Lecture 19 - Hedgehog and Wnt signalling Flashcards
Describe Drosophilia larva struture.
Drosophila larva
* First systematic screen for patterning genes in Drosophila
* Denticles are present across the anterior half of each segment
* Posterior half is smooth
* Nüsslein-Volhard and Wieschaus, identified a group of mutants that affected the patterning within the segments but left the number of segments unaltered
* Segment polarity mutants
* In one mutant the posterior half of each segment failed to develop, resulting in a larva that were entirely covered by denticles
Cuticle phenotypes
* Wingless (Wnt) also identified - similar phenotype
After Hh mutants were discovered they identified the gene and then the protein which was found to be a signaling molecule
Name the different types of hedgehog proteins. Describe the sructure of hedgehog protiens.
Single Hedgehog (Hh) in Drosophila
3 Hhs in vertebrates:
* Sonic (Shh)
* Indian (Ihh)
* Desert (Dhh)
Secreted signalling proteins
Modified by cleavage and addition of cholesterol
Hedgehog processing is autocatalytic
* Inactive precursor (like TGF-beta): Signal peptide 0-82 – Secretory pathway
* 20kda N-terminus is signaling molecule
* 25kda C-terminus catalyzes the cleavage and modification of the N-terminal domain with cholesterol
* Cholesterol is added to the c-terminus of the N-terminal region
Describe the expression of Wg and hh in drosophilia.
hh tethered to the membrane to restrict activity to immediately adjacent cells. In Drosophila, embryo segments are marked out by alternating expression of Wingless and Hedgehog. Wingless’s role is to switch on expression of hedgehog in the adjacent stripe
The boundary between the stripes is called the para-segment boundary. Here tethered hedgehog induces diffusible wingless in the adjacent cell
* Wg is expressed in cells adjacent to Hh expressing cells at the parasegment boundary
* Job of Wingless is to induce Hh expression in adjacent cells – mutual induction process (e.g. mammalian kidney)
* Ensures adjacent stripes at the crucial stage where the segment boundaries must be demarcated
* Accommodates movement of cells in and out of the boundary while still maintaining the demarcation
If a form of Hh that lacks the cholesterol on the c-terminus of the N-fragment is present then it diffuses and switches on wingless in more cells (posterior cells don’t have the receptor)
In other scenarios Hh acts at a distance
Dispatched releases hh from the membrane
Dispatched extracts Hh from the membrane but not via cleavage - the cholesterol remains.
Other partners are required to aid its diffusion
Describe the strucutre and function of patched.
Patched is the Hh receptor
* 12-pass transmembrane domain is more like an ion channel than a receptor
* Unusually its default behaviour is to inhibit the Hh response
* KO (knock out) of patched activates the Hh response (opposite of what you’d expect e.g. Tgf-βR KO inhibits pathway)
* Not clear how this works but it involves smoothened (could be a GPCR)
* and is not a direct physical interaction
Describe hedgehog signalling in drosophila.
In absence of Shh
1. Ptc acts (somehow) to sequester Smo in membrane bound vesicles -> degraded
2. Ci/Cos2/Fu (Fused) complex bound to microtubules through Cos2
3. Ci is phosphorylated by PKA
4. Slimb recognizes Ci-P -> Ci targeted to the proteasome release 75kda Ci75 fragment
5. binds target genes and represses transcription
In presence of Hh:
1. Ptc is unable to sequester Smo
2. Cos2/Fu are recruited by Smo dissociating form the microtubules
3. Ci is released form the complex so can’t be PKA phosphorylated
4. Full length Ci binds CBP and activates expression
Absence of Hh
Patched sequesters Smoothened in membrane bound vesicles
Ci/Cos2/Fu form a complex on microtubules
Ci is phosphorylated by protein kinase A (PKA)
Ci phosphorylation results in targetting to proteosome
75kDa fragment of Ci enters the inhibits transcription
Presence of Hh
Hh binds to its receptor patched
Smo moves to plasma membrane as Ptc repression is removed
C terminal of Smo interacts with Cos2/Fu -> dissociated from microtubules
Cos2/Fu further phosphorylated
Full length (155kDa) unphosphorylated Ci moves to the nucleus
Binds CBP and activates target genes
Describe the induction of neural fates by Shh
Induction of neural fates by Shh (morphogen)
Nervous system forms from ectoderm overlying the notochord as it folds to make the neural tube in a process called neurulation
Notochord secretes Shh
Shh induces floor plate
Floor plate secretes Shh
Motoneurons specified at high Shh
V2 interneurons specified at medium Shh
V1 interneurons specified at low Shh
Can recapitulate in neural tube explant culture
1. Notochord secretes Shh
2. Shh induces floor plate
3. Floor plate secretes Shh
4. Motoneurons specified at high Shh
5. V2 interneurons specified at medium Shh
V1 interneurons specified at low Shh
What is the role of Hh signalling in cancer.
- There are many examples of Hh signalling in developmental processes
- Components of Hh pathway are also often mutated in cancers, especially basal cell carcinoma (BCC)
- Stem cell proliferation in the basal layer of the skin is stimulated by Hh
- Excess of Hh leads to over proliferation of these cells
- As Ptc1 inhibits Hh signaling it acts as a tumor suppressor – heterozygous patched mutations lead to BCC (somatic mutation LOH)
- High frequency of BCC in Ptc-/Ptc+ heterozygotes
- Possibility of anti-cancer drugs based on inhibitors of Hh signalling
Basal cell carcinoma
* High susceptibility in patients with Gorlin’s syndrome – mutation in Ptc
* BCC is not that serious - treated with surgery not metastatic
Cancers shrink when treated with small molecule inhibitors of Hh - possible therapeutic avenue
Describe the general structure of Wnt proteins
- Small secreted signaling proteins similar pathway to Hh
- Wnt name is an amalgamation of Wingless (segment polarity screen) and Int (oncogene identified in mouse)
- Vertebrates have multiple Wnt’s generally between 35-45 kda in size (humans 11-12)
- N-terminal signal peptide – secretory pathway
- Cysteines important as form intra-chain disulfide bonds (stability)
- Like Hh, Wnt is modified with a hydrophobic molecule, in this case a fatty acid – Palmitoleate added to the conserved serine
Addition is carried out by an enzyme called Porcupine
Describe the ‘canonical’ Wnt signalling pathway.
- Largely elucidated in Drosophila by looking at mutants
- Striking similarities with Hh signaling pathway
- Two receptors - Lrp (specific to canonical) and Frizzled
In the absence of Wnt - GSK3 (Kinase) /APC/Axin (Scaffold) phosphorylates β-catenin (TF)
- Subsequently targeted for degradation by Slimb
- Unlike in Hh pathway where Ci is partially degraded here β-catenin is completely degraded
- In absence of β-catenin TCF binds DNA and represses its targets
In the presence of Wnt: - Wnt binds its receptors Frizzled and Lrp
- Conformational changes in the cytoplasmic domains then lead to Phosphorylation of Lrp
- It becomes a high affinity binding partner for Axin sequestering it and dissociating the complex
- The constant degradation of β-catenin then is disrupted as no phosphorylation by GSK3
- Translocates to nucleus, binds TCF -> repressor to activator
Absence of Wnt
* LRP is not phosphorylated
* Β-catenin, GSK3 and APC held in a complex by Axin
* GSK3 phosphorylates β-catenin
* Phosphorylated β-catenin → proteosomal degradation
* TCF represses transcriptional targets
Presence of Wnt
* Wnt binds to LRP and Frizzeled
* LRP’s cytoplasmic tail phosphorylates Axin
* De-stabilisation of the degradation complex results in release of β-catenin
* Β-catenin translocates to the nucleus
β-catenin binds to TCF → complex activates transcription of target genes
What is teh Nieuwkoop centre.
Specification of the Nieuwkoop centre
* The Nieuwkoop centre (dorsal vegetal region) of Xenopus embryo instructs dorsal mesoderm and organizer
* Wnt essential to establishing the Nieuwkoop center
Add β-Catenin mRNA to the ventral side of Vegetal region -> second Nieuwkoop centre and duplicated axis of development
What is the role of Wnt signalling and cancer.
Over-expression of Wnts can cause cancer (original Int-1 gene)
APC is a tumour suppressor (adenomatous polyposis coli) – heterozygotes develop large numbers of polyps in colon.
Mutations which prevent β-catenin degradation found in colon cancer and melanoma
* Wnt expressed by MC’s in the crypt important for driving ISC proliferation
* APC mediates the destruction of β-Catenin
* Loss of APC results in an excess of Wnt signaling
* LOH of APC in patients with a null allele -> no β-Catenin degradation -> large numbers of precancerous polyps in the colon
Also caused by mutations in β-Catenin that prevent degradation of APC
