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Unlike most transforming growth factor-beta (TGF-beta) superfamily members, Vg1 has been shown not to produce gross phenotypic alterations in Xenopus embryos when overexpressed by mRNA injection. Experiments with artificial chimeric constructs and a recently identified second allele of Vg1 suggest that this may be due to unusually stringent requirements for proteolytic processing. We provide biological and biochemical evidence that cleavage by two distinct proteolytic enzymes is required for effective activation of Vg1. We demonstrate a tightly restricted overlap in expression patterns of Vg1 with the proteases required to release the mature peptide. The data presented may account for the long-standing observation that the vast majority of Vg1 protein, in vivo, is present in its unprocessed form. Taken together, these observations provide a plausible mechanism for local action of Vg1 consistent with requirements imposed by current models of pattern formation in the developing body axis.
Fig. 2. Spatial distribution of Vg1 and SPCs in whole-mount hemisected stage 9 Xenopus blastulae. (A) Vg1, (B) Furin, (C) SPC4, (D) SPC6. Embryos are not oriented specifically.
Fig. 3. Spatial distribution of Vg1 and SPCs in sections of stage 9 Xenopus blastulae. Panels show Vg1 expression in red and Furin, SPC4, and SPC6 in green. The double-label images indicate the specific overlapping expression patterns of Vg1with Furin, SPC4, and SPC6 (indicated by white arrows) on the dorsal–vegetal side of the embryo, in the region of the Nieuwkoop center. Adjacent sections were probed with Siamois to confirm dorsal–ventral orientation of the embryos.
Fig. 6. Vegetal expression of Furin and SPC6 can restore a normal dorsal axis to UV-ventralized Xenopus embryos. UV-treated embryos were injected in a vegetal blastomere at the 4 cell (B, D and E) or 8-cell stage (C). (A) Control noninjected, non-UV-irradiated stage 37/38 embryos. (B) UV-irradiated embryos injected with 1 ng GFP mRNA showing DAIs of 0–2. (C) UV-irradiated embryos injected vegetally with 450 pg Furin and 450 pg SPC6 mRNA at the 8-cell stage showing DAIs ranging from 2 to 3. (D) UV-irradiated embryos injected vegetally with 450 pg Furin and 450 pg SPC6 mRNA at the 4-cell stage exhibiting DAIs of 4–5. All embryos were allowed to develop until control non-UV-treated embryos reached stage 37/38. The representative rescued embryos shown in panel D have anterior structures, including eyes, cement gland, and neural crest-derived pigmented cells. (E) The frequency distribution of DAI scores for a representative experiment is shown, each dot corresponds to a single embryo. These experiments were performed six times, in which 0/150 of the control, UV-irradiated embryos had a DAI > 2. (F). Whole mount hybridization in situ of Vg1 mRNA in hemisected control and UV-irradiated stage 9 Xenopus embryos.
gdf1 (Growth Differentiation Factor 1) expression by in situ hybridization in Xenopus laevis stage 9 embryos.
Copyright Elsevier Science
gdf1 (growth differentiation factor 1) expression visualized by immunohistochemistry, in a bisected Xenopus laevis embryo, NF stage 9, dorsal left.
