Vegfc in hematopoiesis

Question 1

What is the Hamada, 2000 phenotype with VEGFR-3 deficient embryos?

Answer 1

Defect in vasculature, anemia, and 50% reduction in blood cells

Question 2

Why do VEGFR-3 deficient mouse embryos show early lethality?

Answer 2

They show a defect in large vessels and severe anemia (before emergence of lymphatic system)

Question 3

What is the Hamada, 2000 phenotype with VEGFR-3 deficient p-sp explants at E9.5?

Answer 3

Much less blood cells than het or WT explants, specifically less erythroid cells and hemtaopoietic progenitors

Question 4

What are p-sp explants cultured on?

Answer 4

OP9 stromal cells

Question 5

In Hamada, 2000 what is the effect of adding either VEGF-A or VEGF-C to p-sp explants?

Answer 5

Increased vasucular bed formation but decreased hematopoietic cells (dose dependent)

Question 6

According to Hamada, 2000…does the expression of VEGF-A, VEGF-C or VEGFR2 change in VEGFR-3 homozygous embryos?

Answer 6

No, according to rt-PCR analysis none of these levels change

Question 7

In Hamada, 2000 they find that VEGFR-3 deficient mice have a larger vascular bed…surprising! Why might this be?

Answer 7

VEGF-C can only signal through VEGFR-2 in the absence of R3, causing aberrant vascular growth

Question 8

In Hamada, 2000 what is the hematopoietic effect of adding VEGFR3-Fc (soluble) to VEGFR-3 deficient systems?

Answer 8

Increased…so when R3 is KO…VEGF-C is binding R2 and decreasing hematopoiesis? But addition of soluble R3 binds all that up allowing for increased hematopoiesis

Question 9

What is the overall finding regarding VEGF-C in hematopoiesis from Hamada, 2000?

Answer 9

Normally, VEGF-C acts through R3 and hematopoiesis is normal, BUT when VEGFR-3 is KO it acts through R2 causing aberrant vascular growth and supressed hematopoiesis.

Question 10

When thinking of Hamada, 2000 findings….under normal conditions VEGF-C signals lightly through R2 and mostly through R3…how does this relate to your work?

Answer 10

Maybe VEGF-C binding through R3 is important in hematopoiesis…need a balance of binding to R2 and R3 to get right amount

Question 11

What does P-Sp stand for

Answer 11

para-aortic splanchnopleural mesoderm

Question 12

What does the P-Sp explant do?

Answer 12

basically makes a vascular bed around it…precursor for AGM..p-sp is earlier stage

Question 13

What is the mouse model used in Fang, 2016?

Answer 13

Vegfc deletion at E7.5, E10.5 or 8 weeks

Question 14

What did Fang, 2016 show with vegfc deletion at E7.5

Answer 14

smaller, pale livers…decreased erythrocytes (enucleated primitive and less definitive) and apoptosis

Question 15

Did Fang, 2016 do CFU on vegfc deleted?

Answer 15

Yes, they showed no difference in colony forming ability

Question 16

What cells were responsible for apoptosis in E14.5 FL? What techniques were done? (Fang, 2016)

Answer 16

Erythrocytes and macrophages….identified by caspase-3 staining in FL and flow cytometry with propidium iodide (PI) and annexin V

Question 17

How does apoptosis by annexin V and PI work?

Answer 17

Binds to phosphotidylsterine residues which are normally hidden in membrane, during cell death they are present on surface….PI enters cells with disrupted membrane intregrity

Question 18

Fang, 2016 showed that there is no effect on E14.5 FL HSCs when VEGF-C is deleted…what is your response to that considering your results?

Answer 18

No deletion is 100% and in hematopoietic system, you just need one HSC to repopulate the entire population… they do not look at AGM they look mainly at FL