L11 - Hedgehog signalling Flashcards
What is hedgehog signalling?
Signalling pathway that transmits information to embryonic cells required for proper cell differentiation
Why is it called hedgehog signalling?
Along the ventral body of Drosophila larvae there are stripes which are denticles
When 1 gene was mutated, you lost the stripe pattern
They thought it looked like a hedgehog so they called it the hedgehog gene
What are the 3 hedgehog homologues?
Indian Hedgehog – IHH
Desert Hedgehog – DHH
Sonic Hedgehog – SHH
Main components of the hedgehog pathway
Hedgehog (Hh) - secreted proteins that act as morphogens
Patched (Ptch) - 12 transmembrane receptor
Smoothened (Smo) - 7 transmembrane protein - Hh signal transducer
What is Sufu?
Suppressor of Fused
Cytoplasmic protein that can also act as a tumour suppressor
Plays critical roles in the production, trafficking & functions of Gli proteins
What are the different types of signalling?
Autocrine
Paracrine
Endocrine
What type of signalling is Hh signalling?
Paracrine signalling is used to establish a graded response in the signal-receiving cells
Why is Hh signalling graded?
Gradient of Hh protein induces different subsets of genes to be transcribed
Gene activation only occurs at certain thresholds, adding specificity to gene transcription
Different concentrations of Hh give different levels of intracellular signalling
What is a morphogen?
A signalling molecule that produces a graded response over a distance
Hh protein is a morphogen
How do you create a concentration gradient?
Morphogen diffuses away from the source
Localised production of an inhibitor that diffuses away from its source and blocks the action of a uniformly distributed inducer
Hedgehog protein structure
The Hh protein is produced in a form that needs to be processed into an active form
C terminus = hog domain
N terminus = hedge domain
Modified in the ER to become active before secretion
How is the hedgehog protein transported from the ER to target cells?
1) HH undergoes autocatalytic cleavage to produce HH-N & HH-C domains
2) HH-C undergoes proteasomal degradation
3) HH-N undergoes autocatalytic modification - cholesterol is added
4) HH-N moves to the cell membrane
5) Acyltransferase skinny hedgehog (SKI) modified HH-N by attaching a palmitic acid group to HH-N
6) This is recognised by transmembrane protein Dispatched (DISP)
What happens once HH-N is recognised by DISP?
HH-N is released by DISP but remains anchored within cell membrane (cholesterol & palmitic acid)
HH-N oligomers interact with the heparan sulphate chains of glypians
- Glypian recruits lipoproteins (make Shh soluble)
- The anchor of glypian is cleaved by phospholipase C-like protein
- HH-N-associated lipoprotein particles (complex) are released
- Diffuses away from the cell to initiate the response in the target cell
Why are lipoproteins essential to Hh transport?
Lipoproteins are essential for making Hh protein soluble to allow it to be released from the cell membrane to move away & go to its target cell
How is Hh secreted?
There are different variations
- HH-N can interact with SCUBE2 & in combination, this allows HH protein to be released from the secretory cell
- HH protein can accumulate on cell membrane & form a soluble multimer & be released
- Involves glypian & association with lipoproteins, allowing it to be soluble & to be released
- Part of the cell membrane buds of with HH protein forming an exovesicle & this can migrate away from the cell
What happens when the Hh pathway is INACTIVATED in INVERTEBRATES?
When theres no HH protein, then Ptch inhibits Smo
Smo is no longer needed so its degraded
A complex forms in the cytoplasm that consists of: • Sufu • Fu • Cos2 • Ci (specific for invertebrates)
Ci becomes phosphorylated by PKA, CKI-alpha & GSK2-beta to form CiR (repressed state)
No gene transcription occurs
What happens when the Hh pathway is ACTIVATED in INVERTEBRATES?
Hh binds and inhibits Ptch so Smo is no longer inhibited
Smo is active and interacts with Fu, Sufu, Cos2 and Ci
Smo is phosphorylated by Gprk2, PKA & CkI-alpha
Ci isn’t phosphorylated so is released from the complex to form CiA (active state)
CiA enters the nucleus and binds DNA inducing genes involved in cell patterning & differentiation
What controls the levels of signalling & gene induction in invertebrates?
Ratio of CiR/CiA
How is Hh signalling different in vertebrates & invertebrates?
Signalling in primary cilium in vertebrates
GLI proteins (vertebrates) are similar to Ci proteins (invertebrates)
Patched and Smo both involved
Different types of cilia
Cilia of the airways - motile
Primary cilium - non-motile
• Hh pathway requires primary cilia
What happens when the Hh pathway is INACTIVATED in VERTEBRATES?
No response in target cell as no transcription of the Hh responsive genes
Ptch1 inhibits Smo by inhibiting its translocation to the cilia
Smo is degraded in endosomes
Gli-FL binds Sufu
Gli-FL becomes phosphorylated by GSK3-beta, CK1 & PKA
This complex is processed in the proteasome to form Gli3-R
What is Gli3-R?
A transcriptional repressor that inhibits Hh target gene expression
Repressed form
What is Gli3-FL?
Gli3 full length protein
What is Gli3-A?
Gli3 active form
What is PKA?
Protein kinase A
What is CK1?
Casein kinase 1
Caseins are phosphoproteins
What is GSK3-beta?
Glycogen synthase kinase 3β
What happens when the Hh pathway is ACTIVATED in VERTEBRATES?
Transcription of the Hh responsive genes
Hh binds Ptch1 & Ptch1 is degraded
Smo is activated by phosphorylation by PKA, CK1 & Gprk2
Smo accumulates in the membrane of the primary cilia & migrates up
Initiates the dissociation of Gli3-FL & Sufu, reducing Gli3-R activity
Gli3-FL moves to the cilia tip and converted to activator form Gli3-A
Gli3-A moves to the nucleus, where it acts as a transcriptional activator to induce Hh target gene expression
What is Gprk2?
G protein-coupled receptor kinase 2
What controls the levels of signalling & gene induction in vertebrates?
Ratio of Gli3R/Gli3A
It controls several processes during organogenesis
What is organogenesis?
Process by which the three germ tissue layers of the embryo, which are the ectoderm, endoderm, and mesoderm, develop into the internal organs of the organism
Variations of the Hh pathway
PTCH1 & PTCH2
• Both interact with Smo to maintain it in an inactive state when no Hh is present
• Differential expression patterns
GLI1 - full length TF
GLI2 & GLI3
• Can act as positive or negative regulators
• determined by post transcriptional and post translational processing
Target genes • Proliferation • Apoptosis • EMT • Stem cell self renewal
Why is reciprocal signalling between mesenchyme & epithelium essential for limb development?
On the edge of the limb bud there is an epithelial thickening known as the apical ectodermal ridge which is very important for limb bud growth.
Fgf8 (strongly expressed within AER) is secreted into the underlying mesenchyme which is essential for proliferation and this is why the limb bud grows out
Role of morphogens in development
Dorsoventral - Wnt
Proximodistal - Tbx, FGF genes
Anteroposterior - Shh
What is dorsoventral?
Back to front
Wnt as a morphogen
What is proximodistal?
Tip of appendage to where it joins the body
Tbx, FGF genes as morphogens
What is anteroposterior?
Head to tail
Shh as a morphogen
What is an appendage?
A projecting part of an invertebrate or other living organism, with a distinct appearance or function
What is the AER?
Apical ectodermal ridge
Acts as a major signaling center to ensure proper development of a limb
What happens if you place an additional AER on the limb bud of a chick embryo?
An additional wing is produced
What happens if you remove the AER on the limb bud of a chick embryo?
No wings develop
What happens if you replace the AER on the limb bud of a chick embryo with FGF-bead?
A normal wing is produced
FGF is highly expressed in the AER
This confirms that is is FGF that is important - activity of FGF8 is sufficient ti mimic the activity of the AER
What happens if you remove the mesenchyme cells below the AER on the limb bud of a chick embryo?
No wing is produced
Shows signal from underlying mesenchymal cells is what is really important, and this is where sonic Hh plays its role
What is ZPA?
Zone of polarising activity
An area of mesenchyme that contains signals which instruct the developing limb bud to form along the anterior/posterior axis
Shh is required in the ZPA
Importance of cilia for Hh signalling
By removing cilia, nothing is there to respond to Hh signalling pathways sp anything that requires Hh signalling pathway is disrupted
Removal leads to defects such as polydactyly, shortened bones & craniofacial defects
Role of Shh in body axis
Paraxial mesoderm is forming segmented structures called the somites either side of the neural tube
Notochord sits under ventral part of the NT
Notochord and floor plate strongly express Shh
From this region, sonic then forms the concentration gradient so it is acting as a morphogen
How does Shh centralise the neural tube to pattern the neurones?
Shh secreted by the notochord
Forms a concentration gradient from floor plate
BMP4 – opposing gradient from roof plate
Generates specific relative amounts of each signalling molecule at a certain position.
Gradients specify different classes of interneurons
What gradients cause different classes of interneurons?
Low concentration – dorsal interneurons
High concentration – motor neurones
Very high – induce floor plate cells and V3 interneurons
Distinct cell fates at different distances from the floor plate
EVC & EVC2 genes
Map to chromosome 4p16
Are arranged in a head to head configuration
Share a very small promoter region
When EVC and EVC2 are mutated in humans, it results in Elliz-van Crevald syndrome
What happens when you get a mutation in either the EVC or EVC2 genes?
When EVC and EVC2 are mutated in humans, it results in Elliz-van Crevald syndrome
What is Elliz-van Crevald syndrome?
A recessive condition and allelic with Weyers acrofacial dysostosis, a disorder showing dominant inheritance
Caused by mutations in EVC or EVC2
Elliz-van Crevald syndrome phenotypes
Skeletal defects
Heart defects
Teeth defects
What is the role of EVC and EVC2?
They enhance the cellular response to Hh signalling
How do EVC & EVC2 enhance the cellular response to Hh signalling?
- In the presence of Hh ligand Smo translocates to primary cilia
- The Evc/Evc2 complex binds to Smo
- The Evc/Evc2/Smo complex facilitates diffusion of Gli3-FL from Sufu
- The Gli3-FL/Gli3-R ratio changes – more Gli3-FL traffics along the microtuble
- Increased levels of Gli3-FL enter the nucleus & activate expression of Hh target genes
Absence of Dhh (desert hedgehog) in mice
Results in:
• Defects in testis - infertility
• Disrupted formation of connective tissue sheath around peripheral nerves
Absence of Ihh (Indian hedgehog) in mice
Results in:
• Reduced chondrocyte proliferation
• Failure of osteoblast development during enchondral bone formation
Absence of Shh in mice
Results in:
• Defects in the establishment and maintenance of midline structures such as the notochord and floorplate
• Absence of distal limb structures
• Cyclopia, holoprosencephaly
• Absence of ventral cell types within the neural tube
• Absence of the spinal column and most of the ribs
• Defects in lung and oesophagus
Induced expression of Shh in the skin of mice
Results in:
• Induction of basal cell carcinomas (BCCs)
Ectopic & increased expression of SHH has been found in a variety of different human tumours
Mutations in the human Shh
Lead to a reduction in gene activity
Not complete loss of SHH as not compatible with life
Holoprosencephaly (HPE) – a cephalic disorder in which the prosencephalon (the forebrain of the embryo) fails to develop into two hemispheres
Single median central incisor (manifestation of mild holoprosencephaly)
Syndromes/diseases caused by defects in the hedgehog signalling pathway
Basal cell nevus syndrome (BCNS) – Gorlin syndrome
Greig cephalopolysyndactyly syndrome (GCPS)
Mutations in SUFU or SMO
What is the non-canonical Hh pathway?
Gli-independent mechanisms
What are the 2 types of non-canonical Hh pathway?
Type 1 - downstream of Smo
Type 2 - independent of Smo
Non-canonical Hh pathway
Type 1 - downstream of Smo
Modulates Ca2+ and actin cytoskeleton
- Shh bind Ptch, couples Gi proteins (G)
- Activates small GTPases RhoA and Rac1 – control of actin cytoskeleton
- And active Smo stimulates Ca2+ release from the ER and activates PI3K and PLC-δ generates IP3 leading to the release of Ca2+
Function in chemotaxis and cell migration through actin rearrangement
Proliferation via calcium induced ERK activation
Non-canonical Hh pathway
Type 2 - independent of Smo
Shh binds Ptch
Disrupts interaction between Ptch and cyclinB1 (part of cell cycle)
Leads to increase in cell proliferation and survival
