Lecture 10 - Programmed Cell Death in Development

Question 1

what is necrosis?

Answer 1

accidental death of cells and living tissue.
Generally, results in swelling and bursting of cell.
Release of digestive enzymes from lysosome.
Provokes a local inflammatory response

Question 2

what is apoptosis?

Answer 2

is a regulated process.
The cell usually becomes round.
Chromatin condenses and then is fragmented.
Cell releases “eat me” signals.
Membrane-bound apoptotic bodies form.
Apoptotic bodies are phagocytosed.
* Cellular contents are not released to surrounds – no inflammation.

• Apoptosis is a developmental decision like any other – like
  differentiation etc. But the cell dies – in a highly regulated manner.
• Creates gaps between fingers, kills nerves with poor synapses etc.

Question 3

what are nucleosomes (DNA in chromatin)?

Answer 3

• Inter-nucleosomal DNA fragmentation is a classic sign of
  apoptosis in mammalian cells.
• Chromosomal DNA is cut by a non-specific nuclease during
  Apoptosis. This is irreversible and ensures cell dies!

Question 4

what are Mouse TGF-Beta family gene knockouts?

Answer 4

Mice and Humans have multiple TGF-Beta genes.
Mouse Tgfβ2-/-Tgfβ3-/- double gene knock-outs.
During limb formation, apoptosis is significantly
reduced.
TGF-β is a critical extrinsic regulator of apoptosis in
the developing limb – helps make digits.

Question 5

what is p53?

Answer 5

• p53 is a tumour suppressor gene – protects against cancer.
• p53 protein is a multifunctional integrator of cellular damage detection/signalling.
• P53 integrates multiple stress/damage sensing systems in the cell – and
  can activate the death pathway.
• The Unfolded Protein Response can activate apoptosis.
• Loss of growth factor stimulation can cause apoptosis.
• Activation of TGF-beta signalling can drive apoptosis in the cell that
  receives this signal (very context-dependant**).
• **TGF-beta signalling ONLY causes apoptosis in some specific
  circumstances. P53 is a much more general trigger for apoptosis.

Question 6

what are the 3 types of DNA damage?

Answer 6

• Moderate genome damage – induce DNA repair – return to normal
• Severe genome damage (e.g. telomere dysfunction) – senescence
• Severe genome damage plus hyperproliferation - apoptosis

Question 7

How does a cell decide to Apoptose?

Answer 7

There are many different inducers of apoptosis. Most commonly a
response to something going wrong – these include:
DNA damage, oncogene activation, unfolded proteins, loss of growth
factor stimulation.

Question 8

how is TUNEL staining used in apoptotic cells?

Answer 8

Chromatin becomes fragmented during apoptosis.
This can be used to detect apoptosis.
TUNEL staining - directly labels the free ends of broken DNA strands.

Apoptosis plays a critical role in limb morphogenesis (the generation of shape)
* Massive cell death in the mesenchyme of the interdigital spaces accompanies the
formation of free digits.

Question 9

what are types of motor neurons?

Answer 9

• ~20,000 motor neurones innervate chick leg from spine.
• ~ half die soon after formation.
• Neurone survival depends on synapse with muscle.
• Initially several motor neurones synapse single muscle fibre.
• Competition between synapses – more powerful input
  destabilises less powerful.
• Selection for strong synapses.

Question 10

what are the cellular changes during apoptosis?

Answer 10

Membrane “blebbing”
Formation of Apoptotic Bodies – “eat me” signals
Engulfment by phagocytes
DNA fragmentation

Question 11

Apoptosis - lymphocyte development homeostasis

Answer 11

1. Removing T and B lymphocytes that recognise “Self”.
2. Removing T and B lymphocytes that don’t recognise anything.
3. Removing the excess of B lymphocytes produced.
4. Removing proliferated B lymphocytes after infection.

Apoptosis plays a critical role in lymphocyte development and
homeostasis.
* Enhanced lymphocyte apoptosis can cause immunodeficiency
through cell loss.
* Conversely, inhibition of apoptosis can lead to development of
autoimmunity or lymphoma.

Question 12

what is the Conservation of central death pathway – C. elegans to mammals?

Answer 12

EGL-1 is orthologous to BH3-only, CED-9 to BCL-2, CED-4 to APAF-1 and CED-3 to CASPASES.
Caspases are a class of cysteine protease – digest proteins. Active caspases and nucleases
promote apoptosis – they are the EXOCUTIONERS. Mammals have multiple caspases.

• CED-9/BCL-2 are anti-apoptotic BCL-2 family members.
• EGL-1/BH3-only are pro-apoptotic BCL-2 family members.
• The balance of activity between pro and anti-apoptotic members makes the
  life/death decision.
• CED-4/APAF-1 activate downstream caspases.
• Release of Cytochrome C from the mitochondria is a common method of
  triggering the death side of this decision in mammals.

The C. elegans CED-9 and human Bcl-2 proteins are similar in sequence.
Homology – similarity due to common evolutionary origin
* CED-9/BCL-2 protects against cell death – block apoptosis.
* Bcl-2 was identified as an oncogene.
* Oncogenic Bcl-2 is over-expressed in B-cells - a translocation places the gene
adjacent to immunoglobulin heavy-chain gene enhancer.
* Causes follicular lymphoma (B-cell lymphoma). B-cells that CANNOT commit
suicide by apoptosis.