Cousin H , Abbruzzese G , McCusker C , Alfandari D .

DE016289 NIDCR NIH HHS, R01 DE016289 NIDCR NIH HHS

	Fig. 1. ADAM13 knock down in the neural tube does not affect morphant crest migration. (A) Embryos were injected with mRNA encoding GFP alone (Ctl) or together with antisense morpholino for ADAM13 alone (MO13) or ADAM13 and 19 (2MO). The compounds were injected either in one cell at the 2-cell stage (for graft) or in one dorso-animal cell at the 8–16 cell stage (for targeted injection). The graft procedure was carried out at stage 15 and the embryos were scored for their lack of CNC migration at tailbud stage (26–28) for both assays. The pictures show the typical result obtained for each case. The histogram represents the percentage of embryos with no CNC migration normalized to embryos injected with GFP alone and corresponds to the mean of seven (targeted injection) and at least six (graft) independent experiments. The number of embryos scored is as follows. For grafts: Ctl; n=42, MO13; n=25, 2MO; n=61. For targeted injection: Ctl; n=248, MO13; n=258, 2MO; n=281. While 2MO inhibits CNC migration at similar levels for both the grafts and the targeted injection assays, MO13 alone is 5 times more efficient at inhibiting CNC migration in the targeted injection assay than the graft assay. (B) Embryos were injected in one cell at the 2-cell stage with either GFP (Ctl donor), GFP and MO13 (MO13 donor) or RFP and MO13 (MO13 recipient). At stage 15, three types of grafts were performed: 1-GFP expressing CNC were grafted into non-injected embryos (Ctl->NI), 2-GFP+MO13 injected CNC were grafted into non-injected embryos (MO13->NI), 3-GFP+MO13 injected CNC were grafted into embryos injected with RFP+MO13 (MO13->MO13). The pictures show the typical result obtained for each case. The histogram represents the percentage of embryos with no CNC migration normalized to embryos injected with GFP alone and corresponds to the mean of three independent experiments, representing the scoring of 20 embryos for each case. Results show that morphant CNC display a statistically identical level of migration inhibition whether they are grafted into a wild type embryo or a morphant embryo. The error bar represents standard deviation from the mean. ⁎=Statistically different from controls (p<0.05). Arrowheads point to the segments of the CNC. m: mandibular, h: hyoid, b1 and b2: branchial.
	Fig. 2. Manually separating CNC from the surrounding tissue partially rescues CNC migration induced by ADAM13 knock down. (A) Schematics of the experimental procedure. Either GFP mRNA alone, or a mixture of GFP mRNA and ADAM13 morpholino was injected into the dorso-animal cell at the 8-cell stage. At stage 15, embryos were sorted for their expression of GFP and underwent one of the following procedures. 1—The ectoderm was peeled off (ecto peel). 2—The ectoderm was peeled off and the crest lifted away from the underlying mesoderm (ecto+meso peel). 3—The CNC were excised from the embryo and placed back in (autograft). 4—After the procedure, the ectoderm was pulled back on and left to heal. The embryo was left to develop until tailbud stage and scored for their lack of CNC migration. (B) The histogram represents the percentage of embryos with no CNC migration normalized to embryos injected with GFP alone and corresponds to the mean of three independent experiments. As previously reported, ADAM13 KD significantly inhibits CNC migration in the targeted injection assay. The ectoderm peel procedure did not affect this inhibition. However, the ectoderm+mesoderm peel and the autograft procedures rescued CNC migration to levels not statistically different from the GFP control. The number of embryos scored was as follows: GFP; n=159, MO13; n=154, MO13 ecto peel; n=47; MO13; n=45; MO13 autograft; n=43. The error bar represents standard deviation from the mean. ⁎=Statistically different from GFP (p<0.05).
	Fig. 3. ADAM13 function is dispensable for CNC migration in vitro. (A) ADAM13 and 19 knock down has no effect on CNC migration in vitro. Embryos were injected in one cell at the two-cell stage with a mixture of GFP mRNA and either control morpholino (Ctl), ADAM13 morpholino (MO13) or ADAM13 and 19 morpholino (2MO). At stage 15, embryos were sorted and CNC explants placed on fibronectin-coated 96-well plates. For each case, pictures of typical explants are shown before migration (t=0), at the end of the sheet migration phase (t=6 h) and at the end of the single cell migration phase (t=16 h). Morphant CNC cells were capable of migrating in vitro in a pattern indistinguishable from the control CNC. (B) ADAM13 knock down in CNC is efficient and prevents Cadherin-11 cleavage. Twenty CNC were dissected at stage 15 and placed on FN substrate for 2 h before cell surface proteins were biotinylated. Proteins were extracted and immunoprecipitated sequentially using a goat anti-ADAM13 antibody (gA13), the mouse mAb to Cadherin-11 (1B4) and the mouse mAb to integrin β1 (8C8). Both gA13 and 1B4 recognize the cytodomains of ADAM13 and Cadherin-11, respectively. ADAM13 knock down completely abolishes the expression mature ADAM13 at the cell surface (ADAM13, arrowhead). The full length Cadherin-11 protein is still strongly expressed (Cad-11, arrowhead a) but is no longer cleaved (Cad-11, arrowhead b). Integrin α5β1 cell-surface expression is unaffected by ADAM13 knockdown.
	Fig. 4. ADAM13 function is not cell autonomous. Embryos were injected in one cell of two-cell stage embryos with both GFP mRNA and morpholino against ADAM13 and 19 (GFP+2MO) or with RFP mRNA. At stage 15, CNC expressing the lineage tracer were dissected and grafted into non-injected embryos in either one of the following configurations. Either the RFP expressing explant was grafted ventrally to the morphant crest (Wt leader and 2MO follower) or the RFP expressing crest was grafted dorsally to the morphant crest (Wt follower and 2MO leader). Pictures represent a typical example of each type of grafted embryo before (stage 15) and after migration (stage 26). The histogram represents the percentage of failed migration of each type of CNC (Wt or 2MO) for each grafting configuration. Controls (Ctl) correspond to the graft of GFP expressing CNC alone. Student t-tests between the 2MO CNC grafted alone and all other experimental cases were performed. The experiments show that the co-grafting of wild type CNC significantly rescues morphant CNC migration. The configuration of the graft has no impact on the efficiency of the rescue. The co-grafted morphant CNC had no effect on the ability of wild type CNC to migrate. The histogram represents the average of 7 independent experiments. The number of embryos scored was as follows: Ctl; n=42, 2MO alone; n=40, 2MO leader+Wt follower; n=38; 2MO follower+Wt leader; n=33 n=47. The error bar represents standard deviation from the mean. *=Statistically different from 2MO alone (p<0.05).
	Fig. 5. ADAM 13 metalloprotease function is required crest autonomously. Embryos were injected in one cell of 2-cell stage embryos with the following mixture. Donor embryos were injected with RFP as a lineage tracer and either the morpholinos against ADAM13 and 19 alone (2MO) or the two morpholinos plus the mRNA encoding the cytoplasmic domain of ADAM13 (2MO+C13). The recipient embryos were injected with a mixture of GFP and either a form of ADAM13 lacking the cytoplasmic domain (δCyto) or the cleaved form of Cadherin-11 (EC1-3). At stage 15, embryos were sorted for their expression of lineage tracer, CNC explants taken out from donor embryos (RFP, 2MO or 2MO+C13) and grafted into various recipient embryos (NI, δCyto or EC1-3). The embryo was left to develop until tailbud stage and scored for their lack of CNC migration. The histogram represents the percentage of embryos displaying no CNC migration normalized to embryos injected with RFP alone and corresponds to the mean of three independent experiments. Results show that morphant crest migration is not rescued if the metalloprotease activity of ADAM13 is not expressed by the CNC themselves (2MO->δCyto). The expression of the cytodomain of ADAM13 in the morphant crest partially rescues CNC migration (2MO+C13->NI). This partial rescue is not enhanced when these CNC are grafted in embryos expressing δCyto (2MO+C13->δCyto) confirming that ADAM13 can exert its metalloprotease function only when expressed by the CNC. The rescue is complete when the 2MO+C13 CNC are grafted in embryos expressing EC1-3 (2MO+C13->EC1-3). The number of embryos scored was as follows: RFP->NI; n=15, 2MO->NI; n=14, 2MO->δCyto; n=16, 2MO+C13->δCyto; n=15. The error bar represents standard deviation from the mean. *=Statistically different from 2MO->NI (p<0.05).
	Fig. 6. The partial knock down of Cadherin-11 does not rescue CNC migration of ADAM13 morphants. Embryos were injected at the 1 cell stage with either ADAM13 morpholino (MO13 1 ng) alone or together with various doses of morpholino against Cadherin-11 (MOC11) ranging from 5 ng to 25 ng. At stage 20, the proteins of 10 embryos equivalent were extracted, immunoprecipitated with either the cadherin-11 mAb 1B4 or ADAM13 rabbit pAb 6615F and blotted with the cadherin rabbit pAb C11 and the ADAM13 goat pAb 877 respectively. The knock down of ADAM13 alone leads to an increase of the full-length form of Cadherin-11 that can be decreased by a partial cadherin-11 knock down using 5 ng of MOC11. (B) Embryos were injected at the 8-cell stage with either ADAM13 morpholino (MO13 1 ng) alone or together with various doses of morpholino against Cadherin-11 (MOC11) ranging from 1 ng to 5 ng. The mRNA encoding GFP was either co-injected or injected alone as lineage tracer. Embryos were raised until stage 28 and scored for their lack of CNC migration. The histogram represents the percentage of embryos displaying no CNC migration normalized to embryos injected with RFP alone and corresponds to the mean of four independent experiments. The partial knock down of Cadherin-11 with either 1 or 2.5 ng of MOC11 did not significantly rescue the inhibition of CNC migration induced by ADAM13 knock down. The co-injection of 5 ng of MOC11 produced a significantly more severe inhibition than ADAM13 knock down alone. The error bars represent standard deviation from the mean. The number of embryos scored for each case ranged between 207 and 218. *=Statistically different from MO13 (p<0.05).
	Fig. 7. CNC cells can migrate through 20 μm openings. Migration of CNC through small openings. Photographs of explants (10×) taken from time-lapse movies are represented. The width of the obstacle is 200 μm (horizontal arrows) while the opening is 20 μm. Frames from non-injected control (NI) or morphant (MO13) CNC are presented at 0, 3 and 6 h of migration. The arrowhead points to the leading edge of cells migrating in the tunnel. The mask used to obtain the substrates is represented below. The white sections represent the walls of the chambers (200 μm high). Each of the two rectangular chambers has four opening for each of their long side ranging from 15 to 30 μm). The center of the rectangles are filled with SDF-1-coated beads and sealed by covering it with a small coverslip fixed with vacuum grease. Explants are placed with their ventral (leading) edge facing each opening.

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