Neurogenesis

Question 1

Define the two diff types of cell divisions

Answer 1

Symmetric divisions: generate two daughter cells that have equivalent fates (e.g. two stem cells or two cells that differentiate).
Asymmetric divisions: generate two daughter cells that have different fates (e.g. one stem cell and one cell that differentiates).

Question 2

totipotent

Answer 2

can generate every cell type of the animal, including extraembryonic tissues (placenta and yolk sac).

Question 3

Pluripotent

Answer 3

can generate all cell types of the adult animal but not those of the extraembryonic tissue

Question 4

Multipotent

Answer 4

can generate a wide range of different cell types associated with a specific tissue (e.g. CNS)

Question 5

Unipotent

Answer 5

Can only generate one cell type

Question 6

Proliferative divisions

Answer 6

generates one or more daughter cells that remain in the cell cycle (i.e. are mitotically active)

Question 7

Differentiative divisions

Answer 7

generates one or more daughter cells that exit the cell cycle and begin to differentiate (e.g. into neurons or glia)

Question 8

Direct neurogenesis:

Answer 8

stem cell divides to produce at least one neuron.

Question 9

Indirect neurogenesis

Answer 9

stem cell divides to produce at least one “intermediate progenitor”. This subsequently divides symmetrically to generate two neurons.

Question 10

Anatomy of the developing cortex

Answer 10

• Ventricular zone: proliferative region
  
  • Preplate: contains pioneer neurons that ultimately migrate to form marginal zone (MZ). Also splits to generate subplate.
  • Subventricular zone: additional proliferative region. Splits and becomes sup plate and marginal
  • Cortical plate: where newly born neurons migrate to. Becomes grey matter of cortex.
    Fibre layer (or intermediate zone): contains axons. Becomes white matter of adult brain. Neurons migrate through this region to reach cortical plate.

Question 11

Evidence that Reelin is expressed in CR cells

Answer 11

• 1995 Mikoshiba lab generated antibody against Reelin.
• Found to be highly expressed in Marginal Zone cells of developing cortex.
• Localises to Cajal–Retzius cells in this region.
  Cajal–Retzius cells of MZ secrete Reelin during cortical development.

Question 12

Evidence that ApoER2 and VLDLR are expressed on migrating cortical neurons

Answer 12

• E.g. Riboprobes against AP-RR36 (part of the ApoER2 sequence).
• Labels migrating cortical neurons but not proliferating cells.
  receptors on developing neurons and reelin by caja r cells on margin

Question 13

Evidence that loss of reelin affects cortical layering

Answer 13

• Neurons born first settle in deeper layers (layer 6, then 5 and so on)
  
  • Neurons born later migrate past early born neurons to stablish superficial layers (e.g. layers 2,3)
  • So the layers of the cortex are populated in an inside-out fashion
  • This is inverted in reelin mutants

Question 14

Evidence that loss of reelin signalling affects cortical layering

Answer 14

• In situ hybridization against:
  
  • ER81: marker of early born neurons
  • Cux2: marker of late born neurons.
  • Reelin mutant has reversed organisation of neurons (early born neurons near basal surface, late born nearer apical surface).
  • Early born neurons scatter in reelin mutant and very few sit at the upper superficial layer of cortex

Question 15

Evidence that reelin regulates somal translocation without affecting glial guided locomotion

Answer 15

• Early born neurons do not use glial guided locomotion
• Instead they migrate via somal translocation alone
• E12.5 mice: early born neurons of Dab1 (downstream effector of reelin) mutants fail to undergo somal translocation.
• Dab function don’t translocate: 1 stay in intermediate zone above svz, 2 all processes that would touch the basal region of brain are absent

Question 16

Evidence of the effects of defects in somal translocation stage

Answer 16

• Time lapse imaging of GFP labelled WT and Dab1 mutant neurons undergoing somal translocation.
• WT cells extend process that attaches to MZ. Then undergo somal translocation.
• Dab1 mutant cells also extend process that attach to MZ. However, somal translocation fails to occur.
• Instead, neurons retract process and adopt multipolar morphology (and cell bodies end in deeper layers of cortex).

Question 17

Evidence of the effect of Later born neurons during glial-guided locomotion stage of migration

Answer 17

Express GFP in migrating cortical neurons. Study morphology and migration patterns as neurons move towards final locations.

During embryonic development : WT and Dab1 mutant neurons have migrated towards MZ and extended processes to make contact with it.

In early posnatal life there are marked defects: - Control neurons retain bipolar morphology and maintain contact with MZ.
However, Dab1 mutant neurons adopt multipolar morphology and lose contact with MZ. They also have disorganised Golgi apparatus.

• Suggests that something happens during final stages of migration (i.e. somal translocation stage).
  Mutant basal processes (retracted and sit in cortex) become retracted and morphology changes, sit deeper in incortex retracted and sit in cortex

Question 18

Experiment investigating whethersomal translocation of layer II/III neurons account for inverted cortical layering?

Answer 18

• At E14.5 electroporate early born cells with GFP.
  One day later, at E15.5 electroporate later born neurons with mCherry. Also introduce Dab1–RNAi vector into these cells. - The later born neurons will have reduced Dab1, earlier born neurons will not.
  Time-lapse imaging to monitor migration of early born (green) and later born (red) neurons at E18.5, P1.5 and P7.

Mutant: At P7, somal translocation has failed and later born neurons are intermingled with early born neurons.

Control:

Question 19

Why does somal translocation fail?

Answer 19

• Reelin activates Dab1.
  
  • Dab1 then activates several downstream proteins
  • Limk1
  • Akt1
  • Rap1
• Limk1 important for controlling actin stability.
• Atk1 and Rap1 important for controlling neuronal adhesion.
• Express dominant forms of these proteins in migrating cortical neurons.
• Rap1 knockdown has marked effect on somal translocation.

Rap1 is important for localisation of the cell adhesion molecule cadherin on the surface of developing neurons.

Expressing dominant-negative form of cadherin in late born cortical neurons causes detachment from MZ and somal translocation failure.
-Overexpressing cadherin in Rap1 knockdown neurons rescues this defect.
suggests that loss of rap1 results in loss of cadherin localisation