Regeneration

Question 1

in what scenarios may regeneration occur?

Answer 1

• Some cells generated in embryogenesis live as long as the organism itself e.g. neurons, heart muscle
• Other cells are replaced continuously from stem cells e.g. blood, epithelia
• Some cells are replaced/reformed after injury e.g. muscle fibres

Question 2

what is regeneration?

Answer 2

Regeneration is the possibility of the fully developed organism replacing organs or appendages by growth or repatterning of existing tissue

Question 3

is regeneration linked to the organism’s complexity?

Answer 3

no, regeneration is not linked to the complexity of the organism
- Simple rotifers and nematodes do not have regenerative powers whatsoever. In contrast certain “complex” vertebrates like newts can regenerate surprisingly well

Question 4

what are the two types of regeneration?

Answer 4

1. morphallaxis
2. epimorphosis

Question 5

what is morphallaxis?

Answer 5

repatterning of tissue without growth
- existing cells change their fate to reform the lost structure/tissue
- cells do not grow
- organism will be smaller as no growth has occurred, and the positional value will change

Question 6

what is epimorphosis?

Answer 6

repatterning of tissue with growth
- new cells are added to grow back the missing structure
- positional value will not change
- organism will retain its size

Question 7

what is an example of an organism which undergoes morphallaxis?

Answer 7

hydra
- Hydra grows continuously, therefore cells have to change their positional values constantly
- repatterning also occurs during reproduction -> regenerative capacity

Question 8

what are hydra?

Answer 8

• hydra have a simple structure
• a mouth region called hypostome which is surrounded by tentacles an elongated body column, consisting of a gastric region, a budding region where next hydras are formed by asexual budding, and a basal disc

Question 9

how do hydra grow?

Answer 9

Hydra grows continuously, and therefore in order to maintain its size, cells need to be lost
- therefore cells have to change their positional values, repatterning also occurs during reproduction
- This occurs at the tip of the tentacles, at the basal disc and also by asexual budding, leading to the formation of a new individual
- they must continuously change their positional values
- the cells are flexible and know where they are to adapt their fates accordingly

Question 10

how can the position of cells in hydra be visualised as they are always changing?

Answer 10

visualised by labeling cells in a particular region and following their fate
- shows that the cells move continuously in the body

Question 11

is growth required for hydra regeneration?

Answer 11

no, hydra regenerate via morphallaxis, so repatterning without growth occurs

Question 12

what are the 2 gradients involved in hydra head regeneration?

Answer 12

1. gradient in the positional value (PV)
   - This positional value determines the head-inducing ability (1 high, 5 low) and it also determines the level of resistance to a head inhibitor
2. head inhibitor gradient
   - The head inhibitor is produced in the head itself
   - Gradient goes from head where inhibitor is high, and down to the basal disc

Question 13

how can the presence of a head inhibitor gradient be shown?

Answer 13

Graft experiment:
- The presence of a head inhibitor can be shown by transplanting a piece of region 1 tissue to an intact hydra in the upper part of the body column
- Head inhibitor from the existing head will prevent this graft from forming a second head
- If the head from the recipient is removed, the source of head inhibitor is lost and now a graft at the same position can form a second axis
- It is expected that the concentration of head inhibitor falls when tissue is further removed from the head.
- If piece of region 1 tissue is transplanted to region 6 at the lower part of the body column of an intact hydra, the level of head inhibitor is insufficient to prevent head formation by the graft, so a head at the bottom will form

Question 14

how can the effect of the positional value/head inducing capacity be analysed?

Answer 14

if you take region 1 just below the head, and transplant it below the head of an intact hydra nothing will happen.
- However if the head of a hydra is removed, and after 6 hours region 1 is transplanted into the body column of a host at a similar position as in the previous experiment, it can induce a head, showing that it has gained a stronger head inducing capacity, so the PV has become higher

A piece of region five, from lower down the body column cannot do this after 6 hours, one needs to wait for 30 hours before this region has a similar head-inducing capacity
- From region 5, there is a smaller PV so smaller head-inducing capacity and have to wait longer

If the head is removed the source of inhibitor is lost and this will result in an increase in positional values.
- In regions where this occurs the leftover tissue with the highest positional value will reach the “head-value” first and will start producing inhibitor again, thus reestablishing the original situation only in a smaller body, and retaining the original polarity in the body column.

Question 15

what signalling molecules determine the positional value?

Answer 15

Wnt and beta-catenin

Question 16

how are Wnt and beta-catenin involved in PV and head formation?

Answer 16

• Wnt is expressed in the hydra head and is also activated in a regenerating tip
• Inhibition of GSK3-beta (negative regulator of Wnt signalling) leads to upregulation of nuclear beta-catenin and thereby activation of the Wnt pathway and head regeneration
• If you do this in hydra all regions of the body acquire characteristics of the head organiser, which can be shown by grafting.

Question 17

what is an example of an organism which undergoes epimorphosis?

Answer 17

Urodele = tailed amphibians
- urodele are the masters of regeneration in the vertebrate kingdom

Question 18

what body parts can urodele regenerate?

Answer 18

dorsal crest, limbs, retina, lens, jaw, tail

Question 19

how does lens regeneration in urodele occur?

Answer 19

Lens regeneration occurs from the iris, meaning cells have to transdifferentiate

Question 20

how does urodele limb regeneration occur at the point of amputation?

Answer 20

In limb regeneration, regeneration occurs from a level that is appropriate to where the cut was made
- A distal amputation only regenerates distal structures, whereas a proximal amputation only regenerates proximal structures

Tissues know which tissue needs to be reformed – tissue knows where the PV has been removed

Question 21

how does urodele regeneration occur?

Answer 21

Regeneration occurs by growth of new tissue, therefore it is of the epimorphic type
- If you take the limb as an example, regeneration involves production of various tissue types like bone, muscle, blood vessels, and skin

Question 22

what is the process of urodele limb epimorphosis?

Answer 22

1. After amputation, epithelial cell migration occurs over the wound site – regeneration is dependent on this
2. Once the epithelium has covered the wound, cells below the epithelium dedifferentiate from dermis, cartilage, muscle to form a blastema
   - Multinucleate muscle cells can revert to mononucleate cells in cell culture under the influence of thrombin, which is a protease
3. blastema can then generate appropriate limb structures to reform the amputated limb

Question 23

how does dedifferentiation of muscle cells occur in urodele epimorphosis?

Answer 23

• Dedifferentiation of muscle cells involves expression of Msx1, a homeobox transcription factor, and inactivation of the Rb gene (by phosphorylation), which inhibits proliferation of muscle under normal circumstances.
• The presence of thrombin is crucial for dedifferentiation and may have wider significance, lens regeneration also requires local activation of thrombin.

Question 24

how do generation and regeneration occur on different scales?

Answer 24

• generation of a limb during embryogenesis and regeneration must be different and occur on different scales
• morphogen gradients may have to work on a 10x larger scale in regeneration
• certain genes expressed in regeneration are not expressed in embryogenesis

Question 25

can blastema cells transdifferentiate completely in epimorphosis?

Answer 25

blastema cells have limited transdifferentiation capacity, as the cells are only partially dedifferentiated
- apart from dermis ability to differentiate to cartilage, the rest of cells remain true to type
- muscle to muscle, Schwann cell to Schwann cell, epidermis to epidermis

cell fate tends to stay the same

Question 26

what are the 3 key rules of regeneration?

Answer 26

1. limb regeneration is always distal to the wound, according to the PV at the site of the cut
2. regeneration is not just replacing missing parts, there is a global view of the situation
3. blastemas have morphogenetic autonomy after transplant

Question 27

how is regeneration not just replacing missing parts, and is instead a global view?

Answer 27

If the distal limb of a newt is amputated and the stump is inserted into the flank (to establish vascular connections) and the limb is subsequently cut again, the anterior limb will regenerate the missing distal parts as expected
- importantly, the remaining stump will also regenerate a distal limb, with a mirror image humerus, and not a proximal humerus
– So, the wound blastema “reads” the local positional value and generates more distal positional values, it does not determine what is missing.
- So as a result a second limb with two radii and ulnae is formed

Question 28

how do blastemas have morphogenetic autonomy after transplantation?

Answer 28

A blastema also has considerable autonomy, once formed it can regenerate the structures lost in a different location (dorsal crest for instance)
- This will also happen if a distal blastema is transplanted to a proximal wound.
- The blastema will only form the hand and then an entire limb will form.
- This shows that the site of the wound can sense a discontinuity in positional values between the distal blastema and the cut site.
- The remaining tissue will be formed by intercalary growth from tissue that is just below the transplantation point

Question 29

how do proximal and distal cells organise?

Answer 29

via differential adhesion

Question 30

what is differential adhesion?

Answer 30

• If a distal blastema is combined with a proximal blastema the proximal blastema will start to engulf the distal blastema
• This can be interpreted as a sign the distal cells stick more tightly to one another and that the proximal cells stick more to the distal cells than to themselves
• Differential adhesive properties might also help to explain why distally transplanted cells do not mix with the proximal blastema cells that will form as a result of the intercalary regeneration

Question 31

how do blastemas become proximalised?

Answer 31

Retinoic acid (RA) can proximalise a blastema via Rarδ2, meis homeobox genes and/or upregulation of a GPI-linked protein prod1
- the effect of RA is dose-dependent: the more RA, the more proximal structures will be generated

Question 32

what evidence is there that retinoic acid is involved in proximal regeneration?

Answer 32

A hand was amputated and after amputation the blastema was treated with a high concentration of RA, this led to a proximalisation of the blastema and it went on the regenerate an entire limb at the level of the wrist (humerus, radius, ulna)
- PV value was very proximal

Question 33

how does proximalisation of blastema by retinoic acid occur?

Answer 33

• Proximalisation by RA may occur via Rar-delta2 and upregulation meis homeobox genes and/or prod 1.
• Prod1 is a GPI-linked adhesion protein that is expressed at the highest level in the proximal regenerating blastema, and is induced by RA treatment
• Prod1 may play a role in the differential adhesion
• Prod 1 antibodies could prevent the engulfment that is normally seen when proximal and distal blastemas are opposed

Question 34

what is required for regeneration of a limb to occur?

Answer 34

nervous innervation
- unless the limb had no nerve in the first place

if a limb is denervated and then amputated, the limb will not regenerate, as the nerve is required

Question 35

what is nAG?

Answer 35

Newt “Anterior Gradient” (nAG) is a protein that can replace the nerve in supporting outgrowth, by binding to prod1
- nAG is expressed in nerve sheath in response to wounding
- nAG can rescue regeneration of a denervated limb

Question 36

does an innervated vs denervated limb express nAG?

Answer 36

Innervation leads to downregulation of epidermal nAG

Aneurogenic limb: persistent epidermal nAG expression

Question 37

how does regeneration occur in cockroaches?

Answer 37

• Sensing discontinuities in positional values at local level (site of cut)
• Intercalation to regenerate missing ones, irrespective of overall structure
• Segments of a leg contain similar positional values
• Graft of a mid-tibia to mid-femur will not cause regeneration of intermediate structures

Question 38

can mammals undergo regeneration?

Answer 38

yes, young children and mice can regenerate the very tip of their fingers - if a fingertip is amputated distal to the nail bed the tip can regenerate, however if the nail bed is also removed regeneration will fail

PNS axons and CNS axons can be regenerated

liver, ribs and heart can also regenerate

Question 39

how can PNS axons be regenerated in mammals?

Answer 39

• in the peripheral nervous system, axons can be regenerated, however if the neuron itself is lost no regeneration occurs.
• Regeneration occurs along the original pathway of the axon and is stimulated by Schwann cells along the way.

Question 40

how can CNS axons be regenerated in mammals?

Answer 40

• In the CNS little regeneration occurs
• neurons actually try to send out new axons but they appear to collapse it is thought that astrocytes and oligodendrocytes of the CNS provide a non-permissive environment.
• It has actually been found that implanted Schwann cells in the CNS can promote axon growth.
• Schwann cells secrete neurotrophic factors which promote regeneration
• One assumption that may explain this difference is that the CNS is a very delicate and elaborate system that could be disrupted easily if new axonal could form without difficulty.

Question 41

how can ribs regenerate in mammals?

Answer 41

Ribs have been reported regenerate as long as the periosteum, the membrane that surrounds the ribs, is left intact.
- This is often exploited by surgeons doing reconstructive surgery, ribs can be source of transplantable bone material.

Question 42

what happens to cardiomyocytes when the heart is damaged?

Answer 42

Cardiomyocyte are present when heart is damaged, but they don’t divide;
- Progenitors are present, but not used for repair
- Leads to scar formation and hypertrophy – maladaptive (scarring causes more issues)

Question 43

how does ventricular regeneration occur in zebrafish?

Answer 43

• Not identical to embryonic heart development
• MsxB and MsxC expressed
• Regeneration dependent on dedifferentiating muscle cells
• Endocardium/Epicardium are involved
• Neuregulin may be a signal from the epicardium, inducing proliferation in myocardium
• There is a balance between scarring and regeneration - zebrafish Mps1 mutants show scarring rather than regeneration

Question 44

what is the process of heart regeneration in zebrafish?

Answer 44

1. After resection of the heart it will bleed profusely but soon a blood clot will form preventing the circulation from coming to a complete standstill.
2. Very soon after injury the endocardium will be activated and start expressing certain genes, Raldh2 for instance.
3. In addition the epicardium will become activated, expand rapidly and start to cover the wound. This covering of the wound actually looks quite like what happens after limb amputations in newt.
4. The epicardium will signal to the muscle myocardium below causing dedifferentiation and proliferation to regenerate the missing tissue
   - There will be vascularization of this new tissue
5. Newly forming muscle expresses FGF and epicardial cells respond to this signal by delaminating and invading the regenerate and reform blood vessel that will help to restore a functional ventricle.
6. The growing muscle will lead to dissolution of the clot and after about 30 days the process will be complete.

Question 45

what is neuregulin?

Answer 45

neuregulin is an EGF-like factor expressed by the epicardium to stimulate cardiomyocyte cell division and growth/proliferation of the myocardium
- Neu is an important mitogen for cardiomyocytes in zebrafish
- in uninjured zebrafish hearts, Neuregulin can drive cardiomyocyte proliferation (hyperplasia) and result in cardiomegaly

Question 46

can mice regenerate their heart?

Answer 46

neonatal mice can regenerate their heart in a similar way as zebrafish but loose this ability soon after birth

Question 47

why do mice lose their ability to regenerate their heart after birth?

Answer 47

loss of regeneration in mice with correlates with reduction in erbb2 expression which is required for Neu signal transduction
- erbb2 is a coreceptor for this Neu.
- Thus one speculation is that mammal lose their ability to regenerate due to loss of responsivity to Neuregulin.

Question 48

can restoration of erbb2 sensitivity for Neu restore heart regeneration?

Answer 48

a transgenic mouse that had a doxycycline inducible-dominant active form of erbb2 was created.
- This transgene was then induced for some time after an infarction was created in these mice
- interestingly they were able to induce cardiomyocyte proliferation and a significant improvement in function, and could show a smaller scar area.