Development and molecular characterization of adult and larval eyes in Platynereis dumerilii (Polychaeta, Annelida, Lophotrochozoa) [Elektronische Ressource] / presented by Keren Guy

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 45
• Langue: English
• Poids de l'ouvrage: 29 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences






















Presented by
Keren Guy

Born in: Ramat-Gan, Israel
Oral-examination: 2.12.2008

Group leader: Dr.Detlev Arendt

Referees: Prof. Thomas Holstein
Dr. Jan EllenbergDevelopment and molecular

characterization of adult and larval

eyes in Platynereis dumerilii

(Polychaeta, Annelida,
Lophotrochozoa)




PhD Student: Keren Guy

Group Leader: Detlev Arendt

PhD Examiners:

Prof. Thomas Holstein
Dr. Jan Ellenberg
Dr. Darren Gilmour
Dr. Jochen Wittbrodt Table of content

Abstract 1

Zusammenfassung 2

Introduction
1.1 Overview 5
1.2 Variety of eyes 6
1.2.1 The basic units of the eye 8
1.2.1.1 Photoreceptor cells 8
1.2.1.2 Pigment cells and pigment synthesis 9
1.2.2 The phototransduction cascade in Ciliary versus Rhabdomeric 11
photoreceptors
1.2.3 The lens 12
1.3 Cell type comparison and molecular fingerprint concept 15
1.4 Platynereis dumerilii as a model organism to study evolution and 15
development
1.4.1 Why choosing a Lophotrochozoa as a model organism? 16
1.4.2 Platynereis dumerilii (Polychaeta, Annelida) as model
organism–In general 17
1.4.3 For eye development and eye evolution 17
1.4.4 Eye development of Platynereis dumerilii 18
1.4.5 Introducing the investigated specimen – Platynereis
embryos and life cycle 19
1.5 Molecular regulation of visual systems development 21
1.5.1 Key regulators of eye development 21
1.5.1.1 The homeobox containing genes: Otx 21
1.5.1.2 Pax family of transcription factors 21
1.5.1.3 Prox1/Prospero 23
1.5.1.4 The paired like homeobox gene: Rx 23
1.5.2 The retinal determination gene network 25
1.5.3 The Hedgehog signaling pathway 29
1.5.3.1 Pathway overview 29
1.5.3.2 Hh pathway in eye development 31
1.5.3.3 The use of Cyclopamine for inhibiting the Hh pathway 34
Results
2.1 Identification of Platynereis eye specific genes 37
2.1.1 EST collection screen for molecular markers of Platynereis 37
Eyes 2.1.1.1 The identification of several eye markers: 42
Synaptotagmin, Tryptophane 2,3 dioxygenase,
Sepiapterin Synthase A, FVRI
2.1.2 Construction of a brain-eye specific library 44
2.1.3 Eyes absent 3’ RACE 44
2.1.3.1 Eyes absent expression pattern 45
2.1.4 Prox1 cloning and expression (collaboration) 45
2.1.5 MITF cloning attempts 49
2.1.6 Lens protein identification project 49
2.1.7 r-opsin II cloning 50
2.1.7.1 r-opsin II expression 51
2.1.7.2 r-opsin II southern blot 51
2.1.8 BAC screening 52
2.2 Assignment of different eye markers to different cell types 54
of the larval eyes
2.2.1 The early expression of three LE markers: 58
Synaptotagmin, FVRI, Tryptophan 2,3 dioxygenase.
2.3 Assignment of different eye markers to different cell types 60
of the adult eyes
2.4 Establishing a molecular fingerprint of Platynereis adult 63
and larval eyes
3 A role for the hedgehog signaling pathway in Pdu eyes development 74
3.1 The expression of Hh, Gli-1, Smo and sufu 74
3.2 Cyclopamine inhibition results 76
Discussion
3.1 Platynereis adult versus larval eyes 84
3.1.2 Molecular fingerprint of Platynereis adult and larval eyes 85
3.1.3 Molecular differences between adult and larval eyes 86
3.1.3.2 Platynereis larval eye r-opsinII 86
3.1.4 The ancestral single cell eye hypothesis 87
3.1.5 Comparing Platynereis and Drosophila eyes MFP 88
3.2 The fine structure of Platynereis adult and larval eyes 90
basic units: rPRC and PCs
3.3 Larval and adult eyes respond differently to hedgehog antagonist 91
3.3.1 Differences in Hh signaling between Drosophila and Mammals 92
3.4 Prox1 cloning and expression (collaboration) 93
3.5 Pax genes expression in Platynereis adult and larval eyes 94
3.6 Candidate regulators for Platynereis adult eye development 95 3.7 The function of Platynereis adult and larval eyes 96
Materials and Methods
4.1 Platynereis dumerilii animals and embryos 99
4.1.1 Platynereis dumerilii culture 99
4.1.2 Platynereis dumerilii embryos handling 99
4.2 Gene cloning 99
4.2.1 General gene cloning techniques 99
4.2.2 r-opsinII cloning 103
4.2.3 EyeA 3’ RACE 104
4.3 Whole mount in-situ hybridization 105
4.3.1 General protocol - single probe detection with 105
Acetylated tubulin antibody and DAPI staining
4.3.2 Double fluorescent in-situ hybridization protocol 108
4.3.3 Double detection: fluorescent probe and DIG probe 109
4.4 Cyclopamin inhibitions 111
4.5 construction of brain-eye specific library 113
4.5.1 Trizol extraction of total RNA 113
® 4.5.2 mRNA isolation using Dynabeads 114
4.5.3 cDNA library synthesis and cloning 114

References