Early steps in ventral nerve cord development in chelicerates and myriapods and formation of brain compartments in spiders [Elektronische Ressource] / von Carola Maria Döffinger

Carola Hähnel-Mesnard

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2010
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Early steps in ventral nerve cord development
in chelicerates and myriapods and formation of
brain compartments in spiders



Dissertation
zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften
im Fachbereich Biologie


Eingereicht an der Johannes Gutenberg-Universität
Mainz
von Diplom Biologin
Carola Maria Döffinger

Geboren am 19.09.1979
in Böblingen



Mainz, März 2010













Dekan:


1. Berichterstatterin:

2. Berichterstatter:


Datum der mündlichen Prüfung:
07.06.2010
INDEX OF CONTENTS
Index of contents

1 Introduction ..............................................................................1
1.1 Early neurogenesis in arthropods .................................................................2
1.1.1 Formation of neural precursors........................................................2
1.1.2 Neuroblast lineages versus neural precursor groups ......................7
1.1.3 Identity of neural precursors ............................................................8
1.1.3.1 DV patterning of the developing nervous system in
vertebrates and invertebrates .............................................10
1.1.4 Marker gene expression in postmitotic differentiating neural cells.12
1.2 Brain development in arthropods ................................................................13
1.2.1 Organisation of the brain ...............................................................15
1.2.2 Brain development in insects.........................................................17
1.3 Aims............................................................................................................18
2 Materials and Methods ..........................................................20
2.1 Equipment...................................................................................................20
2.2 Chemicals20
2.3 Biomolecular methods ................................................................................20
2.3.1 Phenol-chloroform precipitation.....................................................20
2.3.2 Ethanolic precipitation ...................................................................20
2.3.3 Gel electrophoresis........................................................................21
2.3.3.1 DNA-Gel .............................................................................21
2.3.3.2 RNA-Gel22
2.3.4 Working with RNA .........................................................................22
2.3.5 RNA-Isolation ................................................................................22
I INDEX OF CONTENTS
2.3.6 cDNA synthesis .............................................................................23
2.3.7 PCR...............................................................................................24
2.3.7.1 PCR reactions.....................................................................24
2.3.8 5‘ PCR ...........................................................................................25
2.3.9 Cloning ..........................................................................................25
2.3.9.1 Preparation of vector...........................................................25
2.3.9.2 insert............................................................25
2.3.9.3 Ligation ...............................................................................26
2.3.9.4 Transformation with electrocompetent cells........................26
2.3.9.5 tion with chemocompetent cells27
2.3.9.6 Colony PCR ........................................................................27
2.3.9.7 Bacterial cultures ................................................................28
2.3.9.8 Isolation of plasmid DNA from bacteria...............................28
2.3.10 Generation of RNA probes ............................................................28
2.3.11 Generation of double-stranded RNA interference..........................29
2.3.12 Sequences and Primers30
2.4 Animal stocks..............................................................................................30
2.4.1 Achaearanea tepidariorum animals ...............................................30
2.4.2 Cupiennius salei animals...............................................................31
2.4.3 Glomeris marginata animals..........................................................31
2.5 Preparation of embryos for in situ hybridisation and
immunohistochemistry ................................................................................32
2.5.1 Dechorionisation............................................................................32
2.5.2 Fixation..........................................................................................32
2.5.2.1 Cupiennius and Achaearanea for Phalloidin staining..........32
2.5.2.2 for in situ hybridisation .....................................32
2.5.2.3 Glomeris for Phalloidin staining ..........................................33
II INDEX OF CONTENTS
2.5.2.4 Glomeris for in situ hybridisation.........................................33
2.5.3 Devitellenisation ............................................................................33
2.6 Injection of dsRNA interference ..................................................................33
2.6.1 RNAi injection in Cupiennius salei embryos ..................................33
2.6.2 Parental RNAi in Glomeris marginata............................................34
2.7 Immunohistochemistry ................................................................................35
2.7.1 Phalloidin staining..........................................................................35
2.7.2 Antibody staining ...........................................................................35
2.7.3 In situ hybridisation........................................................................36
2.8 Documentation and evaluation of Phalloidin staining, in situ hybridisation
and antibody staining ..................................................................................39
2.8.1 Flat preparations of embryos.........................................................39
2.8.2 Documentation ..............................................................................39
2.8.3 Evaluation......................................................................................39
3 Results ....................................................................................40
3.1 Neural precursor identity in chelicerates and myriapods.............................40
3.1.1 Number and arrangement of neural precursor groups in the
chelicerates Achaearanea tepidariorum, Cupiennius salei and
the myriapod Glomeris marginata..................................................40
3.1.2 In the expression domain of the DV patterning gene msh in
Glomeris marginata seven to eleven identified NPGs are
recruited.........................................................................................44
3.1.3 Two to six NPGs are recruited in the expression domain of the
DV patterning gene ind in Glomeris marginata..............................46
3.1.4 The expression of msh and ind seem to be mutually exclusive in
the leg segments of Glomeris marginata .......................................48
3.1.5 msh and ind might overlap in the anterior
segments of Glomeris marginata...................................................50
III INDEX OF CONTENTS
3.1.6 RNA interference experiments did not reveal reliable data about
the gene functions of msh and ind in Glomeris marginata.............53
3.1.7 All NPGs of rows f and g and some NPGs of row a are recruited
in the expression domain of the segment polarity gene engrailed
in Cupiennius salei ........................................................................54
3.1.8 Eleven NPGs are recruited in the expression domain of the DV
patterning gene msh in Cupiennius salei.......................................55
3.1.9 The homologue for the Drosophila DV patterning gene ind could
not be identified in Cupiennius salei ..............................................61
3.1.10 The differentiation marker even-skipped is expressed in a small
cell cluster in the NE of the posterior segments in Cupiennius
salei ...............................................................................................62
3.1.11 islet is expressed in several cell
clusters in the lateral half of the NE in Cupiennius salei ................64
3.1.12 The DV patterning gene msh is involved in conferring specific
identities to the NPGs in Cupiennius salei.....................................66
3.1.12.1 msh expression is reduced by the injection of double-
stranded msh RNA interference..........................................67
3.1.12.2 msh does not influence the number and arrangement of
NPGs ..................................................................................69
3.1.12.3 msh plays a role in neural precursor identity.......................71
3.2 Brain development in chelicerates ...............................