VEGF and IHH rescue definitive hematopoiesis in Gata-4 and Gata-6-deficient murine embryoid bodies.
OBJECTIVE
Murine embryonic stem cells can be differentiated into embryoid bodies (EBs), which serve as an in vitro model recapitulating many aspects of embryonic yolk sac hematopoiesis. Differentiation of embryonic stem cells deficient in either Gata-4 or Gata-6 results in EBs with disrupted visceral endoderm (VE). While lack of VE has detrimental effects on hematopoiesis in vivo, it is unclear whether lack of VE affects hematopoiesis in EBs. Therefore, we compared Gata-4 null (G4N) and Gata-6 null (G6N) EBs with wild-type EBs to assess their ability to commit to hematopoietic cells.


METHODS
EB VE formation was examined using cell-sorting techniques and analysis visceral endoderm gene expression. Hematopoietic progenitor potential of EBs cultured under various conditions was assessed using colony-forming assays.


RESULTS
Definitive erythroid, granulocyte-macrophage, and mixed colonies were significantly reduced in G4N and G6N EBs compared to wild-type EBs. Vascular endothelial growth factor (VEGF) expression and secretion were also reduced in both G4N and G6N EBs, consistent with VE serving as a site of VEGF production. Addition of exogenous VEGF(165), to EB cultures completely rescued definitive colony-forming cells in G4N and G6N EBs. This rescue response could be blocked by addition of soluble Flk-1 Fc to EB cultures. Similarly, addition of exogenous Indian hedgehog to EB cultures also recovers the diminishment in definitive hematopoiesis in a reversible manner.


CONCLUSIONS
These results suggest that the absence of VE in G4N and G6N EBs does not prevent emergence of definitive progenitors from EBs. However, the decreased level of VEGF and Indian hedgehog production in VE devoid G4N and G6N EBs attenuates definitive hematopoietic progenitor cell expansion.
Pierre
Monique
M
Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Yoshimoto
Momoko
M
Huang
Lan
L
Richardson
Matthew
M
Yoder
Mervin C
MC
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D004707:Endoderm / Q000166:cytology / Q000196:embryology; D050991:GATA4 Transcription Factor; D050993:GATA6 Transcription Factor; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D053823:Hedgehog Proteins / Q000096:biosynthesis* / Q000235:genetics; D006411:Hematopoiesis, Extramedullary / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics; D015017:Yolk Sac / Q000166:cytology / Q000196:embryology
D050991:GATA4 Transcription Factor; D050993:GATA6 Transcription Factor; D053823:Hedgehog Proteins; D042461:Vascular Endothelial Growth Factor A; C514061:ihh protein, mouse; C467480:vascular endothelial growth factor A, mouse