Wu C and Muslin AJ (2002), Role of 14-3-3 proteins in early Xenopus develo...

XB-ART-6383

Mech Dev 2002 Nov 01;1191:45-54. doi: 10.1016/s0925-4773(02)00287-3.

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Role of 14-3-3 proteins in early Xenopus development.

Wu C , Muslin AJ .

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14-3-3 proteins are intracellular dimeric phosphoserine/threonine-binding molecules that participate in signal transduction and checkpoint control pathways. 14-3-3 proteins are required for normal eye development, brain function, and terminal patterning in Drosophila melanogaster, but the role of 14-3-3 proteins in vertebrate development is undefined. In this work an unphosphorylated peptide inhibitor of 14-3-3, R18, was used to determine the role of 14-3-3 proteins in Xenopus embryonic development. Biochemical analysis demonstrated that R18 was specific and efficient at attenuating global 14-3-3 activities in Xenopus embryos. Microinjection experiments showed a requirement for 14-3-3 function in mesodermal specification. Inhibition of 14-3-3 resulted in embryos with axial patterning defects and reduced expression of mesodermal marker genes. These phenotypic defects were caused by impaired fibroblast growth factor signaling in R18-injected embryos. These results establish the importance of 14-3-3 proteins in vertebrate embryonic development.

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Species referenced: Xenopus
Genes referenced: chrd fgf2 gsc myod1 raf1 tbxt wnt8a

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	Fig. 1. Expression and function of GST-R18 fusion protein in Xenopus embryos. (A) GST-R18 fusion protein binds to 14-3-3 in embryos. Xenopus two-cell embryos were injected with 20 ng RNA encoding GST, GST-R18, or the mutant fusion protein GST-R18M. Protein lysates were generated at embryonic stage 14 (Nieuwkoop and Faber, 1967). In the upper panel, protein lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with an anti-GST antibody or with an anti-pan-14- 3-3 antibody (anti-14-3-3b). In the lower panel, protein lysates were added to glutathione-sepharose 4B beads to immobilize GST-containing proteins and washed four times. Bound proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with an anti- GST antibody (upper panel) or with an anti-pan-14-3-3 antibody (anti-14-3- 3b). Note the slightly slower mobility of the GST-R18 and GST-R18M fusion proteins compared to GST alone in the immunoblot. (B) Injection of RNA encoding GST-R18 results in prolonged expression of GST-R18 protein that binds to 14-3-3 in vivo. Twenty nanograms of GST-R18 RNA was injected into two-cell stage embryos. Whole embryo lysates at the indicated embryonic stages were generated and analyzed by anti-GST immunoblotting to determine GST-R18 protein levels after RNA injection (upper panel). Parallel lysates were utilized in GST pull-down assays as described earlier in (A).
	Fig. 2. The GST-R18 fusion protein efficiently inhibits 14-3-3 activity in Xenopus embryos. (A) Depletion of 14-3-3 protein by GST-R18 in vivo. Xenopus two-cell embryos were injected with RNA encoding GST or GSTR18 and protein lysates were obtained from stage 14 embryos. Each lysate was divided into two and half the lysate was incubated with glutathionesepharose 4B beads for 4 h, separated by centrifugation, and the supernatant was collected. The control half of the lysate (Lys) and the supernatant precleared by glutathione-sepharose beads (GST-PD) were separated by SDSPAGE and analyzed by anti-pan-14-3-3 (upper panel) or anti-GST immunoblotting (lower panel). Note the reduction of 14-3-3 protein in GST-R18- injected embryonic lysates after glutathione-sepharose chromatography. (B) The GST-R18 fusion protein inhibits the association of 14-3-3 with Raf-1 in Xenopus oocytes. Oocytes were injected with 40 ng of RNA encoding GST or GST-R18. Oocyte protein lysates were obtained 24 h after control or RNA injection and lysates were analyzed by anti-Raf-1, anti-pan-14-3-3, or anti-GST immunoblotting (upper 3 panels). Anti-Raf-1 (C-12, Santa Cruz Biotech) immunoprecipitates obtained from whole cell lysates were immunoblotted with anti-Raf-1 or anti-pan-14-3-3 antibodies (lower 2 panels).
	Fig. 3. The GST-R18 fusion protein binds specifically to 14-3-3 proteins. Embryos were injected with 20 ng of RNA encoding GST, GST-R18M, or GST-R18 at the two-cell stage. Protein lysates of control or RNA injected embryos were obtained from stage 14 embryos. Protein lysates were added to glutathione-sepharose beads, washed, and adherent proteins were separated by SDS-PAGE and visualized by silver staining. The bracket indicates the specific proteins bound to the R18 moiety of the GST-R18 fusion protein, running at the apparent molecular weight of ,29 kDa.
	Fig. 4. Phenotypic abnormalities in GST-R18 injected embryos. (A,B) Failure of blastopore lip closure in neurula-stage (stage 15) GST-R18 injected embryos. Two-cell embryos were injected into both blastomeres with RNA encoding GST-R18M (A) or GST-R18 (B). Each blastomere was injected with 20 ng of RNA. (C,D) Truncation of posterior structures in tadpolestage (stage 40) GST-R18 injected embryos. Two-cell embryos were injected in both blastomeres with RNA encoding GST-R18M (C) or GST-R18 (D). Each blastomere was injected with 20 ng of RNA. (E,F) Reduced and disorganized skeletal muscle development in stage 40 GSTR18 injected embryos. Two-cell embryos were injected in both blastomeres with RNA encoding GST-R18M (E) or GST-R18 (F). Each blastomere was injected with 20 ng of RNA. Embryos were fixed, embedded in paraffin, sectioned and probed with muscle-specific antibody 12/101 (Kintner and Brockes, 1984). Skeletal muscle was visualized with a red substrate (indicated by arrows). (G,H) Bent anteroposterior axis in stage 40 embryos injected unilaterally with GST-R18. Two-cell embryos were injected in one blastomere with 20 ng RNA encoding GST-R18M (G) or GST-R18 (H).
	Fig. 5. Analysis of marker gene expression in gastrula stage (stage 10.25) embryos injected with GST-R18 or GST-R18M RNA. (A) GST-R18 inhibits Xbra expression. (A,B) Four-cell stage embryos were injected equatorially in all four blastomeres with 10 ng of RNA per blastomere. Note the reduced Xbra expression in the GST-R18 injected embryo (B). (C,D) Two-cell embryos were injected equatorially in one blastomere with 20 ng of RNA per blastomere. Note the reduced Xbra expression on the injected side (indicated by arrows) of GST-R18 injected embryos (D). (E,F) Four-cell embryos were injected equatorially into two blastomeres with 10 ng of RNA per blastomere. Note the reduced Xbra expression in two of the four quadrants (indicated by arrows) in the GST-R18 injected embryos (F). (G,H) GST-R18 inhibits XmyoD expression. Two-cell embryos were injected equatorially in one blastomere with 20 ng of RNA per blastomere. Note the reduced XmyoD expression on the injected side (indicated by arrow) of the GST-R18 injected embryo (H). (I,J) GST-R18 inhibits Xwnt8 expression. Two-cell embryos were injected equatorially in one blastomere with 20 ng of RNA per blastomere. Note the reduced Xwnt8 expression on the injected side (indicated by arrow) of the GST-R18 injected embryo (J). (K,L) GST-R18 does not inhibit goosecoid expression. Four-cell embryos were injected equatorially in two blastomeres with 10 ng of RNA per blastomere. (M,N) GST-R18 does not inhibit chordin expression. Four-cell embryos were injected equatorially in two blastomeres with 10 ng of RNA per blastomere.
	Fig. 6. 14-3-3 activity is required for FGF-induced mesoderm induction in Xenopus animal caps. Forty nanograms of RNA encoding GST, GST-R18, or X14-3-3z was injected in the animal pole of all cells of four-cell embryos. Animal caps were dissected from stage 9 embryos and incubated in the presence or absence of bFGF (50 ng/ml) for 3 h. Total RNA was purified from animal caps and used in RT-PCR experiments with established primers. EF-1a expression was analyzed as a control. RNA purified from whole embryos at stage 14 was used as a positive control. Note that GST-R18, but not GST, blocked FGF-induced Xbra gene expression in animal caps. Injection of RNA encoding x14-3-3 z rescued the inhibition of Xbra expression in GST-R18 injected animal caps.