Biology 624 - Developmental Genetics Lecture #9 -Blood Vessel Formation

Blood vessel formation is important in: Embryogenesis Pregnancy Wound healing 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 Pathway FromShibuya, 2006

VEGF Signaling Pathway Flk-1 Flk-1 Flt-1 VEGF Cell Division + Migration + Survival + Cell Division - Migration + Angioblast Endothelial Cell

ES cell mutant vascular phenotype mimics mutant phenotype in vivo Wild-type Flk-1-/- Flt-1-/-

Vascular development - differentiation of angioblasts Mesoderm angioblasts Hemangioblast Hematopoietic cells

Embryonic vascular differentiation via the hemangioblast Figures\Chapter15\DevBio7e15130.jpg

Vascular development - differentiation of angioblasts Mesoderm angioblasts Hemangioblast Hematopoietic cells

Single cell fate mapping in fish gives Evidence for the hemangioblast FITC = descendants of single cell at 40% epiboly From Vogeli et al, 2006

Circulatory System of a 4-Week Human Embyro Figures\Chapter15\DevBio7e15090.jpg

Embryonic vascular development Sprouting Angiogenesis Recruitment of mural cells and Remodeling Vasculogenesis angioblasts proliferation migration VSMCs/pericytes differentiation assembly primitive vessel mature blood vessel branching vascular plexus

Vasculogenesis VEGF bFGF angioblasts primitive vessel differentiation assembly primitive vessel

Angiogenesis VEGF/neuropilin Slit/robo Netrin/unc Ephrin/eph Notch/delta proliferation migration sprouting branching vascular plexus primitive vessel

Retinal angiogenesis occurs outward from optic nerve postnatally From Gerhardt et al, 2003

Tip cell vs. stalk cell Tip cell: Does not proliferate Extends numerous filopodia Expresses markers- VEGFR2, PDGFB, Dll4, etc Stalk cell: Proliferates Has few filopodia Down-reglates tip cell markers From Gerhardt et al, 2003

Tip cell characteristics From Gerhardt et al, 2003 Tip cell characteristics Express PDGFB No proliferation Actin-rich filopodia

VEGF pathway influences tip cells From Gerhardt et al, 2003

VEGF Signaling Pathway Flk-1 Flk-1 Flt-1 VEGF Cell Division + Migration + Survival + Cell Division - Migration + Angioblast Endothelial Cell Soluble flt-1 isoform selectively rescues migration/branching

Emerging flt-1 mutant sprouts differ from WT sprouts Local sprout guidance measures are rescued by sFlt-1 but not mFlt-1 Chappell et al. (2009) Dev Cell 17, 377

Local sprout guidance measures are perturbed In retinas with reduced Flt-1 activity Flt-1 neutr ab Adeno-Cre flt-1flox/flox Chappell et al. (2009) Dev Cell 17, 377

1:1 Day 0 Day 8 Wild-type ES cells flt-1-/- ES cells Mosaic blood vessels “Hanging drop” EBs

Local sprout guidance measures are rescued by sFlt-1 but not mFlt-1 in lateral base areas Chappell et al. (2009) Dev Cell 17, 377

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

Lumen formation in developing blood vessels HUVEC Zebrafish Kamei et al, 2006

Discontinuous lumens form and then connect in developing blood vessels Kamei et al, 2006

Intersomitic vessels appear composed of 4-7 cells and intercellular junctions Mosaic analysis Junction marker stain Blum et al, 2008

Putative structure of zebrafish intersomitic vessels Blum et al, 2008

The mouse DA forms via Cord Hollowing Strilic et al, 2009

Guidance pathways in vascular development From Klagsbrun and Eichmann, 2005

Remodeling Ang PDGF branching vascular plexus mature blood vessel Recruitment of Smooth muscle /pericytes branching vascular plexus mature blood vessel

Ephrin/Eph specifies arteries vs. veins Figures\Chapter15\DevBio7e15161.jpg

A-V formation in fish requires Vegfa Saggital sections of zebrafish over time show that Vegfa knockdown enhances CV formation at the expense of DA Herbert et al, 2009, Science Transverse sections show that Vegfa perturbations enhance CV formation at the expense of DA, while opposite for PI3K inh.

Blocking flow down-regulates arterial markers and up-regulates venous markers From le Noble et al, 2004