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Fig. 8. Xenopus Sp1 is modified by sumoylation and plays a significant role in eye development. (A-D) Sp1 expression in developing Xenopus at stage 28 (A) and stage 32/33 (B-D). Sp1 expression was analyzed by whole mount in situ hybridization (A and B) followed by sectioning (20 μm) (C, D). Sp1 expression is indicated by purple signal (A-D). White arrows indicate the eyes (A, B). Scale bar, 200 μm (C), 100 μm (D). (E) Structures of HA-tagged Sp1(1-723) and HA-tagged Sp1(17-723), which lacks the sumoylation target Lys (K16) residue. (F) Plasmid DNAs encoding each protein represented in (F) were introduced into 293T cells followed by SDS-PAGE and Western blot. (G) Structure of fusion protein, sumo-Sp1-GR. (H-J) Xenopus embryos injected with sumo-Sp1-GR RNA that were harvested at stage 37/38. DEX or DMSO treatment after the optic vesicle was formed (st.25-28) (H-J). Injected side is indicated by β-gal activity in blue (H, I). Differences in eye diameter between the injected side and the uninjected side were classified as indicated in the right side of (J) and the ratio of each class to the total number are indicated (J). (K-N) Embryos were injected with sumo-Sp1-GR RNA and harvested at stage 32/33 followed by in situ hybridization after collecting embryos which were injected at left side. DEX or DMSO treatment was applied after the optic vesicle was formed (st.25-28) (K-N). Same embryos are presented at different magnitudes (K, M) and (L, N). Yellow arrows indicate the location of the eyes (M, N). (O-S) Embryos were injected with synthetic RNAs, harvested and processed at stage 37/38 (O, P), stage 41 (Q-S). Injected RNAs are indicated below (O, P) or on the left side (Q, R) of each panel. DMSO or DEX treatment was applied around stage 23. DMSO-treated (O) and DEX-treated (P) embryos. Green arrow indicates reduced eye (P). Injected side is indicated by β-gal activity in blue (O, P). Embryos were labeled at stage 41 with BrdU for 1 h before harvest and subsequently immunohistochemistry was performed with anti-BrdU antibody with retinal sections (Q, R). Scale bar, 100 μm (Q). Yellow arrows indicate BrdU-incorporating cells outside the CMZ (R). Data are means ± s.e.m. (S). n = 3 eyes, 9 sections (DMSO), n = 3 eyes, 7 sections (DEX) (**P = 0.028, Student's t-test) (S). |
