L12 - Wnt signalling in development & disease Flashcards

Question

Cytonemes in transporting Wnt

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

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

Answer 5

Dishevelled (Dvl) is the branch point between the 3 Wnt pathways This will determine what is activated downstream

Answer 6

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

Answer 7

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

Answer 8

Wnt signalling is transduced through β-catenin dependent pathway β-catenin acts as a key transcriptional co-activator Activated by Wnt1, Wnt3a, Wnt8

Answer 9

``` Cell proliferation Cell fate Programmed cell death Maintenance of stem cells Development of neuronal circuits ```

Answer 10

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

Answer 11

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

Answer 12

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

Answer 13

Members of the homeobox family (TF) Genes expressed in the development of the embryo Cellular proliferation genes Wnt signalling components

Answer 14

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)

Answer 15

Axin, APC, GSK3-beta & CK1-alpha GSK3-beta & CK1-alpha phosphorylate β- catenin for degradation

Answer 16

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

Answer 17

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

Answer 18

Wnt signalling is transduced via regulation of intracellular Ca levels Inhibits the Wnt/β-catenin signalling

Answer 19

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

Answer 20

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

Answer 21

Signalling through small G-proteins via actin cytoskeleton remodelling Activated by Wnt4, Wnt5a, Wnt11

Answer 22

* 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

Answer 23

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

Answer 24

``` – Directly modulate the cytoskeleton – Actin polymerisation – Activate gene transcription – Cell polarity – Cell migration ```

Answer 25

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

Answer 26

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

Answer 27

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

Answer 28

Scrb Pk Vangl2 CElsr1

Answer 29

Celsr1 Fz (where the Wnt will bind) Dvl

Answer 30

Causes NT & convergent extension defects Can also cause heart defects

Answer 31

PCP is required for the correct positioning of cilia

Answer 32

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

Answer 33

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

Answer 34

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

Answer 35

Modulate the signalling by secreted Wnt ligand Interfere with ligand-receptor complex formation – block Wnt signalling

Answer 36

sFRP (secreted Frizzled related proteins) family WIF (Wnt inhibitory factor) DKK (Dickkopf)

Answer 37

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

Answer 38

Bind to Wnt to prevent it binding to Frizzled Inhibit canonical & non-canonical Wnt signalling

Answer 39

Bind to co-receptor LRP5/6 in canonical pathway Prevents FZD-LRP6 dimerisation

Answer 40

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

Answer 41

Wnt7a – diffusible morphogen secreted by dorsal ectodermal cells Wnt7a induces expression of homeobox gene Lmx1 in underlying mesoderm adjacent to dorsal surface

Answer 42

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

Answer 43

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

Answer 44

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

Answer 45

Brachdactyly type B (BDB1) Robinow syndrome (RRS)

Answer 46

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

Answer 47

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

Answer 48

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

Answer 49

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

Answer 50

Increased activation of the pathway Cell proliferation is continuous, ultimately resulting in tumour formation Abnormal cell proliferation

Answer 51

Underexpression of secreted inhibitors Increased sensitivity to Wnt ligands Increased pathway activation Cell proliferation is continuous, ultimately resulting in tumour formation