Sunday, August 12, 2012

What is Danio rerio?


Zebra Fish as a Model Organism

Zebra fish are small (3–4 centimeters) tropical freshwater fish long popular among aquarium hobbyists. In the early 1970s, George Streisinger at the University of Oregon established zebra fish as a model organism in order to exploit a variety of experimental advantages. Zebra fish have a relatively short generation time of approximately three months. Large clutches of embryos can be obtained from a single mating (typically 100 to 300 fertilized eggs), and a large number of progeny can be maintained in a moderately small space. Embryos develop external to the mother, are relatively large (about 0.5 millimeters), and entirely lack pigment throughout early development, making them transparent. Thus development from fertilization to hatching can be directly observed under a dissecting microscope. Such efficient observation of developmental phenotypes has permitted genetic analysis of mutants that has led to the identification of genes required for embryogenesis in vertebrates.





















Embryonic Development of Zebra Fish

Sunrise stimulates females to lay eggs, which are protected by a tough proteinaceous chorion. Males fertilize the eggs externally, and embryogenesis proceeds very rapidly. Highly regular and spatially oriented cleavage occurs about one to two hours postfertilization. Division continues, and a blastula of thousands of cells emerges by five hours. Gastrulation occurs in five to ten hours. Segmentation occurs from ten to twenty-four hours. Organogenesis begins within thirty-six hours. Larvae hatch from the chorionic sac between forty-eight and seventy-two hours, swimming and feeding independently by around seventy-two hours.




Mutagenesis Screens

A main advantage of zebra fish as a model organism to study embryonic development in vertebrates is the exploitation of forward genetics. Mutations are introduced at random in parental genomes, and their progeny are screened for phenotypic abnormalities during embryogenesis. Upon identification of a phenotypically abnormal individual, true breeding mutant strains are generated from which the mutant gene may be isolated and identified by molecular cloning. This approach permits identification of genes required for proper embryonic development.


Embryonic development had previously been studied in the mouse, but because mammalian embryogenesis takes place inside the mother, it is difficult to observe embryonic phenotypes, and impossible to do so without sacrificing the subject, precluding a mutational analysis. Zebra fish, however, permit efficient mutagenesis screens of many thousands of individuals.


The first such screens in zebra fish were carried out in the early 1990s in the laboratories of Christiane Nüsslein-Volhard (who shared the Nobel Prize for similar screens in
Drosophila melanogaster
) at the Max Planck Institute for Developmental Biology in Tübingen and Wolfgang Driever at Massachusetts General Hospital. These screens led to the identification of genes required in blood, kidney, heart, and brain formation, patterning of the dorsal-ventral and anterior-posterior body axes, and other aspects of embryonic development.




Techniques and Experimental Manipulation in Zebra Fish

Since Streisinger first developed zebra fish as a model organism, many more techniques have been elaborated. Generation of transgenic zebra fish is well established. DNA molecules containing transgenes are injected into early embryos, where they integrate into the genome. Transgenes passed on to progeny cells that give rise to germ cells will be stably transmitted to offspring. This technique allows scientists to investigate gene function by reverse genetics. Another technique used to investigate gene function is to decrease expression of a gene by injecting morpholino antisense oligonucleotides to “knock down” expression of the gene either by inhibiting pre-mRNA processing or by inhibiting translation. By removing most of the gene function from the organism, an experimenter can determine if the function of the gene is required for embryonic development, and what phenotype appears in the absence of that gene’s function. Finally, technologies for fully eliminating or “knocking out” gene function are becoming available.


The zebra fish genome has been completely sequenced, greatly aiding in the identification of genes required for embryonic development. Moreover, the genomes of three other fish species have also been completely sequenced: two puffer fish (Fugu rubripes and Tetraodon nigroviridis) and medaka (Oryzias latipes). Sequence comparisons have allowed the identification of sequence elements that are conserved among these species and are therefore probably required for conserved biological processes in these organisms. Comparison of these genomes with the human genome has allowed genomicists to identify genes that are widely conserved among vertebrates. Finally, medaka has emerged as a complementary model organism to zebra fish. Comparative investigations have allowed geneticists to identify both conserved and divergent genetic pathways in vertebrate embryonic development.


Cell lineage tracing in zebra fish is accomplished by injecting fluorescent dye into single cells in the early embryo. The dye is passed on to daughter cells after subsequent divisions, so cell lineage and cell migration can be observed as development progresses. Likewise, by expressing fluorescent proteins, individual cells can be visualized in live transgenic embryos and larvae.




Impact

Zebra fish bring experimental advantages previously associated with invertebrate model organisms to bear on vertebrate biology. Studies in zebra fish, as a well-established model organism, have identified many genes demonstrated to be required for vertebrate embryonic development. The zebra fish model has allowed for the identification of critical developmental genetic pathways that have been conserved throughout evolution even in higher vertebrates, including humans. This system has also begun to show promise as a platform for rapid small molecule screening for drug discovery, and as a model in which to investigate the molecular basis of behavior in vertebrates and the genetic and environmental bases of human disease.




Key Terms



embryogenesis

:

the development of a fertilized egg into a fully formed embryo and ultimately into a free-living juvenile




forward genetics

:

the investigation of gene function starting with a mutant phenotype and proceeding to identify a mutated gene




morpholino

:

a nucleic acid analog used experimentally to reduce expression of a gene of complementary DNA sequence





mutagenesis


:

the introduction of DNA mutations, such as by chemicals or radiation; used experimentally to screen for mutations in genes required for a particular biological process




organogenesis

:

the development of internal organs during embryogenesis




reverse genetics

:

the investigation of gene function starting with an identified gene and proceeding to manipulate it experimentally in order to observe a potential phenotype





transgenic


:

referring to an individual carrying a DNA sequence not occurring naturally in that species





Bibliography


Bryson-Richardson, Robert, Silke Berger, and Peter Currie. Atlas of Zebrafish Development. London; Waltham: Academic/Elsevier, 2012. Digital file.



Carver, Ethan A., and Charles A. Lessman. Zebrafish: Topics in Reproduction, Toxicology and Development. New York: Nova, 2014. Digital file.



Detrich, H. William, Monte Westerfield, and Leonard I. Zon. The Zebrafish: Cellular and Developmental Biology. 3d ed. Amsterdam: Elsevier, 2011. Digital file.



Detrich, H. William, Monte Westerfield, and Leonard I. Zon. The Zebrafish: Genetics, Genomics, and Informatics. 2d ed. San Diego: Elsevier, 2004. Print.




Development 123 (1996): 1–481. Print.



Harper, Claudia, and Christian Lawrence. The Laboratory Zebrafish. Boca Raton: CRCP, 2011. Print.



Nüsslein-Volhard, Christiane, and Ralf Dahm. Zebrafish: A Practical Approach. New York: Oxford UP, 2002. Print.



Westerfield, Monte. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio). 5th ed. Eugene: U of Oregon P, 2007. Print.

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