Cousin H , Abbruzzese G , Kerdavid E , Gaultier A .

R01 DE016289-01A2 NIDCR NIH HHS, R01 DE016289-02 NIDCR NIH HHS, R01 DE016289-03 NIDCR NIH HHS, R01 DE016289-04 NIDCR NIH HHS, R01 DE016289-05 NIDCR NIH HHS, R01 DE016289 NIDCR NIH HHS

	Fig.1. ADAM13 and 19 Cooperate during CNC Migration(A) Schematic representation of the grafts. Donor embryos are injected in one cell at the 2-cell stage with GFP, or combinations of GFP, MO against ADAM13 and 19 and mRNA encoding various ADAM constructs. At stage 15, CNC are dissected and grafted into host embryos. CNC migration is visualized using a fluorescent microscope.(B) Fluorescence photographs of representative grafted embryos at stage 24. A positive migration is only counted if cells have progressed ventrally in one or more of the posterior arches.(C) Histogram representing the analysis of CNC migration in at least three independent experiments. The error bars correspond to the standard deviation (SD). n: number of embryos.
	Fig.2. ADAM13 Cytoplasmic Domain Translocates to the Nucleus(A) Schematic representation of ADAM13 proteolytic processing (left). The Pro-domain is cleaved during transit to the cell surface (1). A second extracellular cleavage within the cysteine-rich domain has also been documented (2), releasing the active protease. The cytoplasmic domain is cleaved by GS (3). The GFP-fusion protein corresponding to the ADAM13 cytoplasmic domain and the various mutants are represented (right).(B) Western blot of ADAM13 cytoplasmic domain. 293T Cells were transfected with GFP, GFP-C13, and wild-type ADAM13 (A13). Cells were fractionated to generate a cytoplasmic (C) and a nuclear (N) fraction. Fractions were analyzed by western blot using a polyclonal antibody to the cytoplasmic domain of ADAM13 (arrowhead) (Alfandari et al., 1997). All membrane-bound fragments of ADAM13 are found both in the cytoplasmic fraction (plasma membrane) and in the nuclear fraction (ER attached to the nuclear membrane). The nuclear protein PARP is found in the nuclear fraction, while GAPDH is in the cytoplasm.(C) ADAM13 was immunoprecipitated from 10 embryos using a goat polyclonal antibody. Noninjected embryos (NI) at stage 12 (gastrula) and stage 18 (neurula) are compared with sibling embryos injected with MO13. The ADAM13 protein was detected using 6615F.(D) Cos-7 cells transfected with ADAM13 were treated for 4 hr with GS inhibitors (+, ILCHO 50 μM or DAPT 10 μM).(E) Localization of GFP-fusion proteins expressed in XTC cells. Hoechst was used to detect the nuclei in cells transfected with GFP-C13NES.See also Figure S2.
	Fig.3. ADAM13 Cytoplasmic Domain Is Critical for Cranial Neural Crest Cell Migration(A) Lateral view of representative embryos injected with RFP and a control MO (CMO) or with MO13 (see also Figure S4). The white arrows point to the fluorescence observed in the three main CNC segments (from left to right, Mandibular Hyoid and Branchial).(B) Histogram representing the percentage of embryos in which CNC migration was inhibited by either MO13 (black bars) or MO13 and MO19 (2MO, gray bars). The numbers on the right indicate the number of embryos scored for each construct (schematic representation to the left). Statistical significance (at least three independent experiments) was calculated using a Student's t test p < 0.05. Significant rescue is indicated with an asterisk (*), and error bars are SD.(C) Western Blot from Cos-7 cells transfected with various ADAM13 constructs and the protocadherin PAPC. The cell extract (Cell) and the glycoprotein purified from the culture supernatant (Media) were probed with an antibody to the extracellular domain of PAPC. The cell extract was also probed with the 7C9, a monoclonal antibody to the cysteine-rich domain of ADAM13.(D) Fluorescence images of live XTC cells transfected with GFP-fusion proteins corresponding to the various ADAM cytoplasmic domains. The ADAM number and the species are indicated (xl; Xenopus laevis, dr; Danio rerio, mm; Mus musculus).(E) Histogram representing the rescue experiments described in (A), using a complementation assay. The total number of embryos scored in at least three independent experiments is indicated on the right (N). Asterisk indicate significant rescue (p < 0.05), error bars are SD. The species name is indicated according to standard nomenclature (ce; Caenorhadbitis elegans, md; Monodelphis domestica).
	Fig.4. The Cytoplasmic Domain of ADAM13 Regulates Gene Expression(A) Representative genes were selected from the microarray data. The average fold change between the control MO and MO13 (MO13) or between MO13 and the rescue MO13 plus GFP-C13 (MO13+C13) is represented as a Log2. Error bars represent the SD. Four groups of genes are presented. Xmc (Xenopus Marginal Coil), pafr (Platelet-activating factor receptor), capn8-a (Calpain8-a), wnt3-A (Wingless-type MMTV integration site 3A), and pou50 (Pou-homeobox gene 50) are decreased in CNC injected with MO13 and are partially rescued by GFP-C13. nudc (Nuclear distribution gene C), rad1 (Cell cycle check point), and vamp8 (Vesicle-associated membrane protein 8) are increased in CNC lacking ADAM13, and rescued by GFP-C13. dusp18 (Dual specificity Phosphatase 18) and mek-2 (Erk Kinase) are increased in CNC with MO13, while camK1 (Calcium/calmodulin-dependent protein kinase 1) and dap (Death-associated protein kinase 1) are decreased, with no significant effect from GFP-C13.(B) Histogram representation of quantitative PCR data. Amplification was performed from cDNA isolated from CNC of embryos injected with either MO13 or a combination of MO13, plus the cytoplasmic domains of ADAM9 (blue), ADAM13 (red), or ADAM19 (green). Values are represented as the Log2 fold change compared with MO13 alone. Error bars correspond to the SD between triplicates. (myt1: myelin transcription factor 1, fst: follistatin, nubp1: nucleotide binding protein 1, inta6: Integrin alpha 6).(C) Complementation assays with embryos injected with MO13 plus δCyto alone or with MMP13, Calpain8-a, or Xmc. CNC migration was also measured in embryos injected with a MO to Calpain8-a or a DN Calpain8-a construct (Capn8-aC105S). The total number of embryos is given (n). Error bars correspond to the SD. Statistical analysis was done using a Student's t test. Asterisk represents p values <0.01, for (A) or <0.05 for (B). Representative examples are given in (D).

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