The Planarian Model System
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Jochen Rink, Ph.D.
MPI‐CBG
Dresden
Germany
Planarian Flatworms
S. mediterranea
Planarian Phylogeny
1.: Planarians represent an understudied branch of animal life
Peculiarities of Planarians
2.: Planarians don’t have a fixed body size
1.6
1.2
Width (mm)
Starvation
Feeding
0.8
0.4
~50x size difference
20 mm
3.: Planarians do not age
0.4 mm
0
2
4
Length (mm)
6
8
4.: Planarians are Masters of Regeneration
0 days
18 fragments
1 mm
2 days
14
days
20 4days
4
days
14 days
1 mm
111
mm
mm
mm
0.3 mm
Complete animals from random fragments
Stem
Stem
cellsCells
Planarian Anatomy
Pharynx
Gut
FAZ Grafik
Protonephridia
CNS
Protonephridia
Nishimura et al.
The Planarian Model System
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Regeneration
Organogenesis
Stem Cells
1.: Regeneration
The BIG Questionsions
How does the remaining tissue sense what’s missing?
How is new tissue made in an adult organism?
Integration of old and new tissue?
Why can some animals regenerate, but not others?
Planarians as morphogenesis system
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Molecules: Which patterning pathways pattern the cardinal body axes?
Regulatory logic: Self-organization of the patterning system?
Signaling &. Mechanics: Definition of proportions?
Growth &. De-growth: Mechanisms of global scale adjustments?
Planarians as Regeneration system
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Complete regeneration within 2 weeks
Cheap and easy to maintain
Planarians: The Yeast of Regeneration
What is the global positioning system defining planarian bodyplan shape?
1.: Axial patterning during regeneration
A, P?
2 days
4 days
10 days
2 days
6 days
12 days
Day 1
A, P?
A, P?
0.5 mm
A, P?
A: Qualitative only?
1.: Intrinsic A/P patterning
in tissues.
2.: Quantitative A/Ppattern &. integration
B: Qualitative AND Quantitative?
The Planarian Tool Kit
Signaling library
RNAi-mediated loss of function
+ Sequenced genome
http://smedgd.neuro.utah.edu/
= Candidate gene approach
RNAi-screening
1. Trunks
2. Lateral Slivers
3. Intact animals
β-catenin: Central regulator of A/P polarity
Wnt
RNAi:
RNAi:
Decreased β-Catenin activity: Head specification
Increased β-Catenin activity: Tail specification
β-Catenin-1 acts as switch in A/P-patterning
“head”
“tail”
Concept: Bi-stable signaling network &. “organizer” formation
Science, 2008
III: Orchestrating Regeneration
Embryogenesis: INVARIANT starting point
Regeneration: ARBITRARY starting point:
www.xenopus.com
www.eb.tuebingen.mpg.de/departments/3-genetics/drosophila/uwe-irion/
Body plan morphogenesis
Body plan morphogenesis
Self-organization from
Robustness
Self-organizing patterning systems
Starvation
Feeding
~50x size difference
20 mm
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A
0.4 mm
Width (mm)
2.: Allometric growth &. degrowth
1.
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1.
2
0.
8
0.
4
0
2
4
6
Length (mm)
8
“Patterning” from a homogenous starting point
Dynamic “Regeneration” of patterns
Scaling- size matching between pattern and tissue
How to re-build a worm from a random fragment?
1) Self organizing signaling networks
2) Organizers
3) Long-Range interactions
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(Current) Hypothesis
Regeneration from arbitrary pieces:
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Global positioning system
Local positioning systems governing
organogenesis
Protonephridia
The planarian excretory system
Campbell & Reece (2005)
α-Ac.Tub-antibody, depth coded
Need for cell-type specific markers
Molecular anatomy of protonephridia
Inx10
CAVII-A
DNAH-β3
Molecular anatomy of protonephridia
Inx10/CAVII-A/aTub-antibody
Inx10/DNAH-β3/aTub-antibody
Protonephridia are complex epithelial organs
Regenration:
De novo organogenesis or outgrowth from existing structures?
Inx10 proto-tubule
Onset CAVIIA
Branching
Branching
Branch extension
Inx10 CAVII‐A, aTub Inx10 CAVII‐A, aTub
Control
Protonephridia Regeneration
Regen. complete
De Novo Regeneration
Origin of prototubule?
Molecular regulators of differentiation and Morphogenesis?
Control
Inx10(RNAi)
inx10(RNAi)
RNAi of protonephridial genes causes bloating
Edema formation:
Bloating
Protonephridia
= accumulation
areofrequired
tissue water?
for osmoregulation
Decrease
of transepithelial
osmolarity
gradient should
rescue
Edema formation
as screen
for protonephridial
defects
RNAi of an EGF- receptor causes edema formation
EGFR-5(RNAi)
Uncut
EGFR-5 domain structure
D.m. EGFR
Regenerated Fragments
head
H.s. ERBB-4
S.m. EGFR-5
trunk
tail
Function within protonephridia or indirect physiological effect?
EGFR-5 is specifically expressed in Protonephridia
EGFR-5 is expressed in flame cells
Function: cell fate specification and/or morphogenesis?
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I.: Intact phenotypes
Effects on marker expression
EGFR-5(RNAi) phenotype progression after 3 feedings
EGFR-5(RNAi)
W.T.
EGFR-5 is required for the maintenance of flame cells
I.: Intact phenotypes
Morphological defects
EGFR-5(RNAi)
Day 19
Day 14
Day 10
Day 6
Day 3
Control
RNAi of EGFR-5 correlates with collapse of proximal branches
Regeneration-induced organogenesis to understand ontogeny
EGFR-5 is required for Protonephridia morphogenesis
Late phenotype (d14)
EGFR-5
Control
Flame cell quantification in regenerates: (3 feeds -> amp. vs. 3 feeds -> amp -> inj.)
1.: Early branching- and branch extension defects
II.: Posterior Miss-Projections in the head and coil formation in tail
III.: RNAi dose dependent reduction in flame cell numbers
GFR-5: required for flame cell maintenance/formation and branching morphogenes
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Summary
Flame cells as “tip motors” in P.N. Branching Morphogenesis
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Outlook 1: Exretory system evolution
Similar structure/function: Convergent evolution or evolutionary homology?
Vertebrates: Metanephridium
Planaria: Protonephridium
Nephrin EGFR-E a-Tubulin
Scimone et al., 2011
Molecular components of the filtration diaphragm are conserved
Early cell fate specification is conserved
Proto- and Metanephridia: Common evolutionary origin
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Outlook 2: Scaling
1.: Allometric growth &. degrowth
2.: 14 or 15 flame cells/proximal unit
Starvation
Feeding
~50x size difference
20 mm
0.4 mm
Width (mm)
1.6
1.2
0.8
0.4
0
2
4
6
Length (mm)
8
How is this number determined so precisely?
Scaling of organ capacity: Addition/removal of entire units?
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Organogenesis from a single type of pluripotent stem cells?
III: Planarians as Stem Cell Model
Stem cells:
Self‐renewal + ability to differentiate into several cell types
Medical importance
Regenerative therapies, Cancer, Ageing …
Planarians
Regeneration requires cell division: Likely stem cell involvement
Regeneration from random pieces: Pluripotent stem cells?
Regeneration in adults: Adult stem cells?
Immortal animals = immortal stem cells?
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A brief history of “Neoblasts”
1930s: Coining of term to summarize pre‐1900’s light microscopic observations
1950’s: “Neoblasts” are radiation sensitive‐ irradiated animals no longer
regenerate, are at first entirely normal, ventral curling ~d12, lysis ~d21.
1960’s: Electron‐microscopy/Cytology: “Neoblasts are the only dividing cells”
PROBLEM: From then on, all dividing cells are neoblasts
2000: BrdU, H3P to visualize “Neoblasts”
2002: Piwi genes: Insitu‐marker for “Neoblasts”
A brief history of “Neoblasts”
2005: A FACS protocol to isolate stem cells 2007: Stem cell progeny markers A brief history of “Neoblasts”
BUT:
STILL: Neoblast = dividing cell, hence: “Dividing cells are the only cells that divide in planarians
Are Neoblasts a HOMOGENOUS population of pluripoten stem cells or a HETEROLOGOUS population of lineage restricted stem cells?
Neoblasts are pluripotent stem cells
Wagner et al., Science, 2011
A S R1 R2 R3
“Neoblasts” ARE pluripotent
Neoblasts versus vertebrate stem cells:
High density in adult tissues
Constantly active
Give rise to all cell types
Neither signs of ageing nor cancer
Planarians as emerging stem cell model
1.: Pluripotency: Is “Stemness” Conserved in Planarians?
What, on the moleculat level, bestows cells
With pluripotency and indefinite self‐renewal
Capacity?
Planarians contain homologues of the pluripotency core network
RNAi of the planarian Oct4 homologue causes stem cell loss (L. Gentile)
Current key question: Conservation of the network
2.: Stem Cell Lineage Organization
Category I
Category I
Stem Cell
Stem Cell
1
Category II
Category II
2
1.: Are there transit amplifying cells in planarians?
2.: Lineage commitment: How and where?
2.: Stem Cell Lineage Organization
Where do eye precursor cells arise during regeneration?
Precursors arise far from target side
Differentiation starts already in “stem cell compartment”
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2.: Stem Cell Lineage Organization
tyrosinase
• How does a stem cell daughter decide to become an eye precursor:
Stochastic (cell intrinsic) or signal mediated (cell extrinsic) mechanisms?
• Do all stem cells produce the same ratio of daughter cells?
• How flexible are differentiation ratios?
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MPI-CBG, Dresden www.mpi-cbg.de
Postdoctoral Research Opportunities!
Rink Lab
Shang-Yun Liu
Tom Stueckemann
Sarah Mansour
Varnesh Tiku
Felix Becker
Sánchez Lab
Alejandro Sánchez Alvarado
Hanh Ti-Kim Vu
MPI-CBG
Planarians: A cell collective
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“Neoblasts”: Current questions
Only 7/130 transplant recipients survived: Technical reasons or very low proportion of true stem cells amongst “neoblasts”?
• Stem cell/progenitor dynamics: Markers for transient amplifying populations?
• What is the niche of the pluripotent stem cells?
• “Stemness” in planarians: Evolutionarily conserved or a different animal?
Single population of pluripotent stem cells: All organs compete for the same progenitor pool
Progenitors arise AWAY from their target tissues
Signaling: “Need replacement signal” from tissue to stem cell pool? Specific/Generic?
Differentiation: When and how is cell fate specified?
Cell biology: Coordinating progenitor movements/integration into target tissue
Growth/degrowth: GLOBAL and proportional gain/loss of cells
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The Blastema
2 days
1 mm
• White, unpigmented tissue underneath wound epithelium
• Does not form in irradiated animals: Formation stem cell dependent
• BUT Blastema relatively devoid of cell division, MAINLY postmitotic cells
Current dogma: Blastema formed by DIVERSION of stem cell progenitors generated elsewhere
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The Blastema
The blastema is required for re‐formation of lost tissues (“Epimorphosis”)
AND for the re‐shaping of pre‐existing tssues (“Morphallaxis”)
BMP‐signaling: Required for blastema formation
No blastema = no regeneration/body re‐shaping 14d tail fragment
14d tail fragment
BUT: Potentially back‐up pathways even in absence of blastema…
Blastema questions:
• What are the cues for its formation?
• What determines Blastema identity?
• By which principles does the blastema re‐build missing tissues?
Old tissues
Blastema
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III: Orchestrating Regeneration
molecular decision making
1.: Heal
2.: Repair
3.: Regenerate
The BIG Regeneration Questions:
How does the tissue know what’s missing?
Scale‐matching of old‐ and new?
Functional Integration of old‐ and new?
Dissecting shape &. Proportions
1.: Amputation/Regeneration
Constant width, varied1d
length
Varied
4d length
2d width, constant
2d
WntP-2
2d
4d
6d
15d
10d
2.: Allometric growth &. degrowth
Starvation
Feeding
Width (mm)
1.6
1.2
0.8
0.4
~50x size difference
20 mm
0.4 mm
0
2
4
6
Length (mm)
3.: Extremely plastic body plan morphology
…unique experimental opportunities
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Orchestrating Regeneration
A, P?
2 days
4 days
10 days
2 days
6 days
12 days
Day 1
A, P?
A, P?
0.5 mm
A, P?
A: Qualitative only?
1.: Intrinsic A/P polarity
in tissues.
2.: Quantitative A/Ppattern &. integration
B: Qualitative AND Quantitative?
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Midline specification is linked to A/P-patterning
CNS
Slit1
Control
Hh
Hh
Hh
Bambi-3
Hh signaling has no direct effect on midline
Midline defects correlate with incomplete A/P-patterning
Midline targeting A/P-extremes?
Hh
A/P‐extremes induce ectopic midlines
bCat‐RNAi: Ectopic Heads
Sfrp1
Slit1
APC‐RNAi: Ectopic Tail
Slit1
Fz4
A/P-extremes are necessary &. sufficient for midline initiation
Ptc(RNAi) induced cyclopia due to incomplete A/P-patterning
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Anchoring to A/P-extremes as elegant solution
to the midline alignment problem
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“Tail”
“Head”
“Edge”
III.: Development of planarian transgenesis tools
1. Constructs
2. Delivery
1.: Deep Sequencing
Ubiquitous promoters
EF1α
2.: Primary cell culture
3. Transplant‐rescue of
irradiated worms.
3.: Transplant-rescue
Quantitative 96W-assay/
media screen
Codon Usage
Otto Guedelhoffer
5’/3’-UTR
Reporters
(βGal, TOMATO)
VSV-G pseudotyped Lenti-v.
Transposons
Viruses/Electroporation
In-vivo selection (Zeocin)
Tools to follow cell dynamics in vivo
Tools to follow signaling dynamics in vivo
Re-building the “whole” from random fragments
Constant width, varied length
Varied width, constant length
2d
2d
2 days
4 days
14 days
6d
4d
15d
10d
1 mm
βCat RNAi
APC RNAi
Assumptions:
I.: Discrete signaling centers (head/Tail/Edge/D/V)
II.: Centers as self-organizing “organizers”
III.: Shape/Proportions via feedback between organizer networks
What are the self-organizing signaling networks?
How is the regenerative response tuned to what is missing?
Polarity dependent changes in gene expression
1.: Array experiment
Amputation
Dissection
RNA isolation
Micro-Array/
Deep sequencing
“Ant.”
Xh
“Post” “R-Lat” “L-Lat” “Cut”
2.: Analysis (Collaboration with Andreas Beyer) “Head”
“Edge”
expression change
expression change
“Tail”
t
All
Lat + Ant + Post
Ant
Post
t
Conceptual challenges of midline specification
1.: Specification of a straight line?
2.: Alignment with the long axis of the fragment to make it the “midline”?
Planarians can regenerate the midline
Ventral
Midline marker: Slit1
V
D
1d
2d
4d
6d
8d
Dorsal
t0
1.: Midline marker regeneration initiates asymmetrically.
2.: Anterior midline extends first, posterior only after Pharynx regeneration
3.: Lateral growth centres midline
Molecular control of midline regeneration?
Planarian Hedgehog functions in midline specification
Anterior Marker (Sfrp1)
Ptc(RNAi)
Posterior Marker (Fz4)
tailless
Planaria
half-tail
Control
Control
Vertebrates
Cyclopamineheadless
Cyclops
USDA-Agricultural Research Service
two-tail
Danio Labs, S Roy and F Muller
Planarian Cyclopia: Gain of function phenotype (Vertebrates: l. o’f)
Partial phenotype
Occurs within a series of A/P-patterning defects
Ontology of midline defects?