Questions for discussion about leg regeneration

Please look at the following youtube videos of the same time-lapse video of a newt leg regeneration.

Please notice the following quote from the second of these web sites.

"Cells in and near the limb stump dedifferentiate to form a mass of stem-like cells that can produce all the specialized tissues of the limb , such as muscles, nerves and blood vessels."

The underlining is added by me, to direct your attention toward a false implication that each dedifferentiated cell can re-differentiate into any of the cell types listed. In other words, any of the "stem-like" cells could differentiate into whichever cell type is needed at any given location. The stem-like cells that differentiate down the middle would (because of their locations?) differentiate into cartilage, the cells located where muscles are needed would (because of where they are) differentiate into muscle cells. However, notice the choice of the word "produce", instead of "become". What does that imply? Perhaps an unwillingness to be explicitly wrong, even while misleading.

A reader could easily conclude that what we need to achieve leg regeneration is human stem cells, in the sense of undifferentiated cells that can be stimulated to differentiate into whichever cell type is needed at whatever position they are located. If that were true, then the key medical problems would be: (#1) To cause some human cells to lose their differentiation AND (#2) become able to re-differentiate into many different cell types, and discover signal proteins ("myo-D" has been discovered) that can cause re-differentiation into muscle cells, and other signal proteins that cause differentiation into skeleton ("Bone Morphogenetic Protein" has also been discovered).

In contrast to those hopes, the results of cell-labeling experiments (by Trygve Steen, Elizabeth Hay, and others.) consistently show that dedifferentiated muscle cells re-differentiate only into muscle cells, and dedifferentiated cartilage cells re-differentiate into cartilage cells (Only? Mostly?)

Maybe someone will discover a treatment that causes REAL De-Differentiation of limb cells? By that I mean, production of cells, any one of which can differentiate into a cartilage cell, or into a muscle cell, or a skin cell, or a blood vessel cell.

How might that be accomplished? Transforming certain genes into their cells? Inhibition or RNA synthesis for an extended time period? Us your imagination?

Spatial signals of some kind (probably proteins that were first discovered in Drosophila) would control re-differentiation of the "stem-like" cells. The shapes, locations and connections of muscles, bones etc. would be controlled by whatever signals stimulate re-differentiation of the "stem" like cells. In other words, at whatever locations muscles are needed, the stem-like cells at those locations would be stimulated to differentiate into muscle cells. Likewise, skeleton would regenerate by stimulation of cells at the right locations to differentiate into chondrocytes.

That's the future apparently hoped for by those axolotl researchers in Gainesville, Florida, who got the $2,600,000.00 special NIH grant. Perhaps their grant proposal did not cite Steen's or Hay's discoveries.

If cells that de-differentiate always re-differentiate back to being the same cell type (chondrocytes form 80% from de-differentiated chondrocytes; myocytes form 100% from dedifferentiated myotubes) this suggests leg regeneration results from cell rearrangement, rather than differentiation.

In that case, should grants support studies of methods by which differentiated cells rearrange?

Notice the similarity to the history of sorting-out of dissociated sponge and hydroid cells? Wilson thought cells were switching from one cell type to another; and later that new sponge tissues were made of previously undifferentiated archeocytes (spare "to be used in an emergency" cells) which many people might now called "stem cells".


Given that dedifferentiating limb cells (with an exception of many cartilage cells re-differentiating from dermal cells) mostly don't switch from being one cell type to another, then what function is accomplished by their apparent de-differentiation?

Muscle cells refuse to synthesize DNA except while uninucleate; so they can't grow in number unless they cleave back into individual (one nucleus per muscle cell) cells.

Maybe mature chondrocytes (cartilage cells) can't form new cartilages, of smaller sizes and different shapes than the femur that they had been parts of?

Matrix secretion is supposed to be a driving force in cartilage growth.

Remember those regular geometric patterns of chondrocytes in growing legs? I wonder if similar patterns are formed in cartilages that regenerate. (Based on their causation being different.)

Visualize an experiment in which cartilage and muscles are dissected out of the tail, or the head, of a newt, and grafted to the site from which a leg had been cut off: what would regenerate, if anything.

If you had a salamander whose chondrocytes contained a protein that had an attached green fluorescent protein (and no other differentiated cell type had any gfp), then how could you use such salamanders to study the mechanisms by which salamanders regenerate their legs?


More topics for discussion:

Possible reasons why Mammals (& also Reptiles, Birds and Frogs) can't regenerate legs, but Salamanders can regenerate legs.

I) Mammal, Bird and Reptile skeletons are made of bone; Salamander skeletons are made of cartilage. Maybe cartilage is (for some unknown reason) easier to regenerate.

II) Higher blood pressure and metabolism causes more rapid bleeding to death, in mammals relative to salamanders, which removes the evolutionary advantage of leg regeneration.

III) Salamanders can still crawl (slither) rapidly without legs, and are still able to eat. Mammals would die before their legs had time to regenerate. This reduces the evolutionary advantage of being able to regenerate.

IV) The much larger sizes of mammal legs may require molecular signals to go so much larger distances. (Distances to far for whatever signals control regeneration.)

V) Mammal, Bird etc. myotubes may not be able to cleave into single cells, as is required for muscle cells to synthesize DNA and reinter the cell cycle, forming more muscle cells.

V 1/2) Subdivision into individual muscle cells may be necessary for new muscles to form ("muscles" in the anatomical sense, like "the trapezius")

VI) Maybe pattern-forming mechanisms (whatever they are) continue to exist in salamanders all their lives, but these mechanisms soon get turned off in mammals.

* Supporting this, indirectly:
1) Frog tadpoles can regenerate their legs about as well as salamanders, but then lose this ability.
2) Human children can regenerate the most distal joints of their fingers, unless bandaged.

VII) Mammal etc. wounds heal by stronger contraction that in amphibia, and mesenchymal cells form strong, dense scars. Scar tissue may block diffusion of "morphogen" signal chemicals needed to stimulate and/or control regeneration of limb tissues.

VIII) Salamanders are the only vertebrates whose embryonic limb buds don't have (and don't need) apical ectodermal ridges. Maybe if a thickening or up-folding of the epidermis could (somehow!) be surgically created on the surface of a mammal limb stump, then it might stimulate regeneration. (there is a particular transcription factor located in AERs and also fin-folds of fish this should be looked for in mammals, and tested to find out if it stimulates limb regeneration.

* Surgical removal of apical ectodermal ridges causes limb elongation to stop. The earlier the removal, the shorter the leg. Nobody has a real explanation why thickenings in the outer layer of the skin are needed for legs and fins to develop. The AER looks somewhat like a fin. Its mechanical cause is not known. It may be the result of contractions being propagated in the anterior-posterior direction.

* Legs extending from the body are smaller (approximate) cylinders extended at right angles to a larger cylinder. Because the surfaces of cylinders are stretched circumferentially with twice the tension as in their longitudinal axis, therefore development of limb buds requires local changes in physical tension in the developing skin (somewhat analogous to how budding occurs in hydra). Apical ectodermal ridges may be part of this local re-direction of physical tension. Its shape must occur for some reason, related to the ability of vertebrate legs to develop.


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