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The small ubiquitin-related modifier (SUMO) is a member of the ubiquitin-like protein family, and SUMO conjugation (SUMOylation) resembles ubiquitination. Despite many SUMOylation target proteins being reported, the role of this system in vertebrate development remains unclear. We inhibited the function of Xenopus SUMO-1 (XSUMO-1) using a morpholino antisense oligo against XSUMO-1 (XSUMO-1-MO) to clarify the role of SUMOylation. XSUMO-1-MO inhibited normal axis formation in embryos and elongation of activin-treated animal caps. The expression of several mesoderm markers was reduced by XSUMO-1-MO. We measured activin-like activity by using a reporter construct containing a multimer of activin-responsive elements from the Goosecoid promoter, [DE(6x)Luc]. This assay showed that XSUMO-1-MO directly inhibited activin/nodal signaling. Furthermore, XSUMO-1-MO inhibited ectopic axis formation induced by XSmad2, and XSmad2/4 mRNA could not rescue the axis elongation defect induced by XSUMO-1-MO. These results suggested that XSUMO-1 is required for normal axis elongation, at least partly mediating activin/nodal signaling.
Figure 4. Suppression of endogenous expression of Xbra by XSUMO-1-MO. A-F: Embryos were coinjected with indicated RNA or MO and lacZ (lineage tracer) into the dorsal marginal zone at the 8-cell stage. No injection: Nothing (A), MO + mRNA: coinjected with 40 ng XSUMO-1-MO and 1 ng myc-XSUMO-1 mRNA (D and F), and 40 ng Control MO or XSUMO-1-MO (B, C, and E) was injected. Red and blue indicate the injected area and Xbra-expressing area, respectively. Xbra expression was inhibited in the XSUMO-1-MO-injected area (C and E, arrow). This inhibition was rescued by coinjection with myc-XSUMO-1 mRNA (D and F). A-D: Dorsal is up, vegetal pole is front. E and F: Animal pole is up, dorsal is front. All embryos shown were at the early gastrula stage (stage 11).
Figure 6. XSmad2 and XSmad4 did not rescue the phenotype induced by XSUMO-1-MO. A-D: XSUMO-1-MO inhibited ectopic axis formation induced by XSmad2. Injection of XSmad2 into the ventral marginal zone caused ectopic axis formation while control MO did not affect the secondary axis formation induced by XSmad2 (A and B, arrow). XSUMO-1-MO inhibited axis formation induced by XSmad2 (C). Wild-type embryos (D). E: Axis duplication frequency by injection of XSmad2 and by coinjection of XSmad2 and control MO or XSUMO-1-MO. Over 20 ng XSUMO-1-MO strongly inhibited ectopic axis formation induced by 1,000 pg XSmad2. F: Frequency of axis elongation error by XSUMO-1-MO and by coinjection with XSUMO-1-MO, XSmad2, and XSmad4. XSUMO-1-MO was coinjected with increasing amounts of XSmad2 mRNA (0, 250, 500, and 1,000 pg) or XSmad2 and XSmad4 mRNA (500 pg each). C.MO, control MO. Severe defects are shown in black, medium defects in gray, and mild defects in white. G-L: XSmad2 did not rescue inhibition of Xbra expression by XSUMO-1-MO. Embryos were coinjected with indicated RNA or MO and lacZ (lineage tracer) into the dorsal marginal zone at the 8-cell stage. Then, expression of Xbra was investigated by WISH. 500 or 1,000 pg XSmad2 mRNA and 40 ng XSUMO-1-MO were injected (H-J, respectively). XSUMO-1-MO (40 ng) was coinjected with 500 or 1,000 pg XSmad2 mRNA (K and L, respectively). No-inj., Injected nothing (G). Red and blue indicate the injected area and Xbra-expressing area, respectively.
