5. DEVELOPMENT OF BLOOD



Mesonephric
tubules
Blood cells

primitive hematopoiesis
ventral mesoderm
Fish gastrulation movie

8h embryo
ICM
2d embryo circulation
scl, gata1/2, lmo2 - transcriptional factors that specify early hematopoietic tissue (gata2
expressed also in ectoderm)
RBI – rostral blood islands
ICM – inner cell mass
5-somite stage embryo – hematopoiesis starts officially
gata1 promoter-driven GFP = early erythroid lineage
  dorsal aorta

MYELOPOIESIS STARTS IN RBI @ 10-SOMITE STAGE
spi1 – marker of myeloid lineage
rostral
caudal
spi1 detected by
Fast Red riboprobe
scl – early hematovascular cell fate
fli1 – early vascular fate
nkx2.5 – heart fate

      7.5 dpc mouse embryo (late gastrulation stage)
8.5 dpc – yolk sac encloses entire embryo
blood islands merge to form vascular channels
LacZ driven by b-globin promoter
(X-gal staining – primitive erythroblasts)
bl - blood islands
ec - ectoplacental cone
ac - amniotic cavity
te - trophectoderm
al - allantois
eem - extraembryonic mesoderm (blue)
ve - visceral endoderm
e - embryonic ectoderm

 AORTA-GONAD MESONEPHROS (AGM)
Development of intra-embryonic hemogenic site
 fetal liver
 hemogenic site
 aorta
HSC
A- aorta
NO- notochord
AV- axial vein
e - endothelial cell
A
 lateral plate mesoderm
ICM
 Rohon-Beard neurons
 RUNX1 riboprobe

 RUNX1 (CBFA2, AML1) is necessary for ‘definitive’ hematopoiesis to start at AGM
Runx1+/-
 E11.5: dark- hematopoietic cells
only a few primitive erythrocytes left – likely a carry-over from the ‘primitive’ hematopoiesis
Runx1-/-

PRIMITIVE vs. DEFINITIVE
      EXTRAEMBRYONAL vs EMBRYONAL





BONE MARROW NICHE IS CRITICAL FOR HEMATOPOIESIS



 COLLAGEN TYPE-X DELETION
wt
Col X-/-
 normal hematopoiesis
aplasia
wt
Col X-/-
wt
Col X-/-
   thymus
   spleen

THE HUNT FOR HEMANGIOBLAST AND ITS HEM- ANGIO-DIFFERENTIATION



HEMANGIOBLAST
scattered among endothelial and HSC in heart field of the gastrula
ventral marginal cells of the heart field, n- notochord

      Endothelial progenitor
HEMANGIOBLAST or HAEMOGENIC EPITHELIA, or BOTH?


       blast colony-forming cell



Hemangioblast culture
Angiopoietin 1 rec.
Integrin alpha 2B
tight adherent structure

Tie2hic-KIT+CD41_ can generate hematopoietic precursors
endothelial markers
             endothelia in matrigel
Tie2hic-KIT+CD41_ are present in vivo by FACS
Tie2hic-KIT+CD41_ are present in vivo by IHC

Runx1-/-
Blast movies 3, 4
Runx-/- movie

TRANSCRIPTIONAL FACTORS IN BLOOD DEVELOPMENT



GATA-1 - binds DNA via a zinc finger motif
Dracula fish – loss-of-function mutation in GATA1 – impaired erythroid differentiation


Spadetail/TBX16
DNA binding domain derived from the prototype gene called transcription factor T
impaired erythroid but not
myelopoietic differentiation

Moonshine
TIM-family of transcriptional factors
‘primitive’ – survival of HSC                  ‘definitive’ – cardiomegaly and impaired
red                                     cell differentiation
   erythrocyte    proerythroblast

WHEN SOMETHING GOES WRONG WITH BLOOD






AML1 (RUNX1)



-no hematopoiesis in liver
-only primitive erythrocytes in peripheral blood, no platelets


ACUTE MYELOID LEUKEMIA









Sca1 – locus active in hematopoietic stem cells in adult mice



CHRONIC MYELOID LEUKEMIA
  bcr
  abl
  bcr-abl
mice transplanted with patient bone marrow







Am J Med. 1977 Jul;63(1):125-30. Chronic myelocytic leukemia: clonal origin in a stem cell common
to the granulocyte, erythrocyte, platelet and monocyte/macrophage. Fialkow PJ, Jacobson RJ,
Papayannopoulou T.