L5 - Hh and Wnt Signalling Flashcards
What are Hh and Wnt signalling involved in?
Patterning tissues
Development of organs
Regulation of stem cell fate/cell division
Maintenance and initiation of cancer
How was Hh discovered?
Discovered by Nüsslein-Volhard and Wieschaus - 1980
In Drosophila
Segment polarity gene
How was Wnt discovered?
Wg + int = Wnt
In mice
How do Hh and Wg interact in Drosophila embryos?
Hh acts in a reciprocal loop with Wg
During segmentation in flies – Hh and Wg maintain each other’s expression in an autoregulatory loop
- Genes are dependent on one another
- Loss of Wg = loss of Hh expression
What are the different types of Hh in multicellular animals?
Sonic Hedgehog, Desert Hedgehog, Indian hedgehog
No Hh in C.elegans
What are the different types of Wg in multicellular animals?
18 Wnts
Hh signalling method
- Hh translated with an N-terminal signal sequence - targets them to secretory pathway
- Signal sequence is removed and the protein undergoes autoproteolytic cleavage
- Catalysed by the C-terminal of the protein - C-terminus of N-terminal coupled to cholesterol
- N-terminus of N-terminal coupled to Palmitoyl group
- Both cholesterol and palmitate are hydrophobic
- Render Hh insoluble in water and target it to membranes
Palmitoyl and cholesterol are both?
Hydrophobic
Hh signalling - short range
Hydrophobicity of Hh would make it impossible for it to leave cell membrane
Only allow signalling to neighbouring cells
Hh signalling - long range
Action of Dispatched protein and Scube glycoproteins important
- Help load Hh molecules on lipoprotein particles
Cytonemes
In the ECM - heparan sulfate proteoglycans
Wt signalling method
- Produced with a signaling sequence that is cleaved off when it enters the secretory pathway
- Modified by palmitoylation – palmitoleic acid modification of ser209
- Palmitate is hydrophobic
- Hydrophobicity makes Wnt insoluble in water
Wt signalling - long range
Lipoprotein particles or cytonemes - present ligand to other cells
Wntless 7 pass transmembrane protein – helps Wnt get to membrane
In the ECM - heparan sulfate proteoglycans
What are cytonemes?
Long cellular protrusions
Wnt producing cell use them to touch other cells and signal to them to change their behavior
Hh signalling model
Signal transduction model
Proposed by Hooper and Scott - 2005
Hh signalling - Ptc gene
12 pass transmembrane protein
Can bind hedgehog
Acts in a negative way - continuously inhibits a positively acting component - Smo - when the ligand is absent
Hh signalling - Smo gene
7 pass transmembrane protein
Acts in a positive way
What sort of complex does Ptc and Smo form?
Not present in a stoichiometric complex
What was discovered about Ptc in Drosophila experiments?
Single Ptc molecule can inhibit a large number of Smo molecules
What does Ptc regulate?
Subcellular distribution and stability of Smo
Trafficking of Smo to a compartment where Smo gets degraded
What does Ptc do if no Hh is present?
Ptc stops Smo reaching cell surface
What does Ptc do if Hh is present?
Ptc binds to HH and they both get internalized and degraded
Smo gets trafficked to cell surface
What 3 changes does Smo go through?
Relocation
Accumulation
Phosphorylation
If no Hh in cilia?
Ptc1 is localised to the cilium of the cell and smo is excluded from this territory
Hh binding to Ptc is removed from cilium
Allows smo to accumulate and initiate signaling
What is Ci?
A transcription factor
If no Hh which two complexes keep Ci out of the nucleus?
Costal2 (scaffold protein) and fused serine threonine kinase
Ci and suppressor of fused gene (gene without clear domains)
What conditions have to be met for 3 other kinases to act on Ci?
The complex costal2 and fused serine threonine kinase has to be bound to Smo
What are the 3 kinases that act on Ci?
Casein kinase I
Protein kinase A
Glycogen synthase kinase 3 beta
The long form transcription factor Ci is?
A transcriptional activator
It is processed under the influence of the 3 kinases to a shorter form
This happens via Slimb and involves ubiquitination
The short form transcription factor CiR is?
A transcriptional repressor
Hh target genes that are actively repressed
What happens to the interaction of Ci with the 3 kinases when a ligand is present?
When there is ligand present, the interaction of Ci with the three kinases is blocked
A full length Ci is released – actively promotes transcription of target genes
How does Hh signalling act on its own pathway in a negative way?
Induction of Ptc
Reduced level of Hh activation
How does Hh signalling act on its own pathway in a positive way?
Induction of Gli1
Gli cannot be proteolyzed into a repressor
What is the role of Hh in Drosophila wing patterning?
In drosophila wing imaginal disk Hh is expressed in posterior compartment
It diffuses into anterior compartment where it induces expression of decapentaplegic
This helps to pattern the wing
What is the role of Hh in vertebrate neural development?
Neural tube cells differentiate into different neurons depending on how long and how much Shh
Sources of Hh protein
- Notochord
- Floor plate
What is the role of Hh is AP patterning of the limb bud?
Hh is active is in the posterior limb bud where it forms the zone of polarizing activity
A region that can confer posterior identity to and outgrowth of the forming limb
Loss of Hh signalling diseases
Holoprosencephaly – loss of ventral brain structures so eyes fuse
Cyclopamine - can inhibit Smo and block the Hh signal
Polydactyly – extra digits (misregulation)
Syndactyly (misregulation)
Gain of Hh signalling diseases
Basal cell carcinoma
Medulloblastoma
Rhabdomyosarcoma
Inactivation of Ptc1 or Sufu - tumor suppressor genes
Activating mutations of Smo - Smo is a protooncogene
Genetic diseases related to Hh
Gorlin syndrome
Large numbers of BCCs
Due to heterozygosity for Ptc1 (defective patch)
Cancer treatment by Smo inhibitors
GDC-0449 - treated patients
Initially good result
But tumor cells acquire resistance by a mutation in Smo
Wnt signalling method
- Beta catenin is continually produced and is bound by destruction complex
- Beta catenin is phosphorylated by Gsk3 beta and CK1a
- Phosphorylated beta catenin is recognised by Slimb/BetaTrcp and ubiquitinated
- Ubiquitinated beta catenin is destroyed by the proteasome - freeing destruction complex
- Wnt binds to its receptors frizzled and arrow/lrp5/6 and brings them together - initiates signal
- Dishevelled is recruited to the Fz receptor and phosphorylated
- Leads to recruitment of the destruction complex
- Dsh binding axin, phosphorylation of arrow/lrp5/6 by gsk3b creating binding sites for axin
- Slimb is lost from complex - As a result Beta catenin that is produced will still bind the complex and become phosphorylated
- But is will not be ubiquitinated - remain bound to destruction complex
- The system clogs and newly produced beta catenin will start accumulating
- Beta catenin enters nucleus to displace groucho to start activating transcription
What is the role of Wnt signalling in Drosophila?
Expressed at D/V boundary of wing
Required for patterning and outgrowth
What is the role of Wnt signalling in C.elegans?
Regulation of neuronal fate leading to migration of two neuroblasts
QRd and QLd
What does the small intestine consist of?
Crypts and villi
What cells occupy the villi?
Differentiated enterocytes, enteroendocrine cells and goblet cells
What cells occupy bottom of the crypt?
Paneth cell
What cells occupy higher positions?
Stem cell
Where is Wnt expressed in crypts and villi?
Wnts are expressed by the stroma below the intestinal crypts
What happens if you block Wnt signalling in the intestine?
If you block Wnt signaling it will cause loss of stem cells in the colon crypt
Wnt signalling is required for maintenance of the stem cell compartment
What can cause ectopic Wnt signalling?
Ectopic Wnt signalling can occur by loss of APC
What can ectopic Wnt signalling cause?
Familial adenomatous polyposis Autosomal dominant Many polyps in colon and rectum Occasional loss of remaining APC - Results in activation of Wnt
What other types of cancer are caused by Wnt?
Sporadic colon cancer Breast cancer Ovarian and uterine cancers Melanomas Prostate cancer
What disease can Wnt cause?
Wnt3-tetra-amelia - absence of limbs Bone diseases - LRP5 – gain of function point mutant - Increased bone density - Other LRP5 – loss of function mutations - Decreased bone density Axin2 - tooth agenesis (missing teeth)
When Wnt and Hh work via alternative pathways what is this called?
Non-canonical signalling
What does Shh activate in myocytes/adipocytes?
Activates Smo-Ca Ampk signalling
What does Smo-Ca Ampk signalling stimulate?
Stimulates metabolic reprogramming towards aerobic glycolysis
- Glucose –> Lactate + 2 ATP
- Acidic
What do cyclopamine and GDC0449 do?
Certain inhibitors of canonical Hh signaling activate non-canonical AMPK signaling
In what other way was the effect of cyclopamine and GDC0449 discovered?
Patients taking hedgehog inhibitors were suffering from serious side effects - weight loss and muscle cramps
What impacts the planar cell polarity/convergent extension pathway in vertebrates?
Fz, Dsh, Rho, Rac
What impacts the planar cell polarity/convergent extension pathway in zebrafish?
Wnt11 and Wnt5 mutants show defects
- AP axis fails to elongate properly
What impacts the planar cell polarity/convergent extension pathway in Drosophila?
Hair on wings
