8. VL Flashcards
Zebrafish and Morphogenesis
Zebrafish (Danio rerio)
- The species arose in the Ganges region in eastern India, and commonly inhabits streams, canals, ditches, ponds, and slow-moving or stagnant water bodies, including rice fields (with large differences in temperature, pH levels, oxygen content, turbidity of water).
- Natural habitat: the zebrafish is found in streams and rivers of the Himalayan region (India, Pakistan, Bangladesh, Nepal, Burma).
What did George Streisinger (1927-1984)?
Production of clones of homozygous diploid zebrafish
Advantages of the Zebrafish as a vertebrate model system
• Excellent genetics
Short generation period Largeeggclutches
• Extrauterine and transparent development
• Rapid mode of development
• Early development is possible without functioning organs
• Ease to perform experimental embryological manipulations
• Cheap maintenance costs
Advantages of the Zebrafish as a vertebrate model system
- Circulatingwater system with temperature between 24°-28°C
- ~5adultfish/liter
- Matings are possible 1x/week: approx. 100- 500 eggs
Survival of the early zebrafish embryo
does not
require a functional heart
Cardiac troponin T is essencial in sarcomer assembly and cardiac contractility
TnT = Tropomyosin-binding subunit
TnC = Ca2+-binding subunit
TnI = inhibitory subunit
2 days after fertilization is heart not so differentiated (blood can flow in 2 directions), 4 days after fertilization more differentiated with Valve leaflets and Trabeculae
Myocardium and Endocardium getting more differentiated
Oscillatory blood flow induces Klf2 (regulator) at the cardiac cushions
These 3 people got nobel prize for research with zebra fish
Christine Nüsslein-Volhard, Edward B. Lewis, Eric F. Wischaus
Mutagenesis can be caused with
Ethylnitrosourea
N-ethyl-N-nitrosourea (ENU) is a highly potent mutagen. For a given gene in mice, ENU can induce 1 new mutation in every 700 loci. It is also toxic at high doses.
The chemical is an alkylating agent, and acts by transferring the ethyl group usually to thymine in nucleic acids.
Classical F2 Generation Screen in Zebrafish
einen WT fish mit ENU treated male: +/+ x m/+ (in der F1) random crossing in F2 generation (50% sind reine WT, 50% sind +/m)
F3 generation: 25% +/+, 50% +/m; 25% m/m
How can you create a haploid zebrafish?
+/+ und ENU treated fish –> m/+ (hier: UV treated sperm: 50% m and 50% +
zebrafish
George Streisinger: Generating the toolbox for classical genetics •Establishment of fish maintenance
•Fish breeding and husbandry
•Mutagenesis protocols
•Production of haploid embryos
•First cloning of an animal model
•Establishment of different types of genetic screens •Mapping strategies for mutations
•Transplantation methods (conditional mutations for cell autonomy studies)
Transplantation mthods
Cell transplantation: Robert Ho and Donald Kane (1990)
Termes for transplantation methods
• Fate indicates what a cell will normally develop into if not challenged with another environment (based on its position within the embryo)
• Determination refers to a stable internal state of a cell that will not change even when transplanted into another environment
• A cell is specified when it develops into a particular fate even when kept in
isolation
One-eye pinhead genes function in ….
mesoderm and endoderm formation in zebrafish and interacts with no tail
is it possible to create a maternal -zygotic mutant zebrafish? How?
by germ line replacement (by cell transplation)
Zebrafish genom
• Genome size (haploid state):1,412 Gb distributed among 25 chromosomes
• Zebrafish genome project by the Sanger Institute (“reference genome”)
based on the standard fish strain Tübingen was published online:
• http://www.ncbi.nlm.nih.gov/genome/annotation_euk/Danio_rerio/104/
• About 71% of the 20,479 protein-coding genes of Homo sapiens have
orthologs in the zebrafish genome. In turn, 69% of the 26,206 protein-coding
zebrafish genes have human counterparts.
• The increased gene number in the zebrafish genome originated from a
whole-genome duplication during teleost evolution that was followed by a
loss of many orthologs.
• Human genes can have more than one zebrafish ortholog.
• Mutations in the two orthologs accumulate mostly in regulative regions (with
more restricted expression patterns compared to their human orthologs)
ZFIN-Zebrafish Model Organism Database
