Project 1: Mechanism of Cranial Neural Crest Cell Migration

(NIH DE016289)

Proper cranial neural crest (CNC) cell migration is essential for the construction of the face, jaws and their peripheral nervous system connections. Despite this importance, little is known about how neural crest cell migration is regulated. During Xenopus embryo development the expression of ADAM 13 (a protein containing A Disintegrin And Metalloprotease) correlates with the migration of the cranial neural crest cells from the lateral border of the neural plate to the ventral anterior station where they eventually form facial structures (Alfandari et al., 1997). Our on-going analyses of cranial neural crest cells expressing a dominant negative form of ADAM13 suggest that ADAM13 promotes and/or directs their migration in two of the three possible pathways. Our working hypothesis is that ADAM13 cleaves a protein that normally restricts cranial neural crest cell migration. This protein may either be inserted in the migration path as a stop signal to prevent cell passage or be expressed at the cranial neural crest cell surface to hold the cells in place as an anchor. To test these hypotheses and analyze whether other ADAM and related metalloproteases may also be involved in cranial neural crest cell migration we propose the following specific Aims. This proposal has three Aims to understand 1) if cells missing ADAM13 protein can use other ADAM and related metalloprotease to migrate, 2) if ADAM13 functions as a "drill" to open migration pathways, 3) if ADAM13 cuts an anchor that attaches cranial neural crest cells to their environment. Using specific morpholino oligonucleotides, we can prevent translation of ADAM proteins including ADAM13 in embryos and test how cranial neural crest cells migrate. This can be compared to the migration of cells in which ADAM 13 function is blocked (using drug inhibitor). Using grafts we will test whether cranial neural crest cells missing ADAM13 activity can follow cells that have ADAM13. Finally, we will test if ADAM13 can cleave proteins that are known to anchor cells down. The proposed studies will increase our understanding of events that govern normal formation of the face, an essential step towards diagnosing and treating conditions that lead to abnormal development. Furthermore, information about ADAM contributions to cell migration could lead to new understanding of the function of these proteins in various cancer and metastasis. In particular these protein (ADAM) are likely to be involved in the escape of cells from the original tumor to new sites. 

We have developped assays that allow us to test CNC migration both in vivo ; CNC expressing a fluorescent marker together with either wild type ADAM13 (left) or a dominant negative form (right) are grafted into host embryos. and in vitro ; CNC are placed onto artificial substrate composed of bovine fibronectin (left) or a fusion protein containing the central cell binding domain of fibronectin expressed in bacteria (right).

Project 2: Implication of ADAM related Metalloproteases in Horse Laminitis:

Hatch

    Laminitis is an inflammation of the laminae that attaches the hoof of a horse to the major bone in the foot (third phalanx). It is a very common disease that affects both professionals of the horse industry as well as private owners. To date there are over 270 entree in the medline virtual library related to horse laminitis showing the wide interest of researcher in this field.  Laminitis can be caused by a large number of otherwise unrelated illness (digestive disturbance or excessive high impact work) that have in common to activate an inflammatory response. Why is the laminae a preferential target of this large-scale inflammation remains unknown. Laminitis progresses rapidly (a few hours) starting with a noticeable lameness and can in the most severe cases cause a complete immobility of the affected horse. In these cases in the absence of efficient treatment, the only alternative is euthanasia. The 2 main hypothesis concerning the initial causes of laminitis are 1) a closure of the blood vessel that irrigates the laminae causing cell necrosis and 2) a local inflammation of the laminae that recrui