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Fig. 2. Sipa1l3 is required for Xenopus ocular development. (A) Loss of Sipa1l3 through injection with Sipa1l3 MO leads to smaller and deformed eyes (white arrowheads) at stage 42, including a disturbed RPE (red arrowheads) in comparison with uninjected side and control MO (CoMO) injected embryos. Scale bars: 500 µm in dorsal views; 1000 µm in ventral views; 100 µm in detail and section views. (B) Quantification of data in A reveals an abnormal eye phenotype in a dose-dependent manner upon Sipa1l3 deficiency (dark gray columns). The Sipa1l3 MO phenotype was rescued by co-injecting a full-length rat sipa1l3 RNA (black column) but not a mutated rat sipa1l3 R1491* RNA (red column). The sipa1l3 R1491* construct reflects the nonsense point mutations identified in human patients (Evers et al., 2015). (C) Measurement of the eye area size at stage 42. Red circles indicate the eye areas. Scale bars: 1000 µm in upper row; 200 µm in lower row. (D) Quantification of the data in C revealed a significant reduction in eye size upon Sipa1l3 depletion compared with control. Wild-type rat sipa1l3 but not sipa1l3 R1491* RNA restores the microphthalmia phenotype. (E) Lens area measurement of cryaa-stained embryos at stage 36 upon loss of Sipa1l3 compared with uninjected side and control MO-injected embryos. Note that the cryaa staining is not absent. Red circles indicate the lens area. Scale bar: 250 µm. (F) Quantification of the data in E revealed a significant reduction in lens size upon Sipa1l3 depletion. (G) Sipa1l3 MO injection does not reduce the expression of celf1 and cryba1 (black arrowheads). Scale bars: 100 µm in upper row; 50 µm in lower row. (H) DAPI staining on cryosections revealed nuclei in the lens center upon loss of Sipa1l3 (injected side, white arrowhead) whereas the uninjected side shows no nuclei in the lens center. The dotted line indicates the lenses. Scale bar: 50 µm. RPE, retinal pigmented epithelium; 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≤0.001; ****P≤0.0001. P-values were calculated by a nonparametric, one-tailed Mann–Whitney rank sum test. |
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Fig. 4. Loss of Epha4 phenocopies the loss of Sipa1l3 in Xenopus.
(A) Loss of Epha4 through injection with Epha4 MO phenocopies the eye phenotype upon Sipa1l3 deficiency including smaller and deformed eyes (white arrowheads) with disturbed RPE (red arrowheads) in comparison with uninjected side and control MO (CoMO).
(B) Quantification of the data shown in A. The abnormal eye phenotype is rescued by epha4 RNA co-injection (black column).
(C) Measurement of eye area size at stage 42. Red circles indicate eye areas. Scale bars: 1000 µm in upper row; 200 µm in lower row.
(D) Quantification of the data in C revealed a significant reduction in eye size upon Epha4 depletion. Epha4 RNA restores the microphthalmia phenotype upon Epha4 depletion.
(E) Lens area measurement of cryaa-stained embryos at stage 36 upon loss of Epha4 compared with uninjected side and control MO-injected embryos. Red circles indicate lens areas. Scale bar: 250 µm. Note that cryaa staining is not absent.
(F) Quantification of the data in E revealed a significant reduction in lens size upon Epha4 depletion. (
G) Epha4 MO injection does not reduce celf1 and cryba1 expression (black arrowheads). Scale bars: 100 µm in upper row; 50 µm in lower row.
(H) DAPI staining on cryosections revealed nuclei in the lens after loss of Epha4 (injected side, white arrowhead), compared with internal control (uninjected side). The dotted lines indicate the lenses.
Scale bar: 50 µm. 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≤0.001. P-values were calculated by a nonparametric, one-tailed Mann–Whitney rank sum test. |
