TASK 4: The Zebrafish Information Network

The Zebrafish Information Network (www.zfin.org) is a database that stores data from zebrafish research. Using the search bar, you can research specific mutations to find more information.

Question 4. Pigmentation gene mutation  [2 marks].

In task 3, you phenotyped the pigment mutants.  What is the full name of the gene responsible (i.e. the affected gene) for the nacre mutation? (Hint: search for nacre and look at first result under gene/transcript.)  What is the function of this gene?  

[2 marks]

Question 8:Experimental scenario  [5 marks]

Clinicians have identified a cohort of human patients with abnormally small kidneys that have a mutation in a gene named PODRICK. The podrick gene is highly conserved in zebrafish. The clinicians approach you for a collaboration to determine whether mutation of podrick can cause kidney defects.  

Firstly, you want to know whether podrick protein is present in the developing kidney.

TASK 5: Observation of fluorescent zebrafish

Using transgenic approaches, specific organs and tissues can be labelled with a fluorescent marker. Since zebrafish embryos are known for their transparency, labelling tissues with fluorescent markers allows in vivo time-lapse imaging.

Task 3: Zebrafish forward genetic screens [8 marks]

Genome-wide genetic screens for phenotypes can identify new gene candidates and reveal novel mechanistic pathways that control development of tissues and organs. So-called “forward genetics screens” employ ENU-induced mutagenesis to produce random mutations in the genome. Phenotypes are observed and through breeding and mapping the affected gene can be identified. Two large scale ENU-mutagenesis zebrafish genetic screens performed in the mid-1990s produced thousands of mutations affecting development and organogenesis. 

Question 3: 

3a) Take a photograph of the wild type fish to compare your mutants. (1 mark)

3b) Take a photograph of the nacre mutant zebrafish and identify the defect/s compared to the wild type fish. (2 marks; one mark for the image and one for the labelling of defect.)

3c) Take a photograph of the casper mutant zebrafish and identify the defect/s compared to wild type fish. (3 marks; one mark for each correct drawing and correct labelling of defects - note, plural!)

3d) Take a photograph of the leopard

mutant zebrafish and identify

the defect/s compared to wild type fish.   ( 2 marks; one mark for the image and one for labelling of defect.)

Combine your images for Q3 into a single figure together with

labels indicating each zebrafish (ppt is a good place to do this).  Upload the

Question 6. Mutant Pigment genes [3 marks]. 

Why do nacre and casper mutants display melanophores in their eyes despite having no melanophores elsewhere? (Hint: compare the gene name to other vertebrate homologues!) 

[3 marks]

Question 5. Fish Pigment cells [6 marks].

Chromatophores are pigment cells found in the hypodermis

of the zebrafish skin.  

- Name the THREE types of chromatophores (hint, do an

online search).

-Where along the length of the adult fish are these chromatophores

located?

(6 marks; one mark each for correct pigment name, and

one mark each for location.)

Question 8: 

Have the paper on Slugh at hand (can be found on Moodle, under mouse prac B). There may also be hard copies of the paper in the lab.

a) What type of protein is Slugh?  [1 mark]

b) Where in the cell would you expect mature Slugh protein, carrying out its function,  to be located?  [1 mark]

c) What is the phenotype of Slugh knockout animals?  [2 marks]

In situ hybridisation.

What is the difference between a sense and anti-sense probe? What is the purpose of the sense probe?                                                    

[2 marks]

At

E12.5 of mouse development, the lung is composed of two cell types; the

epithelial cells and the surrounding mesenchymal cells. You are interested in

identifying the genes expressed at this stage of development in these two

different cell types at a global level using RNA-seq (RNA-sequencing). Using

the information you have been given in the lectures and this practical session

(both weeks) answer the following questions.

Question 11:  [9 marks].

a) Describe a genetic approach you could use to distinguish and separate these two cell types from the other. (Not simply endogenous gene expression).  Use dot points to outlines the steps involved in making an appropriate mouse model. Hint: knock-in.  (4 marks) 

b) Using the approach you outlined in (a) above, name the specific method that you could use to separate these two cell types from one another so that you can extract RNA from each cell type, to perform your RNA-seq study.  (1 mark).

c) You have performed the RNA-seq and find that a gene called Vegf (Vascular endothelial growth factor) is highly expressed in the lung epithelial cells. Vegf is a secreted signalling molecule (growth factor). Using your RNA-seq data from the two populations, what information could you seek to support the idea that Vegf signaling is involved in lung development?  (Hint: Recall the signaling lectures. What do signalling molecules act on?)  (2 marks).

d) Name a method you could use to validate your finding that Vegf is highly expressed in lung epithelium and to visualise its expression in the embryonic lung.  (2 marks).

Question 10:  Sox9 in situ hybridisation [ 2 marks]

Some mouse embryos are available that have been subjected to whole mount in situ hybridisation for the gene, Sox9. These will be handed out during the practical. Based on the expression pattern, what role might this gene play in development? What might happen in human embryos if this gene had a loss-of-function mutation?            

 [2 marks]