12. regenerative biology 2 Flashcards
what was discovered in 2001?
that there soluble factors in serum that are important for muscle regernation - particularly in re-entry to the cell cycle
how do you get more proliferation when growing muscle cells in culture?
plate them at low density - this way they are not contact inhibited
what is required for de-differentiation regeneration of myofibres in vivo in newts? and why is this?
direct clipping - cutting off the end of fibres
- they need to be physically damaged to trigger cell cycle re-entry
>need to trigger start of programmed cell death in order to regenerate
what happens when newt skeletal muscle is fragmented (breaking up of multinuclear structure)?
programmed cell death response - involves caspase response, this may act on cells or be released from cell (they are not sure)
>from this, this paper were able to get terminally differentiated skeletal muscle cells into regernative progenitors
what happens when skeletal muscle cells are injured?
most of them will die
>some don’t die and re-enter cell cycle and de-differentiate and contribute to regenerating muscle
what two things do axolotl regernating limbs need to do in order for there to be cell cycle re-entry by differentiated cells ?
> trigger a programmed cell death response
>down regulate TS p53
if p53 is not down regulated what happens?
cell cycle re-entry cannot be triggered = no regeneration/impaired regeneration
why do we not want to interfere with p53 in humans?
it is regarded as the ‘guardian of the genome’ - interfering with this will lead to cancer
>this may be one of the reason why we cannot regenerate
a paper want to see what pushed myotubules to divide, what did they do? what protein did they identify?
screened blastema secreted proteins for effects on division of cultures myotubes
- to see how salamanders re-enter the cell cycle safely
>MLP (MARKS-like protein)
what did they do to show that MLP is important during regernation?
- did a big cloning screen
- saw the effect on dissociated cells
- purified active protein
- made antibodies to see where it was
- KO
- added it ectopically
MLP is very good at inducing the blastema. what can it induce?
significant increase in length of blastema
what was seen when anti-MLP was used to block it affect in a wide range of regernating tissues?
blocking it has an affect on dividing nuclei - MLP upregulates division
>this shows it is widely used in axolotl to regenerate tissue
what is it proposed that the ‘serum factor’ may do?
this might P Rb allowing it to release E2F and drive proliferation
what do we know about the S phase re-entry factor in serum?
it is being cleaved by thrombin - serum and thrombin can trigger cell cycle re-entry
is thrombin cleaving a factor in the serum or on the cells? and how was this determined?
thrombin cleaves something in the serum
>pre-incubate the serum with thrombin, inhibit thrombin then treat cells with the serum - this was shown to trigger cell cycle re-entry
what can the S phase re-entry factor also stimulate cell cycle in?
newt iris pigmented epithelial cells
what might being able to identify this S-phase cell cycle re-entry factor determine?
why salamanders can re-grow limbs and we cannot
>may also allow for us to do mammalian regernation
mouse muscle cell line was treated with serum or newt blastema extract, what happened?
cells immediately upregulated genes like fos and c-myc, left G0 and entered G1
>they resist entry into cell cycle and do not enter S phase
some people are born without limbs, what might we want to be able to do?
kick start embryonic development in that part of the body again
how does embryonic development in salamander differ from regeneration?
> embryonic development doesn’t need nerves
>blastema division and growths needs nerves (depends on nerve derived factors)
what can be used to induce ectopic limbs in salamanders?
re-direct the nerve, damage the tissue and the nerve will generate a limb to grow in a ectopic position
what are some of the nerve derived factors? and what implication does this have on humans?
BMP and FGF drive blastema cell proliferation
>we will need to think about nerve derived factors if we ever get to this stage in humans.
what protein sets up a proximodistal gradient and what is this set up by?
Prod1 - this is set up by MEIS
where is Prod1 found?
only in salamanders not in humans
