Lecture 5- Hh and Wnt signalling

Question 1

How were Hh and Wnt first discovered as patterning mutants in Drosophila?

Answer 1

• Wnt and Hh genes were first defined as mutations that showed defects in drosophila segmentation
• Loss of Hh gene causes a segment polarity phenotype whereby the naked cuticle is lost and the embryo is fully coated with denticles

Question 2

What type of gene is Hh?

Answer 2

Segmental polarity gene

Question 3

How is the Hh ligand formed and how does it diffuse?

Answer 3

1. Hh genes are transcribed with an N-terminal signal sequence that targets them to the secretory pathway
2. The signal sequence is removed and then the protein undergoes an autoproteolytic cleavage catalysed by the C-terminal part of the protein
3. Concurrently with this cleavage the C-terminus of the N-terminal part is coupled to a cholesterol molecule
4. Another modification is made at the N-terminus where a palmitoyl group is added (palmitoylation)
5. Both cholesterol and the palmitate are strongly hydrophobic and will render hedgehog insoluble in water and target it to membranes
6. This hydrophobicity of the signaling component would make it impossible for the molecule to leave the cell membrane and only would normally only allow signaling to directly neighboring cells
7. The action of the Dispatched, Scube glycoproteins and HSPGs in the ECM are important for long range signaling
8. They may help load hedgehog molecules on lipoprotein particles. Alternatively, cytonemes might be involved and these molecules might be needed for the function of these cytonemes

Question 4

How is the Wnt ligand formed and how does it diffuse?

Answer 4

1. Wnt genes are transcribed with an N-terminal signal sequence that targets them to the secretory pathway
2. The signal sequence is removed and then the protein undergoes an autoproteolytic cleavage catalysed by the C-terminal part of the protein
3. Wnt is modified by palmitoylation in cys77 and a palmitoleic acid modification of ser209
4. The hydrophobicity of the modifications make Wnt insoluble in water
5. Lipoproteins or cytonemes are involved in presenting the ligand to other cells
6. Wntless may also be involved in getting Wnt to the membrane for interaction with other cells

Question 5

How do Wnt and Hh express themselves in Drosophila?

Answer 5

• Hh acts in a reciprocal loop with Wnt
• During the segmentation patterning, Hh and Wnt maintain each others expression in an autoregulatory expression
• Therefore the loss of Wnt will also lead to the loss of Hh expression and give similar phenotypes

Question 6

What are cytonemes and how are they involved Wnt diffusion/signalling?

Answer 6

• Cytonemes are long cellular protrusions that the Wnt producing cells use to touch other cells and signal to them to change their behaviour
• Wnt ligand is exposed at the tip of the cytonemes, moved away from the cell to touch other cells
• This induces and activates the Wnt signalling pathway

Question 7

Explain the Wnt diffusion handover mechanism

Answer 7

1. A HSPG protein called Dlp has been identified
2. Dlp can bind to Wnt by binding it first with heparin sulphate molecules and then binding Wnt to its palmitate binding pocket
3. The pocket shields the hydrophobic palmitate and allows Wnt to bind
4. The molecule is coupled to the membrane via a GPI link, allowing them to be motile and able to diffuse over the cell membrane
5. Wnt may also be handed over from one Dlp to another neighbouring Dlp and therefore diffuse along the membrane

Question 8

What are they 2 ways in which Wnt diffuses?

Answer 8

1. Cytonemes

2. Handover mechanism

Question 9

Why do Wnt and Hh ligands struggle to diffuse in aqueous environments?

Answer 9

They are hydrophobic

Question 10

How is the Hh signal received?

Answer 10

1. The patched protein can bind hedgehog
2. Patched acts in a negative way in that it continuously inhibits a positively acting component called smoothened when the ligand is absent
3. Smoothened is inactive when Hh is not bound as it is inhibited by patched

Question 11

What are the two crucial TM proteins in the Hh pathway?

Answer 11

Patched (12 pass TM) and smoothened (7 pass TM)

Question 12

How do these Ptc and Smo proteins work at the membrane in Drosophila?

Answer 12

o Experiments in drosophila have shown that Ptc and Smo do not work “one-to-one”

o Instead, a single Ptc molecule can inhibit a large number of Smo molecules

o Ptc somehow regulates the subcellular localisation and stability of Smo

o In the absence of Hh, Ptc somehow keeps Smo from getting to the cell surface/membrane

o It is thought to regulate the trafficking of Smo to a compartment where Smo gets degraded

o When Ptc binds to Hh, they both get internalized and degraded, and Smo now gets trafficked to the cell surface and therefore can activate/start a signaling pathway

o Overall there are three change that occur to smoothened: relocation, accumulation and phosphorylation

Question 13

How do these Ptc and Smo proteins work at the membrane in mammals?

Answer 13

o Similar to Drosophila but it appears that the primary cilium plays a role in the signaling process

o In the absence of Hh signal, ptc1 is localised to the cilium of the cell and Smo is excluded from this territory, prevented from entering and activating the signaling pathway

o As a result of Hh binding to Ptc it is removed from the cilium allowing Smo to accumulate there and initiate signaling

Question 14

How has the importance of cilium for Hh signalling in mammals been discovered?

Answer 14

o Discovered in mice where mutations that disrupt cilia formation were found to impair Hh signaling.

Question 15

How does Ptc inhibit Smo?

Answer 15

o Recent structure analysis using CryoEM and and other work have revealed that Ptc works as a pump, depleting the inner leaflet of the lipid bilayer of cholesterol, keeping Smo in a silent state

o Hh it thought to inactivate Ptc allowing cholesterol accumulation in the inner membrane surface leading to Smo activation

Question 16

What happens inside the cell in Hh signalling?

Answer 16

1. Under the influence of the first complex that is bound to Smo, three other kinases can act on Ci
2. They form complex consisting of casein kinase I, protein kinase A and glycogen synthase kinase 3 beta
3. The transcription factor Ci (a transcriptional activator) is processed under the influence of these kinases to a shorter form via Slimb this involves ubiquitination
4. The short form acts as a transcriptional repressor and is called CiR
5. In this way Hh target genes are actively repressed. When there is no Hh around, there is active repression of Hh target genes
6. When there is ligand/high levels of Hh present, the interaction of Ci with the three kinases is somehow blocked, and a full length Ci is released that will actively promote transcription of target genes
7. This activation could involve phosphorylation
8. It is thought that phosphorylation of sufu by fused promotes formation of the active form of Ci.
9. SuFu and PKA are negative regulators of Hh pathway

Question 17

What are the two complexes that keep Ci responsible for keeping Hh signalling out of the nucleus?

Answer 17

Complex 1: contains costal2 that acts as a scaffold protein and fused

Compex 2: contains Ci and sufu

Question 18

How can Hh signalling act on its own pathway?

Answer 18

Negative feedback: induction of the Ptc gene, which is limits the level of activation

Positive feedback (vertebrates): autoregulatory target gene is gli1. Gli1 is always an activator of the Hh signal (it can’t be proteolyzed to a repressor) this is essentially a “feedforward” response.

The induction of patched is also seen in drosophila, but Ci is not induced.

Question 19

Where is the Hh pathway activated in Drosophila?

Answer 19

o Segment polarity and wing patterning

o In the drosophila wing imaginal disk hedgehog is expressed in the posterior compartment and diffuses to the anterior compartment where it induces expression of decapentaplegic

o This molecule in turn helps to pattern the A-P axis of the wing

Question 20

How is Shh involved in vertebrate development?

Answer 20

1. Shh protein is secreted by the notochord and ventral floor plate of the spinal cord an becomes distributed in a gradient across the ventral half of the spinal cord
2. Different transcription factor genes are activated by the distinct concentrations of Shh experienced by neural progenitors located at different positions within the spinal cord which then differentiate into distinct neuronal subtypes
3. Thus, Shh protein acts as a morphogen in the ventral spinal cord and patterns the neural tube

Question 21

How is Hh involved in limb development?

Answer 21

1. Involved in A-P patterning of the limb
2. Hh is active is in the posterior limb bud where it forms the ZPA
3. ZPA can confer posterior identity to the forming limb and is also involved in its outgrowth

Question 22

Give an example of a congenital disease caused by loss of Hh function and the chemical that causes it?

Answer 22

Holoprosencephaly: caused by loss of ventral brain structures and can lead to eyes fusing

Caused by cyclopamine which inhibits Smo and blocks the Hh pathway

Question 23

What happens if there is too gain of function/miss-regulation of Hh in the limbs?

Answer 23

Extra digits can form or can syndactyly

Question 24

Give examples of cancers that are the result of defects in Hedgehog signalling. Which genes in the Hedgehog pathway are the main causative genes and which type of mutations (activating or loss-of-function) are responsible?

Answer 24

Basal cell carcinoma, medullobastoma, or rhabdomyosarcoma

Patched (and Sufu) loss of function mutations (tumor suppressor genes)

Activating mutations in Smo (a proto-oncogene)

Question 25

What were the results of cancers treated with the Smo inhibitor GDC-0449?

Answer 25

Initially good results but tumour cells acquire resistance by a mutation in Smo

Question 26

What happens in the receiving cells when there is no Wnt ligand?

Answer 26

1. Beta catenin (transcription factor) in the Wnt pathway is continuously produced
2. Beta catenin is bound by destruction complex (composed of APC/Axin/CK1/GSK3 beta) and phosphorylated by Gsk3 beta and CK1a
3. Phosphorylated beta catenin is recognised by Slimb/BetaTrcp (part of the SCF ubiquitination complex) and ubiquitinated
4. Ubiquitinated beta catenin is destroyed by the proteasome, freeing up the destruction complex
5. TCF in the nucleus is bound by a transcriptional repressor called Groucho when there is no Wnt

Question 27

What happens in the receiving cells in the presence of Wnt ligand?

Answer 27

1. Wnt binds to its receptors frizzled and arrow/lrp5/6 and brings them together, this initiates the signal/downstream pathway
2. Dsh is recruited to the Fz receptor and phosphorylated
3. This somehow leads to recruitment of the destruction complex, possibly by dsh binding axin
4. Lead to phosphorylation of arrow/lrp5/6 by gsk3b creating binding sites for axin and further phosphorylation events
5. Importantly, it is thought that Slimb is somehow lost form this destruction complex.
6. As a result Beta catenin that is produced will still bind to the destruction complex and become phosphorylated
7. But instead of being ubiquitinated and degraded, it will remain bound to the destruction complex
8. Beta catenin will start accumulating and enter the nucleus to displace groucho to start activating transcription for Wnt target genes

Question 28

What is the role of Wnt signalling in Drosophila?

Answer 28

1. Wnt signaling in drosophila is involved in segmentation and wing formation
2. Wg is expressed at the D/V boundary of the wing disk and its function is required there for patterning and outgrowth of the future wing.

Question 29

How is Wnt involved in intestinal stem cells?

Answer 29

• The epithelium of both intestines is continuously renewed by stem cell division
• Stem cells divide slowly but produce rapidly dividing transit amplifying cells thereby creating a continuous flow of cells from the crypt upwards
• Wnts are expressed by the stroma below the intestinal crypts
• If you block Wnt signaling in the gut it will cause depletion of stem cells in the colon crypt, indicating that Wnt signaling is required for maintenance of the stem cell compartment
• Ectopic activation of Wnt can cause over proliferation probably due to overproduction of stem cells.

Question 30

Explain the role of Wnt in cancer

Answer 30

o Ectopic Wnt signaling can occur due to loss of the APC gene (protein in the destruction complex).

o Patients heterozygous for APC loss of functions mutations suffer from familial adenomatous polyposis.

o They suffer from a large amount of polyps in their intestine, these polyps are the result of sporadic loss of the single remaining wild type copy of the APC gene, resulting in activation of Wnt signaling in such a cell and increased proliferation is the result

o Over time these cells can accumulate further mutations and can become malignant tumors

o Therefore the APC gene is a classic example of a tumour suppressor gene.

o More than 90% of colon cancers have an activated Wnt pathway often biallelic loss of APC.

o Activation of the Wnt pathway has been noted in many other tumors including breast and ovarian cancer, melanomas, prostate and various other cancers

Question 31

Give examples of diseases associated with Wnt

Answer 31

1. Absence of limbs: Wnt3-tetra-amelia
2. Bone disease: LRP5-g.o.f mutations increases bone density
3. Bone disease: LRP5-l.o.f mutations decrease bone density
4. Tooth loss/agenesis: Axin2

Question 32

Give an example of non-canonical Wnt and Hh signalling

Answer 32

Wnt: planar polarity or convergent extension

Hh: Ca2+ Ampk signalling or reprogramming towards aerobic glycolysis in myocytes and adipocytes.

Question 33

Explain the non-conical Wnt signalling example: planar polarity or convergent extension

Answer 33

o In vertebrates the pathway involves: Wnt11, Wnt5, Frizzled, Vang11/2, Celsr, Prickle, Inversion, Dishevelled, Rho and Rac

o Wnt signaling components are involved in directing planar cell polarity and convergent extension movements

o An example are the hairs in a drospohila wing

o In zebrafish wnt11 and wnt5 mutants show clear defects in convergent extension movements

o Such defects will lead to very short and wide embryos, as the AP axis fails to elongate properly

Question 34

Explain the non-conical Hh signalling example: Ca2+ Ampk signalling or reprogramming towards aerobic glycolysis in myocytes and adipocytes

Answer 34

o One important effect may be via Smo on Calcium, and activating AMPK which has been observed in adipocytes and myocytes

o Activation of this has a profound effect on cellular metabolism stimulating aerobic glycolysis, where glucose is used as an energy source but without fully “burning” it in the mitochondria

o Instead it leads to the production of lactate, which leaves the cell and will acidify the extracellular medium

o This aerobic glycolysis is also a feature of many tumour cells and this is known as the Warburg effect

o It was found that certain inhibitors (antagonists) of the canonical Hh pathway were in fact activators (agonists) for this noncanonical AMPK signaling pathway