Prelude: Evolution and Development of Limb Reduction in the Australian Skink Hemiergis

Nadia Rosenthal

EMBL European Molecular Biology Laboratory Mouse Biology Programme

Rosenthal@embl-monterotondo.it

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!