L19: Hedgehog and Wnt signalling

Question 1

Discovery of segment polarity in Drosophila

Answer 1

• Nusslein-Volhard and Wieschaus screened mutagenased Drosophila to identify mutations that acted on particular aspects of dev
• Wt larvae have denticles on anterior half, smooth on posterior half
• Identified a grp of mutants that affected the patterning within the segments but left the number of segments unaltered (Hedgehog and wingless)

Question 2

Basics of the Hh in Drosophila

Answer 2

• 3 Hhs in vertebrates (Sonic, Indian, Desert)
• Secreted signalling proteins
• Modified by cleavage and addition of cholesterol to N-terminal region (processing is autocatalytic)
  -> inactive precursor

Question 3

Processing of Hh

Answer 3

• Cleavage occurs between Glycine-257 and Cysteine-258
• Sulphur on Cysteine side chain attacks peptide bond, converts into thioester
• Hydroxyl grp on Cholesterol can then form an ester bond -> completes cleavage
• Palmitoyl fatty acid addition to N-terminus makes Hh hydrophobic at both ends, embedded in cell membrane
  -> restricts activity to immediately adjacent cells

Question 4

How do wg and hh interact?

Answer 4

• 2-way induction
• Wingless’s role is to switch on expression of hedgehog in the adjacent stripe
• Parasegment boundary found between the two segments; tethered Hh induces diffusible Wg in the adjacent cell
• Ensures adjacent stripes at the crucial stage where the segment boundaries must be demarcated (accommodates movement of cells in and out of the boundary while still maintaining the demarcation)

Question 5

Role of Dispatched protein

Answer 5

• Extracts Hh from PM (NOT cleavage; cholesterol intact)
• Other partners required to aid its diffusion

Question 6

Hh receptor

Answer 6

• Patched (Ptc); 12 pass TM protein, inhibits Hh response
  -> KO of Ptc activities activates Hh response
  -> known to indirectly involve Smoothened rec.

Question 7

Hh signalling pathway - in absence of Hh

Answer 7

• Ptc acts to sequester Smo in membrane bound vesicles in cytosol to be degraded
• Ci phosph. by PKA (part of Ci/Cos2/Fu complex bound to m.tubs)
• Allows Slimb to recognise pCi, Ci75 (75 KDa) released after targeting for degradation by proteosome
  -> binds target gene, represses tr.

Question 8

Hh signalling pathway - in presence of Hh

Answer 8

• Hh ligands binds and inhibits Ptc; Smo not sequestered, can move to PM
• Cos2/Fu recruited by C-terminal of Smo, dissociating from m.tubs
• Ci released, not phosh. by PKA
  -> Full length Ci binds CBP and activates expression

Question 9

Induction of neural fates by Shh (in the formation of the Nervous System)

Answer 9

• NS forms from ectoderm overlying the notochord; folds to make the neural tube in a process called neurulation
• Notochord secrets Shh; Shh induces floor plate
• Floor plate secretes Shh…
  HIGH: Motoneurons
  MED: V2 interneurons
  LOW: V1 interneurons
• Process can be recapitulated in neural tube explant culture

Question 10

Hh and cancer

Answer 10

• Basal cell carcinoma
• Misregulation results in over-proliferation in basal layer of skin
• Excess in Hh
• As Ptc1 inhibits Hh signalling it must act as a tumour suppressor (heterozygous ptc mutations lead to BCC)
  -> potential for anti-cancer drugs that target Hh signalling pathway
• NOT very deadly

Question 11

Wnt general structure

Answer 11

• Small secreted signalling protein; amalgamation of Wg and Int
• Conserved cysteine (intra-chain disulphide bonds), conserved serine (pamitoleate attachment by Porcupine)
• Modified w/ hydrophobic fatty acid

Question 12

Wnt signalling in absence of Wnt

Answer 12

• GSK3/APC/Axin scaffold phosph. b-catenin
• Targeted for degradation by Slimb (complete, NOT partial)
• TCF in nucleus binds DNA, represses targets

Question 13

Wnt signalling in presence of Wnt

Answer 13

• Wnt binds Frizzled and Lrp; conformational change -> phosph. of Lrp
• Becomes high affinity binding partner for Axin sequestering it and dissociating the complex
  -> no degradation of b-catenin as GSK3 is not phosph.
• b-Catenin translocates to nucleus, binds TCF -> repressor to activator

Question 14

Evidence of Wnt in NK centre induction

Answer 14

• Adding b-catenin mRNA to the ventral side of vegetal region
  -> 2nd NK centre and duplicated axis of development

Question 15

Wnt signalling in cancer

Answer 15

• Overexpression of Wnts can cause cancer
• APC is a tumour suppressor; heterozygotes develop large numbers of polyps in colon
• Mutations which prevent b-catenin degradation found in colon cancer and melanoma
• V. deadly type of cancer

Question 16

ISC function (and maintenance by MCs)

Answer 16

• Colon is an epithelium maintained by ISCs
• Crypt; Intestinal stem cells sit at bottom, divide infrequently, producing ‘transit amplifying cells’ (found in proliferative zone)
  -> new cells pushed up from the base, old cells lost form top of crypt
• Mesenchymal cells surround epithelium (mesodermal origin); maintain ISC population by producing Wnt and BMP)

Question 17

Formation of polyps (Wnt signalling mutation in colon)

Answer 17

• APC (adenomatous polyposis coli) mediates destruction of b-catenin
• Loss of APC results in excess Wnt
• LOH of APC in patients w/ null allele
  -> no b-catenin degradation, over-proliferation of TACs, large no.s of pre-cancerous polyps in colon
• Also produced by mutations in b-catenin that prevent degradation of APC

Question 18

Holoprosencephaly and Shh

Answer 18

• Deficiency in Shh signalling pathway
• Embryonic forebrain fails to sufficiently divide
  -> single-lobed brain structure, severe skull and facial defects

Question 19

Notum in Wnt signalling

Answer 19

(Kakugawa et al.)
- Notum inhibits Wnt signalling; removes acyl group which is crucial in binding to Frizzled rec.