Lecture 5- Hh and Wnt signalling Flashcards
(34 cards)
How were Hh and Wnt first discovered as patterning mutants in Drosophila?
- 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
What type of gene is Hh?
Segmental polarity gene
How is the Hh ligand formed and how does it diffuse?
- Hh genes are transcribed with an N-terminal signal sequence that targets them to the secretory pathway
- The signal sequence is removed and then the protein undergoes an autoproteolytic cleavage catalysed by the C-terminal part of the protein
- Concurrently with this cleavage the C-terminus of the N-terminal part is coupled to a cholesterol molecule
- Another modification is made at the N-terminus where a palmitoyl group is added (palmitoylation)
- Both cholesterol and the palmitate are strongly hydrophobic and will render hedgehog insoluble in water and target it to membranes
- 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
- The action of the Dispatched, Scube glycoproteins and HSPGs in the ECM are important for long range signaling
- 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
How is the Wnt ligand formed and how does it diffuse?
- Wnt genes are transcribed with an N-terminal signal sequence that targets them to the secretory pathway
- The signal sequence is removed and then the protein undergoes an autoproteolytic cleavage catalysed by the C-terminal part of the protein
- Wnt is modified by palmitoylation in cys77 and a palmitoleic acid modification of ser209
- The hydrophobicity of the modifications make Wnt insoluble in water
- Lipoproteins or cytonemes are involved in presenting the ligand to other cells
- Wntless may also be involved in getting Wnt to the membrane for interaction with other cells
How do Wnt and Hh express themselves in Drosophila?
- 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
What are cytonemes and how are they involved Wnt diffusion/signalling?
- 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
Explain the Wnt diffusion handover mechanism
- A HSPG protein called Dlp has been identified
- Dlp can bind to Wnt by binding it first with heparin sulphate molecules and then binding Wnt to its palmitate binding pocket
- The pocket shields the hydrophobic palmitate and allows Wnt to bind
- The molecule is coupled to the membrane via a GPI link, allowing them to be motile and able to diffuse over the cell membrane
- Wnt may also be handed over from one Dlp to another neighbouring Dlp and therefore diffuse along the membrane
What are they 2 ways in which Wnt diffuses?
- Cytonemes
2. Handover mechanism
Why do Wnt and Hh ligands struggle to diffuse in aqueous environments?
They are hydrophobic
How is the Hh signal received?
- The patched protein can bind hedgehog
- Patched acts in a negative way in that it continuously inhibits a positively acting component called smoothened when the ligand is absent
- Smoothened is inactive when Hh is not bound as it is inhibited by patched
What are the two crucial TM proteins in the Hh pathway?
Patched (12 pass TM) and smoothened (7 pass TM)
How do these Ptc and Smo proteins work at the membrane in Drosophila?
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
How do these Ptc and Smo proteins work at the membrane in mammals?
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
How has the importance of cilium for Hh signalling in mammals been discovered?
o Discovered in mice where mutations that disrupt cilia formation were found to impair Hh signaling.
How does Ptc inhibit Smo?
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
What happens inside the cell in Hh signalling?
- Under the influence of the first complex that is bound to Smo, three other kinases can act on Ci
- They form complex consisting of casein kinase I, protein kinase A and glycogen synthase kinase 3 beta
- The transcription factor Ci (a transcriptional activator) is processed under the influence of these kinases to a shorter form via Slimb this involves ubiquitination
- The short form acts as a transcriptional repressor and is called CiR
- In this way Hh target genes are actively repressed. When there is no Hh around, there is active repression of Hh target genes
- 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
- This activation could involve phosphorylation
- It is thought that phosphorylation of sufu by fused promotes formation of the active form of Ci.
- SuFu and PKA are negative regulators of Hh pathway
What are the two complexes that keep Ci responsible for keeping Hh signalling out of the nucleus?
Complex 1: contains costal2 that acts as a scaffold protein and fused
Compex 2: contains Ci and sufu
How can Hh signalling act on its own pathway?
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.
Where is the Hh pathway activated in Drosophila?
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
How is Shh involved in vertebrate development?
- 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
- 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
- Thus, Shh protein acts as a morphogen in the ventral spinal cord and patterns the neural tube
How is Hh involved in limb development?
- Involved in A-P patterning of the limb
- Hh is active is in the posterior limb bud where it forms the ZPA
- ZPA can confer posterior identity to the forming limb and is also involved in its outgrowth
Give an example of a congenital disease caused by loss of Hh function and the chemical that causes it?
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
What happens if there is too gain of function/miss-regulation of Hh in the limbs?
Extra digits can form or can syndactyly
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?
Basal cell carcinoma, medullobastoma, or rhabdomyosarcoma
Patched (and Sufu) loss of function mutations (tumor suppressor genes)
Activating mutations in Smo (a proto-oncogene)
