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PRIMER ON REGENERATION
A. Definition, Related Phenomena, Occurrence
1. Regeneration may be defined as the process that allows a multicellular organism to
re-grow and re-organize lost body parts [Stages of salamander leg regeneration].
2. Regeneration is related to asexual reproduction, such as the budding of Hydra or the
ability of plants to form stolons, or “runners”.
3. Regeneration is also related to cloning, i.e., the production of multiple individuals that
are genetically identical. For example, whole fertile plants can be regenerated, and
thus cloned, from single differentiated cells. This has been reported by different
investigators using potato leaf [ABD 7.10a,b], carrot phloem, and tobacco stem pith
cells.
4. However, the specific feature of regeneration is that it serves to restore a specific
body part in its original size, polarity, handedness, etc. Regeneration therefore entails
not only growth but also the exchange of signals that tell the regenerate how much to
grow and how to reorganize.
5. Species differ widely in their ability to regenerate.
a. Plants are well known for their ability to regenerate from isolated leaves or stem
pieces, or even from single differentiated cells as mentioned earlier.
b. Simple animals like sponges and polyps are good at regeneration and asexual
reproduction.
c. Among the vertebrates, amphibians have great powers of regeneration while
reptiles, birds and mammals generally are not good at this.
6. In mammals, the ability to regenerate also differs from organ to organ: Liver
regenerates from as little as 25% of its original mass while other organs do not
regenerate or are very limited in this capacity.
B. Morphallaxis and Epimorphosis
1. When a Hydra is cut in two pieces, two complete organisms will be regenerated, both
smaller than the parental hydra [Hydra regeneration]. Once regeneration is
completed, the two Hydra grow until they reach the size of their original parent.
Growth requires cellular proliferation but it occurs after, not before, the
reorganization process.
2. The type of regeneration seen in Hydra is called morphallaxis (Gk. morphe, shape;
allassein, to change). It refers to the construction of lost body parts by remodeling
the remaining tissue. In this type of regeneration, little or no cellular proliferation
takes place during reconstruction.
3. Planarians (flatworms) are simple animals with bilateral symmetry and anteroposterior polarity. When a planarian is cut in half, medially or transversely, each part
can form a new animal [Planarian Regeneration]. In the damaged region, a
growing mass of cells is formed that takes on the correct shape – be it of a head or a
tail. Gradually the cells become differentiated again and begin their normal functions.
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4. The type of regeneration seen in planarians is called epimorphosis (Gk. epi, over,
upon). It requires active cellular proliferation prior to the replacement of the lost
body part. Epimorphosis can be further subdivided into dedifferentiationdependent and dedifferentiation-independent subclasses.
a. Planarians regenerate using a dedifferentiation-independent mechanism in which
preexisting stem cells, known as neoblasts, begin to proliferate and migrate to the
injured site. These cells then form a mass of proliferating cells, known as the
regeneration blastema, which will later differentiate into the specialized cells that
comprise the regenerated structure. This mechanism is very effective in
planarians: pieces amounting to less than 1% of the original volume can
regenerate an entire animal.
b. The normal turnover of tissues in mammals also resembles the dedifferentiationindependent subclass of epimorphosis. For example, mammals renew their bone,
muscle, epithelia of skin and gut, and blood cells from adult stem cells.
c. Salamanders regenerate lost body parts through dedifferentiation-dependent
epimorphosis. Here, differentiated cells reverse the normal developmental
process and form a regeneration blastema of cells that once again look embryonic
and divide actively. These dedifferentiated cells later redifferentiate to form the
regenerated structure or organ.
C. Limb Regeneration in Amphibians
Tailed amphibians are known for their ability to regenerate limbs and other body parts.
1. When a salamander limb is cut off, epidermal cells spread over the wound and form
an apical ectodermal cap. Underneath the cap, all stump tissues undergo
dedifferentiation and form a regeneration blastema. The blastema gives rise to the
mesodermal tissues (including bone and muscle) of the regenerate while the
ectodermal cap forms the epidermis.
a. Cells from all stump tissues (skin, muscle, bone) contribute to the regeneration
blastema. However, if the blastema cells are killed by radiation, an implanted
piece of (un-irradiated) cartilage will develop into a limb with bone, muscle, and
skin. This indicates that cartilage cells can give rise to muscle and skin.
b. Regeneration of a limb from a blastema resembles embryonic development of a
limb from a limb bud.
c. The regenerated limb comprises the parts distal to the cut surface [ABD 21.10].
d. The regenerate receives complex patterning information, which can be modeled
by the polar coordinate model [ABD 21.16].
2. Salamander limb regeneration is nerve-dependent.
a. If the nerves of a limb are cut shortly after amputation and proximal to the
amputation surface, no regeneration occurs.
b. After denervation, limb regeneration can be rescued by injecting the blastema
with a plasmid encoding newt anterior gradient (nAG) protein, which acts as a
ligand to Prod 1, a cell surface protein of blastema cells. nAG is expressed in
nerve sheaths and apical ectodermal cap cells of amputated but not denervated
limbs [Kumar et al. (2007) Fig. 1].
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D. Regeneration after Autotomy
Geckos and various other animals can regenerate tails and other body parts after a selfamputation process called autotomy (Gk. auto, self; tomnein, to cut).
1. Geckos, lizards, and some salamanders that are captured by the tail will shed the end
of the tail and thus be able to flee. The detached tail will continue to wriggle, creating
a deceptive sense of continued struggle and distracting the predator's attention from
the fleeing prey animal. Later, the animal can partially regenerate its tail over a period
of weeks. The new section will contain cartilage rather than bone, and the skin may
be discolored compared to the rest of the body [Tail regeneration after autotomy].
2. Autotomy in lizards is enabled by special zones of weakness located at regular
intervals in the vertebrae. Essentially, the lizard contracts a muscle to fracture a
vertebra rather than break the tail between two vertebrae. Sphincter muscles in the
tail then contract around the caudal artery to minimize bleeding.
3. Crabs, lobsters, spiders, and other invertebrates can also sever and regenerate lost
appendages.
E. Sequential Polymorphism
Cell differentiation is usually irreversible, and differentiated cells do not normally retool
for other functions. However, there are exceptions to this rule. Some cells, as part of
their normal development, undergo dramatic changes in structure and function called
sequential polymorphism. Other cells demonstrate this ability under experimental
conditions.
1. The follicle cells of insect ovaries carry out two different functions at subsequent
stages of development [ABD 7.3].
a. During vitellogenesis, follicle cells are tall columnar cells involved in the
accumulation of yolk proteins in the oocyte.
b. During the subsequent formation of the eggshell (chorion), the same follicle cells
assume a flat shape and synthesize large amounts of different chorion proteins.
2. When the lens of the eye is removed surgically from the newt Triturus viridescens,
iris cells can transform into a new lens [ABD 7.4].
a. The process involves a series of events in the iris cells including shape changes,
loss of pigment, mitosis, RNA synthesis, shaping of a new lens, and synthesis of
large amounts of lens-specific proteins (crystallins).
b. These events are not a recapitulation of normal lens development, which occurs in
a lineage of cells that would otherwise form epidermis.
F. Regeneration and Transdetermination in Imaginal Disks
In normal development, overt cell differentiation is often preceded by a stage of
commitment, or determination, when cells are still looking embryonic but will no longer
regulate, i.e., depart from their fate.
1. In Drosophila and other holometabolous insects, metamorphosis involves resorption
of most larval organs while adult structures are formed from bags of epithelial cells
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called imaginal disks. (This term is derived from imago, the technical term for the
adult stage of insects.) Legs, wings, etc. are formed from pairs of imaginal disks, the
anal and genital structures from one disk [ABD 6.18; 6.17].
2. The cells of all imaginal disks look embryonic and alike. However, each disk is
already determined to develop according to fate, legs disks into legs, wing disks into
wings, and so forth.
3. In serial transplantation experiments, [Hadorn photo] tested the stability of the
determined state of imaginal disks that were cut and allowed to regenerate.
a. Adult flies were used as walking culture tubes for implanted segments of imaginal
disks [ABD 6.19]. When the hosts reached the end of their life, the grown
implants were retrieved, cut to their original size, and transplanted to new hosts.
b. To test the determined state of the transplanted disk fragments, these were
inserted into grown larvae. When the larval hosts metamorphosed, so did the
implanted disk fragments. The adult structures formed by implants were removed
from the now adult hosts, inspected microscopically, and identified as parts of a
leg, wing, etc.
c. In an experiment that started with a genital imaginal disk and extended over more
than 100 serial transplantations, the regenerating disk fragments were initially true
to fate in forming only genital and anal structures. However, from the 8th transfer
generation on, antenna and leg structures appeared in addition to analia and
genitalia [ABD 6.20]. (Hadorn called his two senior graduate students back to the
lab on Christmas Eve to witness the discovery.)
d. The disk fragments in which the switches to antenna and leg had occurred, the
same structures were found in subsequent test implants. These imaginal disk
parts now behaved as if they were determined to form antenna or leg. Hadorn
called this phenomenon transdetermination.
e. Further investigations by Hadorn and his students showed that transdetermination
happened in distinct steps, each occurring with certain probabilities for going
forward (and sometimes reverse) [ABD 6.21]. For instance, it took at least three
distinct steps for genital imaginal disk cells to transdetermine to thoracic cells.
f. Transdeterminations are reminiscent of the phenotypes caused by mutations in
homeotic genes, such as bithorax or Antennapedia. However, clonal analysis and
other observations indicate that transdetermination is not caused by mutation or
by delayed determination.
g. Transdetermination is best explained by changes in the expression of homeotic
genes that begin in single cells or small groups of cells and are passed on clonally.
It can be modeled by assuming that homeotic gene expression is controlled by a
set of bistable control circuits. In the simplest case, each control circuit could
consist of two genes, A and B, encoding transcription factors that reinforce
expression of their own gene and inhibit expression of the other gene [ABD 6.19].
h. Bistable control circuits could be “flipped” by disturbances like excessive
regeneration [ABD 16.20]. If the determination of Drosophila imaginal disks
were controlled by five binary control circuits, each transdetermination step
(except between antenna and leg) could be accounted for by “flipping” a single
control circuit.
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G. The Role of Transcription Factors in Cell Differentiation
If indeed cell differentiation is controlled by bistable circuits of gene expression, then
“flooding” cells with transcription factors that they do not normally produce may “derail”
them from their normal pathway of differentiation.
1. Cultured fibroblast were transfected with cDNA encoding MyoD, a transcription
factor characteristic of developing skeletal muscle. The treated fibroblasts behaved
like myoblasts, fusing into myotubes, synthesizing myosin, and proceeding to form
skeletal muscle fibers.
2. Similar results were obtained with other cDNAs representing the MyoD family of
transcription factors [ABD 20.18]. These factors share a basic helix-loop-helix
(bHLH) domain, which functions in the formation of dimers, the transcriptionally
active protein structure. The myogenic bHLH proteins, depending on their
heterodimeric partner, can be activating or inhibiting, which makes them well suited
for acting in bistable control circuits [ABD 20.19]. Indeed, the bHLH proteins
interact with many other regulatory proteins in controlling the growth, patterning, and
differentiation of skeletal muscle [ABD 20.20].
H. Dedifferentiation or Transdifferentiation?
Different modes of regeneration are intertwined with the ways cells change their range of
potency. Morphallaxis, as seen in Hydra, involves the remodeling of existing tissue.
This may occur by extensive reshuffling and re-patterning of differentiated cells, or by
transdifferentiation, that is, direct change of cells from one differentiated state to another.
Dedifferentiation-dependent epimorphosis, as seen in salamander limb, involves a return
of differentiated cells to an embryonic, or undifferentiated state. From there a cell may
return to its previous differentiated state or switch to a new one. The question of
transdifferentiation versus dedifferentiation has been approached in vivo as well as in
vitro.
1. Bjornson et al. (1999, Science 283: 534-537) injected mouse neural stem cells, which
normally form neurons or glia cells, into the blood stream of mice that had been
heavily X-irradiated to destroy their hematopoietic system. Nevertheless, the
recipient mice survived, and newly formed blood cells in their spleens were shown
unequivocally to be derived from the injected brain cells. Circumstantial evidence
suggested that injected neural stem cells had turned into blood stem cells, which in
turn had given rise to the full spectrum of blood cells [ABD 20.22]. However, other
ways of deriving blood cells from neural stem cells were not excluded.
2. While the exact steps from brain cells to blood cells in the Bjornson experiment
remain to be elucidated, the result has been game-changing
a. for research because it demonstrates that the potency of adult stem cells is not
restricted to their fates in their normal niches
b. for medical cell replacement therapies because adult stem cells from easily
accessible tissue (such as fat) in a patient may be used to derive isogenic
progenitor cells of different tissues for which adult stem cells may be hard to
obtain
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3. Zhou et al. (2008, Nature 455: 627-632) reprogrammed pancreatic exocrine cells to
endocrine (β) cells in adult mice [Zhou et al. Fig.1].
a. They caused the formation of new β-cells by injecting into the pancreas a
combination of three β-cell-specific transcription factors (Ngn3, Pdx1, and Mafa).
b. The induced β-cells were indistinguishable from endogenous β-cells in size,
shape, and ultrastructure. They also secreted insulin and reduced hyperglycemia.
However, they did not organize into the islets characteristic of native β-cells.
c. A transient expression of the inducing factors was sufficient to convert exocrine
cells to a stable new β-cell state.
d. Continuous 5-bromodeoxyuridine (BrdU) labeling and other observations
indicated that the induced β-cells did not divide extensively and did not revert to a
dedifferentiated state for an appreciable time.
e. It should be noted that pancreatic exocrine and β-cells differ in their adult
structure and function but are developmentally related cell types. They share their
embryonic origin from pancreatic endoderm as well as many of their epigenomic
markers. Conversion between pancreatic exocrine and β-cells may therefore
require less chromatin remodeling than transitions between other cell types.
f. In conclusion, the authors suggest differentiated cells can be transformed directly
into a developmentally related cell type without reversal to a pluripotent stem cell
state.
4. New work reviewed by Chambers and Studer (2011, Cell 145: 627-630) shows that
cultured fibroblasts can be turned directly into neurons, cardiomyocytes, or blood
cells. This was done by transforming the fibroblasts with combinations of a few
transcription factors characteristic of the target cells. The speed and the yields of
these transformations indicate that the target cells did not pass through intermediate
states of high pluripotency.
5. In summary, there is overwhelming evidence that cell differentiation, in the course of
normal development as well as regeneration, begins with the synthesis of an
appropriate set of transcription factors, which initiate a hierarchy of differential gene
expression. These changes in gene expression patterns may be promoted by
a. epigenetic changes from a closed to an open chromatin conformation. Such
changes would facilitate the binding of key transcription factors to their
recognition sites in nuclear DNA.
b. high concentrations of key transcription factors. These would keep the chromatin
conformation open in the appropriate regions due to the dynamic nature of DNAprotein interactions.
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