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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. |
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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. |
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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. |
