L9 : Elucidation of Wnt Signalling Pathways

Question 1

What is the role of convergence and extension during gastrulation?

Answer 1

C-E are coordinated cell movement types that drive formation of the rostro-caudal axis

Convergence - cells move towards the midline
Extension - Cells intercalate and lengthen the body axis

Question 2

What 2 behaviours contribute to C-E in the axis?

Answer 2

1. Collective migration (A-P axis)
   - Cells cohesively stick together and move as one
2. Cell intercalation (A-P + M-L axis)
   - Cells rearrange leading to narrowing and lengthening

Question 3

What are some fates and movement types of the axial mesoderm during early gastrulation?

Answer 3

• Collective migration -> predchordal plate
• Cell intercalation -> notochord and somitic mesoderm

Both act as key signalling centres for neural and axis development

Question 4

What is the silberblick mutant and how was it investigated?

Answer 4

slb disrupts C-E movements, particularly of axial mesoderm (prechordal plate)

In situ hybridisation studies indicated incomplete extension of axis responsible for phenotype

Question 5

How was the silberblick gene identified?

Answer 5

-> Forward genetic screen (start with phenotype)
-> Positional cloning (mapping)
-> Candidate gene approach (test known genes, Wnt11 = strong candidate)
2 mutant alleles of slb phenotype with premature stop codons confirmed Wnt11 = slb

Question 6

Describe the expression of Wnt11

Answer 6

Paraxial head mesoderm and germ bud/ring
Expression reduced in Slb mutants (particularly head mesoderm)

Question 7

How did phenotype rescue confirm Wnt11 = Slb?

Answer 7

Injecting normal Wnt11 rescued the phenotype

Question 8

What are Wnts?

Answer 8

Proto-oncogene
- identified in vertebrates as Int-1
- discovered in same gene as drosophila Wingless

Diffusible signalling molecule that can act on cell via receptor and downstream pathways

Question 9

How was this experiment limited?

Answer 9

EXPAND

Question 10

Describe the canonical Wnt pathway?

Answer 10

1. Wnt8 signals via cell surface receptor Frizzled (Fz)
2. Wnt8 binding induces conformational change in Fz causing downstream activation of Dishevelled (Dsh)
3. Of 3 Dsh domains, DIX and PDZ inhibit the activity of kinase GSK3
4. Suppressed GSK3 prevents phosphorylation of TF B-catenin within cytoplasm
5. B-catenin is therefore able to enter nucleus and interact with other TFs
6. Activating transcription of various specific genes, which control cell fate

Question 11

Describe the non-canonical Wnt/PCP pathway?

Answer 11

1. Wnt11 signals via cell surface receptor Frizzled (Fz)
2. Wnt11 binding induces conformational change in Fz causing downstream activation of Dishevelled (Dsh)
3. Of 3 Dsh domains, DEP and PDZ activate RhoA/Rac signalling pathways
4. Activates transcription of target genes, leading to morphogenesis

Question 12

How did researchers determine which signalling pathway is affected in Slb?

Answer 12

Overexpression (injection) of activated B-catenin did not rescue Slb embryo phenotypes
- Rules out canonical signalling

Overexpression of PDZ/DEP rescued Slb mutant embryos
- Wnt11 is non-canonical signalling pathway with involvement in morphogenesis
- Concludes PCP signalling involved in axis formation in vertebrates

Question 13

What is the role of Wnt/PCP in gastrulation and how was this tested?

Answer 13

Cells need to know which way to polarise in order to undergo C-E and prechordal plate migration
Mutants in other species show cell polarity defects (eg. mouse ear hair follicles)

Cell transplantation
- Slb -/- cells in slb -/- ambryos do not converge and extend properly
(same for WT in Slb hosts)

Question 13

How does incorrect prechordal plate location affect development?

Answer 13

Incorrect localisation key signalling centre (also affects notochord)
Due to defective C-E caused by Wnt11 (polarity) absence causes defects seen in Slb mutants
- Does not pattern overlying ectoderm correctly