Neural Development Flashcards
How is a coordinate system set up in the maternal oocyte?
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)
What are the stages of developing a combinatorial code in an embryo?
- Maternal cytoplasmic polarity
- Gap genes
- Pair-rule genes
- Segment polarity genes and homeotic (Hox) genes.
Encouraged by the French flag model
What is the French Flag model? How is it achieved?
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.
Outline the stages of nervous system development (rough):
- Gastrulation allowing mesoderm creation
- Invagination to form neural tube
- Induction of the floor plate
- Separation of mesencephalon (brain) from spinal cord.
What are the molecules involved in setting up a polarised neural tube dorso-ventral?
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
What are the molecules involved in setting up a polarised neural tube anterior-posterior?
At dorsal posterior:
- Wnt highest conc.
- Binds frizzled receptor
At dorsal anterior:
- Sfrp1 highest
At ventral anterior:
- Wnt4 and 7b
What are neural crest cells and what do they form?
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….
What is the experimental evidence for the creation of a dorsal-ventral axis in an embryo?
- 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
What evidence led to the discovery of neural inducing genes?
- 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
What is the general formula for how a signalling molecule induces a response? Use RA as an example.
- Signal
- Receptor
- Pathway
- TF (genes)
E.g. RA -> RAR -> pathway -> RARE (response element) modulating hox gene transcription.
What evidence suggests that neural tissue is the default? What does activin do?
- 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.
What evidence suggests the role of the notochord?
- 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
What are hox genes? Give evidence.
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
How is the neural tube specialised to form the hindbrain and spinal cord? (4 points)
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
Describe the structure of the growth cone:
- Axon terminates in the central domain (houses organelles + microtubules)
- Transition zone
- Peripheral domain with filo and lamellipodia
What is the experimental evidence for growth cone movement being independent from the cell body?
- 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)
How do growth cones move forward?
- 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)
How is the direction of growth cone speed determined?
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
What is the evidence for microtubules being pushed into the GC forward?
- 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.
What evidence is there for filopodia extending and exerting a tensile force on the growth cone?
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)
What is the clutch hypothesis?
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.
What are the principle mechanisms that alter the speed of retrograde flow and allow turning of the growth cone?
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
What are the intracellular cascades responsible for turning of the GC?
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.
What are the insect cuticle burning experiments and what do they show?
- 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.
Do pioneer axons have a special ability?
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?)
How do neurons navigate?
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)
What are the two models explaining the change in reaction to the floor plate after crossing the midline?
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
How is the midline arranged and which genes control the decision of GCs to cross (or not)?
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.
What are neural crest cells?
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.
Which molecules affect GC identity?
- FGF2 induces cholinergic and adrenergic neuron formation
- Dorsalin (along with other dorso-ventral cues) induces neural crest formation
Give examples of molecular markers which neurons use to navigate:
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
How does the birth position (and time) of a motor neuron affect its final pathway and destination?
- Decide which neurons will be motor neurons (by [shh]) = early born neurons (in LMC)
- These early born neurons produce RA
- 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
- Means mLMCs express Islet1 factor and retinaldehyde dehydrogenase-2 (RALDH2) while lLMCs express TF Lim1
- This changes the behaviour of neuron groups which progressively gets more disparate
How is spatial visual information retained in the retinotopic map?
- 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)
How is the behaviour of a developing motor neuron changed to map motor system?
- 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
How do BF1 and BF-2 help to map the visual system spatially?
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
What do the striped carpet assays show?
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
Which experiments suggest that retinal axons retain their original identity?
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)
What is the timeline of the NMJ formation?
- Post-synaptic pre-pattern: AChR receptors begin to aggregate at presumptive synaptic regions
- Growth cones arrive at site
- AChRs aggregate at innervation site
- Agrin deposited (MuSK) activated; triggering intramuscular cascade
- ACh released from nerve terminal diffuses across
- Binding of ACh to AChRs causes Ca2+ influx
- Synaptic refinement occurs: extra-synaptic AChRs disperse through internalisation
What changes occur after growth cone arrives at the NMJ?
- Extra filopodia retract (localisation)
- Growth cone changes to bouton-like shape
- AChR clusters aggregate and move due to wnt signals from pre-synaptic side
How does Agrin help to make precise synapses?
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)
What is the organisation of the agrin receptor complex?
Which molecules are involved?
- 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
What is the experimental evidence for AChR clustering prior to GC arrival?
- AChR labelled with ▫ α-bungarotoxin (binds strongly)
- Under functional microscopy their existence is seen before meeting
How was agrin identified?
- 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
What evidence is there that agrin is anti-dispersal rather than synapse inducing?
- 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)
What are the different ways that synapses are aligned in the CNS?
- 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)
How do different scaffolding and trans-synaptic formation molecules during synapse formation dictate the type of synapse?
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.
