Continuing experiments to assess loss-of-function in the MMP-14 protein on the migration of neural crest cells, wild-type embryos were treated with a chemical inhibitor. Instead of localized effects from microinjections, these inhibitors are introduced through solution and therefore affect the entire embryo. In contrast to EDTA experiments performed earlier in which all metalloproteinases were targeted, these inhibitors are highly specific and therefore will be more effective in bringing about changes in activity given that their activity is not occupied by other proteins. Prinomastat hydrochloride inhibits both the solitary activity of MMP-14 and its proteolytic ability to activate MMP-2, from which previous studies have shown plays a large role in matrix digestion. NSC 405020 inhibits the sole activity of MMP-14 by interacting with the PEX domain and inhibiting MMP-14’s activity to dimerize, to interact with receptors, and to activate signaling pathways, but leaves its proteolytic activity unaffected. Thus, the use of these two inhibitors is to dissect the importance of MMP-14’s ability to activate MMP-2 compared with its solo activity.
Hypothesis:
Regardless of concentration, embryos treated with Prinomastat hydrochloride will feature greater abnormalities in distance of neural crest migration and migratory stream segregation due to the importance of the activation of MMP-2, which this inhibitor blocks.
Experimental Design:
For both inhibitors, sample populations of embryos were treated with 0.5mM, 1mM, and 2mM concentrations of their respective inhibitor. Prinomastat hydrochloride solid was soluble in 0.1x MMR, and thus these embryos were compared against an MMR control group. NSC 405020 was soluble in DMSO (an organic solvent), and thus embryos treated with this inhibitor were compared against a DMSO control. Treatment was administered at Stage 17 and the embryos were allowed to develop until stage 24, in which they were fixed and processed using in situ hybridization for sox10 antisense probe.
Results:
Hypothesis:
Regardless of concentration, embryos treated with Prinomastat hydrochloride will feature greater abnormalities in distance of neural crest migration and migratory stream segregation due to the importance of the activation of MMP-2, which this inhibitor blocks.
Experimental Design:
For both inhibitors, sample populations of embryos were treated with 0.5mM, 1mM, and 2mM concentrations of their respective inhibitor. Prinomastat hydrochloride solid was soluble in 0.1x MMR, and thus these embryos were compared against an MMR control group. NSC 405020 was soluble in DMSO (an organic solvent), and thus embryos treated with this inhibitor were compared against a DMSO control. Treatment was administered at Stage 17 and the embryos were allowed to develop until stage 24, in which they were fixed and processed using in situ hybridization for sox10 antisense probe.
Results:
Figure 1. To distinguish MMP2- dependent and –independent effect of MMP14 on neural crest migration, two chemical inhibitors are applied to the embryos at stage 16, after neural crest specification but before their migration. Embryos are collected at stage 24 and subject to in situ hybridization with sox10 antisense probe. Prinomastat hydrochloride (dissolved in 0.1x MMR) inhibits the activities of both MMP-14 and MMP-2, while NCS 405020 (dissolved in DMSO) targets the PEX domain of MMP14 thus inhibits MMP-14’s activity to dimerize, to interact with receptors, and to activate signaling pathways, but not its proteolytic activity to activate MMP-2. Neural crest migration was inhibited by both inhibitors to different degree. Lateral view of embryos are shown with dorsal to the top and anterior to the left.
Discussion:
Prinomastat hydrochloride was observed to have a much more dramatic impact on neural crest migration, in which all embryos regardless of concentration were severely affected, with neural crest streams featuring next to no migration or segregation. NCS 405020 was still shown to impact migration, but to a lesser degree, some of which could possibly be attributed to its dissolution in DMSO (Figure 2). Thus, it is reasonable to predict that the synergistic activity of MMP-14 and MMP-2 is critical to the proper migration of neural crest cells, given that the inhibitor that blocked MMP-2 activation (Prinomastat hydrochloride) resulted in universally severe abnormalities.
Prinomastat hydrochloride was observed to have a much more dramatic impact on neural crest migration, in which all embryos regardless of concentration were severely affected, with neural crest streams featuring next to no migration or segregation. NCS 405020 was still shown to impact migration, but to a lesser degree, some of which could possibly be attributed to its dissolution in DMSO (Figure 2). Thus, it is reasonable to predict that the synergistic activity of MMP-14 and MMP-2 is critical to the proper migration of neural crest cells, given that the inhibitor that blocked MMP-2 activation (Prinomastat hydrochloride) resulted in universally severe abnormalities.