Limb Repair and Regeneration

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Limb Repair and Regeneration
Ricardo Gutierrez Biology 680 February 25, 2008
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-Morphallaxis- lost body parts are replaced by
remodeling of remaining part. -Occurs in
Planarians -Epimorphosis- requires active cell
proliferation before replacement -Occurs in
limb and fin regeneration, urodele amphibian and
teleost fish
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Epimorphosis characterized by formation of a
blastema after wound healing
http//www.eb.tuebingen.mpg.de/departments/3-genet
ics/zebrafish/christopher-antos/regeneration
Zebrafish caudal fin after wound
healing BlBlastema, WeWound epidermis
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Blastema is a mass of undifferentiated cells
capable of growth and regeneration resulting from
dedifferentiation of cells.
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Zebrafish used as model organism for studies
regeneration
Most studies done on caudal fin -more accessible
for surgery and manipulations
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• Fin regeneration divided into four successive
  steps
• Wound healing
• Blastema formation
• Regenerative outgrowth concomitant to
  differentiation
• Patterning of blastema

http//www.eb.tuebingen.mpg.de/departments/3-genet
ics/zebrafish/christopher-antos/regeneration
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• Fins consist of two distinct ontogenic origins
• Endoskeleton derived from endochondral bone
  located at base of fin
• Exoskeleton derived from dermal origin located
  in the external part of the fin

Regeneration is limited to the exoskeleton. Bone
of each ray is called lepidotrichia
Nature 445, 311-314
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Wound healing

• Wound closure results from rapid apical migration
  of epithelial cells.
• A dense mass of cells called the wound epidermis
  or apical epidermal cap form within 12 to 18
  hours.
• Migration of epithelial cells occur from close to
  the wound to several segments proximal to the
  wound.
• There is no cellular proliferation

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Two distinct layers are formed -An outer layer
with compact flat cells -Basal layer with
cuboidal cells. -The rest of the cells form a
loose multilayered mass of cells between the two
distinct layers
Cytoplasmic protrusions occur from the basal
epithelial layer into the underlying mesenchymal
cells. Geraudie and singer showed that exchange
between epithelial and mesenchymal cells
necessary for blastema formation
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Poss et al showed that wnt 5 and lef 1 are
activated by epithelial cells lining the
mesenchymal cells
Other studies have shown that epithelial-mesenchym
al interactions are required for organ formation.
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Balstema formation
In urodele amphibians dedifferentiation of
firbroblast-like cells occurs leading to
proliferation. There is also proliferation of
Scleroblasts, which line the hemirays and
endothelial cells.
-Scleroblast play a role in bone
repair -Endothelial cells are involved in
revascularization of he fin.
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It has also been proposed that stem cells could
lead provide cells for the formation of the
blastema along with the dedifferentiation of
cells.
The only stem cells that have shown to develop in
zebrafish are melanocytes from unpigmented
precursor.
Mesenchymal cells also migrate from distant
locations and become part of the blastema.

• Akimenko and Smith conclude that there are two
  cellular mechanisms
• Cell dedifferentiation
• Activation of reserve cells.

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FGF
Fibroblast growth factor (FGF) plays a role in
amphibian limb regeneration.
FGF signaling has been shown to be necessary in
blastema formation
Poss et al. (2000b) used SU 5402, an FGFr1
inhibitor, following amputation. -SU 5402
prevented blastema formation , but did not affect
wound healing. -SU 5402 did not prevent
disorganized aspect of cells where the blastema
would have formed.
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Msx
Msx homeobox gene family also involved in
epithilial-mesenchymal interactions
Msx products play a role in organogenesis Han et
al. (2003) demonstrated that msx is required for
distal digit regeneration in fetal mise.
Msx proteins are used in signaling pathways
involving Bmp, Fgf and SHH
During blastema formation msxa and msxd
transcripts restricted to the epithelial
cap -msxb and msxc are localized in
proliferating cells in the blastema
SU5402 also inhibit expression of msxb and msxc
Han et al. (2003)
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-Studies show that the nervous system also plays
a critical role in fin and limb regeneration
-resection of the nerves in the brachial plexus
prevents blastema formation and outgrowth, but
does not affect wound healing.
-Factors involved with regeneration and the
nervous system are still unknown.
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In urodele, medium to late blastema buds do not
require nerves for limb formation, but are
required for the early blastema formation.
http//www.mun.ca/biology/desmid/brian/BIOL3530/DB
_Ch13/DBNRegen.html
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Outgrowth of the regenerate
During regenerative outgrowth the blastema
becomes divided into two populations with
different cell cycles -Proximal blastema (PB)
cells -proliferate at a faster rate than during
blastema formation -Distalmost blastema
(DB) -very low cycling to a
nonproliferation rate.
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Only PB cells express proliferating cell nuclear
antigen (PCNA).
Proliferation is associated with modifications of
msxb expression. Msxb becomes restricted to DB
during outgrowth
Suggested that msxb-expressing cells establish a
boundary providing direction for regenerative
outgrowth
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Poss et al. demonstrated that mps1 are
coexpressed with msxb in the blastema -During
regenerative outgrowth mps1 and msxb segregate.
-msxb in the DB, poor proliferation -mps1 in
PB, high poliferation -This suggests that mps1
required to maintain high proliferation in the PB.
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Differentiation and pattern Formation
Differentiation starts while the blastema
continues the outgrowth phase. -Differentiation
starts with cells located at the proximal part of
the PB.
-Blastema cells lining the epithelial tissue and
located between the PB and the patterning zone
differentiate into scleroblasts.
-Scleroblast will secrete the bone matrix forming
the lepidotrichia.
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-Bone matrix will mineralize in a proximal-distal
direction
-the bones regenerate by formation of new
segments. -Scleroblasts at the joint express
evx1, suggesting that evx1 involved in joint
formation.
-expression of hoxa11 and hoxa13b in scleroblasts
suggests that these factors are involved in bone
cell differentiation.
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Tetrapod limb regeneration
Regeneration can be broken into a sequence of
stages from amputation to a fully developed
regenerative limb
Time need varies with urodele specie and size of
organism
Dr. Susan Bryant, Univ. of Calif., Irvine
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As with zebrafish after amputation epidermis
migrates to cover the wound (WE) and an apical
epidermal cap (AEC) forms.
Formation of the blastema is dependent on factors
produced by the WE and AEC.
Regeneration progresses from proximal to distal
and from anterior to posterior.
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All tetrapods regenerate at some stage in the
life history of the organism.
Regenerative ability is intrinsic property of
limb cells and not hormonal or other changes in
animals
In studies young limb buds grafted to
regenative-incompetent older host regenerate,
whereas older limb buds grafted to young
regenative-competent host are unable to
regenerate.
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Loss of regenerative ability in Xenopus is
associated with inability reactivate expression
of genes involved in growth and patter formation,
such as Shh.
Treatment of regeneration-incompetent Xenopus
limbs with Fgf partially rescues regenerative
ability.
In mammals the ability to regenerate the most
distal structures, terminal phalanx digits, is
retained.
If the wound is sutured the regenerative response
is inhibited.
Han et al. (2003)
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Regeneration requires two steps.
First step involves formation of the blastema,
preparation phase I. Second step involves the
redevelopment of the limb, redevelopment phase II.
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Stages of development are comparable to those of
regeneration, during the phase II.
Hoxa9 diagonal, hoxa13 and hoxa9 black
Both hoxa9 and hoxa13 are expressed on in the
autopod, while only hoxa9 is expressed in the
zeugopod.
Phase I gene expression is unique to regeneration
Phase I Phase II
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Cell contributions to blastema formation
-Nerves and blood vessels are formed by the
structures that are left behind do to amputation.
-As mentioned before a nerve supply is required
for regeneration to occur.
-Skeletal tissues have a minor role in
contributing to the blastema. -early studies
concluded that periosteum and perichondrium
contributed to the blastema
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Muscle tissue becomes mononucleated in the
blastema through an unknown mechanism. -the
cells contribute mostly to regenerating muscle,
but some form cartilage
This suggests that there is some
transdifferentiation.
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Connective tissue fibroblasts have the greatest
contribution in number of cells and control of
growth and pattern formation.
On average fibroblast contribute about 42 of
cell mass in the blastema.
Fibroblast tissues are the only mature limb
tissues the influence pattern formation
determining final regenerate orientation.
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Positional information is demonstrated by
induction of growth and pattern formation.
A) Control. B) Graft from the left hindlimb bud
was placed onto a right regenerating hindlimb
stump
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http//8e.devbio.com/printer.php?ch1id184
-Results of grafting blastema cells from anterior
and posterior positions within the limb
demonstrated that cells from different limb
positions express different cell surface
molecules -If cells express similar cell surface
molecules there is no outgrowth.
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Fibroblast cells show high degree of development
and physiological plasticity, they are basically
blastema cells. -have ability to give rise to
many cell types. -Studies of bone induction
using BMP target fibroblast that are responsive
to BMP signaling. -It has not been presumed that
fibroblasts go through dedifferentiation then
transdifferentiation into chondrocytes or
osteocytes.
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Reference
Han, M et al. (2003) Digit regeneration is
regulated by Msx1 and BMP4 in fetal mice.
Development 130, 5123-5132