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

2. Blood vessel formation is important in:
Embryogenesis
Pregnancy
Wound healing
Disease

3. Tumor vascularization is a major step in tumor progression

4. Diabetic retinopathy (excess blood vessels in the eye) is a major cause of blindness

5. VEGF Signaling Pathway
FromShibuya, 2006

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

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

8. Vascular development - differentiation of angioblasts
Mesoderm
angioblasts
Hemangioblast
Hematopoietic cells

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

10. Vascular development - differentiation of angioblasts
Mesoderm
angioblasts
Hemangioblast
Hematopoietic cells

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

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

13. 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

14. Vasculogenesis VEGF bFGF angioblasts primitive vessel differentiation
assembly
primitive vessel

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

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

17. 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

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

19. VEGF pathway influences tip cells
From Gerhardt et al, 2003

20. 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

21. 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

22. 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

23. 1:1 Day 0 Day 8 Wild-type ES cells flt-1-/- ES cells
Mosaic blood vessels
“Hanging drop” EBs

24. 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

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

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

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

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

29. Putative structure of zebrafish intersomitic vessels
Blum et al, 2008

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

31. Guidance pathways in vascular development
From Klagsbrun and Eichmann, 2005

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

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

34. 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.

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