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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).
Movie of CNC expressing the EC1-3 fragment of Cadherin-11 (For NIH review). Note the segment (red arrowhead) that retracts movie.
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 recruits leucocytes and activates matrix
metalloproteases which destroys the collagen-rich laminae. Laminitis is
already a focus for our department with Dr. Samuel Black's laboratory
investigating the factors responsible for the local inflammation. His
team in a collaboration with 2 other teams in Georgia and Australia
have already accumulated evidences that leucocytes were targeted to the
laminae during the early stage of laminitis. They also
collected evidences that 2 matrix metalloproteases (MMP2 and 9) were
up-regulated in the laminae resulting in detectable level of degraded
collagen in the blood stream. The expertise of my team is focused on
another family of metalloproteases, the (protein containing A
Disintegrin and A Metalloprotease domain), involved both in regulating
inflammation by activating cytokines, and in remodeling extracellular
matrices. ADAM can both remove defective extracellular matrix proteins
and promote deposition of new proteins. We feel that the
combination of Dr. Black and collaborator insight on inflammation, and
horse physiopathology combined with my expertise in extracellular
matrix and metalloproteases should provide a great environment
for the understanding of horse laminitis.