Cell Death Flashcards
is there a relationship btwn targets of innervation and the pool of innervating neurons?
remove limb bulb (target) from one side of chick embryo
-results: many fewer neurons in the region of the spinal cord that would originally internervate the ‘missing’ limb
limb ablation and limb addition
-ablation resulted in smaller ganglia with removal of target
-addition (supernumerary) resulted in a larger ganglia
what did Hamburger postulate about muscle and skin
muscle and skin made 2 different, specific agents/stimuli that control the development of their respective nerve centers
how do ‘agents’ from each target reach their respective nerve centers? (recruitment model)
agents travel retrogradely to their respective nerve centers (brachial lateral motor columns and spinal ganglia)
-regulate the development of corresponding centers
how do the agents regulate the development of these centers? (recruitment model)
-nerve centers send axons to their respective target areas
-axons would explore the size of the target area and send ‘agent’ to cell bodies
-promote the differentiation of adjacent undifferentiated cells, would then send more axons to target
-‘recuritment’ of undifferentiated cells would proceed until target is saturated with nerves
recruitment model
targets of innernation ‘recruit’ naive cells in the spinal cord/spinal ganglia adjacent to the neuronal cell bodies to become neurons
-‘recruitment’ factors acquired by axons and determine cell fate of naive cells
reduced and extra target material
remove limb bud: fewer neurons
additional limb bud: more neurons
Hamburger’s model
- decline in neuronal numbers is because many neurons that are initially present degenerate following ablation->atropy causes reduced numbers, not recruitment
- cell death occurs not only after limb ablation, but as an ordinary and normal feature of embryonic development
what is the molecular mechanism by which targets can regulate neuronal cell death?
neuronal survival requires acquisition of a target derived ‘survival factor’ which is present in limiting quantities, leading to competition for the survival factors by the pool of internervating neurons
-axons need to reach threshold of survival factor to survive, or they will die
what are the predictions of Hamburger’s model
- number of neurons arriving at single target increases, the number of survivors should stay constant, % of survivors should decrease
- # of neurons arriving at single target decrease, # of survivors should stay constant, % of survivors should increase
Mouse sarcoma 180
as it grows it gets invaded by nerves
-DRG/sympathetic ganglia closest to tumor get very large
-both sensory and sympathetic neurons invade tumor, but not other neurons (motor neurons)
is the survival factor a diffusable/ soluble substance?
Exp: put tumor on other side of allantoic membrane instead of inside embryo (no direct contact w/ embryo)
-survival factor diffuses everywhere
-results: ganglia was still enlarged when placed on other side of membrane
-conclusion:survival factor is likely a diffusable factor
fractionation procedures
-starting material is run through resin to fractionate
-fractons are assayed to identify desired molecules
-fractionate with diff resin->assay again (repeat)
what was the result of adding the survival factor to a chick dorsal root ganglia (halo assay)?
-when survival factor was added-> lots of extended neurites (halo)
-when no survival factor was added->dead ganglia
phosphodiesterase experiement: is the survival activity a protein or nucleic acid?
-if it was a protein there should have been a halo
-if it was a nucleic acid, there should be no halo
-results: there was a HUGE halo
what happened when a negative control (buffer) was added with phosphodiesterase (PDE)?
there was a huge halo
-this wasn’t expected because the control shouldn’t have any survival factor
-made researchers ask what the starting material that PDE was purified from->snake venom
what is a better homolog for snake venom producing organs?
male mose salivary glands
if we add extra NGF, neuronal cell death should decline and neuronal survival should increase (prediction #1)
Addition of NGF
-increased neuronal survival
-chemotaxis-growth of axons to site of injection
NGF is sufficient to promote neuronal survival
if we remove endogenous NGF, neuronal cell death should increase and neuronal survival should decrease (prediction#2)
immunosympathectomy: remove sympathetic nerves
-resulted in first ever use of function blocking anitbodies
-is there a halo with the function blocking Ab-> NO
FB Ab resulted in complete destruction of sympathetic NS, partial destruction of sensory SN and no effect on motor neurons
NGF must by synthesized in targets in proportion to the levels of innervation (prediction#3)
increased NGF levels and density of innervation supports the idea that production of NGF by target organs determines density of innervation
NGF must be synthesized in targets at the right time in development (prediction #4)
-NGF mRNA and protein synthesis both start around the time that the axon arrives to cell body
-heart still synthesizes NGF, despite no growth cones arriving->NGF isn’t induced by arrival of growth cones
how did researchers make larger quantities of NGF?
1.isolation of NGF cDNA as a probe to detect NGF mRNA on RNA blots
2.improved immunological assays to detect NGF proteins using monoclonal antibodies
what target derived trophic factors act in the CNS?
brain derived neurotrophic factors
-65% amino acid homology to NGF
-both are neurotrophins
are there more members of the neurotrophin family?
sequenced highly conserved regions shared btwn NGF and BDNF
-found that NT3 and NT4 were homologous
