Establishment of combinatorial genetics in zebrafish for analyzing cellular mechanisms of hindbrain development [Elektronische Ressource] / Martin Distel

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• Biologie

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 20 Mo

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TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Entwicklungsgenetik




Establishment of combinatorial genetics in zebrafish for analyzing
cellular mechanisms of hindbrain development




Martin Distel






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. S. Scherer

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

2. Univ.-Prof. Dr. A. Gierl

3. Univ.-Prof. A. Schnieke, Ph. D.





Die Dissertation wurde am 14.12.2009 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 16.02.2010 angenommen.










Establishment of combinatorial genetics in zebrafish for analyzing
cellular mechanisms of hindbrain development






Kumulative Arbeit





Martin Distel





















Abstract
Abstract
Zebrafish embryos have become a popular model system for the study of developmental
biology, mainly because of their transparency and external development, which allow for in
vivo investigations and easy manipulation. However, some methods are not well developed
for zebrafish. In my PhD project, I have thus addressed the need for combinatorial genetics,
imaging of opaque adult fish and cell type-specific subcellular imaging.
The Gal4-UAS combinatorial genetic system provides spatial and temporal control over gene
expression and thus enables one to perform sophisticated genetic studies. I adapted the Gal4-
UAS system to zebrafish by creating optimized Gal4 activators and UAS effector constructs,
which show good bio-tolerance and non-mosaic transactivation. My quantification of the
expression activity of these activator/effector combinations provides a rational basis for the
prediction of expression levels and thus allows to model concentration dependent effects.
Subsequently, I generated 60 tissue specific transgenic Gal4 activator strains in a self-
reporting enhancer trap approach biased for the central nervous system. As these strains
express a red fluorescent protein in Gal4 expressing cells, embryos of these strains can be
readily used to study the development of the respective tissues in vivo by time-lapse
microscopy. Furthermore, these strains reliably transactivate any UAS-dependent transgene
and thus represent a valuable resource for reverse genetic studies. To be able to plan and to
analyse Gal4 mediated transgene expression experiments, I analysed the transactivation
kinetics of an enhancer trap strain expressing optimized Gal4 (KalTA4) in rhombomeres 3
and 5 (r3/5). KalTA4 expression was found to be present transiently at early embryonic stages
and was restricted to r3/5. In order to follow the fate of r3/5, I generated an effector strain,
Kaloop, which is capable of prolonging reporter gene expression in r3/5 until adulthood in a
KalTA4 mediated self-maintaining feedback loop. Using this Kaloop strain, I showed that the
rhombomeric organisation of the zebrafish hindbrain persists until adult stages, similar to frog
or chick. Furthermore, I found that the secondary octaval nucleus (SON) is derived from
egr2b expressing cells of r5. This demonstrates that the Kaloop effector strain can be used in
combination with tissue specific Gal4 activator strains to explore the relationship between
embryonic and adult structures. As the established Gal4 strains are capable of transgene
activation they can be used to study gene functions in a tissue specific manner in both gain
and loss of function experiments. In a collaborative effort the r3/5 specific enhancer trap
strain was used to rescue the phenotype of lunatic fringe morphants, which showed increased
neurogenesis, specifically in r3/5. This experiment confirmed the hypothesis that Lunatic
I Abstract
Fringe is required to maintain cells in a progenitor state in the zebrafish hindbrain.
Additionally, Gal4 strains were successfully used to generate tumor models. By tissue specific
overexpression of human oncogenic HRAS-G12V we generated a glioma model with fast
onset of tumorigenesis, as well as a melanoma model strongly resembling human melanoma
based on its morphological, molecular, genetic and epigenetic properties. These models will
be used for screens for compounds effective against cancer and for mechanistic in vivo studies
of tumorigenesis and disease progression.
Ideally, the progression of tumors in such models would be followed in a live animal. In
contrast to optical methods, which suffer from light scattering, photoacoustic imaging can
visualize structures deep within highly scattering tissue. Photoacoustic imaging is based on
the conversion of absorbed light into ultrasound, which can be detected outside the tissue.
However, this technique had not previously been applied to visualize fluorescently labelled
structures. In collaboration with the Institute of Biological and Medical Imaging at the
Helmholtz Center Munich, we established multispectral opto-acoustic tomography (MSOT), a
variation of photoacoustic imaging, in order to visualize fluorescent protein expressing cells.
Using MSOT, we could resolve the fluorescently labelled vertebral column and the crista
cerebellaris of adult transgenic Gal4 fish in vivo. Together with Gal4-UAS genetics this
technique allows one to investigate and manipulate processes like tumorigenesis, adult
neurogenesis or regeneration in the living organism.
Finally, the Gal4-UAS system can also be used to study a biological process at the subcellular
level. Here, I established a Gal4 based subcellular labelling system for the in vivo
characterization of migration of tegmental hindbrain nuclei (THN) neurons. Multiple
subcellularly targeted fluorescent proteins simultaneously expressed under UAS control were
used to observe the dynamics of cell organelles in migrating cells within live zebrafish
embryos. My subcellular analysis of THN neuron migration revealed that, in contrast to the
classic model of neuronal migration, the centrosome is not permanently positioned ahead of
the nucleus, but instead is overtaken by the nucleus in a saltatory manner during tangential
migration. Furthermore, the position of the centrosome does not determine the site of axon
outgrowth in THN neurons in vivo as previously observed in cultured hippocampal neurons.
Gal4 mediated subcellular labelling can be readily combined with Gal4 mediated
overexpression of dominant-negative variants of proteins, which in the future will allow for
functional studies into the roles of candidate proteins in migration and axonogenesis with a
direct in vivo read-out. Thus Gal4 mediated subcellular labelling promises to merge the fields
of developmental biology and cell biology into a new field of in vivo cell biology.
II Abstract
Zusammenfassung
Der embryonale Zebrabärbling ist aufgrund seiner Transparenz und externen Entwicklung, die
einfache genetische Manipulationen und in vivo Untersuchungen erlauben, ein beliebter
Modellorganismus für entwicklungsbiologische Studien geworden. Dennoch wurden einige
Methoden bisher nicht ausreichend für eine Anwendung im Zebrabärbling entwickelt. In
dieser Arbeit habe ich ein System für kombinatorische Genetik für die Anwendung im
Zebrabärbling und Methoden, sowohl zur Untersuchung von fluoreszenzmarkierten Geweben
im lebenden adulten, als auch der Dynamik von Zellorganellen im lebenden embryonalen
Zebrabärbling etabliert.
Kombinatorische genetische Systeme, wie etwa das Gal4-UAS System, haben sich als
wertvolle Werkzeuge für genetische Studien in Drosophila erwiesen. Das Gal4-UAS System
erlaubt räumliche und zeitliche Kontrolle über die Expression von Transgenen und ermöglicht
somit komplexe genetische Experimente. Ich habe das Gal4-UAS System für seine
Anwendung im Zebrabärbling adaptiert und optimierte Gal4 Aktivatoren und
Effektorkonstrukte generiert, deren Expression im Zebrabärbling keinen schädlichen Einfluss
auf seine Entwicklung hat und die nicht-mosaike Transaktivierung zeigen. Die
Quantifizierung der Expressionsstärken verschiedener Aktivator/Effektor Kombinationen
ermöglicht zudem die Planung und Durchführung von Experimenten, bei denen
unterschiedliche Mengen eines Transgens exprimiert werden sollen, um konzentrations-
abhängige Effekte zu studieren.
Mit diesem optimierten Gal4 System habe ich durch einen „Screen“ nach regulatorischen
Elementen 60 gewebsspezifische transgene Gal4 Aktivator Linien erzeugt, von denen die
Mehrzahl Gal4 in Geweben des zentralen Nervensystems exprimiert. Gal4 exprimierende <