Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of ...

273 Pages·2012·9.96 MB·English

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
Maria Antonietta Tosches
born in Foggia, Italy
Oral-examination: .....................

Development and function
of brain photoreceptors
in the annelid Platynereis dumerilii
Referees: Dr. Darren Gilmour
Prof. Dr. Jochen Wittbrodt

“Quelli che s’innamoran di
pratica sanza scienzia son come
’l nocchier ch’entra in navilio
senza timone o bussola, che mai
ha certezza dove si vada.”
Leonardo da Vinci
(Anyone who loves practice without
theory is like a sailor going aboard a
ship without rudder or compass and
havingnoideawhereheisgoing.)

cknowledgments
A
Thisworkwasmadepossiblebythecontributionandthesupportofmanypeople,andhere
I want to express all my gratitude for them.
First, I wish to thank Detlev Arendt, for giving me the opportunity to work in his lab,
for the supervision and the support during this work, and for all our inspiring discussions,
which contributed so much to my scientific maturation in these years.
I also want to thank my thesis advisory committee at EMBL, Prof. Jochen Wittbrodt, Dr.
DarrenGilmourandDr. AnneEphrussi,fortheircommentsandvaluableadviceduringthis
work.
Moreover, IwouldliketothankGasparJekelyforhiscriticalfeedback, forveryinteresting
discussions, for hosting the preparation of a neuropeptide antibody and for sharing unpub-
lished data. Thanks also to Kristin Tessmar-Raible, for discussing data on the melatonin
system, and for sending me her opsin antibody.
I am especially grateful to Nicola Kegel: we worked together on the Rx project - that
she had initiated - during my first months in the lab, and she taught me all the secrets of
Platynereis injections. Thanks also to Marianna Karageorgi and Nathalie Tisch, for helping
me with special commitment and enthusiasm during their internships in the lab.
Here, I would like to thank the EMBL GeneCore and ALMF, for providing assistance and
facilitiesformyexperiments. ThanksalsototheIT-Services,forrescuingmydatamorethan
once...
I feel very lucky because in these years I had the opportunity to work with many fantastic
people,thepastandpresentmembersoftheArendtlab. ThankstoMetteHandberg-Thorsager
andAntonellaLauriforbeinggreatbenchmates,andforworkingtogetheronthedevelopment
of new techniques for our unusual model system, with all the excitement and sometimes the
frustration that this brings. I am indebted to Heather Marlow and Pavel Vopalensky: our
vii
stimulating discussions on larval evolution and photoreceptors contributed significantly to
theinterpretationofmydata. ThankstoOlegSimakovandTomasLarsson,fortakingcareof
thePlatynereisgenomicandtranscriptomicresources,whichweresoimportantforthiswork.
I am grateful to Raju Tomer, who developed the PrImR resource, and assisted the generation
of new average expression patterns. Thanks to Antje Fischer, who had always the patience
to answer my naive questions on zoology and evolution. Moreover, I wish to thank Diana
Hofmannfortakingcaresowelloftheanimalcultures,andinparticularthemutantworms,
and Heidi Snyman for being a special “lab navigator” in these years. Thanks also to Fay
Christodoulou, for being so much supportive, and for constantly spreading her enthusiasm
and energy. Finally, I want to thank all the past and present members of the lab - the list
would be so long here! - for the pleasant and stimulating working atmosphere. A special
thanks to Silvia Rohr for translating the Summary of this thesis.
During these years in Heidelberg, I could count on the support of my “old” friends, espe-
cially those who by chance moved in the neighborhood, and of new friends here at EMBL. I
want to thank them all for the support, and for having a great time together outside the lab.
My special gratitude belongs to Daniele. Thanks for tolerating my bad days and for
enjoying with me the good ones. Thanks also to my parents, my sister and my brother.
Despite the long distances, you have always been truly supporting and understanding. It’s
been a long way... and you have always been by my side.
viii
able of contents
T
Summary 1
Zusammenfassung 3
I Introduction 5
1 Deciphering the evolution of nervous systems 7
1.1 Diversity of nervous systems in the animal kingdom . . . . . . . . . . 7
1.2 The annelid Platynereis dumerilii: a model system for studying brain
evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.2.1 Life cycle and early development of P. dumerilii . . . . . . . . . 10
1.2.2 Advances of P. dumerilii as a model system . . . . . . . . . . . . 13
1.3 Homologies of brain regions and cell types in Bilateria . . . . . . . . . 13
1.3.1 Conservation of brain regions along the anterior-posterior and
the dorso-ventral axes . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3.2 Conservation of cell types . . . . . . . . . . . . . . . . . . . . . . 15
1.3.3 The division of labour model for nervous system evolution . . 17
1.4 Studying marine larvae uncovers the earliest steps in brain evolution. 20
2 Evolution of photoreceptor cell types and non-visual light detection 21
2.1 Ciliary and rhabdomeric photoreceptors . . . . . . . . . . . . . . . . . 21
2.1.1 Phototransduction cascades . . . . . . . . . . . . . . . . . . . . . 23
2.1.2 Opsin families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.2 Non-visual light detection in animals . . . . . . . . . . . . . . . . . . . 27
2.2.1 Illuminance detection and circadian rhythms in protostomes . 28
2.2.2 Illuminance detection and circadian rhythms in vertebrates . . 30
ix
2.3 The evolution of the vertebrate pineal and melatonin system . . . . . . 32
2.3.1 The pineal complex: luminance detection, melatonin release
and shadow response . . . . . . . . . . . . . . . . . . . . . . . . 32
2.3.2 CSF contacting neurons and deep brain PRCs . . . . . . . . . . 36
2.3.3 Early evolution of pineal and retina . . . . . . . . . . . . . . . . 37
2.4 Roles of Rx in the development of ciliary photoreceptors . . . . . . . . 39
2.5 Aim of the thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
II Results 43
3 Development of Platynereis dorsal brain 45
3.1 Morphological landmarks in the dorsal brain . . . . . . . . . . . . . . . 45
3.2 Timing of cell cycle exit and differentiation of the main cell types of
the dorsal brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.3 Time-lapse imaging reveals the early steps of brain development . . . 51
3.3.1 First insights in Platynereis neurogenesis . . . . . . . . . . . . . 55
3.4 Connecting lineages to early expression patterns and cell types in the
developing episphere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.4.1 The dorsal midline and the asymmetric serotoninergic cell. . . 60
3.4.2 The lateral serotoninergic cells and the adult eye region . . . . 63
3.4.3 The ciliary photoreceptors and the medial photoreceptive region 65
4 Establishment of techniques to study gene function in Platynereis 67
4.1 The genomic structure of the rx gene. . . . . . . . . . . . . . . . . . . . 68
4.2 The problem of single nucleotide polymorphisms (SNPs) . . . . . . . . 69
4.3 Validation of the knockdown efficiency of the rxin1ex2 MO . . . . . . . 69
4.4 Establishment of rx knock out lines with the zinc finger nucleases
technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.4.1 Selection of an rx ZFN pair and validation of its mutagenicity 73
4.4.2 Establishment of rx knockout lines . . . . . . . . . . . . . . . . . 75
5 Functions of Rx during the development of the dorsal brain 79
5.1 General considerations on the analysis of morphant phenotypes at
24hours post fertilization (hpf) and 48hpf . . . . . . . . . . . . . . . . . 79
5.2 Rx is necessary to the differentiation of ciliary photoreceptors . . . . . 80
5.3 Roles of Rx in the development of the brain serotoninergic system . . 84
x