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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
Cell signaling, endothelial migration, and zebrafish: a simplified model for angiogenesis
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
Zebrafish embryos are optically clear and develop rapidly
QuickTime™ and aPhoto - JPEG decompressor
are needed to see this picture.
From Karlstrom and Kane, 1996
From Yancopoulos et al., 2000
Model of Tumor Angiogenesis
Novel Angiogenic
Factors Candidate Anti-Tumor
Agents
Advantages of Studying Angiogenesis in Zebrafish
Angiogenesis is a conserved vertebrate-specific function
Analysis in living embryos
2.7 dpf
QuickTime™ and aH.264 decompressor
are needed to see this picture.
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
Advantages of Studying Angiogenesis in Zebrafish
Microangiography: analysis of blood flow in living embryos
The intersegmental vessels form by sprouting angiogenesis
QuickTime™ and aPhoto - JPEG decompressor
are needed to see this picture.
ve-cadherin expression identifies primitive endothelial cells in the early zebrafish embryo
Primary angiogenesis in the trunk and tail are apparent at 24 hpf
ve-cadherin in situ hybridization
Each intersomitic vessel is composed of three endothelial cells
fli1-egfp transgenic embryo at 2 dpf
QuickTime™ and aCinepak decompressor
are needed to see this picture.
QuickTime™ and aCinepak decompressor
are needed to see this picture.
fli1/gfp embryos allow the behavior of individual cells to be followed during primary angiogenesis
Movies from Brant Weinstein’s lab at the NIH
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
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
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
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%
Syndecan-2 VEGF/VEGFR1&2
erm1
?
F-actin
?
?
erm1 may associate with Syndecan-2 during vascular formation to transmit VEGF-signaling
Migration
VEGFR2 (flk1)
Hypothesis I: endothelial migration is dependent on the concentration of VEGF
VEGF
The embryonic midline influences vasculogenesis and angiogenesis by inducing VEGF expression
Lawson et al., 2001
VEGF is required for the correct number of endothelial cells
ve-cadherin expression
Vasculogenesis is dependent on VEGF in zebrafish embryos
Wt VEGF MO
3 dpf
QuickTime™ and aH.264 decompressor
are needed to see this picture.
VEGF-A is required for vasculogenesis in zebrafish
Microangiography allows high resolution mapping of mature vessels.
Nasevicius et al., 2000
Migration of the intersegmental vessels is severely affected in VEGF-Aknockdown embryos at 2 dpf
Wt VEGF-A
Migration
VEGFR2 (flk1)
Endothelial migration is dependent on the concentration of VEGF
VEGF
VEGFR2 (flk1)
VEGF
Wt VEGF MO
Formation of the intersegmental vessels by sprouting angiogenesis requires VEGF
Zebrafish ve-cadherin expression at 48 hpf
Planar transcytosis
Argosomes
Cytonemes
Restricted diffusion
Gradients can be set up and interpreted in many different ways
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
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.
Migration
VEGFR2 (flk1)
Endothelial migration is dependent on the concentration of VEGF and VEGFR2
VEGF
VEGFR2 (flk1)
VEGF
wt flk1a and flk1b MO
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
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
Migration
VEGFR2 (flk1)
Endothelial migration is dependent on the concentration of VEGF, VEGFR2, and Syndecan-2
VEGF Syndecan2 presenting cells
VEGFR2 (flk1)
VEGF
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
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
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
Migration
VEGFR2 (flk1)
Endothelial migration is dependent on the concentration of VEGF and VEGF requires Syndecan2 for signaling
VEGF Syndecan2 presenting cells
Mass action law
Biochemical equations
Role of cell cycle and cell movement equations
Cell movement
Full model equations