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Limb regeneration: fact or (science) fiction?

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Editor's Introduction 

Molecular Basis for the Nerve Dependence of Limb Regeneration in an Adult Vertebrate. Kumar et al.
 
annotated by Jason Librande
 
Human limb regeneration has been a common theme throughout fiction. The Amazing Spider-Man makes good use of this idea by telling the story of a scientist who, while trying to regenerate his arm, is turned into a lizardlike creature, not unlike the salamanders he was studying in his lab. Despite this dramatization, salamanders are used to study regeneration as they possess amazing regenerative ability. This research study attempts to identify why, at the molecular level, nerves are required for limb regeneration. Results from this study provide insights into vertebrate regeneration and bring human limb regeneration further away from science fiction and closer to science fact. 

Abstract 

The limb blastemal cells of an adult salamander regenerate the structures distal to the level of amputation, and the surface protein Prod 1 is a critical determinant of their proximodistal identity. The anterior gradient protein family member nAG is a secreted ligand for Prod 1 and a growth factor for cultured newt blastemal cells. nAG is sequentially expressed after amputation in the regenerating nerve and the wound epidermis—the key tissues of the stem cell niche—and its expression in both locations is abrogated by denervation. The local expression of nAG after electroporation is sufficient to rescue a denervated blastema and regenerate the distal structures. Our analysis brings together the positional identity of the blastema and the classical nerve dependence of limb regeneration.
 

Report 

Limb regeneration occurs in various species of salamander and offers important insights into the possibilities for regenerating a complex structure in adult vertebrates (1). Regeneration proceeds from the limb blastema, a mound of mesenchymal stem cells that arises at the end of the stump. A blastema always regenerates structures distal to its level of origin; a wrist blastema gives rise to the hand, whereas a shoulder blastema gives rise to the arm (2). Distal blastemal cells are converted to more proximal cells by exposure to retinoic acid or other retinoids over a relatively narrow range of concentration (34). This finding led to the identification of Prod 1, a determinant of proximodistal (PD) identity that is expressed at the cell surface as a glycosylphosphatidylinosotol (GPI)–anchored protein of the Ly6 superfamily (5). Its expression is graded (P > D) in both normal and regenerating limbs (6), and distal cells of the larval axolotl blastema are converted to more proximal cells following focal electroporation of a plasmid expressing Prod 1 (7). We have suggested that a ligand for Prod 1 could be an important player in PD identity (5).
 
The stem cell niche for limb regeneration has been studied intensively, and the key tissues are the regenerating peripheral nerves and the wound epidermis (8). The severed axons retract after amputation and then regenerate back along the nerve sheath and into the blastema. Axonal regeneration can be prevented or arrested by transecting the spinal nerves at the base of the limb, distant from the amputation level (9). The generation of the initial cohort of blastemal cells occurs in a denervated limb, but the growth and division of these cells depends on the concomitant regeneration of peripheral axons (10). Both motor and sensory axons have this activity, and it is independent of impulse traffic or transmitter release (1112). If a peripheral nerve is cut and deviated into a skin wound, it can even evoke the formation of an ectopic appendage (1314). Limb regeneration is abrogated if the blastema is denervated during the initial phase of cellular accumulation, but denervation after the mid-bud stage allows the formation of a regenerate (15). The wound epidermis is not required to support proliferation during the first week of regeneration in an adult newt, but it is critical for subsequent division (16).
 
It remains unclear which molecules are responsible for the activity of the nerve and wound epidermis (8). The candidates considered to date include neuregulin (1718), fibroblast growth factor (19), transferrin (20) and substance P (21). In no case has it been demonstrated that a rigorously denervated blastema can be rescued such that it regenerates to form digits. We have identified a secreted protein that is a ligand for Prod 1 and a growth factor for limb blastemal cells. It is induced after amputation as axons regenerate along the nerve sheath, and then appears in the wound epidermis. The expression in both locations is abrogated by denervation. Most notably, the expression of this protein can rescue the denervated limb blastema and support regeneration to the digit stage.
 
Identification of nAG protein as a ligand of Prod 1. We performed a yeast two-hybrid screen with the 69 amino acid newt Prod 1 protein (without N or C terminal signal/anchoring sequences) as bait and with prey libraries derived from both normal newt limb and limb blastema. In a search for potential extracellular ligands, two secreted proteins were identified as positives from the screen and subsequent control experiments (Fig. 1A). One was a newt member of the family of anterior gradient proteins, originally defined by the XAG2 protein, which is expressed in the cement gland of the Xenopus tadpole (Fig. 1B) (22). These proteins have a single thioredoxin fold with a secretory signal sequence (23). They are expressed in secretory epithelia and have been identified as elevated in various examples of rodent and human cancer (2425).
 
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Fig. 1.  Identification of nAG protein as a ligand for Prod 1. (A) Yeast two-hybrid assay illustrating the interaction between nAG and Prod 1. (B) Consensus Bayesian phylogenetic tree of representative members of the AG family of secreted proteins, highlighting nAG, the founder member XAG2, and the human AG2, which is up-regulated in several examples of cancer. (C) Pull-down assay with epitope-tagged forms of nAG and Prod 1 purified after bacterial expression. Lane 1, connective tissue growth factor (CTGF) beads; 2, nAG beads; 3, control beads + Prod 1; 4, CTGF beads + Prod 1; 5, nAG beads + Prod 1. Note that Prod 1 is only pulled down in lane 5. (D) Secretion of nAG after transfection of Cos 7 cells. Cos 7 cells were transfected with a plasmid expressing the myc-tagged nAG, or RFP, as control. The medium was analyzed by Western blotting with antibody to myc. The central lane is the nAG-transfected sample, the right is the RFP, and the left is the molecular weight markers. (E) Reaction of myc-tagged nAG at the surface of Prod 1 transfected mouse PS cells. nAG-conditioned medium derived as in (D) was reacted at 4°C with live PS cells transfected to express Prod 1. Note the purple reaction product at the membrane junction between the two cells (arrow). Scale bar, 50 μm.
 
A candidate

Previous studies have clearly shown that Prod1 is likely a membrane-bound protein that acts as a receptor. Naturally, if something is a receptor it must have something that binds to it or acts as ligand. The main purpose of these experiments was to identify exactly what binds to Prod1 in hopes that this molecule plays a role in limb regeneration.

Criteria for candidates

Determining what binds to a receptor can be an arduous process, especially in this case, where the authors had no clear leads. However, Prod1 being a membrane-bound protein involved in limb regeneration does narrow down the possibilities. The authors needed to find a protein that was:

1. Secreted: For that protein to bind to Prod1 it must be secreted to the outside environment or else it would never reach Prod1. Cytoplasmic proteins would not be good candidates.

2. Expressed during limb regeneration: A good candidate is likely expressed in higher levels during limb regeneration as opposed to normal homeostasis.

Although these criteria do make things slightly easier, the number of proteins that could bind to Prod1 was still enormous. To find the ligands or proteins that bind to Prod1 they needed to perform some sort of screen. That is, they needed to look at a large number of potential hits in the simplest fashion possible. The most common technique when dealing with this type of problem is the yeast two-hybrid screen (Y2H).

Y2H

In short, the Y2H screen allows one to determine what other proteins bind to your protein of interest. In this case, what proteins bind to Prod1. The amazing thing about this technique is that an extremely large number of proteins can be screened. Theoretically, all proteins expressed in the salamander could be screened to see if they interact or bind with Prod1.

In Y2H, yeast are normally transformed with two plasmids. One plasmid expresses the “bait” protein (e.g., Prod1) and the other expresses the “prey” (e.g., whatever binds to Prod1). What makes the system work is that the bait and prey are fused to other proteins. That is, they are combined with another protein to make a longer one with new functions. The bait is fused to a DNA-binding domain and the prey is fused to an activator domain. That means that the bait will now bind to a certain region of DNA when it normally wouldn’t. Similarly, the prey wouldn’t normally activate a gene, but now, with the activator domain fused, it will. The magic happens when the bait and prey bind together. The activator domain-prey protein is not usually within reach of the gene it activates, however when it binds to the bait it comes into close proximity with that gene and is able to activate it. Remember, the bait with its DNA-binding domain is bound to a predetermined region of DNA. The gene activated by this complex is usually some sort of reporter that either provides antibiotic resistance or produces some color in the presence of chemicals. For a diagram of this, please see: http://www.wormbook.org/chapters/www_biochemmolbio/biochemfig1.jpg

DB is the DNA-binding domain.
AD is the activating domain.
X is the bait protein fused to the DNA-binding domain.
Y is the prey protein fused to the activator domain.

Using special molecular cloning techniques, scientists are able to create many plasmids expressing different prey proteins. By these means they are able to screen a large number of proteins to find out which ones have interactions with Prod1 using their Y2H screen.

For an animation overview of Y2H screens, see: http://www.sumanasinc.com/webcontent/animations/content/yeasttwohybrid.html

Panel A

Y2H screens are usually an enormous amount of work, but do not produce any data that look nice in a scientific publication. As a result, the authors show only the main finding, that the protein nAG appears to bind to Prod1.

They show a picture of a petri dish coated with media (food for the yeast) on which they grow. Three streaks or lines of yeast are shown, each one of a different genotype: nAG plasmid only, Prod1 plasmid only, and both nAG and Prod1 plasmids.

Because both prey and bait are needed to activate the reporter gene, only the yeast with both plasmids are able to do that. In this case, the yeast turn blue in the presence of certain chemicals and activation of the reporter gene.

Panel B

Now that nAG has been identified as a likely binding partner for Prod1, the authors wanted to see whether other vertebrates also have proteins similar to nAG. You can imagine that findings from this would be highly relevant to human health. If humans have a protein similar to nAG perhaps we could possibly regenerate limbs, too!

The authors produced a phylogenetic tree comparing the relatedness of nAG with proteins found in other organisms and saw that many other organisms appear to have proteins very similar to nAG.

Coimmunoprecipitation and immunoblotting

Although the Y2H screen provides good evidence that Prod1 binds to nAG, it is still important to confirm these results by other means. The Y2H screen is prone to false-positive results. Here the authors check to see whether these proteins come out of solution together (coimmunoprecipitate), which would further suggest that they bind each other.

To do this, the authors expressed nAG or Prod1 in bacterial cells and then “lysed” or broke those cells open. Now, for all proteins, DNA and RNA are free floating in solution because there are no longer cell walls and plasma membranes keeping them in.

This mixture, called lysate, is then mixed with small beads. These beads are unique though in that they are covered in nAG or Prod1 protein. Thus, if nAG in the lysate does bind to Prod1 we would expect nAG to be bound to the Prod1 beads. Next, centrifugal force can then be applied to separate the solution by size. The bead-Prod1-nAG complex will be at the bottom of the tube and the rest of lysate can be thrown away.

Finally, the authors can perform an immunoblot, or western, to separate the proteins by size and to further identify them using antibodies. A video explaining immunoblots can be found at: https://www.youtube.com/watch?v=v-O103PLhm8

An additional video can be found at: https://www.thermofisher.com/us/en/home/life-science/protein-biology/pro…

Panel C

The authors performed coimmunoprecipitation and immunoblotting as previously described.

Lane 0/Far-left lane: This is the “ladder” for the blot. One of the purposes of immunoblotting is to separate proteins by size, so you need some way to determine the size of your proteins. A ladder contains predetermined proteins of various sizes. Because you know the sizes of the proteins in the ladder ahead of time, you can compare the protein you are studying with the ladder. This is how the authors generate the kilodalton sizes of proteins (25, 37, 50, etc.) next to the blot.

Lane 1: This lane had CTFG beads run by themselves. Because they blotted for antimyc we would expect there to be no nAG present, but because CTGF has a myc tag added to it we see a band between 37 and 50 kilodaltons. Two bands that are likely immunoglobulins are also present. These proteins are a part of the beads and are an artifact of using this method and can be disregarded.

On the whole, this lane acts as a negative control. As expected, no protein of the right size is detected on the blot.

Lane 2: This lane had nAG-bound beads run alone. This lane acts as a positive control because we will expect there to be nAG present in this lane regardless of bead binding. When probing for antimyc (the nAG protein is fused to a “myc” tag), we see a band show up that is indicated by the arrow.

Lane 3: Control beads were mixed with lysate from cells expressing Prod1. These control beads should not pick up any nAG because it is not present in the lysate or bound to the bead. As a result, there is no nAG band present. This acts as an additional negative control.

Lane 4: CTGF beads were incubated with Prod1 lysate. Because nAG is not present in this lysate we still do not see a band correlating nAG. However, there is a return of the CTGF band between 37 and 50 kilodalton that was present in Lane 1.

Lane 5: nAG beads were mixed with Prod1 lysate. In this lane we can see the appearance of a band correlating to nAG as indicated by the arrow. This is expected because the bead is coated in nAG protein. However, if you look at the blot on the right where they performed the same experiment, but used an antibody toward Prod1 instead of nAG (antimyc), you can see there is a band for Prod1 present. This shows that Prod1 did in fact bind to nAG beads.

Panel D

The authors wanted to determine whether their myc-nAG fusion protein is secreted as with the wild-type nonfusion protein. Results from this study played a large role with how the authors performed experiments for Figure 6.

Left lane: Ladder indicating the size of proteins.

Middle lane: Medium from Cos7 cells expressing myc-nAG. Because nAG is a secreted protein we would expect these cells to release nAG out into the environment. In this instance, the medium these cells are grown in are the outside environment. When this medium is immunoblotted we see reactivity as indicated by the arrow.

Right lane: Medium from Cos7 cells expressing red fluorescent protein. This acts as a negative control.

Panel E

Mouse PS cells had a plasmid introduced so that they express Prod1 protein on the surface of their plasma membrane. nAG was then added to the medium these cells were growing in and interactions between the two proteins were allowed to happen.

These cells were then washed multiple times such that only nAG bound to Prod1 would be leftover. Any unbound nAG would be removed by the washing steps.

Antibodies toward nAG were then added to the medium where they could then bind to the nAG protein. These antibodies had enzymes fused to themselves, which allows for a blue-purple color to show up in the presence of certain chemicals.

Thus, only areas where there are bound antibodies to nAG will you see color. Any unbound antibodies, like unbound nAG, will be washed away.

 Conclusion

All these experiments have shown that nAG is likely a ligand or binding partner for Prod1. Through multiple experiments and techniques the authors have supplied good evidence for follow-up studies of nAG in the living organism.

 
Epitope-tagged versions of bacterially expressed nAG and Prod 1 were found to complex together in a standard pull-down assay (Fig. 1C). When myc-tagged nAG was expressed after transfection of mammalian Cos 7 cells, it was secreted and then detected in immunoblots using two different antibodies directed at non-overlapping sequences (Fig. 1D). The conditioned medium was reacted with live mouse PS cells transfected so as to express the anchored newt Prod 1 on the surface. The binding of nAG to the surface was detected by phosphatase-labeled secondary antibodies (Fig. 1E) and was absent in untransfected PS cells or after reaction with control medium from mock transfected Cos 7 cells.
 
Nerve-dependent expression in regeneration. The expression of nAG protein was analyzed by reacting sections of the newt limb with the two antibodies, and these gave comparable results (fig. S1). In the normal limb, there was weak staining of a subset of glands in the dermis (fig. S2), but after amputation the distal end of the nerve sheath reacted strongly, as illustrated by a longitudinal section at 5 days after amputation [post-amputation (pa)], corresponding to the early dedifferentiation stage (26) (Fig. 2A and fig. S3). We analyzed cross sections of the sheath by staining for both nAG and the Schwann cell marker HNK1, and the nAG was expressed in the Schwann cells but not in axons (Fig. 2B). The wound epidermis was initially negative during regeneration, but after day 10 pa it reacted in glandular structures, as shown at day 12 pa, corresponding to the early bud stage (26) (Fig. 2Cand fig. S4).
 
p2.jpg
 
Fig. 2.  Expression of nAG after amputation of the adult newt. (A) Longitudinal section of a blastema at day 5 pa, stained with antibodies to nAG (green). Note the strong reaction of the nerve sheath and lack of reaction in the WE. The dotted line indicates the position of the amputation plane. (B) Cross section of a nerve sheath in a blastema at day 10 pa, stained for nAG (green), the Schwann cell marker HNK1 (red), and nuclei (blue). (C) Longitudinal section of a blastema at day 12 pa, stained with antibodies to nAG and showing nAG positive glands (arrow). NS, nerve sheath; BL, blastema; WE, wound epidermis. Scale bars: (A) 200 μm; (B) 50 μm; (C) 250 μm.
 
When and where?

nAG is now a reasonable candidate for binding to Prod1. The next logical experiments would be to determine when and where we see nAG expressed. If nAG is expressed somewhere odd, like in the intestines, but not the regenerating limb, it may not be a good protein to study further.

 Immunohistochemistry

Very much like immunoblotting, immunohistochemistry relies on antibodies. However, unlike immunoblotting, the secondary antibody instead of being fused to horseradish peroxidase or a similar enzyme, is fused to a fluorophore. This fluorophore, when hit with a certain wavelength of light, emits a fluorescent signal. Through these means one can determine where the antibody is bound when viewed under a microscope by looking for the light emitted.

Also, because tissues from animals are usually very thick, they have to be cut into thin slices or “sectioned.” These sections can then be laid down on microscope slides and stained using the antibodies.

Through these means scientists are able to visualize proteins and their dynamic lives.

For an image of antibody-binding during immunohistochemistry, see: http://sites.lafayette.edu/neur401-sp10/files/2010/04/untitled.jpg

For an explanation of how fluorophores work, see: https://www.thermofisher.com/us/en/home/life-science/protein-biology/pro…

For a video demonstrating this protocol, see: https://www.youtube.com/watch?v=5AcrhhHmQZs

Panel A

The authors wanted to know what happens to nAG expression when the salamander limb is amputated. Experiments shown in the supplementary figures demonstrated that there is low/no expression of nAG in the intact limb.

Figure 2A clearly shows a dramatic increase in protein levels of nAG as evidenced by the green staining seen in the picture. More nAG protein is present, so a great amount of antibodies bind to it, causing an increase in fluorescence intensity. The results of this experiment show that the nerve sheath is highly stained.

Panel B

Since the previous figure showed nAG expression in the nerve sheath during amputation the authors wanted to determine what specific cells in the nerve sheath exactly express nAG. To do this they use a Schwann cell marker HNK1. They performed immunohistochemistry using their HNK1 antibody, but the color fluorophore they use is different. Instead of green color, they use red fluorophore. This allows them to visualize both HNK1 and nAG in the same sample. Furthermore, they use a DNA stain that fluoresces blue.

The results from this experiment show that the Schwann cells are stained green in this nerve sheath cross section. However, the axons do not appear to stain. Of note is that this experiment was performed 10 days postamputation.

Panel C

Experiments were performed at later time points postamputation and the authors noticed expression in the glands of the wound epidermis. This was not seen in earlier time points. As a result, this suggest that expression of nAG starts in the nerve sheath and after time it starts being expressed in the wound epidermis.

 Conclusion

Using immunohistochemistry the authors were able to show expression and localization of nAG in the regenerating limb. They initially saw expression only in the Schwann cells of the nerve sheath. Though, after more time passed they saw expression of nAG in gland cells underlying the wound epidermis as well.

These results suggest that nAG likely plays some role in nerve-dependent regeneration. The fact that it is found in cells closely affiliated with nerve and the wound epidermis suggests a role in this process.

 
Newts were denervated by cutting the spinal nerves at the brachial plexus of the right limb and then amputated on both sides. The nerve sheath in the innervated limb showed strong expression at day 8 pa, whereas the sheath on the denervated side showed no reactivity (Fig. 3, A and B). Interestingly the expression in the wound epidermis was also dependent on the nerve. Figure 3C shows a low power image of the wound epidermis on the innervated side with nAG positive glands clearly visible, whereas the contralateral limb showed no reactivity and no glandular structures (Fig. 3D). We conclude that the nAG protein is expressed in the key niche tissues early in regeneration and that expression in both locations is abrogated by denervation.
 
p3.jpg
 
Fig. 3. Expression of nAG in the early blastema depends on innervation. (A) Cross section of a nerve sheath on the innervated side at day 8 pa, and (B) cross section of a sheath on the contralateral denervated side, both stained in parallel with antibodies to nAG. (C) Longitudinal section of a blastema on the innervated side at day 13 pa, showing nAG-positive glands (arrow). The inset shows two glands at higher magnification. (D) Section of the contralateral denervated epidermis, with the amputation plane (dotted line) and the blastema. Scale bars: (A) and (B), 100 μm; (C) and (D) 500 μm.
 
Nerve dependence?

As stated before, salamanders are unable to regenerate their limbs when they are denervated. Consequently, the molecules that underlie this are likely not present when the nerve goes away. No nerve, no molecule. Becauase nAG is likely, the authors wanted to determine what exactly happens to nAG expression when you denervate the animal.

Panels A and B

This figure shows immunohistochemistry using an antibody toward nAG. Panel A shows the nerve sheath of an animal with an intact nerve and Figure B shows the other side of the animal, which has been denervated.

Panels C and D

These panels are very similar to Figure 3A and B, but this time they focus on the wound epidermis. The top figure shows the control limb and the bottom panel shows the denervated limb on the same animal. No expression of nAG is seen in the wound epidermis of the denervated limb.

 Conclusion

These results show that nAG, itself, is nerve dependent. When the limb is denervated expression dramatically decreases compared with the control limbs. These data further suggest that nAG is the likely molecule underlying nerve-dependent regeneration.

 
Activities of nAG on the denervated blastema. To deliver the protein to the adult newt limb, we electroporated plasmid DNA into the distal stump at day 5 pa. In trial experiments, red fluorescent protein (RFP) was strongly expressed in about 30 to 50% of the mesenchymal cells in this region (Fig. 4A) and persisted for up to 3 weeks. We expressed nAG from a plasmid with the N terminal signal sequence, and the protein was readily detectable both in the electroporated cells (Fig. 4B) and after secretion in the extracellular space of the early regenerate. Because this procedure appeared to deliver the protein effectively, we denervated animals on the right side, amputated both limbs, and then electroporated the nAG plasmid or empty vector on the denervated side. At day 8 after electroporation, we sectioned the distal limbs on both sides and stained with the nAG antibodies. None of the animals electroporated with the control vector showed the appearance of nAG positive glands in the wound epidermis, but five out of six animals electroporated with the nAG plasmid showed the induction of nAG positive glands (Fig. 4C). Therefore, the delivery of this protein to a denervated blastema can induce these elements in the wound epidermis.
 
p4.jpg
 
Fig. 4.  Delivery of nAG protein to regenerating newt limbs. (A) RFP expression at the end of the limb stump at day 10 pa after electroporation at day 5 pa. (B) Expression of nAG in cells of the limb after electroporation of nAG plasmid at day 7 pa. The section was stained with antibodies to nAG (green). (C) Section of a nAG positive gland in the WE after electroporation of nAG plasmid into a denervated limb blastema at day 5 pa and analysis at day 17 pa. (D) Experimental design for assaying activity of nAG on the denervated blastema. Newts were denervated and amputated before electroporation on the denervated side with either vector or nAG plasmid DNA. (E) Representative animals at day 40 pa from the two groups of an experiment outlined in (D). The yellow star indicates the position of the initial denervation. Scale bars: (B) and (C), 250 μm.
 
nAG to the rescue

The authors now believe that nAG is probably an important molecule in limb regeneration. However, they still need to test this idea. To do this they, they denervate the salamander, amputate, and then exogenously add back in nAG. Remember, nAG is not present in the denervated limb as shown by Figure 3. In this sense the authors are performing a rescue experiment. In other words, they are trying to see whether adding back nAG will allow the limb to regenerate.

 Electroporation

Electroporation is technique by which a scientist can cause a cell to take up DNA found in the external environment. Often times the scientist is trying to overexpress a protein or insert a DNA sequence not normally found in that organism.

This technique is actually very intuitively named. “Electro” is in reference to the electrical force applied to the cells during this technique. “Poration” refers to how this electrical current causes holes in the plasma membrane to form. The holes generated by electroporation are large enough for things like plasmids to enter, which is extremely useful for introducing exogenous DNA. Eventually, the holes of the cell close up and the alien DNA is now trapped inside the cell where it can instruct machinery to make mRNA and proteins.

The authors use a version of this technique called focal-point electroporation, which allows for electroporation of tissues and not individual cells. This technique is especially useful for electroporating tissues such as those that make up the regenerating limb.

For an animation of electroporation as it used in bacteria, see: http://www.auburn.edu/academic/classes/biol/3020/iActivities/ch08/NEanim…

Although this is different from focal-point electroporation, the principle is still the same.

Panels A, B, and C

These experiments show an excellent test for efficiency for the electroporation procedure used in these set of experiments. In figure 4A, the authors demonstrate that 30% to 50% of cells had and expressed their plasmid. Furthermore, they show that the red fluorescent protein expressed from this plasmid is still present 3 weeks later, demonstrating that expression is long-lived.

Figures 4B and C are very similar, but instead of electroporating a red fluorescent plasmid into the salamander, they instead use a nAG plasmid. They then follow up on nAG’s expression using immunohistochemistry to show where nAG is expressed. Taken together, these experiments demonstrate that focal-point electroporation is an efficient way to ectopically express proteins in salamanders.

Panel D

This shows the setup for the experiments performed for Figure 4E.

Panel E

Using the experimental setup from 4E, the authors show that electroporation of a nAG plasmid rescues the phenotype of denervated animal (i.e., they regenerate limbs).

Left salamander: Both limbs are amputated, but only the right limb is denervated. The right limb is electroporated with a control plasmid. As expected, this animal is able to regenerate its left limb, but not its denervated right limb.

Right salamander: Both limbs are amputated, but only the right limb is denervated. The right limb is electroporated with a nAG plasmid. The only difference between the right salamander and the left is what is electroporated. In this case, electroporation of nAG is able to rescue the phenotype and one can clearly see the regenerated limb on the right side of the animal.

 Conclusion

The authors have demonstrated that expression of nAG in denervated limbs is able to allow for limb regeneration. This is an important finding that continues to hammer away at the idea that nAG underlies the molecular mechanisms underpinning nerve-dependent regeneration. Here the authors show that nAG is necessary for proper limb regeneration.

 
To determine whether nAG can rescue the nerve dependence of limb regeneration, groups of animals were denervated on the right side, and then amputated bilaterally (Fig. 4D). At day 5 pa, the right limb was electroporated either with nAG plasmid or with empty vector. The animals were allowed to regenerate, and the progress of limb regeneration was monitored up to day 40 pa. Two representative newts are shown at day 40 inFig. 4E. The position of the initial denervation is marked with a yellow star. The newt on the left has regenerated its control left limb, whereas the right denervated limb has failed to regenerate. In some animals, the axons may subsequently regenerate from the level of the star to the amputation plane, but denervated adult newt blastemas undergo fibrosis and other tissue changes that stop them from making a delayed regenerative response (27). All animals electroporated with the vector resembled the left newt in Fig. 4E. The right animal has also regenerated on the control left side, but the expression of nAG has rescued the denervated blastema and regeneration has proceeded to the digit stage. We analyzed the animals of different batches at day 30 to 40 pa, and half of the nAG-electroporated animals showed digit-stage regeneration (Fig. 4E). Some animals regenerated more slowly and were not included as reaching digit stage, whereas others did not regenerate, possibly because of the variability in the nAG expression level observed after electroporation of plasmids into adult limbs.
 
Limbs rescued by nAG expression were sectioned and stained with antibodies, along with their contralateral control limbs. After staining with antibody to acetylated tubulin, which stains peripheral nerves, the rescued limb showed few labeled profiles, whereas the control limb was densely innervated (Fig. 5, A and B). This result also indicates that nAG did not rescue the denervated blastema by enhancing the rate of nerve regeneration. These limbs were usually atrophic compared with the contralateral controls (Fig. 4E), and Fig. 5, C and D, shows sections stained with antibody to myosin. The experimental limbs had less muscle than the innervated controls, and it appears that the dependence of skeletal muscle on its innervation (28) was not satisfied by substituting nAG. It is clear, however, that the nerve requirement for completion of the PD axis was met in these animals.
 
p5.jpg
 
Fig. 5.  Nerve and skeletal muscle are deficient in nAG-rescued limbs. Rescued limbs were analyzed along with the contralateral innervated limbs, generally at mid-radius/ulna level. Sections of innervated (A) and rescued (B) regenerate limbs were stained with antibody to acetylated tubulin. Sections of innervated (C) and rescued (D) limbs were stained with antibody to skeletal myosin to label muscle. M, muscle. Scale bars, 200 μm.
 
Necessary vs. sufficient

Although the authors have shown that nAG is necessary for proper limb regeneration, they haven’t shown whether nAG by itself is sufficient to regenerate it properly.

That is, they want to know whether there are any abnormalities in the denervated limb rescued with nAG.

Panels A and B

Immunohistochemistry for acetylated-tubulin is performed on normal (A) and rescued (B) limbs.

In the limbs rescued with nAG electroporation, there appears to be much less acetylated tubulin staining, which shows that there is much less peripheral nervous system tissue in the nAG rescued limb.

Panels C and D

A similar experiment to Figure 5A and B was performed but with an antibody toward myosin. This stains skeletal muscle in the samples.

Like Figure 5B, much less staining is found in the rescued limbs.

This demonstrates that skeletal muscle was not regenerated to as great a level as in a normal regenerated limb.

 Conclusion

Clearly, there are some major differences between regeneration in the normal limb versus the nAG rescued limb. At the least, in regards to nervous and muscle tissue.

Of note, results from these experiments also show that nAG works by some means other than simply regenerating the nervous tissue.

If it did regenerate the nerves, those nerves could potentially then send the signals for a correct regenerative response.

The muscle follows along with this because it, too, is nerve-dependent in regard to regeneration.

If the nerve is severed or destroyed, the skeletal muscle will be unable to regenerate.

Likewise, the results we see in this experiment point to lack of proper nerve formation, which leads to lack of skeletal muscle regeneration.

 
nAG acts as a growth factor for cultured blastemal cells. It is difficult to understand the events underlying cell division in limb mesenchyme because of the complexity of epithelial-mesenchymal interactions in development and regeneration (2930). To determine whether nAG acts directly on limb blastemal cells to stimulate their proliferation, the wound epidermis was removed from limb blastemas, and the cells were dissociated and allowed to attach to micro-wells in serum-free medium before maintenance in medium containing 1% serum (Fig. 6A). These cultures were reacted under live conditions with antibody to Prod 1, and ∼70% of the cells were specifically stained on their cell surface. The cells were incubated with medium from Cos 7 cells transfected with a nAG plasmid or with a control plasmid. The nAG protein was detected in the medium after immunoblotting under both reducing and nonreducing conditions as a band at 18 kD (fig. S5). The mean stimulation index for S-phase entry, as determined by bromodeoxyuridine (BrdU) pulse labeling, was 8.3 ± 3.3 fold (SD as determined in eight independent experiments) (table S1). All cell preparations were responsive to nAG; an example is shown in Fig. 6, B and C. This evidence supports the view that nAG can rescue the denervated blastema by acting directly on blastemal cells to stimulate their proliferation and, therefore, that it mediates the nerve-dependent growth of the early regenerate.
 
p6.jpg
 
Fig. 6.  Activity of nAG on cultured newt limb blastemal cells. (A) Blastemal cells in dissociated culture at 10 days after plating. (Band C) Microwell cultures of blastemal cells that were analyzed for S-phase entry promoted by (B) control concentrated Cos 7 cell conditioned medium or (C) nAG-transfected medium processed in parallel. The cells were pulse labeled with BrdU, fixed and stained for nuclei (blue) or BrdU uptake (green). Scale bars: (A) 200 μm; (B) and (C), 1 mm.
 
 How?

nAG has now been shown to be an extremely important molecule in the regenerating limb.

Addition of nAG is able to rescue the phenotype of denervated limb and allow for it to regenerate.

Now that the role of nAG in limb regeneration has been well-studied, what is left is finding out the mechanism behind how nAG works.

Primary cell culture

To determine the mechanism of nAG, the authors use a technique called primary cell culture.

This technique takes cells directly from the animal they are studying where they can then be grown in a petri dish or flask.

Unlike normal cell culture, though, these cells are usually not immortal. In other words, they will eventually cease to grow or die off.

The benefit of this though is that these cells are usually much more biologically relevant to what is going on in the living organism compared with an immortal cell line.

Panel A

This picture simply shows the cells growing in culture. Notice that they are for the most part separated from each other.

These cells were taken from the limb blastema and chemically separated or “dispersed.”

BrdU labeling

Whenever new DNA is synthesized, new nucleotides are used to synthesize the strands.

BrdU is an artificial nucleotide similar to thymidine. It is useful because it can be artificially added to cells to measure how many cells are making new strands of DNA.

Usually fluorescent antibodies, as in immunohistochemistry, are used to visualize BrdU nucleotides incorporated into the strands of DNA. Through these means one is able to determine how many cells are getting ready to divide. A nondividing cell is not synthesizing new DNA and would not incorporate the BrdU nucleotides.

Panels B and C

Primary limb blastemal cells were cultured in either control medium or nAG containing medium and pulsed with BrdU. Cells with the nAG containing medium were significantly brighter in fluorescent signal compared with the control, demonstrating that these cells were actively preparing to divide.

 Conclusion

With much more BrdU taken up in the nAG exposed cells, it appears as though the authors have a mechanism behind nAG’s function.

It appears as though nAG stimulates cells to grow and divide. Imagining this in the living organism, one can imagine that nAG signals cells of the amputated limb to start growing and dividing to reproduce the amputated limb.

Although these data will have to followed up in the actual salamander, it does leave a path down which the authors can continue their studies.

 
Our identification of nAG as a ligand for the PD determinant Prod 1 has underlined that patterning and cell division are linked at the molecular level. We envisage that PD identity is manifested by the quantitative gradation of Prod 1 (6) andthat nAG has no role in specifying that identity but rather acts through Prod 1 to promote cell division. Blastemal growth is stimulated in experimental confrontations of cells differing in positional identity–for example in PD intercalation, in which a wrist level blastema is grafted onto a shoulder stump (3132)–and this is always dependent on the presence of the nerve. In a recent study of supernumerary limb formation in the axolotl, the deflection of the brachial nerve into a skin wound provided a growth stimulus to form an ectopic blastema or “bump”; such bumps only progress to form limbs if a piece of skin is grafted from the contralateral skin to the wound site so as to provide dermal fibroblasts of disparate identity (14). It is interesting that dermal fibroblasts express Prod 1 and that this expression is up-regulated by retinoic acid (6).
 
Two previous studies on AG proteins are relevant to the present results. First, the human AG2 protein was used as bait in a yeast two-hybrid assay and found to complex with a GPI-anchored protein called C4.4, which is associated with metastasis (2433). This protein has two Ly6-type domains that are related in sequence to urokinase-type plasminogen activator receptor (34). The degree of relatedness between the three-dimensional structures of Prod 1 and C4.4 domains is not yet resolved, but taken together these results suggest that functional interactions between AG proteins and this class of small Cys-rich protein domains may be conserved.
 
In the second study, it was found that overexpression of the XAG2 protein in early cleavage stage Xenopusembryos could induce formation of an ectopic cement gland that expressed XAG2 (22). We find that expression of nAG induces formation of nAG-positive glands in the denervated newt wound epidermis. After amputation, nAG appears first in the Schwann cells of the distal nerve sheath and then in glands in the wound epidermis. If axonal regeneration is prevented by denervation, neither the Schwann cell nor the glandular expression is detected. Our results suggest that nAG is released by the distal sheath and induces the formation of glands in the wound epidermis. It appears that the secreted nAG acts directly on the limb blastemal cells. It is unclear how the regenerating axons act on the sheath cells, although the membrane form of neuregulin is a candidate, in view of its importance for such interactions (35). The nerve dependence of regeneration offers a distinction between limb development and regeneration, because the outgrowth of the limb bud is not dependent on its innervation (36). Nonetheless, the ingrowth of the nerve is critical for establishing the nerve dependence, as shown in elegant transplantation experiments on axolotl larvae (37). The identification of nAG offers a new opportunity to study the mechanisms underlying this switch.
 
Nerve dependence of regeneration is conserved in phylogeny. It has been studied in regeneration of the fish fin, the taste barbel in catfish, the arms of crinoid and asteroid species in echinoderms, and the body axis in annelids (2838). In most vertebrate appendages, the density of innervation is lower than in salamanders, and Singer suggested that this is a primary determinant for the loss of regenerative ability, for example in mammals (939). This hypothesis now seems unlikely because there are other variables apparently curtailing regeneration (1). It is notable that the expression of a single protein can rescue limb regeneration in an adult animal (Fig. 4E), and this finding underlines that the blastema is an autonomous unit of organization for which there is no obvious mammalian counterpart. We have suggested that one approach for regenerative medicine would be to understand the specification of the blastema at a level of detail that would allow it to be engineered in mammals (1). The local delivery of permissive regulators such as nAG could then evoke formation of the appropriate structures without the need for subsequent intervention.
 
Supporting Online Material
 
Materials and Methods
Figs. S1 to S5
Table S1
References
 
References and Notes
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