Lecture notes for Friday, April 8, 2016



Limb regeneration in salamanders

I) Newts and many other kinds of salamanders can regenerate their legs.

No other vertebrates can do this, except partial regeneration of limbs in frog tadpoles before metamorphosis.

Humans can sometimes regenerate the last digit of fingers and toes, IF the wound is NOT stitched up (which is standard practice).

Regenerating newt legs "re-grow" all the bones (i.e. cartilages), muscles, tendons, blood vessels, connective tissue and skin.

In the stump of the cut limb, cartilage, muscle and other cell type become indistinguishable, "dedifferentiating" back to what looks like their early embryonic state, forming a mass of cells called a blastema, in which rapid cell growth and mitosis replaces the lost cells.
Skeletal muscle cells, which are syncytial, subdivide back to individual cells before resuming mitosis.

It would be interesting to know whether this subdivision of multi-nucleate muscle cells has the same contractile-ring mechanism as the ordinary cytokinesis that follow mitosis.
The myoblasts then undergo many cycles of mitotic cell divisions, and fuse back together.

II) Grafting of cartilage and muscle labeled with radioactive labeled DNA, and also the use of genetic markers like nucleolus number, consistently show that each cell remains "loyal" to their original differentiated cell type.
In other words the dividing cells in the blastema that had been cartilage cells re-differentiate only into cartilage, & the descendants of muscle cells only become muscle.

Most experimenters had not expected this, which make the results even more persuasive.

It also contradicts expectations that regeneration should have the same basic mechanism as the original formation of limbs, except if the original development was by sorting out of cells whose differentiation had already been decided, which nobody considers possible.

Limb regeneration is not thought of as a special case of sorting out by differentiated cells, I guess because there is no deliberate dissociation into single or randomly-mixed cells.

Nevertheless, consider that cartilage cells from which the humerus had previously been constructed separate into what seem to be undifferentiated cells, and then re-differentiate as the radius, ulna digits, wrist bones, as well as replacements for the lost parts of the humerus.

Furthermore, consider that the many muscles of the regenerated leg are made of cells that had been parts of different muscles of the upper limb.

Likewise: skin cells, blood vessels, connective tissue & nerves.
All are replaced by descendants of the same kinds of the cells in the stump of the cut limb.

III) Salamanders are the only vertebrates whose limb buds do not form an apical ectodermal ridge.

However, during regeneration of salamander, the stumps develop an epithelial thickening which is well-formed, but not elongated in the A-P axis (unlike the AER)

I can't figure out how to interpret this combinations of odd facts, but it must mean something.

IV) If you cut off just the "hand" part of a newt leg, then just that part will regenerate.
If you cut the leg off at the elbow or knee, then just the missing part will regenerate.
If you cut off the whole leg, than all the parts of will regenerate.

BUT: If you cut off just the "hand" part, and then paint the cut surface with retinoic acid, then a whole new leg will regenerate, starting over at the shoulder, and forming an extra elbow, etc.

AND: Newts can regenerate their tails.
BUT if you cut off the tail, and paint the cut surface with retinoic acid, guess what regenerates instead!

The more of a newt leg you cut off, the faster tissues regenerate; So that the total time to completion of regeneration is about the same when you cut off the whole leg as when you cut off just the tip.

V) a) The spaces between fingers and toes are created by the programmed cell death.

So the web of a duck's foot is the primitive condition.
A chicken's foot has separate toes because programmed cell death has removed the tissues in between.

b) The bases of limbs are "sculpted" by regions of programmed cell death.

c) The neck is narrowed by large areas of programmed cell death.

d) The space between your gums and your cheek and limb tissue is created by programmed cell death.

e) The self-destruction of tadpole tails is a classic example of programmed cell death.

f) >98% of lymphocytes self-destruct by programmed cell death. (normally)
(in part, this serves to kill anti-self lymphocytes, whose binding sites fit some normal body protein)

g) Defense against viral infections is achieved by inducing programmed cell death of infected cells.

h) Graft rejection occurs by induction of programmed cell death, by mechanisms that are meant to induce death of virally-infected cells.
(As if the grafted cells were mistaken for self-cells infected with viruses.)

i) Many viruses defend themselves by producing proteins that mimic the body's own inhibitors of programmed cell death. (specifically mimics of the bcl-2 protein, which will be discussed below.

j) Much of the death of heart cells during heart attacks, and of brain cells during strokes is claimed to be programmed cell death (setting off self-destruction enzymes), rather than simple destruction.

Therefore, chemicals that inhibit programmed cell death might save many lives.

k) Many anti-cancer drugs are now believed to act by inducing programmed cell death, somewhat selectively in cancer cells, but not so much in normal cells.

Conversely, a whole series of carbon chains attached to phosphates were designed to reduce osteoporosis by either strengthening bone or promoting more bone formation, but are now believed to stimulate programmed cell death in osteoclasts (but unfortunately in lots of other cell types, in addition to osteoclasts, with nasty side-effects)

l) One specific form of cancer is known to be caused (mostly) by too much synthesis of a particular protein named bcl-2, which localizes around mitochondria, and blocks release of reactive molecules (that release being part of how cells self-destruct in programmed cell death.)

B-Cell Lymphoma-2, because this was the second protein to be discovered by close studies of chromosome translocations in human victims of non-Hodgkins lymphoma. There are other proteins called bcl-1, and bcl-3 ; the latter is a cyclin.

Any gene that gets translocated to the chromosome site next to an antibody gene will get made in large amounts in B-lymphocytes, who are "trying" to make antibodies. Note that the same translocation in any other differentiated cell type might have no effect.

m) A large percentage of nematode embryonic cells are eliminated by programmed cell death. (Mutant worms in which this cell death doesn't occur are almost normal. It isn't at all clear what good it does the worm to have so many programmed cell deaths!

A nematode gene was found that is very similar to the human bcl-2 proteins, and replacement of this worm gene by the human bcl-2 gene restores normal development!

n) Another nematode gene, without which programmed cell death will not occur, was found to code for an inactive precursor of a protein-digesting enzyme, called a caspase. Part of the molecule blocks the active site, but can be digested off by a caspase from which its block has already been removed. Thus, activation of one caspase molecule will activate others, in a chain reaction, and they digest the cell from the inside out. Almost all cells of multicellular animals have millions of caspase enzymes dissolved in the cytoplasm.

o) Embryologists had been studying programmed cell death since ~1900 but the word "apoptosis" wasn't invented until 1980.
[There are disagreements about whether the second P is silent, or not: Apo_Tosis or A Pop Tosis The latter is probably correct. But be prepared for someone to correct you, no matter which way you pronounce it.]

Necrosis is what you call it when cells just die. (Especially if pieces of dead cell are left) Apoptosis is when they self-destruct.

p) Higher plants have a different kind of self-killing method, that they use to resist infections by induction of death in areas surrounding germs, so as to wall them off. Prof. Dangl is a world leader in research on this important phenomenon.

q) For many years, researchers guessed that there would be several different mechanisms of programmed cell death in animal cells, but apparently caspases are used for all the different examples, from making gaps between fingers to killing anti-self lymphocytes and virally-infected cells.

The greatest researcher on programmed cell death was Prof. Glucksman, of Cambridge University who was a friend of mine and a very wise man, and used to show me slides of programmed cell deaths.



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