Zebrafish Model of Cancer

Question 1

What family are zebrafish members of?

Answer 1

The minnow family (Cyprinidae)

Question 2

What is the latin name for zebrafish?

Answer 2

Danio rerio

Question 3

What are zebrafish?

Answer 3

Small freshwater fish native to Northern India/Bangladesh

Question 4

How did Streisinger die in 1984?

Answer 4

In a scuba diving accident

Question 5

What did Streisinger develop methods for?

Answer 5

Generating homozygous diploid clones of zebrafish

Question 6

What year were zebrafish first included in a publication in Nature?

Answer 6

1981

Question 7

Describe the progression of zebrafish studies since the first papers were published on them?

Answer 7

Studies using zebrafish have grown exponentially

Question 8

How long did it take for zebrafish to become the second most widely used model in bioscience research?

Answer 8

In around 20 years

Question 9

Between what years was there development and dissemination of zebrafish methodologies?

Answer 9

1984-1992

Question 10

Describe the pros of using zebrafish as models for human cancers

Answer 10

Fast
Large scale
Cheap
High resolution

Question 11

Describe the pros of using mice as models for human cancers

Answer 11

High resolution

Question 12

Describe the cons of using mice as models for human cancers

Answer 12

Time consuming

Expensive

Question 13

Describe the pros of using bacteria as models for human cancers

Answer 13

Fast

Large scale

Question 14

Describe the cons of using bacteria as models for human cancers

Answer 14

Resolution limited

Cheap

Question 15

What did the Zon lab in March 2011 use zebrafish for?

Answer 15

High-throughput screen of chemicals that might suppress melanoma

Question 16

What was George Streisinger’s hobbies?

Answer 16

He was a tropical fish enthusiast who studied his fish in his own collection to find a potentially useful model

Question 17

Why did George Streisinger want to find a new model organism?

Answer 17

He found that mice are too complex and microorganisms are too simple

Question 18

Where did George Streisinger work?

Answer 18

At the University of Oregon

Question 19

Who is George Streisinger and what did he work on?

Answer 19

Geneticist

Worked on T4 bacteriophage genome in the 1960s and 1970s

Question 20

What did George Streisinger begin to do in the 1980s?

Answer 20

Began to look for more complex systems to study genetic regulation of development

Question 21

Give 4 advantages of the zebrafish life cycle

Answer 21

Transparent embryos
Rapid development
Spawn at dawn
100’s of eggs produced per female

Question 22

What did scientists do prior to Streisinger’s death in order to promote the utility of zebrafish as a genetic model?

Answer 22

They ran courses

Question 23

Give an example of another Oregon scientist who had become interested in harvesting the power of the zebrafish model prior to Streisinger’s death

Answer 23

Charles Kimmel

Question 24

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?

Answer 24

True

Question 25

Since around what year have many cancer biologists turned their attention to using zebrafish for cancer research?

Answer 25

2000

Question 26

Do immature (embryonic/larval) zebrafish develop tumours?

Answer 26

Rarely

Question 27

How big is a larval fish at 4 days old?

Answer 27

~4mm

Question 28

In immature zebrafish, how can precocious tumours be induced?

Answer 28

With transgenic approaches

Question 29

Can immature zebrafish be readily zenografted with human tumour cells?

Answer 29

Yes, the graft is not rejected

Question 30

In immature zebrafish, what does their optical transparency permit?

Answer 30

In vivo imaging of tumour growth/metastasis

Question 31

What does the small size of immature zebrafish permit the high throughput chemical screening of?

Answer 31

Carcinogens/chemical tumour suppressors

Question 32

In vivo imaging approaches can be used to resolve what in early-stage zebrafish?

Answer 32

Intravasation and metastasis

Question 33

Why are fish better for high throughput in vivo screens than mouse models?

Answer 33

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

Question 34

What types of tumour can adult zebrafish spontaneously develop?

Answer 34

Almost any type of tumour

Question 35

How can tumours be readily induced in adult zebrafish?

Answer 35

Carcinogen exposure Zenograft Gene knockout/insertion (fast and easy)

Question 36

Adult fish tissues are opaque so in vivo imaging is not possible, true or false?

Answer 36

False, in vivo imaging is still possible to an extent when using pigmentation mutants

Question 37

What have forward genetic screens of zebrafish identified?

Answer 37

New oncogenes

Question 38

What did the Hopkins lab develop in 2004 using forward genetics?

Answer 38

Mass mutagenic screening

Question 39

Outline the mass mutagenic screening approach to identify new oncogenes

Answer 39

1. Inject 1000s of eggs with a retrovirus 2. Virus randomly inserts into the genome 3. Outcross and screen for mutant phenotypes 4. Sequence mutated genes

Question 40

What did Amsterdam et al. use the mass mutagenic screening approach in 2004 to identify?

Answer 40

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

Question 41

Give an example of a zebrafish liver oncogene

Answer 41

apc

Question 42

Give an example of a zebrafish intestine oncogene

Answer 42

apc

Question 43

Give an example of a zebrafish ocular oncogene

Answer 43

pten

Question 44

Give examples of a zebrafish nerve sheath oncogene

Answer 44

tp53 | msh

Question 45

Give an example of a zebrafish leukemia oncogene

Answer 45

c-myc

Question 46

Give an example of a zebrafish melanoma oncogene

Answer 46

BRAF

Question 47

Give an example of a zebrafish neuroendocrine oncogene

Answer 47

MYCN

Question 48

Adult zebrafish can shed new clues on the formation of what?

Answer 48

Melanoma

Question 49

Imaging metastases in vivo with zebrafish has shown that what compounds work together to cause metastases?

Answer 49

VEGF and RhoC

Question 50

Over the past 20 years the zebrafish has become a credible and powerful model for studying cancer, true or false?

Answer 50

False, only over the past 12 years

Question 51

Give an example of a rapid, reliable method for studying cancer in zebrafish

Answer 51

The insertion of transgenes

Question 52

Outline the method for the easy insertion of transgenes in zebrafish

Answer 52

1. Inject one-cell stage eggs with DNA 2. Raise to adulthood (3 months) 3. Outcross and test for germline transmission 4. Widely used technique for introducing oncogenes (e.g. RNUX1/c-myc/bcl2/Notch/BRAF) into fish

Question 53

What is c-myc?

Answer 53

A transcriptional regulator

Question 54

What are mutations in c-myc associated with?

Answer 54

Cancer (highly prevalent in Burkitt's lymphoma)

Question 55

What is BRAF a member of?

Answer 55

The RAF kinase family

Question 56

What is BRAF?

Answer 56

A serine/threonine kinase that participates in the RAS/RAF sigalling pathway

Question 57

What is BRAF critical for?

Answer 57

Normal development

Question 58

Without BRAF, what syndrome develops?

Answer 58

Cardio-facio-cutaneous (CFC) syndrome

Question 59

What are the symptoms of CFC?

Answer 59

Metal retardation Heart defects Distinct facial features

Question 60

What are gain of function mutations in BRAF during later life often associated with?

Answer 60

Melanoma (but only in around ~50% of cases)

Question 61

What substitution is present in 90% of cancers involving BRAF?

Answer 61

Valine is substituted with glutamine at codon 600

Question 62

In 2005, what did Leonard Zon and colleagues used zebrafish to ask?

Answer 62

Whether the mutation of BRAF alone was sufficient to induce melanoma

Question 63

What must BRAF work cooperatively with?

Answer 63

Other gene mutations

Question 64

What locus other than BRAF is also commonly associated with melanoma?

Answer 64

CDKN2A

Question 65

What does the CDKN2A locus contain coding regions for?

Answer 65

p16 and p19 which are components of the Rb and p53 tumour suppressor pathways

Question 66

How did Patton et al. produce a fish with the melanoma in 2005?

Answer 66

Generated BRAF-V600E/p53 double mutants by injecting mutated BRAF DNA into loss-of-function p53 mutant embryos and then raised until adulthood

Question 67

What did Patton et al. conclude from their research in 2005?

Answer 67

That melanoma depends on a gain of function mutation in BRAF coincident with the loss of p53 tumour suppressor pathway

Question 68

What is the role of methyltransferases?

Answer 68

They regulate the methylation of chromatin

Question 69

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

Answer 69

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

Question 70

What happens to melanoma progression when leflunomide is administered in combination with the BRAF inhibitor PLX4720?

Answer 70

Melanoma progression is completely arrested

Question 71

What type of cancer has zebrafish research provided new insights into?

Answer 71

The metastasis of breast cancer