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Discuss the regeneration of amphibian limbs
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A blastema is the starting point of limb regeneration, while a limb bud
is the starting point of normal limb development within an embryo
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The blastema is also a collection of
mesenchymal cells that will proliferate and differentiated in order to regenerate a near
identical replica of the original limb generated during development
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Limb bud formation occurs in the amphibian embryo at four discrete positions along the
anteroposterior axis, due to the levels of Homeotic (Hox) gene expression, e
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the forelimbs
develop at the anterior region of HoxC-6 expression (Gilbert, 2000)
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By the time cells migrate and enter the limb bud they are tissue restricted
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These cells
proliferate and migrate to accumulate at the site of limb bud formation causing an outgrowth
which is covered with a simple overlying ectoderm
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Within a couple of
hours of amputation the wound begins to heal forming a ‘wound epithelium’ (Stoick-Cooper
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et al
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Like the limb bud, the blastema is an outgrowth of mesenchymal cells that are
already tissue restricted and covered by an epithelium
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, 2014)
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In both cases the ultimate goal is for
undifferentiated mesenchyme to proliferate and differentiate to form the fully functional limb,
whether for the first time or through regrowth
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A
B

Limb development compared to limb regeneration

Figure 1A – shows the outgrowth of the limb bud and origins of the underlying mesenchymal cells
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The cells from the myotome region of the somites will form the muscle of the limb
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Cells migrating from neural crest will form the
Schwann cells and melanocytes of the limb (Nacu and Tanaka, 2011)
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Figure 1B - shows the outgrowth of the forming blastema and the origins of the mesenchymal cells
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Cells from schwann cells will form schwann cells and cells
derived from cartilage will form cartilage
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(Adapted from Kragl et al
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The cells in an adult amphibian have already undergone differentiation, therefore in order to
reprogram and reform amputated structures, the cells must dedifferentiate back into
undifferentiated mesenchymal cells (King et al
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The injury site induces metabolic
changes and remodelling of underlying tissues, so when the wound epithelium fully closes a
blastema starts to form from dedifferentiated cells from a number of various adult tissues;
dermis, cartilage, muscle and Schwann cells
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, 2009)
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Due to modern experimental techniques, e
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cell fate mapping using GFP integration
in both urodeles and anuran, this has been proven incorrect
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,
2009)
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The same is true for Schwann cell origins only giving rise to Schwann cells, and
cartilage only giving rise to cells with restricted cartilage lineage during regeneration
(Muneoka et al
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This shows that the mesenchymal cells of the blastema do not lose
all their former characteristics and retain a memory of their origin
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These cells
can form dermis, cartilage and tendons
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All the cells within the blastema, although they have reverted back to an
undifferentiated state, are still lineage restricted based upon embryonic origin (Maki et al
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This lineage restriction is present in both the blastema and the limb bud
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Studies have shown that blastema formation has an initial dependency upon
nerves, compared to the limb bud which is initiated via retinoic acid and subsequent Hox
expression levels with no nerve dependency
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This AEC is equivalent to the apical ectodermal ridge (AER)
in the limb bud, as they both originate from epidermis and form a multi-layered epithelium at
the distal tip of the outgrowth
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, 2010)
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Fgf-8 is
expressed in the AER of the limb bud and the basal layer of the AEC in the blastema
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, 2001)
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Cells in the PZ measure the length of time they are
exposed to Fgf signals from the AER, in order to commit to differentiating into specific
structures in a proximal to distal direction
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Fgf-8, in the AER, induces the expression of a morphogen called
sonic hedgehog (Shh), in a signalling region localised to the posterior part of the AER called
the zone of polarising activity (ZPA) (Capdevila and Belmonte, 2001)
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In turn, the
establishment of the ZPA and expression of Shh sets up a feedback loop between the AER
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and ZPA, maintaining proliferation of the cells underneath
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Also included in this feedback
loop is the secreted factor, wingless-7a (Wnt-7a)
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En-1 is an inhibitor of Wnt-7A, setting up the dorsoventral axis (figure 2)
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, 1995)
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Figure 2- shows a limb bud with
its key signalling centres; the ZPA,
AER and DE (dorsal ectoderm)
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(Taken from Yamamoto et al
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This describes a large similarity between limb regeneration and limb development, as the
outgrowth of both is reliant upon the key signalling molecules mentioned above
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, 1997)
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The signals from the AER initiate differentiation of cells in the limb bud,
while the signals from the AEC are required for the dedifferentiation of adult tissue cells,
followed by the differentiation of these blastema cells into functioning, regenerated limb
structures
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The blocking of any of the vital signalling
pathways above, leads to lack of regeneration of amputated limbs (Poss, 2010)
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This is
secreted from the AEC and is needed for the proliferation of the underlying mesenchymal
cells (Kawakami et al
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It turns out nAG is a ligand for a protein found in the
salamander called Prod1
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The crystal structure of Prod1 shows that this protein present in
salamanders does not have a homolog in mammals, making it potentially salamanderspecific (Garza- Garcia et al
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Just as positioning along the proximodistal axis is vital in forming a functional limb, so is the
restriction of positional memory of the cells at the site of amputation
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This positional memory was determined in studies via the
transplantation of an upper limb blastema into an amputated site lower down the arm
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This positional identity of cells along with the proximodistal axis can also be viewed via
specific molecular markers to see contrasts between development and regeneration
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As cells progress distally, expression of Hoxa genes
fall into specific regions, with Hoxa13 being a ‘hand’ marker
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This suggests that unlike in the developing limb,
during regeneration, distal structures are formed first
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An important question
regarding limb regeneration is how does this re-establishment of gene expression occur?
Studies have been conducted relating regeneration of limbs to mammalian induced
pluripotent stem cells (iPSC), and how gene expression is reverted in those cells to convert
them back to an undifferentiated state in vitro
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Gene transcription is regulated via epigenetic mechanisms that affect the
chromatin structure of DNA
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, 2010)
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In
order for iPSCs to be generated, this system needs to be reversed, removing silencing
modifications
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By epigenetic modifications the chromatin structure
can be loosened allowing access of transcription factors to promotor regions
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, 2007)
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Blastema cells, although undergo dedifferentiation, are not, as
previously discussed, reverted back to a completely pluripotent state
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As seen, the ability of amphibians to regenerate amputated limbs is similar to the limbs
original development in many aspects, although extra mechanisms are needed due to the
increased complexity of the surrounding adult tissues in regeneration
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This
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process potentially has many regenerative medicine implications
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A potential
answer to this could lie in the salamander protein Prod1, which mammals do not possess
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The development of nerve-induced supernumerary limbs in the adult
newt,Triturus viridescens
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1958;102:555–581
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Patterning mechanisms controlling vertebrate limb
development
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2001;17:87-132
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Growth and differentiation of a long
bone in limb development, repair and regeneration
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2014;56:410–424
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Solution structure and
phylogenetics of Prod1, a member of the three-finger protein superfamily implicated in
salamander limb regeneration
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2009;4(9)
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Formation of the Limb Bud
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6th edition
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Han MJ, An JY, Kim WS
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Dev Dyn
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Kawakami Y, Esteban CR, Raya M, Kawakami H, Marti M, Dubova I, Belmonte JCI
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Khavari DA, Sen GL and Rinn JL
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King MW, Neff AW, Mescher AL
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Kragl M, Knapp D, Nacu E, Khattak S, Maden M, Epperlein HH, Tanaka EM
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Maden M
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Mok GF, Sweetman D
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Muneoka K, Fox WF, Bryant SV
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1986;116(1):256-260
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Limb regeneration: a new development? Annu Rev Cell Dev
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Okita K, Ichisaka T and Yamanaka S
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Nature
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Poss KD
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Satoh A, Makanae A and Wada N
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Dev Bio
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Shaikh N
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Doctoral thesis, UCL
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Singer M
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Noda M, The transformation suppressor gene Reck is required for postaxial patterning in
mouse forelimbs
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2012;1:458-466
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Nerve-blastema interactions induce
fibroblast growth factor-1 release during limb regeneration in Pleurodeles waltl
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1997 Feb;39(1):15-22

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