Luận án tiến sĩ: Intercellular adhesion and pathfinding molecule T-cadherin in the development of the nervous system

UNIVERSITY OF CALIFONIA, SAN DIEGO Intercellular Adhesion and Pathfinding Molecule T-cadherin in the Development of the Nervous System A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Neurosciences by Harper C VanSteenhouse Committee in Charge: Professor Barbara Ranscht, Chair Professor Nicholas Spitzer, Co-Chair Professor Samuel Pfaff Professor Eric Turner Professor Anthony Wynshaw-Boris 2007 UMI Number: 3244741 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion.

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Box 1346 Ann Arbor, MI 48106-1346 Copyright Harper C VanSteenhouse, 2007 All rights reserved. The dissertation of Harper C VanSteenhouse is approved, and it is acceptable in quality and form for publication on microfilm: Co-Chair Chair University of California, San Diego 2007 iii Try to learn something about everything and everything about something. Thomas Henry Huxley LV Table of Contents M2000 11. nh n TT TT ng ng KT KT TT TK KH KT KT kg rà XI 190192510 122727757 Ô ÔỔốố.-lAal ăn XI Awards and HOTOTS.

ch tà XII Abstract of the Dissertation 0 1. XIV lÔÌ)0i500880ii900/6i10 2 1018. 1 Adhesion molecules as guIdance CU€S. HH ng key 3 Cadherin family of cell adhesion molecuÌeS.

5 Patterned development of motor pathWAWS. cv Hs HH HH key 8 In OVO electroporation. chu 17 Chapter 2: Role of T-cadherin in development of segmentation of spinal motor TET VES. TT nọ HH kh 22 T-cadherin is a repulsive cue f†o mofOr aXONS 1n VIẨTO.

cày 26 Transgenic T-cadherin expression by chicken embryo sclerotome cells in Ectopic T-cadherin in anterior sclerotome establishes an ectopic repulsive rˆ005ã¡080101910/1901100 TT. 35 T-cadherin repulsion is mediated by a homotypic interaction with axonal I0.aaIIẠẶnn 39 RNAi induced knockdown of T-cadherin in chicken embryo motor neurons in ovo results in aberrant axons entering the posterior SJI9š01001 52111777. 43 Normal T-cadherin expression pattern is not required for normal gross pathfinding of motor neurons into the limb and to major muscle ¡010. 54 Materials and MethOdS.

HH HH TH HH kh kh kp 60 Chapter 3: Structure and function of T-cadherin adhesive domain. TT nọ HH kg 67 T-cadherin dimer interface mutants are expressed normally on the cell SULPACE II. 69 T-cadherin dimer interface mutants do not confer intercellular homophilic adh€SIOT. HT TH TH Hàn HH kề 72 Pro-T-cadherin expressed on the cell surface is susceptible to convertase DTOf€OlYyfIC CÏ€AVØ€.

HH HT TT TT TT ng 11 11g 1 kg kg 1 1 cv. 79 vị Role of alternative proteolytic processing of T-cadherin for adhesivity and intermolecular In†€FACfOTI. sees ccccaneeccecauscescuseeceseueescesauescsanens 83 Chapter 4: Functional interaction of T-cadherin with other cadherin family IMCMDETS. Gv ng KT KHE kn 93 Role of T-cadherin expression in combinatorial modulation of other cadherins: CHO cell morpholOBV.cc c1 cv xxx xe 96 Role of T-cadherin expression in combinatorial modulation of other Cadherins: adhesivity.ccccccccsccccesssseececsssseeeeeeeesteeeeeeesssseesessseeeeeeeeaes 101 Role of T-cadherin expression in combinatorial modulation of other cadherins: Agøregation and SeøT€ØatIOI.

cá che 106 Role of T-cadherin in the cleavage of N-cadherin. c2 112 Materials and MethOdlS. HT nh nh kg 117 DISCUSSIOTN. Q0 HH ng TT ng TH Ki TH HH HH 119 Chapter 5: Concluding ReIaTS.- 1111133111111 112 111 1E kg như 122 Ra.

122 T-cadherin as a pathfinding cue: simultaneous “adhesion” and “repulsion”. 127 T-cadherin interaction with other cadherin family members.- 130 Species differences in T-cadherin expression paffern.cccccc + 138 Implications for regeneration and neuraÌ r€DAIT. 141 vil List of Figures Figure 1-1: Domain structure of T-cadherin and comparison with other classical 680195111727 da. 7 Figure 1-2: Motor axon pathways from neural tube to muscle targets in limb and 04205722757 a4.

12 Figure 1-3: In ovo electroporation Into embryonic chieken selerotome. 15 Figure 1-4: Expression vector and RNAI used for In ovo electroporation. 16 Figure 2-1: T-cadherin Induces motor neuron ørowth cone collapse. 30 Figure 2-2: In ovo electroporation and ectopic co-expression of T-cadherin and S60580189I540i0 222.

34 Figure 2-3: Motor axons avoid anterior sclerotome cells ectopically expressing T- CACHETIN. ốắ 38 Figure 2-4: T-cadherin 1s a homotypIe repulsIV€ CUe€ 1n VIẨTO. cà, 42 Figure 2-5: T-cadherin is a homotypic repulsive cue fo mofor axonS In OVO. 47 Figure 2-6: T-cadherin expression perturbation doesn’t cause any gross motor axon pathfinding errors during growth through the limb.«‹<+<++>: 53 Figure 3-1: Surface expression of T-cadherin dimer interface mufanfs.- 71 Figure 3-2: T-cadherin mediated homophilic intercellular adhesion is abrogated when critical dimer interfaces residues are mufafed.

che 75 Figure 3-3: T-cadherin dimerization is required for homotypic neurite outgrowth mi i0. 78 viii Figure 3-4: Western blot of T-cadherin.- 1111122 kg kg nhu 81 Figure 3-5: T-cadherin processing by the proprotein convertase Furin. 82 Figure 3-6: L929 cells transfected with wildtype T-cadherin or TcadXmut 34003-11010/20/0VdiiầidiđiiiiiiiiaiẢŸẢ. 85 Figure 4-1: T-cadherin expression makes CHO cells rounder, and is dominant to elongation due to N-cadher1n.

cc c2 c2 vn S1 9 1111118211111 1111k ng 1x sec 99 Figure 4-2: Summary results of cell-substrate adhesion assays between various combinations of T- and N-cader1n. - - - c1 vs ng vi ret 105 Figure 4-3: T-cadherin expressing cells sepregate from both Type-I and Type-lI cadherin expressing celÏS II VIẨFO. c1 vn v2 v9 111118821111 ng ren 111 Figure 4-4: N-cadherin 1s present as a shorter form when co-expressed with T- 68In9yii0ii0905i900211 1e. 115 Figure 4-5: Western blot of N-cadherin immunoprec1pIfaf€S.

cày 116 Figure 5-1: Summary of electroporation experiments reSuÌfs.-+- 124 Figure 5-2: Schematic of continuum of kinetics between adhesive and signaling INTELACTIONS 2. 128 Figure 5-3: Model of T-cadherin interaction with other cadherins on a growth i10 ái.- 135 Figure 5-4: Possible pathway for T-cadherin to control N-cadherin induced T€UTIf€ OU{ĐTOWWẨH,. HS TT ng TH ng ng Hit 137 Ix Acknowledgements I have been blessed with many valued mentors and role models in both my personal and scientific lives. I wish to first thank my family for their unwavering support and guidance through all of my endeavors.

I know they were proud of all of my achievements, but I wish my grandparents could see this manuscript. Special appreciation goes to my fiancé, Tracey, for her daily support and enthusiasm for everything we pursue. I am deeply indebted to my major advisor, Dr. Barbara Ranscht, for her direction and encouragement in my development as an independent scientist and provision of an environment of intellectual curiosity and integrity.

The members of my committee also provided instrumental guidance during the planning and execution of many of the experiments described. The members of the Ranscht Lab have all contributed immensely to my learning and to my life—thank you all. I am thankful to Catherine Krull and Yaxiong Chen for assistance with electroporation technique, Samuel Pfaff and Nicolas Girard for help with backlabeling technique, electroporation vectors and antibodies as well as many helpful discussions, and to Elena Pasquale for ephrinB-1 antibody. 1 will always appreciate Dr.

Birgit Zipser and Dr. Laura Symonds for providing my first introductions to the satisfying and entertaining aspects of scientific research and preparing me for a life in science. Chapter 2 in part is being prepared for publication as T-cadherin is a homotypic repulsive axon pathfinding cue that directs segmented motor neuron outgrowth by VanSteenhouse H and Ranscht B. The dissertation author is the primary investigator on this paper.

Chapter 3 in part is being prepared for publication as Untitled by Ciato C, VanSteenhouse H, Ranscht B and Shapiro L. The dissertation author is the primary investigator on this portion of the collaborative paper. xi Vita 2007 Doctor of Philosophy, University of California, San Diego 2002-2007 Graduate Student Researcher, University of California, San Diego 2006 Teaching Assistant, HDP110, Brain and Behavioral Development, University of California, San Diego 2001-2002 Graduate Research Assistant, Laboratory of Birgit Zipser, Ph., Departments of Physiology and Neuroscience at Michigan State University 2001 Bachelor of Science, Michigan State University, East Lansing, MI Publications VanSteenhouse HC, Ranscht B. T-cadherin is a homotypic repulsive axon pathfinding cue that directs segmented motor neuron outgrowth.

Ciatto C, VanSteenhouse HC, Ranscht B, Shapiro L. VanSteenhouse HC, Ranscht B. Cadherins as Regulators of Specific Motor Neuron Connectivity. Abstract Viewer and Itinerary Planner.

Washington, DC: Society for Neuroscience, 2004. xil VanSteenhouse HC, Horton ZA, Goodman MB, O’Hagan R, Tai M-H, Zipser B. Cell type-specific glycosylations in C. Submitted, Revision in progress.

VanSteenhouse HC, Horton ZA, Goodman MB, O’ Hagan R, Tai M-H, Zipser B. Cell type-specific glycosylations in C. Society for Neuroscience 32nd Annual Meeting. Baker M, VanSteenhouse HC, Tai M-H, Huang L, Johansen J, Johansen KM, Xu Y, Hollingsworth RI, Zipser B.

Constitutive and Developmentally Regulated Glycosylations of CAMs Mediate Sequential Steps in Synaptic Targeting. 4th International Symposium on Organogenesis: Molecular Control of Neuronal Organogenesis. Ann Arbor, MI. VanSteenhouse HC, et al.

Symposium on Transcriptional Regulatory Mechanisms. East Lansing, MI. Awards and Honors 2003-2006 UCSD Genetics Training Program grant 2003-2004 Merck & Co. fellowship 2002 NSF Graduate Research Fellowship honorable mention XII ABSTRACT OF THE DISSERTATION Intercellular Adhesion and Pathfinding Molecule T-cadherin in the Development of the Nervous System by Harper C VanSteenhouse Doctor of Philosophy in Neurosciences University of California, San Diego, 2007 Professor Barbara Ranscht, Chair Professor Nicholas Spitzer, Co-chair XIV Adult animals exhibit an amazing array of behaviors controlled by an exquisitely complex nervous system.

Axon pathfinding is an essential component to the development of a fully functioning nervous system. Axon pathfinding cues are molecules in the tissue surrounding growing axons that instruct directionality of axonal outgrowth leading to their proper trajectory from cell body to target. Cell adhesion molecules are one class of proteins that function as contact attractant or repellent pathfinding cues. This dissertation examines the contribution to axon pathfinding of T-cadherin, a member of the cadherin family of adhesion molecules.

Both in vitro and in vivo, T-cadherin is found to be a contact repellant pathfinding cue. Not only is T-cadherin a cue in the environment, it signals to T- cadherin on growth cones in a homotypic manner. Soluble T-cadherin collapses growth cones of motor neuron explants. T-cadherin substrates inhibit neurite outgrowth of wildtype—but not knock-out—spinal neurons.

T-cadherin expressed on motor neurons and in posterior sclerotome of chicken embryos directs the outgrowth of motor neurons exclusively through the anterior sclerotome. T-cadherin’s properties of homophilic adhesion and inhibition of neurite outgrowth require dimerization, and both functions can be blocked by a single point mutation disrupting this dimer formation as predicted by structural studies. The presence of the pro-domain of T- cadherin also appears to disrupt normal T-cadherin function. T-cadherin acts in a dominant negative manner over N-cadherin function in several in vitro assays.

T-cadherin co-expression negates N-cadherin induced cellular morphology, causes abnormal cell aggregation and segregation, and abrogates strong XV N-cadherin homophilic adhesion. T-cadherin may have dominance by inducing the cleavage of N-cadherin. When T- and N-cadherin are coexpressed in the same cell, a portion of the N-cadherin is detected as a smaller species than when expressed alone. Thus, T-cadherin is shown to be a homotypic repulsive axon pathfinding cue, which may be functioning though an interaction with other cadherins.

This interaction may explain T-cadherin’s signaling abilities—in spite of being GPI-anchored—by modulating the signal transduction abilities that normally induce neurite outgrowth as a function of the other cadherin’s interactions. Xvi Chapter 1: Introduction Axon Pathfinding The adult nervous system displays a high order of structural specificity that is generated during development. The specific positioning of neurons, and the specific connections they make to their target cells is essential for a properly functioning nervous system.