XB-ART-6191Dev Biol November 15, 2002; 251 (2): 395-408.
The E3 ubiquitin ligase GREUL1 anteriorizes ectoderm during Xenopus development.
We have identified a family of RING finger proteins that are orthologous to Drosophila Goliath (G1, Gol). One of the members, GREUL1 (Goliath Related E3 Ubiquitin Ligase 1), can convert Xenopus ectoderm into XAG-1- and Otx2-expressing cells in the absence of both neural tissue and muscle. This activity, combined with the finding that XGREUL1 is expressed within the cement gland, suggests a role for GREUL1 in the generation of anterior ectoderm. Although GREUL1 is not a direct inducer of neural tissue, it can activate the formation of ectopic neural cells within the epidermis of intact embryos. This suggests that GREUL1 can sensitize ectoderm to neuralizing signals. In this paper, we provide evidence that GREUL1 is an E3 ubiquitin ligase. Using a biochemical assay, we show that GREUL1 catalyzes the addition of polyubiquitin chains. These events are mediated by the RING domain since a mutation in two of the cysteines abolishes ligase activity. Mutation of these cysteines also compromises GREUL1''s ability to induce cement gland. Thus, GREUL1''s RING domain is necessary for both the ubiquitination of substrates and for the conversion of ectoderm to an anterior fate.
PubMed ID: 12435366
Article link: Dev Biol
Genes referenced: actc1 actl6a ag1 egr2 hoxb9 ncam1 nrp1 odc1 otx2 rnf128 snai2 tubb2b
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|FIG. 1. GREUL1 converts epidermis into cement gland and neural tissue in whole embryos. Embryos were injected with 500 pg of GREUL1 mRNA into one blastomere of two-cell Xenopus embryos. At late neurula stage (19�21), the embryos were stained by in situ hybridization for tissue-specific markers. (A, B) Laterally viewed, XAG-1-stained embryos, injected (A) compared with uninjected (B). The inset in (B) shows an anterior view of the same embryo. (C, D) Laterally viewed, Xotx2-stained embryos, injected (C) compared with uninjected (D). (E) Dorsally viewed, injected and Nrp1-stained embryos. The injected side, marked by an arrow, can be clearly distinguished from the uninjected side. (F) Embryos injected at the one-cell stage with 500 pg of GREUL1 and stained for N-tubulin. (G) 500 pg GREUL1-injected embryos stained for c-actin, showing normal somite development. In (H), the embryos were also injected with lacZ and stained for -galactosidase activity (red) prior to in situ hybridization for slug. Dashed lines separate control and GREUL1/lacZ-injected sides. The control side is oriented toward the top. (I�L) One-cell-stage GREUL1-injected embryos stained by in situ hybridization for both XAG-1 (magenta) and GREUL1 (blue�green). (I) was injected with 1 ng of GREUL1, and (J, K) were injected with 500 pg of GREUL1. (L) An uninjected control. (M) An expanded view of a portion of the embryo in (K), showing a few ectopic XAG-1 dots that appear to be outside of the blue�green GREUL1-expressing region.|
|FIG. 2. GREUL1 induces XAG-1 and Xotx2 from naı�ve ectoderm. A total of 1 ng, 500 pg, or 250 pg of GREUL1 was injected into the prospective ectoderm of a one-cell Xenopus embryo. The injected ectoderm and noninjected control ectoderm were explanted at the blastula stage and aged until stage 20. (A) The explants and a whole embryo were then analyzed by RT-PCR for NCAM, Krox20, HoxB9, c-actin, and the loading control EF-1 . The RT indicates the whole embryo processed without addition of reverse transcriptase. (B�K) Explants and uninjected whole embryos (B,G), were stained for Xotx2 (B�F) and XAG-1 (H�K), by in situ hybridization. For Xotx2 (B�F), the bottom of the figure shows the number of explants exhibiting patches of darkly staining cells/the total number of explants analyzed. 1 ng and 500 pg were significantly different from the control at P values of less than 0.05, using a Williams corrected G-test for independence.|
|FIG. 3. GREUL1 makes ectoderm competent to respond to neural-inducing signals. (A) Animal cap explants were cut at stage 8�9 and transplanted into the lateral flanks of stage 14 embryos. At stage 28, transplants were stained with Nrp1 and lacZ. (B) GREUL1-overexpressing grafts were cut from the lateral flanks of stage 14 embryos and grafted into uninjected controls. At stage 28, the graft is visible by its punctuate pattern of Nrp1 expression. (C) GREUL1-overexpressing neural tubes were grafted into uninjected control embryos and analyzed for Nrp1 expression. Nrp1-positive cells outside the graft were not observed (red arrow).|
|FIG. 4. XGREUL1 is expressed in the cement gland, cranial placodes, and the pronephros. Whole-mount in situ hybridization of Xenopus embryos using XGREUL1 antisense and sense probes. (A) Eight-cell-stage embryos; upper embryo showing GREUL1 expression in the animal hemisphere, the lower embryo is a sense control. (B) Anterior view of stage 20 embryos exhibiting cement gland staining, lower embryo is the sense control. Embryos were cleared with benzyl benzoate:benzyl alcohol (1:1). Stage 27 (C), stage 39 (D), and stage 42 (E) embryos expressing XGREUL1 in the lateral line system, the pronephros, the olfactory placode, and the otic vesicle. (F) RT-PCR showing GREUL1 expression during Xenopus development and using ODC as a control. Abbreviations: cg, cement gland; m, migratory primordium of middle trunk line; pAD, anterodorsal lateral line placode; pd, pronephric duct; g, pronephric tubules; pOl, olfactory placode; ot, otic vesicle.|
|FIG. 7. GREUL1 functions as an E3 ubiquitin ligase and the RING domain is necessary for both E3 activity and XAG-1 induction. (A) Comparison of the GST fusion constructs to wild type GREUL1. Abbreviations: TM, transmembrane domain; RING, RING finger domain; wt, wild type; *, point mutation (cysteine replaced by glycine); the triangle represents the signal peptide cleavage site. (B) Upper panel: Western-blot with FLAG antibody visualizing FLAGtagged ubiquitin chains, ranging from 25 to 250 kDa. The Apc2/Apc11 (Apc2/11) complex was used as a control. Lower panel: Western blot with GST antibody showing the actual amount of GSTC-term and GST C1C2 added to the above reactions. 10, 30, and 100 ng of total purified GSTC-term and 10, 30, 100, 300, and 900 ng of total purified GST C1C2 was used in each reaction. Only 30 and 100 ng of GSTC-term were able to catalyze ubiquitination. The size of the protein band is 49 kDa. (C, D) A functional RING domain is necessary for XAG-1 induction. 1 ng of either wild type GREUL1 (D) or C1C2 GREUL1 (C) was injected into two-cell embryos and stained by in situ hybridization for XAG-1. Ectopic XAG-1 dots are not apparent in the C1C2 GREUL1-injected embryos.|
|rnf128 (ring finger protein 128) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, anterior view, dorsal up.|
|rnf128 (ring finger protein 128) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 39, lateral view, anterior right, dorsal up.|