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BGM2002 CS > L12 - Wnt signalling in development & disease > Flashcards

L12 - Wnt signalling in development & disease Flashcards

1
Q

Why are the Wet proteins important?

A

Important in animal development & disease

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2
Q

What processes does Wnt signalling regulate?

A

Tissue patterning

Cell proliferation

Cell migration

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3
Q

What does Wnt signalling do in adults?

A

Regulates stem cell maintenance in tissue homeostasis

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4
Q

How is Wnt signalling involved in disease?

A

Deregulation of Wnt signalling is associated with cancer

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5
Q

What are the 3 Wnt signalling pathways?

A

Canonical Wnt/Beta-catenin pathway

Non-canonical Wnt/Calcium pathway

Planar cell polarity pathway

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6
Q

What is the canonical Wnt/Beta-catenin pathway?

A

Stabilization of β-catenin in response to ligand binding

Leads to regulation of gene transcription

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7
Q

What is the non-canonical Wnt/Calcium pathway?

A

Ability of Wnt and Frizzled proteins to cause increase in intracellular calcium

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8
Q

What is the planar cell polarity pathway?

A

Controls the polarity of cells in a planar tissue

Regulates the cytoskeleton

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9
Q

What is common to all 3 of the Wnt signalling pathways?

A

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

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10
Q

Where do the 3 pathways differ?

A

Downstream of dishevelled

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11
Q

Where does the name Wnt come from?

A

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

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12
Q

What are Wnt proteins produced by?

A

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)

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13
Q

What are Wnt proteins?

A

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)

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14
Q

What modification sites does Wnt have?

A

Potential glycosylation and lipid modification sites

Lipid targets Wnt to the membrane

Glycosylation ensures proper folding and secretion

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15
Q

Wnt secretion from the ER to the Golgi

A

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

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16
Q

What happens when theres no porcupine?

A

Loss of Porcupine – retention of Wnt protein inside the ER – no secretion

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17
Q

Wnt secretion from the Golgi to the PM?

A

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

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18
Q

What is Wntless (WIs)?

A

Multipass transmembrane protein

Wls evolutionarily conserved from worm to man

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19
Q

What happens to the WI once Wnt is transferred to the PM?

A

Wls recruited back to the Golgi by multiprotein retromer complex, where it can pick up more Wnt

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20
Q

What happens if theres no WIs?

A

Loss of Wls – Wnt proteins fail to reach the plasma membrane

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21
Q

How is Wnt secreted to Wnt-responsive cells?

A

Lateral diffusion

Transport proteins

Exovesicles

Cytonemes

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22
Q

Lateral diffusion in transporting Wnt

A

Short range

HSPGs (Heparan sulfate proteoglycans) facilitate lateral diffusion of Wnt ligand into receiving cell

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23
Q

Transport proteins in transporting Wnt

A

Long range

Wnt signalling is bound & solubilised by extracellular lipid binding proteins & transported to receiving cell

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24
Q

Exovesicles in transporting Wnt

A

Long range

Exovesicles shuttle Wnt ligands on their surfaces & deliver to receiving cell

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25
Q

Cytonemes in transporting Wnt

A

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

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26
Q

Cell communication involving Wnt

A

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

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27
Q

Graded expression of Wnt

A

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

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28
Q

Frizzled receptors

A

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

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29
Q

Dishevelled

A

Dishevelled (Dvl) is the branch point between the 3 Wnt pathways

This will determine what is activated downstream

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30
Q

What are the 3 protein conserved domains in dishevelled?

A

DIX
PZD
DEP

Activation of one pathway sequesters Dishevelled to one location, leaving it unavailable for the other pathway

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31
Q

What domains are important in the canonical pathway?

A

DIX

PZD

32
Q

What domains are important in the non-canonical pathways?

A

PZD

DEP

33
Q

Dishevelled mutants in Drosophila

A

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

34
Q

Canonical Wnt / β-catenin Signalling

A

Wnt signalling is transduced through β-catenin dependent pathway

β-catenin acts as a key transcriptional co-activator

Activated by Wnt1, Wnt3a, Wnt8

35
Q

Canonical Wnt / β-catenin Signalling

Functions in:

A 
Cell proliferation
Cell fate
Programmed cell death
Maintenance of stem cells
Development of neuronal circuits
36
Q

Canonical Wnt / β-catenin Signalling

In the presence of Wnt:

A

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

37
Q

Canonical Wnt / β-catenin Signalling

What is LRP5/6?

A

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

38
Q

Canonical Wnt / β-catenin Signalling

What is beta catenin?

A

β-catenin is a transcription co-activator, controls gene expression

39
Q

Canonical Wnt / β-catenin Signalling

Wnt responsive genes

A

Members of the homeobox family (TF)

Genes expressed in the development of the embryo

Cellular proliferation genes

Wnt signalling components

40
Q

Canonical Wnt / β-catenin Signalling

In the absence of Wnt?

A

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)

41
Q

Canonical Wnt / β-catenin Signalling

What is the destruction complex made of?

A

Axin, APC, GSK3-beta & CK1-alpha

GSK3-beta & CK1-alpha phosphorylate β- catenin for degradation

42
Q

Non-canonical Wnt pathways

A

Wnt / Calcium Pathway & Planar Cell Polarity (PCP) Pathway are both important for convergence extension movements required for vertebrate body plan organisation

43
Q

What is convergent extension?

A

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

44
Q

Wnt/Calcium pathway

A

Wnt signalling is transduced via regulation of intracellular Ca levels

Inhibits the Wnt/β-catenin signalling

45
Q

Wnt/Calcium pathway

What happens?

A

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

46
Q

Wnt/Calcium pathway

Roles in zebrafish

A

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

47
Q

Planar Cell Polarity (PCP) Pathway

A

Signalling through small G-proteins via actin cytoskeleton remodelling

Activated by Wnt4, Wnt5a, Wnt11

48
Q

Planar Cell Polarity (PCP) Pathway

Functions:

A
  • 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
49
Q

Planar Cell Polarity (PCP) Pathway

In the presence of Wnt:

A

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

50
Q

Planar Cell Polarity (PCP) Pathway

What are the downstream pathways?

A 
– Directly modulate the cytoskeleton 
– Actin polymerisation 
– Activate gene transcription 
– Cell polarity 
– Cell migration
51
Q

Planar Cell Polarity (PCP) Pathway

PCP mutants in vertebrates

A

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

52
Q

How can a cell be able to move?

A

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

53
Q

Core PCP proteins

A

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

54
Q

What PCP proteins are at the front of the cell?

A

Scrb
Pk
Vangl2
CElsr1

55
Q

What PCP proteins are at the back of the cell?

A

Celsr1
Fz (where the Wnt will bind)
Dvl

56
Q

What happens when theres disruptions in the PCP signalling pathway?

A

Causes NT & convergent extension defects

Can also cause heart defects

57
Q

How is PCP involved with cilia?

A

PCP is required for the correct positioning of cilia

58
Q

PCP & the correct positioning of the cilia

A

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

59
Q

PCP & the correct positioning of hairs in the inner ear

A

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

60
Q

What are non-canonical pathways?

A

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

61
Q

Inhibition of Wnt secretion

A

Modulate the signalling by secreted Wnt ligand

Interfere with ligand-receptor complex formation – block Wnt signalling

62
Q

How can you modulate the signalling by secreted Wnt ligand?

A

sFRP (secreted Frizzled related proteins) family

WIF (Wnt inhibitory factor)

DKK (Dickkopf)

63
Q

sFRP (secreted Frizzled related proteins) family

A

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

64
Q

WIF (Wnt inhibitory factor)

A

Bind to Wnt to prevent it binding to Frizzled

Inhibit canonical & non-canonical Wnt signalling

65
Q

DKK (Dickkopf)

A

Bind to co-receptor LRP5/6 in canonical pathway

Prevents FZD-LRP6 dimerisation

66
Q

Role of Wnt canonical signalling in the limb bud – Wnt7a

A

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

67
Q

What is Wnt7a?

A

Wnt7a – diffusible morphogen secreted by dorsal ectodermal cells

Wnt7a induces expression of homeobox gene Lmx1 in underlying mesoderm adjacent to dorsal surface

68
Q

What happens when theres no Wnt7a in mice?

A

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

69
Q

Role of Wnt canonical signalling in the limb bud – Wnt5a

A

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

70
Q

Role of Wnt/β-catenin signalling in limb bud

A

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

71
Q

Human mutations in the PCP pathway & limb development

A

Brachdactyly type B (BDB1)

Robinow syndrome (RRS)

72
Q

How is the neural tube and the spinal cord patterned during development?

A

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

73
Q

What can be the cause of colorectal cancer?

A

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

74
Q

Colorectal cancer

APC loss of function mutation

A

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

75
Q

Colorectal cancer

β-catenin gain of function mutation

A

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

76
Q

Colorectal cancer

Overexpression of Fzd or Wnt

A

Increased activation of the pathway

Cell proliferation is continuous, ultimately resulting in tumour formation

Abnormal cell proliferation

77
Q

Colorectal cancer

Mutation in sFRP

A

Underexpression of secreted inhibitors

Increased sensitivity to Wnt ligands

Increased pathway activation

Cell proliferation is continuous, ultimately resulting in tumour formation

BGM2002 CS (14 decks)

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