Haematopoietic System Development

Question 1

Bone marrow function

Answer 1

Main site of haematopiesis

Question 2

Thymus

Answer 2

Main site of T lymphocyte development

Question 3

Spleen function

Answer 3

Blood depot and filter

Question 4

Hameatopoietic system hierarchy

Answer 4

Highest - Haematopoietic
Can proliferate and differentiate give rise to downstream blood cells

CFU-S (splenic) (myeloid)

CFU-C (culture) (myeloid)

Common myeloid progenitor gives rise to myeloid cells
Common lymphoid progenitors give rise to lymphoid cells

Question 5

HSC functional assay

Answer 5

Isolate bone marrow
Transplant into irradiated mouse (ablated all HSCs and other progenitors)

Then if HSC present after 3.5 months all haematipoetic cells in recipient will be derived from donor marrow
Reconstituted the haematopoietic system

Question 6

CFU-S functional assay

Answer 6

Is more commuted progenitor than HSC
Transplantation into irradiated mouse will not reconstitute haematopoietic system long term
BUT
will produce colonies of myeloid cells in spleen
Each colony made from 1 transplanted cell (hence cfu)
Colony forms because cell survives but is corrupted in some way idk???

Question 7

CFU-C functional assay

Answer 7

Colony Is also result of differentiation of one cell
C = culture
Plate cells from bone marrow into dish in semisolid medium with growth factors and they will produce colonies w diff morphologies of myeloid cells

Most committed from three discussed

Question 8

Modes of HSC division

Answer 8

Asymmetric- maintenance of HSCs - homeostasis

Symmetric - expansion of HSCs (eg regeneration of haematopoietic system)

Symmetric division - expansion of progenitors

Mode gone with depends on conditions
Eg expansion in order to replenish blood cells

Question 9

Foetal liver

Answer 9

Intermediate organ
Develops at early stage
Main haematopoietic organ prior to bone marrow development

Contains blood cells filling foetal liver parenchyma

Question 10

CFU-S and yolk sac

Answer 10

Cfu-s only in yolk sac at E8
Then start to appear in F liver and circulation
Was believed that it was HSC source then (wrong !!!!)

Was thought then travel to liver after
First cfu-s seen at E10 there

Question 11

The AGM

Answer 11

Aorta-gonad-mesonephros

Was shown that embryo had no CFU-S at E8
Yolk sac actually had v few CFU-S

Have blood cells at day 8 but no cfu-s

When testing whole embryo instead of just yolk sac
Note colonies came from body of embryo than the yolk sac
Appeared at similar time - E9 instead of 7/8

Question 12

When are HSCs first detectsble

Answer 12

HSCs detectable by transplants only appear at E10.5-11.5
So how can the blood cells in yolk sac/embryo body be there on earlier days

Question 13

Haematopoietic sites in embryo

Answer 13

Yolk sac
Chorion (becomes placenta)
Alontois
Foetal liver
AGM

Question 14

How can blood cells in yolk sac appear in absence of HSCs?

Answer 14

Two alternative models

Model 1- two haematopoietic hierarchies exist
One transient and one permanent
Embryonic ones exist earlier but willl expire
And the definitive hierarchy coming from HSCs appear a bit later

Model 2- have common progenitor that gives rise to embryonic hierarchy and also to the definitive adult haematopoietic hierarchy

Question 15

Model 1 or model 2?

Answer 15

One thing to note - yolk sac has large nucleated erythrocytes
V diff to adult ones
So adult peripheral erythrocytes are coming from HSCs while yolk sac ones come from yolk sac

MODEL 2 IS NO LONGER SEIOUSLY CONSIDERED - 1 IS PREFERABLE MODEL

Question 16

Why is model 1 preferable? (2 separate haematopoietic systems - ephemeral embryonic and definitive adult)

Answer 16

Graft developing quail body onto chick embryo - replacing chick body
Discriminate chick v quail cells by nucleus structure
Before hatching shown that majority of cells in circulation were quail in origin (body)
Despite the fact that first blood cells come from yolk sac (chick tissue)
Supporting fact that 2 diff ones arise

Dye injected into xenopus blastocyst cells
Track origin of dorsal aorta
And the ventral blood island (yolk sac equivalent)
Can see even at early stage the origins of these tissues are v diff
Backs up 2 separate haematopoietic system model

Question 17

Which hameatopoietic system comes from which region (at least in xenopus)

Answer 17

Transient embryonic one from yolk sac

Permanent definitive one comes from aorta gonad mesoneohros region AGM

Question 18

Pros of mouse model (even tho zebra fish development is more visible)

Answer 18

Mammalian model
Inbred mines
Sequences genomes/good genetics
Accessible genome for manipulation (transgenesis, KO, ES cells)
Developed functional assays

Question 19

Cons of mouse model over zfish

Answer 19

Develop inside mother. - harder to see - limited experimental access
More expensive to keep
Obtaining sufiicient numbers if embryos for certain experiments can be problematic

Question 20

Quantitating on of HSCs assay

Answer 20

Limiting dilution analysis
Cells in H system migrate so looking at sections can be hard to find origin

Can count colonies on sleep

Or can do LDA
Can calculate no of HSCs in certain region
Prepare cell suspension
Then transplant into cohort of mice at certain dilution where some mice survive and some do not
One HSC is sufficient for rescue of HP system - dead mouse didn’t even receive one

Means that nice that survived statistically likely only got 1 HSC (or maybe a couple)
Can use the number of surviving mice to count the number of HSCs in the cell suspension transplanted and hence the region it was prepared from

Question 21

Emergence of HSCs in embryonic tissues

Answer 21

Comparing AGM and yolk sac
No significant difference between them at E10.5
Increased LDA rescue of rats at same amount by E11.5

So is it AGM or YS producing the HSCs

Do transplantation assay to see when they are maturing (can’t rescue when immature ig)
Maturation from their embryonic precursors
Jumps from total of 4 in embryo at day 11 to 130 day 12
Way too fast for proliferation (cell cycle too slow)
So must be maturing from another cell type already pregnant

Question 22

Where to HSCs mature from?

Answer 22

AGM region (and not the yolk sac) can autonomously generate definitive HSCs

Question 23

3 waves of haematopoiesis in embryo

Answer 23

1 - maturation and gradually disappear
But recently shown that tissue macrophages (or circulating ones) arise from yolk sac??)

2- more potent erythroid progenitors appearing at day 8 (1 day later than w1)

Wave 3- HSCs appear at day 10.5-11.5

HSCs will take month to begin producing blood cells
Need some for embryo
So need to have the early blood cells for function in embryo

Question 24

Haemangioblast concept

Answer 24

Blood island in yolk sac at E7
Made of cells called haemangioblasts - arise clonally
Form chick yolk vasculature

Haemangioblasts give rise to vasculature
Both endothelial and blood cells

Blood islands are clonal in origin

Question 25

Haemangioblasts in mouse

Answer 25

Found at E7.5 Going in area if mesoderm formation Can generate 3 separate lineages Endothelial Blood Smooth muscle actin+ cells (this one controversial) Mostly accepted as bipotential Endothelium Blood cells

Question 26

Haematogenic endothelium

Answer 26

In dorsal aorta Look at bottom (ventral side) Cells sitting there stained for haematopoietic markers Cells bud from the ventral endothelium Endothelial to haematopoietic transition Formation of these intra-aortic haematopoietic clusters coincides with HSC appearance HSCs emerge from here

Question 27

Cell fate experiments for HSC emergence from embryonic endothelium

Answer 27

Cre recombinase attached to VE-cad promoter specific to endothelial cells And silent LacZ with sites for recombinase Cre inactive - cell stain red Recombinase active. - removes stop on LacZ - LacZ stained Trace the lineages from the endothelial cells: At E10 only endothelium labelled Then blood labelled at E14 - so the later HSC blood coming from endothelium lineage

Question 28

Generation of CFU-S and HSC in Reaggregate culture

Answer 28

Can disocciate AGM region and then reaggregate it in a tight reaggregate Becoming like a disorganised explant Can culture this Gey large numbers of CFU-S generated in culture from it About 150 of them per AGM region

Question 29

Pre definitive progenitors of HSC markers

Answer 29

Ve-cad+ CD45+

Question 30

Step wise development of HSC hierarchy

Answer 30

Endothelium CD41 stage in Pro-HSC E9.5 CD43 stage in Pre-HSC I E10.5 CD45 stage in Pre-HSC II E11.5 CD45 definitive HSC E11.5 All ve-cad+

Question 31

CD43- cells in dorsal aorta

Answer 31

Are only in the dorsal aorta SO Pre-HSCs sit in the floor of the dorsal aorta

Question 32

Runx1

Answer 32

General haneatopoietic cell marker

Question 33

CD45 staining

Answer 33

CD45 - haematopoietic marker Ve-cadherin - endothelial marker Captures transition from endothelium like cells to haematopoietic cell In intra-aortic haematopoietic cluster - in endothelial cells - cells not round (endothelial character) but express CD45 Captured in recess of becoming haematopoietic cell Partway between both

Question 34

Ckit, CD31

Answer 34

Tyrosine kinase Principle marker for adult HSCs CD31 endothelial marker

Question 35

Relationship between haemangioblast adn haematogenic endothelium

Answer 35

Haemangioblast gives rise to haematogenic endothelium This haematogenic endothelium can give rise to either haematopoietic cells or structural endothelium

Question 36

Blood origin germ layer

Answer 36

Mesoderm Marked by brachuary expression Can use this to label mesoderm formation/presence at diff stages

Question 37

Etv2/Er7 TF

Answer 37

Specifies haematopoietic and endothelial lineages BMP, NOTCH, and WNT signalling involved in inducing Etv2 expression Leads to Flk1 expression Etv2 expression is transient Labels dorsal aorta Expressed during early vasculature formation Induces Flk1 Etv2 KO prevents development of blood vessels (endothelial and haematopoietic lineages blocked)

Question 38

Flk1

Answer 38

Induced by Etv2 TF expression Cell surface receptor for faculae endothelial growth factor VEGF Weakly marks early mesoderm and strongly marks late mesoderm KO mice lack endothelium Flk1-GFP - reporter in blood islands at E7 E10.5 shows network of vessels marked by Flk1 reporter

Question 39

Stem cell leukaemia gene (SCL/Tal-1) Lmo2

Answer 39

Essential for haematopoietic specification - KO = no blood Vessels present (endothelium) but no blood Lmo2 mutant has similar phenotype Is a TF KO = endothelium but no blood So there is a hierarchy of TFs and secreted molecules that play a role in different time points in haematopoietic development

Question 40

RUNX1

Answer 40

Essential for adult type but not yolk sac type haematopoiesis KO - foetal liver discoloured and only parenchymal cells no blood But no difference to WT in yolk sac Selective blocking of development of HP system from dorsal aorta No intra-aortic clusters Liver is anemic and does not give rise to CFU-S/C, HSCs in transplantation

Question 41

Intra aortic clusters and Runx1

Answer 41

Are Runx1+ Clusters stil maintain Allen VE-cad expression after budding but begun expressing RUNX1 too Runx1 required for endothelial to haematopoietic transition EHT

Question 42

What step wise stage of HSC development does Runx1 deletion block

Answer 42

Take each stage Delete Runx1 from it See if they can become HSCs in culture Runx1 is required pre-HSC type I and II stages

Question 43

C-myb

Answer 43

Required for foetal liver haematopoiesis TF Needed for development of adult haneatopoietic system KO dies later than runx1 KO And while the runx1 KO blocks HSC development completely c-myb KO give push in to development but very quickly differentiate so there is no self renewing of HSCs

Question 44

C myb KO vs Runx1 KO

Answer 44

Cmyb - presence of HSCs but quickly all differntiate Runx1 - no HSCs at all

Question 45

4 haneatopoietic waves in zfish

Answer 45

W1 - primitive macrophages W2- primitive erythrocytes W3- erythro-myeloid cells W4 - multipotent progenitors and HSCs arise from dorsal aorta Dorsal aorta formed at 18hr stage - there is a cohort of primitive erythrocyte cells Erythromyeloid cells come from posterior end

Question 46

Runx1 staining in zfish embryo

Answer 46

Intra aortic clusters

Question 47

EHT in zfish

Answer 47

Flat cell becomes curved Gradually becomes haematopoietic cell on outside of aorta and go in Different to mice where intra aortic clusters are multicellular and are inside aorta (possible due to having more space - aorta bigger)

Question 48

CD41 marker, Runx1 and EHT

Answer 48

Cells undergoing EHT gain the CD41 haematopoietic marker Upregulated Runx1 required for successful EHT In zfish runx1 mutant - cell tries to curve and become rounded but it bursts in absence of runx1 (seeing this directly is benefit of zfish model)

Question 49

External factor for formation of blood cells

Answer 49

Blood follow induced shear stress induced formation of blood cells Induced notch signalling (important for aorta specification and initiation of HSCs) If flow is different can cause problems by giving different TF profile

Question 50

Initiation of HSCs in developing dorsal aorta

Answer 50

PLPM- posterior lateral plate mesoderm Expression of ETS factors in upper parts of naive mesoderm indicates Haemangioblast specification area ETS + GATA2 induce Flk1 expression Then there is VEGFA signal - binds Flk1 - induces SCL Cells from here come to midline where the dorsal aorta will form VEGFA expressed in somites Interactions between these moving cells to midline and Jam1/Jam2 adhesion molecules on somite cell surfaces (notch signalling important for this) Throug these interactions the cells arrive at dorsal aorta where they become HSCs

Question 51

How is dorsal aorta polarised along dv axis

Answer 51

Shh signalling from notochord above dorsal aorta BMP signalling from below Opposite from the neural tube where BMP above and Shh from notochord below (important for nerve formation in diff parts of NT) Countergradients if these signalling molecules polarise the dorsal aorta BMP4 signalling inhibition needed later on for HSC maturation tho

Question 52

Reciprocal inductive interactions in the AGM region experiment

Answer 52

Dissect dorsal and ventral regions of dorsal aorta Make one half GFP in each So can see which half certain cell come from Countergrafients of Shh in dorsal Does this shh affect differentiation in ventral? Take either dorsal or ventral region GFP+ and put it with non GFP ventral Use just central part as control Need both dorsal and vebtral parts for proper HSC development? So interactions between dorsal and ventral regions lead to induction of HSC sun embryo

Question 53

Downregulation if BMP signalling in HSC development

Answer 53

BMP countergradient needed for dv polarisation And other earlier events starting from mesoderm formation Noggin - secreted antagonist if BMP4 - labels intra aortic cluster BMP4 seen in area just below IAClusters But IAC itself is protected from BMP signalling (visualised using smad1,5,8)

Question 54

Antagonists of BMP4

Answer 54

BMPER Noggin Shh upregulaion increases HSC differentiation ????? Something about interactions of dorsal region with the ventral one SCF not a bmp4 antagonist but is also important in this group of early HSC development molecules

Question 55

Stem cell factor SCF and early HSC development

Answer 55

Ligand for cKIT cell surface receptor Deletion of SCF causes progressive loss of HSCs during development SCF expression centrally polarised in dorsal aorta Then the intra aortic clusters express cKIT - receptor for SCF

Question 56

NOTCH signalling and arterial development

Answer 56

Notch signalling defines arterial but not venous endothelium and therefore is necessary for HSC production Downrefulation of notch is needed for successful progression from arterial endothelium into definitive HSCs Sox17 upregulates notch - repression haematopoietic fate Also binds runx1 and gata2 repression haematopoietic fate Ablation of sox17 increase HSC production

Question 57

Mature CD45+ cells in AoV

Answer 57

Macrophages and neutrophils Enriched in AoV (ventral aorta?)

Question 58

Pro inflammatory signalling and HSC emergence

Answer 58

Positively upregulates it TNF-alpha and IFN-gamma proinflammatory cytokines So 3 major factors of emergence: TNF alpha IFN gamma Blood flow

Question 59

Zfish macrophages

Answer 59

Primitive macrophages Migratory below ventral part of dorsal aorta Patrol the HSCs Support their migration to vein - secrete matrix metalloproteinases to loosen ECM DISCLAIMER - remember the zfish IACs are single felled and are on outside of ventral side of dorsal aorta Undergo EHT then move down to vein where they enter circulation Vein is located ventral to the dorsal aorta