3/4 heart and blood vessels

Question 1

How did Peter Nieuwkoop (1969) discover the lateral plate mesoderm

Answer 1

Mesoderm is usually made in marginal zone, he wanted to know why.
Cut animal cap off top of xenopus embryo and stuck it onto the vegetal cells.
Animal cap produced mesoderm but normally makes epidermis
Demonstrated existence of vegetal inducing signals and importance of blastocoel as a negative signalling space (protects animal cap cells from signals from vegetal cells)
Can grow animal cap cells in culture, get cluster of skin cells – Jim Smith identified signal as TGF beta signal Nodal which sets off differentiation pathway – grow animal cap cells with beta signal, got cells that would normally form skin to form skeletal muscle and notochord

Question 2

Describe signalling in emergence and determination of the LPM

Answer 2

LPM originates during gastrulation following specification of mesoderm
Early LPM formation is influenced by BMP and Notch signalling pathways that co-ordinate
patterning of AP and DV axes
High levels of BMP signalling in the ventral domain of the embryo chiefly specifies the mesoderm territory that form the LPM in all vertebrates – highly conserved among vertebrates

Question 3

What does the LPM go on to become?

Answer 3

Heart, blood vessels, blood, body cavity lining, limb bones.
Somatic/somatopleuric mesoderm (mesothelium)
Splanchnic/splanchnopleuric mesoderm (cardiovascular system)

Question 4

What are the different genes expressed in different compartments of the LPM?

Answer 4

Draculin - strongly expressed in blood cells and entire LPM
Imo2 - expressed in blood cells
Slcl - important for development of blood endothelium
Pax2.1 - master regulator of kidney development
Hand2 - TF important in specification of heart progenitor cells, expressed throughout posterior of embryo

Question 5

Describe components of vertebrate hearts

Answer 5

4 chamber heart
Inner endocardial lining and an outer myocardium (muscle layer which provides the force to push)
Cushion cells form valves
Purkinje fibres form sinoatrial node
Epicardium = outer cell layer where coronary arteries develop to supply blood to the heart muscles
Most is from lateral plate mesoderm but some contribution from neural crest cells

Question 6

Describe the circulation in drosophila

Answer 6

Drosophila has an open circulatory system – hemolymph is released into a body cavity
A single dorsal vessel that consists of a single layer of contractile cells acts as a heart

Question 7

What are the steps of mammalian cardiac function?

Answer 7

1. Formation of the primitive heart tube
2. Cardiac looping
3. Cardiac remodelling and septation

Question 8

Describe formation of the primitive heart tube.

Answer 8

Need initial slow function to be able to increase in efficiency as development refines their function.
First heart field will become future left ventricle and both atria. Second heart field will become outflow tract and future right ventricle.
1st heart field generates a scaffold which is added to by the second heart field and cardiac neural crest
Outflow tube is contribution from cardiac neural crest – everything else is mesoderm
Chick: cells migrate to head, cardiogenic mesoderm forms from head end, heart progenitors from primitive blood vessels move to the midline where they fuse to form the linear primitive heart tube and primitive pumping action of the heart starts
Zebrafish: medial migration of heart progenitors, vascular progenitors (angioblasts) and blood progenitors from anterior lateral mesoderm (ALM)

Question 9

Describe cardiac looping

Answer 9

Need to separate into chambers.
Atrium moves up and behind ventricles to sit above them.
Some is coordinated by tissues surrounding heart, others is cell intrinsic coordination
Process occurs while heart is beating and pumping blood around the bodye

Question 10

Describe cardiac remodelling and septation

Answer 10

Ventricular septum primordia migrate anteriorly to separate left and right ventricles
Atrial septum primordia migrate down to separate left and right atria.
Cushion cells form atrioventricular valves
The aortic and pulmonary valves are derived from neural crest and endocardial cells.
Cardiac neural crest migration to branchial arches and outflow tract (primitive arterial connection). CNCC in outflow tract region contribute to conotruncal ridges which fuse and spiral down outflow tract, sealing off each section of the heart and forming the conotruncal septum. Process is critical for separation of the aortic and pulmonary tracts and to connect each tract to its corresponding ventricle, allowing us to have double circulation with established connection

Question 11

If some TF’s are required for multiple stages of heart development, how are they regulated?

Answer 11

Either expressed in temporal manner and only active at specific time points or expressed more broadly but expression is tightly controlled

Question 12

Describe Nkx2.5 as a TF involved in the genetic control of heart development.

Answer 12

Expressed at multiple stages
Mutants in drosophila don’t have a heart > drosophila TF is essential for heart development.
In humans, it causes atrial septal defects - more redundancy for formation of the heart than flies do.
Its vertebrate homologue Nkx2.5 expressed in heart progenitors of the bilateral heart field but is not essential for heart development

Question 13

Describe Mesp1 being expressed in the mouse embryo

Answer 13

Expressed in mesodermal precursors from E6.5. Expressed earlier in heart progenitors but turned off about E7.5.

As cells leave primitive streak, they switch off Mesp1 gene.
Homozygous mutations form endocardial and myocardial cells but develop cardia bifida (cells in bilateral heart fields fail to migrate to midline to form one medial heart, instead 2 bilateral hearts develop). Bit of redundancy between Mesp1 and 2 but KO both fail to specify

Question 14

Describe how Wnt and BMP are involved in genetic control of heart development in Chicks

Answer 14

BMP present laterally, notochord expresses BMP antagonists medially (repress signalling)
Wnt signalling in posterior, Wnt antagonists in anterior
Overlaying signals shows cardiogenic mesoderm forms laterally and anteriorly
BMP signalling induces Nkx2.5 and Mesp1  activate gene network needed for heart development;
Mesp1 also represses the expression of other developmental programmes
Because the pathways are effectively redundant, pathways and development of the heart still occurs with the loss of one of the genes

Question 15

Which other TFs control myocardial differentiation within the heart field?

Answer 15

Gata4 promotes myocardial differentiation
Antagonistic action of Tbx5 and Tbx20 leads to specification of left and right ventricle and the proper formation of the ventricular septum between the two chambers

Question 16

Describe the ENU (N-ethyl-N-nitrosourea) mutagenesis screen for cardia bifida in zebrafish

Answer 16

ENU (nasty alkaline agent before CRISPR/Cas9) randomly mutates DNA - mutagenesis screens in zebrafish identified several mutants that displayed it
Once carried a mutation in the gene miles apart (mil – results in 2 atria and 2 ventricles with one of each on opposite sides of the embryo because they couldn’t get to the midline) – located in gene that encodes a G protein coupled receptor for Sphingosine-1-phosphate expressed in endoderm (if knockout, endoderm doesn’t migrate properly and get heart issues as a secondary effect of failure in gut looping)
This receptor is expressed by the endoderm (cell non-autonomous requirement for mil in cardiomyocyte migration)
In the mouse FoxP4 encodes a TF that is expressed in foregut endoderm

Question 17

What happens to the ductus arteriosus and foramen ovale at birth?

Answer 17

Increased blood pressure in left side of heart closes foramen ovale.
Dropping levels of prostaglandins cause muscles around ductus arteriosus to close duct.
Failure to close holes makes heart inefficient

Question 18

Describe the intermediate steps of angioblasts becoming vessels

Answer 18

Angioblasts
Differentiation and migration
Coalesce and adhere to each other forming cell-cell junctions.
Tube formation (lumenisation and formation of a basal lamina)
Tube stabilisation (attraction of other cell types such as mural cells, pericytes, smooth muscle cells)

Question 19

Describe vasculogenesis

Answer 19

De novo formation of BV from angioblasts, mesodermal progenitors of endothelial cells.
Mural (vessel walls) cells are pericytes and vascular smooth muscle cells.
Haematopoiesis and vasculogenesis occur in close proximity in the mammalian YS

Question 20

What is the definition of a haemangioblast?

Answer 20

Bipotent progenitors for blood and endothelial cells. They are single cells isolated from embryoid bodies and form the posterior primitive streak of the mouse embryo

Question 21

What receptors do angioblasts express?

Answer 21

VEGFR2 (aka Flk1) - major morphogen that’s the key regulator of formation of complex vascular networks.
Ligand VEGFA secreted by neighbouring cells - can induce EC survival, migration, proliferation and differentiation (context dependent).
Targeted deletion of both copies of Flk1/VEGFR2 gene in mouse causes loss of vessel formation and embryonic lethal at E9.5
KO one copy of VEGFA ligand gene leads to similar phenotype, haploinsufficiency, lack all RBC

Question 22

Describe the genetic and physiological determinants of arterial vs venous distinction

Answer 22

Eph/ephrin interact ions are involved in cell sorting and cell segregation in many different tissues
Tight boundaries between different cell types are formed within the developing brain in this way
Notch1 single and Notch1/4 double mutants display defects in vessel maturation
Notch signalling is active in zebrafish posterior lateral mesoderm and guides arterial differentiation
Notch signalling is activated in angioblasts within PLM before they begin their migration to the midline to form the first BV by Vasculogenesis

Question 23

How is notch involved in specifying arterial endothelial cells and vein identity?

Answer 23

Endothelial cells that receive strong VEGFA and strong Notch become arterial. ECs that do not receive a notch signal become venous. In the absence of notch signalling, differences between arteries and veins cannot be established and vessel network doesn’t mature.

COUP-TFII is TF expressed in veins but not arteries in mice. Mutants displayed ectopic expression of arterial genes (Ephrinb2) in veins and loss of venous genes (Ephb4). Transgenic expression of Myc tagged COUP-TFII in mouse arteries inhibits expression of the VEGF receptor NP1 and Notch ligand Jag1. –> Demonstrated active repression of arterial genes in arteries by misexpressing COUP-TFII (normally not expressed in arteries)

Question 24

Define angiogenesis

Answer 24

Growth and expansion of the existing primitive blood vessel network

Question 25

Describe what is meant by sprouting angiogenesis

Answer 25

Independent and/or dependent on haemodynamic flow. Formation of sprout from existing vessel reaching across to another vessel before fusing and lumenising. Main route which vessels remodel to form their complex vascular network. Associated with change of morphology, behaviour of endothelial cells within tubular endothelium. Adopts invasive and migratory phenotype with filopodia protrusions which sense environment

Question 26

Describe what is meant by intussusceptive angiogenesis

Answer 26

dependent upon haemodynamic flow AKA splitting angiogenesis. Vessel splits into two vessels. Pillars form, associated with deposition of extracellular matrix. Cells of blood vessel sense flow patterns and respond differently

Question 27

Describe how VEGF guides angiogenic sprouting

Answer 27

Tip cells (with invasive migratory morphology) migrate towards VEGF secreted ligands. Chronic high VEGF induces ectopic filopodia formation in stalk cells (adopt tip cell identity to be able to do the migration too). Expression of soluble VEGFR1s/Flt1 (cleaved from tyrosine kinase domain) acts like molecular sink to soak up all the VEGF ligand without activating downstream stuff if cell is expressing Flt1. Blocks ectopic filopodia formation. Sprout cell proliferation is dependent on higher VEGF concentration – simple model implied vascular network formation depended on balance of 2 different qualities of VEGF signal (gradient and concentration) Vascular network formation is a highly organised and stereotypes process which this simplistic VEGF model can’t fully explain the necessary control required to co-ordinate the process.

Question 28

Why was VEGF first known as BPF or Vascular permeability factor?

Answer 28

Blood vessels become really permeable if they see too much VEGF - main reason tumour vasculature is really leaky because of the shedding of loads of VEGF in tumour

Question 29

Describe how Notch and Dll4 are involved in angiogenic sprouting.

Answer 29

Dll4 = delta-like 4 (ligand). Loss of single copy of Dll4 in mouse YS increases vessel density and disrupts vascular network formation. Loss of both copies further increases vessel density alongside gross developmental abnormalities. Dll4 expressed at right place right time intracellularly in tip cells at migrating front. When blocking cleavage of Notch1 receptor, get highly dense network and more tip cells in notch KO. In developing zebrafish arteries, suppression of notch signalling is associated with tip cell identitity and is required cell autonomously within ECs. Inhibiting notch signalling results in hyperbranching phenotype of segmental arteries. Activated signalling gave opposite effect of fewer vessels. Transplanting WT endothelial into WT embryo able to form tip cells/artery/vein, inhibit notch and only form tip cells but not artery or vein. Notch signalling limits endothelial cell proliferation in developing arteries

Question 30

Describe the steps from vessel receiving VEGF signal to inhibiting neighbouring stalk cells

Answer 30

Vessel receives VEGF signal Activates VEGFR2 Activates expression of Dll4 within cell Inhibits ability of neighbouring stalk cells to respond to that VEGF Overall, - Elevated VEGF = increased angiogenesis - reduced Dll4 = increased angiogenesis

Question 31

Describe segmental artery sprouting

Answer 31

Looks like ladder, gaps between sprouting arteries correspond to somites. Sprout dorsally until reach dorsal tube then sprout laterally. Stereotypical sprouting Ordered and regular Segmental arteries have limited variation in endothelial cell number

Question 32

Define vascular remodelling

Answer 32

Optimisation and refinement of vascular network to maximise efficiency

Question 33

Describe how fluid shear stress sensing controls vascular remodelling.

Answer 33

FSS = force of RBCs bashing into walls of vessels and causing stretch forces which are detected. PECAM1, VEGFR2and3/Flk2and3 are all part of mechanosensory complex which can sense how much flow is going through the vessel - also has cadherin and other proteins. With cerebral cavernous malformation (CCV), complex doesn't work properly and cells can't respond to the flow resulting in pathological remodelling and abnormal chaotic connection of arteries and veins - sometimes get sacs full of blood that are prone to rupture. Can sense no flow because of blockage and cause expansion of neighbouring vessel to adapt to increased blood flow. High/laminar shear protects against abnormal remodelling – maintains ECs in quiescent state, limit endothelial-mesenchymal transition (EndmT) Low/disturbed shear promotes remodelling – activates invasive endothelial phenotype promotes EndmT – like atherosclerotic plaque in bends or point of Y in Y shaped vessel Sustained deviations away from the set point induces remodelling

Question 34

How does blood flow coordinate formation of a vascular network?

Answer 34

Sub-intestinal venous plexus (SIVP) allows embryo to obtain nutrients from yolk during ealry development like mammalian vitelline veins. Vessel remodelling occurs at same time blood circulation enters plexus. Angiogenic sprout regression is dependent upon blood flow - withour blood flow, continue to migrate all the way round so plexus meets the other side of the embryo and meet there - not good. Blood flow promotes regression of angiogenic sprouts: tracked endothelial cells in developing vascular plexus imaged over time; tracks overlapped during migration. Blood flow coordinates cell migration during sprout regression to support plexus expansion – reverse migration, blood flow tells endothelial cells which way to migrate – absence of blood flow keep migrating down – halving the number of cells with same length vessel makes it thinner and worse at getting blood cells through

Question 35

Describe how lymphatic vessels form using similar mechanisms to those employed by blood vessels despite having a distinct structure and function to BV.

Answer 35

Originate from venous endothelium Lymphatic vessels form via lymphangiogenesis. Mammalian: Vein specific signalling pathway --> lymphatic sprouting via major morphogen VEGFC (bind to vegfr3) --> aggregation of platelets which block hole and allow two vessels to separate without blood getting into the lymphatic vessels