Download BIOLOGY 624 Fall 2009 DEVELOPMENTAL GENETICS

BIOLOGY 624 Fall 2009
DR. VICTORIA BAUTCH
DEVELOPMENTAL GENETICS
Lecture #9: BLOOD VESSEL FORMATION – Tues 12/1/09 and Thurs
12/3/09
Reading: Gilbert Ch. 15, p. 482-489 (with reservations – not particularly
well done);
PAPER DISCUSSION for Tues 12/1: Ghabrial AS and Krasnow MA (2006).
Social interactions among epithelial cells during tracheal branching
morphogenesis. Nature 441, 746-749.
PAPER DISCUSSION for Thurs 12/3: Hellstrom et al, 2007, Dll4 signalling
through Notch1 regulates formation of tip cells during angiogenesis.
Nature 445, 776-780.
(Click here to access the Powerpoint)
A. BLOOD VESSEL FORMATION (BVF) is the process whereby
mesodermal cells in vertebrate embryos form tubes that will allow for
the movement of blood, the diffusion of oxygen and nutrients, and the
removal of CO2 and waste. It also will be an entry point into the body for
immune cells. It is important in (Slide 2):
1.
2.
3.
4.
Embryogenesis
Pregnancy
Wound-healing
Many diseases:
a. tumors use signaling pathways to induce tumor vessels that allow a cancer
to grow. The vessels can also provide a conduit for metastasis of the tumor
cells (Slide 3).
b. Many diseases are accompanied by chronic inflammation – this means that
the immune system is locally activated. This often sets up inappropriate
angiogenesis, since local inflammatory cells produce positive angiogenic
factors – example, rheumatoid arthritis
c. Diabetes is an ever-growing metabolic disease (with consequences for
development as well) that is accompanied by dysfunctional vessels that lead
to blindness (diabetic retinopathy) and limb amputation (from ischemia) (Slide
4).
d. Finally, there is a lot of excitement about the idea that new blood vessel
generation might be therapeutically useful in REGENERATIVE MEDICINE –
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for example, it may be possible to provide the right signals to regenerate
cardiac vessels to provide oxygen to heart muscle, thus avoiding bypass
surgery.
e. HOWEVER, to be able to think about implementing (d) above we need to
understand in detail how the body controls blood vessel formation (BVF).
B. Major steps of Blood Vessel Formation
In all phases of blood vessel formation, the VEGF signaling pathway is
important to get information to the target cells (slide 5). The parts of the
pathway involved in development include a major ligand, VEGF-A, and
two high affinity receptors, flk-1 (VEGFR-2) and flt-1 (VEGFR-1) (Slide 6).
There are also two co-receptors, NPN1 and NPN2. Genetic deletion of
any of the major components of the pathway leads to embryonic lethality
due to disruption of vascular development (Slide 7), and in fact deletion
of just one of the VEGF-A alleles is embryonic lethal, showing a strong
requirement for the correct amount of ligand.
1. Specification of Angioblasts – precursors to endothelial cells
a. All blood vessels are comprised of endothelial cells, and many also have
other cell types (see below).
b. Angioblasts derive from mesoderm, and it seems that most mesoderm is
capable of producing angioblasts, tho need proper signals to do so (Slide 8).
c. There are TWO pathways to angioblast formation:
1. via HEMANGIOBLAST, a common precursor of both endothelial and
hematopoietic stem cells – this lineage may predominate in the yolk sac
(Slide 9).
2. directly from mesoderm to ANGIOBLAST – this lineage probably
predominates within the embryo (Slide 10).
3. Stainier lab published a seminal paper in Nature (Kamei et al, 2006) that
laser marked individual cells and descendants to show for first time that
hemangioblast exists in vivo, but also showed that the hemangioblast gives
rise to only a small percentage of endothelial cells in zebrafish, rest seem to
come directly from mesoderm (Slide 11)
d. Angioblasts are specified, differentiated into endothelial cells and
organized into vessels in BOTH the embryo proper and the yolk sac (in
amniotes), and the sets of vessels are interconnected via angiogenesis (see
below) (Slide 12).
e. Specification is probably regulated by:
-- BMP signal
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-- VEGF signal
-- FGF signal
-- activin signal
--scl transcription factor
at least these factors are important to form a hemangioblast in vitro. Little
solid experimental evidence exists regarding specification in vivo, due to either
redundancy (FGFs) or multiple roles (VEGF).
f. The next steps occur sequentially in a given vessel, but are happening at
the same time during embryogenesis, and include vasculogenesis,
angiogenesis, and remodeling (Slide 13).
2. Vasculogenesis (Slide 14):
a. Once angioblasts are specified, they can either:
--migrate, coalesce into a vessel, then differentiate into endothelial cells, or
--coalesce and differentiate without further migration
b. Differentiation to endothelial cells involves VEGF signaling and probably
bFGF signaling as well.
3. Angiogenesis (Slide 15):
a. Once the first vessels are formed, they almost always expand and form
interconnections via sprouting angiogenesis – in this process endothelial cells
migrate from parent vessels as a sprout, then fuse with other vessels or
sprouts, and form new vessels.
b. Recently the Drosophila trachea model has been used as a paradigm in
the mammalian blood vessel system – Gerhardt et al used a retinal model of
angiogenesis to propose a tip cell and stalk cells:
--retinal angiogenesis occurs postnatally in rodents (Slide 16)
--Gerhardt et al defined several properties that distinguish tip vs. stalk cells in
the retinal vasculature: proliferation, filopodia, and markers (Slides 17, 18)
--HOWEVER, unlike in the fly there is no evidence that vascular tip cells are
genetically distinct – what does this mean?
--VEGF/VEGFR are expressed in appropriate places and shown by extensive
manipulations to affect tip cell guidance (Slide 19)
--next paper discussion asks the question: how do some cells become tip cells
and others stalk cells? THEME OF HETEROGENEITY
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c. Angiogenesis involves both CELL DIVISION and MORPHOGENESIS, and
regulated VEGF signaling is important in both of these events. How these
processes are co-ordinated during blood vessel formation, and the role of the
flt-1 (VEGFR-1) receptor, is an area of active interest in our lab (Slides 20-25)
d. LUMEN FORMATION also occurs, and very recent data suggests that
apical membrane biogenesis is an important mechanism (Slide 26, 27).
e. Recently some elegant studies labeled cell-cell junctions and found
evidence for 4-7 endothelial cells/vessel in the fish ISV (Slide 28). The lumen
model was based on one cell! This suggested a new model and that the
lumens form by INTERCELLULAR contacts (Slide 29).
f. Even more recently (Stirlic et al, 2009) a study of dorsal aorta (DA)
formation in the early mouse embryo showed that cells first form a closed
cord, then rearrange to form a lumen. Model posits at least two stages, one
dependent on VEGF and one not (Slide 30).
g. There are also numerous other signaling pathways involved in GUIDING
the angiogenic process, so that it occurs in the right places, and most
pathways are also known to guide in the nervous system (Slide 31):
1. semaphorin/neuropilin
2. ephrin/eph
3. slit/robo
4. netrin/unc/DCC
4. Remodeling (Slide 32):
a. This process is associated with the recruitment of mural cells – these are
vessel cells that are not endothelial – ie smooth muscle and pericytes. These
cells reinforce some vessels by providing a nearby source of survival factors
such as VEGF, they also down-regulate expansion and promote quiescence.
b. Recruitment of mural cells for remodeling requires:
--Angiopoietin (Ang) signaling through its receptor Tie2
--PDGF signaling through the PDGF receptor
Loss of either pathway prevents recruitment and leads to embryonic lethality.
C. VESSEL IDENTITY
1. At some point vessels decide to be either arteries or veins (or associated
capillaries). How does this happen?
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2. Originally thought that the onset of flow and the pressure differences led to
formation of arteries vs. veins – or a response to physiological stimulus
3. However, several years ago, Wang et al. (1998) showed that future arteries
expressed ephrin B2, and future veins expressed its ligand, EphB4, BEFORE
the start of blood flow. This showed that some aspects of vessel identity are
genetically hard-wired and not a result of physiological input (Slide 33).
4. There are now several arterial markers: ephrin B2, neuropilin-1 (NRP-1),
Notch3, DLL4; and venous markers: EphB4, neuropilin-2 (NRP-2)
5. A recent study in zebrafish (Herbert et al, 2009) showed that initially one
vessel forms along the main body axis, and that the cells respond to VEGF
signaling differentially to sort via migration to form the vein (Slide 34).
6. Recently, Eichmann and colleagues (le Noble et al, 2004) did some
elegant experiments showing that, although arteries and veins are initially
genetically determined, flow is critical to their continued identity – what this
means is that they could change the marker expression of arteries into veins
by blocking flow in the avian yolk sac (Slide 35).
Optional reading:
Reviews:
Horowitz A and Simons M. (2008). Branching morphogenesis. Circ Res 103,
784-795.
Le Noble F, Fleury V, Pries A, Corvol P, Eichmann A, and Reneman RS.
(2005). Control of arterial branching morphogenesis in embryogenesis: go
with the flow. Cardiovasc Res 65, 619-628.
Klagsbrun M and Eichmann A. (2005). A role for axon guidance receptors and
ligands in blood vessel development and tumor angiogenesis. Cyt & Growth
Factor Rev 16, 535-548.
Weinstein BM. (2005). Vessels and nerves: marching to the same tune. Cell
120, 299-302.
Lacaud G, Keller G and Kouskoff V. (2004). Tracking mesoderm formation
and specification to the hemangioblast in vitro. Trends Cardiovasc Med 14,
314-317.
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Articles:
Le Noble F, Moyon D, Pardanaud L, Yuan L, Djonov V, Matthijsen R, Breant
C, Fleury V and Eichmann A. (2004). Flow regulates arterial-venous
differentiation in the chick embryo yolk sac. Development 131, 361-375.
Wang HU, Chen ZF and Anderson DJ. (1998). Molecular distinction and
angiogenic interaction between embryonic arteries and veins revealed by
ephrin-B2 and its receptor Eph-B4. Cell 93, 741-753.
Lu X,… and Eichmann A. (2004). The netrin receptor UNC5B mediates
guidance events controlling morphogenesis of the vascular system. Nature
432, 179-186.
Kamei M, Saunders WB, Bayless KJ, Dye L, Davis GE and Weinstein BM.
(2006). Endothelial tubes assemble from intracellular vacuoles in vivo. Nature
442, 453-456.
Vogeli KM, Jin S-W, Martin GR, Stainier DYR. (2006). A common progenitor
for haematopoietic and endothelial lineages in the zebrafish gastrula. Nature
443, 337-339.
Gerhardt H…Betsholtz C (2003). VEGF guildes angiogenic sprouting utilizing
endothelial tip cell filopodia. J Cell Biol 161, ….
Blum Y, Belting H-G, Ellertsdottir E, Herwig L, Luders F, Affolter M. (2008).
Complex cell rearrangements during intersegmental vessel sprouting and
vessel fusion in the zebrafish embryo. Dev Biol 316, 312-322.
Herbert SP,…Stainier DYR. (2009). Arterial-venous segregation by selective
cell sprouting: an alternative mode of blood vessel formation. Science 326,
294-298.
Chappell JC, Taylor SM, Ferrara N, Bautch VL. (2009). Local guidance of
emerging vessel sprouts requires soluble Flt-1 (VEGFR-1). Dev Cell 17, 377386.
Strilic B….Lammert E. (2009). The molecular basis of vascular lumen
formation in the developing mouse aorta. Dev Cell 17, 505-515.
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