Lecture 17- Neural crest III

Question 1

Summary of neural crest cells so far, so lectures I and II?

Answer 1

• Methods that can be used to track neural crest while they migrate
• Cranial neural crest: Cranial ganglia neurons and glia (with placode cells), Schwann cells. Mesenchymal derivatives: connective tissues such as bone and cartilage of the face. Pigment cells.
• Vagal neural crest: overlaps with cranial and trunk. Gives rise to cardiac neural crest and enteric neural crest.
• Trunk neural crest: Ventrally migrating cells form neurons and glia in dorsal root and sympathetic ganglia, Schwann cells, adrenal medulla and pigment cells. Dorsolaterally migrating cells form pigment cells.
• Sacral neural crest: neurons and glia in parasympathetic ganglia and some enteric neurons in colon.

Question 2

What sort of molecules do neural crest cells use for cell-cell adhesion? (this is 3. Cell-cell adhesion and interaction)

Answer 2

• neural crest cells express a variety of receptors to interact with other cells and extracellular matrix
  a) cadherins: require Ca2+ to brind to another cadherin= Homophilic binding
  b) integrins-binds to things on the cells like laminin or Fibroconectin= Heteriophilic binding
  c) CAMS= Homophilic binding

Question 3

What is the evidence that neural crest cells need cell-cell interaction and some adhesion to migrate?

Answer 3

• Neural crest cells interact with each other and the environment.
• Time lapse shows many neural crest cells migrate in chains (have some connections but not too tight)
• During migration the neural crest cells make transient connections with each other, and can signal to each other(they swap partners during migration
• can use these connections to signal to eachother, can also exchange cytosplasmic material= give each other information)

-Neural crest cells can even exchange cytoplasmic material during these connections.

• must have some level adhesion to migrate, too much is bad!
• transient connections are optimal

Question 4

What sort of migration is observed in neural crest cells?

Answer 4

• chain like migration= observed in many part of neural crest mogration= in cranial and migration along the gut
• cranial migration in chicken embryo= come out of neural tube, work out direction, first cells= pioneering cells (have different gene expression to the cells behind them= so info passed between)
• leading= high levels of integrin and matrix proteases
• follwoing= Ncam and cadherins higher= these are the adhesion molecules
• communication by touch, cell contact to follow neighbour, cell contact with ectoderm
  1. cell acquire direction after exiting the neural tube
  2. Cell-cell contact maintainst directed migration (high expression at Ncams, Fgfrs, Cadherins, EphAs)
  3. Cell-induced gradient drives directed migratuin (high expression of VEGFr2, MMP2, Integrings, Adam33)

Question 5

What is the example of cell-cell adhesion and interaction we have to know?

Answer 5

-many adhesion molecules expressed by cells in the gut, one is L1-CAM

• incubating with anti-L1 antibody, inhibits L1CAM, then migration is slower and neural crest cells don’t migrate as far as they should and chain formation is lost
• more single, unconnected cells in the culture

Question 6

What is the example we need to know for growth factor signalling and its effects on neural crest migration?

(4. Growth factor signalling)

Answer 6

• growth factor signalling affects neural crest migration
• GDNF-Ret signalling is neccessary for enteric neural crest migration along the gut
• GDNF binding to the GFRa1 receptor activates Ret

(GDNF= glial cell line derived neurotrophic factor; GFRalpha1=ligand binding molecule, Ret= receptor tyrosine kinase)

• GDNF binds to receptor on neural crest cells in the gut GFRapha1= that activates Ret
• Ret undergoes phosphorylation and contributes to lot of intracellular signalling

Question 7

What role does GDNF signalling play in enteric nervous system development?

Answer 7

• GDNF is expressed by the gut mesenchyme
• Ret and GFRalpha1 are expressed by enteric neural crest cells
• GDNF or Ret knockout mice have no enteric neurons
• Ret +/- mice have no defects in the enteric nervous

system

• Some humans with mutations in a single copy of Ret have Hirschsprung’s disease
• Ret GDNF signalling is crucial to enteric neural crest migration
• crucial for their survival
• differences in sensitivity in mice and humans, mice heterozygous for Ret are fine, some humans do have Hirschprung’s disease

Question 8

How does GDNF signalling affect enteric neural crest? (4)

Answer 8

GDNF signalling in the gut affects enteric neural crest:

1. Migration
2. Survival
3. Proliferation
4. Neuronal differentiation

Question 9

What type of cells are neural crest cells?

Answer 9

-mesenchymal

Question 10

Why is cell matrix adhesion required for neural crest migration?

(5. Cell-matrix adhesion)

Answer 10

• As mesenchymal cells, neural crest interact with the 3-dimensional matrix, which contains many types of extracellular matrix molecules.
• Examples of extracellular matrix are fibronectin, collagen and laminin.
• Molecules in the matrix can be attractive, permissive or inhibitory to migration, depending on the receptors expressed by the neural crest.

Integrins are transmembrane proteins that interact with many extracellular molecules.

-lot of the receptors here are integrins,
pioneer cells express higher level of integrins

Question 11

What determines what Integrins bind to?

Answer 11

• What alpha and beta chains they have
• integrins interact with the cytoskeleton and pathways that can affect cell survival, migration etc.

Question 12

What is the example of cell-matrix adhesion we have to know?

(5. cell-matrix adhesion)

Answer 12

• loss/ or lack of beta1 integrin impedes neural crest migration along the hindgut, neural crest cells do not reach the anal end
• loss of beta1 integrin inhibits ability to migrate in fibronectin/tenascin C matrix
• the mouse hindgut is rich in fibroconectin and tenascin C
• tenascin C inhibits migration of enteric neural crest (stop signal)
• fibroconnectin stimulates migration of enteric neural crest cells= go signal (but not if integrin beta1 knockouts)
• in wild-type overall effect is to promote migration through the hindgut
• in beta1 integrin mice, migration doesn’t occur, this is as the beta1 integrin binds to fibronectin and promotes the migration

Question 13

How does matrix metalloprotease activity affects neural crest migration?

(6. Matrix metalloprotease activity)

Answer 13

1. Matrix metalloproteases (MMPs) cleave or digest proteins, including extracellular matrix molecules and cell adhesion molecules.
2. Examples are ADAM proteins and MMPs.
3. MMP activity is also involved in the EMT process.
4. During cranial neural crest migration in Xenopus, ADAM13 activity is essential for migration.
   - MMP are involved in getting away from the neural tube
   - example is ADAM 13= essential for migration of neural crest in Xenopus

Question 14

What are ADAMs and what can they do?

(6. Matrix metalloprotease activity)

Answer 14

• transmembrane proteins with multiple domains, including metalloproteinase (MP) and cytoplasmic domains (CD)
• ADAM13 can cleave both cadherin-11 (cell-cell adhesion molecule expressed by neural crest) and fibronectin (extracellular matrix).
• ADAM13 also acts intracellularly in Xenopus cranial neural crest.

Question 15

What happens when you knock ADAM13 out in a Xenopus embryo?

Answer 15

• Knocking down ADAM13 by morpholino (MO13) inhibits neural crest migration.
• Separating the neural crest from the mesoderm and ectoderm partially rescues this inhibition (to test if ADAM13 is cleaving the pathway for the neural crest cells to go through, they did it manually, created space for the neural crest cells= partially rescued the neural crest migration)
• Therefore, one action of ADAM13 is to cleave/remodel extracellular matrix (eg fibronectin).

Question 16

What can rescue the neural crest migration in ADAM13 knockouts?

Answer 16

• Neural crest migration is blocked by morpholinos to ADAM13 and ADAM19 (2MO) (then migration blocked almost completely 70% block)
  1. Neural crest migration is partially rescued by the cytoplasmic portion of ADAM13 (C13) (can rescue, by adding the cytoplasmic domain of ADAM13 (and the morpholinos of blue bit) from 70% to 40% inhibition)
  3. Neural crest migration is rescued by the cytoplasmic portion of ADAM13 and the cleaved domain of cadherin 11 (EC1-3). (this is the extracellular domain of cadherin 11 (the one that ADAM normally cuts and frees it up))

Question 17

What are the roles of ADAM13 in Xenopus cranial neural crest migration? (3)

(6. Matrix metalloprotease activity )

Answer 17

1. Cleaves/remodels extracellular matrix (fibronectin):cutting fibronectin,
   - ADAM13 clears space for the cells to migrate
2. Cleaves cadherin-11 (reduce cell- cell adhesion? Signalling?): this helps to reduce cell-cell adhesion
3. Intracellular signalling -regulates transcription of other genes:cytoplasmic domain, (ADAM13 gets cleaved by another enzyme) and the c domain regulates transcription of other genes

Question 18

What is the contact inhibition of locomotion? (7. contact inhibition of locomotion)

Answer 18

• contact inhibition of locomotion is involved in the initial migration of cranial neural crest in Xenopus
• Contact inhibition of locomotion results in cells retracting their processes and change direction following contact with each other. This results in the cells moving away from each other.
• only seen in initial migration of Xenopus (not seen in other species)
• when cells contact each other, at point of contact signalling= causes processes that make them move away from each other= results in cell dispersal

Question 19

What does this show?

Answer 19

• the cells at the front= not inhibited at the front
• the following ones inhibited
• where no point of contact the cell can move

(contact inhibition of locomotion)

Question 20

Between what cells does contact inhibition of locomotion occur (Xenopus only)?

Answer 20

• between neural crest cells (homotypic) but not between neural crest cells and other cell types (heterotypic).
• has to only work between neural crest cells, wouldn’t work if they ran away from all the cells they encounter
• neural crest cells do not migrate over each other, they do over other cells

Question 21

What are the molecular basis of contact inhibition of locomotion in cranial neural cells of Xenopus?

Answer 21

• PCP pathway is involved in contact inhibition of locomotion
• can block PCP using a dishevelled protein (dominant negative form)
• to prove that PCP is involved in the contact inhibition
• have protrusions where free space, when block dishevelled have protrusions in every direction

test:

1. Expressed a dominant negative form of dishevelled to inhibit PCP pathway
2. In control explants, only leading cells were highly polarized
3. Cells expressing the dominant negative form of dishevelled were not polarized and extended protrusions in all directions

Question 22

What happens when cells contact each other (Xenopus only)?

Answer 22

• neural crest cells are polarised by contact with one another, this leads to cells dispersion
• N cadherin= cell adhesion: inhibit protrusions Rho A

Question 23

Summary of neural crest migration:

Answer 23

• Multiple mechanisms are involved in neural crest migration:
• Different populations of neural crest have different goals/requirements. (cranial and trunk: populate discrete locations. Enteric: populate as migrate)
  
  • The idea of contact-inhibition of locomotion appears to contradict the role of cell-cell adhesion, and chain migration.
• Contact-inhibition of locomotion may be specific to some species (Xenopus), whereas in other species or populations cell-cell adhesion and guidance is more critical.
• Activation of receptors in different regions of a cell leads to activation or repression of particular signalling pathways.
• This can lead to extension or retraction of particular lamellipodia, adhesion to a substrate or cell, and ultimately affects the direction of cell migration.

Question 24

What factors influence the differentiation of neural crest?

Answer 24

Environment/gene expression prior to migration:

a) rostrocaudal differences
b) differences between neural crest cells emigrating from the neural tube (timing of migration)

Environmental factors encountered during migration or at the target site:

• growth factors or signals from adjacent tissues

Question 25

What is an example of a difference in gene expression in neural crest cells at different rostrocaudal levels?

Answer 25

• Hox genes
• Neural crest at different rostrocaudal/anterior-posterior levels express different genes prior to and during migration.
• Neural crest at different rostrocaudal levels contribute to different tissues.
• Can neural crest from different rostrocaudal levels compensate for each other? Can you put them in each other’s locations? Will they do what they should?

Question 26

True/false: Neural crest from different rostrocaudal levels have different intrinsic abilities

Answer 26

• true
• Use chick/quail chimaera technique to transplant segments of trunk neural crest (neural tube) to the vagal or cranial level.
• Trunk neural crest (red) at the vagal level contribute to the vagus nerve (vn) but not enteric ganglia: weren’t able to do everything!
• differences in the plasticity of cranial and trunk neural crest cells

Question 27

What happens when you tranplant cranial neural cres cells to the trunk?

Answer 27

• form normal trunk derivatives such as dorsal root ganglia and sympathetic ganglia
• form connective tissue and ectopic cartilage (not supposed to do that here)
• so they can form some of the things trunk neural crest cells should but also create some that cranial neural crest cells should

Question 28

What happens when you tranplant trunk neural cres cells to cranial levels?

Answer 28

• when mixed with cranial neural crest migrate appropriately (not on their own)
• differentiate into a variety of tissues
• do not form normal cranial neural crest structures such as appropriately patterned ganglia and skeletal structures.
• more limited in their ability than the cranial neural crest cells

Question 29

What determines the identity of trunk neural crest derivatives?

Answer 29

• Trunk neural crest give rise to derivatives depending on the timing of migration
• The timing of migration of trunk neural crest correlates with their final destination.
• changes in gene expression depending on the pathway tehy are in
• qRT-PCR of cells migrating in different pathways showed differences in gene expression to cells just emerging from the neural tube.
• There are several differences in gene expression between cells in the dorsolateral pathway and those migrating in the ventral pathway prior to emigration (eg: c-kit in mice, loss of FoxD3).
• Early migrating cells (in the ventral pathway) express neural markers ectopically then undergo apoptosis when they are grafted into the dorsolateral (melanocyte) pathway.
• Ventrally migrating neural crest display some plasticity in fate when challenged.

Question 30

What is the plasticity of neural crest like?

Answer 30

• progenitors respond to local cues (restrictions but also some plasticity)
• Early migrating cranial neural crest form connective tissues in branchial arches.
• Late migrating cranial neural crest form neurons and glia in the trigeminal ganglia.
• Early neural crest transplanted to late neural crest and vice versa differentiate appropriate to their location.
• But by 24 h after arrival in their location they lose this plasticity.(after that they are restricted, cannot change)
• Therefore, cranial neural crest display multipotency (as a population) and become fate-restricted by the environment of their final location.

Question 31

What is special about neural crest cells along nerves?

Answer 31

• multipotent
• cranial neural crest, parasympathetic ganglia
• do not form those if nerves are missing
• looks like the neural crest cells go along the nerve to form the ganglia
• Cranial neural crest along nerves give rise to cranial parasympathetic ganglia. The ganglia are absent when the nerves are missing.

Question 32

How might the navigation of neural crest cells along nerves happen?

Answer 32

• time lapse of how this may be happening
• nerve first and cells migrate along the nerve, then change their gene expression as they go along
• and as they finish migration, change the gene expression again

-initially multipotent progenitor cells, then make a decision, ganglion or schwann cell and that is it

Question 33

What will become of neural crest cells that remain associated with nerves and those that don’t?

Answer 33

• In the trunk, neural crest that remain associated with nerves will form Schwann cells. Neural crest that become separate from the nerve in the skin form melanocytes.
• again just showing that they are progenitors initially , then decide fate based on environment

Question 34

What controls the differentiation of neural crest?

Answer 34

• Neural crest cells can be destined to migrate to particular destinations and consequently give rise to particular derivatives based on their rostrocaudal level and the timing of migration.
• Trunk neural crest cells cannot substitute for cranial or vagal neural crest. Ventrally migrating trunk neural crest cannot survive in the dorsolateral pathway.
• But, many neural crest display plasticity of cell fate. Neural crest cells can respond to their environment and changes in the environment and differentiate appropriately.

Question 35

How do neural crest cells determine size and shape of facial features?

Answer 35

• by carrying specific-specific information
• Neural crest cells in the branchial arches are induced to make skeletal structures by signals from the environment (overlying epithelium)
• Reciprocal interspecies transplantations of beak neural crest cells between quail and duck embryos

Question 36

What was the experiment with quail and duck facial features?

Answer 36

• Neural crest cells carry species specific information that determines size and shape of facial features
• Quail neural crest cells in duck embryos (“qucks”) exhibited quail-like beaks
• “Duails” developed beaks typical of ducks

Question 37

What are the neural crest stem cells?

Answer 37

• have been found and observed, in some places where migration is finished they remain stem cell like= cells in skin
• Neural crest cells may not fit the true definition of stem cells, perhaps best described as progenitor cells.
• When challenged, many neural crest cells display plasticity, and are multipotent.
• Neural crest stem cells have been isolated from embryonic and adult tissues, such as teeth, dorsal root ganglia and skin.
• NCSCs have also been derived from human and mouse embryonic stem cells.
• These cells can be cultured, differentiated into a variety of cell types (neurons, glia, pigment cells, cartilage etc) and tested for use in treating diseases.