lecture 32

Question 1

What have mutations in Wnt/PCP signalling revealed?

Answer 1

• 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

Question 2

What is Dishevelled?

Answer 2

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

Question 3

What is Wnt/PCP signalling?

Answer 3

• 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

Question 4

What are the cytosolic proteins associated with Stbm and Fmi?

Answer 4

• Pk

Question 5

What are the cytosolic proteins associated with Fz and Fmi

Answer 5

• Dsh/Dvl and Dgo

Question 6

What is the Wnt-PCP pathway?

Answer 6

• 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

Question 7

What are mutations in Wnt/PCP signalling?

Answer 7

• 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)

Question 8

How do we identify PCP components?

Answer 8

• 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

Question 9

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

Answer 9

• 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

Question 10

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

Answer 10

• 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

Question 11

What can be summised based on these studies?

Answer 11

• 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)

Question 12

What specifies A-P axis?

Answer 12

organiser transplant experiments show that:

• properties of the organiser change with time
• early organiser induces head structures
• late organiser induces tail structures

Question 13

What does the organiser secrete?

Answer 13

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

Question 14

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

Answer 14

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

Question 15

What do BMP antagonists do?

Answer 15

• 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

Question 16

What is the hypothesis of gradients and the Hox code?

Answer 16

• 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

Question 17

What is L-R patterning?

Answer 17

• 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

Question 18

What is the importance of mutations in cilia genes?

Answer 18

• 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

Question 19

What are cilia defects in humans?

Answer 19

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)

Question 20

What is the consequence of mutations in cilia genes?

Answer 20

• 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

Question 21

What is nodal flow and nodal expression?

Answer 21

• 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

Question 22

How does L-R patterning occur in the chick?

Answer 22

• 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

Question 23

What does activin do?

Answer 23

• antagonises Shh on the right hand side

Question 24

What is the main pathway on the left hand side?

Answer 24

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

Question 25

What leads to asymmetric distrubution?

Answer 25

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