. (B) Control side of an embryo coinjected with Wnt4-MO and HAWnt-4 mRNA. The inset illustrates normal SGLT-1L expression in the pronephros. (C) Injected side of the embryo shown in (B). The enlargement of the bordered area reveals substantial expression of SGLT-1L in the pronephros. Note that Beta-gal-expressing cells (arrowhead) are adjacent or colocalize with SGLT-1L-expressing cells (arrow). Scale bars: 600 um (A); 120 um (insets).
FIG. 7. Disruption of pronephric tubule formation by Wnt4-MO injection. Single V2 blastomeres of eight-cell-stage embryos were injected either with 5 ng Wnt4-MO (A) or Wnt4(mp)-MO (B). cytBgal mRNA (0.1 ng) was coinjected to allow identification of the MO-injected side. Embryos were fixed at stage 33/34, processed for cytBgal activity, and hybridized for SGLT-1L expression. Transverse sections (A, 1.5 um; B, 3 um) of plastic-embedded embryos were cut at the level of the pronephros. Selected sections counterstained with Toluidine Blue and basic fuchsine (A, B) are shown with enlargements of the pronephric tubule region of control (A', B') and injected (A", B") sides. (A', B') Well-developed pronephric tubule epithelia (arrowheads) were present on the control sides. (A") In contrast, no pronephric tubules could be detected when Wnt4-MO was injected. An asterisk indicates the position where tubules would normally form. (B") Injection of Wnt4(mp)-MO did not affect development of pronephric tubules (arrowheads). Scale bars: 250 um (A, B); 75 um (A', A", B', B").
