L12 - Wnt signalling in development & disease Flashcards
Why are the Wet proteins important?
Important in animal development & disease
What processes does Wnt signalling regulate?
Tissue patterning
Cell proliferation
Cell migration
What does Wnt signalling do in adults?
Regulates stem cell maintenance in tissue homeostasis
How is Wnt signalling involved in disease?
Deregulation of Wnt signalling is associated with cancer
What are the 3 Wnt signalling pathways?
Canonical Wnt/Beta-catenin pathway
Non-canonical Wnt/Calcium pathway
Planar cell polarity pathway
What is the canonical Wnt/Beta-catenin pathway?
Stabilization of β-catenin in response to ligand binding
Leads to regulation of gene transcription
What is the non-canonical Wnt/Calcium pathway?
Ability of Wnt and Frizzled proteins to cause increase in intracellular calcium
What is the planar cell polarity pathway?
Controls the polarity of cells in a planar tissue
Regulates the cytoskeleton
What is common to all 3 of the Wnt signalling pathways?
Wnt
Frizzeled
Dishevelled (Dvl)
All 3 pathways are all regulated and activated by Wnt proteins, all utilise receptor frizzled (transmembrane membrane) and they all involve recruitment of dishevelled
Dishevelled is in the cytoplasm and is recruited towards the membrane
Where do the 3 pathways differ?
Downstream of dishevelled
Where does the name Wnt come from?
In 1987 found the int1 gene was the homologue of Drosophila segment polarity gene wingless (Wg)
Name Wnt is a fusion of Wg and int
What are Wnt proteins produced by?
Produced and secreted by a defined subset of cells
Concentration gradient of diffusion of Wnts when they are produced is really important (influences how target cells will respond)
What are Wnt proteins?
Highly conserved secreted signalling molecules
Wnt proteins are ~40kDa (350-400 amino acids)
Contain high number of cysteine residues responsible for ensuring proper folding and secretion (disulphide bonds)
What modification sites does Wnt have?
Potential glycosylation and lipid modification sites
Lipid targets Wnt to the membrane
Glycosylation ensures proper folding and secretion
Wnt secretion from the ER to the Golgi
1) N-terminal signal peptide directs protein to ER
2) In ER, signal peptide is cleaved off
3) Wnt protein modified by addition of sugars and lipids (palmitoleate) by Porcupine
Lipid modification is palmitoylation of cysteines
Porcupine is required for transfer of Wnt from ER to Golgi
What happens when theres no porcupine?
Loss of Porcupine – retention of Wnt protein inside the ER – no secretion
Wnt secretion from the Golgi to the PM?
Wnt secretion requires Wntless (Wls)
1) Wls binds to Wnt – requires lipid attachment
2) Transfer of Wnt protein to the cell surface via secretory vesicles
3) Secretion of Wnt proteins
What is Wntless (WIs)?
Multipass transmembrane protein
Wls evolutionarily conserved from worm to man
What happens to the WI once Wnt is transferred to the PM?
Wls recruited back to the Golgi by multiprotein retromer complex, where it can pick up more Wnt
What happens if theres no WIs?
Loss of Wls – Wnt proteins fail to reach the plasma membrane
How is Wnt secreted to Wnt-responsive cells?
Lateral diffusion
Transport proteins
Exovesicles
Cytonemes
Lateral diffusion in transporting Wnt
Short range
HSPGs (Heparan sulfate proteoglycans) facilitate lateral diffusion of Wnt ligand into receiving cell
Transport proteins in transporting Wnt
Long range
Wnt signalling is bound & solubilised by extracellular lipid binding proteins & transported to receiving cell
Exovesicles in transporting Wnt
Long range
Exovesicles shuttle Wnt ligands on their surfaces & deliver to receiving cell
Cytonemes in transporting Wnt
Long range
Cytonemes are thin, cellular projections that are specialized for exchange of signalling proteins between cells
Wnt ligand is transported through cytonemes that extend from the emitting cell to the receiving cell
Cell communication involving Wnt
Wnt signals can act locally (neighbouring cell) or can form concentration gradients across tissues (up to 20 cell diameters away)
Signalling can be paracrine and autocrine
Wnt-responsive cells respond to signal and modulate the expression of downstream genes
Graded expression of Wnt
Wnt antagonists expressed in opposing gradient to maintain anterior structures or stem cells
At the posterior end, there is a high level of Wnt
Whereas, towards the anterior end, there is less Wnt
This allows the patterning of the different structures in the embryo
Frizzled receptors
Frizzled is the receptor that Wnt will bind to in the receiver cell
Seven transmembrane receptor
Cysteine-rich extracellular domain – binds to Wnt proteins
Three intracellular Dishevelled (Dvl) binding motifs
Dishevelled
Dishevelled (Dvl) is the branch point between the 3 Wnt pathways
This will determine what is activated downstream
What are the 3 protein conserved domains in dishevelled?
DIX
PZD
DEP
Activation of one pathway sequesters Dishevelled to one location, leaving it unavailable for the other pathway
What domains are important in the canonical pathway?
DIX
PZD
What domains are important in the non-canonical pathways?
PZD
DEP
Dishevelled mutants in Drosophila
The head of the Drosophila has hairs that are polarised and facing one direction
In the dishevelled mutant, the hairs are disorganised and are pointing in lots of different directions
Again, in the eye of the drosophila, the ommatidia in the wt eye are all pointing in a single direction but in the dishevelled mutant, they are all pointing in different directions, so polarity is lost
Canonical Wnt / β-catenin Signalling
Wnt signalling is transduced through β-catenin dependent pathway
β-catenin acts as a key transcriptional co-activator
Activated by Wnt1, Wnt3a, Wnt8
Canonical Wnt / β-catenin Signalling
Functions in:
Cell proliferation Cell fate Programmed cell death Maintenance of stem cells Development of neuronal circuits
Canonical Wnt / β-catenin Signalling
In the presence of Wnt:
1) Wnt binds Frizzled & LRP5/6 (co-receptor)
2) Dishevelled & axin recruited to the membrane
3) β-catenin accumulates in cytoplasm and translocates to the nucleus
4) β-catenin binds to Transcription Factors TCF/LEF
5) Transcription of 50 Wnt-responsive genes
Canonical Wnt / β-catenin Signalling
What is LRP5/6?
Co-receptor for the Wnt/βcatenin pathway
Wnt binds to FZD and LRP5/6
Binding of Wnt induces GSK3β to phosphorylate LRP5/6
Phosphorylated LRP5/6 is then able to bind to Axin
Canonical Wnt / β-catenin Signalling
What is beta catenin?
β-catenin is a transcription co-activator, controls gene expression
Canonical Wnt / β-catenin Signalling
Wnt responsive genes
Members of the homeobox family (TF)
Genes expressed in the development of the embryo
Cellular proliferation genes
Wnt signalling components
Canonical Wnt / β-catenin Signalling
In the absence of Wnt?
1) Dishevelled is not activated – not recruited to the membrane, so it stays within the cytoplasm
2) Formation of the destruction complex – Axin, APC, GSK3-beta & CK1-alpha
3) GSK3-beta & CK1-alpha phosphorylate β- catenin
4) Targets β-catenin for degradation – cannot accumulate in the cell, it cannot translocate to the nucleus and it cannot activate gene transcription
5) No gene transcription of Wnt responsive genes
6) TCF/LEF transcription factors bind to transcription inhibitors (eg. Groucho)
Canonical Wnt / β-catenin Signalling
What is the destruction complex made of?
Axin, APC, GSK3-beta & CK1-alpha
GSK3-beta & CK1-alpha phosphorylate β- catenin for degradation
Non-canonical Wnt pathways
Wnt / Calcium Pathway & Planar Cell Polarity (PCP) Pathway are both important for convergence extension movements required for vertebrate body plan organisation
What is convergent extension?
Cells intercalate with each other and move toward the midline
Result in the narrowing and lengthening of the embryo
Disruption leads to short fat embryos
Are regulated by a highly conserved signalling pathway
Wnt/Calcium pathway
Wnt signalling is transduced via regulation of intracellular Ca levels
Inhibits the Wnt/β-catenin signalling
Wnt/Calcium pathway
What happens?
1) Binding of Wnt5a / Wnt11 to the Fz receptor and co-receptor Ror1/2 which sits on the cell membrane
2) Ror1/2 will bind to dishevelled which will activate a G protein.
3) Induces Ca2+ release inside the cell
4) Activates Protein Kinase C (PKC) and calcium/calmodulin-dependent kinase II (CAMKII)
5) Activates transcription factors including NFAT
6) Upregulation of target gene expression
7) Bring about changes in cell fate and cell movement
Wnt/Calcium pathway
Roles in zebrafish
When Wnt5a is removed or mutated, the zebrafish have a much shorter tail
They haven’t undergone the conversion extension; they haven’t elongated out
This was shown to be because of a reduction in calcium (no calcium released into cell) so there was no increase in calcium
Planar Cell Polarity (PCP) Pathway
Signalling through small G-proteins via actin cytoskeleton remodelling
Activated by Wnt4, Wnt5a, Wnt11
Planar Cell Polarity (PCP) Pathway
Functions:
- Organisation of multicellular structures
- Tissue remodelling
- Control of polarised cell migration
- Coordinated cell movements
- Disruption of PCP can lead to developmental defects and disease
- Highly studied in Drosophila
Planar Cell Polarity (PCP) Pathway
In the presence of Wnt:
1) Binding of Wnt to the Fz receptor, initiates signalling
2) Fz recruits Dishevelled
3) Celsr1 and Vangl2 become recruited
4) These bind Scribbled and Prickle in the cytoplasm
5) Dishevelled will bind to Diego and Daam1
6) Forms a highly conserved core complex
7) Activates the small GTPases RhoA & JNK to activate downstream effectors
8) Lead to an increase in gene transcription or cytoskeletal changes
Planar Cell Polarity (PCP) Pathway
What are the downstream pathways?
– Directly modulate the cytoskeleton – Actin polymerisation – Activate gene transcription – Cell polarity – Cell migration
Planar Cell Polarity (PCP) Pathway
PCP mutants in vertebrates
In a planar cell polarity mutant (mutated Dishevelled), the hairs on Drosophila are not polarised and are all pointing in different directions
This is the same on a mouse - same impact, two completely different organisms
How can a cell be able to move?
In order for a cell to move, the front and the back of the cell have to be defined
To achieve this, there are a group of proteins sitting at the front and a different group of proteins sitting at the back
The planar cell polarity core proteins play a role in this
Core PCP proteins
The co-proteins sit in different parts of the cell
In the front of one cell there is Scrb, Pk, Vangl2 and CElsr1
At the back of a neighbouring cell, which will allow it to bind to, there is Celsr1, Fz (where the Wnt will bind) and Dvl
The binding of the front of one cell and the back of another cell will activate downstream components
This sets up the polarity of the cell
What PCP proteins are at the front of the cell?
Scrb
Pk
Vangl2
CElsr1
What PCP proteins are at the back of the cell?
Celsr1
Fz (where the Wnt will bind)
Dvl
What happens when theres disruptions in the PCP signalling pathway?
Causes NT & convergent extension defects
Can also cause heart defects
How is PCP involved with cilia?
PCP is required for the correct positioning of cilia
PCP & the correct positioning of the cilia
At the bottom of a cilia is the basal body
The PCP pathway is really important in the positioning of this
PCP ensures all basal bodies are pointing in the same direction
PCP & the correct positioning of hairs in the inner ear
Hair cells in ears respond to movement and are essential for balance
Each has a cilium pointing in the same direction
Mutations in core proteins (Vangl2, Celsr1, Dvl2) all cause hair misorientation
These are disorganised when PCP is disrupted
What are non-canonical pathways?
Non-canonical pathways are β-catenin independent
Two non-canonical pathways
• Wnt calcium pathway
• Planar cell polarity pathway
Can have direct effects on the cytoskeleton or via gene transcription
Regulate cell proliferation, polarity and cell movements
Inhibition of Wnt secretion
Modulate the signalling by secreted Wnt ligand
Interfere with ligand-receptor complex formation – block Wnt signalling
How can you modulate the signalling by secreted Wnt ligand?
sFRP (secreted Frizzled related proteins) family
WIF (Wnt inhibitory factor)
DKK (Dickkopf)
sFRP (secreted Frizzled related proteins) family
Bind to Wnt to prevent it binding to Frizzled receptor
Domain resembles the Wnt binding cysteine rich domain of Fz receptors hence binds to Wnt
WIF (Wnt inhibitory factor)
Bind to Wnt to prevent it binding to Frizzled
Inhibit canonical & non-canonical Wnt signalling
DKK (Dickkopf)
Bind to co-receptor LRP5/6 in canonical pathway
Prevents FZD-LRP6 dimerisation
Role of Wnt canonical signalling in the limb bud – Wnt7a
Wnt pathway is involved in the patterning of the dorsal-ventral side of the limb bud
Wnt7a is expressed in the dorsal side of the limb bud
Lmx1 encodes a TF essential for specifying dorsal cell fates in the limb
Engrailed antagonises Wnt7a to define ventral region of limb bud
What is Wnt7a?
Wnt7a – diffusible morphogen secreted by dorsal ectodermal cells
Wnt7a induces expression of homeobox gene Lmx1 in underlying mesoderm adjacent to dorsal surface
What happens when theres no Wnt7a in mice?
Loss of Wnt7a function in the mouse resulted in embryos with ventralized paws having sole pads on both surfaces, showing that Wnt7a is necessary for specifying dorsal cell fates in the distal limb
Role of Wnt canonical signalling in the limb bud – Wnt5a
Growth of limb bud requires organised, polarised orientated cell division controlled by Wnt5a.
When Wnt5a is deleted, the mesenchymal cells are a lot smaller, they have lost the polarity and they are not stretching out. When they map the movement of the cells, they all move in different directions
Wnt5a is expressed in the distal mesenchyme underneath the AER in both chick and mouse
Loss of Wnt5a activity also results in reduced proliferation of the progressive zone underneath the AER
Role of Wnt/β-catenin signalling in limb bud
In the beta-catenin mutant, the limbs are a lot shorter than in the wt
This illustrates that different components of the same pathway are involved in development of one structure in the mouse embryo
Human mutations in the PCP pathway & limb development
Brachdactyly type B (BDB1)
Robinow syndrome (RRS)
How is the neural tube and the spinal cord patterned during development?
By the antagonistic activities of Shh & Wnt
Counteracting gradients of Shh, secreted from the floorplate (FP), and Wnt/BMP, derived from the roof plate (RP) induce the concentration-dependent differentiation of precursor cells along the dorso-ventral (D-V) axis.
The specific combinations of transcription factors induced by Shh and Wnts generate a cell identity code that specifies the neural progenitor subtypes
What can be the cause of colorectal cancer?
Loss of function mutation in APC (in absence of Wnt signal)
Gain of function of mutations in β-catenin (prevents phosphorylation)
Overexpression of Frizzled or Wnt signals
Mutation in sFRP
Colorectal cancer
APC loss of function mutation
Destruction complex cannot form properly
Even with no Wnt binding, there is accumulation of cytosolic β-catenin
β-catenin translocates into nucleus
Constitutive expression of Wnt/ β-catenin dependent genes
Leads to abnormal proliferation
Colorectal cancer
β-catenin gain of function mutation
Prevents phosphorylation of β-catenin
Prevents degradation of β-catenin
Accumulation of cytosolic β-catenin
Abnormal activation of gene expression
Destruction complex tries to form, but because beta-catenin is unable to be phosphorylated, it doesn’t bind the complex and does not get degraded
Colorectal cancer
Overexpression of Fzd or Wnt
Increased activation of the pathway
Cell proliferation is continuous, ultimately resulting in tumour formation
Abnormal cell proliferation
Colorectal cancer
Mutation in sFRP
Underexpression of secreted inhibitors
Increased sensitivity to Wnt ligands
Increased pathway activation
Cell proliferation is continuous, ultimately resulting in tumour formation
