Xenopus ADAM13 and ADAM19 Are Important for Proper Convergence and Extension of the Notochord
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University of Massachusetts Amherst ScholarWorks@UMass Amherst Open Access Dissertations 2-2011 Xenopus ADAM13 and ADAM19 are Important for Proper Convergence and Extension of the Notochord Russell David Neuner University of Massachusetts Amherst Follow this and additional works at: https://scholarworks.umass.edu/open_access_dissertations Part of the Cell Biology Commons, and the Molecular Biology Commons Recommended Citation Neuner, Russell David, "Xenopus ADAM13 and ADAM19 are Important for Proper Convergence and Extension of the Notochord" (2011). Open Access Dissertations. 337. https://scholarworks.umass.edu/open_access_dissertations/337 This Open Access Dissertation is brought to you for free and open access by ScholarWorks@UMass Amherst. It has been accepted for inclusion in Open Access Dissertations by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. XENOPUS ADAM13 AND ADAM19 ARE IMPORTANT FOR PROPER CONVERGENCE AND EXTENSION OF THE NOTOCHORD A Dissertation Presented By RUSSELL D. NEUNER Submitted to the Graduate School of the University of Massachusetts, Amherst in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY February 2011 Molecular and Cellular Biology © Copyright by Russell Neuner 2011 All Rights Reserved XENOPUS ADAM13 AND ADAM19 ARE IMPORTANT FOR PROPER CONVERGENCE AND EXTENSION OF THE NOTOCHORD A Dissertation Presented By RUSSELL D. NEUNER Approved as to style and content by: ________________________________________ Dominique Alfandari, Chair ________________________________________ Rafael Fissore, Member ________________________________________ Rolf Karlstrom, Member ________________________________________ Jesse Mager, Member ______________________________________ Barbara A. Osborne, Director Molecular and Cellular Biology Program DEDICATION I dedicate this dissertation to everyone who has helped me over the years. To those who have helped shape me into the person that I am today. To everyone that has supported me through the triumphs and tragedies of my life. I cannot express how sincerely grateful I am to have each of you by my side. This dissertation is dedicated to my father David Neuner, my mother Janet Allen, my brother Scott Neuner, my sister Stephanie Luster and the rest of my extended family especially my loving grandparents, Fred and Doris Neuner. I also dedicate this dissertation to the special people in my life; Martha Espy, my loving and supportive fiancé Lindsay Grigg, the entire Grigg family and my life-long friends Eric Harmon and Luke Woolley. For Everything – Thank You! ACKNOWLEDGMENTS There are a lot of people to acknowledge through my journey here at UMass. I would like to start by thanking everyone from the Alfandari research team. I recognize Dominique Alfandari and Helene Cousin for their dedication to my growth and development as a scientist. Your relentless support has instilled values that will continue to grow throughout the rest of my scientific career. For that, I sincerely thank you for everything. I would also like to thank Kate McCusker and Julian Sosnik for their support as previous lab mates and as friends. My experience with Mike Coyne, Erin Kerdavid, Wei Ding and Genevieve Abbruzzese has been great and it has been an honor to be part of this research team. I would like to acknowledge my thesis committee; Rolf Karlstrom, Rafael Fissore and Jesse Mager. I greatly appreciate your help, constructive criticism and enduring support over the years. To all of my friends outside of the lab, especially Ankit Vahia and Nick Griner – Thank you. You have brought a tremendous amount of happiness and laughter to my life. I would also like to acknowledge the Molecular and Cell Biology Program and the Veterinary and Animal Science Department for fostering an outstanding environment for my graduate studies here at UMass. v ABSTRACT XENOPUS ADAM13 AND ADAM19 ARE IMPORTANT FOR PROPER CONVERGENCE AND EXTENSION OF THE NOTOCHORD FEBRUARY 2011 RUSSELL D. NEUNER, B.S., PENNSYLVANIA STATE UNIVERSITY Ph.D., UNIVERSITY OF MASSACHCUSETTS AMHERST Directed by: Professor Dominique Alfandari Gastrulation is a fundamental process that reorganizes the primary germ layers to shape the internal and external features of an early embryo. Morphogenetic movements underlying this process can be classified into a variety of different types of cellular movements. I will focus on investigating in this thesis two types of cell movements in the dorsal mesoderm; mediolateral cell intercalation and convergence and extension. During gastrulation, mesoderm cells send protrusions to gain traction on neighboring cells and the surrounding extracellular matrix; a process called mediolateral cell intercalation. Mesoderm cells use this type of cell movement to converge and extend the dorsal mesoderm tissue during gastrulation; a process called convergence and extension. These morphogenetic movements are essential to form the early embryo and are important for later development. There are a number of different proteins involved in regulating the morphogenetic movements during gastrulation. The Planar Cell Polarity Signaling Pathway helps establish individual cell polarity and is activated in dorsal mesoderm cells undergoing convergence and extension. In addition, dorsal mesoderm cells migrate by using integrin receptors and the surrounding extracellular matrix to correctly position the mesoderm in the embryo. I will focus my efforts on analyzing the function of ADAM proteins during Xenopus laevis gastrulation. The ADAM family of metalloproteases is important for a variety of biological processes. ADAM proteins function as ectodomain sheddases by cleaving membrane bound proteins involved in signal transduction, cell-cell adhesion, vi and cell-extracellular matrix adhesion. I will focus on investigating the roles of two ADAM family members; ADAM13 and ADAM19 during gastrulation. Both ADAM13 and ADAM19 are expressed in the dorsal mesoderm during gastrulation. Throughout early embryonic development, ADAM13 is expressed in the somitic mesoderm and cranial neural crest cells. ADAM19 is expressed in dorsal, neural and mesodermal derived structures such as the neural tube, notochord, the somitic mesoderm, and cranial neural crest cells. Since ADAM13 and ADAM19 are expressed in similar tissues, I investigated if both proteins functionally interacted. I show that a loss of ADAM13 protein in the embryo reduces the level of ADAM19 protein by 50%. In the opposite experiment, a loss of ADAM19 protein in the embryo reduces the level of ADAM13 protein by 50%. This suggests that both ADAM13 and ADAM19 are required to maintain proper protein levels in the embryo. This might be explained through their physical interaction in a cell. The ADAM19 Proform binds to the ADAM13 Proform in cultured cells. Through domain analysis, I show that ADAM19 binds specifically to the cysteine-rich domain of ADAM13. When co-overexpressed in a cell, the level of Mature ADAM13 (compared to the Proform) is reduced suggesting a complex form of regulation. I propose a few models that discuss how ADAM19 may function as a chaperone to stabilize and regulate the further processing of ADAM13 protein. Some of the unpublished work discussed in this thesis focuses on the roles of ADAM13 and ADAM19 in the dorsal mesoderm during gastrulation. Specific emphasis is made on investigating the axial mesoderm during notochord formation. I show that ADAM19 affects gene expression important for the A-P polarity of the notochord while ADAM13 does not. The changes in gene expression can be partially rescued by the EGF ligand Neuregulin1, a known substrate for ADAM19 in the mouse. ADAM13 and ADAM19 are important for convergence and extension movements of the axial mesoderm during gastrulation. Specifically, a loss of ADAM13 or ADAM19 causes a delay in mediolateral cell intercalation resulting in a significantly wider notochord compared to control embryos. These defects occur without affecting dishevelled intracellular localization or the activation of the PCP signaling pathway. However, a loss vii of ADAM13 or ADAM19 reduces dorsal mesoderm cell spreading on a fibronectin substrate through 51 integrin. To conclude, the work presented in this thesis focuses on the similarities and differences of ADAM13 and ADAM19 in the early embryo. Although ADAM13 and ADAM19 are required for normal morphogenetic movements during gastrulation, my data suggests they have different functions. ADAM13 appears to function in regulating cell movements while ADAM19 appears to function in regulating cell signaling. I propose a few models that discuss how each ADAM metalloprotease may function in the dorsal mesoderm and contribute to convergence and extension movements during gastrulation. viii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ...................................................................................................v ABSTRACT ....................................................................................................................... vi LIST OF TABLES ............................................................................................................ xii LIST OF FIGURES ......................................................................................................... xiii CHAPTER I. INTRODUCTION ............................................................................................................1 Abstract ....................................................................................................................1