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]

TASK 2. Staging zebrafish embryos

Question 2.   

Staging zebrafish

embryos or larvae. [

You have a series of dishes containing zebrafish embryos or larvae at three different developmental stages. For each dish compare the embryos to the staging series images (see lab practical manual staging series ). Through this comparison and by counting the somite number, determine the stage of fishes in each dish. 

[3 marks; 1 mark per dish]

Task 1: Anatomy of Zebrafish embryos 

Observe the basic anatomy of the zebrafish embryos, using the dish containing the fish at 4dpf (dpf = days post-fertilisation).  

Question 1.  Zebrafish embryos. [7 marks.]

• Eye
• Pectoral fin 
• Yolk
• Spinal chord 
• Tail fin 
• Otic vesicle (Hint: Look up "optic vesicle" online) 

(7 marks; 1 mark for the image, 6 marks for the labelling).