REGULATION OF THE BMP SIGNALLING PATHWAY BY BMPl-RELATED METALLOPROTEASES FIONA CLAIRE WARDLE A Thesis Submitted for the Degree of Doctor of Philosophy at the University of London 1998 Department of Anatomy and Developmental Biology University College, London. ProQuest Number: 10016134 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10016134 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Bone Morphogenetic Proteins (BMPs) 2-8 are members of the TGFp superfamily of secreted signalling molecules. During embryonic development BMPs are involved in many processes including cell fate determination, morphogenesis, growth and programmed cell death, all of which are essential for normal development. BMP activity may be regulated in variety of ways. Of particular interest is the finding that three Xenopus proteins. Noggin, Chordin and Follistatin are able to bind BMP4 and prevent it activating its receptor (Piccolo et al., 1996; Zimmerman et al., 1996; Fainsod et al., 1997). Inhibition by Chordin can be alleviated by Xolloid, a BMP 1-related metalloprotease that cleaves Chordin (Piccolo et al., 1997). Similarly, in the Drosophila embryo Dpp, a BMP2/4 homologue, is inhibited by the Chordin homologue, Sog, but activated by Tolloid, a BMP 1-related metalloprotease (Marqués et al., 1997). Since BMPl- related metalloproteases are present during development of many animal species, an attractive hypothesis is that release from inactive complexes by these metalloproteases is a general mechanism for regulating BMP activity. Mesodermal patterning during Xenopus development, a process known to require BMP activity, is used as a test system to investigate the role of BMP 1-related metalloproteases in BMP signalling. Recent experiments have shown that overexpression of XBMPl and Xolloid partially ventralizes dorsal mesoderm (Goodman et al., 1998). In contrast, expression of the sea urchin BMP 1-related metalloprotease, SpAN, completely ventralizes dorsal mesoderm. This ventralizing activity requires a functional BMP signalling pathway, indicating that these metalloproteases may be activating endogenous BMPs in the Xenopus embryo. In addition, SpAN, XBMPl and Xolloid inhibit the activity of Chordin and SpAN is also inhibits Noggin. To further test the action of these metalloproteases, putative dominant-negative constructs were made, lacking the metalloprotease domain but retaining the protein-protein interaction domains. These truncated constructs dorsalize ventral mesoderm, consistent with the truncated protein binding its target, but being unable to process it. Finally, two sea urchin TGF(3s expressed during early sea urchin development, Univin and suBMP2/4, were tested in the Xenopus embryo and found to be functionally homologous to Vgl and BMP4 respectively, suggesting that similar molecules may act in both sea urchins and Xenopus to pattern the early embryo. TABLE OF CONTENTS ABSTRACT..........................................................................................................................2 TABLE OF CONTENTS......................................................................................3 FIGURES AND TABLES.....................................................................................8 ABBREVIATIONS........................................................................................................10 ACKNOWLEDGMENTS....................................... 13 Chapter 1: GENERAL INTRODUCTION 1.1 The Transforming Growth Factor (3 Family .............................................................. 14 1.1.1 Sub-groups of the TGFp superfamily .................................................................................. 14 1.1.1.1 The TGFp subgroup ...........................................................................................................14 l.l.l.ii The activin subgroup ...................................................................................................... 15 1.1.1.111 The nodal-related subgroup ........................................................................................... 15 l.l.l.iv The 60A and Dpp subgroups .........................................................................................15 1.1.1.v The Vgl subgroup ........................................................................................................... 16 1.1.2 The structure of TGFPs ..........................................................................................................16 1.1.3 TGpp signalling pathways ....................................................................................................17 1.1.3.1 Receptors .............................................................................................................................17 1.1.3.Ü Signal transduction ........................................................................................................... 18 1.1.4 Control of TGFp activity .........................................................................................................19 1.2 Zinc Metalloproteases ...................................................................................................... 20 1.2.1 The matrix metalloprotease family of zinc metalloproteases (matrixins)...................21 1.2.2 The astacin family of zinc metalloproteases ..................................................................... 22 1.2.2.1 Domain structure of astacin metalloproteases ..............................................................22 1.2.2.Ü BM PI-related metalloproteases ...................................................................................... 23 1.2.2.111 Activity of astacin family metalloproteases .................................................................24 1.3 TGFps in Development ....................................................................................................24 1.3.1 The early development of Xenopus embryos .....................................................................24 1.3.2 The three-signal model ......................................................................................................... 25 1.3.3 Mesoderm induction ..............................................................................................................26 1.3.4 Dorsoventral patterning of mesoderm in Xenopus ..........................................................28 1.3.5 Dorsoventral patterning in the early Drosophila embryo ................................................ 30 1.4 Sea Urchin Development .................................................................................................31 1.4.1 Early development of the sea urchin embryo ...................................................................32 1.4.2 Cell fate specification in the sea urchin embryo ...............................................................33 1.4.2.1 Fate maps ............................................................................................................................. 33 1.4.2.Ü Inductive interactions ....................................................................................................... 33 1.5 Summary......................................................................................................................................... 36 Chapter 2: MATERIALS AND METHODS 2.1 Materials ................................................................................................................................51 2.1.1 Vectors ..................................................................................................................................... 51 2.1.2 DNA constructs ....................................................................................................................51 2.1.3 Solutions and media ............................................................................................................52 2.2 Methods ...............................................................................................................................60 2.2.1 Xenopus techniques ...............................................................................................................60 2.2.1.1 Obtaining XeAio/JMj eggs ................................................................................................ 60 2.2.1.Ü In vitro fertilization ........................................................................................................ 60 2.2.1.111 Dejelling eggs ...............................................................................................................60 2.2.1.iv Microinjection of mRNA into Xenopus eggs .............................................................60 2.2.1.v Culturing embryos .......................................................................................................61 2.2.1.vi Staging and scoring embryos .....................................................................................61 2.2.1.VÜ Microdissection of embryos