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

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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

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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

Advantages of Studying Angiogenesis in Zebrafish

Microangiography: analysis of blood flow in living embryos

The intersegmental vessels form by sprouting angiogenesis

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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

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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

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

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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