Functional Analysis of ADAM13 during Xenopus embryogenesis.

University Of Massachusetts Amherst, Amherst MA

Investigators

Abstract

Cell migration is one of the most important processes of embryo development as it controls both the shape and the cell composition of organs. During the early phase of organogenesis cells at the border of the neural plate separate and migrate into the head to form bones, cartilage, muscle and ganglia of the face. These cells, present in all vertebrates, are called Cranial Neural Crest (CNC). While much is known in birds, mammals and amphibians about the pathways of CNC cell migration and the derivative tissues that they contribute to, much less is known about the precise molecules that control this migration. Recent works suggest that an emerging family of cell surface proteins (ADAM) with both proteolytic and adhesive capabilities may play a role in cell migration. These studies have shown that ADAM metalloprotease can cleave proteins, in vitro, known to affect cell adhesion and migration (e.g. Cell adhesion molecules and Chemokines). While this is a compelling evidence, there is no direct link between ADAM cleavage of these proteins and a natural cell migration process in vertebrate embryos. ADAM13 is expressed at the surface of the migrating CNC cells. Using a dominant negative approach, it was shown that its proteolytic function is required for CNC migration in vivo. The mechanism by which this ADAM controls cell migration remains unknown. The current proposal will investigate this mechanism. Recent work has shown that ADAM13 can bind and cleave the extracellular matrix protein (ECM) fibronectin (FN). This protein is both required and sufficient to promote CNC cell migration in vitro. The domain responsible for the interaction with FN is the adhesive (disintegrin and cysteine-rich domain) region of ADAM13. These results suggest that the adhesive region of ADAM proteins may selectively bind to the metalloprotease substrate. Surprisingly, while efficient in vivo, both the dominant negative form of ADAM13 and a metalloprotease inhibitor effective on most ADAM (including ADAM13) do not prevent CNC cell migration in vitro. These results are the base for the current proposal. In the first Aim, grafts and explants will be used to examine how ADAM13 controls cell migration. In the second Aim potential ADAM13 substrate will be tested to identify proteins which cleavage by ADAM13 is critical for CNC migration. Results from the proposed study will better the understanding of craniofacial development, as well as provide insights into the role of ADAM metalloproteases in natural and cancer related cell invasion. CNC migration is recognized as an excellent model system in which to dissect the process that regulates cell movements. In this model, the precise moment when cells acquire motility, the path that they will follow and the tissues that they invade are known. This project will be developed at the University of Massachusetts (Amherst) in the department of Veterinary and Animal Sciences. It will involve both graduate and undergraduate students working side by side with the PI. The model system, the frog embryo, is one of the best to lead students in experimental appreciation of embryogenesis because large number of eggs can be obtained and the relative large size of the embryos allows easy micro manipulation including injection, dissection and grafts. Because of the abundance of embryos, biochemical approaches can also be undertaken. For these reasons, the scope of students that can be trained in the laboratory is extremely wide and includes areas of Cell and Developmental biology as well as Biochemistry. In the future the PI hope to develop a higher-level undergraduate classical embryology course including laboratory and using Xenopus embryos.