Zebrafish Model of Cancer Flashcards
What family are zebrafish members of?
The minnow family (Cyprinidae)
What is the latin name for zebrafish?
Danio rerio
What are zebrafish?
Small freshwater fish native to Northern India/Bangladesh
How did Streisinger die in 1984?
In a scuba diving accident
What did Streisinger develop methods for?
Generating homozygous diploid clones of zebrafish
What year were zebrafish first included in a publication in Nature?
1981
Describe the progression of zebrafish studies since the first papers were published on them?
Studies using zebrafish have grown exponentially
How long did it take for zebrafish to become the second most widely used model in bioscience research?
In around 20 years
Between what years was there development and dissemination of zebrafish methodologies?
1984-1992
Describe the pros of using zebrafish as models for human cancers
Fast
Large scale
Cheap
High resolution
Describe the pros of using mice as models for human cancers
High resolution
Describe the cons of using mice as models for human cancers
Time consuming
Expensive
Describe the pros of using bacteria as models for human cancers
Fast
Large scale
Describe the cons of using bacteria as models for human cancers
Resolution limited
Cheap
What did the Zon lab in March 2011 use zebrafish for?
High-throughput screen of chemicals that might suppress melanoma
What was George Streisinger’s hobbies?
He was a tropical fish enthusiast who studied his fish in his own collection to find a potentially useful model
Why did George Streisinger want to find a new model organism?
He found that mice are too complex and microorganisms are too simple
Where did George Streisinger work?
At the University of Oregon
Who is George Streisinger and what did he work on?
Geneticist
Worked on T4 bacteriophage genome in the 1960s and 1970s
What did George Streisinger begin to do in the 1980s?
Began to look for more complex systems to study genetic regulation of development
Give 4 advantages of the zebrafish life cycle
Transparent embryos
Rapid development
Spawn at dawn
100’s of eggs produced per female
What did scientists do prior to Streisinger’s death in order to promote the utility of zebrafish as a genetic model?
They ran courses
Give an example of another Oregon scientist who had become interested in harvesting the power of the zebrafish model prior to Streisinger’s death
Charles Kimmel
The interest in zebrafish as a model organism gained critical mass and many groups across the US and Europe began working with the fish, true or false?
True
Since around what year have many cancer biologists turned their attention to using zebrafish for cancer research?
2000
Do immature (embryonic/larval) zebrafish develop tumours?
Rarely
How big is a larval fish at 4 days old?
~4mm
In immature zebrafish, how can precocious tumours be induced?
With transgenic approaches
Can immature zebrafish be readily zenografted with human tumour cells?
Yes, the graft is not rejected
In immature zebrafish, what does their optical transparency permit?
In vivo imaging of tumour growth/metastasis
What does the small size of immature zebrafish permit the high throughput chemical screening of?
Carcinogens/chemical tumour suppressors
In vivo imaging approaches can be used to resolve what in early-stage zebrafish?
Intravasation and metastasis
Why are fish better for high throughput in vivo screens than mouse models?
Once a candidate drug is identified and tested on a mouse model, accurate results cannot be observed until several years later and then there is 98% probability of failure
What types of tumour can adult zebrafish spontaneously develop?
Almost any type of tumour
How can tumours be readily induced in adult zebrafish?
Carcinogen exposure
Zenograft
Gene knockout/insertion (fast and easy)
Adult fish tissues are opaque so in vivo imaging is not possible, true or false?
False, in vivo imaging is still possible to an extent when using pigmentation mutants
What have forward genetic screens of zebrafish identified?
New oncogenes
What did the Hopkins lab develop in 2004 using forward genetics?
Mass mutagenic screening
Outline the mass mutagenic screening approach to identify new oncogenes
- Inject 1000s of eggs with a retrovirus
- Virus randomly inserts into the genome
- Outcross and screen for mutant phenotypes
- Sequence mutated genes
What did Amsterdam et al. use the mass mutagenic screening approach in 2004 to identify?
12 mutant lines that had increased incidence of cancer
11 of these were mutations in ribosomal genes, which had not been previously associated with cancer
Give an example of a zebrafish liver oncogene
apc
Give an example of a zebrafish intestine oncogene
apc
Give an example of a zebrafish ocular oncogene
pten
Give examples of a zebrafish nerve sheath oncogene
tp53
msh
Give an example of a zebrafish leukemia oncogene
c-myc
Give an example of a zebrafish melanoma oncogene
BRAF
Give an example of a zebrafish neuroendocrine oncogene
MYCN
Adult zebrafish can shed new clues on the formation of what?
Melanoma
Imaging metastases in vivo with zebrafish has shown that what compounds work together to cause metastases?
VEGF and RhoC
Over the past 20 years the zebrafish has become a credible and powerful model for studying cancer, true or false?
False, only over the past 12 years
Give an example of a rapid, reliable method for studying cancer in zebrafish
The insertion of transgenes
Outline the method for the easy insertion of transgenes in zebrafish
- Inject one-cell stage eggs with DNA
- Raise to adulthood (3 months)
- Outcross and test for germline transmission
- Widely used technique for introducing oncogenes (e.g. RNUX1/c-myc/bcl2/Notch/BRAF) into fish
What is c-myc?
A transcriptional regulator
What are mutations in c-myc associated with?
Cancer (highly prevalent in Burkitt’s lymphoma)
What is BRAF a member of?
The RAF kinase family
What is BRAF?
A serine/threonine kinase that participates in the RAS/RAF sigalling pathway
What is BRAF critical for?
Normal development
Without BRAF, what syndrome develops?
Cardio-facio-cutaneous (CFC) syndrome
What are the symptoms of CFC?
Metal retardation
Heart defects
Distinct facial features
What are gain of function mutations in BRAF during later life often associated with?
Melanoma (but only in around ~50% of cases)
What substitution is present in 90% of cancers involving BRAF?
Valine is substituted with glutamine at codon 600
In 2005, what did Leonard Zon and colleagues used zebrafish to ask?
Whether the mutation of BRAF alone was sufficient to induce melanoma
What must BRAF work cooperatively with?
Other gene mutations
What locus other than BRAF is also commonly associated with melanoma?
CDKN2A
What does the CDKN2A locus contain coding regions for?
p16 and p19 which are components of the Rb and p53 tumour suppressor pathways
How did Patton et al. produce a fish with the melanoma in 2005?
Generated BRAF-V600E/p53 double mutants by injecting mutated BRAF DNA into loss-of-function p53 mutant embryos and then raised until adulthood
What did Patton et al. conclude from their research in 2005?
That melanoma depends on a gain of function mutation in BRAF coincident with the loss of p53 tumour suppressor pathway
What is the role of methyltransferases?
They regulate the methylation of chromatin
Outlined the methods and findings of the experiment conducted by White et al. in 2001 in which chemical agents that inhibited neural crest formation in developing zebrafish embryos were screened
Used mitf:GFP line which expresses GFP in precursors of melanocytes and carried out a mass screen tested on around 2000 compounds and then tested efficacy of leflunomide in a mouse xenograft model
Found that leflunomide, an anti-arthritic drug suppressed proliferation of neural crest cells and suppresses tumour growth
What happens to melanoma progression when leflunomide is administered in combination with the BRAF inhibitor PLX4720?
Melanoma progression is completely arrested
What type of cancer has zebrafish research provided new insights into?
The metastasis of breast cancer
