Comparative molecular and morphogenetic characterisation of larval body regions in the polychaete annelid Platynereis dumerilii [Elektronische Ressource] / vorgelegt von Patrick R. H. Steinmetz

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2006
• Nombre de lectures: 54
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Comparative molecular and morphogenetic
characterisation of larval body regions
in the polychaete annelid Platynereis dumerilii
Dissertation
zur Erlangung des Doktorgrades der Naturwissenschaften
Doctor rerum naturalium
(Dr. rer. nat.)
dem Fachbereich der Biologie
der Philipps-Universität Marburg
vorgelegt von
Dipl.-Biol. Patrick R. H. Steinmetz
aus Luxemburg, Großherzogtum Luxemburg
Marburg/Lahn, März 20062
Vom Fachbereich Biologie
der Philipps-Universität Marburg als Dissertation am
angenommen.
Erstgutachterin: Prof. Dr. Monika Hassel
Zweitgutachterin: Prof. Dr. Renate Renkawitz-Pohl
Tag der mündlichen Prüfung:
Erklärung
Ich versichere hiermit, dass ich meine Dissertation
“Comparative molecular and morphogenetic characterisation of larval body regions in
the polychaete annelid Platynereis dumerilii”
selbst und ohne unerlaubte Hilfe verfasst und mich dabei keiner anderen Hilfsmittel
als der von mir ausdrücklich bezeichneten Quellen und Hilfen bedient habe und dass
ich die Dissertation in der vorliegenden oder einer ähnlichen Form bei keiner anderen
Hochschule zu Prüfungszwecken eingereicht habe.
Marburg, den 31.März 2006
Dipl.-Biol. Patrick Steinmetz3
Comparative molecular and morphogenetic charac-
terisation of larval body regions in the polychaete an-
nelid Platynereis dumerilii4
Acknowledgments
I am very thankful to Dr. Detlev Arendt for all his support that helped me to become a
PhD student in his lab, for his supervision, help, and sharing his endless scientific
knowledge. I would also like to thank Dr. Jochen Wittbrodt for his financial support
during part of my time as a PhD student. I thank Prof. Dr. Monika Hassel for her sup-
port, help and for reviewing this thesis. I thank Dr. Jochen Wittbrodt, Prof. Dr.
Monika Hassel and Dr. Elena Conti for helpful comments, criticism and discussions
as members of my thesis advisory committee. I also thank Prof. Dr. Renkawitz-Pohl
for accepting to review this thesis and all the other members of my defence committee
at the University of Marburg.
I acknowledge the people from the Ministère de la Culture, de l’Enseignement Supé-
rieur et de la Recherche, especially Pierre Decker and Josiane Entringer, for the allo-
cation of a “bourse formation-recherche” that made this thesis possible.
I want to thank all past and present members of the Arendt team for their support,
critical comments and help, especially Dr. Carola Burgtorf, Dr. Fabiola Zelada and
Dr. Detlev Arendt for their previous work on the Platynereis otx, gbx, hox1, and six3
genes, without which considerable parts of this thesis would not have been possible,
Dr. Kristin Tessmar-Raible for technical help, helpful discussion and constructive
criticism and Heidi Snyman for keeping the Platynereis worms happy and alive as
well as for technical help and personal support. I thank Isabelle Philipp (Innsbruck)
for a SP600125 protocol.
I also thank Jens Rietdorf, Timo Zimmermann and Stefan Terjung from the Advanced
Light Microscopy Facility (ALMF) at EMBL for their initiation, help and support in
confocal microscopy and Dr. Martina Rembold (Wittbrodt lab) for very helpful hints
and comments on time-lapse analysis.
I would like to thank the librarians at the EMBL Szílard library, especially David
Wesley for digging out ancient books and publications.5
I express my thanks for materials to Prof. Dr. Michael Brandt (BODIPY dyes), Dr.
Angel Nebreda (α-tubulin antibody) and Dr. Jochen Wittbrodt (medaka cDNA).
Last but not least, I am very thankful to my family for their support and the following
people for the good moments I have shared with them outside the lab (in order of ap-
pearance): Tom Fleming, Solveig Frick, Inês Baptista, Heike Link, Christiane Jost,
Will Norton and Corina Guder.6
Table of contents
1 INTRODUCTION............................................................................................................................. 13
1.1 UNDERSTANDING THE EVOLUTION OF ANIMALS BY STUDYING THEIR DEVELOPMENT............ 13
1.2 PLATYNEREIS DUMERILII AS A MODEL ORGANISM TO STUDY EVOLUTION AND DEVELOPMENT .
.................................................................................................................................................... 13
1.3 THE LIFE CYCLE OF PLATYNEREIS DUMERILII........................................................................... 15
1.4 LARVAL MORPHOLOGY OF THE PLATYNEREIS DUMERILII TROCHOPHORE............................... 16
1.5 EARLY DEVELOPMENT, EPIBOLY AND GASTRULATION IN PLATYNEREIS DUMERILII............... 18
1.6 MOLECULAR REGIONALISATION OF THE NEUROECTODERM IN BILATERIA.............................. 22
1.6.1 Six3 orthologues regionalise the most rostral neuroectoderm in Bilateria....................... 24
1.6.2 Otx as regionalisation marker for anterior head structures in Bilateria........................... 25
1.6.3 Gbx genes specify the neuroectoderm posterior of the otx expressing territories in
Bilateria............................................................................................................................................... 26
1.6.4 Hox1 as regionalisation markers in bilaterian brains........................................................ 27
1.6.5 Engrailed is a conserved marker of segment boundaries in arthropods and Platynereis ....
............................................................................................................................................... 28
1.7 MOLECULAR CHARACTERISATION OF THE DEVELOPING MESODERM IN PLATYNEREIS........... 28
1.7.1 The role of twist in mesoderm development........................................................................ 29
1.7.2 The mesodermal patterning role of myoD orthologues...................................................... 30
1.7.3 The role of mef2 orthologues as mesodermal differentiation genes .................................. 30
1.7.4 Troponin I as a marker gene for differentiated muscle cells.............................................. 31
1.8 THE EVOLUTION OF GASTRULATION MOVEMENTS IN BILATERIA............................................. 32
2 MATERIAL AND METHODS ....................................................................................................... 36
2.1 TECHNICAL EQUIPMENT ............................................................................................................ 36
2.2 STANDARD CLONING VECTORS AND BACTERIAL STRAINS........................................................ 36
2.3 PLATYNEREIS DUMERILII CULTURE........................................................................................... 36
2.4 LIQUID AND SOLID BACTERIAL CULTURE MEDIA AND BUFFERS............................................... 37
2.5 ANTIBODIES ............................................................................................................................... 38
2.6 SINGLE COLOUR WHOLE-MOUNT IN SITU HYBRIDISATION (WMISH)...................................... 38
2.6.1 Probe preparation................................................................................................................ 38
2.6.2 Hybridisation procedure...................................................................................................... 39
2.7 DOUBLE COLOUR FLUORESCENT IN SITU HYBRIDISATION........................................................ 40
2.8 IMMUNOHISTOCHEMISTRY ........................................................................................................ 42
2.9 BRDU ASSAY ............................................................................................................................. 42
2.10 VISUALISING F-ACTIN BY PHALLOIDIN STAINING..................................................................... 43
2.11 IN VIVO STAINING OF CELLULAR OUTLINES BY BODIPY564/570 ........................................... 43
2.12 TIME-LAPSE RECORDINGS.......................................................................................................... 43
2.13 MORPHOMETRIC MEASUREMENTS............................................................................................. 447
2.14 INCUBATIONS IN NOCODAZOLE, CYTOCHALASIN B AND SP600125....................................... 44
2.15 GENERAL GENE CLONING STRATEGY ........................................................................................ 44
2.15.1 Cloning of novel fragments............................................................................................. 44
2.15.2 Rapid amplification of cDNA-ends (RACE) of existing fragments ............................... 45
322.16 SYNTHESIS OF P-LABELED PROBES......................................................................................... 46
2.17 SOUTHERN BLOTS AND COLONY-LIFTS FROM BACTERIAL PLATES........................................... 46
2.17.1 High stringency Southern Blots and colony-lifts ........................................................... 46
2.17.2 Low stringency Southern Blots with heterologous probes ............................................ 47
2.18 POLYMERASE CHAIN REACTIONS .............................................................................................. 47
2.18.1 Reaction mixtures for cloning of novel fragments for twist, strabismus and dachsous ...
.......................................................................................