zebrafish model

Zebra Fish – Are they perfect?

Taxonomy

Kingdom: Animalia

Phylum: Chordata

Class: Actinopterygii

Order: Cypriniformes

Family: Cyprinidae

Genus: Danio

Species: rerio

General Features Benefits

Appearance-Dimension ~4 cm-Salient distinguishable features of male and female-Often transparent adult bodies

Large number can be kept easily and cheaply in lab

Good model for visualization of cellular activity

Habitat-Fresh water fish- Tropical fish

Universally available

Feeding-Omnivorous Low cost of maintenance

Reproduction-Female spawns every 2-3 days-Breeds all year round-Several hundreds of eggs produced in single clutch-External fertilization

Large number of offspring- good for batch variation studies

Easy availability of eggs

General Features

Life cycle

• Total life span: 42-66 months

• Good model for developmental studies because of transparency at early stages

Genetics

Genetics • Largest set of vertebrate genome so far sequenced

• Eudiploid

• Unique features of chromosomal DNA– Unique repeat content– Scarcity of pseudo-genes– Chromosome 4 is enriched with zebra fish specific genes and sex-

determining genes

• Mitochondrial genome completely sequenced

Genetics

• According to a paper published in Nature, 70 per cent of protein-coding human

genes are related to genes found in the zebrafish (Danio rerio), and 84 per cent of

genes known to be associated with human disease have a zebrafish counterpart.

Mutagenesis

• Model for First mutagenesis studies

• Homozygous strain production by using genetically inactivated sperm

http://zfin.org/zf_info/zfbook/chapt7/7-11.gif

Mutagenesis

• Exon Disruption Insertional Mutagenesis

• Retroviral Insertional Mutagenesis

• Transposon Insertional Mutagenesis

http://genomebiology.com/2007/8/S1/S9/figure/F1

• Knockdown techniques

Transgenics

Transgenics

• Easy visualization of tumor development (in heart, brain, lateral vessels, and intestinal tube), cell migration, fluorescent probes, pigmented cells

• One can monitor a disease without harming the organisms

Casper Strain:

Homozygous recessive mutants of mitfa gene (associated to melanophores) & an unnamed gene (associated to iridophores)

- Easy to manipulate: easy mRNA, DNA, fluorescent tag micro-injection and their visualization-

Development-Egg size ~ (0.7mm)

- Optically transparent : easy to study embryonic development (epiboly, gastrulation, segmentation), growth delay, malformations or morphological abnormalities

Development

Development

http://www.masonposner.com/research/projects/projects.htm

Development

Pigmentation and melanocyte development studies

SLC24A5

- required for melanin production

- orthologous gene is present in humans also

http://www.sciencemag.org/content/310/5755/1782.full.pdf

Development

Cancer Research

• Shares most of their organs with mammalian counterparts

• Differently aged animals each offers distinct advantages for cancer-relevant phenotypes

Cancer Research

Regeneration• Regeneration of

– fins, skin, heart in adult– brain in larvae– photoreceptor cells and retinal neurons

following injury

Developmental Dynamics 226:202–210, 2003

Toxicology

Why using Zebrafish?

-Very small size

- High fertility

- High predictivity for Human

- Embryo transparency and fast development

Zebrafish Embryo Toxicity Test (ZEFT) : Robust and sensitive model

Toxicology

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3148099/figure/F2/

• Typical chemical induced malformations in Zebrafish

Zebrafish- A model Organism

Freezing???

Last common ancestor with humans was 445 million years ago: far more remote from humans than other animal models such as rodents

Zebrafish Vs. Humans

Disadvantages:-

• Several mammalian organs are not present in the zebrafish, including breast tissue, lungs, and prostrate. Skin lacks some specific cellular components found in humans.

• ectothermic (cold-blooded): physiology not identical to humans

• Fish are greatly influenced by their environment and many environment variables, including temperature, lighting, population density, water quality and nutrition, must be tightly controlled in order to accurately interpret data obtained

Addressing Specific Issues

Gene expression• A known problem with gene knockdowns is

that, because the genome underwent a duplication after the divergence of toleost fishes.

It is not always easy to silence the activity of one of the two gene paralogs reliable due to complementation by the other.

Genome sequencing• In 2009, researchers at the Institute of Genomics and

Integrative Biology in Delhi, India, announced the sequencing of the genome of a wild zebrafish strain, Containing 1.7 billion genetic letters.

Comparative analysis with the zebrafish reference genome revealed over 5 million single nucleotide variations and over 1.6 million insertion deletion variations.

• It is difficult to draw evolutionary conclusions because it is difficult to determine whether base pair changes have adaptive significance via comparisons with other vertebrates' nucleotide sequences.

Research Limitations

Zebrafish Model for Toxicology and Toxicologic Pathology Research

• Shortage of Basic Data on Zebrafish Pathology. There are scant baseline data on zebrafish pathologic lesions in infectious and noninfectious diseases.

• Many lines of knockout mice for which the inactive genes are not related directly to the immune system still are immunodeficient and at high risk for opportunistic infection

Infectious Diseases of Zebrafish

• Two infectious diseases that commonly occur in well- managed zebrafish colonies are microsporidiosis ( infects the central nervous system, cranial and spinal nerves, and skeletal muscle of zebrafish) and mycobacteriosis.

• We are uncertain whether vertical transmission of microsporidia can occur in zebrafish. Unfortunately, these parasites are very difficult to inactivate and remove from recirculating husbandry systems.

• Piscine mycobacteriosis remains a tenacious problem in aquarium fish colonies. It most often occurs as an opportunistic infection in fish over 1 year of age. The sources of infection and epizootiology are not well defined. Currently no effective chemopreventative or therapeutic regimens are defined for fish.

• Because mycobacterial antigens are potent immune adjuvants, these agents can seriously confound research in disease resistance or immune responses.

Animal models of human disease zebrafish swim into view

• First, human pathogens cause disease at 37°C, whereas zebrafish are maintained at 28°C; it might not be possible, therefore, to study some pathogens at this lower temperature. So, most host–pathogen interactions that could be studied using this approach might be fish-specific.

• Finally, crucial gaps remain in the comparative physiology of the zebrafish immune system, which might impinge on the validity and usefulness of modelling human infections in zebrafish.

Neurobiology and development

Zebrafish as a model to study the neurodevelopmental causes of autism?

• First, the genetic and neuroanatomical defects that cause autistic disorders in humans are largely unknown. Thus, it is not completely clear what aspects of zebrafish brain development should be investigated in a model of autism.

• Second, the constellation of behaviors that define autism may not be suitably represented in the repertoire of non-human behaviors.

Transgenic zebrafish model for blood-brain barrier development

• The BBB, initially regarded as static and rigid, have now been proven to be dynamic structures with both paracellular and transcellular pathways capable of rapid modulation in response to physiological or pathological signals.

• One of the major limitations in this regard has been the absence of an easily regulated in vivo system that allows alterations of these barriers.

• In 2012, Australian scientists published a study revealing that zebrafish use a specialised protein, known as fibroblast growth factor, to ensure their spinal cords heal without glial scarring after injury.

• If similar processes occur , absence of the glial scar has been associated with impairments in the repair of the blood brain barrier???????

Cancer Research

Influence of Diet and Husbandry Systems on Spontaneous Neoplasia

• Zebrafish colonies fed commercial diets and maintained in standard recirculating systems have different patterns of neoplasia and spontaneous pathologic lesions than those observed in the Core Fish Facility of the Marine/Freshwater Center at Oregon State University.

• But systemic mechanism of this spontaneous neoplasia, its molecular basis and gene expression variance has not been characterized, challenging its suitability to cancer model or toxicity research.

The Mystery of Hepatic Megalocytosis in Zebrafish

• In diagnostic cases from around the world and in about 50% of groups of broodstock from standard husbandry systems feeding commercial diets, show mild to moderate hepatocyte megalocytosis with karyomegaly.

• The toxicant sources causing megalocytosis are uncertain.

• Problems: Reducing early life stage survival, longevity, reproductive potential, immune competence, and disease resistance.

Hematology Research

Zebrafish in hematology: sushi or science

• Anatomy:

• Different morphology of blood cells, eg, erythrocytes and thrombocytes are nucleated.

• Different gross anatomy, eg, what is the equivalent of the marrow stroma?

Physiology:

• Lack of cell markers/antibodies

• Lack of hematopoietic cell lines

• Lack of biochemical reagents, eg, purified cytokines

• Lack of in vitro differentiation system (hematopoietic cell culture assays)

• Lack of inbred strains, eg, for transplantation studies; to facilitate gene mapping.

Other Areas:

The Problem of Skewed Sex Ratios in Cohorts of Zebrafish

• Problems with skewed sex ratios in cohorts of zebrafish can interfere with natural breeding, and can complicate studies such as carcinogen or other toxicant bioassays where balanced sex ratios in control groups are desired.

• In contrast to mammalian species, sex determination in fish is more flexible, often reversible, and less dictated by genetic factors.

• The relative influences of and interactions between environment, genetics, and toxicants in sex determination in zebrafish are not yet clearly defined.

Zebrafish-Based Small Molecule Discovery

• Lack of phenotypic fluidity. Only extremes phenotypes (subtle or distorted are seen in general).

• Therefore, despite a few notable successes, the benefits of whole-organism, phenotype-based small molecule discovery have been overshadowed by the practical limitations of the approach.

Ethanol Disorder of embryo• Several human ethanol disorders are difficult or impossible to

model in this species (e.g. cardiac septation defects)

• Different scales of phenotypic response to ethanol in case of humans and zebrafish.

Logistics Issue• A further challenge will be to maintain the ongoing development of

community-based resources for zebrafish research. (ZFIN)

• The main pressure points will be stock centers and the sequencing of the zebrafish genome, both of which will require continued ongoing investment to provide the necessary quality of material and sufficient access to it.

Conclusion: