Haematopoietic System Development Flashcards
Bone marrow function
Main site of haematopiesis
Thymus
Main site of T lymphocyte development
Spleen function
Blood depot and filter
Hameatopoietic system hierarchy
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
HSC functional assay
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
CFU-S functional assay
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???
CFU-C functional assay
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
Modes of HSC division
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
Foetal liver
Intermediate organ
Develops at early stage
Main haematopoietic organ prior to bone marrow development
Contains blood cells filling foetal liver parenchyma
CFU-S and yolk sac
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
The AGM
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
When are HSCs first detectsble
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
Haematopoietic sites in embryo
Yolk sac
Chorion (becomes placenta)
Alontois
Foetal liver
AGM
How can blood cells in yolk sac appear in absence of HSCs?
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
Model 1 or model 2?
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
Why is model 1 preferable? (2 separate haematopoietic systems - ephemeral embryonic and definitive adult)
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
Which hameatopoietic system comes from which region (at least in xenopus)
Transient embryonic one from yolk sac
Permanent definitive one comes from aorta gonad mesoneohros region AGM
Pros of mouse model (even tho zebra fish development is more visible)
Mammalian model
Inbred mines
Sequences genomes/good genetics
Accessible genome for manipulation (transgenesis, KO, ES cells)
Developed functional assays
Cons of mouse model over zfish
Develop inside mother. - harder to see - limited experimental access
More expensive to keep
Obtaining sufiicient numbers if embryos for certain experiments can be problematic
Quantitating on of HSCs assay
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
Emergence of HSCs in embryonic tissues
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
Where to HSCs mature from?
AGM region (and not the yolk sac) can autonomously generate definitive HSCs
3 waves of haematopoiesis in embryo
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
Haemangioblast concept
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
Haemangioblasts in mouse
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
Haematogenic endothelium
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
Cell fate experiments for HSC emergence from embryonic endothelium
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
Generation of CFU-S and HSC in Reaggregate culture
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
Pre definitive progenitors of HSC markers
Ve-cad+
CD45+
Step wise development of HSC hierarchy
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+
CD43- cells in dorsal aorta
Are only in the dorsal aorta
SO Pre-HSCs sit in the floor of the dorsal aorta
Runx1
General haneatopoietic cell marker
CD45 staining
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
Ckit, CD31
Tyrosine kinase
Principle marker for adult HSCs
CD31 endothelial marker
Relationship between haemangioblast adn haematogenic endothelium
Haemangioblast gives rise to haematogenic endothelium
This haematogenic endothelium can give rise to either haematopoietic cells or structural endothelium
Blood origin germ layer
Mesoderm
Marked by brachuary expression
Can use this to label mesoderm formation/presence at diff stages
Etv2/Er7 TF
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)
Flk1
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
Stem cell leukaemia gene (SCL/Tal-1)
Lmo2
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
RUNX1
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
Intra aortic clusters and Runx1
Are Runx1+
Clusters stil maintain Allen VE-cad expression after budding but begun expressing RUNX1 too
Runx1 required for endothelial to haematopoietic transition EHT
What step wise stage of HSC development does Runx1 deletion block
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
C-myb
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
C myb KO vs Runx1 KO
Cmyb - presence of HSCs but quickly all differntiate
Runx1 - no HSCs at all
4 haneatopoietic waves in zfish
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
Runx1 staining in zfish embryo
Intra aortic clusters
EHT in zfish
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)
CD41 marker, Runx1 and EHT
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)
External factor for formation of blood cells
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
Initiation of HSCs in developing dorsal aorta
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
How is dorsal aorta polarised along dv axis
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
Reciprocal inductive interactions in the AGM region experiment
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
Downregulation if BMP signalling in HSC development
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)
Antagonists of BMP4
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
Stem cell factor SCF and early HSC development
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
NOTCH signalling and arterial development
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
Mature CD45+ cells in AoV
Macrophages and neutrophils
Enriched in AoV (ventral aorta?)
Pro inflammatory signalling and HSC emergence
Positively upregulates it
TNF-alpha and IFN-gamma proinflammatory cytokines
So 3 major factors of emergence:
TNF alpha
IFN gamma
Blood flow
Zfish macrophages
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
