Molecular and cellular characterisation of neurogenesis control and addiction behaviour in zebrafish [Elektronische Ressource] / Katharine Joy Webb

Kwebb

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN


Lehrstuhl für Entwicklungsgenetik





Molecular and cellular characterisation of neurogenesis control
and addiction behaviour in zebrafish

Katharine Joy Webb


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.






Vorsitzender: Univ.-Prof. Dr. A. Gierl

Prüfer der Dissertation: 1. Univ.-Prof. Dr. W. Wurst

2. Univ.-Prof. A. Schnieke, Ph.D.
(Univ. of Edinburgh, UK)


Die Dissertation wurde am 20.04.2009 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 22.11.2009 angenommen. Abstract

Abstract

Neurogenesis control is crucial for the correct development of the nervous system from early
embryonic stages to the fully developed adult organism. Premature differentiation not only
prevents the formation of later-born cell types – it also causes disorganization of the shape
and cytoarchitecture of the brain. In addition, progenitors that are maintained life-long
provide sources for brain plasticity and regeneration and there is increasing evidence that
neurogenesis plays a role in mood and behaviour in the fully developed adult organism. This
project uses the advantages of zebrafish as a model for embryonic development, but also as a
model for use in behavioural studies. The main aim of my PhD work is to add to the
understanding of the molecular and cellular processes that maintain neural progenitors within
the vertebrate central nervous system (CNS) and also to set the basis for understanding the
impact of neurogenesis on brain physiological processes such as reward and drug
reinforcement. To this end I isolated and characterised a novel member of the zebrafish
Hairy/Enhancer of Split (hairy/E(spl)) family – her8a. In addition, I also investigated the
molecular mechanisms of reward and drug reinforcement, through the characterisation of a
mutant that fails to respond to amphetamine, no addiction (nad). Analysis of the mutant
reinforces the link between behaviour and neurogenesis.

The neural plate of the early embryo is divided into areas of neurogenesis (‘proneural
clusters’) and areas where neurogenesis is actively inhibited (‘progenitor pools’). In the
proneural clusters neurogenesis promoting genes, such as neurog1, are expressed in a salt and
pepper pattern with members of the Hairy/E(spl) factors, such as her4 (Takke et al., 1999). In
these proneural clusters neurogenesis is controlled through the process of lateral inhibition.
Outside the proneural clusters, neurogenesis is actively inhibited in the progenitor pools,
which are characterised by the expression of certain hairy/E(spl) genes, such as her3, her5,
her9 and her11/him. For example, the midbrain-hindbrain domain of the vertebrate
embryonic neural plate displays neuronal differentiation organised around a neuron-free zone
at the midbrain-hindbrain boundary (mhb). Forced neurogenesis in this area prevents the
continued expression of genes defining mhb identity. The morpholino-mediated knockdown
of her5 causes ectopic neurogenesis, and thus loss of the medial mhb progenitor zone. Basic
Helix Loop Helix (bHLH) transcription factor family members, such as Her5, form hetero- or
homo-dimers in order to carry out their functions as repressors or activators. The elucidation
of which bHLH factors form dimers with Her5 would provide an insight as to the mechanisms
of the Her5 neurogenesis inhibition. To this end a yeast-2-hybrid experiment was carried out
in order to identify factors that bind to Her5. I cloned and characterised the expression of the
most promising candidates from the screen. One factor, Her8a, is particularly promising, as it
is expressed at the mhb from before the start of segmentation. Its expression, while broad in
the early embryo, becomes increasingly restricted and it is only expressed in proliferation
zones in the adult brain. Experiments using morpholino mediated knockdown of her8a and
the overexpression of her8a establish Her8a as a novel negative regulator of neurogenesis in
the embryonic midbrain-hindbrain domain and a manuscript describing this work is currently
in preparation. her8a’s sensitivity to Notch changes throughout development – at early stages
her8a does not require Notch for its expression, however it is sensitive to Notch signalling.
At later stages her8a requires Notch for its expression. This indicates that the ability of her
genes to respond to Notch is not fixed, but they respond according to their cellular context.
Abstract

no addiction (nad) is a dominant mutation that was isolated in a screen for its failure to show
conditioned place preference response to amphetamine in our laboratory. My task was to
characterise this mutant at the molecular level in order to contribute to the understanding of
the mechanisms leading to reward and drug addiction. This work has been compiled in an
article, currently under revision at Genome Biology. To this end I devised a series of
microarray experiments that were then combined to specifically isolate genes implicated in
both the non-response to amphetamine in the wildtype as well as the failure of the mutant to
respond to amphetamine – referred to in this work as the “reward pool”. I analysed this pool
using Gene Ontology (GO) enrichment analysis and network analysis. Network analysis
linking proteins according to function is based on experimentally derived protein-protein
interactions through literature curation. As there are comparatively less abstracts on zebrafish
than on mammalian subjects, commercial network analysis software does not yet provide a
large number of links. Therefore, in collaboration with the Institute for Bioinformatics and
Systems Biology, I participated in the development of a database - zfishDB
(http://mips.gsf.de/zfishdb/) - that uses zebrafish information as well as information derived
from the mammalian homologues of zebrafish genes. The bioinformatics analysis of the
microarray results implicates for the first time the reuse of developmental transcription factors
in reward and drug reinforcement events. In addition, I used the bioinformatic analysis to
choose a subset of genes for validation using qPCR and in situ hybridisation. In situ
hybridisation revealed that a subset of these genes is down-regulated in neurogenic zones
upon amphetamine administration. This lead to a further project, with investigated the
influence of amphetamine on proliferation and differentiation in the adult brain. Here I was
able to show that amphetamine leads to premature differentiation of adult progenitors.

In summary, this thesis contributes to a greater understanding of neurogenesis inhibition and
to the molecular cascades involved in reward/drug reinforcement. It provides the basis for
further studies looking at the mechanisms of the function of the chosen candidate genes and
studies looking at the effects of amphetamine on neurogenesis in the adult. Zusammenfassung

Zusammenfassung

Die Steuerung der Neurogenese ist kritisch für die richtige Entwicklung des Nervensystems
von den frühen embryonalen Stadien zum voll entwickelten ausgewachsenen Organismus.
Verfrühte Differenzierung kann nicht nur die Ausformung später entstehender Zelltypen
verhindern- es verursacht auch die Disorganisation der Form und Zellarchitektur des ganzen
Gehirns. Zusätzlich stehen die Ursprungszellbestände lebenslang als Quellen für
Gehirnplastizität und Regeneration zur Verfügung und es gibt zunehmend mehr Hinweise
darauf, dass die Neurogenese eine Rolle bei Stimmung und Verhalten ausgewachsener
Organismen spielt. Das Hauptziel meiner Doktorarbeit ist ein Beitrag zum Verständnis
darüber, wie Ursprungszellbestände spezifiziert und aufrechterhalten werden. Zu diesem
Zweck habe ich ein neues Mitglied der Zebrafisch "Hairy/Enhancer of Split" Familie - Her8a-
isoliert und charakterisiert. Zusätzlich habe ich die molekularen Mechanismen von
Belohnung und Drogenbestärkung untersucht, durch Charakterisierung einer nicht auf
Amphetamin reagierenden Mutante - no addiction (nad).

Die neural plate des frühen Embryos ist aufgeteilt in Gebiete mit Neurogenese ("proneurale
Clusters") und Gebiete, in denen Neurogenese aktiv unterdrückt wird
("Vorläuferzellpopulationen"). In den proneuralen Clusters werden Neurogenese befördernde
Gene wie ngn1 in einem gesprenkelten Muster mit Mitgliedern der hairy/E(spl) Genen wie
her4 exprimiert. In diesen proneuralen Gruppierungen wird Neurogenese durch den Prozess
lateraler Unterdrückung gesteuert. Außerhalb der proneuralen Gruppierungen wird die
Neurogenese aktiv in den Vorläuferreservoirs unterdrückt, die durch die Expression
bestimmter h