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Flashcards in lecture 32 Deck (25)
1

What have mutations in Wnt/PCP signalling revealed?

- Wnt signalling has been implicated in convergent extension
- pathway does not involve beta-catenin
- involves the non-canonical Wnt/Frz/Dvl
- activation of Dvl but on different domains
- Wnt/PCP pathway regulates cytoskeleton and Jnk
- mutations in PCP pathway disrupt convergent extension and gastrulation
- Rho/Rac tends to modify actin cytoskeleton
- gene transcription via JNK (JNK is a MAPK)
- these modifications are what is involved in planar cell polarity formation

2

What is Dishevelled?

- activated in both canonical and non-canonical wnt pathways
- DIX and PDZ domain activated in canonical
- PDZ and DEP involved in non-canonical

3

What is Wnt/PCP signalling?

- Wnt/PCP pathway is complex
- antagonistic to Wnt/Beta-catenin signalling
- NB: results in asymmetric distribution of transmembrane and cytoplasmic proteins
- e.g. Fz and Fmi or Stbm and Fmi
- as a consequence different cytosolic proteins associating with these transmembrane proteins


- range of transmembrane proteins in PCP e.g. flamingo, Vangl2, coreceptor: knypek
- whole complex of downstream proteins discovered
- Dvl interacts with Rac and Rho --> cytoskeletal modification through other small GTPases such as Cdc42
- can activate gene transcription via JNK
- one of the hardest pathways to figure out because doesn't always involve gene transcription
- time lapse photography

- this asymmetric distribution critical to knowing left and right

4

What are the cytosolic proteins associated with Stbm and Fmi?

- Pk

5

What are the cytosolic proteins associated with Fz and Fmi

- Dsh/Dvl and Dgo

6

What is the Wnt-PCP pathway?

- specific Wnt-Fz combinations activate PCP pathway
- Van Gogh/Strabismus
- Dishevelled recruits Daam1, which activates RhoA/Rok (or Rac)
- results in cytoskeletal re-arrangements
- pathway also activates Jnk to regulate gene expression (targets not known)
- main effects seem to be via cytoskeleton and regulation of membrane protein localisation

7

What are mutations in Wnt/PCP signalling?

- Wnt/PCP pathway mutants (e.g. trilobite/stbm) fail to extend A-P axis
- mutations in PCP pathway disrupt convergent extension and gastrulation movements (but not cell fate)
- mouse orthologue (Vangl2) mutation has defects of tail (looptail)

- silberblick (slb) = wnt11 mutant
- pipetail (ppt) = wnt5 mutant
- subtle convergent extension phenotype
- double mutant leads to more servere phenotype (--> act redundantly in Wnt-PCP signalling pathway)

8

How do we identify PCP components?

- mutants are often identified by expression patterns and that they phenocopy and interact with slb (wnt11) and ppt (wnt5) mutants
- e.g. def6, a novel GEF that regulates Rho GTPase
- experiments involve injecting capped mRNA or morpholinos (antisense)
- def6 morpholinos: phenotype becomes more and more severe as you inject more

9

Does knock-down of the candidate gene rescue or synergise the slb (wnt11) or ppt (wnt5) phenotypes?

- injection of Def6 morpholino (MO) synergises the CE defects observed in wnt11 morphants (equivalent to slb mutant)
- what does this tell you about def6?
- synergises with wnt11 and therefore probably functions in that pathway

- mild phenotype when only one or the other
- more severe phenotype when both

10

Does over-expression of the candidate gene rescue the slb (wnt11) or ppt (wnt5) phenotypes?

- injection of capped Def6 mRNA rescues the CE movement defects observed in Wnt5b morphants (equivalent to ppt mutant)
- not complete rescue
- therefore downstream
- novel player is this signalling pathway

11

What can be summised based on these studies?

- hypothesize that def6 acts on RhoA to increase its activation
- increased RhoA activity activates downstream PCP signalling

- def6 is a GEF
- would this approach work for RhoA itself?
- if you over-express RhA would you get effects? (probs not)

12

What specifies A-P axis?

organiser transplant experiments show that:
- properties of the organiser change with time
- early organiser induces head structures
- late organiser induces tail structures

13

What does the organiser secrete?

- Wnt/BMP antagonists
- XWnt8 an BMPs can ventralise mesoderm
- head organiser (mesendoderm) tissue expresses Frzb (Wnt antagonist)
- trunk organiser (notochord) expresses chordin (BMP antagonist)

14

In the A-P axis, what are Wnt/BMP antagonist gradients?

Wnt and BMP antagonists:
- specify head and trunk tissues
-- head - low Wnt signals
-- trunk - low BMP signsl
-- tail - high Wnt signals
- notochord-derived BMP antagonists induce the neural tube (neurulation)

- posterior to anterior gradient of wnt
- ventral to dorsal gradient of BMPs

15

What do BMP antagonists do?

- neurulation

A. normal neurula embryo showing formation of neural tube following gastrulation
B. depletion of all BMP antaginists (chordin, noggin) prevents specification of neural tissue
C. depletion of BMPs expands neural tissue specification
D. depletion of all BMPs coverts all ectoderm to neural tissue

Sox2 = early neural marker

wnt/bmp antagonism specifies neurulation in the overlying ectodermal tissue

16

What is the hypothesis of gradients and the Hox code?

- posterior-anterior gradients of Wnt, BMP, FGF and retinoic acid are thought to activate the Hox genes (via Cdx genes)
- some evidence from chick and mouse
- mechanisms not yet clearly understood

17

What is L-R patterning?

- in the developing chick embryo, asymmetric expression of genes
- cerberus (secreted protein), nodal (secreted protein), Pitx2 (tf)
- L-R asymmetry is particularly important for our internal organs

18

What is the importance of mutations in cilia genes?

- mutations in various genes, particularly cilia-related genes lead to randomisation of L-R axis
- cilia, present in the "node" (node is analagous to blastopre lip, organiser tissue) of mouse and chick, function to control L-R axis
- mouse mutant (situs inversus viscerum, iv) --> immotile cilia (dynein gene)
- experiments reversing ciliary flow in the node, reverse L-R axis

19

What are cilia defects in humans?

respiratory epithelium - cilia
- genetic defect of cilia (dynein genes) --> immotile cilia
--> recurrent respiratory diseases in children
- 50% of Kartagener's syndrome patients have situs inversus (organs on the wrong side)

20

What is the consequence of mutations in cilia genes?

- cells in the mouse node produce vesicles (NVP) that contain Shh, retinoic acid
- release stimulated by FGF and moved by vortical movement of central cilia to LHS
- NVP stimulate calcium release on LHS - mechanosensors (peripheral cilia)

- either growth factor mediated
- or mechanosensor that detect flow that release calcium

21

What is nodal flow and nodal expression?

- lhs
- beating nodal cilia (xenopus, zebrafish, mice, humans) in node drive asymmetric distribution of growth factors or activate calcium channels on LHS
- asymmetry then induces activation of nodal expression, which results in asymmetric development (Pitx2)

- activinn and sonic hedgehog on the right hand side

- pitx2 required for heart development on the left hand side of the embryo

22

How does L-R patterning occur in the chick?

- no cilia detected in the chick primitive streak - passive mechanism?
- asymmetric distribution of H+/K+ATPase - mechanism unknown
- leads to membrane depolarisation and Ca2+ release
- activates Notch signal and nodal expression on LHS
- activin signalling on the RHS antagonises shh; Shh/Nodal on the LHS

- development of two mutually antagonistic signalling cascades
- RHS: BMP/Activin --> FGF (right side programme) but antagonises Shh
FGF8 antagonises cerberus
- LHS: shh/BMP --> Nodal --> Pitx2 (left side programme)
- Midline: Lefty maintains the L-R boundary

23

What does activin do?

- antagonises Shh on the right hand side

24

What is the main pathway on the left hand side?

- Shh
--> nodal
--> Pitx2
--> correct heart looping

25

What leads to asymmetric distrubution?

- nodal monocilia beating to create vortical flow --> unidirectional fluid flow