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BACKGROUND: Notch signaling in pronephros development has been shown to regulate establishment of glomus and proximal tubule, but how Notch signal works on competency of pronephric anlagen during the generation of pronephric components remains to be understood.
RESULTS: We investigated how components of pronephros (glomus, proximal tubule, intermediate tubule, distal tubule, and connecting tubule) were generated in Xenopus embryos by timed overactivation and suppression of Notch signaling. Notch activation resulted in expansion of the glomus and disruption of the proximal tubule formation. Inhibition of Notch signaling reduced expression of wt1 and XSMP-30. In addition, when Notch signaling was overactivated at stage 20 on, intermediate, distal, and connecting tubule markers, gremlin and clcnkb, were decreased while Notch down-regulation increased gremlin and clcnkb. Similar changes were observed with segmental markers, cldn19, cldn14, and rhcg on activation or inhibition of Notch. Although Notch did not affect the expression of pan-pronephric progenitor marker, pax2, its activation inhibited lumen formation in the pronephros.
CONCLUSIONS: Notch signal is essential for glomus and proximal tubule development and inhibition of Notch is critical for the differentiation of the intermediate, distal, and connecting tubule.
Figure 1. Gene markers used in this study and expression domain in pronephric compartment. To identify each part of Xenopus pronephros, gene markers shown above were used in this study. Definition of pronephric segment was shown according to Raciti et al. (Raciti et al., 2008).Download figure to PowerPoint
Figure 2. Notch signaling regulates glomus development. Either 2 ng of NICD+hGR (constitutive active form of Notch) or X-Su(H)1DBM+hGR (dominant-negative form of Notch) was injected into a ventrovegetal blastomere at eight-cell stage and then embryos were treated with dexamethasone at indicated stages. Whole-mount in situ hybridization was performed at st.35 to detect wt1 expression (dark brown). ß-galactosidase was used as a tracer (blue). Overexpression of Notch increased wt1 expression. Conversely, dominant-negative form of Notch decreased wt1.Download figure to PowerPoint
Figure 3. Notch regulates proximal tubule formation. Either 2 ng of NICD+hGR or X-Su(H)1DBM+hGR was injected as described in Figure 1 and then embryos were treated with dexamethasone at indicated stages. Whole-mount in situ hybridization was performed at st.35 to detect XSMP-30 expression (dark purple). ß-galactosidase was used as a tracer (blue). Overactivated Notch disrupted proximal tubule formation marked by XSMP-30. On the other hand, down-regulation of Notch repressed XSMP-30 expression.Download figure to PowerPoint
Figure 4. Overactive Notch decreased gremlin expression. Either 2 ng of NICD+hGR or X-Su(H)1DBM+hGR was injected as described in Figure 1 and then embryos were treated with dexamethasone at indicated stages. Whole-mount in situ hybridization was performed at st.35 to detect gremlin expression (dark brown or purple). ß-galactosidase was used as a tracer (blue). The length of the tubule of embryos injected with NICD+hGR was measured. Lu and Li represent the length of the tubule on uninjected side or injected side. Li/Lu indicates the ratio of length of the tubule. Li/Lu was correlated with the stage at which embryos were treated by dexamethasone.Download figure to PowerPoint
Figure 5. Notch down-regulates clcnkb expression. Either 2 ng of NICD+hGR or X-Su(H)1DBM+hGR was injected as described in Figure 1 and then embryos were treated with dexamethasone at indicated stages. Whole-mount in situ hybridization was performed at st.35 to detect clcnkb expression (dark purple). ß-galactosidase was used as a tracer (blue). Notch decreased clcnkb expression as gremlin expression in embryos injected with NICD+hGR. Conversely, dominant-negative form of Notch enhanced clcnkb expression.Download figure to PowerPoint
Figure 6. Repression of Notch signaling is necessary for differentiation of the intermediate, distal, and connecting tubule. Either 2 ng of NICD+hGR or X-Su(H)1DBM+hGR was injected as described in Figure 1 and then embryos were treated with dexamethasone at indicated stages. Whole-mount in situ hybridization was performed at st.35 to detect cldn19, cldn14, and rhcg expression (dark purple). ß-galactosidase was used as a tracer (blue). Overexpression of Notch led to down-regulation of cldn19 and cldn14 expression. rhcg expression was disappeared when Notch signal was activated. X-Su(H)1DBM+hGR augmented these segmental markers.Download figure to PowerPoint
Figure 7. Notch signaling does not affect pax2 expression in the intermediate, distal, and connecting tubule of Xenopus pronephros. Either 2 ng of NICD+hGR or X-Su(H)1DBM+hGR was injected as described in Figure 1 and then embryos were treated with dexamethasone at indicated stages. Whole-mount in situ hybridization was performed at st.35 to detect pax2 expression (dark purple). ß-galactosidase was used as a tracer (blue). Embryos injected with NICD+hGR and treated with dexamethasone at st.20 showed the expansion of pax2-positive cells around proximal tubule (red arrowhead). In other conditions, expression pattern of pax2 was not affected.Download figure to PowerPoint
Figure 8. Notch down-regulation is necessary for the formation of lumen structure. Histological analysis was performed to examine lumen structure. A: Embryos after whole-mount in situ hybridization with gremlin probe (as in Fig. 4). Two broken lines indicate the plane of section (B; anterior side) and (E; posterior side). B: Transverse section at the anterior side of gremlin-detected embryos. Arrow indicates NICD+hGR-injected side. Dark blue gremlin-positive cells are seen in both uninjected and injected side (red arrowheads, C (uninjected side) and D (injected side)). C: High magnification photomicrograph of uninjected side. The lumen structure was observed. D: High magnification photomicrograph of injected side. The gremlin-positive cells did not form lumen. E: Transverse section at the posterior side. Arrow indicates NICD+hGR-injected side as described above. Gremlin-positive cells were not seen in injected side (red arrowheads, F (uninjected side) and G (injected side). F: High magnification photomicrograph of uninjected side. Lumen was formed at the center of gremlin-positive cells. G: High magnification photomicrograph of injected side. Neither gremlin-positive cells nor lumen structure were observed. H–J: Hematoxylin and eosin staining of NICD + hGR-injected embryos. H: Transverse section of NICD + hGR-injected embryos. Arrow indicates NICD + hGR-injected side. Lumen formation was not observed in injected side (red arrowheads, I (uninjected side) and J (injected side). I: High magnification photomicrograph of uninjected side. The lumen formation was observed. J: High magnification photomicrograph of injected side. No lumen formation was seen in injected side. Scale bar = 1 mm in A; 0.1 mm in B,E,H; 10 µm in C,D,F,G,I,J.Download figure to PowerPoint
Figure 9. Notch activity regulates differentiation of pronephric compartments. Notch signal is required for glomus formation because Notch activation or inhibition leads to the increase or decrease of wt1 expression. Proper Notch activity is critical for normal differentiation of the proximal tubule. In the intermediate, distal, and connecting tubule formation, down-regulation of Notch is essential for functional maturation and lumen formation.Download figure to PowerPoint
