Lecture 5

Question 1

How was hh first discovered?

Answer 1

discovered as a mutant in drosophila which had no naked cuticle between the bands= is a segment polarity gene

Question 2

How was Wnt discovered and how did it get its name?

Answer 2

The wg gene was activated by the integration of the Lnt1 tumour virus in mice

Question 3

Describe wg’s role in drosophila

Answer 3

Is part of the Wnt family- is a polarity gene required for the segmentation in drosophila. It works in a reciprocal loop with hh- they depend on eachother’s expression= no hh means no wg- they have similar phenotypes when mutated

Question 4

Describe the orthologues of hh and Wnt in drosophila and vertebrates

Answer 4

Vertebrates have shh, dhh and ihh. They have 18 Wnt orthologues and 7 in drosophila. Vertebrates are more complex

Question 5

Describe how hh is produced in the cell

Answer 5

1) Has an N terminus signal sequence that targets it to the secretory pathway
2) It has a proteolytic domain on the C-terminus. Once the signal terminus is cleaved it undergoes auto-proteolysis catalysed by the C terminus
3) cholesterol is added
4) palmityl group is added= very hydrophobic which directs it to the membrane

Question 6

How is hh able to signal over a long distance?

Answer 6

dispatched (12TMD) and scube help load hh into lipoproteins or cytonemes which require HSPGs

Question 7

How is wnt produced in the cell?

Answer 7

1) signal sequence is cleaved once enters secretory pathway
2) palmitoylation at cys77
3) palmitoeic acid modification at ser 209 which makes Wnt insoluble

Question 8

How is wnt secreted?

Answer 8

Put in lipoproteins/ cytonemes with the aid of HSPGs and possible aid of Wntless in the membrane

Question 9

What are cytonemes and give an example on how they work?

Answer 9

Long, thin, cellular protrusions that look like filopodia. They touch Wnt receiving cells- e.g. Wnt8gT-p

Question 10

Describe hh signalling in drosophila

Answer 10

Usually ptc (12TMD) inhibits smo (7TMD)- continuously degrades it. When hh binds to ptc they are both internalised and degraded= allows smo to travel to the cell membrane and be phosphorylated

Question 11

How does mammalian hh signalling differ to invertebrates?

Answer 11

They have cilia which ptc1 sits in and excludes smo from this area- used for signalling as when KO cilia= impaired hh signalling

Question 12

What 2 complexes keep Ci out of the nucleus?

Answer 12

1) Ci+ Costal 2+ fused

2) Ci+ sufu

Question 13

What is complex 1 bound to and how does it repress hh target genes?

Answer 13

It is bound to smo and consists of GSK3, CK1 and PKA. Under the influence of slimb they ubiquitinate Ci which results in its partial proteolysis= shorter form of the TF which actively represses hh target genes

Question 14

How does hh signalling promote activation of hh target genes?

Answer 14

The kinase activity in complex1 is somehow turned off causing the release of active Ci. Also phosphorylation of sufu by fused can result in the formation of active Ci.

Question 15

How is the hh pathway regulated?

Answer 15

1) Negative feedback= ptc is a negative regulator of hh
2) positive feedback= Gli is a product of hh signalling which acts to activate the hh signal and can’t be proteolyzed into a repressor

Question 16

How is the wing disc patterned?

Answer 16

Hh is expressed on the posterior side which diffuses anteriorly- activates decapentaplegic (BMP/TGFB like genes)

Question 17

How is hh used for neural patterning?

Answer 17

It is expressed in the floor plate and notochord and acts as a morphogen to pattern different cell types

Question 18

How does hh pattern the limb bud?

Answer 18

Shh is secreted and produced in the ZPA and induces posterior identity and drives pattern formation of digits

Question 19

What effects does hh loss of function have in terms of disease?

Answer 19

Causes holoprosencephaly which can occur due to cyclopin ingestion which is an inhibitor of smo (hh pathway)

Question 20

What effects does hh overexpression/misexpression have in terms of disease?

Answer 20

It can cause polydactyly or syndactyly. Can cause basal cell carcinoma and other cancers due to ectopic activation- arise due to lack of ptc or sufu

Question 21

Why do people develop gorlin’s syndrome?

Answer 21

people with heterozygous for ptc1 can obtain a mutation in their healthy copy causing tumour formation

Question 22

How can cancers that occur from hh misregulation be treated and what are its limitations?

Answer 22

Can use smo inhibitors which reduces the amount of most cancers but cancer cells acquire resistance due to mutation in smo receptor

Question 23

What happens when Wnt signalling isn’t active?

Answer 23

WNt receptor= frizzled and arrow (drosophila)
Beta catenin is in a destruction complex with APC, axin, CK1 and GSK3- CK1 and GSK3 phosphorylate beta catenin causing it to be recognised by slimb and targeted for ubiquitination where it is degraded by a proteasome

Question 24

Describe canonical Wnt signalling

Answer 24

1) Wnt binds to fz and arrow/LPR5 and 6 and brings them together= causes phosphorylation
2) fz recruits dsh and phosphorylates it causing it to recruit the destruction complex via axin binding sites
3) Slimb will be lost from the complex and beta catenin will still be phosphorylated but not broken down and will remain the in the destruction complex
4) When new b-catenins are produced they accumulate and eventually enter the nucleus and bind to TFC to dissociate groucho and act as a transcriptional activator

Question 25

What are the roles of Wnt signalling?

Answer 25

1) Wg in drosophila is expressed at the D/V boundary of the wing plate for D/V patterning
2) neurons in C.elegans- Wnt activates mab 5 homeobox in Qld neurons which cause it to migrate posteriorly and causes Qrd to migrate anteriorly- KO= move anteriorly, ectopic expression= move more posteriorly
3) are embryonic lethal if mutant vertebrate

Question 26

Describe Wnt’s role in intestinal homeostasis and describe the intestinal cells

Answer 26

Have a crypt with slow dividing stem cells and Paneth cells at the very bottom. endocrine, enterocytes and goblet cells are at the top of the villus and rapidly dividing transit amplifying cells (from stem cells) move upwards. No wnt= no renewal/ migration. Ectopic expression= overproliferation

Question 27

How does wnt cause cancer and give an example?

Answer 27

Over activation of the wnt pathway results in multiple polyps in the crypt of intestines e.g. familial adenomatous polyposis- caused by loss of WT APC (TSG).

Question 28

Give 3 examples of diseases caused by disruptions in Wnt signalling

Answer 28

1) Loss of limbs due to mutations in Wnt3a
2) Higher bone density due to Lrp5 gain of function mutation
3) familial tooth agenesis caused by loss of function mutations in axin2

Question 29

Describe the non-canonical hh pathway and how it was discovered

Answer 29

Inhibition of canonical hh pathway activates non. Hh activates smo bound to Ca2+ in myocytes and adipocytes which activates the AMPK pathway- stimulates anaerobic glycolysis which acidifies the EC medium. Discovered due to acquired muscle cramps and weight loss in patients that were taking hh inhibitors

Question 30

What is non-canonical wnt signalling involved in and how was this discovered?

Answer 30

Planar hair cell polarity (direction of hair growth) and convergent extension movements. Zebrafish wnt 11 and 5 mutants displayed short and wide embryos due to defect in extending A/P axis