Abbruzzese G et al. (2014), GSK3 and Polo-like kinase regulate ADAM13 funct...

XB-ART-51154

Mol Biol Cell 2014 Dec 15;2525:4072-82. doi: 10.1091/mbc.E14-05-0970.

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GSK3 and Polo-like kinase regulate ADAM13 function during cranial neural crest cell migration.

Abbruzzese G, Cousin H, Salicioni AM, Alfandari D.

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ADAMs are cell surface metalloproteases that control multiple biological processes by cleaving signaling and adhesion molecules. ADAM13 controls cranial neural crest (CNC) cell migration both by cleaving cadherin-11 to release a promigratory extracellular fragment and by controlling expression of multiple genes via its cytoplasmic domain. The latter activity is regulated by γ-secretase cleavage and the translocation of the cytoplasmic domain into the nucleus. One of the genes regulated by ADAM13, the protease calpain8, is essential for CNC migration. Although the nuclear function of ADAM13 is evolutionarily conserved, it is unclear whether the transcriptional regulation is also performed by other ADAMs and how this process may be regulated. We show that ADAM13 function to promote CNC migration is regulated by two phosphorylation events involving GSK3 and Polo-like kinase (Plk). We further show that inhibition of either kinase blocks CNC migration and that the respective phosphomimetic forms of ADAM13 can rescue these inhibitions. However, these phosphorylations are not required for ADAM13 proteolysis of its substrates, γ-secretase cleavage, or nuclear translocation of its cytoplasmic domain. Of significance, migration of the CNC can be restored in the absence of Plk phosphorylation by expression of calpain-8a, pointing to impaired nuclear activity of ADAM13.

???displayArticle.pubmedLink??? 25298404
???displayArticle.pmcLink??? PMC4263450
???displayArticle.link??? Mol Biol Cell
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Species referenced: Xenopus laevis
Genes referenced: adam10 adam13 adam17 adam19 adam9 cadm1 capn8 cdh11 gsk3a gsk3b itgb1 pcdh8.2 plk1 plk2 plk3 plk4 snai2 sox10 twist1
???displayArticle.antibodies??? Adam13 Ab1 Adam13 Ab2 Adam13 Ab3 Adam13 Ab4 Gsk3a/b Ab1 Itgb1 Ab1 Pcdh8.2 Ab1 phospho-Adam13 Ab5
???displayArticle.morpholinos??? adam13 MO3 adam19 MO2

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	FIGURE 1: Phosphorylation sites are required for ADAM13 function during CNC migration. (A) Fluorescence images showing representative embryos for the injections of RFP alone, the knockdown with 2MO, or the rescue with wild-type ADAM13. Arrows indicate the position of the three migration segments (from left to right: hyoid, branchial, mandibular). (B) Histogram of targeted injection assays testing the ability of ADAM13 phosphomutants to rescue CNC migration in ADAM13/19-deficient embryos (2MO) from at least three independent experiments. Values are percentages of embryos with no CNC migration, normalized to 2MO + wild-type ADAM13. The error bars correspond to the SD. n, number of embryos scored for each case. Statistical significance of rescue: p < 0.05, *p < 0.01.
	FIGURE 2: GSK3 and Plk can phosphorylate ADAM13. (A) Western blots for Plk1 (68 kDa) and GSK3Î² (47 kDa) on protein extract from 27 dissected CNC (CNC) or one total embryo equivalent at the same stage as dissection (Tot St17). (B) Western blot showing phosphorylation of ADAM13. The main forms of ADAM13 protein are represented at the top. The pro form (P) contains the prodomain and is inactive, the mature form (M) is proteolytically active, and cleavage of the metalloprotease domain results in a shorter form (D) that appears to be the principal phosphorylated form of ADAM13. Nuclear Cherry (nCherry, negative control), ADAM13 (A13), or the nonphosphorylatable mutant (A13-Plk/A) was expressed in HEK293T cells. Cells expressing ADAM13 were treated overnight with dimethyl sulfoxide (DMSO), Plk inhibitor (Plk Inh), or GSK3 inhibitor (Gsk Inh). All of the samples were first immunoprecipitated with a monoclonal antibody to ADAM13 (mAb 4A7), followed by Western blot using the phospho-ADAM13 antibody to the Plk phosphorylation site (P-A13) or the rabbit polyclonal to the ADAM13 cytoplasmic domain 15F for the total ADAM13 protein (Tot). (C) Autoradiograph of immunoÂprecipitated ADAM13 produced in HEK293T cells phosphorylated by purified active GSK3.
	FIGURE 3: GSK3 activity is critical for ADAM13 in CNC cell migration. (A) In situ hybridization using a probe to detect the CNC marker slug in neurula-stage embryos (st. 14), showing that induction is not affected by GSK3-DN. Injected sides of each embryo are on the left (asterisk). (B) Histogram representing the percentage of embryos with no CNC migration in a targeted injection assay from at least five independent experiments. Values are normalized to injection of RFP alone. Error bars are SD. n, number of embryos scored. p < 0.01, **p < 0.001. (C) Fluorescence images showing typical result for each case in the targeted injection assay in B. A defect in migration is scored by the absence of RFP-labeled cells within the migration pathway.
	FIGURE 4: ADAM13 requires Plk activity in the CNC. (A) Histogram showing the percentage of embryos with inhibited CNC migration in a targeted injection assay. Values are normalized to injection of GFP alone and are from at least three independent experiments. Error bars are SD. n, number of embryos scored. p < 0.05, *p < 0.01. (B) In situ hybridization detecting both of the CNC markers Sox10 and Twist in early tailbud embryos (st. 21), showing that migration, but not induction, is decreased by Plk-DN. Injected side of each embryo is on the left. (C) Analysis of CNC migration from the in situ hybridizations in B, showing the percentage of embryos for each case with severe, weak, or no defect in CNC migration on the injected side compared with the noninjected side of each embryo.
	FIGURE 5: Phosphorylation of ADAM13 does not affect its proteolytic activity. (A) Western blot from Cos-7 cells transfected with the protocadherin PAPC and various ADAM13 constructs. The glycoproteins purified from the conditioned media were probed with an antibody to the extracellular domain of PAPC or with 7C9, a monoclonal antibody to the cysteine-rich domain of ADAM13. E/A is a catalytically inactive variant of ADAM13. (B) The histogram represents the percentage of embryos displaying no migration from targeted injection of cadherin-11 mRNA alone or together with the ADAM13 variants. Values are normalized to RFP alone. Error bars are SD. n, number of embryos scored from at least three independent experiments. p < 0.05, *p < 0.01.
	FIGURE 6: ADAM13-dependent regulation of calpain8 expression depends on ADAM13 phosphorylation. (A) ADAM13 was immunoprecipitated (IP) from 20 embryos using a goat polyclonal antibody g821. Noninjected embryos (NI) at stage 18 (neurula) are compared with sibling embryos injected with MO13 or MO13 plus MO-resistant mRNA encoding ADAM13-Plk/A or wild type. ADAM13 protein was detected by Western blot using 6615F. The polyvinylidene fluoride membrane was cut below 25 kDa and the two halves probed separately. The cleaved cytoplasmic domain (Cyto) is observed at 17 kDa for both wild-type and Plk/A ADAM13. Samples of the input for the IP were analyzed by Western blot with the 8C8 antibody for Î²1 integrin as a loading control. (B) Fluorescence images showing the localization of GFP-fusion proteins (green) expressed in Cos-7 cells along with membrane-bound mCherry (red). GFP is observed uniformly throughout the cytoplasm and nucleus, whereas GFP-C13 and the phosphodeficient mutants all accumulate strongly in the nucleus. (C, D) Analyses of targeted injection assays displaying the loss of CNC migration as a percentage of embryos. (C) MO13 + MO19 (2MO) was coinjected with mRNA encoding ADAM13 lacking its cytoplasmic domain (Î´Cyto) plus either GFP-C13 wild type or phosphomutants. Values are normalized to the rescue with GFP-C13 wild type, and Student's t tests were performed to compare values to 2MO + Î´Cyto. (D) 2MO was injected with ADAM13-Plk/A or ADAM13-Gsk/A mRNA alone or together with Capn8 mRNA. Inhibitions are normalized to RFP, and Student's t tests were performed against 2MO. Error bars are SD from four or more independent experiments. n, number of embryos scored. p < 0.01, *p < 0.005.
	FIGURE 7: Successive phosphorylation of ADAM13. Proposed model depicting successive phosphorylation of ADAM13 by which GSK3 primes ADAM13 at two sites (S752 and S768; step 1) for subsequent phosphorylation at a second site on ADAM13 (T833; step 2). NLS, nuclear localization signal.
	Figure S1: Representative embryos from each targeted injection experiment are provided. For each condition, one embryo in which CNC are observed in the migration pathway and one in which they are absent are provided. Each embryo was injected at the 8-cell stage with a lineage tracer (RFP) in a dorsal animal blastomere that is predicted to contribute to the cranial neural crest cells.
	Figure S2: ISH of rescued embryos. Lateral views of tailbud stage embryos stained with Sox10 and Twist probes. The right panels correspond to the side injected with the mRNA (Aâ€™-Eâ€™). Embryos were injected at the one cell stage with MO13 and 19 and at the 8-cell stage in one dorsal animal blastomere with the various ADAM13 constructs and a fluorescent lineage tracer. Embryos were sorted using the lineage tracer (left/right/dorsal anterior expression), and processed for in situ hybridization using a combination of Sox10 and Twist probes. The lengths of CNC segments were measured on each side or each embryo. The percent rescue (green value) corresponds to the percentage of embryos with a 20% or more increase in segment length on the side injected with each mRNA compared to the contralateral side (2MO alone). The percentage of inhibition (red value) corresponds to a 20% decrease in the length of CNC segment on the side injected with each mRNA compared to the contralateral side. The total number of embryos scored from 3 independent experiments is: A13 N=59 (Aâ€™), A13-Plk/A N=51 (Bâ€™), A13-Plk/D N=64 (Câ€™), A13-Gsk/A N=52 (Dâ€™), A13-Gsk/D N=69 (Eâ€™). These data confirm that the phospho-mimetic mutants A13-Plk/D and A13-Gsk/D can rescue CNC positioning, while A13-Gsk/A does not. In this assay the A13-Plk/A provides a similar rescue as the wild type ADAM13, but also worsened CNC positioning more frequently (22%).
	Figure S3: ISH of grafted embryos. Embryos were injected at the 2-cell stage either with RFP alone or MO13, MO19 and A13-Plk/A and RFP mRNA. CNC were grafted at stage 15 into sibling non-injected host embryos and grown until tailbud stage as previously described (Alfandari et al., 2001). Fluorescent photographs were taken prior to fixation and ISH were performed with a combination of Sox10 and Twist probes.
	Figure S4: ISH versus lineage tracer. Variability of phenotypes in fluorescence-based methods to track CNC migration (grafts or targeted injection) and in situ hybridization methods to track CNC positioning (ISH). In the first method, cell position can be visualized in the same embryo over time to test cell migration. In the second method, embryos are fixed either before or after migration and stained for CNC markers giving the position of CNC. When reagents that inhibit migration of all CNC without affecting gene expression are used, the results from both techniques are similar. On the other hand, when reagents inhibit a subset of CNC and the cells that are inhibited turn off CNC markers, the inhibition is obvious in lineage tracer experiments but not with ISH techniques. One example of ADAM13 and 19 inhibition is given on the right. The majority of grafted CNC (red) did not migrate (yellow oval), but a small percentage of cells can be seen to migrate in spite of the absence of ADAM13 and 19 (white arrowhead). The ISH of the same embryo reveals a weaker but normal positioning of CNC. Note that the majority of the cells that did not migrate are not obviously stained by ISH.
	FIGURE 1:. Phosphorylation sites are required for ADAM13 function during CNC migration. (A) Fluorescence images showing representative embryos for the injections of RFP alone, the knockdown with 2MO, or the rescue with wild-type ADAM13. Arrows indicate the position of the three migration segments (from left to right: hyoid, branchial, mandibular). (B) Histogram of targeted injection assays testing the ability of ADAM13 phosphomutants to rescue CNC migration in ADAM13/19-deficient embryos (2MO) from at least three independent experiments. Values are percentages of embryos with no CNC migration, normalized to 2MO + wild-type ADAM13. The error bars correspond to the SD. n, number of embryos scored for each case. Statistical significance of rescue: p < 0.05, *p < 0.01.
	FIGURE 2:. GSK3 and Plk can phosphorylate ADAM13. (A) Western blots for Plk1 (68 kDa) and GSK3β (47 kDa) on protein extract from 27 dissected CNC (CNC) or one total embryo equivalent at the same stage as dissection (Tot St17). (B) Western blot showing phosphorylation of ADAM13. The main forms of ADAM13 protein are represented at the top. The pro form (P) contains the prodomain and is inactive, the mature form (M) is proteolytically active, and cleavage of the metalloprotease domain results in a shorter form (D) that appears to be the principal phosphorylated form of ADAM13. Nuclear Cherry (nCherry, negative control), ADAM13 (A13), or the nonphosphorylatable mutant (A13-Plk/A) was expressed in HEK293T cells. Cells expressing ADAM13 were treated overnight with dimethyl sulfoxide (DMSO), Plk inhibitor (Plk Inh), or GSK3 inhibitor (Gsk Inh). All of the samples were first immunoprecipitated with a monoclonal antibody to ADAM13 (mAb 4A7), followed by Western blot using the phospho-ADAM13 antibody to the Plk phosphorylation site (P-A13) or the rabbit polyclonal to the ADAM13 cytoplasmic domain 15F for the total ADAM13 protein (Tot). (C) Autoradiograph of immunoprecipitated ADAM13 produced in HEK293T cells phosphorylated by purified active GSK3.
	FIGURE 3:. GSK3 activity is critical for ADAM13 in CNC cell migration. (A) In situ hybridization using a probe to detect the CNC marker slug in neurula-stage embryos (st. 14), showing that induction is not affected by GSK3-DN. Injected sides of each embryo are on the left (asterisk). (B) Histogram representing the percentage of embryos with no CNC migration in a targeted injection assay from at least five independent experiments. Values are normalized to injection of RFP alone. Error bars are SD. n, number of embryos scored. p < 0.01, **p < 0.001. (C) Fluorescence images showing typical result for each case in the targeted injection assay in B. A defect in migration is scored by the absence of RFP-labeled cells within the migration pathway.
	FIGURE 4:. ADAM13 requires Plk activity in the CNC. (A) Histogram showing the percentage of embryos with inhibited CNC migration in a targeted injection assay. Values are normalized to injection of GFP alone and are from at least three independent experiments. Error bars are SD. n, number of embryos scored. p < 0.05, *p < 0.01. (B) In situ hybridization detecting both of the CNC markers Sox10 and Twist in early tailbud embryos (st. 21), showing that migration, but not induction, is decreased by Plk-DN. Injected side of each embryo is on the left. (C) Analysis of CNC migration from the in situ hybridizations in B, showing the percentage of embryos for each case with severe, weak, or no defect in CNC migration on the injected side compared with the noninjected side of each embryo.
	FIGURE 5:. Phosphorylation of ADAM13 does not affect its proteolytic activity. (A) Western blot from Cos-7 cells transfected with the protocadherin PAPC and various ADAM13 constructs. The glycoproteins purified from the conditioned media were probed with an antibody to the extracellular domain of PAPC or with 7C9, a monoclonal antibody to the cysteine-rich domain of ADAM13. E/A is a catalytically inactive variant of ADAM13. (B) The histogram represents the percentage of embryos displaying no migration from targeted injection of cadherin-11 mRNA alone or together with the ADAM13 variants. Values are normalized to RFP alone. Error bars are SD. n, number of embryos scored from at least three independent experiments. p < 0.05, *p < 0.01.
	FIGURE 6:. ADAM13-dependent regulation of calpain8 expression depends on ADAM13 phosphorylation. (A) ADAM13 was immunoprecipitated (IP) from 20 embryos using a goat polyclonal antibody g821. Noninjected embryos (NI) at stage 18 (neurula) are compared with sibling embryos injected with MO13 or MO13 plus MO-resistant mRNA encoding ADAM13-Plk/A or wild type. ADAM13 protein was detected by Western blot using 6615F. The polyvinylidene fluoride membrane was cut below 25 kDa and the two halves probed separately. The cleaved cytoplasmic domain (Cyto) is observed at 17 kDa for both wild-type and Plk/A ADAM13. Samples of the input for the IP were analyzed by Western blot with the 8C8 antibody for β1 integrin as a loading control. (B) Fluorescence images showing the localization of GFP-fusion proteins (green) expressed in Cos-7 cells along with membrane-bound mCherry (red). GFP is observed uniformly throughout the cytoplasm and nucleus, whereas GFP-C13 and the phosphodeficient mutants all accumulate strongly in the nucleus. (C, D) Analyses of targeted injection assays displaying the loss of CNC migration as a percentage of embryos. (C) MO13 + MO19 (2MO) was coinjected with mRNA encoding ADAM13 lacking its cytoplasmic domain (ΔCyto) plus either GFP-C13 wild type or phosphomutants. Values are normalized to the rescue with GFP-C13 wild type, and Student's t tests were performed to compare values to 2MO + ΔCyto. (D) 2MO was injected with ADAM13-Plk/A or ADAM13-Gsk/A mRNA alone or together with Capn8 mRNA. Inhibitions are normalized to RFP, and Student's t tests were performed against 2MO. Error bars are SD from four or more independent experiments. n, number of embryos scored. p < 0.01, *p < 0.005.
	FIGURE 7:. Successive phosphorylation of ADAM13. Proposed model depicting successive phosphorylation of ADAM13 by which GSK3 primes ADAM13 at two sites (S752 and S768; step 1) for subsequent phosphorylation at a second site on ADAM13 (T833; step 2). NLS, nuclear localization signal.

References [+] :

Abu-Elmagd, Frizzled7 mediates canonical Wnt signaling in neural crest induction. 2006, Pubmed, Xenbase

	FIGURE 1: Phosphorylation sites are required for ADAM13 function during CNC migration. (A) Fluorescence images showing representative embryos for the injections of RFP alone, the knockdown with 2MO, or the rescue with wild-type ADAM13. Arrows indicate the position of the three migration segments (from left to right: hyoid, branchial, mandibular). (B) Histogram of targeted injection assays testing the ability of ADAM13 phosphomutants to rescue CNC migration in ADAM13/19-deficient embryos (2MO) from at least three independent experiments. Values are percentages of embryos with no CNC migration, normalized to 2MO + wild-type ADAM13. The error bars correspond to the SD. n, number of embryos scored for each case. Statistical significance of rescue: p < 0.05, *p < 0.01.
	FIGURE 2: GSK3 and Plk can phosphorylate ADAM13. (A) Western blots for Plk1 (68 kDa) and GSK3Î² (47 kDa) on protein extract from 27 dissected CNC (CNC) or one total embryo equivalent at the same stage as dissection (Tot St17). (B) Western blot showing phosphorylation of ADAM13. The main forms of ADAM13 protein are represented at the top. The pro form (P) contains the prodomain and is inactive, the mature form (M) is proteolytically active, and cleavage of the metalloprotease domain results in a shorter form (D) that appears to be the principal phosphorylated form of ADAM13. Nuclear Cherry (nCherry, negative control), ADAM13 (A13), or the nonphosphorylatable mutant (A13-Plk/A) was expressed in HEK293T cells. Cells expressing ADAM13 were treated overnight with dimethyl sulfoxide (DMSO), Plk inhibitor (Plk Inh), or GSK3 inhibitor (Gsk Inh). All of the samples were first immunoprecipitated with a monoclonal antibody to ADAM13 (mAb 4A7), followed by Western blot using the phospho-ADAM13 antibody to the Plk phosphorylation site (P-A13) or the rabbit polyclonal to the ADAM13 cytoplasmic domain 15F for the total ADAM13 protein (Tot). (C) Autoradiograph of immunoÂprecipitated ADAM13 produced in HEK293T cells phosphorylated by purified active GSK3.
	FIGURE 3: GSK3 activity is critical for ADAM13 in CNC cell migration. (A) In situ hybridization using a probe to detect the CNC marker slug in neurula-stage embryos (st. 14), showing that induction is not affected by GSK3-DN. Injected sides of each embryo are on the left (asterisk). (B) Histogram representing the percentage of embryos with no CNC migration in a targeted injection assay from at least five independent experiments. Values are normalized to injection of RFP alone. Error bars are SD. n, number of embryos scored. p < 0.01, **p < 0.001. (C) Fluorescence images showing typical result for each case in the targeted injection assay in B. A defect in migration is scored by the absence of RFP-labeled cells within the migration pathway.
	FIGURE 4: ADAM13 requires Plk activity in the CNC. (A) Histogram showing the percentage of embryos with inhibited CNC migration in a targeted injection assay. Values are normalized to injection of GFP alone and are from at least three independent experiments. Error bars are SD. n, number of embryos scored. p < 0.05, *p < 0.01. (B) In situ hybridization detecting both of the CNC markers Sox10 and Twist in early tailbud embryos (st. 21), showing that migration, but not induction, is decreased by Plk-DN. Injected side of each embryo is on the left. (C) Analysis of CNC migration from the in situ hybridizations in B, showing the percentage of embryos for each case with severe, weak, or no defect in CNC migration on the injected side compared with the noninjected side of each embryo.
	FIGURE 5: Phosphorylation of ADAM13 does not affect its proteolytic activity. (A) Western blot from Cos-7 cells transfected with the protocadherin PAPC and various ADAM13 constructs. The glycoproteins purified from the conditioned media were probed with an antibody to the extracellular domain of PAPC or with 7C9, a monoclonal antibody to the cysteine-rich domain of ADAM13. E/A is a catalytically inactive variant of ADAM13. (B) The histogram represents the percentage of embryos displaying no migration from targeted injection of cadherin-11 mRNA alone or together with the ADAM13 variants. Values are normalized to RFP alone. Error bars are SD. n, number of embryos scored from at least three independent experiments. p < 0.05, *p < 0.01.
	FIGURE 6: ADAM13-dependent regulation of calpain8 expression depends on ADAM13 phosphorylation. (A) ADAM13 was immunoprecipitated (IP) from 20 embryos using a goat polyclonal antibody g821. Noninjected embryos (NI) at stage 18 (neurula) are compared with sibling embryos injected with MO13 or MO13 plus MO-resistant mRNA encoding ADAM13-Plk/A or wild type. ADAM13 protein was detected by Western blot using 6615F. The polyvinylidene fluoride membrane was cut below 25 kDa and the two halves probed separately. The cleaved cytoplasmic domain (Cyto) is observed at 17 kDa for both wild-type and Plk/A ADAM13. Samples of the input for the IP were analyzed by Western blot with the 8C8 antibody for Î²1 integrin as a loading control. (B) Fluorescence images showing the localization of GFP-fusion proteins (green) expressed in Cos-7 cells along with membrane-bound mCherry (red). GFP is observed uniformly throughout the cytoplasm and nucleus, whereas GFP-C13 and the phosphodeficient mutants all accumulate strongly in the nucleus. (C, D) Analyses of targeted injection assays displaying the loss of CNC migration as a percentage of embryos. (C) MO13 + MO19 (2MO) was coinjected with mRNA encoding ADAM13 lacking its cytoplasmic domain (Î´Cyto) plus either GFP-C13 wild type or phosphomutants. Values are normalized to the rescue with GFP-C13 wild type, and Student's t tests were performed to compare values to 2MO + Î´Cyto. (D) 2MO was injected with ADAM13-Plk/A or ADAM13-Gsk/A mRNA alone or together with Capn8 mRNA. Inhibitions are normalized to RFP, and Student's t tests were performed against 2MO. Error bars are SD from four or more independent experiments. n, number of embryos scored. p < 0.01, *p < 0.005.
	FIGURE 7: Successive phosphorylation of ADAM13. Proposed model depicting successive phosphorylation of ADAM13 by which GSK3 primes ADAM13 at two sites (S752 and S768; step 1) for subsequent phosphorylation at a second site on ADAM13 (T833; step 2). NLS, nuclear localization signal.
	Figure S1: Representative embryos from each targeted injection experiment are provided. For each condition, one embryo in which CNC are observed in the migration pathway and one in which they are absent are provided. Each embryo was injected at the 8-cell stage with a lineage tracer (RFP) in a dorsal animal blastomere that is predicted to contribute to the cranial neural crest cells.
	Figure S2: ISH of rescued embryos. Lateral views of tailbud stage embryos stained with Sox10 and Twist probes. The right panels correspond to the side injected with the mRNA (Aâ€™-Eâ€™). Embryos were injected at the one cell stage with MO13 and 19 and at the 8-cell stage in one dorsal animal blastomere with the various ADAM13 constructs and a fluorescent lineage tracer. Embryos were sorted using the lineage tracer (left/right/dorsal anterior expression), and processed for in situ hybridization using a combination of Sox10 and Twist probes. The lengths of CNC segments were measured on each side or each embryo. The percent rescue (green value) corresponds to the percentage of embryos with a 20% or more increase in segment length on the side injected with each mRNA compared to the contralateral side (2MO alone). The percentage of inhibition (red value) corresponds to a 20% decrease in the length of CNC segment on the side injected with each mRNA compared to the contralateral side. The total number of embryos scored from 3 independent experiments is: A13 N=59 (Aâ€™), A13-Plk/A N=51 (Bâ€™), A13-Plk/D N=64 (Câ€™), A13-Gsk/A N=52 (Dâ€™), A13-Gsk/D N=69 (Eâ€™). These data confirm that the phospho-mimetic mutants A13-Plk/D and A13-Gsk/D can rescue CNC positioning, while A13-Gsk/A does not. In this assay the A13-Plk/A provides a similar rescue as the wild type ADAM13, but also worsened CNC positioning more frequently (22%).
	Figure S3: ISH of grafted embryos. Embryos were injected at the 2-cell stage either with RFP alone or MO13, MO19 and A13-Plk/A and RFP mRNA. CNC were grafted at stage 15 into sibling non-injected host embryos and grown until tailbud stage as previously described (Alfandari et al., 2001). Fluorescent photographs were taken prior to fixation and ISH were performed with a combination of Sox10 and Twist probes.
	Figure S4: ISH versus lineage tracer. Variability of phenotypes in fluorescence-based methods to track CNC migration (grafts or targeted injection) and in situ hybridization methods to track CNC positioning (ISH). In the first method, cell position can be visualized in the same embryo over time to test cell migration. In the second method, embryos are fixed either before or after migration and stained for CNC markers giving the position of CNC. When reagents that inhibit migration of all CNC without affecting gene expression are used, the results from both techniques are similar. On the other hand, when reagents inhibit a subset of CNC and the cells that are inhibited turn off CNC markers, the inhibition is obvious in lineage tracer experiments but not with ISH techniques. One example of ADAM13 and 19 inhibition is given on the right. The majority of grafted CNC (red) did not migrate (yellow oval), but a small percentage of cells can be seen to migrate in spite of the absence of ADAM13 and 19 (white arrowhead). The ISH of the same embryo reveals a weaker but normal positioning of CNC. Note that the majority of the cells that did not migrate are not obviously stained by ISH.
	FIGURE 1:. Phosphorylation sites are required for ADAM13 function during CNC migration. (A) Fluorescence images showing representative embryos for the injections of RFP alone, the knockdown with 2MO, or the rescue with wild-type ADAM13. Arrows indicate the position of the three migration segments (from left to right: hyoid, branchial, mandibular). (B) Histogram of targeted injection assays testing the ability of ADAM13 phosphomutants to rescue CNC migration in ADAM13/19-deficient embryos (2MO) from at least three independent experiments. Values are percentages of embryos with no CNC migration, normalized to 2MO + wild-type ADAM13. The error bars correspond to the SD. n, number of embryos scored for each case. Statistical significance of rescue: p < 0.05, *p < 0.01.
	FIGURE 2:. GSK3 and Plk can phosphorylate ADAM13. (A) Western blots for Plk1 (68 kDa) and GSK3β (47 kDa) on protein extract from 27 dissected CNC (CNC) or one total embryo equivalent at the same stage as dissection (Tot St17). (B) Western blot showing phosphorylation of ADAM13. The main forms of ADAM13 protein are represented at the top. The pro form (P) contains the prodomain and is inactive, the mature form (M) is proteolytically active, and cleavage of the metalloprotease domain results in a shorter form (D) that appears to be the principal phosphorylated form of ADAM13. Nuclear Cherry (nCherry, negative control), ADAM13 (A13), or the nonphosphorylatable mutant (A13-Plk/A) was expressed in HEK293T cells. Cells expressing ADAM13 were treated overnight with dimethyl sulfoxide (DMSO), Plk inhibitor (Plk Inh), or GSK3 inhibitor (Gsk Inh). All of the samples were first immunoprecipitated with a monoclonal antibody to ADAM13 (mAb 4A7), followed by Western blot using the phospho-ADAM13 antibody to the Plk phosphorylation site (P-A13) or the rabbit polyclonal to the ADAM13 cytoplasmic domain 15F for the total ADAM13 protein (Tot). (C) Autoradiograph of immunoprecipitated ADAM13 produced in HEK293T cells phosphorylated by purified active GSK3.
	FIGURE 3:. GSK3 activity is critical for ADAM13 in CNC cell migration. (A) In situ hybridization using a probe to detect the CNC marker slug in neurula-stage embryos (st. 14), showing that induction is not affected by GSK3-DN. Injected sides of each embryo are on the left (asterisk). (B) Histogram representing the percentage of embryos with no CNC migration in a targeted injection assay from at least five independent experiments. Values are normalized to injection of RFP alone. Error bars are SD. n, number of embryos scored. p < 0.01, **p < 0.001. (C) Fluorescence images showing typical result for each case in the targeted injection assay in B. A defect in migration is scored by the absence of RFP-labeled cells within the migration pathway.
	FIGURE 4:. ADAM13 requires Plk activity in the CNC. (A) Histogram showing the percentage of embryos with inhibited CNC migration in a targeted injection assay. Values are normalized to injection of GFP alone and are from at least three independent experiments. Error bars are SD. n, number of embryos scored. p < 0.05, *p < 0.01. (B) In situ hybridization detecting both of the CNC markers Sox10 and Twist in early tailbud embryos (st. 21), showing that migration, but not induction, is decreased by Plk-DN. Injected side of each embryo is on the left. (C) Analysis of CNC migration from the in situ hybridizations in B, showing the percentage of embryos for each case with severe, weak, or no defect in CNC migration on the injected side compared with the noninjected side of each embryo.
	FIGURE 5:. Phosphorylation of ADAM13 does not affect its proteolytic activity. (A) Western blot from Cos-7 cells transfected with the protocadherin PAPC and various ADAM13 constructs. The glycoproteins purified from the conditioned media were probed with an antibody to the extracellular domain of PAPC or with 7C9, a monoclonal antibody to the cysteine-rich domain of ADAM13. E/A is a catalytically inactive variant of ADAM13. (B) The histogram represents the percentage of embryos displaying no migration from targeted injection of cadherin-11 mRNA alone or together with the ADAM13 variants. Values are normalized to RFP alone. Error bars are SD. n, number of embryos scored from at least three independent experiments. p < 0.05, *p < 0.01.
	FIGURE 6:. ADAM13-dependent regulation of calpain8 expression depends on ADAM13 phosphorylation. (A) ADAM13 was immunoprecipitated (IP) from 20 embryos using a goat polyclonal antibody g821. Noninjected embryos (NI) at stage 18 (neurula) are compared with sibling embryos injected with MO13 or MO13 plus MO-resistant mRNA encoding ADAM13-Plk/A or wild type. ADAM13 protein was detected by Western blot using 6615F. The polyvinylidene fluoride membrane was cut below 25 kDa and the two halves probed separately. The cleaved cytoplasmic domain (Cyto) is observed at 17 kDa for both wild-type and Plk/A ADAM13. Samples of the input for the IP were analyzed by Western blot with the 8C8 antibody for β1 integrin as a loading control. (B) Fluorescence images showing the localization of GFP-fusion proteins (green) expressed in Cos-7 cells along with membrane-bound mCherry (red). GFP is observed uniformly throughout the cytoplasm and nucleus, whereas GFP-C13 and the phosphodeficient mutants all accumulate strongly in the nucleus. (C, D) Analyses of targeted injection assays displaying the loss of CNC migration as a percentage of embryos. (C) MO13 + MO19 (2MO) was coinjected with mRNA encoding ADAM13 lacking its cytoplasmic domain (ΔCyto) plus either GFP-C13 wild type or phosphomutants. Values are normalized to the rescue with GFP-C13 wild type, and Student's t tests were performed to compare values to 2MO + ΔCyto. (D) 2MO was injected with ADAM13-Plk/A or ADAM13-Gsk/A mRNA alone or together with Capn8 mRNA. Inhibitions are normalized to RFP, and Student's t tests were performed against 2MO. Error bars are SD from four or more independent experiments. n, number of embryos scored. p < 0.01, *p < 0.005.
	FIGURE 7:. Successive phosphorylation of ADAM13. Proposed model depicting successive phosphorylation of ADAM13 by which GSK3 primes ADAM13 at two sites (S752 and S768; step 1) for subsequent phosphorylation at a second site on ADAM13 (T833; step 2). NLS, nuclear localization signal.