Jose Rafael E. Clemente       

One cannot help but marvel at the healing of a torn skin of the healing of a broken bone. The healing process is a process of regeneration which involves increase in cell mass and volume. Thus regeneration, which ultimately results in the restoration of, lost tissues must involve biosynthesis of nucleic acids as well as cellular proteins and other components. It must be a well-regulated process otherwise if everything regenerated there would be no death and if there were no regeneration; there would be no life.
Not all organs regenerate. Those that easily regenerate are the liver, kidney, pancreas, adrenal, and the salivary glands. Studies with these organs revealed that critical event in regeneration is the conversion of repressed to de repressed DNA.
It has been shown that a lysine-add-rich histone disappears in partially hepatectomized rat at the onset of regeneration and reappears after regeneration has been completed. The disappearance of this histone is associated with the onset of DNA synthesis and the reappearance is associated with the stage when DNA, RNA and protein biosynthesis have reached the peak. Similar observations were reported with experimentations with the regeneration of pancreas and the salivary gland.
It is proposed that normally these histones interact with double stranded DNA with the amino groups interacting with the phosphate backbone of DNA. These interactions would not allow unwinding of the strands for replication and duplication. When the histones are phosphorylated, methylated, or acetylated, conformational changes occur leading to the removal of the histones from the DNA. This has been shown when increased phosphorylation, methylation, and acetylation of histones that are detached from the DNA were observed.
Studies with regenerating rat liver showed an increase in histone acetylation during the first five hours of hepatectomy. It was also separately that within an hour after hepatectomy histones of the liver were phosphorylated. Methylation of histones reached a peak at about the same time, as the peak of DNA synthesis implying that methylation of histones was an event occurring parallel to or after the synthesis of DNA. With studies of rat pancreas acinar cell regeneration after ethionine administration, it was shown that a lysine-rich histone fraction decreased considerably during regeneration, and returned to control values after regeneration. Phosphorylation of F1 fraction of histone was elevated shortly after the peak of DNA and RNA synthesis. (Syllangco et al 1987)

How cells differentiate:

          All of the cells of an organism contain the same genes, which is stored in its DNA. Studies in frogs have shown that when cells specialize, the genetic information are preserved in the DNA. However only the genes that are required by the specializing cell is expressed. This can be done experimentally by removing the nucleus of a specialized cell and transplanting it to an egg cell or stem cell, which have been a denucleated. In this procedure an entire organism will eventually develop.

          Looking at it, the intact cell is very stable and once a cell has differentiated or specialized, it will not redifferentiate or specialized to another type of specialized cell. An example is a cardiac muscle cell. It cannot differentiate into a liver cell or other type of cell because its genes are already expressed and the other genes stored in the DNA have been repressed.

          These are principles that determine a specialization by a cell. The localization principle model, this is focused that there are other determinants in the molecular level that determines the cell type a germ cell would be. One cell might contain a number of different determinants and produce different types of specialized cells. This gives the idea that cells in the body communicate through signal transduction and receptor interaction to send messages during its development.

Germ Cells

Being a germ cell means being able to have descendants which can become any cell type in the body (a quality called totipotency). We don't yet know much about the molecular basis of this ability. One gene, called octamer binding (OCT4), is important in this context. It is expressed in the unfertilised egg and continues to be expressed for a short time after fertilisation. At that stage, the cells which will form the body's somatic cells switch it off, and only those which will result in germ cells continue to express it. Somatic cells are not totipotent, although their nuclei can, as Dr Gurdon described, be persuaded to become totipotent by putting them back into an unfertilised egg - something that was thought not to be possible in mammals.

Mammalian Development:

          Germ cells are cells whose daughter cells all differentiate to sperms and egg cells, and forms the very link of the generation to the next generation. Also called the seed that carries the information of the past generation and passes it to the next generation, by expressing it and making the cell proliferate to become the next in line. Germ cells arise in many ways; this depends on what type of animal it has been extracted. A good example of this is the fruit fly in which forms the localization model. The mRNA programs the stem cells to become germ cells from the start.

          In other animals like mice, the process is different. It happens through cell interaction. Also the germ cells which are found in a particular location inside a cluster of cells, which are implanted in the uterus of the animal, hence it is highly selective. Also the daughter cells that migrated to the same location as of the mother cell had the same fate, but the cells that did not follow or migrated outside of the position before maturity was not able to differentiate to germ cells. (Mc Laren 1999)

          These factors that stimulate specialization of the cells are still not known and research is still on going.

Tissue transplantation

If all cells have the same genes, there could come a time in the future when it will be possible to switch any cell to any other cell. The result could be a tissue or organ transplantation.Researchers are only just beginning to understand how cells switch from one type to another, and for the moment it is difficult to achieve. However, in future, it would be possible to imagine taking some embryo or stem cells from a newborn human, finding ways of proliferating those cells in vitro, and freezing samples permanently. Later in life when the person needs particular cells to replace damaged or diseased ones, some of the store could be unfrozen and made to differentiate to the required type. We'll need to know much more about the way organs are formed, in their greater complexity than single cells, for this to work; but presuming that research is successful, tissue or organ transplantations could result.

          During development, axons reach their targets before the vast majority of Schwann cells are born.  However, after nerve injury, resident Schwann cells autophagocytose their myelin, and remain associated with their basal lamina until the neonacient axons re-enter the distal nerve, where they become myelinated.  We believe that an understanding of the molecular events surrounding the intrinsic differences between development and regeneration is critical for the rational design of therapeutics to treat the diseased tissue.  We have made significant headway in this area, and have identified genes that are expressed by the Schwann cell and are upregulated, as part of the regeneration-specific cascade.

Mouse genetics is a powerful tool that we have exploited to study the transcriptional events that regulate in neuron-glial interactions.  In specific, we have described many of the Schwann cell-executed transcriptional events that occur during PNS promyelination.  Promyelination begins at or about the time of Schwann cell cell-cycle arrest, but prior to the onset of myelin-specific gene expression.  We have identified the expression and function of two members of the POU family of transcription factors as being critical in both establishing and maintaining myelin gene expression.  We are presently working to understand the inter-relationship between these protein in both development and regeneration. (Mescher 1998)

Regenerative growth:

          Researches are done to see tissue interactions during development that affect cell proliferation and its growth. We now focus on the regeneration of an amphibian limb. After its amputation it is able to regenerate completely. The growth and regeneration of its tissues are dependent on the effect of the nerves, the nature of the regenerative factors that induces its regrowth is still not known.

          Peripheral nerves contain large amounts of iron-transport proteins called transferring which is found in the plasma and has been found to have an activity in activation of the cell cycle of regeneration. Investigation of hypotheses have shown that nerves release transferring and this protein could have involved in the neural stimulation for the regeneration of the limb. (Mescher 1999)

          Experiments done on cultured regenerating limb tissues have shown that transferring is transported axonally  in nerves which are released from the ends of regenerating axons. Molecular methods were used to examine expression of the transferring gene in the peripheral nerve cells of the proliferating limbs.(Kiffmeyer et al 1991)

In addition to the investigations on the role of nerves in regeneration, a new study in collaboration with other Indiana University regeneration biologists will look for newly synthesized gene products important for limb regeneration.

These projects on the control of regenerative growth offer graduate students the opportunity to learn and gain experience with a variety of experimental methods, including microscopy, cell and organ culture, immunochemistry, as well as gene cloning and other basic molecular techniques. (Mescher 1996)

Regeneration

Professor Jeremy Brockes, from University College London, talked about why mammals cannot regenerate damaged limbs, organs and tissues to the same extent that frogs and other non-mammalian vertebrates can. Deer can regrow their antlers but our ability to regenerate significant structures is very limited. Regeneration is a biological mechanism which occurs in animals with the same body plan as ourselves. Some amphibians have particularly remarkable regenerative powers. Aquatic salamanders and newts are the only adult vertebrates which can regenerate a limb, and they can regenerate other structures as well. Newts can regenerate their upper and lower jaws and teeth, eye tissues (lens, iris and retina), and heart tissue. The evolutionary development of animals shows that the ability to regenerate tissues is widespread. However some species that can regenerate are closely related to others that cannot; and researchers do not yet understand why this should be. At the moment, there is no known single process or mechanism which can explain regenerative abilities. One key aspect of the process has however been identified. Newts and salamanders can locally reverse the differentiated state, so that when tissue is lost or injured, differentiated cells are able to go back into the cell cycle, divide and redifferentiate. This seems quite different from a stem cell-type mechanism, and it seems the closest candidate researchers have to an underlying mechanism.

Newts

The newt heart has a single ventricle chamber and two atria. If a section of the ventricle is removed, the newt rapidly seals off the ventricle with a blood clot. The heart muscle cells in the area around the clot then go back into the cell cycle and through several rounds of division. This does not happen in an adult mammalian heart! The newt lens can be removed. The new lens grows progressively over several weeks: black, pigmented cells in the iris diaphragm go back into the cell cycle, divide, lose their pigment and turn into lens cells. The newt also has a mound of cells at the end of the limb stump which can regenerate the limb or jaw. If the mound of cells is transplanted into another part of the animal, it will form a new limb there. This is not like the totipotency described earlier. It is a much more restricted reversal of differentiation. The cells retain their identity as limb precursor cells but they go through several rounds of division.

Conclusion

Will humans ever be able to regenerate limbs? The newt's strategy of locally reversing the state of differentiation is a powerful one, so it is worth looking at whether it could be made to work for people too. Through focusing on newt muscle cells in culture, researchers have thrown some light on the mechanism which allows the cells to re-enter the cell cycle. A very important controlling player in this process is the retinoblastoma protein. It is involved in locking the cells out of the cell cycle so that the differentiated cells no longer divide. In culture, the newt can inactivate this protein, so that the cells go back into the cell cycle. The same protein can be inactivated through genetic mutation in mammalian muscle cells. When this is done, these cells act in rather the same way as those of the newt. The same change can be induced in culture by using serum - the soluble fraction of clotted blood - which cells normally encounter in the context of wound healing. Newt cells are responsive to some signal in serum which induces them to re-enter the cell cycle. As far as researchers can tell, the signal is generated in the wounding response in all vertebrates. However, differentiated newt cells respond to it whereas mammalian cells do not. It seems then that the mechanisms involved in regulating the differentiated state are rather similar in the differentiated newt cell and its mammalian counterpart.Researchers hope that by studying the process that allows differentiated cells to go back into the cell cycle, they will come to understand how this pathway operates, and why it does not operate in mammals. In future this knowledge may turn out to be helpful in tissue replacement.