Lecture 19

Question 1

Drosophila larva

Answer 1

• Denticles present across anterior half of each segment
• Posterior half smooth
• In mutant, posterior segment failed to develop, covered in denticles

Question 2

How many hedgehog proteins in Drosophila?

Answer 2

Single hedgehog (Hh) in Drosophila

Question 3

Hedgehog proteins in vertebrates?

Answer 3

Sonic (Shh)

Indian (Ihh)

Desert (Dhh)

Question 4

What are hedgehog proteins and how are they modified?

Answer 4

• Secreted signalling proteins
• Modified by cleavage and addition of cholesterol

Question 5

Explain hedgehog processing

Answer 5

Inactive precursor - Signal peptide 0-82 - Secretory pathway

20kDa N-terminus is signalling molecule

25kDa C-terminus catalyses cleavage - modifies N-terminus with cholesterol

Cleavage occurs between glycine-257 and cysteine-258

• Sulphur on cysteine attacks peptide bonds - converts into thioester
• Hydroxyl group on cholesterol forms ester bond - completes cleavage
• Palmitoyl fatty acid added to N-terminus - Hh hydrophobic at both ends
• Cholesterol embeds Hh in cell membrane

Question 6

Explain why hh is tethered to the membrane

Answer 6

• Restrict activity to immediately adjacent cells

Question 7

Parasegment boundary

Answer 7

• Boundary between stripes is parasegment boundary
• Tethered hedgehog induces diffusible wingless in adjacent cell

Question 8

What occurs when Hh N-domain lacks cholesterol?

Answer 8

Freely diffusible

Switches on more wingless in more cells

Question 9

Explain dispatched Hh from membrane

Answer 9

• Dispatches from membrane, not via cleavage - cholesterol remains
• Other partners required to aid diffusion

Question 10

Explain Patched (Ptc)

Answer 10

Hh receptor

• 12-pass transmembrane domain
• Usually inhibits Hh response
• KO of patched activates Hh response
• Could involve smoothened (Smo)

Question 11

Hedgehog signalling in Drosophila

Answer 11

• Absence of Shh, Ptc acts to sequester Smo in membrane bound vesicles -> degraded
• Ci/Cos2/Fu complex binds microtubules through Cos 2

Ci phosphorylated by PKA

Slimb recognises Ci-P -> Ci targeted to the proteasome release 75kDa Ci75 fragment

Binds target genes, represses transcription

Question 12

What occurs in the Drosophila hedgehog pathway in presence of Hh

Answer 12

• Ptc unable to sequester Smo
• Cos2/Fu recruited by Smo - disassociate from microtubules
• Ci released from complex - can’t be PKA phosphorylated
• Full length Ci binds CBP - activates expression

Question 13

Explain induction of neural fates by Shh

Answer 13

• Nervous system forms ectoderm - overlying notochord
• Folds to make neural tube in neurulation

-Notochord secretes Shh

• Shh induces floor plate
• Floor plate secretes Shh
• Motorneurons specified at high Shh
• V2 interneurons at medium Shh
• V1 interneurons at low Shh

Question 14

Hh signalling and cancer

Answer 14

Components in Hh pathways can mutate

Stem cell proliferation in basal layer of skin stimulated by Hh

Excess Hh - Over-proliferation

• Ptc1 inhibits signalling - tumour supressor
• High frequency of BCC in ptc-/ptc+

Possibility of anti-cancer drugs - inhibitors of Hh

Question 15

Who are at high risk of basal cell carcinoma, and why?

Answer 15

People with Gorlin’s syndrome - mutation in Ptc

• BCC not that serious - treated with surgery
• Cancers shrink when treated with small molecule inhibitors of Hh

Question 16

Structure of Wnt proteins

Answer 16

Vertebrates have multiple Wnt’s between 35 and 45 kDa

N-terminal signal peptide

Cysteine important - forms intra-chain disulphide bonds

Wnt modified with hydrophobic palmitoleane on serine

Addition carried out by porcupine

Question 17

Wnt signalling pathway in absence of Wnt

Answer 17

1. Largely elucidated in Drosophila by looking at mutants
2. Lrp and Frizzled used
3. GSK3/APC/Axin phosphorylate beta catenin
4. Targeted for degradation by Slimb
5. Beta-catenin fully degraded
6. TCF binds DNA and represses targets

Question 18

Wnt signalling pathway in presence of Wnt

Answer 18

Wnt binds Fizzled and Lrp

Phosphorylation of Lrp by conformational structure change

High affinity for Axin sequestering it and disassociation from complex

Degradation of beta-catenin disrupted - No phosphorylation by GSK3

Translocates to nucleus and binds TCF -> repressor to activator

Question 19

What does Nieuwkoop centre do?

Answer 19

Instruct dorsal mesoderm and organizer

Wnt essential to estanlishing centre

Adds beta catenin mRNA to ventral side of Vegetal region -> second Nieuwkoop centre and duplicated axis of development

Question 20

What occurs when Wnt is over-expressed

Answer 20

Cancer

Question 21

What can cause colon cancer and melanoma

Answer 21

Mutations preventing beta catanin degradation

Question 22

Wnt signalling with cancer

Answer 22

Wnt expressed by MC’s in crypt important for ISC proliferation

APC mediates destruction of beta-catenin

Excess Wnt signalling

LOH of APC in patient with null allele -> No beta catenin degradation -> Large numbers of precancerous polyps in colon

• Mutations in Beta-catenin that prevent APC degradation