Hedgehog signalling

Question 1

What is hedgehog (mainly in drosophila and what it was discovered as by Nusslein and Wieschause 1980s)

Answer 1

Segment polarity gene

Question 2

Mutations in hedgehog in drosophila causes what?

Answer 2

Defects in where the naked cuticle is lost creating a hedgehog phenotype

Question 3

How does Hh act with Wg?

Answer 3

Acts in a reciprocal loop
They maintain each others expression in an autoregulatory loop
Hh directly upregulates Wg transcription
Wg controls the expression of engrailed (en)
These genes are dependent of each other so the loss of one causes the loss of another, giving a similar phenotype

Question 4

What are the ligand proteins involved in the hedgehog signalling pathway?

Answer 4

Hh (Shh, Ihh, and Dhh)
Skinny hedgehog (Hedgehog actyltransferase)
Dispatched (in both)
? (Scuber gene)
Heparan sulfate proteoglycans (in both)

Question 5

Is this pathway found in all metazoans?

Answer 5

No eg not in c.elegans but is found in some

Question 6

Why do vertebrates have more homologs for the dropsohila genes involved?

Answer 6

They have undergone genome duplications so there are several genes in drosophila which have more than one homolog

Question 7

How is the hedgehog ligand formed?

Answer 7

Transcribed with an N-terminal signal sequence which targets the pathway
This is removed by autoproteolytic cleavage catalysed by the c terminal
New c terminus is then coupled to cholesterol molecules
N terminus has palmitoyl added performed by skinny hedgehog or Hhat
Molecule can not leave the cell due to hydrophobicity
so the action of scube genes and dispatched is required for it to leave the cell

Question 8

How is it thought that dispatched and scube genes MIGHT work?

Answer 8

They may help create multimeric Hh signalling particles that have their hydrophobic tail buried in the particle
They could also help load hedgehog molecules on lipoproteins

Question 9

What have heparan sulfate proteoglycans been shown to be required for?

Answer 9

Long range diffusion of hedgehog molecules

Question 10

What are the receptors involved in the hedgehog pathway?

Answer 10

Patched (1, 2, Hedgehog interacting protein Hhip)
Smoothened (same in both)
Ihog/Boi (CDO/brother of CDO, Growth arrest factor GAS1)

Question 11

What is the action of ptc on smoothened in drosophila?

Answer 11

Regulates the subcellular distribution and stability of Smo
A single Ptc molecule can inhibit a large number of Smo particles
In the absence of Hh Ptc stops Smo getting it to the surface - it is thought to regulate the trafficking of Smo to a compartment where Smo gets degraded

Question 12

What happens when Ptc binds to Hh?

Answer 12

Ptc is internalised and degraded so Smo is trafficked to the surface

Question 13

What are the three changes which can occur to Smo?

Answer 13

Relocation
Accumulation
Phosphorylation

Question 14

In mammals what is thought to play a primary role in mammals?

Answer 14

Cellular Antennae/ Cilia

Question 15

In the absence of Hh where is Ptc located in mammals?

Answer 15

Localised to the cilium of the cell

Smo is therefore excluded from this territory

Question 16

Ptc has a homology to which genes?

Answer 16

Prokaryotic RND

Niemann -Pick protein

Question 17

It is thought that Ptc may do what?

Answer 17

May pump a small molecule in to cells that inhibits Smo or pump out of cells a small molecule that activates Smo

Question 18

What normally inhibits Smo?

Answer 18

Cyclopamine

Could block the activator of Smo

Question 19

What is the role of hedgehog interacting protein?

Answer 19

It is most likely acting to mop up free hedgehog, so it prevents it from reaching the patched receptor keeping the signal down

Question 20

CDO, BOC and GAS1 is most likely to act as what?

Answer 20

A co-receptor promoting the binding of Hh signal to patched and thereby promoting a signal

Question 21

What are the trasnduction genes in the Hh pathway?

Answer 21

Fused (STK36 or CDC2/1)
Suppressed of fused (same in both)
Costal (Kif7)
? (Dzip1 and Talpid 3)
Slimb (BetaTRCP)
Protein kinase A (same in both)
Casein kinase 1 (same in both)
Glycogen synthase kinase B (same in both)

Question 22

Briefly describe the intracellular pathway in Drosophila

Answer 22

a) Two complexes exist to keep Ci out of the nucleus
- Costal 2 -Kinesin molecule, that acts as a scaffold protein and fused a serine threonine kinase
- Ci and Sufu
b) Under the influence of the first complex binding to Smo 3 other genes act on Ci to process it in to a shorted form via slimb
4) The short form is a transcriptional repressor

Question 23

What are the 3 genes which act on Ci in the complex bound to Smo?

Answer 23

Casein kinase 1
Protein kinase A
Glycogen synthase kinase 3 beta

Question 24

What happens when low levels of Hh is present?

Answer 24

The PKA/GSK3/CK1 complex dissociates from Smo so active repression is lost

Question 25

What happens at high concentrations of hedgehog?

Answer 25

It acts through the complexes to release full length Ci that will actively promote the transcription of target genes
May involve some type of phosphorylation
Phosphorylation of Sufu by fused promotes the formation of the active form of Ci

Question 26

What are the 3 vertebrate homologs of Ci?

Answer 26

Gli 1, 2 and 3

Question 27

Which vertebrate homolog of Ci does not get cleaved to a shorter repressor?

Answer 27

Gli1

Question 28

Describe the negative feedbacks of Hh signalling

Answer 28

• Patched = suppresses the levels of signaling inside the cell and the extra amount of ptc binds extracellular Hh and acts to restirct diffusion of the ligand = steeper gradient
• Hhip is induced to limit diffusion of Hh
• Downregulation of DCO, BOC and GAS1

Question 29

Describe the positive feedbacks of Hh signalling

Answer 29

Gli1 is always an activator of the Hh signal as it can’t be processed in to a repressor - this is essentially a feedforward loop

Question 30

Describe Hh in wing patterning

Answer 30

In the wing imaginal disc hedgehog is epxressed in the posterior compartment and diffuses to the anterior
Here it induces the expression of decaplentaplegic = patterns the wings

Question 31

Describe Hh in neural development

Answer 31

Neural tube cells differentiate in to different types of neurons depending on how much Shh they recieve
In verterbrates the notochord and floorplate expresses Shh and patterns the neural tube (ventralisation)

Question 32

Loss of Hh can lead to what malformation?

Answer 32

Holoprosencephaly

Question 33

Misregulation of Hh in limbs can cause what?

Answer 33

Syndactyly

Question 34

What are the shared features of Hh and Wg?

Answer 34

Both ligands are modified by 2 hydrophobic molecules and depend on HSPG and TM proteins to move away from the producing cell
Both are multimers and lipoproteins