Biology 624 - Developmental Genetics

Lecture #9 -Blood Vessel Formation

Blood vessel formation is important in:

1.Embryogenesis2.Pregnancy3.Wound healing4.Disease

Tumor vascularization is a major step in tumor progression

Diabetic retinopathy (excess blood vessels in the eye) is a major

cause of blindness

VEGF Signaling VEGF Signaling PathwayPathway

FromShibuya, 2006

VEGFVEGF

FlkFlk-1-1

AngioblAngioblastast

FlkFlk-1-1

FltFlt-1-1

VEGFVEGF

VEGF Signaling VEGF Signaling PathwayPathway

Cell Division +Cell Division +Migration +Migration +Survival +Survival +

Cell Division -Cell Division -Migration +Migration +

Endothelial Endothelial CellCell

ES cell mutant vascular ES cell mutant vascular phenotype mimics mutant phenotype mimics mutant

phenotype in vivophenotype in vivo

Wild-Wild-typetype

Flk-1-/-Flk-1-/- Flt-1-/-Flt-1-/-

angioblasts

Vascular development - differentiationVascular development - differentiationof angioblastsof angioblasts

Mesoderm

Hemangioblast

Hematopoietic cells

Embryonic vascular differentiationEmbryonic vascular differentiationvia the hemangioblastvia the hemangioblast

angioblasts

Vascular development - differentiationVascular development - differentiationof angioblastsof angioblasts

Mesoderm

Hemangioblast

Hematopoietic cells

Single cell fate mapping in fish givesSingle cell fate mapping in fish givesEvidence for the hemangioblastEvidence for the hemangioblast

From Vogeli et al, 2006

FITC = descendants of single cell at 40% epiboly

Circulatory System of a 4-Week Human Embyro

VasculogenesisVasculogenesis

angioblasts

differentiation assembly

SproutingSproutingAngiogenesisAngiogenesis

primitive vessel

proliferation migration

branching vascular plexus

VSMCs/pericytes

Recruitment of muralRecruitment of mural cells and Remodelingcells and Remodeling

mature blood vessel

Embryonic vascular developmentEmbryonic vascular development

angioblasts primitive vessel

differentiation assembly

VasculogenesisVasculogenesis

VEGFVEGFbFGFbFGF

branching vascular plexus

primitive vessel

proliferation migrationsprouting

AngiogenesisAngiogenesis

VEGF/neuropilinVEGF/neuropilinSlit/roboSlit/roboNetrin/uncNetrin/uncEphrin/ephEphrin/ephNotch/deltaNotch/delta

From Gerhardt et al, 2003

Retinal angiogenesis occurs outward fromRetinal angiogenesis occurs outward fromoptic nerve postnatallyoptic nerve postnatally

From Gerhardt et al, 2003

Tip cell vs. stalk cellTip cell vs. stalk cell

Tip cell:Tip cell:1.1. Does not proliferateDoes not proliferate2.2. Extends numerous Extends numerous

filopodiafilopodia3.3. Expresses markers- Expresses markers-

VEGFR2, PDGFB, Dll4, VEGFR2, PDGFB, Dll4, etcetc

Stalk cell:Stalk cell:1.1. ProliferatesProliferates2.2. Has few filopodiaHas few filopodia3.3. Down-reglates tip cell Down-reglates tip cell

markersmarkers

Tip cell characteristicsTip cell characteristics

No proliferationNo proliferation

Actin-rich filopodiaActin-rich filopodia

Express PDGFBExpress PDGFB

From Gerhardt et al, 2003

VEGF pathway influences tip cellsVEGF pathway influences tip cells

From Gerhardt et al, 2003

VEGFVEGF

FlkFlk-1-1

AngioblAngioblastast

FlkFlk-1-1

FltFlt-1-1

VEGFVEGF

VEGF Signaling VEGF Signaling PathwayPathway

Cell Division +Cell Division +Migration +Migration +Survival +Survival +

Cell Division -Cell Division -Migration +Migration +

Endothelial Endothelial CellCell

Soluble flt-1 isoform selectively rescues migration/branching

Emerging flt-1 mutant sprouts differ from WT sprouts

Chappell et al. (2009) Dev Cell Chappell et al. (2009) Dev Cell 1717, 377, 377

Local sprout guidance measures are rescued by sFlt-1 but not mFlt-1

Local sprout guidance measures are perturbed

In retinas with reduced Flt-1 activity

Flt-1 neutr ab

Adeno-Cre

flt-1flox/

flox

flt-flt-11flox/floxflox/flox

flt-flt-11flox/floxflox/flox

Chappell et al. (2009) Dev Cell Chappell et al. (2009) Dev Cell 1717, 377, 377

Wild-type ES cells flt-1-/- ES cells

Day 8

Day 0

Mosaic blood vessels

“Hanging drop” EBs

1:1

Local sprout guidance measures are rescued by sFlt-1 but not mFlt-1 in

lateral base areas

Chappell et al. (2009) Dev Cell Chappell et al. (2009) Dev Cell 1717, 377, 377

Local Sprout Local Sprout Guidance Guidance

-- “pushes” the nascent sprout away from parent vessel -- utilizes soluble Flt-1 generated by parent vessel

Lumen formation in developing

blood vessels

Kamei et al, 2006

HUVEC Zebrafish

Discontinuous lumens form and then connect

in developingblood vessels

Kamei et al, 2006

Intersomitic vessels appear composed of

4-7 cells and intercellular junctions

Blum et al, 2008

Junction marker stain

Mosaic analysis

Blum et al, 2008

Putative structure of zebrafish intersomitic vessels

The mouse DA forms via Cord Hollowing

Strilic et al, 2009

Guidance pathways in vascular developmentGuidance pathways in vascular development

From Klagsbrun and Eichmann, 2005

branching vascular plexus

Recruitment ofSmooth muscle/pericytes

mature blood vessel

RemodelingRemodeling

AngAngPDGFPDGF

Ephrin/Eph specifies arteries vs. veinsEphrin/Eph specifies arteries vs. veins

Saggital sections of zebrafishover time show that Vegfa knockdown enhances CV formation at the expense of DA

Herbert et al, 2009, Science

Transverse sectionsshow that Vegfa perturbations enhance CV formation at the expense of DA, while opposite for PI3K inh.

A-V formation in fish requires VegfaA-V formation in fish requires Vegfa

Blocking flow down-regulates arterial markers and up-regulates venous

markers

From le Noble et al, 2004