Garmon T , Wittling M , Nie S .

R00 DE022796 NIH HHS , R00 DE022796 NIDCR NIH HHS, R00 DE022796 GF

             metallopeptidase activity

	Figure 2. MMP14 regulates the migration of cranial neural crest cells. (A) Embryos receiving MMP14 RNA (0.5ng), MMP14-MO (15ng), or both in one of the dorsal animal cells were fixed at early tailbud stages and the migration of neural crest cells was examined by the expression of neural crest marker gene Sox10. While MMP14 overexpression led to premature migration of neural crest cells (arrowheads; n=15/25), loss of MMP14 resulted in reduced neural crest migration (arrow; n=16/27. This a e resued  oepressig MMP RNA ithout the ’-UTR (0.2ng; n=16/20). When MMP14 was inhibited by a specific inhibitor NSC405020 (1mM), the migration of neural crest cells was also impaired (arrow; n=15/24). (B) The efficiency of MMP14-MO was validated. 0.5ng of (UTR)MMP14-mCherry or MMP14-mCherry was coinjected with 15ng of MMP14-MO into 2-cell stage embryos. The translation of (UTR)-MMP14-mCherry, but not MMP14-mCherry was efficiently inhibited by MMP14-MO.
	Figure 3. MMP14 activity affected the gelatin degradation by neural crest cells. Cranial neural crest explants were dissected and plated on Cy3-labelled gelatin matrix and their ability in gelatin degradation was analyzed 40 hours later. (A) Fluorescent, DIC, and merged images are shown. While adding MMP14 promoted gelatin degradation, addition of MMP14-MO alone or together with chemical inhibitors impaired the degradation of gelatin matrix. Objective, 40x. (B) The relative area of matrix degradation was compared with total cell area. 9-16 explants from two experimental repeats were summarized. 15ng of MMP14-MO decreases the relative degradation area significantly. While the addition of 0.2mM NSC405020 did not change the rate of matrix degradation, the addition of 0.2mM Prinomastat further impaired the degradation of gelatin matrix.
	Figure 4. MMP14 is required cell-autonomously for cranial neural crest migration. (A-H) Grafting experiment was performed to determine where MMP14 and MMP2 are required for neural crest cell migration. The heads of grafted embryos were shown with anterior to the right with the source of the donor NC and the host embryo indicated. The color of the text indicates whether EGFP-CAAX (green) or mCherry (red) was used as a lineage tracer. The results showed that the migration of cranial NCCs was affected only when MMP14 was knocked down in cranial neural crest and/or when MMP2 was knocked down in head mesenchyme. (I) The farthest distance of neural crest cell migration in each transplanted embryo was measured relative to the entire D-V axis of the embryo and summarized in the bar graph. While MMP14-MO in neural crest cells or MMP2-MO in cranial mesenchyme reduced the relative distance of migration to 28.5% or 62.1%, MMP14-MO in neural crest cells and MMP2-MO in mesenchyme together reduced the relative distance of neural crest migration to 18.9%. Error bars, s.d. Student t-test was performed and * marked conditions with significantly reduced neural crest migration (p<0.01).
	Figure 5. MMP14 regulates neural crest EMT in culture. (A) Cranial neural crest explants were dissected at stage 13 and cultured on FN-coated coverslip. The migration of neural crest explants was followed by photograph at sibling stages 13, 20, and 24 for premigratory, onset of migration, and late migration stages. Control explants spread at stage 20 and migrated extensively both as a coherent sheet and as individual cells at stage 24. Overexpression of MMP14 led to further migration and cell separation at stage 20, while MMP14-MO led to delayed spreading and migration. Both MMP14-MO and pan-MMP inhibitor Prinomastat (0.2mM) inhibited the separation of neural crest cells from each other. (B) The ratio of neural crest explant spreading was calculated by comparing the area it covered at the end vs. at the start of the experiment. Error bars, s.d.
	Developmental DynamicsFigure 4. MMP14 is required cell-autonomously for cranial neural crest migration. (A-H) Grafting experiment was performed to determine where MMP14 and MMP2 are required for neural crest cell migration. The heads of grafted embryos were shown with anterior to the right with the source of the donor NC and the host embryo indicated. The color of the text indicates whether EGFP-CAAX (green) or mCherry (red) was used as a lineage tracer. The results showed that the migration of cranial NCCs was affected only when MMP14 was knocked down in cranial neural crest and/or when MMP2 was knocked down in head mesenchyme. (I) The farthest distance of neural crest cell migration in each transplanted embryo was measured relative to the entire D-V axis of the embryo and summarized in the bar graph. While MMP14-MO in neural crest cells or MMP2-MO in cranial mesenchyme reduced the relative distance of migration to 28.5% or 62.1%, MMP14-MO in neural crest cells and MMP2-MO in mesenchyme together reduced the relative distance of neural crest migration to 18.9%. Error bars, s.d. Student t-test was performed and * marked conditions with significantly reduced neural crest migration (p<0.01). Figure 5. MMP14 regulates neural crest EMT in culture. (A) Cranial neural crest explants were dissected at stage 13 and cultured on FN-coated coverslip. The migration of neural crest explants was followed by photograph at sibling stages 13, 20, and 24 for premigratory, onset of migration, and late migration stages. Control explants spread at stage 20 and migrated extensively both as a coherent sheet and as individual cells at stage 24. Overexpression of MMP14 led to further migration and cell separation at stage 20, while MMP14-MO led to delayed spreading and migration. Both MMP14-MO and pan-MMP inhibitor Prinomastat (0.2mM) inhibited the separation of neural crest cells from each other. (B) The ratio of neural crest explant spreading was calculated by comparing the area it covered at the end vs. at the start of the experiment. Error bars, s.d. Figure 6. MMP14 regulates neural crest EMT and migration through regulating the expression levels of N-cadherin and E-cadherin. Cranial neural crest explants were fixed around stage 22 and immunohistochemistry analysis was performed against N-cadherin and E-cadherin. (A-C)Control explants, A ad A’, B ad B’ sho the epressio of E-/N-cadherins in the same explants. In control, E-cadherin is highly expressed at cell-cell contacts in the periphery of the explant, where cells extend pronounced membrane protrusions. Higher level of N-cadherin expression is often detected on the surface of inner cells. (D-F) When MMP14 was overexpressed, decreased level of E-cadherin was detected, especially in cells separated from each other. High levels of N-adheri epressio as deteted further ito the eter of the eplat. D ad D’, E ad E’ are different channels of the same explant. (G-I) When MMP14-MO was injected, the expression of N-cadherin was increased and it overlapped with the expression of E-cadherin. E-cadherin expression was detected in both periphery cells and in inner cells. Wide arrow in each panel indicates the direction of explant migration. Small arrows point to individual cell or small cell clusters. Scale bar=20m.

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