Zebrafish Module

Zebrafish (Danio rerio) are a small tropical fish native to southeast Asia. With the goal to employ genetics for understanding the development of the nervous system, George Streisinger recognized in the 1970s the potential of zebrafish and established it as a model for vertebrate genetics. Since then they have become a prominent vertebrate model for basic genetic and developmental biology and behavioural studies, and more recently for phenotype-based drug screening. This is due to the ease of embryological and genetic manipulation and their small size and relatively simple husbandry.

Large scale genetic screens have identified hundreds of mutants to study a wide variety of biological processes, most prominently developmental biology, as well as models for human disease. The CRISPR/Cas9 genome editing revolution enabled the generation of targeted gene knock-out and knock-in zebrafish lines, enhancing the reverse genetics potential of zebrafish in basic and biomedical research.

In contrast to other genetic vertebrate systems, zebrafish have the capacity to regenerate the majority of tissues, including for instance the heart and sensory hair cells, making them exciting models to investigate why humans have lost this potential.

A unique feature among vertebrates is that zebrafish embryos and larvae are transparent and together with their small size allow the live observation of dynamic developmental and regenerative processes, including those of internal organs. The availability of a plethora of transgenic fluorescent reporter lines for various cells and tissues (e.g. heart, blood vessels, liver, sensory hair cells and neuronal subpopulation), cellular compartments (e.g. membrane, nucleus, cytoskeleton), as well as intra-cellular signaling indicators (e.g. calcium-signaling) and more recently optogenetic tools has opened the door to imaging fast dynamic events at subcellular resolution.

In this module students will learn how to handle, stage and anesthetise zebrafish embryos, including micro-injection at the one-cell stage. A large variety of RNAs, DNAs for fluorescent labelling of single cells or subcellular structures will be available for injection. Likewise, different methods for interfering with gene function will be introduced: antisense morpholino oligonucleotides for gene knockdown and CRISPR/Cas9 genome editing for gene inactivation. The latest insights into the biology and possible advantages and disadvantages of either technique will be discussed. Moreover, students will be able to stimulate or block major signalling pathways during embryo development in a temporally precise fashion by drug incubation. In addition, students will learn to transplant labelled cells between embryos, a powerful method to test cell autonomous and non-autonomous gene functions and cell behaviours. Utilizing the transparency of the embryos, students will be taught how to live-image entire embryos, as well as dynamic processes such as cell migration of fluorescently labelled cells using the latest models of state-of-the-art microscopes and equipment, such as light-sheet, spinning disc and confocal microscopes. Using this diverse set of tools students will design and investigate their own research question.