Neural Development

Question 1

How is a coordinate system set up in the maternal oocyte?

Answer 1

All continuous gradients:

Dorsal-ventral: ‘dorsal’ molecule concentrated at ventral side

Anterior-posterior: ‘bicoid’ at anterior; retinoic acid (RA) and FGF at posterior.

Animal-vegetal: specific transcripts (e.g. in Xenopus = Vg1)

Question 2

What are the stages of developing a combinatorial code in an embryo?

Answer 2

1. Maternal cytoplasmic polarity
2. Gap genes
3. Pair-rule genes
4. Segment polarity genes and homeotic (Hox) genes.

Encouraged by the French flag model

Question 3

What is the French Flag model? How is it achieved?

Answer 3

Where the difference in concentration of a substance is unimportant until a threshold reached.

• Switches on combinations of TFs
• Resolution increased by : +ve feedback on itself and -ve feedback on its neighbour.

Question 4

Outline the stages of nervous system development (rough):

Answer 4

1. Gastrulation allowing mesoderm creation
2. Invagination to form neural tube
3. Induction of the floor plate
4. Separation of mesencephalon (brain) from spinal cord.

Question 5

What are the molecules involved in setting up a polarised neural tube dorso-ventral?

Answer 5

Ventral:
- BMPs
- Activin
- Dorsalin

Dorsal:
Notochord induces floor plate which produces:
- Shh (binds patched receptor)
- Anti-BMPs (noggin; chordin; follistatin) –> These bind BMP molecules stopping them bind their receptor; reducing activation of TGF-β

Question 6

What are the molecules involved in setting up a polarised neural tube anterior-posterior?

Answer 6

At dorsal posterior:
- Wnt highest conc.
- Binds frizzled receptor

At dorsal anterior:
- Sfrp1 highest

At ventral anterior:
- Wnt4 and 7b

Question 7

What are neural crest cells and what do they form?

Answer 7

Collection of multipotent stem cells formed proximal to the neural tube and epidermis (driven by FGF signalling)

Form:
- Neuronal cells (enteric gut neurons; sympathetic ganglia; glial cells; sweat glands)
- Non-neuronal cells (pigment cells; cartilage cells; skeletal elements like teeth)

Shown by ABCD syndrome = albinism, gut neuron disorder, deafness….

Question 8

What is the experimental evidence for the creation of a dorsal-ventral axis in an embryo?

Answer 8

• Grafting a second dorsal tip on an embryo induces a second neural axis
• Grafted tip able to recruit new cells
• BMP at dorsal; sonic hedgehog (shh) at ventral

Question 9

What evidence led to the discovery of neural inducing genes?

Answer 9

• UV light inhibits development of dorsal structures (including neural)
• Lithium inhibits development of ventral structures (hyperdorsal)
• mRNA extracted from hyperdorsal embryo can ‘rescue’ UV treated one

Led to discovery of neural genes: noggin and chordin

Question 10

What is the general formula for how a signalling molecule induces a response? Use RA as an example.

Answer 10

1. Signal
2. Receptor
3. Pathway
4. TF (genes)

E.g. RA -> RAR -> pathway -> RARE (response element) modulating hox gene transcription.

Question 11

What evidence suggests that neural tissue is the default? What does activin do?

Answer 11

• Injection of mRNA for non-functional activin receptor
• Led to induction of neural tissue (that is default)

Activin changes fate of cells to not become neural.
- Follistatin inhibits activin where neural tissue desired.

Question 12

What evidence suggests the role of the notochord?

Answer 12

• Transplantation of a second ectopic notochord onto neural tube leads to ectopic neurons
• Removal of notochord leads to hugely reduced neural tissue induction

Notochord induces floor plate which then leads to the induction of neural tissue

Question 13

What are hox genes? Give evidence.

Answer 13

An independent set of TF genes defining a particular region of the body (rhombomere = morphological subdivision).

• Deletion of Hoxa1 causes loss of rhombere r5 but others unaffected

Question 14

How is the neural tube specialised to form the hindbrain and spinal cord? (4 points)

Answer 14

Activation
- Specialises forebrain

Stabilisation
- Specialises neural and forebrain states

Transformation
- Caudalies tissue forming hindbrain and spinal cord

Sympathetic ganglia arise from the neural crest; parasympathetic from the trunk

Question 15

Describe the structure of the growth cone:

Answer 15

• Axon terminates in the central domain (houses organelles + microtubules)
• Transition zone
• Peripheral domain with filo and lamellipodia

Question 16

What is the experimental evidence for growth cone movement being independent from the cell body?

Answer 16

• Retinal ganglion cells labelled and time-lapsed movement
• Growth cone continues to navigate for hours after separation from cell body (contained)
• Continues to respond to attractive/repulsive cues (local translation of proteins can occur; just not central)

Question 17

How do growth cones move forward?

Answer 17

• Axons extend their microtubules into the distal tips pushing the GC forward
• Filopodia extend, exerting a tensile force on the GC
• Direction: determined by balance of F-actin retrograde flow and myosin based filament retraction (and the proximal recycling of filamentous actin in the transitional zone)

Question 18

How is the direction of growth cone speed determined?

Answer 18

Determined by speed of retrograde flow which is reliant on:
- F-actin assembly rate
- Myosin based filament retraction
- When both in equilibrium no growth

Must be stabilised by microtubule insertion:
- Faster flow increases speed of microtubule shunting out of filopodia
- Thus reduced retrograde encourages stabilisation of microtubules

Question 19

What is the evidence for microtubules being pushed into the GC forward?

Answer 19

• MT fluorescently labelled
• An area photobleached with a laser.
• The spot remains stationary but the tubules move forward, suggesting new MTs are pushed forward rather than synthesised at the tip.

Question 20

What evidence is there for filopodia extending and exerting a tensile force on the growth cone?

Answer 20

Evidence for filopodia myosin/actin extension:
- Addition of cytochalasin-β (actin depolymeriser) disrupts the growth (slower) and pathfinding ability of the GC.
- Washing it away allows re-activation of growth cone (suggests continued polymerisation)

Evidence for tensile force:
- Picking up a single filopodia causes the GC to jerk in the opposite direction (due to loss of the tensile force)

Question 21

What is the clutch hypothesis?

Answer 21

The link between the actomyosin cytoskeleton (the molecular clutch)
- Altered by the adhesiveness of the surface

Depends on isoform of the integrins expressed:
- Concanavalin-α = very adhesive substrate
- Laminin and fibronectin promote outgrowth
- Changed by context: chick retinal ganglion cells start preferring laminin then switch to fibronectin with decreased α6-subunit expression.

Question 22

What are the principle mechanisms that alter the speed of retrograde flow and allow turning of the growth cone?

Answer 22

Asymmetrical flow encourages turning:

• Assembly of actin at the leading edge
• Translocation of monomers by myosin motors away from leading edge
• Enzyme mediated disassembly/recycling in the transition zone

Question 23

What are the intracellular cascades responsible for turning of the GC?

Answer 23

Balance between cAMP and cGMP:

Attractive cue:
- Upregulates cAMP
- Amplified by CICR
- Initiates VAMP2 mediated vesicle exocytosis

Repulsive cues:
- Upregulates cGMP
- Low amplitude Ca2+ release
- Calcineurin activated
- Clathrin mediated endocytosis to retract GC

Mutual inhibition between cAMP and cGMP signalling facilitates decisiveness.

Question 24

What are the insect cuticle burning experiments and what do they show?

Answer 24

• Burning a small section allows compensation and regrowth back to CNS
• Excessive burning – axons end in spiral. Need other axons as scaffold substrate

Shows self organising properties during development and regeneration.

Question 25

Do pioneer axons have a special ability?

Answer 25

Ti1 neurons make initial path. - Important for first time BUT other neurons still manage if pioneers ablated - Suggests all have innate pathfinding ability - Suggests others have potential to be pioneers but happy following - More important for neurons crossing a segmental boundary (to cross or not to cross?)

Question 26

How do neurons navigate?

Answer 26

Stepping stones: provide physical landmarks - Particularly across segmental boundary - Physical guidance (e.g. radial glia to crawl along) Guidance cues: provide chemical landmarks (balance of attractive and repulsive cues) - Secreted molecules (gradients) - Surface ligands and receptors (e.g. integrins) **Reaction depends on context (which TFs activated -- think robo and com)**

Question 27

What are the two models explaining the change in reaction to the floor plate after crossing the midline?

Answer 27

Changes GC responsiveness after initial exposure to floor plate (attractive than repulsive after signpost) Shh model: shh induces responsiveness to semaphoring repulsion during midline crossing Timer switch model: upregulation of 14-3-3 suppresses PKA

Question 28

How is the midline arranged and which genes control the decision of GCs to cross (or not)?

Answer 28

Midline provides symmetrical line and is arranged like ladder -- provides decision point. Organised by ventral cells which an secrete repulsive midline signal (Slit) - Robo gene discourages crossing Commissure gene encourages crossing (temporarily to allow one cross) - When mutated no GCs cross - Antagonises robo to desensitise GC from Slit.

Question 29

What are neural crest cells?

Answer 29

A collection of progenitor cells proximal to the dorsal epidermal layer that give rise to critical areas of the craniofacial and peripheral nervous system. Induced by dorsalin following neurulation.

Question 30

Which molecules affect GC identity?

Answer 30

- FGF2 induces cholinergic and adrenergic neuron formation - Dorsalin (along with other dorso-ventral cues) induces neural crest formation

Question 31

Give examples of molecular markers which neurons use to navigate:

Answer 31

Floor vs. roof plate: - shh provides initial attractive gradient towards floor plate - BMPs repulse from roof plate - Neutrin provides 'carpet' for axons to grow along (attractive and facilitates movement) Cerebellum: - Neurons crawl along particular radial glia (fan out from ventricles) - Use αv integrin on glia cells to bind

Question 32

How does the birth position (and time) of a motor neuron affect its final pathway and destination?

Answer 32

1. Decide which neurons will be motor neurons (by [shh]) = early born neurons (in LMC) 2. These early born neurons produce RA 3. RA gradient signals to late born motor neurons to change their gene expression dependent medial-lateral position – mLMCs are early born; lLMCs are late born 4. Means mLMCs express Islet1 factor and retinaldehyde dehydrogenase-2 (RALDH2) while lLMCs express TF Lim1 5. This changes the behaviour of neuron groups which progressively gets more disparate

Question 33

How is spatial visual information retained in the retinotopic map?

Answer 33

- Retinal ganglion cells project to specific regions in tectum - Relative spatial relationships and axonal terminations conserved - Determined by original anatomic location (BF-2 on temporal side; BF-1 on nasal side) - BF changes reaction to engrailed in tectum (highest concentration in posterior which is attractive to BF-1)

Question 34

How is the behaviour of a developing motor neuron changed to map motor system?

Answer 34

- Achieved by adhesion matching of GC to surface (using cell adhesion molecules (CAMs)) and guidance cues (e.g. netrins/semaphorins). - Leads to attractive and repulsive cues - E.g. Expression of guidance receptor Eph-B1 (ventral) or Eph-A4 (dorsal) causes choice between dorsal or ventral limb

Question 35

How do BF1 and BF-2 help to map the visual system spatially?

Answer 35

Due to expression of genes: BF-2 on temporal side; BF-1 on nasal side - Changes reaction to Engrailed expression in tectum (highest in posterior so attractive to BF-1 expressing neurons) - Engrailed-2 repels temporal axons and attracts nasal axons - Taken up by nasal axons as TF - Temporal neurons go to anterior (repulsion from posterior area by ephin expression) - Nasal to posterior

Question 36

What do the striped carpet assays show?

Answer 36

Alternate stripes of posterior and anterior tectum: - Posterior has high engrailed-2 concentration - Posterior induces ephin expression Shows temporal axons are very anterior preferring while nasal have little preference. - Temporal repelled by ephin expression

Question 37

Which experiments suggest that retinal axons retain their original identity?

Answer 37

Sperry's chemo affinity Map: turning eye upside-down leads to upside-down view of world (neurons do not re-navigate) Replacing dorsal half of retina with a ventral from a donor results in all axons going to same place (act the same)

Question 38

What is the timeline of the NMJ formation?

Answer 38

1. Post-synaptic pre-pattern: AChR receptors begin to aggregate at presumptive synaptic regions 2. Growth cones arrive at site 3. AChRs aggregate at innervation site 4. Agrin deposited (MuSK) activated; triggering intramuscular cascade 5. ACh released from nerve terminal diffuses across 6. Binding of ACh to AChRs causes Ca2+ influx 7. Synaptic refinement occurs: extra-synaptic AChRs disperse through internalisation

Question 39

What changes occur after growth cone arrives at the NMJ?

Answer 39

- Extra filopodia retract (localisation) - Growth cone changes to bouton-like shape - AChR clusters aggregate and move due to wnt signals from pre-synaptic side

Question 40

How does Agrin help to make precise synapses?

Answer 40

Ensures both sides are functionally and spatially in register: - Deposited by postsynaptic side to protect desired AChRs during synaptic refinement - Activates MuSK which degrades calpain (preventing AChRs from being degraded by calpain)

Question 41

What is the organisation of the agrin receptor complex? Which molecules are involved?

Answer 41

- Lpr-4: part of the Agrin receptor complex which binds MuSK – assists in pre-synaptic differentiation - MuSK: muscle specific receptor tyrosine kinase triggers AChR clustering - Rapsyn: scaffolding protein

Question 42

What is the experimental evidence for AChR clustering prior to GC arrival?

Answer 42

- AChR labelled with ▫ α-bungarotoxin (binds strongly) - Under functional microscopy their existence is seen before meeting

Question 43

How was agrin identified?

Answer 43

- Used the electric organ of a torpedo ray (a giant cholinergic synapse) as source of basal lamina to purify - Agrin identified as necessary for synaptogenesis (maintenance) - Agrin knockout in mice leads to impaired NMJ formation

Question 44

What evidence is there that agrin is anti-dispersal rather than synapse inducing?

Answer 44

- AChR clusters form without Agrin (e.g. in genetic mutants) - Live imaging of NMJ formation shows AChR clustering before and in the absence of motor neuron innervation - Removal of dispersal agent and Agrin (in mutants) results in synapse formation (no dispersal signal; no anti-dispersal needed)

Question 45

What are the different ways that synapses are aligned in the CNS?

Answer 45

- Bidirectional organisation (e.g. neurexin pre-synaptic and neuroligin post-synaptic link to mutually co-activate each other) - Anterograde organisers (post-synaptic receptor with pre-synaptic signalling molecules secreted) - Retrograde organisers (pre-synaptic receptor with post-synaptic signalling molecules e.g. FGF7 binds to FGFR) - Glial derived organisation (e.g. astrocyte secretes neuroligin1)

Question 46

How do different scaffolding and trans-synaptic formation molecules during synapse formation dictate the type of synapse?

Answer 46

Scaffolding: - Gephyrin in glycine terminals - Stargazin for glutamate terminals - Rapsyn for NMJs Signalling molecules: - E.g. a retrograde organised synapse with an FGFR on the pre-synaptic side will become inhibitory if FGF7 binds or excitatory if FGF22 binds.