Di cư tế bào neural crest sọ trong phôi chim: Nghiên cứu vai trò EPH-A4 và Ephrin-A5

CRANIAL NEURAL CREST CELL MIGRATION IN THE AVIAN EMBRYO AND THE ROLES OF EPH-A4 AND EPHRIN-AS Thesis by Carole Chih-Chen Lu In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy California Institute Of Technology Pasadena, California 2007 (Defended September 21, 2006) UMI Number: 3244121 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 Carole C. Lu © 2007 Carole Chih-Chen Lu All Rights Reserved Carole C. Lu iii ACKNOWLEDGEMENTS My relationship with developmental biology began in 1998, when | took my first developmental biology class. To me, it was love at first sight.

Since arriving at graduate school, the path has been mostly uphill, and there are many people who have supported, motivated, and kept me on the path. | thank my advisor and mentor, Scott E. Fraser, for his guidance and support all these years. Scott provides a unique research environment where graduate students are given freedom to pursue scientific questions.

He is also incredibly supportive and has (what must be) an infinite supply of optimistic advice. | thank the members of my committee, Marianne Bronner-Fraser, Paul Sternberg, and David Chan for scientific guidance and expertise. The Fraser lab is intimate because it is filled with friendly and helpful colleagues. | thank Paul Kulesa, who thoroughly and patiently showed me the ropes and provided me with a solid foundation.

| thank Sean Megason, Helen McBride, Elaine Bearer, and Andrew Ewald for invaluable scientific discussions and knowledge. | thank Michael Liebling for expertise with image analysis software, David Koos for advice in molecular biology and histology, Tatyana Demyanenko for advice in histology, and Aura Keeter for invaluable technical support. | thank Mary Flowers for being our lab mom, Gary Belford and Sonia Collazo for their skilled computer technical support. | thank Chris Waters and Dan Darcy for maintaining our confocal systems.

| also thank Julien Vermot, Le Trinh, Robia Pautler, Christie Canaria, Max Ezin, Mat Barnet, Larry Wade, Magdalena Bak-Maier, David Wu, Rusty Lansford, Mary Dickinson, Liz Jones, and Arian Forouhar for being awesome lab mates. | thank Marianne Bronner-Fraser for a wonderful teaching assistant experience and scientific discussions. The Bronner-Fraser lab next door graciously helps us with reagents and discussions. Specifically | thank Meghan Adams, Sujata Bhattacharyya, Vivian Lee, Martin Basch, Martin Garcia-Castro, Tatjana Sauka-Spengler, Ed Coles, Lisa Taneyhill, and Meyer Barembaum.

My fellow classmates were invaluable during graduate school. Specifically, | thank Gloria Choi, Pei-Yun Lee, and Magdalena Bak-Maier for the fun that we had when we studied for and passed our quails. Toby Rosen, Karli Watson, Xavier Ambroggio, and Johannes Graumann are great fun. | thank Sean Megason and Bernadine Tsung-Megason for adventures outside of lab.

| thank Stuart Freed for not throwing out my pots at the pottery studio and for years of therapeutic pottery. Lu iv | thank my dear sisters, Connie Lu and Cathy Lu, for providing me with many photographic opportunities and material for funny stories. | thank Attila Kovacs for feeding Connie. My parents, Kuen and Dong-Chih Lu, have consistently kept me on the path to graduation.

Lastly, | thank Alok J. Saldanha, my husband, for his love, brilliance, and enthusiasm. Alok is passionate about the world and is the best life partner that | could have ever hoped for. In memory of Eric Tse and Ben Edelson.

In memory of Molly. Lu Vv ABSTRACT The neural crest is a transient population of cells that migrate away from the dorsal neural tube in the vertebrate embryo. As the developing hindbrain constricts into rhombomeres, cranial neural crest cells migrate in three discrete streams adjacent to even-numbered rhombomeres, rhombomere 2 (r2), r4, and r6. To test the role of intrinsic versus extrinsic cues in influencing an individual cell’s trajectory, we implanted physical barriers in the chick mesoderm, distal to emerging neural crest cells (NCCs).

We analyzed spatio-temporal dynamics as NCCs encountered and responded to the barriers by using time-lapse confocal microscopy and cell tracking analysis. The majority of NCCs were able to overcome physical barriers. Even though the lead cells become temporarily blocked by a barrier, follower cells find a novel pathway around a barrier and become de novo leaders of a new stream. Quantitative analyses of cell trajectories find cells that encounter an r3 barrier migrate significantly faster but less directly than cells that encounter an r4 barrier, which migrate normally.

NCCs can also migrate into normally repulsive territory as they reroute. These results suggest that cranial neural crest cell trajectories are not intrinsically determined. NCCs can respond to minor alterations in the environment to retarget a peripheral destination. Both intrinsic and extrinsic cues are important in patterning.

We then tested the role of Eph/ephrin signaling on cranial neural crest migration by ectopically expressing full-length ephrin-A5 ligand; a truncated, Carole C. Lu vi constitutively active EphA4 receptor; and a truncated, kinase-dead EphA4 receptor within migratory neural crest cells. Ectopic expression of ephrin-A5 specifically causes the r6 subpopulation of neural crest cells to have truncated migration but does not affect directionality, suggesting that the r6 neural crest cells properly follow guidance cues. Our results support a role for ephrin-A5 in regulating the extent of migration.

Ectopic expression of constitutively active, truncated EphA4 causes NCCs to migrate aberrantly around the otic vesicle. Pathfinding errors are accompanied by changes in migratory behavior, with the NCCs migrating faster but with less directionality. Expression of a truncated, kinase-dead version of EphA4 also leads to pathfinding errors. Our results suggest Eph activity is involved in guidance and extent of migration.

Lu vii TABLE OF CONTENTS 10,9)160)0)08-i89. Vv TABLE OF CONTENTS1.ccccccccccccteecceceseeenecceeceecereensecneeeceeceecnee sees cneecaeeseecneensnesecesnessaeesaesnateniecnas vii LIST OF ïiic6 1. x CHAPTER 1: Introduction nh 6. 1 AVIAN CIDIYO oo.ccccccccecstcccntetesceneceaecneecneeceecneeeeesaeecaaesaaeceeseeseeteeceeeaaeecinecieetieesueesieenieetieenenesieatinees 1 Cranial neural crest cells: what are they and why are they ímporfanIf?.

2 Stereotypical pattern of mÍQFAflOH. HH HH KH ng TH nh kg 4 Possible intrinsic and @XtrinSiC mecha'liSIT. ánh HH HH HH HH HH rệt 5 Goal 0o. 14 CHAPTER 2: Time-lapse analysis reveals a series of events by which cranial neural crest cells reroute around physical 8-0-1.

22 Materials and MethOdlS. -- ác các HH HH KH HH HH KH KT cờ 27 0. 36 Ack'owledg6Im6FS.- cá HH HH HH HH HH HH T11 tá HT TH TC CC Cu crkt 41 San nẽnốố ố ốốốốốố. 45 CHAPTER 3: Time-lapse analysis of perturbations of ephrin-A5 and EphA4 during cranial neural crest Migration in the AVIAN embryO.

cu cece e cence ecee cece eeseseeecaeeecaececeeeeseeaeeseaeeeceeeeteeeeseaeteaiess 55 “Í>0ttPnnnn. 57 Materials And Method’ PS nhanh cố ốốố. 73 Tu i00 6s 1-7-0 ng cố ốốố. 88 CHAPTER 4: Summary and Future Direcfions.

HH hưu 108 SJ/////12a080PP0P7Ẽ7Ẽ7Ẽ85858Ẻ86 h6. 108 7//0/(29)/(-0/00/TT TT 0 no 3ẦÝÊŸỶỶ. Lu ix LIST OF FIGURES Figure 1.0: Stereotypical pattern OF miQrafÍOH. nen HH HT HH ng go Hà nh Hà ha 11 Figure 2.1: Foil barriers are ineffective at blocking Cranial neural cr@Sf.2: Leaders and followers change when a population of neural crest cells encounters a -/-EPPPSn.3: Neural crest cells that are blocked behind the barrier tend to migrate faster than cells Chat YO APOUNC UNC DAITIOL oo.

eee ccctccctscstcenscssecceeeeseesseccsessnecssecuseesasecsecaissssucssessaasessseseseaseceeessceensease 49 Figure 2.4: Neural crest cells are able to migrate into the r3 repulsive zone by migrating on top of CCH OMNOL eee eccecccneenceeececeeetetecsscansnecsaeeseseneesesesuesassauseassnssaesascuassascnsssasaaseasvasesatestcasecaterssatsaseatarasas 57 Figure 3.1: Differential expression of EphA4 and ephrin-A5 within the cranial region of an HH12 09165 PP®-.2: Ectopic ephrin-A& expression leads to fewer NCCs in BA3 inifially.3: Neural crest cells ectopically expressing ephrin-A5 do not migrate to BA3.4: Ephrin-A5+ r6 NCCs do not migrate tO BAS.5: EphA4(int) and EphA4(kd) causes mismigration along the otic vesicle.6: Cell morphology, temporal distribution, and Cell-traCkiNg.7: Eph/ephrin concentration and migrafÍOn. - - -- S121 HT HH Hệ, 98 Figure 3.8: Ectopic ephrin-A5 and EphA4 acfÍVÍẨW. ác c1 12 LH HH TY TH HH HH key 100 Figure 3.9: Summary of ephrin-A5 and EphA4 perturbations .cccccccccccessecssssessetesssenssessessenseees 102 Supplementary materials: cell death and Cell Proliferation .0: Distinct mechanisms guide neural crest migration at r4 and rồ. Lu x LIST OF TABLES Table 2.1: Mean velocity and directionality for barriers at r3 and r.

ST Hs he 53 Table 3.1: Cell tracking analysis: cranial neural Crest C@lÏS. -- - - cà HH HH HH Hệ, 103 Table 3.2: Cell tracking analysis: r8 subpopulation of cranial neural crest cellS.3: Rates of cell proliferation and deatÏh. HH HH HH HH HH Hệ, 105 Carole C. Lu xi KEYWORDS avian embryo cell migration cell-tracking confocal microscopy cranial neural crest electroporation EphA4 ephrin-A5 foil barrier particle-tracking time-lapse imaging whole-embryo explant culture Carole C.

Lu 1 CHAPTER 1: Introduction Introduction The process by which we develop out of a single fertilized egg is wonderfully complex. In theory, it is easy to understand— one cell divides into two daughter cells that too go on to divide until there is a population of cells that makes up an entire organism. How do we end up being a complex organism rather than a clump of cells? How do cells become patterned and coordinated into structures? The question of pattern formation is a global one. Cell division is but one aspect of development.

From the cell’s point of view, there are many different choices along the way. Not only can they divide, but also they can die, differentiate, migrate, respond to cues in the environment, secrete cues into the environment, or any combination of the above. All these actions by individual cells need to occur in an orchestrated fashion such that at the end, there is a complete and functional multi-cellular organism. During my tenure as a graduate student, | chose the migration of cranial neural crest cells within the avian embryo as the system in which to address how migration is involved in pattern formation.

Avian embryo The avian embryo has been a classic system for embryological studies since Aristotle (Aristotle, 350 B.E) for a number of reasons. Fresh, fertilized eggs are easy to obtain, available year round, relatively cheap, develop externally, and are easy to handle within a laboratory setting. The Hamburger Carole C. Lu 2 and Hamilton staging series (1951) allows one to conveniently set eggs for a certain amount of time to obtain embryos at the desired developmental stage.

A good anatomical understanding of the embryo is also available (Bellairs and Osmond, 2005). The accessibility and size of the embryo allows many types of microsurgical techniques such as ablation and grafting. Beside these more classic, embryological types of studies, new techniques have allowed us to take advantage of recent advances in molecular and cell biology. We can functionally test the roles of certain proteins or genes by implantation of protein-soaked beads; electroporation of DNA constructs, MRNA, or morpholinos; and viral transfection (Bronner-Fraser, 1996; Itasaki et al., 1999; Momose et al., 1999; Okada et al., 1999; Swartz et al., 2001; Thakur et al.

The chick genome has been sequenced and allows researchers to take advantage of newly available genomic resources (reviewed in Antin and Konieczka, 2005). The chick genome offers an interesting evolutionary perspective since it is positioned between lower vertebrates, such as fish, and higher vertebrates, such as humans. Lastly, since the avian embryo is a vertebrate embryo, many of the things we learn will be relevant to understanding human development. Cranial neural crest cells: what are they and why are they important?