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. 2023 Oct 16;120(43):e2307203120. doi: 10.1073/pnas.2307203120

Fig. 1.

Fig. 1.

The Synthetic Processing (SynPro) system shows that prodomain cleavage can be non-cell-autonomous. (A) The SynPro system comprises an orthogonal secreted protease derived from tobacco etch virus (secTEVp) and a cognate sequence that replaces the endogenous cleavage site of Vg1 (SynPro Vg1, RSRRKR → ENLYFQS). (B) (i) Rescue percentage after 30 hpf of Mvg1 embryos injected with 50 pg of vg1, SynPro vg1, SynPro vg1 and secTEVp, or SynPro vg1 and secTEVp-KDEL mRNAs. (ii) Representative images of 30 hpf Mvg1 embryos for the indicated injection condition. Minor brain and tail defects are noted in embryos transiently rescued with mRNAs of the SynPro system. (Scale bar, 0.5 mm.) (iii) Fluorescence images of Mvg1 embryos at 50 to 60% epiboly that were injected with mRNAs for sfGFP-tagged wild-type Vg1 and secTEVp-sfCherry (Top) or secTEVp-sfCherry-KDEL (Bottom). (Scale bar, 20 μm.) (C) Schematic of transplantation assay. Mvg1 embryos were injected with 50 pg mRNA each of: (i) DONOR: cyc, SynPro vg1, and secTEVp; HOST: none; (ii) DONOR: cyc and SynPro vg1; HOST: secTEVp; (iii) DONOR: cyc and SynPro vg1; HOST: none; (iv) DONOR: none; HOST: secTEVp. All Mvg1 donor embryos were marked by also injecting 50 pg sfGFP mRNA. At high stage, before the onset of Nodal signaling, sfGFP-marked DONOR cells were transplanted to the animal pole of HOST Mvg1 embryos. (D) At 50 to 60% epiboly, chimeric embryos were fixed and immunostained for sfGFP and pSmad2. DAPI, nuclei. (Scale bar, 20 μm.)