Lecture 20 - Cell interactions in neurlal development

Question 1

Describe the process of neuralation.

Answer 1

1. Notochord forms from mesoderm cells soon after gastrulation is complete
   
   2. Signals from notochord cause inward folding of ectoderm at the neural plate
   3. Ends of the neural plate fuse and disconnect to form an autonomous neural tube

Question 2

How does Shh induce nerual fates.

Answer 2

Nervous system forms from ectoderm overlying the notochord
Folds to make the neural tube in a process called neurulation
​Notochord secretes Shh
Shh induces floor plate
Floor plate secretes Shh
Motoneurons specified at high Shh
V2 interneurons specified at medium Shh
V1 interneurons specified at low Shh

Question 3

What are the main neurons foromed during neuronal specification?

Answer 3

Commissural interneurons - Connect neurons to other neurons across the two sides of the body
Dorsal root ganglion neurons - Sensory neurones make connections with receptors in the periphery (e.g. pain and temperature)
Motor neurons - make direct connections with muscles at the neuromuscular junction

Question 4

What is the effect of notch in drosophila?

Answer 4

Specification of neurons

Notch inhibits the neural fate in the neurectoderm of Drosophila (notch mutants have too many neurons - expansion of the neuroectoderm dorsally)
Sensory bristles in Drosophila - made up of four cells (hair cell, socket cell, sheath cell and sensory neuron)
* Wild type - normal notch
* Missing bristles - too much notch
* Extra bristles - not enough notch

Notch (receptor) - DSL (ligand)
* Highly conserved (nematodes to humans)
* Notch - heterodimeric proteins linked noncovalently
○ Many EGF like repeats
○ Ankyrin repeats
* Ligands EGF repeats in extracellular domain
* Involved in many developmental processes
* Can regulate cell proliferation (cancer)

Question 5

Summarise notch signalling.

Answer 5

Notch signalling summary
* Notch proteins are a family of type-1 transmembrane proteins
* The extracellular domains of Notch and its ligands contain EGF repeats
* DSL (delta/serrate/lag2) are the Notch ligands
* Notch and DSL interact through their EGF repeats
* Ligand binding induces cleavage of the Notch intracellular domain (NICD)
* NICD binds CSL (CBD-1/Su(H)/lag1) - Suppressor of hairless
* Notch-CSL activates gene expression

Question 6

How do notch and delta affect cell fate?

lateral inhibition

Answer 6

A single SOP is specified from the cells in a proneural cluster through lateral inhibition
One cells adopts a fate and inhibits the surrounding cells from adopting the same fate
Initially all cells express equal amounts of Notch and Delta
One cell stochastically accumulates more Delta -> suppression of SOP fate in surrounding cells

Question 7

Describe neural specification.

Answer 7

Neuronal specification summary
Notch and Delta initially expressed uniformly in the proneural cluster
Delta increases in the future SOP (stochastic)
Increased Delta increases Notch activation in adjacent cells
Increased Notch signalling increases levels of Supressor of hairless (CSL)
CSL increases expression of enhancer of split proteins
They in turn repress achaete-scute proteins -> less Delta transcribed
Feedback loop further increases Delta in the future SOP
Results in activation of bHLH transcription factors -> neuronal fate

Question 8

Describe axon outgrowth

Answer 8

Axon outgrowth

Growth cones migrate towards correct targets.
* Receptors are on the cell surface
* Actin polymerisation/depolymerisation determines direction of movement

Axons use chemotaxis to find target tissues.
* Short range cues
○ Contact attraction - e.g. Cadherin
○ Contract repulsion - e.g. Ephrin
* Long range cues
○ Contact attraction - e.g. Netrins
○ Contact repulsion - e.g. Semaphorins

Commissural neurons grow towards the floor plate

Question 9

Describe the structure of netrin receptors.

Answer 9

Netrin
* Similar to C. elegans Unc-6 (mutations in Unc-6 gene have defective neural outgrowth)
* Vertebrates have netrin 1 and netrin 2
* Netrin 1 is expressed in floor plate cells
* Act as chemoattractants and chemorepellents (trochlear motoneurons)
* Unc-40 and Unc-5 are netrin receptors

Netrin receptors
* Unc-40
○ large transmembrane protein
○ immunoglobulin and fibronectin repeats
○ commissural neurons fail to grow towards the floor plate in mouse knockouts

	* Unc-5
	○ large transmembrane protein
	○ immunoglobulin repeats 
	○ large intracellular domain
	○ may modify the response of Unc-40. 

Weather Unc-5 is a chemoattractant or repellent depends on gene expression and can change depending on context

Commissural neurons don’t stop growing when they reach their first target (floor plate). Crossing the floor plate alters the chemotaxis of commissural neurons so they no longer move towards netrin.

Question 10

How are synapses made?

Answer 10

• Making synapses
  Neuromuscular junctions are the target for axons.

Agrin
* Released by motor neuron axon terminal
* 200 kDa protein related to laminin
* Incorporated into basal lamina of synapse
* N-terminal half binds to extracellular matrix
* C-terminal half binds to receptors on muscle
* One molecule of Agrin can aggregate 200 Ach (acetyl choline) receptors
* Binds via LRP4 to Musk (Muscle-specific kinase) tyrosine kinase

1. ARIA (neuroregulin) is released from the axon terminal and stimulates ErbB -> increases AChR expression
2. Agrin binds directly to LPR4/Musk
3. Activates Rapsyn (through Rho/Rac)
4. Activated Rapsyn binds to AChR+
5. Resulting in clustering -> ACh binding
6. Calcium release -> contraction
7. Calcium accumulation outside the NMJ represses AChR expression