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Fig. 1. Knockdown of ADAM13 or ADAM19 inhibits Wnt
signaling and NC specification but not NPB formation.
(A) cDNA sequences of adam12 and adam19 showing the
translation start sites (red) and targets of MOs (colored
boxes). (B) Knockdown (KD) of ADAM13 or ADAM19 inhibits
head cartilage formation. Transgenic X. tropicalis snail2-
eGFP embryos were injected in one anterodorsal (D1)
blastomere at the eight-cell stage with the indicated MO
(1.5 ng each) cultured to stage ∼46 and imaged for eGFP
expression. One representative embryo of each injected
group (ventral view) is shown in the upper panels and the
results of five independent experiments are shown in the
lower panel. (C,D) Effects of ADAM13 or ADAM19 KD on NC
and NPB marker expression. Wild-type embryos were
injected in one blastomere at the two-cell stage with the
indicated MO (6 ng each) and cultured to stage ∼12.5.
Embryos were processed for in situ hybridization for the
indicated genes. (E) KD of ADAM13 or ADAM19 inhibits
endogenous Wnt signaling. Transgenic Wnt reporter
embryos were injected as in C,D, cultured to stage ∼12.5 and
imaged for eGFP expression. White arrowheads indicate the
Wnt signal at the NPB. Embryos in C-E are shown in dorsal
view, with anterior at the top. Red asterisks indicate the
injected side. CT, control MO; n, number of embryos scored;
N, number of independent experiments performed;
***P<0.001; NS, not significant.
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Fig. 2. ADAM13 and ADAM19 function in NC specification through different mechanisms. Wild-type embryos were injected into one blastomere at the
two-cell stage with the indicated MO (6 ng each) and mRNA (100 pg each), cultured to stage ∼12.5 and processed for in situ hybridization for the indicated genes.
(A) Effects of ectopically expressed ADAM13 and ADAM19 variants on NC markers. (B) Both wild-type ADAM19 and the protease-dead E/A mutant rescue
the NC specification defects caused by ADAM19 KD. Rescue constructs were generated as described in the Materials and Methods. All embryos are shown
in dorsal view, with anterior at the top and a red asterisk denoting the injected side. U, uninjected; WT, wild type. ***P<0.001.
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Fig. 3. ADAM19 controls ADAM13 protein levels in vivo. (A) Effects of
various MOs on exogenously expressed ADAM13 and ADAM19. Embryos
were injected in one blastomere at the two-cell stage with the indicated RNA
(100 pg each) and MO (6 ng each), and cultured to stage ∼19. Whole-embryo
lysates were processed for western blot (WB) for the C-terminal myc6 tag on
the ADAM constructs. (B) Effects of various MOs on endogenous ADAM13.
One-cell stage embryos were injected with the indicated MO (12 ng each) and
cultured to stage ∼12.5. Embryo lysates were processed for western blot for
endogenous ADAM13. (C) Upregulation of exogenous ADAM13 by coexpressed
ADAM19. Embryos were injected with the indicated transcripts
(100 pg each; the encoded ADAM13 was myc6-tagged and ADAM19 was HAtagged)
and cultured as in A, and lysates were processed for western blot with
an anti-myc antibody. Single and double arrowheads indicate the pro- and
mature forms of ADAMs, respectively.
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Fig. 4. ADAM19 colocalizes and interacts with
ADAM13. (A,B) Colocalization of ADAM13 and ADAM19
in the ER. XTC cells were transfected with 1 µg of plasmid
encoding ADAM19, and immunocytochemistry for
ADAM19 (red), calnexin (green in A) and ADAM13
(green in B), as well as DAPI labeling for nuclei (blue)
were carried out. Arrowheads indicate lamellipodia. (C,D)
Association of ADAM19 with ADAM13. HEK293T cells
were transfected with plasmids (0.5 µg each) encoding
ADAM19 and untagged ADAM13 (C) or different variants
of myc6-tagged ADAM13 (D). Immunoprecipitation and
western blot were carried out with the indicated
antibodies. Single and double arrowheads indicate the
pro- and mature forms of ADAMs, respectively. CR,
cysteine-rich domain; D, disintegrin domain; DC,
disintegrin and cysteine-rich domains; FL, full-length
protein.
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Fig. 5. The cytoplasmic K911 residue is crucial for ubiquitin-proteasome-mediated ADAM13 turnover and regulation by ADAM19. (A,F) Responses of
different variants of ADAM13 to ADAM19 KD. Embryos were injected in one blastomere at the two-cell stage with the indicated RNA (100 pg each) and MO (6 ng
each), and cultured to stage â¼19. Whole-embryo lysates were processed for western blot for the C-terminal myc6 tag on the ADAM constructs. A shorter exposure
was performed for the ÎC mutant in B,E. Ubiquitylation of wild-type ADAM13 and the K911R mutant. HEK293T cells were transfected to express the FLAG-tagged
ubiquitin with and without HA-tagged ADAM13 (B), or myc6-tagged wild-type ADAM13 or the K911R mutant (E). Immunoprecipitation and western blot were
carried out with the indicated antibodies. (C) MG132 protects ADAM13 and rescues the effect of ADAM19 KD on ADAM13. Embryos were injected, dissociated
and cultured in the absence or presence of MG132. Cell lysates were processed for western blot. (D) XTC cells were transfected with 0.5 µg of plasmids encoding
myc6-tagged wild-type ADAM13 or the K911R mutant. Immunocytochemistry for myc (green) and DAPI labeling for nuclei (blue) with images taken in green and
blue channels merged. Single and double arrowheads indicate the pro- and mature forms of ADAM13, respectively; asterisk denotes the autocleavage product.
UB, ubiquitin.
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Fig. 6. The stabilized ADAM13 mutants can rescue the NC specification defects caused by ADAM19 knockdown and induce ectopic snail2 in the ventral
ectoderm. (A,B) Rescue of the reduced sox9 expression caused by ADAM19 knockdown (KD) (A) or ADAM13 KD (B) by various transcripts. Embryos were
injected into one blastomere at the two-cell stage with the indicated RNA (100 pg each) and MO (6 ng each), cultured to stage ∼12.5 and processed for in situ
hybridization for sox9. Embryos are shown in dorsal view, with anterior at the top and a red asterisk indicating the injected side. (C) Induction of ectopic snail2
by different variants of ADAM13. Embryos were injected into one posterior-ventral (V2) blastomere at the eight-cell stage with the indicated RNA (50 pg each)
and MO (1.5 ng each), cultured to stage∼12.5, and processed for in situ hybridization for snail2. Only the injected side is shown, with anterior at the top and dorsal to
the left. Arrows indicate the ectopic snail2 expression. (D) A model for the regulation of ADAM13 stability and NC specification by ADAM19. Crescents indicate
the metalloproteinase domains of ADAMs. See Discussion for an explanation. ***P<0.001; NS, not significant.
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Figure S1. Developmental expression of adam19 in X. tropicalis embryos. Embryos were
fixed at the indicated stages, and in situ hybridization was carried out for adam19. A. Vegetal
view with dorsal at the top. Arrow denotes the dorsal blastopore lip. B. A bisected embryo
shown with dorsal at the top and anterior to the left.
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Figure S2. KD of ADAM13 or 19 inhibits eye field induction in X. tropicalis embryos. A.
Eye phenotypes caused by ADAM13 or 19 KD. Embryos were injected in one anterodorsal (D1)
blastomere at 8-cell stage with the indicated MO (1.5 ng each), cultured to stage ~38, and the
injected side was imaged. One representative embryo of each injected group is shown in upper
panels, and results of 3 independent experiments are graphed in the lower panel. B, C. Effects of
ADAM13 or 19 KD on the expression of eye field markers. Embryos were injected in one
blastomere at 2-cell stage with the indicated MO (6 ng each), cultured to stage ~12.5, and
processed for in situ hybridization for the indicated genes. Embryos in B, C are shown in dorsal
view, with posterior at the top and a red asterisk denoting the injected side. CT, control MO; n,
number of embryos scored; N, number of independent experiments performed; ***, P < 0.001;
NS, not significant (same below).
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Figure S3. ADAM13 inhibits the autocleavage of ADAM19. Embryos were injected in one
blastomere at 2-cell stage with the indicated RNA (100 pg each) and MO (6 ng each), and
cultured to stage ~19. Whole-embryo lysates were processed for Western blot for the C-terminal
myc6 tag on the ADAM19 construct. Single and double arrowheads indicate the pro- and mature
forms of ADAM19, respectively, and asterisk denotes the autocleavage product.
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Figure S4. Effects of exogenous ubiquitin on the functions of different ADAM13 variants in
X. tropicalis embryos. Embryos were injected in one blastomere at 2-cell stage with the
indicated mRNA (100 pg each) and the plasmid encoding FLAG-tagged ubiquitin (UB; 2.5 pg).
The injected embryos were cultured to stage ~12.5, and processed for in situ hybridization for
sox9. All embryos are shown in dorsal view, with anterior at the top and a red asterisk denoting
the injected side.
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Figure S5. The stabilized ADAM13 mutants rescue the NC specification defects caused by
ADAM19 KD. X. tropicalis embryos were injected in one blastomere at 2-cell stage with MO
19-1 (6 ng) and the indicated mRNA (100 pg each) encoding myc6-tagged (A) or untagged (B)
variants of ADAM13. The injected embryos were cultured to stage ~12.5, and processed for in
situ hybridization for the indicated genes. All embryos are shown in dorsal view, with anterior at
the top and a red asterisk denoting the injected side. Arrows point to the ectopic snail2
expression.
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Figure S6. The stabilized ADAM13 mutants rescue the eye field defects caused by
ADAM19 KD. X. tropicalis embryos were injected in one blastomere at 2-cell stage with the
indicated mRNA (100 pg each) and MO 19-1 (6 ng). The injected embryos were cultured to
stage ~12.5, and processed for in situ hybridization for the indicated genes. All embryos are
shown in dorsal view, with posterior at the top and a red asterisk denoting the injected side.
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