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The RING finger ubiquitin ligase seven in absentia homolog 2 (Siah2) was identified in the R7 photoreceptor cells of Drosophila melanogaster, and it regulates the stability of prolyl hydroxylase domains (PHDs), with a concomitant effect on HIF-1α availability in the hypoxia response pathway. We previously reported that the hypoxia response pathway contributes to eye development during the embryonic development of Xenopus laevis. In this paper, the role of Siah2-mediated hypoxia response pathway in eye development of X. laevis embryos was further characterized. Xenopus Siah2 (xSiah2) mRNA was detected in lenstissue and xSiah2 overexpression caused a thickened lens placode, leading to loss of the optic lens. In embryos overexpressing xSiah2, lens marker gene transcription was reduced, suggesting that xSiah2 contributes to lens formation. xSiah2 overexpression decreased Xenopus PHD accumulation and increased Xenopus HIF-1α (xHIF-1α) accumulation. xHIF-1α degeneration with resveratrol restored the optical abnormality caused by xSiah2 overexpression, suggesting that the xSiah2-mediated hypoxia response pathway contributes to lens formation. Moreover, xSiah2 overexpression decreased endothelial-mesenchymal transition (EMT)-related Notch signaling-responsive genes transcription during the invasion of the lens placode. Our results suggest that the hypoxia response pathway plays an important role in the regulation of the EMT via the Notch signaling pathway during lens formation.
Fig. 1. Accumulation of xSiah2 mRNA during the development of Xenopus laevis. (A–G) The accumulation pattern of xSiah2 mRNA during tailbud stages was investigated by whole mount in situ hybridization. Images (A–C) show anterior views of the head of albino embryos hybridized with the sense probe at st. 24, 30, and 38, respectively. Images (D–F) show anterior views of the head of albino embryos hybridized with the antisense probe at st. 24, 30, and 38, respectively. Arrows indicate the lenstissue. Image (G) shows a single eye surgically detached from the embryo and hybridized with the antisense probe at st. 38.
Fig. 2. Effects of xSiah2 mRNA injection on the accumulation of xSiah2, xPHD, and xHIF-1α, eye phenotype, and lens marker transcription. (A) In C1 embryos, synthesized mRNA was not injected; in C2 embryos, GFP mRNA was injected into both dorsal blastomeres at the two-cell stage; in S1 embryos, GFP mRNA was injected into one dorsal blastomere, and Siah2 mRNA into another dorsal blastomere at the two-cell stage; and in S2 embryos, xSiah2 mRNA was injected into both dorsal blastomeres at the two-cell stage. The expression levels of xSiah2, xPHD, and xHIF-1α at st. 30 and 38 were investigated in these embryos by western blotting. The values in the graphs represent the mean ± SD of three experiments. *p < 0.05, **p < 0.01 vs. C2 embryos (B) GFP or xSiah2 mRNA was injected into each dorsal blastomere at the two-cell stage. Comparison of anterior views of the head between the sides of the same embryo injected with GFP mRNA (GFP) and xSiah2 mRNA (Siah2) at st. 30. Arrow indicates the thickened lens ectoderm. Histological difference between the sides of the same embryo injected with GFP mRNA (GFP) and xSiah2 mRNA (Siah2) at st. 38. (C) GFP (GFP) or xSiah2 mRNA (Siah2) was injected into both dorsal blastomeres at the two-cell stage. The transcriptional levels of FoxE3 and β-crystallin at st. 24, 30, and 38 were investigated in these embryos by RT-PCR.
Fig. 3. Effect of resveratrol on the small eye phenotype. (A and B) In the C1 treatment group, GFP mRNA was injected into both dorsal blastomeres at the two-cell stage. In the S1, R1, and R2 treatment groups, xSiah2 mRNA was injected into both dorsal blastomeres at the two-cell stage. Embryos were exposed to resveratrol (40 μM) from st. 12 to 38 in R1 treatment group and from st. 22 to 38 in the R2 treatment group. (A) The expression levels of xHIF-1α at st. 30 and 38 were investigated in these embryos by western blotting. The values in the graph represent the mean ± SD of three experiments. *p < 0.05 vs. S1 treatment group. (B) Percentages of embryos with optical malformations in each treatment group at st. 38. N.D., not detected.
Fig. 4. Effect of xSiah2 mRNA injection on transcriptional levels of Notch signaling-responsive genes. (A and B) GFP or xSiah2 mRNA was injected into both dorsal blastomeres at the two-cell stage. (A) The transcriptional levels of Snail-1 and N-cadherin mRNAs at st. 30 were investigated in the embryos by RT-PCR. Black and white bars indicate the transcriptional levels in GFP mRNA injected embryos (G) and xSiah2 mRNA injected embryos (S), respectively. The values in the graphs represent the means ± SD of three experiments. *p < 0.05 vs. GFP mRNA-injected embryos (B) The transcriptional levels of ESR-1 mRNA at st. 20, 24, 30, and 38 were investigated by RT-PCR. Closed and open circles indicate the transcriptional levels of ESR-1 mRNA in GFP mRNA-injected embryos and xSiah2 mRNA-injected embryos, respectively. The values in the graph represent the means ± SD of three experiments. *p < 0.05, **p < 0.01 vs. GFP mRNA-injected embryos.
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