Expression of Axdazl and Axvh In
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Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2006 Expression of Axdazl and Axvh in Axolotl Germ Cells, Suggest that Regulative Germ Cells Specifications is a Primitive Trait Conserved in the Mammalian Lineage Matthew J. Drum Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES EXPRESSION OF AXDAZL AND AXVH IN AXOLOTL GERM CELLS, SUGGEST THAT REGULATIVE GERM CELL SPECIFICATION IS A PRIMITIVE TRAIT CONSERVED IN THE MAMMALIAN LINEAGE. By MATTHEW J. DRUM A Disse tation submitted to the Depa tment of Biologi.al S.ien.e in pa tial fulfillment of the e/ui ements fo the deg ee of Do.to of Philosophy Deg ee A0a ded1 Sp ing Semeste , 2004 The membe s of the Committee app ove the Disse tation of Matthe0 J. D um defended on Ap il 4, 2004. __________________________ Hen y W. Bass P ofesso Di e.ting Disse tation __________________________ Cathy W. Levenson Outside Committee Membe __________________________ Geo ge W. Bates Committee Membe __________________________ Robe t H. Reeves Committee Membe __________________________ G ego y M. E i.7son Committee Membe App oved1 __________________________ Timothy S. Moe land, Chai , Depa tment of Biologi.al S.ien.e __________________________ Joseph T avis, Dean, College of A ts and S.ien.es The Offi.e of G aduate Studies has ve ified and app ovedthe above named .ommittee membe s. ii ACKNOWLEDGEMENTS I 0ould li7e to a.7no0ledge and than7 the membe s of my .ommittee 8past and p esent91 Dan Ga :a, Bob Reeves, Geo ge Bates, G eg E i.7son, Jim Fadool, B ian C othe , John Teem, and Cathy Levenson fo giving me thei input and di e.tion du ing my time at FSU. Mo e pa ti.ula ly, I 0ould li7e to than7 Han7 Bass fo going ;above and beyond the .all of duty“, and loo7ing afte me ove the last yea and mo e spe.ifi.ally, fo sho0ing me 0hat it means to thin7 and 0 ite li7e a s.ientist. I am p oud to .all him my majo p ofesso and a good f iend. I 0ould li7e to than7 some of my fello0 g aduate students, someof the best guys I have eve had the fo tune of meeting, B ian Ring, E i. Si7o s7i, Tommy Ho.7, Jason Robotham, O ion Vande linde, La0 en.e Le.la e and Steve Mille . I 0ould also li7e to than7 Amy and Lynnette–both of them have ve y spe.ial pla.es in my hea t. My lab mates 0e e a0esome. My t0o little siste s-- Jen and Jess, and the est of the guys, Fe ell, Tim, Anthea, Me iedith, and Hilda made .oming into 0o 7 eve yday an adventu e, I t uly miss them all. I 0ould also li7e to than7 Steve Mille 8again9, Rani Dha ajen, and Ch is Ba.ot fo putting up 0ith my out ageous .loning, and se/uen.ing e/uests, and Ken Taylo , Robin Smith, and Ellen G ange fo ma7ing tea.hing at FSU a pleasu e. Last and .e tainly not least, I must than7 AJ Johnson, fo giving me the oppo tunity to be.ome a membe in his lab and to 0o 7 on a p oje.t that I eally love. AJ 0as the se geant that I 0ould have follo0ed into any battle. He 0o 7ed ha d at FSU, and did some g eat s.ien.e. We spent a lot time togethe , and in all instan.es, he sho0ed me infinite patien.e. I lea ned mo e f om AJ then he 0ill eve 7no0, and I am t uly indebted to him. I also 0anted to than7 my good f iend and .olleague Tom Masi. Tom is gifted s.ientist blessed 0ith g eat hands. ;If the e is some assay o eApe iment that .anBt be done, give it to Tom and he 0ill 0o 7 it out“, this 0as the gene al sentiment not just in the Johnson lab, but in all of Unit 1. Nume ous times I sa0 him selflessly put his 0o 7 do0n to help othe s. He iii is a gene ous and loyal f iend. Tom and I have seen a lot togethe , and he has been li7e a big b othe to me, and I 7no0 that I 0ould not have finished 0ithout him. iv TABLE OF CONTENTS List of Tables ................................................................................................................. vii List of Figu es............................................................................................................... viii Abst a.t............................................................................................................................ A 1. SURVEY OF GERM CELL FORMATION IN MODEL SYSTEMS.......................1 2. CLONING AND CHARACTERIZATION OF AXVH AND THE EXPRESSION IN THE AXOLOTL GERM CELL LINEAGE Ba.7g ound......................................................................................................12 Results..............................................................................................................14 Dis.ussion........................................................................................................1D E. EXPRESSION OF AXDAZL RNA IS WIDESPREAD IN AXOLOTL OOCYTES AND EARLY STAGE EMBRYOS AND GERM CELL SPECIFIC AS GERM CELLS APPROACH THE GONAD Ba.7g ound......................................................................................................27 Results..............................................................................................................2D Dis.ussion........................................................................................................E2 4. EVOLUTON OF THE REGULATIVE GERM CELL FORMING MECHANISM IN TERRESTRIAL VETEBRATES Ba.7g ound......................................................................................................42 Results..............................................................................................................45 Dis.ussion........................................................................................................4I v 5. METHODS .............................................................................................................54 CONCLUSIONS.............................................................................................................71 REFERENCES ...............................................................................................................7E BIOGRAPHICAL SKETCH ..........................................................................................IE vi LIST OF TABLES 1. Vasa orthologs from vario s species ...............................................................58 2. N cleotide se( ences of primers sed for degenerate PCR..........................,4 . Thermocycler conditions for to chdo0n PCR................................................,5 4. N cleotide se( ence of primers sed for DNA se( encing...........................12 5. N cleotide se( ences of primers sed for 52 RACE .....................................12 ,. N cleotide se( ence of primers sed in analysis of RNA by RT4PCR ........15 1. N mber of PCR cycles performed in RT4PCR e6periments ........................80 vii LIST OF FIG8RES 1. Origin and position of primordial germ cells in Xenopus laevis d ring early embryogenesis.......................................................................................15 2. Position of primordial germ cells in a6olotl d ring embryogenesis..........20 .. A6olotl embryo recombination e6periments performed by Nie 09oop d ring the blast la stage ...............................................................................21 4. Ad lt e6pression of Axvh................................................................................1 5. Temporal e6pression of Axvh in the developing embryo ............................2 ,. E6pression of Axvh transcripts in dissected embryos................................... 1. Cell specific Axvh e6pression in migrating PCGs ........................................5 8. E6pression of Axvh in gonadal germ cells....................................................., 5. Dazl ortholog se( ence comparison .............................................................44 10. .2 8TR Comparison bet0een Axdazl and Xdazl..........................................45 11. IS: of the mitochondrial clo d in Xenopus and a6olotl oocytes ...............4, 12. Sectioned IS: of Axdazl in gonadal germ cells ...........................................41 1.. Temporal e6pression of Axdazl d ring embryogenesis ..............................48 14. Sectioned embryo RT4PCR sing Axdazl probe .........................................45 15. Cell specific e6pression of Axdazl in migrating PGCs ................................51 1,. Ectopic ind ction of PGCs from animal caps .............................................55 11. Evol tionary tree depicting type of embryology and mode of germ cell formation ........................................................................................................,0 18. Se( ence of Provh cDNA and predicted protein.........................................,1 viii 15. Germ cell specific e6pression of Provh .......................................................,2 20. Vasa ortholog cladistic reconstr ction ........................................................,. iA ABSTRACT Ho0 ge m .ells a e spe.ified in animal emb yos has been a myste y fo de.ades. Unli7e most developmental p o.esses, 0hi.h a e highly .onse ved, emb yos spe.ify ge m .ells in ve y diffe ent 0ays. The emb yos of seve al p ominent model o ganisms .ontain ge m .ell dete minants 8ge m plasm9 that seg egate to ge m .ell p e.u so s. In othe animals, in.luding mi.e, ge m .ells fo m in esponse to egulative me.hanisms du ing development. It has been suggested that the ge m line is spe.ified simila ly in u odele amphibians. To investigate the simila ity bet0een u odeles and mi.e, I .loned t0o genes f om the salamande Ambystoma mexicanum 8aAolotl9 that a e e/ui