Lecture 5 Hh and Wnt

Question 1

Name the Hh orthologues

Answer 1

Shh, Desert Hh, Indian Hh

Question 2

Name the Wng orthologues

Answer 2

18 different Wnts

Question 3

Describe the Hh signalling pathway in terms of negative feedback

Answer 3

• Activating pathway, activates patched 1 which will is a negative regulator of the pathway and act on itself to limit the level of activation
• Induction of patched1/patched is one of the best known targets of Hh signalling

Question 4

Name negative feedback in vertebrates/invertebrates

Answer 4

• Negative in vertebrates and invertebrates (patched in drosophila, ptch1 in vert)

Question 5

Positive feedback in vertebrates in Hh signalling

Answer 5

• Gli1 is induced
• Gli1 cannot be proteolyzed into a repressor so it is always an activator of the Hh signal and is a “feedforward” response

Question 6

Describe feedback in drosophila

Answer 6

The induction of patched is also seen in drosophila, but Ci is not induced.

Question 7

Draw out the signalling pathway in Hh

Answer 7

see notes

Question 8

Cyclopamine role in Hh

How discovered

Answer 8

Cyclopamine (chemical that can inhibit smoothened and block the hedgehog signal) discovered as sheep eating that plant, veratrum californicum gave birth to cyclopic lambs

Question 9

What is the TF in the Wnt pathway that is continuously produced

Answer 9

Beta-catenin

Question 10

Decribe steps in Wnt signalling, in absence of ligand

Answer 10

• Beta-catenin is the TF in the wnt pathway and is continuously produced
• It is bound by the destruction complex almost immediately
• Beta catenin is phosphorylated by Gsk3 beta and CK1a
• Phosphorylated beta catenin is recognised by Slimb/BetaTrcp (part of the SCF ubiquitination complex) and ubiquitinated
• Ubiquitinated Beta-catenin is destroyed by the proteasome, freeing up the destruction complex

Question 11

Describe Wnt signalling when ligand bound

1. To which receptors
2. What is recruited
3. This then recruits
4. What is lost from this complex
5. What occurs to beta-catenin

Answer 11

• Wnt binds to its receptors frizzled and arrow/lrp5/6 and brings them together to initiate the signal.
• Dishevelled (Dsh) is recruited to the Fz receptor and phosphorylated.
• This somehow leads to recruitment of the destruction complex, possibly by dsh binding axin, phosphorylation of arrow/lrp5/6 by gsk3b creating binding sites for axin and further phosphorylation events.
• Importantly, it is thought that Slimb is somehow lost form this complex.
• As a result Beta catenin that is produced will still bind the complex
• And become phosphorylated
• But is will not be ubiquitinated and degraded, but it will remain bound to the destruction complex. Thus the system clogs…newly produced beta-catenin will start accumulating
• Beta-catenin will enter the nucleus to displace groucho (transcriptional reporessor) to start activating transcription

Question 12

Role of Wnt signalling in drosophila

Answer 12

Segmentation

Patterning and outgrowth of the wing at the D/V boundary

Question 13

Role of Wnt signalling in C.Elegans

Studied by

Answer 13

Regulation of neuronal fate and migration

Studied by analysing the migration of two neuroblasts: the QRd and QLd cells

Question 14

Describe QLd signalling via Wnt

Answer 14

Wnt signalling in the QLd cell induces a homeobox gene mab5 which induces posterior migration.

Question 15

Ectopic signalling in QLd cell vs loss

Answer 15

Thus in the absence of the signal both cell move anteriorly whereas ectopic activation of wnt leads to both cells migrating posteriorly

Question 16

What cells occupy the villi of SI

Answer 16

• Differentiated enterocytes, enteroendocrine cells and goblet cells

Question 17

What cells occupy the crypt of SI

Answer 17

Paneth cell

Question 18

Where are the stem cells located in the SI

Answer 18

Slightly higher than the Paneth cells

Question 19

Where do stem cells reside in the large intestine

Answer 19

Bottom of the crypt

Question 20

How is the epithelium in both intestines renewed

Describe how

Answer 20

Stem cell division
Stem cells divide slowly but produce rapidly dividing transit amplifying cells thereby creating a continous flow of cells from the crypt upwards.

Question 21

Where are Wnts expressed

Answer 21

Wnts are expressed by the stroma below the intestinal crypts.

Question 22

What happens if Wnt signalling is blocked

Answer 22

If you block wnt signaling it the gut it will cause the loss of stem cells in the colon crypt, indicating that Wnt signalling is required for maintenance of the stem cell compartment.

Question 23

What occurs with ectopic activation of Wnt

Answer 23

Ectopic activation of wnt can cause overproliferation probably due to overproduction of stem cells.

Question 24

How can ectopic wnt signalling occur

Answer 24

Loss of APC

Question 25

What disease occurs from loss of function in APC - patients are homo/hetero - type of mutation - from loss of... - forms...

Answer 25

Patients heterozygous for APC loss of functions mutations suffer from familial adenomatous polyposis. Autosomal dominant From loss of remaining WT copy of APC gene resulting in activation of wnt signaling and increased proliferation Many polyps in colon and rectum

Question 26

APC is described as what type of gene

Answer 26

Tumour suppressor gene

Question 27

How does colon cancer also form

Answer 27

Sporadic | GOF for Wnt

Question 28

Other cancers from wnt pathway

Answer 28

• Also, breast cancer, ovarian and uterine cancers, melanomas, prostate cancer can occur from activation of wnt pathway

Question 29

Name 3 diseases from Wnt

Answer 29

``` • wnt3-tetra-amelia (absence of limbs) • Bone diseases - LRP5 gain of function point mutant - More wnt signal  increased bone density - Other LRP5 loss of function mutations  decreased bone density • Axin2 - Severe tooth agenesis (oligodontia) - Multiple teeth missing ```

Question 30

What is non-canonical signalling

Answer 30

Both Wnt and Hedgehog can work via ALTERNATIVE pathways

Question 31

Name non-canonical signalling in Hh

Answer 31

In myocytes/adipocytes Shh activates Smo-Ca2+-Ampk signaling

Question 32

Describe Hh non-canonical signalling

Answer 32

* Metabolic reprogramming towards aerobic glycolysis – glucose is used as an energy source but without fully ‘burning’ it in the mitochondria * Leads to production of lactate which leaves the cell and will acidify the EC medium * This aerobic glycolysis is also a feature of many tumour cells and this is known as the Warburg effect. * Glucose-> Lactate+ 2 ATP

Question 33

Inhibitors of canonical signalling on Hh non-canonical How discovered?

Answer 33

• Certain inhibitors of canonical Hh signalling activate non-canonical AMPK signaling • Interestingly this effect was also discovered because patients taking hedgehog inhibitors were suffering from serious side effects, including weight loss and muscle cramps. Surprisingly, it was found that certain inhibitors (antagonists) of the canonical hedgehog pathway were in fact activators (agonists) for this noncanonical pathway - (cyclopamine, GDC0449)

Question 34

Describe Wnt non-canonical signalling

Answer 34

* Involved in the planar cell polarity/ Convergent extension pathway * e.g. hairs in a drosophila wing * In Drosophila the involvement of the ligand itself is debated however in zebrafish wnt11 and wnt5 mutants show clear defects in convergent extension movements * Such defects will lead to very short and wide embryos, as the AP axis fails to elongate properly * Other common components are Frizzled and dishevelled but downstream of dishevelled other components are involved and rho and rac appear to be effectors. * Polarises cells within a sheet * Drosophila: hairs on wings * Zebrafish: axis elongation