RBPjkappa-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells.
Definitive hematopoiesis in the mouse embryo originates from the aortic floor in the P-Sp/AGM region in close association with endothelial cells. An important role for Notch1 in the control of hematopoietic ontogeny has been recently established, although its mechanism of action is poorly understood. Here, we show detailed analysis of Notch family gene expression in the aorta endothelium between embryonic day (E) 9.5 and E10.5. Since Notch requires binding to RBPjkappa transcription factor to activate transcription, we analyzed the aorta of the para-aortic splanchnopleura/AGM in RBPjkappa mutant embryos. We found specific patterns of expression of Notch receptors, ligands and Hes genes that were lost in RBPjkappa mutants. Analysis of these mutants revealed the absence of hematopoietic progenitors, accompanied by the lack of expression of the hematopoietic transcription factors Aml1/Runx1, Gata2 and Scl/Tal1. We show that in wild-type embryos, a few cells lining the aorta endothelium at E9.5 simultaneously expressed Notch1 and Gata2, and demonstrate by chromatin immunoprecipitation that Notch1 specifically associated with the Gata2 promoter in E9.5 wild-type embryos and 32D myeloid cells, an interaction lost in RBPjkappamutants. Consistent with a role for Notch1 in regulating Gata2, we observe increased expression of this gene in 32D cells expressing activated Notch1. Taken together, these data strongly suggest that activation of Gata2 expression by Notch1/RBPjkappa is a crucial event for the onset of definitive hematopoiesis in the embryo.
Robert-Moreno
Alex
A
Centre Oncologia Molecular, IDIBELL-Institut de Recerca Oncologica, Hospitalet, Barcelona 08907, Spain.
Espinosa
Lluís
L
de la Pompa
José Luis
JL
Bigas
Anna
A
D000818:Animals; D001011:Aorta / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D004730:Endothelium, Vascular / Q000378:metabolism; D005434:Flow Cytometry; D050989:GATA2 Transcription Factor; D018507:Gene Expression Regulation, Developmental; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D017403:In Situ Hybridization; D008024:Ligands; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051881:Receptor, Notch1; D051883:Receptor, Notch2; D000074562:Receptor, Notch4; D011956:Receptors, Cell Surface / Q000378:metabolism*; D051880:Receptors, Notch; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D050989:GATA2 Transcription Factor; C494712:Gata2 protein, mouse; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D008024:Ligands; C106075:NOTCH4 protein, human; C497762:Notch1 protein, mouse; C497765:Notch2 protein, mouse; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C497571:Rbpj protein, mouse; D051881:Receptor, Notch1; D051883:Receptor, Notch2; D000074562:Receptor, Notch4; D011956:Receptors, Cell Surface; D051880:Receptors, Notch; D014157:Transcription Factors