Lecture 6 - Principles of developmental biology (Hedgehog & Wnt signalling) Flashcards
(48 cards)
What are Hedhehog & Wnt signalling?
2 of the major signal transduction pathways that cells use to communicate with each other.
They are involved in patterning many tissues and development of many organs in both vertebrates and invertebrates
Involved in regulation of stem cell fate/cell division and in the maintenance and initiation of cancer
What model is used to research a lot of the Hh & Wnt pathway?
Drosophila
What is Hedgehog?
Segment polarity gene
- this means that mutant of flies, part of segments always missing
What are Hh and Wg required for?
Hh and Wg are required in Drosophila embryo for segmentation.
The 2 signalling pathway maintain each other
How are stripes created?
Hh gene induces expression of Wg gene, creating stripes. Wg also induces Hh.
What does removal of Hh or Wg lead to?
Removal of Hh = loss of Wg and vice versa.
Phenotype of knockout will be the same, no matter which one
How was the Wnt gene discovered?
Wnt gene was independently discovered in mice: Int1 gene (causing tumours): wg+int=wnt
Where are both signals present?
Both present in most multicellular organisms
- vertebrates often have more than 1 orthologue of genes in these signalling pathay.
Hedgehog:
- Sonic Hedgehog (Shh)
- Desert Hedgehog
- Indian Hedgehog
Wg:
- >18 Wnts
What organism doesn’t have a functional Hh signalling pathway?
C. elegans
Explain how Hh ligands are formed
- Hh protein transcribed with N-terminal sequence.
- The sequence organized so that the protein is entered into the secretory pathway, then cleaved off.
- Autoproteolysis - C-terminal of Hh is an enzyme that cleaves itself
- Following cleavage of C-terminal, cholesterol modification occurs on Hh N-terminal part.
- Another modification occurs- PALMITOYLATION.
- Both modifications are hydrophobic. Fatty acids don’t like to be dissolved in water. Targets protein to membrane.
- Hh ligand needs help of DISPATCHED - 12 transmembrane protein - (SCUBE proteins in invertebrates) allow the protein to diffuse away from the producing cell
Explain how Wnt ligands are formed
Have N-terminal sequence - allows entry into secretory pathway
- modifications - palmitoylation & palmitoleic acid modification. These make the protein extremely hydrophobic
- ‘Wntless’ is therefore required to reach membrane and allow protein to leave from producing cell
What are Cytonemes?
cellular, thin protrusion. Shown in zebrafish that Wnt ligand is accumulated at tip of cytonemes. Cytonemes can then move away, touch another cell & activate signalling in Wnt receiving cell.
Effectively touches cell & changes its fate.
What is the ‘handover’ mechanism of Wnt diffusion?
Dlp = Dally-like protein, a Heparan Sulphate Proteoglycan (HSPG)
Dlp has 2 binding sites for Wnt ligand. Using glycan sidechains, it can bind Wnt, but also binding pocket in protein portion - binding Palmutate that is added to Wnt protein.
First glycan sidechain binds Wnt, then palmutate, then next next molecule. This allow it to move along membrane.
Summarise ligands
- Wnt & Hh ligands are hydrophobic and require help to move from cell to cell
- Wnt diffuses by ‘handover mechanisms’ and cytonemes
- Hh - less known but likely similar mechanisms
How is the Hh signal received at the membrane?
2 important receptors:
- Patched (Ptc)
- Smoothened (Smo)
What is Patched and Smoothened work?
- 12 transmembrane protein - Hh receptor - protein binds Hh ligand.
- Works as a constitutive repressor of Smoothened (another transmembrane protein - 7 transmembrane).
- 2 Patched genes (Patched 1 & Patched 2)
Are Ptc & Smo present in a fixed/constant ratio?
Ptc & Smo aren’t present in a stoichiometric complex (not in a fixed or constant ratio). It is more enzymatic - Ptc can inhibit lots of Smo molecules
Ptc regulates the subcellular distribution and stability of Smoothened
What is the effect of binding of Hh on Ptc & Smo?
Binding of Hh to Ptc leads to Ptc becoming internalized and can’t prevent arrival of Smo towards the membrane - Smo activation.
Hh binding leads to Smo relocalisation to membrane, accumulation and then phosphorylation.
What do mammals Cilia do?
Mammals Cilia ‘cilia antennae’ may be focal points for hedgehog sensing.
Lack of cilia = defects on Hh signalling
How does Patched inhibit Smo signalling?
Patched depletes cholesterol from inner membrane leaflet
Patched is a pump - Patched pumps cholesterol from inner leaflet of membrane to outer membrane.
Reduced cholesterol leads to Smo remaining inactive.
Binding of Shh leads to inhibition of pump action. Cholesterol will accumulate in inner leaflet, binding Smo and activating signalling pathway.
This is how Patched can inhibit multiple Smo molecules
What are Hedgehog downstream events (no Hh)?
The nuclear factor (transmitting signal from membrane to nucleus) is held in cytoplasm by 2 complexes:
1st - Cos2, Fused & Ci (Cos2 = kinase-like molecule, Fused, Ci = Gli1,2,3 in vertebrates)
2nd - SuFu & Ci
Under influence of 1st complex, another complex can bind. This complex contains: PKA, CK1 & GSK3.
This complex phosphorylates Ci, leading to partial proteolysis under influence of Slimn. This leads to short-form of Ci.
This is a repressor transcription facotr - supressing Hh target genes
What are Hedgehog downstream effects of (HH bound)?
- 1st complex doesn’t bind.
- Ci becomes releases and enters the nucleus and acts as an activator driving gene expression.
- Fused may interact with SuFu and phosphorylate it, also leading to the release of Ci.
How does Hedgehog negatively act on its own pathway?
One of the best known targets of Hh signalling is Patched1 (vertebrates)/Patched (drosophila).
Hh pathway activation leads to the production of Patched , ensuring activation of pathway occurs at a limited level
How does Hedgehog positively act on its own pathway?
Gli1 is induced
- Gli1 (Ci) cannot be proteolyzed into a repressor (unlike Gli2/3). It will always act as an activator of pathway
