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. 2014 May 21;289(27):18928–18942. doi: 10.1074/jbc.M114.551929

FIGURE 8.

FIGURE 8.

Knockdown of scube3 suppressed FGF target genes and reduced FGF signaling in zebrafish embryos. A, effect of scube3 knockdown on the expression of FGF target genes (fgf8 and myod1) evaluated by whole mount in situ hybridization. The images are the dorsal view of the eight-somite embryos with anterior to the left. In scube3 morphants, fgf8 and myod1 expression was markedly reduced in the somites. Of note, scube3 knockdown specifically suppressed myod1 expression in lateral fast muscle precursors but not adaxial cells that will differentiate into slow muscles. The numbers in the bottom left-hand corners of photographs are the numbers of affected embryos with phenotype similar to what is shown in the image (left column) and the total number of observed embryos (right column). B, ERK activation in control or scube3 knockdown embryos. After Scube3 tMO1 injection, protein lysates (20 μg) derived from embryos (70% epiboly) were blotted with antibody specific to the phosphorylated form (p-ERK) or total ERK protein and quantified by densitometric scanning (mean ± S.E.) from three independent experiments (bottom panel). Note that scube3 knockdown significantly decreased p-ERK expression in a dose-dependent manner, which supports the role of scube3 in FGF signaling during zebrafish development. C and D, forced expression of scube3 or fgf8 mRNA restored myod1 expression in fast muscle precursors of scube3 morphants. C, co-injection of scube3 morphants with scube3 mRNA rescued myod1 expression. Scube3 morphants showed rescued expression of lateral myod1expression in somites when injected with 100 pg of synthetic scube3 mRNA at the one- or two-cell stage. The images are shown as dorsal views with anterior to the left. Quantification data are shown in the bottom panel. D, forced expression of fgf8 mRNA restored myod1 expression in lateral somites. Scube3 morphants showed rescued expression of lateral myod1 expression in somites when injected with 0.0625 pg of synthetic fgf8 mRNA at the one- or two-cell stage. The images are shown as dorsal views with anterior to the left. Quantification data are shown in the bottom panel. The data are means ± S.E. cMO, control MO.