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
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.