Hh and Wnt signalling Flashcards
What are the 2 major signal transduction pathways?
What 4 things are they involved in?
Hh and Wnt
Involved in:
- Patterning of many tissues/development of many organs
- Regulation of stem cell fate/cell division
- Maintenance and initiation of cancer
- SEGMENT POLARITY
Where were Hh and Wg discovered?
In drosophila
How was Hh discovered?
What does this show about Hh?
In Hh mutation:
- Lawn of denticles due to loss of naked cuticle formation
Shows that the normal function of the Hh gene is for the formation of the naked cuticle (between the dentical belts)
What is wg?
What happens if there is a mutation in wg? Why?
Wingless
- Mutant phenotype looks very similar to Hh mutant phenotype
- As Wg and Hh form a mutual feedback loop with each other (both needed for the expression of each other)
- So, when lose Wg expression, lose Hh expression
Where was int1 discovered?
What did this gene do? What did th
In mice
How was Wnt names?
- Int1 was discovered independently to wg in mice
- Has the same function as wg
- Amalgamation of the 2 names to form wnt
Do C.elegans have hh?
No
Do vertebrates have hh?
Yes, have MANY orthologues:
- Shh
- Desert hh
- Indian hh
How many wnt copies are there of the wg gene?
> 18
What domains does the Hh protein have?
- Protein has signal sequence which makes sure the protein goes into the SECRETORY PATHWAY
- Proteolytic domain
What is the function in the proteolytic domain of the Hh protein?
Auto cleaves itself (autoproteolysis)
When is the signal sequence of the protein cleaved off?
Once the protein is in the secretory pathway
How is the Hh ligand created?
- Signal sequence drives the protein into the secretory pathway cleaved off
- Proteolytic domain is cleaved off
- At the same time - cholesterol modification to the C-terminus
- Palmitoylation of the N-terminus
Why is Hh protein very insoluble in water?
It is stuck to the membrane due to:
- Cholesterol modification at the C-terminus
- Palmitolaytion of the N-terminus
Which proteins are required to allow the Hh protein to diffuse and allow long-range signalling?
What do these proteins do?
1) Dispatched
2) Scube proteins
3) HSPG
These proteins help to load the Hh gene into lipoprotein particles that diffuse away from the signalling cell to the receiving cell
Where are Scube proteins present?
In VERTEBRATES only
Instead of lipoproteins, what else can be used to help the signalling molecules move between cells?
Cytonemes
How is the Wnt ligand created?
- Signal sequence drives the protein into the secretory pathway
- 2 modifications then on the protein:
- Palymitolaytion
- Palmitoleic acid
What causes Wnt to be very insoluble in water?
Where do these modifications direct the protein to?
Fatty acid modifications:
- Palymitolaytion
- Palmitoleic acid
These modifications direct the protein to the cell membrane
What proteins help Wnt to diffuse away from the membrane?
1) Wntless
2) HSPG
3) Lipoproteins or cytonemes
What are cytonemes or lipoproteins required for?
Long term signalling
What are cytonemes?
Thin protrusions from the cell
What occurs in cytonemes?
- Signalling molecule accumulates in the tip of the cytonemes
- Cytonemes move away from the cell and touch a receiving cell - to induce the signal in that cell
What occurs at the cell membrane when there is no Hh signalling?
Patched inhibits smoothened:
- Ptc is at the plasma membrane and prevents smoothened from reaching the plasma membrane
Describe Hh signalling at the cell membrane
Hh binds to PATCHED:
- Ptc is internalised and degraded
- Relocation, accumulation and phosphoylation of smoothened at the plasma membrane
- Smoothened can now initiate the signalling complex
What receptor is the positive signalling component of the hh pathway?
Smoothened
How many transmembrane domains does Patched have?
12
How does patched inhibit smoothened?
- One molecules of patched inhibits MANY molecules of smoothened
- Ptc regulates the subcellular distribution and stability of smo in an enzymatic way
In vertebrates, what are the focal points for Hh sensing?
How?
Cilia:
- Accumulate ptc if theres is no shh around
Whn shh around:
- Ptc is redistributed AWAY from the cilium
- Smo can now enter the cilium
- Shh signalling can occur
How were cilia discovered?
In mice:
Mutations in cilia formation - looked similar to mice with a defect in shh signalling
What happens intracellularly when hh is NOT bound to ptc?
TF Ci is kept OUT of the nucleus
What is Ci called in vertebrates?
Gli
How is Ci kept out of the nucleus when there is no hh signalling?
How?
Cos2 and fused:
- Form a complex
- Complex is bound by 3 other kinases which phosphoylate Ci
- Results in the partial PROTEOLYSIS of the TF - forming a shorter form of Ci
- Shorter form of Ci is a transcriptional REPRESSOR
What type of protein is Cos2?
A scaffold protein
What type of protein is fused?
A kinase
What is the phosphorylation of Ci regulated by?
A ubiquitin ligase called SLIMB
What happens intracellularly when Hh is bound?
- Cos2 and fused complex can no longer interact with the 3 kinases
- Ci is no longer broken down
- Ci is phosphorylated at a DIFFERENT position and becomes a transcriptional ACTIVATOR
How many patched genes are there in vertebrates?
Which one is the most important?
2
Patched1 is the most important
How does Hh signalling act on its own pathway?
Negative feedback
Positive feedback
How does the Hh signalling pathway negatively regulate itself?
- One of the Hh targets in patched/patched1
- SO, more Hh = more ptc
- BUT, ptc is a NEGATIVE regulator, so more inhibition of the hh signal
- Limits the level of activation
How does the Hh signalling pathway positively regulate itself?
- Gli1 (in VERTEBRATES) cannot be proteolysed into a repressor
- So is ALWAYS a POSITIVE regulator of transcription
- Gli1 gives hh responses but also helps to FEEDBACK onto ptc1
- Ptc1 will feedback NEGITIVELY on the signal
Where is Hh expressed?
1) Imaginal disc of the drosophila wing
2) Vertebrate shh - neural development
3) AP patterning of the limb bud (in VERTEBRATES)
How is shh important in neural development?
Patterning of the ventral neural tube:
- Released from the notochord and floor plate
- Neural tube cells differentiate into different cell types depending on HOW LONG and HOW MUCH shh they see
How is shh involved in patterning the limb bud in vertebrates?
- Shh is released from the ZPA
- Shh acts in a negative feedback loop with FGF
- Regulates outgrowth of the limbs
What is an inhibitor of the shh pathway?
Cyclopamine
What does inhibition of the shh pathway cause?
1) Holoprosencephaly (a single eye)
2) Polydactyly (extra digits)
3) Syndactayly (webbed digits)
How does inhibition of the shh pathway cause holoprocencephaly?
Shh is important to form the ventral part of the brain that separates the 2 eyes during development
What happens when Hh is inappropriately ACTIVATED?
Cancer formation:
- Basal cell carcinoma (skin cancer)
- Medulloblastoma (brain tumour)
- Rhabomyosarcoma (muscle cancer)
How is basal cell carcinoma caused?
- Inactivation of ptc1 of sufu (negative regulators of the hh pathway)
- Resulting in ACTIVATION of the shh pathway
OR
By the activation of a mutation of smo
What is sufu?
Supressor of fused
fused normally keeps Ci out of the nucleus
How can smo cause BCC?
Smo is a proto-oncogene
What is Gorlin syndrome?
What does it cause and how?
A genetic form of basal cell carcinoma
- Patients are heterozygous for a patched1 mutation
- No ‘back-up’ copy if something goes wrong with the other patched1 receptor
- Predisposition to BCC - these patients have high numbers of BCCs
What is GC-0049?
Is it effective?
A smo-inhibitor - used to treat cancers caused by over activation of shh:
- Initially good results
- BUT, cells soon acquire resistance by a mutation in smo
What are the receptors for wnt?
In vertebrates: frizzled and arrow
What is the receptor arrow called in invertebrates?
LRP5 and LRP6
What is the transcription factor in the Wnt pathway?
When is it transcribed?
Beta-catenin
Transcribed ALL of the time
What happens intracellularly when there is no Wnt present?
Beta-catenin is bound by a huge complex of:
- APC
- Axin
- 2 kinases
What kind of protein is axin?
A scaffold protein
What do the 2 kinases in the complex that binds beta-catenin do?
What does this result in?
Phosphorylate beta-catenin
Phosphorylated beta-catenin is then recognised and tagged for degradation by the ubiquitination protein SLIMB
How is the Wnt signalling pathway activated?
- Wnt binds to BOTH receptors
- Brings the 2 receptors closer together to activate the signalling wnt pathway
What happens when the wnt signalling pathway is activated?
- Dishevelled recruited to frizzled
- Dsh is phosphorylated at many sites
- Attracts the huge complex (which normally binds beta-catenin) to the membrane
- This complex binds to ARROW at the membrane
Binding to the membrane causes the loss of slimb (ubiquitin ligase):
- Beta-catenin is still phosphorylated BUT cannot be broken down
- More Beta-catenin is produced and if enough present - will bind to TCF (DNA-binding factor) in the nucleus
- This displaces GROUCHO which is normally bound to TCF and represses the target genes when there is no wnt present
What are the roles of wnt signalling in drosophilla?
- Segmentation (segment polarity gene)
- Expressed at the DV axis of the wing - required for wing patterning and outgrowth
What is the role of wnt in C.elegans?
Regulation of neuronal fate and neuronal migration
What are the roles of wnt in vertebrates?
1) Intestines
- Renewal process of the vili from stem cells in the cypt (blocked if block wnt signalling)
2) Cancer
What does ectopic wnt signalling lead to?
How?
Cancer
- Loss of APC in the complex
- Can’t down regulate wnt pathway
What is familial adenomatous polyposis?
- Autosomal dominant disease
- Polyps in the colon and rectum
- Heterozygous for this condition - occasional loss of remaining APC
- Causing activation of the wnt pathway and excess stem cells
What are 3 disease caused by disruptions in wnt sigalling?
1) Tertra-amelia (absense of limbs)
2) Bone diseases
3) Axin2
What causes TA?
Loss-of-function mutation in wnt3
What causes bone diseases?
Either:
- LRP5 (GOF) - increase bone DENSITY
- LRP5 (LOF) - Decrease in bone DENSITY
What does Axin2 cause?
Severe tooth ageneis (missing teeth)
What does non-canonical mean?
Not the usual way
Where is non-canonical hh signalling present?
Describe this signalling pathway
In the myocytes/adipocytes:
- Shh activates smo-Ca2+
- Leads to an increase in Ca2+
- Activation of Ampk
- This drives metabolic reprogramming towards AEROBIC GLYCOLYSIS and an increase in lactate
What is ampk?
AMP dependant kinase
What normally happens when sugars are burnt?
How is this different to glycolysis?
Burnt to completion:
- End product being CO2 and H2O
Glycolysis:
- Glucose forms lactate and small amounts of ATP
How was the non-canonical signalling of shh discovered?
- Certain inhibitors of hh signalling (such as cyclopamine and GDC0449) activate the NON-CANONICAL AMP signalling
When the non-canonical shh signalling pathway is activated, what do the patients suffer from?
- Muscle cramps
- Weight loss
What is the non-canonical wnt signalling pathway?
What does this pathway do?
Planar cell polarity/convergent extension pathway Uses: - Wnt 11 - Wnt 5 - Frizzled, dishevelled - Rho, Rac
This pathway polarises cells within a sheet of cells
What does the planar cell polarity/convergent extension pathway do in drosophila?
What occurs in a mutant?
Polarises the hairs on the drosophila wings
In a mutant - the hairs are in all different directions
What does the planar cell polarity/convergent extension pathway do in zebrafish?
What occurs in a mutant?
Convergent extension and axis elongation
In a mutant - no extension of the axis and shorter in the AP direction
