Prelude: Evolution and Development of Limb Reduction in the ...Prelude: Evolution and Development of...
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Prelude: Evolution and Development of Limb Reduction in the Australian Skink Hemiergis
Nadia Rosenthal
EMBL European Molecular Biology Laboratory Mouse Biology Programme
Prelude: Evolution and development of limb reduction in the Australian skink Hemiergis
Juquehy 2005Nadia RosenthalEMBL/Romewww.embl-monterotondo.it
Mike Shapiro (Chaperone)
From Kardong, 1995
Tetrapod limb diversity
Size reduction Element loss
Ancestral Derived
2-3-4-5-4 2-3-4-5-0 0-3-4-5-X X-3-4-5-X
Archosaurs
2-3-3-3-3 X-3-3-3-X X-X-3-3-X X-0-3-0-X
Mammals
2-3-4-5-4 X-2-3-5-3 X-2-3-5-0 X-X-3-5-0
Lizards
Limb and digit reduction (element loss) in squamates (Greer, 1991)
• Occurs in over 50 lizard lineages
• Scincidae: occurs 31 times in 25 lineages
• When digits are lost, “central” digits (III and IV) are conserved
Hemiergis in Western Australia
SVL = 50 mm SVL = 69 mm
SVL = 65 mm SVL = 75 mm
H. initialis (5/5) H. peronii (4/4)
H. peronii (3/3) H. quadrilineata (2/2)
Maximum likelihoodphylogeny of Hemiergis(Reichert and Reeder, in prep.)
• Hemiergis species are closely related
• Ancestral = 5 digits(2-digit)
(5-digit)
Collection Sites: 1997 - 2004
Lizard country
Lizard country?
THIS IS NOT A WOMBAT
Forelimb
Hind limb
Hemiergis limbs always have the same digit numbers
metacarpal
phalanges
carpals
ulnaradius
Limb morphology
I
V
ulnaradius
IV
sharedforelimb
configuration
2
4
3
Hemiergis: intermediate configurations adult
Who is responsible?
Protein expression of:
• Msx-1+2: apoptosis and cell differentiation– Early or increased expression?
• Dlx: distal structures, including AER– Localized loss of expression?
• Shh: proliferation and A/P patterning– Spatial or temporal restriction?
Shh feedback loops in limb bud development
proximal
FGFs
AER
distal
anterior
posterior
SHH
GLI3-R
dHAND
Alx-4
FORMIN
BMPs
GREMLIN
H. quadrilineata (2/2)63 somites
H. peronii (3/3)60 somites
H. peronii (4/4)62 somites
H. initialis (5/5)59 somites
ba
fl hl
n
Shh expression: Stage 30
H. quadrilineata (2/2) H. peronii (3/3) H. peronii (4/4) H. initialis (5/5)
Shh expression: Stage 31/31+
Sta
ge 3
3S
tage
32
H. quadrilineata (2/2) H. peronii (3/3) H. peronii (4/4) H. initialis (5/5)
Sta
ge 3
2-
Shh expression: Stages 32 & 33
H. quadrilineata (2/2) H. peronii (3/3) H. peronii (4/4) H. initialis (5/5)
St.
32Fo
relim
bS
t. 32
Hin
d lim
bS
tage
32+
BrdU staining (cell proliferation): Stage 32
Sta
ge 3
2-S
tage
32-
Summary
Small expression differences give big morphological consequences!
•Common early chondrogenic stages, later divergence•Reduced morphologies are not simple truncations of ancestral programs•Shh: early termination of expression correlated with digit loss (restricted expression: fewer digits, less proliferation)•Shapiro et al, Exp. Zoolog. 2003, 297:48-56
Development 2003
“We classify limb skeletal elements are Shh dependent or independent, with the ulna/fibula and digits (other than digit 1 in the leg) as Shh dependent.”
Supporting data
“Our investigations into the basis of the ectopic Shhexpression identified the enhancer element that drives normal Shh expression in the ZPA. The regulator, designated ZRS, lies within intron 5 of the Lmbr1 gene 1Mb from the target gene Shh. The ZRS drives the early spatio-temporal expression pattern in the limb of tetrapods.”
Human ZRS mutations
The ZRS regulator
Hypothesis: hypomorphic polymorphisms in the ZRS of Hemiergis species produce digit reductions
Time for another trip downunder to collect DNA!!
Main feature: Mechanisms of vertebrate regeneration:what have mammals forgotten?
Reg
ener
atio
n ca
paci
ty
Evolutionary scale
Premise:
Studying regeneration in the context of other organisms can yield insight into our own limited regenerative capacity
Young Scott Fraser bitten by lizard Caravaggio, 1597
Spallanzani (1768) first reported that urodeleamphibians were able to regenerate their limbs after amputation
Regenerative modes
Diff
eren
tiatio
nProliferation Differentiation
Epimorphosisadult structures dedifferentiate to form a mass of cells that respecify(urodele amphibians)
Compensatory Regenerationcells divide but maintain their differentiated functions (mammalian liver)
repatterning of existing tissues with very little new growth(Hydra)
Morphallaxis
Dedifferentiation INJURY
Plasticity and reprogramming of differentiated cells
Invertebrates:bidirectional regeneration
Vertebrates: unidirectional regeneration
distal amputation: mid-radius and ulna
proximal amputation:mid-humerus
•local formation of a growth zone called the blastema
•wound is sealed in approximately 12 hours by migrating epithelial cells to form the wound epidermis
•zone underlying the wound epidermis is enriched by mesenchymal cells re-entering cell cycle: cartilage tissue and muscle cells lose their differentiated features andre-specify their fate as local progenitor cells to proliferate, differentiate and regenerate the new limb
BLASTEMA
WOUND EPIDERMIS
PROLIFERATING MESENHYMAL CELLS
DE-DIFFERENTIATION
RE-PROGRAMMING
NEW LIMB
Regeneration of the newt forelimb after amputation
Fish fins regenerate perfectly
Fish fin regeneration - blastema formation
wFGFWnt5
Msx1
PCNA
Fish fin regeneration markers
Pre-amp
Amp
Day 3
Day 5
Day 6
Newt tail amputation
Muscle fiber dedifferentiation is a major contributor to the regenerating blastema in newts
Tanaka et al
Newt post-miitotic muscle cells can dedifferentiate
serum
Multinucleate myotube Mononucleate cells
Brockes at al
Regeneration biochemical pathway :serum stimulation of newt myotubes triggers Rb phosphorylation/ inactivation and progression to G1 - S phase
BrdU staining after serum stimulation
No vertebrate myotubes respond to serum (exception: mouse cells that lack the retinoblastoma (Rb) gene
“We tend to regard urodele regeneration as an exceptional attribute, since closely related species do not possess this ability…
Newts can do it, why can’t we?
- Jeremy Brockes
The critical hypothesis is that regeneration is a basic, primordial attribute of metazoans…lost secondarily for reasons which are not understood. “
• inadequate expression of genes
• insufficient intracellular signaling pathway
• irreversible expression of of differentiation markers
• structural characteristics that make dedifferentiation impossible
• adaptive immunity: the cost of regeneration?
Dedifferentiation in mammalian regeneration is absent because:
What about deer antlers? (only adult mammalian regenerating organ)
My adaptive immunity is just fine
thanks for asking
Unlike horns which grow from the base, the deer antler growth centre is at the blastema-like tip, where mesenchymal cells undergo a continuous process of cellular differentiation into abundant vascular tissue, cartilage, and skin and bone. - Jo Price, London
1 2 3
SkinPericondriumMesencymeChondroprogenitorsCartilage
PeriosteumBone
Vascularspaces
Cartilage
Deer antler regeneration
Elements of wound healingDisruption of capillaries triggers activation of platelets and clotting cascade
Neutrophils and leukocytes remove bacteria and release a variety of growth factors and cytokines which function to remodel the tissue
Macrophages release factors inducing fibroblasts to produce ECM components
Formation of new blood vessels by transdifferentiation of fibroblasts to myofibroblasts
Scar formation
Embryonic wound healing..
..parallels morphogenetic movements
Dorsal closure(Drosophila)
C-fos RNA, protein
Actin purse string
Can we improve adult mammalian wound healing?
NEED: Clotting cascadeCell survivalNew blood vesselsTissue regeneration
DON’T need:Cell deathChronic inflammationFibrosis leading to scars
..
Insulin-like growth factor-1 (IGF-1):
Circulating forms:• Synthesized predominately in liver after birth• Control lifespan in flies, worms and mice• Promote cancer in mice and man
Local forms:• Important for fetal growth• Induced in response to local injury• Promote proliferation, differentiation, survival,
angiogenesis, neurite outgrowth
Antler growth is stimulated mainly by IGF-1
•Seasonal peak of IGF-1 production coincides with peak of antler growth rate
•Growing antler tissues have high levels of IGF-1 receptor
•Physiological levels of IGF-1 stimulates antler mesenchymal and cartilagenous cell growth
From Sadighi et al, 2001
•Stimulates growth, mediates the effects of GH
•Induces proliferation and subsequently enhances differentiation of skeletal muscle cells in culture
•Is induced in newly replicating myoblasts after ischemic or toxic injury, and by work-induced muscle hypertrophy
Role of IGF-1 in skeletal muscle
IGF-1: one gene, many functions
S S S B B E E E
Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6
C A D
B C E-41A D
B C E-35A D
B C E-41A D
B E-35C A D m
Class 2 IGF-1A
Class 2 IGF-1B
Circulating
Class 1 IGF-1A
Class 1 IGF-1BLocal
-48 AUG
-32 AUG
-22 AUG
mIGF-1
MGF
Nadine WinnMaria Paola Santini
Response of local IGF-1 isoforms to traumatic injury
S S S B B E E E
Exon 1 Exon 5Exon 2 Exon 3 Exon 4 Exon 6
C A D
mMGF
Low expression induced by injury, repressed in repair (Goldspink et al)
B C E-41A D
High expression induced by injury, sustained in repair
mIGF-1 B E-35C A D
1 5 10 days after injury
Muscle-specific mIGF-1 transgene (local form):
• INCREASES postnatal muscle mass/strength• DECREASES fat, protein degradation in disease• AGING: maintains muscle mass/strength (viral delivery)• EXERCISE: enhances anabolic effects• INJURY: accelerates healing, ablates scarring• DYSTROPHY: counters muscle degeneration• ALS: delays paralysis, preserves muscle and CNS• MUSCLE WASTING: blocks it
Does not:• Induce cardiac pathology• Promote cancer
Musaro et al, 2001-2005
Wt mIGF-1
Stacking the deck towards regeneration
Fibrosis Regeneration
Inflammation Cell replacement
Damage
NFkB mIGF-1
Stem cells?
Generating new tissue with adult stem cells
HSC BM, NSC nerve, CSC cardiocyte
MSC, MAPC all HSC nerve, HSC cardiocyte
cardiocyte CSC cardiocyte
Stem cell markers in bone marrow and muscle
CD45 c-kit Sca-1
Bone marrow + + +
Muscle - - +
Stem cell markers increasein injured IGF-1 muscle
CD45 c-kit Sca-1 CD11b Emx2
Muscle ++ + + + -
mIGF-1 muscle ++ ++ +++ ++ ++
Emx2 - a regeneration gene?
•Vertebrate homeobox gene involved in CNS patterning•Affects the symmetry of cell divisions in the cerebral cortex•Regulates proliferation of stem cells in the adult mammalian CNS•Expressed in mesangioblasts (multipotent adult stem cell)•Expression is graded along the developing newt limb axis•Induced in regeneration blastema of amputated newt limbs
ckit/GFP transgenic mouse (after muscle damage)
Transgenes as cell type markers
Stem cell-specifc reporter transgene labels cells as they migrate to damaged tissues
Do bone marrow stem cells migrate to injured muscle?
1.Deplete host bone marrow(irradiation)
2. Reconstitute host with bone marrow SP from ackit/GFP transgenic mouse(tail vein injection of 2000 cells)
3. After 10 days, injure muscle(C TX injection)
4.Analyze muscle by FACS
cKit-GFP
CD
45
wildtype MLC/mIGF-1
Controlmuscle
CTX injectedmuscle
Transgenic mIGF-1 enhances recruitment of ckit/GFP-marked bone marrow cells to damaged muscles
4.2%2.1%
16.2%5.6%
MLC/hAP transgenic marker
Mdx Mdx x mIGF-1
(3weeks after transplantation)
(Bone marrow cells transplanted intravenously)
mIGF-1 promotes efficient BM cell uptake in damaged muscles
Cells from mIGF-1 muscle are multipotent
Sca-1+ sorted cells
fatmuscle heart
TroponinI
Do newts have higher levels of mIGF-1?
We don’t need it,
we got thrombin
Serum*
Multinucleate newt myotubes Mononucleate proliferating cells
Multinucleate mouse myotubes
Serum
don’t respond..
*Thrombin is the active serum component (Brockes et al)
De-differentiation of muscle cells in response to serum: newt myocytes do it, mouse myocytes don’t…
•Regeneration of structures depends on a mechanism to couple acute response to tissue injury or removal with local activation of plasticity in differentiated cells or stem cells
•Lens regeneration in newts occurs exclusively from the dorsal margin
•Thrombin is transiently and selectively activated in the dorsal margin
•Hypothesis: Thrombin activation is the pivotal signal linking tissue injury to the initiation of vertebrate regeneration.
Imokawa & Brockes. Current Biol. (2003)
Selective activation of thrombin is a critical determinate for vertebrate lens regeneration
Thrombin couples acute events following tissue damage with long-term cell plasticity in efficiently regenerating organisms
O = clotting factorsPT = prothrombinT= thrombinY = tissue factorF = plasma protein?Fa = activated FO Nucleus = cycling cell
Imokawa & Brockes, Curr. Biol. 2003
Thrombin is activated by amputation of limb but not lens
Lens
Enzyme overlay Nuclear stain
Limb
Axolotls,a related urodele species, can regenerate the limb but not the lens
Does mIGF-1 render mouse myocyte cultures sensitive to thrombin?
2 months in serum-free media (no thrombin)
Wildtype mIGF-1
Restimulation of proliferation in mIGF-1 transgenic myocyte cultures by serum (contains thrombin and about 1000 other things)
MyoD+ colony MyoD- colony
CELL MEMBRANE
α α
β β
IGF-1 RECEPTOR
B C A D
S S S B B E E EC A D
B C A D
ER/GOLGI
NUCLEUS
EB C A D
Signalling by local IGF-1 Processing - thrombin cleavage site!
Secretion (local milieu)
E
?
Regeneration pathways?
B C A D
Growth
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
Olivier Mirabeau
3. Dedifferentation of muscle to regenerate lost tissues - needs
2. Resident progenitor cells proliferate in response to injury
Modes of mammalian regeneration
1. Circulating cells home to injured tissue
mIGFmIGF--11
Screening for regeneration alleles - can we do it in mice?
Nadine WinnOlivier MirabeauMaria Paola Santini
U. Roma La Sapienza
Antonio Musarò
IRCCS Roma
Giovanna Borsellino
Luca Battistini
Mike ShapiroJim Henken
UWA
ACKNOWLEDGMENTS
Ken AplinRic HowBrad MaryanRob Browne-Cooper
Obrigada!