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Molecular Cell Biology II Signalling During Vertebrate Development II - Hedgehog
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Hedgehog (HH) signalling has strong clinical relevance in embryonic development and stem cell biology,
amongst other fields.Drosophila signalling is well known.
Hedgehog Ligands:
More than 3 found in vertebrates
Sonic hedgehog (SHH) ² well characterised
Desert hedgehog
Indian hedgehog
Essential for positional identity in development and adult tissue homeostasis across species
e.g. Control of digit formation
e.g. brain and spinal cord formation
Also involved in stem cells and cancer in adults
Main molecules involved:
Patched ² 12-pass transmembrane HH receptor
iHog ² Single-pass HH receptor, probably co-receptor of patched
Smoothened ² 7-pass, similar to Frizzled, transduces the HH signal
When there is no HH signal«
Patched keeps Smoothened inactive in intracellular endosomes
Smoothened is regulated by the conformation of patched, which changes when HH binds, which could
regulate the recruitment and degradation of smoothened.
A gene regulator called Cubitusinterruptus (Ci) is processed
in a complex which contains«
PKA (phosphorylates)
GSK3 (phosphorylates)
CK1 (phosphorylates)
Fused kinase
Costal2 (scaffold protein)
Ci is triple phosphorylated in the complex, then
ubiquitinylated and then cleaved in a proteasome, to leave a
small peptide. This then enters the nucleus and interacts
with a co-repressor to repress the HH target genes.
When there is a HH signal«
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Molecular Cell Biology II Signalling During Vertebrate Development II - Hedgehog
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HH binds to Patched and iHog, inhibiting Patched and inducing its endocytosis and degradation
Smoothened is then phosphorylated by PKA &CK1 and moves to the cell surface
Smoothened in the plasma membrane can recruit the Ci complex, Fused and Costal 2
Costal 2 can·t bind the 3 kinases (PKA, GSK3, CK1) so Ci is not phosphorylated or cleaved
Unprocessed full-length Ci enters the nucleus and wil l activated HH target genes
HH Signalling in
Vertebrates:
3 Ci-like gene regulators (Gli1/2/3)
Gli1/2 are not processed and probably act as transcriptional activators
Gli3 is processed and so can keep genes off (-HH) or switch on transcription (+HH)
The primary cilium is a HH signalling centre in vertebrate cells
+HH, activated smoothened and Gli proteins are concentrated in a primary cilium
All of the components of a signalling pathway in one of these extensions p a quick response
Defects in cilia can disrupt Shh signalling, can cause brain defects
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Molecular Cell Biology II Signalling During Vertebrate Development II - Hedgehog
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Processing of HH proteins:
Signal sequences are cleaved
HH precursor is autocatalytically cleaved
C-terminus is modified ² covalently linked to cholesterol
o Errors in this linkage are bad as they can effect the morphological gradient
N-terminus has a fatty acid added ² palmitate by skinny hedgehog
The HH is now fully active after having dual lipid modification
Potential use of lipid modifications«
Targeting lipid rafts
Regulating HH release and trafficking
Multimerisation of HH to increase solubility
Formation of diffusion gradientsetc«
HH release from cells:
Trafficked to cell surface
Can be released as lipoprotein oligomers by 12-pass transmembrane protein Dispatched
Mechanisms of release«
HH multimers are more soluble
Dispatched releases HH
LRP in target cell takes up HH and passes on signal by cell trafficking
Movement through tissue«
Diffusion
Indirect ² via signalling cascades
Via cytonemes (thin finger-like projections from plasma membrane)
HH can act as a morphogen, the effect on cell will vary depending on concentration
Seemingly small changes in [HH] can give hugely different cellular responses
Regulating the amount of HH signal a target receives:
ECM (extra cellular matrix) can bind HH and present it to a receptor
o Or isolate it from receptor
Ptc 2
Hip (Hedgehog Inhibitory Protein)
Gas1 (Growth Arrest Specific gene) binds Shh and promotes signalling
All of these factors will determine how much HH can bind to a receptor
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Molecular Cell Biology II Signalling During Vertebrate Development II - Hedgehog
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Gli Code:
How are the different concentrations of HH binding sensed and converted to effect on cells?
The three Glis in vertebrates can activate / repress gene transcription
Glis are transcription factors with 5 zinc-binding domains
3 Ci-like gene regulators (Gli1/2/3)
Gli1/2 are not processed and probably act as transcriptional activators
Gli3 is processed and so can keep genes off (-HH) or switch on transcription (+HH)
The balance between 1,2,3 will determine the cellular outcome
The cell integrates the activation / repression of all 3 to give the overall result of the HH signal
Levels of each Gli changes over time, so the result of the HH signal will change over time
Regulation of the Gli code«
Other signalling pathways can modify the activity of PKA, CK1 and GSK3 that regulate Gli cleavage to
a repressor form
Ligases can tag Gli with ubiquitin
Proteins can bind to Glis and prevent them from entering the nucleus e.g. SuFu
Wnt/F-catenin can oGli activity etc«
State of Gli activity will influence the binding of other transcription factors
e.g. Nkx2.2 and Pax6 (both involved in cell fate)
Feedback loops can occur with HH target genes, such as Patched
+HH will increase transcription of patched and inhibit further HH signalling
Transcription independent effects of HH:
Cell motility ² important in the spreading of cancer
Chemoattraction ² important for axon growth
Roles of HH signalling:
Ventral fate in nervous system (determining what is top / bottom)
Anterior ² Posterior axis of limb (thumb / little finger)
Vasculogenesis ² important for cancer
Bone / cartilage formation
Lung branching
Maintenance of stem cell populations post-development
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Molecular Cell Biology II Signalling During Vertebrate Development II - Hedgehog
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More detailed example ² Dorso-ventral patterning in the CNS
A floor plate makes HH, which builds up a morphological gradient
Works in combination with other morphological gradients to determine cell fate
HH also acts as a mitogen to ensure the embryo has enough of each cell type
e.g. In the developing cerebellum, HH is used to match the number of Purkinje cells with the number of
granule cells (number matching)
Also regulates stem cell division / number which can be affected by disease / ageing
Due to its role in cell cycle control HH is directly linked to cancer
e.g. Basal cell carcinoma
Gli codes are disrupted
e.g. Brain cancer
Very aggressive form of cancer
Gli code is deregulated
Widespead with severe side effects
Therapeutics:
e.g. cyclopamine binds to Smo and stops downstream HH signal transduction
The chemical is very toxic makes developing foetuses Cyclops
Has been shown to eliminate lung cancer in mice
Crosstalk between Shh and Wnt:
A lot of interaction between the two pathways particularly in limb development
Wnt and HH signalling have many similarities