Wei S , Xu G , Bridges LC , Williams P , White JM , DeSimone DW .

DE14365 NIDCR NIH HHS, HD26402 NICHD NIH HHS , R01 GM094793-20 NIGMS NIH HHS , R01 HD026402-18 NICHD NIH HHS , R01 GM094793 NIGMS NIH HHS , R01 HD026402 NICHD NIH HHS , R01 DE014365 NIDCR NIH HHS

	Figure 1. The Metalloproteinase Activity of ADAM13 Is Required for Xenopus CNC Induction(A) cDNA sequence of adam13 showing the translation start site (red) and targets of MOs 13-1 and 13-3.(B) One blastomere of two-cell stage embryos was injected with MO 13-1 (6 ng) and transcripts encoding myc-tagged ADAMs (ADAM12 or 13: 200 pg; ADAM19: 100 pg). Embryos were cultured for 20 hr and western blot of whole-embryo lysates carried out using an anti-myc antibody.(C) Western blot of stage 11–12 embryo lysates showing KD of endogenous ADAM13 by injection of MO 13-1 at one-cell stage (12 ng MO/embryo; 20 embryos/lane).(D) Head cartilage phenotypes of ADAM13 KD. One anterior-dorsal blastomere of eight-cell stage embryos was injected with the indicated MO (1.5 ng). Embryos were allowed to develop to stage 46 and scored for head cartilage defects. Images were gray-scale inverted for easier visualization of head cartilage. One example of each phenotype is shown in the upper panels, and results of three independent experiments are graphed in the lower panel (error bars represent SDs). *p = 0.03; **p = 0.005; NS, not significant (p = 0.43). See Figure S1D and Experimental Procedures for details on phenotype scoring and statistics.(E and F) Four-cell stage embryos were injected in one anterior-dorsal blastomere with 3 ng MO 13-3, allowed to develop to stage 12.5/13, and then processed by in situ hybridization for snail2 (E) or sox9 (F).(G) Embryos were injected with the indicated MO (3 ng) and “rescue” transcripts (100 pg each), and in situ hybridization performed as in (E). Red asterisks in (D)–(G) denote injected side.Single and double arrowheads in (B) and (C) point to the pro- and mature forms of ADAMs, respectively, and arrows in (D) point to impaired head cartilage structures as compared with the uninjected side. U, uninjected; CT, control MO; n, number of embryos scored (same below).
	Figure 3 ADAM13 controls CNC induction through regulation of EfnB and Wnt signaling (A, B) Embryos were injected as in Figure 1E with transcripts as follows: 200 pg EfnB1 and 100 pg ADAM13 in (A), and 200 pg each in (B) along with 3 ng of control (CT) or 13-3 MO. In situ hybridization was processed for snail2 at stage 12.5/13; injected side is denoted with a red asterisk. (C) Embryos were injected in one ventral-vegetal blastomere at 16-cell stage with the indicated RNA (1.5 pg Wnt8; 100 or 200 pg EfnB1) and MO 13-3 (1.5 ng), and scored for secondary axis formation at tailbud stages. (D) HEK293T cells were transfected with pTopflash or pFopflash, pCMV-β-gal and the indicated constructs, and cultured for ~40 hrs. Cell lysates were processed for luciferase and β-galactosidase assays as controls for transfection efficiency. A representative experiment performed in triplicate is shown here. Results are presented as ratios of TOPFLASH vs. FOPFLASH luciferase activity (both were normalized for β-gal activity), and the value calculated for cells transfected with control plasmids only was set to 1. Error bars represent standard deviations. **: P < 0.001; NS: not signicicant (P = 0.71). U, uninjected.
	Figure 4 Snail2 is downstream of EfnB and canonical Wnt signaling to mediate ADAM13 function in CNC induction (A) Embryos injected at one-cell stage with the indicated MO (12 ng) were cultured to stages indicated, and quantitative RT-PCR carried out using embryo lysates. Relative levels of snail2 mRNA were determined by normalizing to amount of β-actin mRNA and by setting the levels calculated for control MO to 100%. Error bars represent standard deviations. **: P = 0.001 (N = 4) for stage 10.5, P = 0.005 (N = 3) for stage 12.5, and P= 0.008 (N = 3) for stage 19. (B, C) Embryos were injected with 3ng MO and/or 200 pg each transcript as indicated and in situ hybridization performed for sox9, as described in Figure 1F legend. The injected side is denoted with a red asterisk. (D) A model for ADAM13 function in CNC induction. See text for explanation.
	Figure 2. ADAM13 Cleaves EfnB1/B2 in Cultured Cells and X. tropicalis Embryos(A) Cultured HEK293T cells were transfected with DNA constructs (0.5 μg each) expressing EfnB2 (with an N-terminal HA tag) and wild-type or the E/A mutant of each ADAM (with C-terminal myc tags). Western blots of conditioned media (CM) and whole-cell lysates (CL) were carried out using the indicated antibodies (α-HA or α-myc). Arrows indicate the ectodomain of EfnB2 shed by ADAM13; single and double arrowheads indicate the pro- and mature forms of ADAMs, respectively.(B) Conditioned media of HEK293T cells transfected to express EfnB2 and wild-type ADAM13, as in (A), were treated with or without PNGase and processed for western blot with an anti-HA antibody. The deglycosylated product is denoted with an arrow.(C) Cartoon indicating the approximate location of the ADAM13 cleavage site in EfnB2.(D and E) Each of the two anterior-dorsal blastomeres of eight-cell stage X. tropicalis embryos was injected with control (CT) or 13-1 MO (1.5 ng). Embryos were allowed to develop to stage ∼19, the heads collected by dissection, and western blots of head lysates carried out using the C-18 antibody. Relative intensity of EfnB1/B2 was determined by normalizing the western blot density of EfnB1/B2 band against that of β-actin, and by setting the amount calculated for control MO to 100%. Error bars represent SDs calculated for seven independent experiments. ∗p = 0.03.

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