1
Q
whatare the three modules of regeneration?
A
wound healing/induction= injur response which involves the epithelial cell migration and wound closure, inflammation and apoptosis and induction of regeneration
- then there is the blastema formation which involved the formation of blastema porgenitors, dedifferentiation, SC recruitent, transdifferentiation
- then there is the redevelopment: morphogenesis, differentiation, cell growth, migration and the regeneration of the missing sturcture
2
Q
what animal is used to look at regeneration?
A
salamanders
3
Q
what are the 5 main points to be investigated in regeneration?
A
- how is it initiated
- where do the progenitors comes from
- positional identity
- what are the siganls that underly the need for the nerves in regeneration
- how are growth and patterning achieved
4
Q
ow did they start to go about looking into how the genetic response is initiated?
A
- they looked at the wounding response in mammals, you see there are differences in the mice than in the salamander (?)
5
Q
what is the wound response in mammals?
A
- reepithelialisation
- infammation
- ECM deposiiton and remodeeling
6
Q
what was the first signal found to be involved in amputation regen? how was this found?
A
- the amputation triggers a positive current from the injured tissue at the site of the wound
- driven by sodiuam channels and if you add sodium channels or put electrodes either side to abrogate this current- then no regen takes place.
7
Q
how was the finding that a positive current is induced at the site of amputation link to the regen process?
A
- it has been found that the cells hat are involved in wound heeling exhibit strong electrotaxis- sense electric field which stimulates the migration-
8
Q
what stage of regen is dependent on electrical stimulus?
A
re epithelialisation
9
Q
what is the role of re-epithelialisation?
A
it forms the wound epithelium which is an important signalling centre for regeneration. It sends signals into the blastea such as FGF8, FGF24, Shh, Wnt3 and nAG
10
Q
how can yu test the role of reepith?
A
if you remove it and replce with a normal patch of skin then regen stops- essential part of regen process
11
Q
how was the role of apoptosis implicated in ren initially?
A
- looking at hydras which can regen
- upon transection there is an induction of apoptosis
- it was found that if you treat the wound with apoptosis inhibitors - ZVAD - then you inhibit regeneration. In the hydras, apoptotic, WNT releasing cells are released from the would site
12
Q
hat can you use to inhibit apoptosis?
A
ZVAD
13
Q
how did they find that WNT was needed for the proliferation of the cells in the hydra during regen?
A
- you inhibit WNT with an RNAi
14
Q
what is the link between apoptsosi and WNT signalling needed for proliferation in the hydra?
A
the apoptotic cells release WNT which stimulates the proliferation of the cells underneath the site of injury
15
Q
was the role of apoptosis in regen conserved?
A
yes- in zenopus and salamander
16
Q
how did they look into what signals stimulate apoptosis following amputation? what factors did they find?
A
they used the zebrafish which can regen their fins- they took a pharmacological approach and looked at what happened if they treated fish wound fins with different inhibitors of different factors- they found that certain inhibitors had a huge effect- one was one that inhibited ROS, other inhibited apoptosis and others inhibited JNK
17
Q
what is the pathway of amputation stimulating epidermal cell proliferation and blastema formation in the fish ? is this conserved~
A
- amputation stimulates ROS hich stimulates JNK and apoptosis which then both stimulate proliferaiton and blastema. also in hydra, xenopus, salamander. ROS also stimulates the immune response
18
Q
how did they start looking into the role of ROS in the fish injury?
A
they created a transgenic fish - introduced a construct alled hyper into the 1 cell embryo- this encodes a Tf called OXR which is fused to a YFP which fluoresces when the ROS are present . they found there is a strong production of RO within minutes of wounding
19
Q
how were leukocytes linked to ROS induction? explain the experiments
A
trasgenic leukocte mrked fish were looked at with a Hyper construct injection and they saw tht the leukocytes were recruited at a similar time as the upreg of ROS- they then postulated that this was how they were recruited- they tested this by injecting a Mo for duox which is the main producer of ROS in these cels- you see that leads to a reduction in the recruitment of leukocytes
20
Q
how did the finding that leukoctes wee recruited tot he site of injury via ROS production lead to a specific question being asked?
A
- this led to the question of whether the immune response in regeneration was friend of foe - traditionally you associate inflammation with impaired wound healing - this was addressed by targeting the equivalent cell types in the salamnders- macrophages - they selectivley depleted macrophages-chrodomes- liposomes that are selectivley engulfed by macrophages and you can put clodronate disodium salt in the lysosome so that they kill the macrophages ( use DiI to dye the macrophages to se that they are depeltion)- you see that there is perturbed blastoderm formation is they are targetted early in the response but not later in the response
21
Q
what was it thought that the macrophages that are recritmed may be involved in ?
A
the remodelling of the ECm
22
Q
what mammals can be used for wound healing?
A
african spiny mouse, they lose their skin and are super regenerators- can heal wounds very quickly and scar free
23
Q
what can you see when you compare the response of the african spiny mouse wound response to the normal mouse development?
A
- you see that or example the spiny mouse regn its hair follicles and the germ cells from the hair reenter the cell cycle- this mimics what happens in normal skin development in the mouse
24
Q
what tissue is the spiny mouse unable to regenerate?
A
- muscle
25
what tissues can the spiny mouse regen?
- adipose tissue, cartilage and epidermis
26
how was the difference between the normal mouse and the african spiny mouse observed?
someone looked at the skin of the two mice- they found that there is different ECM- lots of collagen 3 and very porous in the ASM and collagen 1 in the normal mouse- shows that remodeeling the ECM is important
27
what type of cell regeneration could be important in the regen response?
- dedifferenitaiton, transdifferentiation or stem cell self renewal
28
explain the different strategies used to determine the origin of progenitors for regeneration
1. tissue graft- one can swap tissues at the ebryonic stage- take take for example the NC from a donor and put them in a host s that all of the SCs will be marked from the donor. You can use the Cre approach in which you can label different tissue types with a reporter and actiavte by tmx when you amputate- then you can see from what tissue- depending on what tisse has been lablled, the different components of the regenerated limb have come from
2. you can add a donor labelled cell and then amputate and see where your labelled cell ends up
29
how were planarias used to show where the progenitors in the wound come from?
- hey can regen veyr well- have neopasts which are highly proliferative- people wanted to know if these neoblasts were fully pluripotent- the are sensitive to radiation- can expose a planaria to irradiation and kill them all and them amputate and they wont be able to regenerate. You can isolate a neoplast and place it into an irradiated planaria - you see that this cell starts to repopulate all of the different cell tissue types- this shows that the neoplasts can produc all of the tissue you can prove this by take a neoplast from an axeual planaria and implnt into the sxual planaria - you see that your sexual gets turned into an asexual you can also do RNAseq to look at different types
30
how was the source of the regen cells in the newt investigated first ? what did this lead to the question of?
- the newt can regen its lens via transdifferentiation of the dorsal cells of the iris epithelial- these cells lose the iris specific expression, they elongate and then generate more copies of themselves and then become lens cells by upreg crystalin genes . so two processes- dediff and then transdiff - mabe this is a key process?
31
so after seeing that the cells in the retina regen from transdiff in the next eye, how did they go about looking at role of this in regen?
- they looked at the muscle- it is hard to break a muscle up into individual components
- you can take muscles fro a limb- label them implant them back into a regen animal and you can see that the initial long muscle cellularises- showing cellular plasticity- you can also label genetically to express somehing like AP phosphotase to have a longer stud- see that when you implant muscle in a regen sturcture- the nuceli start to enter the S pahse- using BrdU- eventally they cellularise and contribute to the regen structure
32
Once they saw that the muscle was able to express plasticity and contirbute to the regen limb, how did they see if this was what was happening in the actal limb?
can use Cre system to label different tissue sturcture's progenitor cells and hten see what the tissue ends up aas after amputation- you see that all stem cells conserve their tisue type of origin (once GFP expressed- all cell clones will express because it is in the genome)- you see that demris makes dermis and SCs make SCs - cartilage makes cartilage
33
how did they prove that stem cells were not involed in the regen of the axolotl and xenopus?
- they used the cell genetic fate map to label tissue types and them did an amputation and saw that the tissues always form their own tissue type
- they saw that muscles in the salamander, when myofibers were cutured and then implanted, they cellularised and became part of the blastuma after amputation
- you can label an adult cell type by driving a cre recombinase fused to ER ad driven by tissue specific promoter (cardiomyocytes) so that when you add tmx- then when ou add tmz then only the cells in the cardioyoctes at a particular poiint will be labelled. In the zebrafish you do this and then you see if any of the progenitors express GFP after ijury- you see that yes all of the cells in the heart are GFP positive and derive from the cardiomyocytes
- this also occurs in the zebrafish osteoblasts- all of the regen bone comes from this
- this is also the case in the next- muscle does not come from pax-5 positive stem cells
34
generally where do the progenitors come from in salamander regeneration?
the tissues dedifferentiate, proliferate annd then redifferentiate
35
how did they look at the mechanisms behind the dedifferentiation process in myotubes?
1. they cultured myotubes with high serum- they found they started to dedifferentiate and enter the cell cycle
2. they found that they start to phosphorylate Rb - this is bound to the TF E2F which is the master TF for S phase reentry- othe phos means that E2F is released and can stimulate reentry into the cell cycle
36
what happens when you put myotubes in high serum culture?
they start to dedifferentiate
37
alongside of RB phosphrylation, what was found to be downrgeulated?
p53
38
how did they visualise the downreg of p53 upon myotube dediff?
- they cultured the myotubes and then did in situ against a p53 target- Gadd45 and they found that its levels go down upon dediff
39
how did they look at the signiicant of p53 down reg in muscle dediff?
- they used a compound which stabilised p53 callled nutlin3a- you disrupt the ability of the nuclei to reenter the s pahse in the myotubes- aybe p53 is involved in regulated p53
40
nce they found that p53 may be involved in dediff of the myotubes, how di dthey test hwether this happened in vivo ?
- the saw that if they stabilises p53 in the blastema (its activity) you cannot form the blastema anymore
41
how did they look into the pathway behind p53 downreg?
they found that in the dediff muscle, the ER pathway was activated- they did this my using inhibitors of the ERK pathway- this prevents the down reg of p53- this prevents phospphorylation of Rb and the entry into the cell cycle
42
how did they show the connection between the ERK signalling and muscle reporters?
they showed that if you inhibit ERK acivatio, you dont get the downreguation of 6 during dediff
43
what was the pathway that stimulats the dediff of the myoutbe?
when you put the myotube on a hih serum culture, it activates ER, this decreases p53 activity which down rgeulates gadd45 which leads to phosphorylation of p-Rb. ERK also activates the suppression of sox6 which somehow causes the phosphorylation of Rb. This leads to cell dcycle re entry
44
how did they look into how the cels in the planatrian know their positional identity after regen?
- they thought that the axis of the A-P determination may be important
- they did an an in situ and asked whether the WNT pathway that s normally involved in patterning the AP as needed during the regen- they used an RNAi against beta catenin and fed it to the planaria- they showed that the tail generates a head rather than a tail (because there is normally high WNT in the tail;- this is the same as in vert)
-
45
how did the look into axis patterning in the vertebrate initially?
- in an early experiment: exposed a regen limb ti RA, one could induce dplications of the proximal elements nd the limb gets longer- this is interesting because Prod-1 is reggulated by RA and is expressed in a concntration gradient down the salamander limb
46
once they had found that RA treatment of the regen limb could induce proximal duplications, and that prod-1 was induced by RA and present in a gradient down the limb, how did they go about looking at the role of prod-1 in regen of the limb? (2)
1. if you remove a proximal blastema and a distal one- the proximal always engulfs the ditsal ( can show this by labelling the two differentl and putting them together in culture
- but if you eliminate prod-1 which PIPLC treatment (inhibitor) you rpevent the engulfment - so this shows that Prod-1 is responsible for soe of the characteristics which are involved in the differences between the proximal and distals
2. you can electroporate the distal cells in the blastema with an GFP consturct and you can see as t regenerates that these cells always end up in the hand- so these cels in the blastema were already specified
- butif you repeat the expeirment usong GFP and also give prod-1 to these cells- these cels strat to give more proximal tissues
These show that the presence of Prod-1 dteermines positional identitiy during limb regeneration
47
hat is interesting about prod-1 in regen?
confined to salamanders- an evolutionary novelty
48
how did they go about looking for proteins which intraced with prod-1? what did they find?
two hybrid screen- nAG
49
where is nAG secreted from and why is this interesting?
from the Schwann cells- interesting for the relationship between the nerves in regeneration- suggests a role for the interaction between nerve regen and limb regen
50
how did they test the role of nAG in regeneration? what did they find?
- they asked what happens if they denervate an animal but restore nAG expression by electorproations-could you restore the regen of the lib that is lost in nAG mutants or less. - they did this by cutting the nerve in one arm, then cutting both limbs after a few days- then electroporate nAG protein into the innervated limb
- they found that you can get fuly regeneration
- this suggests that nAG is normally what comes from the nerve to allow the regen process
51
what is the accessory limb model and what does this tel us about the role of nerve signals in regen?
- if you have an axoltl and make a wound in the fore arm, it will heal the wound perfectly
- if you make the wound and put a nerve pointing towards the wound- it will heal that wound and also start forming a blastema
- whats more, if you make a wound, deviate the nerve and take an equivalent piece of the patch that they took when making hte wound from the counterlateral and place it on top of the wound with the nerve- it forms an entirely new nerve
- so in order to have rgeen- need nerve and positional disparity
52
how was the accessory limb model used to find what can reconstitute the nerves ?
- you hav ethe wound and no deviated nerves and you have the skin patch different- before adding the skin batch, yo can put beads underneath which you can soak in different compounds- and ask whether this can lead to the formation of the accessory limb- see what factors can replace the nerve
- you see the success of a different combination of factors:
- you find that a combination of FGfs and BMPs that are the best for this - sugges that these are either secreted by the nerve of that the nerve induces
53
explain how they investigated whether they could improve regeneration in the post-metamorphic limb? of the xenopus
- normally it just regenerates a little spike not an entire limb
- they asked if they could make the spike into an entire limb
- they took a fiibrin patch which contains larva limb bud cells - if you just put them into the regen limb it doesnt work- if you combine these cells with factors that are implicated in regeneration maybe cn improve regen
- you see tht some cells have migrated outsode of the patch )(they are labelled) and started to mix with the cell tissues- they are proliferating too- after two months you see that the limb is regenerating digits
54
explain how they investigated whether they could improve regeneration of the posterior piece of the planarian?
- if you cut the planaria in the posterior elements- it cannot regenerate
- they looked into the difference between these trunk pieces and head pieces
- they did expression pattern comparisons - at early stages they are very similar but they find differential expression of the WNT signalling pathwayso they tried to knockdown the WNT pathway and see if the tail pieces could regen by feeding beta cat RNAI you can then regenerate its head - maybe the WNT is just too high for anterior head formation
55
explain how they investigated whether they could improve regeneration of the mammalian muscle?
- if you Kdown Rb and a p53 stabilser if you do this trick then you can acivate S phase re entry (phos of Rb is the same as downreg in mammals)- ut eventually they form these weird sturctures and collapse- do not like cellulariation- but if you do it at the tage when the muscle is mononucleated- yo can make it dediff and form progenitors with regen potential if you inject these myocytes into tibialis anterior then these myoctes are capable of fusin to muscles in vivo
56
at affects can regenertaion have on a general organism levels?
- drosophila can regen their imaginal discs - the regen of the wing disc delays development - irradiation signals to the ganglion sturctures in the brain and stimulates the delay of development- can abbrogate the dela and force thse animasl to develop- genetic elimination of key cmponets of RA pathwa- the RA is the signal from the damaged regen tissue which stimulates the expression of ptth inthe brain to cause development delay
- tis shows that rgeen is a regulated event and can control the physiology of the animal
57
what are the ideas in terms of the evolution of regeneration?
Regenera3ve ability is an ancestral property that has been lost in certain species during evolu3on
- Phylogene'c distribu'on of regenera'on, existence of common mechanisms
- Phylogene'cally conserved regenera'on genes that were lost/modified/unable to func'on in regenera'on in non-regenera've species (ie expression of β-catenin in planaria)
II - Regenera3ve ability evolved locally in certain species (convergent evolu3on)
- Could have evolved against a backdrop of higher regenera've ability compared with mammals, but the cri'cal changes are the result of local evolu'on.
- Iden'fica'on of axon-specific regenera'on genes (ie Prod1, newt CCN1)
Mechanisms o f Development
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