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BGM2002 CS > L11 - Hedgehog signalling > Flashcards

L11 - Hedgehog signalling Flashcards

1
Q

What is hedgehog signalling?

A

Signalling pathway that transmits information to embryonic cells required for proper cell differentiation

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2
Q

Why is it called hedgehog signalling?

A

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

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3
Q

What are the 3 hedgehog homologues?

A

Indian Hedgehog – IHH
Desert Hedgehog – DHH
Sonic Hedgehog – SHH

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4
Q

Main components of the hedgehog pathway

A

Hedgehog (Hh) - secreted proteins that act as morphogens

Patched (Ptch) - 12 transmembrane receptor

Smoothened (Smo) - 7 transmembrane protein - Hh signal transducer

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5
Q

What is Sufu?

A

Suppressor of Fused

Cytoplasmic protein that can also act as a tumour suppressor

Plays critical roles in the production, trafficking & functions of Gli proteins

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6
Q

What are the different types of signalling?

A

Autocrine

Paracrine

Endocrine

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7
Q

What type of signalling is Hh signalling?

A

Paracrine signalling is used to establish a graded response in the signal-receiving cells

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8
Q

Why is Hh signalling graded?

A

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

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9
Q

What is a morphogen?

A

A signalling molecule that produces a graded response over a distance

Hh protein is a morphogen

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10
Q

How do you create a concentration gradient?

A

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

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11
Q

Hedgehog protein structure

A

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

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12
Q

How is the hedgehog protein transported from the ER to target cells?

A

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)

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13
Q

What happens once HH-N is recognised by DISP?

A

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

  1. Glypian recruits lipoproteins (make Shh soluble)
  2. The anchor of glypian is cleaved by phospholipase C-like protein
  3. HH-N-associated lipoprotein particles (complex) are released
  4. Diffuses away from the cell to initiate the response in the target cell
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14
Q

Why are lipoproteins essential to Hh transport?

A

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

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15
Q

How is Hh secreted?

A

There are different variations

  1. HH-N can interact with SCUBE2 & in combination, this allows HH protein to be released from the secretory cell
  2. HH protein can accumulate on cell membrane & form a soluble multimer & be released
  3. Involves glypian & association with lipoproteins, allowing it to be soluble & to be released
  4. Part of the cell membrane buds of with HH protein forming an exovesicle & this can migrate away from the cell
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16
Q

What happens when the Hh pathway is INACTIVATED in INVERTEBRATES?

A

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

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17
Q

What happens when the Hh pathway is ACTIVATED in INVERTEBRATES?

A

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

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18
Q

What controls the levels of signalling & gene induction in invertebrates?

A

Ratio of CiR/CiA

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19
Q

How is Hh signalling different in vertebrates & invertebrates?

A

Signalling in primary cilium in vertebrates

GLI proteins (vertebrates) are similar to Ci proteins (invertebrates)

Patched and Smo both involved

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20
Q

Different types of cilia

A

Cilia of the airways - motile

Primary cilium - non-motile
• Hh pathway requires primary cilia

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21
Q

What happens when the Hh pathway is INACTIVATED in VERTEBRATES?

A

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

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22
Q

What is Gli3-R?

A

A transcriptional repressor that inhibits Hh target gene expression

Repressed form

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23
Q

What is Gli3-FL?

A

Gli3 full length protein

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24
Q

What is Gli3-A?

A

Gli3 active form

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25
Q

What is PKA?

A

Protein kinase A

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26
Q

What is CK1?

A

Casein kinase 1

Caseins are phosphoproteins

27
Q

What is GSK3-beta?

A

Glycogen synthase kinase 3β

28
Q

What happens when the Hh pathway is ACTIVATED in VERTEBRATES?

A

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

29
Q

What is Gprk2?

A

G protein-coupled receptor kinase 2

30
Q

What controls the levels of signalling & gene induction in vertebrates?

A

Ratio of Gli3R/Gli3A

It controls several processes during organogenesis

31
Q

What is organogenesis?

A

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

32
Q

Variations of the Hh pathway

A

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
33
Q

Why is reciprocal signalling between mesenchyme & epithelium essential for limb development?

A

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

34
Q

Role of morphogens in development

A

Dorsoventral - Wnt

Proximodistal - Tbx, FGF genes

Anteroposterior - Shh

35
Q

What is dorsoventral?

A

Back to front

Wnt as a morphogen

36
Q

What is proximodistal?

A

Tip of appendage to where it joins the body

Tbx, FGF genes as morphogens

37
Q

What is anteroposterior?

A

Head to tail

Shh as a morphogen

38
Q

What is an appendage?

A

A projecting part of an invertebrate or other living organism, with a distinct appearance or function

39
Q

What is the AER?

A

Apical ectodermal ridge

Acts as a major signaling center to ensure proper development of a limb

40
Q

What happens if you place an additional AER on the limb bud of a chick embryo?

A

An additional wing is produced

41
Q

What happens if you remove the AER on the limb bud of a chick embryo?

A

No wings develop

42
Q

What happens if you replace the AER on the limb bud of a chick embryo with FGF-bead?

A

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

43
Q

What happens if you remove the mesenchyme cells below the AER on the limb bud of a chick embryo?

A

No wing is produced

Shows signal from underlying mesenchymal cells is what is really important, and this is where sonic Hh plays its role

44
Q

What is ZPA?

A

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

45
Q

Importance of cilia for Hh signalling

A

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

46
Q

Role of Shh in body axis

A

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

47
Q

How does Shh centralise the neural tube to pattern the neurones?

A

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

48
Q

What gradients cause different classes of interneurons?

A

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

49
Q

EVC & EVC2 genes

A

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

50
Q

What happens when you get a mutation in either the EVC or EVC2 genes?

A

When EVC and EVC2 are mutated in humans, it results in Elliz-van Crevald syndrome

51
Q

What is Elliz-van Crevald syndrome?

A

A recessive condition and allelic with Weyers acrofacial dysostosis, a disorder showing dominant inheritance

Caused by mutations in EVC or EVC2

52
Q

Elliz-van Crevald syndrome phenotypes

A

Skeletal defects
Heart defects
Teeth defects

53
Q

What is the role of EVC and EVC2?

A

They enhance the cellular response to Hh signalling

54
Q

How do EVC & EVC2 enhance the cellular response to Hh signalling?

A
  1. In the presence of Hh ligand Smo translocates to primary cilia
  2. The Evc/Evc2 complex binds to Smo
  3. The Evc/Evc2/Smo complex facilitates diffusion of Gli3-FL from Sufu
  4. The Gli3-FL/Gli3-R ratio changes – more Gli3-FL traffics along the microtuble
  5. Increased levels of Gli3-FL enter the nucleus & activate expression of Hh target genes
55
Q

Absence of Dhh (desert hedgehog) in mice

A

Results in:
• Defects in testis - infertility
• Disrupted formation of connective tissue sheath around peripheral nerves

56
Q

Absence of Ihh (Indian hedgehog) in mice

A

Results in:
• Reduced chondrocyte proliferation
• Failure of osteoblast development during enchondral bone formation

57
Q

Absence of Shh in mice

A

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

58
Q

Induced expression of Shh in the skin of mice

A

Results in:
• Induction of basal cell carcinomas (BCCs)

Ectopic & increased expression of SHH has been found in a variety of different human tumours

59
Q

Mutations in the human Shh

A

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)

60
Q

Syndromes/diseases caused by defects in the hedgehog signalling pathway

A

Basal cell nevus syndrome (BCNS) – Gorlin syndrome

Greig cephalopolysyndactyly syndrome (GCPS)

Mutations in SUFU or SMO

61
Q

What is the non-canonical Hh pathway?

A

Gli-independent mechanisms

62
Q

What are the 2 types of non-canonical Hh pathway?

A

Type 1 - downstream of Smo

Type 2 - independent of Smo

63
Q

Non-canonical Hh pathway

Type 1 - downstream of Smo

A

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

64
Q

Non-canonical Hh pathway

Type 2 - independent of Smo

A

Shh binds Ptch

Disrupts interaction between Ptch and cyclinB1 (part of cell cycle)

Leads to increase in cell proliferation and survival

BGM2002 CS (14 decks)

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