Utility and use of zebrafish as model for understanding angiogenesis.
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Transcript of Utility and use of zebrafish as model for understanding angiogenesis.
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I. Utility and use of zebrafish as model for understanding angiogenesis.
II. VEGF signaling in zebrafish during angiogenesis.
III. Mathematical modeling of angiogenesis
Cell signaling, endothelial migration, and zebrafish: a simplified model for angiogenesis
Khalid Boushaba, Jeffrey Essner, and Howard LevineIowa State University
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Cell signaling, endothelial migration, and zebrafish: a simplified model for angiogenesis
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Zebrafish as a High-throughput Model for Angiogenesis Research and Therapeutic Development
Large number of offspringOptically clear embryosShort generation timeSmall SizeForward Genetics:
ENU mutagenesisInsertional mutagenesis
Reverse Genetics:Transgenic fishTilling with ENUMorpholino injection
Genomics:Sequenced GenomecDNA projectsMicroarrays
Small Molecule Screens:Predictive of higher vertebratesDelivery by injection or soaking
Carcinogenesis: Aqueous deliverySimilar to human tumors
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Zebrafish embryos are optically clear and develop rapidly
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From Karlstrom and Kane, 1996
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From Yancopoulos et al., 2000
Model of Tumor Angiogenesis
Novel Angiogenic
Factors Candidate Anti-Tumor
Agents
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Advantages of Studying Angiogenesis in Zebrafish
Angiogenesis is a conserved vertebrate-specific function
Analysis in living embryos
2.7 dpf
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Transgenic zebrafish allow analysis of endothelial cells in living embryos
fli1-egfp transgenic embryo at 2 dpf
Dorsal AortaDorsal Aorta(DA)(DA)
Posterior Cardinal Vein(PCV)
Intersegmental VesselsIntersegmental Vessels(Se)(Se)
Dorsal Longitudinal Anastomotic VesselDorsal Longitudinal Anastomotic Vessel(DLAV)(DLAV)
Caudal Vein Caudal Vein Capillary PlexusCapillary Plexus
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Advantages of Studying Angiogenesis in Zebrafish
Microangiography: analysis of blood flow in living embryos
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The intersegmental vessels form by sprouting angiogenesis
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ve-cadherin expression identifies primitive endothelial cells in the early zebrafish embryo
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Primary angiogenesis in the trunk and tail are apparent at 24 hpf
ve-cadherin in situ hybridization
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Each intersomitic vessel is composed of three endothelial cells
fli1-egfp transgenic embryo at 2 dpf
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fli1/gfp embryos allow the behavior of individual cells to be followed during primary angiogenesis
Movies from Brant Weinstein’s lab at the NIH
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Discovery Genomics, Inc.
Karl J. ClarkJon LarsonAidas NaseviciusShannon Wadman Perry B. Hackett
Iowa State University
Hsin-Kai LiaoYing WangDanhua ZhangKatie Lutz
University of Minnesota
Eleanor ChenStephen C. Ekker
Max-Planck Institute - Freiburg
Matthias Hammerschmidt
Angiogenetics, AB
Mats Hellstrom
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Mechanism of Morpholino Phosphoramidate Inhibition
60S60S40S AUGAUGACCGGUAUUAGUCCGGACCUAGUAG•••••••AAAAA40S
40S
60S
Inhibition of Translation
40S AUGAUGACCGGUAUUAGUCCGGACCUAGUAG•••••••AAAAA
40SMPO
Encoded Protein
BASEn
NP
N
O
O
O
N
OO BASEn+1
P
N
O
CH3
CH3
CH3CH3
Antisense oligonucleotidesDesigned as 25 mersBind tightly Resistant to digestionLow toxicityNot RNAseH mediated
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Microinjection : An Efficient MorpholioDelivery System
InjectionSite
Nasevicius andEkker (2000, 2001)
Easy to perform:can inject thousandsof embryos per day
0 hr
1.5 hrs 4 hrs
28 hrs
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Microarray Pre-selection vs. Random Selection
Discovery Genomics, Inc. /AngioGenetics AB Pilot Screen:
Targets were pre-selected basedon microarray data.
16% of genes (8/50) were identified as angiogenesis candidates.
Random ENU Mutagenesis screens:
Genes are mutated randomly with a chemical mutagen in a forward genetic screen (Habeck et al., 2002). Subsequent gene identification is difficult.
0.5% of genes (approximately 1/200) are estimated to affect angiogenesis.DGI/AG Screen
16%
SelectedCandidates
Random Screens SelectedCandidates
0.5%
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Syndecan-2 VEGF/VEGFR1&2
erm1
?
F-actin
?
?
erm1 may associate with Syndecan-2 during vascular formation to transmit VEGF-signaling
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Migration
VEGFR2 (flk1)
Hypothesis I: endothelial migration is dependent on the concentration of VEGF
VEGF
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The embryonic midline influences vasculogenesis and angiogenesis by inducing VEGF expression
Lawson et al., 2001
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VEGF is required for the correct number of endothelial cells
ve-cadherin expression
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Vasculogenesis is dependent on VEGF in zebrafish embryos
Wt VEGF MO
3 dpf
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VEGF-A is required for vasculogenesis in zebrafish
Microangiography allows high resolution mapping of mature vessels.
Nasevicius et al., 2000
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Migration of the intersegmental vessels is severely affected in VEGF-Aknockdown embryos at 2 dpf
Wt VEGF-A
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Migration
VEGFR2 (flk1)
Endothelial migration is dependent on the concentration of VEGF
VEGF
VEGFR2 (flk1)
VEGF
Wt VEGF MO
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Formation of the intersegmental vessels by sprouting angiogenesis requires VEGF
Zebrafish ve-cadherin expression at 48 hpf
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Planar transcytosis
Argosomes
Cytonemes
Restricted diffusion
Gradients can be set up and interpreted in many different ways
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Migration
VEGFR2 (flk1)
Endothelial migration is dependent on the concentration of VEGF
VEGF
VEGFR2 (flk1)
VEGF
Wt VEGF MO VEGF MO + hVEGF
VEGFR2 (flk1)
VEGF
Migration
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VEGF and VEGFR2/flk1
VEGF signaling is conserved during zebrafish vascular development
In zebrafish there are two flk1 genes: flk1a and flk1b.
Simultaneous knockdown of both flk1a and flk1b resembles VEGF-A knockdown embryos.
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Migration
VEGFR2 (flk1)
Endothelial migration is dependent on the concentration of VEGF and VEGFR2
VEGF
VEGFR2 (flk1)
VEGF
wt flk1a and flk1b MO
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Syndecan-2, a heparan sulfate-containing proteoglycan, is essential for angiogenic sprouting of blood vessels
Syn2 MO, fli-1WT fli-1
Chen et al., 2004
?
Syndecan-2 VEGF/VEGFR1&2
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VEGF 121
VEGF 145
VEGF 165
VEGF 183
VEGF 189
VEGF 206
Heparan Sulfate Binding Region
Vascular Endothelial Growth Factor A (VEGF-A)
Robinson & Stringer, 2001
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Migration
VEGFR2 (flk1)
Endothelial migration is dependent on the concentration of VEGF, VEGFR2, and Syndecan-2
VEGF Syndecan2 presenting cells
VEGFR2 (flk1)
VEGF
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Syndecan-2
Phosphoserine
Growth Factorand Receptor
A Cell-autonomous B Cell-autonomous Presentation model Complex model
C Cell-nonautonomous, inside-outside signaling model
Syndecan-2 may function in multiple ways
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Migration
VEGFR2 (flk1)
Endothelial migration is dependent on the concentration of VEGF
VEGF
VEGFR2 (flk1)
VEGF
wt VEGF +Syn2 MO VEGF MO + hVEGF
VEGFR2 (flk1)
VEGF
Migration
VEGFR2 (flk1)
Syndecan2 presenting cells
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Ectodomain
C1 V C2YRMRKKDEGSY DLGERKPSSAAYQKAPTK EFYA
EphB2 PKCEzrin Synbindin
Synectin Syntenin CASK
Phosphorylation sitesSerines and Tyrosines
HS Chains
A
Ezrin
Synectin
F-actin
B C-terminal cytoplasmic domains
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Migration
VEGFR2 (flk1)
Endothelial migration is dependent on the concentration of VEGF and VEGF requires Syndecan2 for signaling
VEGF Syndecan2 presenting cells
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Mass action law
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Biochemical equations
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Role of cell cycle and cell movement equations
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Cell movement
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Full model equations
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