Lecture 5: HH and Wnt signalling Flashcards
How were Hedgehog (Hh) and Wingless (Wnt) first defined?
These were first defined as mutations that showed defects in segmentation in drosophila.
LOF for HH = segment-polarity phenotype i.e., naked cuticles lost => embryo full of denticles => appears spikey.
HH acts in a reciprocal loop with Wnt by maintaining each-others expression in an autoregulatory loop in segment patterning of Drosophila => loss of Wnt = loss of HH expression => LOF studies in both give similar phenotypes.
Vertebrates have SONIC HH, DESERT HH and INDIAN HH.
A Wg gene was also discovered in mice which is activated by integration of a mammary tumor virus: Int1. Wingless + Int1 = Wnt. Vertebrates have over 18 Wg orthologues called Wnts.
Explain how the Hh ligand is formed. Draw a diagram.
- Hh genes transcribed and translated as a long protein with an N-terminal signal sequence peptide which is removed as it enters the vesicular secretory pathway
- The protein in the Golgi undergoes several modification
- Autoproteolytic cleavage of the C-terminus making it active and at the same time:
- New exposed C terminus modified with a cholesterol
- N terminus modified by palmitoylation
- Cholesterol and palmitate are both modifications make the HH ligand very hydrophobic => targeted to membranes in secretory vesicles.
- The hydrophobic molecules need assistance to signal over a long range through the action of Dispatched (12TM protein) and Scube (vertebrates) glycoproteins. They may work by loading the Hh protein onto lipoproteins but the exact mechanism isn’t yet known.
- Suggested that there is cytonemes, Heparan sulfate proteoglycans (HSPGs) and exovesicles in the EC matrix and may assist long range diffusion, as shown in the Wnt pathway. The proposed mechanism of HSPG is through cell-cell handover.
Explain the events that occur on reception of the Hh ligand. Draw a diagram.
Two TM proteins:
Patched (Ptc) = 12TM protein, binds Hh. Similar to dispatched. Constituently inhibits the positively acting Smoothened when Hh is absent by regulating the subcellular distribution and stability of Smo, preventing it from getting to the cell surface. It is thought to traffic Smo to a subcellular compartment for degredation.
Smoothened (Smo) = 7TM protein. Inactive when HH not bound because inhibited by Patched.
- No Hh = Ptc in membrane and Smo in cytosol
- Hh binds to Ptc
- Ptc and Hh degraded, allowing Smo to enter membrane, accumulates and phosphorylates meaning Hh signalling pathway can start
These processes are so tightly coupled that it is currently unknown what causes them.
Mammalian Model
The primary cilium plays a role in the signalling process.
- No Hh = Ptc1 localised to the cilium of the cell where Smo is excluded from
- Hh binds to Ptc1
- Ptc1 and Hh degraded, allowing Smo to accumulate there, initiating signalling
This has been shown in mice with cilium disrupted which results in impaired Hh signalling.
How does Ptc Inhibit Smo?
Ptc works as a pump, depleting the inner leaflet of the lipid bilayer, keeping So in a silent state. Ptc is a transport protein which transports cholesterol from the inner leaflet of the membrane to the outer where it accumulates. When Hh binds Ptc, the motor is blocked, so cholesterol accumulates in the inner membrane surface leading to Smo activation and downstream signalling.
Explain the intracellular Hh signalling cascade. Draw a diagram.
CiAct = TF (activator) responsible for effects of Hh
CiR = TF (repressor) which actively repressed Hh genes in the absence of the ligand
In absence of Hh, three complexes keep Ci out of the nucleus:
- One contains Cos2 (scaffold protein) and Fused (kinase).
- One contains Ci and SuFu (suppressor of Fused).
- One contains Casein Kinase I (CKI), Protein Kinase A (PKA) and Glycogen Synthase Kinase 3 Beta (GSK3).
No Hh = Smo degraded and therefore the IC complex with Fused is active. PKA is a catalytic component and phosphorylates Ci/Gli meaning it is degraded by slimb to a shorter version called CiR through ubiquitination.
In the presence of Hh
- Kinase complex falls apart => Cos2-Fused complex binds to Smo
- Fused and FuSu release Ci (probably by phosphorylation but unsure)
Hh binds to Ptc, removing it from the membrane. Smo is trafficked to the membrane, relocated and phosphorylated. Smo disrupts the complex meaning PKA can no longer phosphorylate Ci. Therefore it does not get degraded to CiR and the normal length Ci is transported to the nucleus to activate target genes.
How is the Hh signalling pathway regulated?
Negative Feedback
Activated Hh target genes include Ptc/Ptc1 => limit level of Smo activation
Positive Feedback
In vertebrates - Gli1 is one of the Ci analogues = autoregulatory target gene meaning it can never be in repressor form. So even in the absence of Shh it can initiate Hh responses.
Give examples of where the Hh pathway is active.
Drosophila:
- Segmentation
- Wing patterning – expressed in posterior compartment of imaginal disc that gives rise to the wing. Diffuses to the anterior compartment to induce decapentaplegic which patterns the A-P axis of the future wing
Vertebrate:
- SHH expressed in the notochord and floor plate which pattern the neural tube. Different types of neurons expressed depending on how much SHH received
- Limb patterning - SHH forms the zone of polarizing activity. Expressed in the posterior part of the limb and diffuses to the anterior to pattern the A-P axis. Also in a loop with FGF to promote limb outgrowth.
- Very few tissues where SHH doesn’t play a role.
Explain how the Wnt ligand is formed. Draw a diagram.
- N-terminal signaling sequence targets the protein for the secretory pathway.
- Palmitoylation on cystine 77
- Palmitoleic acid modification of ser209
- The hydrophobicity of the modifications makes Wnt insoluble in water => targeted to membrane
- Lipoprotein particles or cytonemes are involved in assisting long term signalling to other cells
- Wntless (7TM protein) may be involved in getting Wnt to the membrane and allowing it reach other cells
- A HSPG called Dlp: ‘handover mechanism of Wnt diffusion’. Wnt binds Dlp at the hydrophobic pocket – can laterally diffuse over the cell membrane via handing over between Dlp proteins
Cytonemes = long cellular protrusions which the Wnt-producing cell uses to touch other cells and signal to them to change their behaviour. Wnt ligand exposed at the tip of the cytonemes.
Explain the downstream events in Wnt signalling. Draw a diagram.
The TF in Wnt signalling is Beta-catenin which is continuously produced.
Receptors = Frizzled and Arrow
When there is no Wnt ligand
- Bcatenin bound by a destruction complex of = Axin (scaffold) + APC + kinases Ck1 and GSK3 + Slimb
- Phosphorylation of Bcatenin by GSK3beta and CK1a so it can be recognised by Slimb, a ubiquitination complex.
- Slimb ubiquitinates Bcatenin => Bcatenin degraded
- In the nucleus, near Wnt target genes, TCF is constituently bound by Wnt target promotors but also by Groucho, a repressor
When there is Wnt ligand
- Wnt binds frizzled and Arrow, bringing the receptors together, forming a complex
- Complex formation drives downstream events (probably)
- Disheveled (Dsh) recruited and becomes phosphorylated leading to further phosphorylation of the receptors that bind further kinases via Axin.
- Phosphorylation of Arrow by GSK3beta creates a binding site for Axin leading to further phosphorylation events
- Slimb lost from destruction complex.
- Bcatenin still binds destruction complex and becomes phosphorylated
- Because there is no Slimb to ubiquitinate it for degradation, it clogs the complex system
- Newly produced, now free, Bcatenin accumulates in the cytoplasm
Bcatenin enters nucleus, displaces Groucho and acts as transcriptional activator for Wnt target genes
Give examples of Wnt signalling.
Drosophila:
- Segmentation
- Wing patterning (D-V boundary required for patterning and outgrowth) i.e., wingless
C. elegans:
- Regulation of neuronal fate
Vertebrates:
Most prominent role of Wnt signalling in vertebrates = epitheliam replaced and renewed all the time. Stem cells located in small intestine in a crypt. They divide slowly and generate a set of highly proliferative progenitors which replace cells in the gut epithelium. Similar process in colon. Wnts are expressed by the stroma below the intestinal crypts. If you block Wnt signaling it the gut it will cause depletion of stem cells in the colon crypt, indicating that Wnt signalling is required for maintenance of the stem cell compartment. Ectopic activation of Wnt can cause over proliferation probably due to overproduction of stem cells
Explain the phenotype seen in holoprosencephaly.
Phenotype = cyclopic embryo - eyes fuse.
Hh signalling has a role in patterning the forebrain.
Caused through LOF mutation - deregulation of Hh signalling.
Explain the phenotype seen in syndactyly.
Extra digits.
Hh has a role in limp patterning.
LOF mutation - mis regulation of Hh
Explain the phenotype for basal cell carcinoma.
Skin cancer (most common form).
LOF in Ptc1 or Sufu (tumour suppressor genes) means activation of the pathway.
GOF in Smo (proto-oncogene) make it insensitive to negative regulation.
Target for new therapeutics – have worked in past but tumour cells acquire resistance.
Explain the phenotype seen in Gorlin syndrome.
Large numbers of BCCs. HH (genetic disease). Heterozygous for LOF allele of Ptc1 mutation => one functional copy => chance of sun damage more likely.
Explain the phenotype seen in familial adenomatous polyposis (cancer).
Large numbers of polyPs in their intestine.
More than 90% of colon cancers have an activated Wnt pathway.
Heterozygous for loss of APC gene leads to ectopic Wnt signalling. PolyPs are the result of sporadic loss of the WT copy of the APC gene, resulting in activation of Wnt signalling, increasing proliferation. Overtime these cells accumulate further mutations and become malignant tumours. APC is a tumour suppressor gene.
Explain activating and deactivating bone diseases.
Wnt signalling.
Arrow GOF mutant = more Wnt signalling => Increased bone density
Arrow LOF mutant = less Wnt signalling => Decreased bond density
