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Fig. 6. Sipa1l3 and Epha4 influence early eye specification. (A,C) WMISH at stage 13 revealed that Sipa1l3 (A) or Epha4 (C) function is required for proper rax and pax6 expression whereas sox3 is not affected. Red arrowheads indicate reduced marker gene expression at the injected side. Scale bars: 1000 µm. (B,D) Quantification of the data in A,C. (E,G) Knockdown of Sipa1l3 (E) and Epha4 (G) resulted in significantly reduced rax and pax6 expression domains (red arrowheads) compared with internal control as well as control MO-injected embryos at stage 23. Scale bars: 500 µm in overview; 250 µm in close-up views. (F,H) Quantification of the data in E,G. (I) rax RNA restores the Sipa1l3 MO-induced ocular phenotype. Red circles indicate eye areas. Scale bars: 500 µm in dorsal, lateral views; 250 µm in detail views. (J) Quantification of the data in I. (K) rax RNA rescues the Sipa1l3 MO-induced microphthalmia phenotype. (L) rax RNA restores the Epha4 MO-induced ocular phenotype. Red circles indicate eye areas. Scale bars: 500 µm in dorsal, lateral views; 250 µm in detail views. (M) Quantification of the data in L. (N) rax RNA rescues the Epha4 MO-induced microphthalmia phenotype. N, number of analyzed embryos in total; n, number of independent experiments. Error bars indicate standard error of the means (s.e.m.); *P≤0.05; **P≤0.01. P-values were calculated by a nonparametric, one-tailed Mann–Whitney rank sum test. |
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Fig. 7. Epha4 and Sipa1l3 act through non-canonical Wnt signaling. (A) Loss of Sipa1l3 function (red arrowhead) is rescued by co-injecting dnLEF RNA at stage 42. Red circles indicate eye areas. (B) Quantification of the data in A. (C) Injection of dnLEF RNA restores the microphthalmia phenotype induced by Sipa1l3 downregulation. (D) Marker gene reduction (red arrowhead) at stage 23 upon loss of Sipa1l3 is rescued by dnLEF RNA. Scale bar: 200 µm. (E) Quantification of the data in D. (F) axin2 is upregulated upon Sipa1l3 or Epha4 depletion as shown by qPCR using cDNA of Xenopus neuralized ACs at stage 13. (G) Split YFP complementation assay. The PDZ domain of rat Sipa1l3 interacts with Xenopus Dsh. For negative controls, the interaction with unrelated proteins (CapZa and Pes1) was analyzed. (H) Loss of Sipa1l3 (red arrowhead) is rescued by dshδdix RNA co-injection. Red circles indicate eye areas. Scale bar: 1000 µm in dorsal and lateral view; 200 µm in detail view. (I) Quantification of the data in H. (J) Injection of dshδdix RNA restores the microphthalmia phenotype induced by Sipa1l3 downregulation. (K) Loss of Epha4 function (red arrowhead) is rescued by dshδdix RNA co-injection. Red circles indicate lens areas. Scale bar: 1000 µm in dorsal and lateral view; 200 µm in detail view. (L) Quantification of the data in K. (M) Injection of dshδdix RNA restores the microphthalmia phenotype induced by Epha4 depletion. (N) Scheme of the predicted mechanism in the wild-type situation. Interaction of Epha4 and Sipa1l3 leads to normal ocular development by blocking Wnt/β-catenin signaling and activation of the non-canonical Wnt pathway. (O) Scheme of the predicted mechanism in the Sipa1l3 loss-of-function situation. Sipa1l3 deficiency results in eye defects by upregulation of β-catenin and axin2. N, number of analyzed embryos in total; n, number of independent experiments; ng, nanogram. Error bars indicate standard error of the means (s.e.m.); *P≤0.05; **P≤0.01. P-values were calculated by a nonparametric, one-tailed Mann–Whitney rank sum test. |
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Figure S2: Retinal lamination is affected upon Sipa1l3 and Epha4 loss of function in Xenopus.
Sipa1l3 as well as Epha4 depletion results in disorganized retinal layers in the eye including rosette-like structures formed by displaced photoreceptor cells from the outer layer (black arrowheads) to the inner layers (red arrowheads) of the retina. Following marker genes were used to stain for retinal cell types at stage 42: arr3 and rho to determine photoreceptor cells; pax6 to detect amacrine and ganglion cells; vsx1 to visualize bipolar cells and pou4f1 to stain for ganglion cells. B qPCR experiments using cDNA of Sipa1l3 or Epha4 depleted stage 42 eyes revealed a down-regulation of rho, whereas the other analyzed marker genes were slightly up-regulated. CoMO injected embryos were used as control. Gapdh was used for normalization. n, number of independent experiments; ng, nanogram. Error bars indicate standard error of the means (s.e.m.); * p0.05; ** p0.01. P-values were calculated by a nonparametric, one-tailed Mann-Whitney rank sum test. |
