Vegf regulates embryonic erythroid development through Gata1 modulation

Abstract

To determine the role of vascular endothelial growth factor (Vegf) in embryonic erythroid development we have deleted or overexpressed Vegf specifically in the erythroid lineage using the EpoR-iCre transgenic line in combination with Cre/ loxP conditional gain and loss of function Vegf alleles. ROSA26 promoter-based expression of the Vegf 164 isoform in the early erythroid lineage resulted in a differentiation block of primitive erythroid progenitor (EryP) development and a partial block in definitive erythropoiesis between the erythroid burst-forming unit and erythroid colony-forming unit stages. Decreased mRNA expression levels of the key erythroid transcription factor Gata1 were causally linked to this phenotype. Conditional deletion of Vegf within the erythroid lineage was associated with increased Gata1 levels and increased erythroid differentiation. Expression of a ROSA26-based GATA2 transgene rescued Gata1 mRNA levels and target genes and restored erythroid differentiation in our IntroductionErythrocytes or red blood cells (RBCs) fulfill the essential functions of facilitating gas exchange in the lungs and meeting tissue oxygen demands. They are continuously renewed from undifferentiated, selfgenerating cells throughout life in a tightly controlled growth process termed erythropoiesis. This process is part of the larger hematopoietic program that is defined by the production of all blood cells from hematopoietic stem cells (HSCs). These cells originate from different embryonic sites during development and are generated in close association with the vascular endothelium. The first hematopoietic cells detected during mouse embryonic development are the primitive erythroid cells of the yolk sac (YS) that are produced from precursors present in this tissue between embryonic days E7.0 and E8.25 and enter the circulation at E9.0. The definitive progenitors, that contain cells from the YS and the aorta-gonado-mesonephros (AGM) region, 1 reach the liver at E10.5 and give rise to the first definitive progenitors which subsequently colonize the bone marrow and spleen in the adult mouse. 2 Vascular endothelial growth factor (Vegf) is a secreted growth factor that mediates its biologic effects predominately by binding to either one of two transmembrane tyrosine kinase receptors, Vegfr-1(Flt1) and Vegfr-2(Flk1 or Kdr) as well as to the coreceptor Neuropilin1 (Nrp1). VEGF/VEGFR signaling pathways are essential for vascular and hematopoietic cell development. Indeed, heterozygous Vegf (ϩ/Ϫ) deficient embryos are devoid of most endothelial and hematopoietic cells in the blood islands and die around E10 as a consequence of severe cardiovascular abnormalities. 3,4 Gain of function studies showed that ubiquitous inducible expression of VEGF during early development leads to a paucity of mature RBCs. 5 During erythropoiesis in the developing YS Vegf is expressed in the YS mesoderm and extraembryonic visceral endoderm. 6 Using diploid and tetraploid embryo complementation approaches in conjunction with a Vegf hypomorphic all...

BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity

“…Vegf (Miquerol et al, 1999) and Ihh (Dyer et al, 2001) are both expressed by the VE of mouse embryos. Gene targeting studies have established a requirement for VEGF in primitive erythropoiesis (Carmeliet et al, 1996; Ferrara et al, 1996) that is mediated, at least in part, through Gata1 (Drogat et al, 2010). IHH protein can substitute for VE in embryo explant cultures and may function through activation of Bmp4 (Dyer et al, 2001).…”

Beta-Actin Is Involved in Modulating Erythropoiesis during Development by Fine-Tuning Gata2 Expression Levels

“…Genotyping was done by fluorescent microscopy followed by a LacZ PCR. Actb +/− mice were crossed with EpoR-iCre Tg/+ [21] mice and with ROSA26-hGata2 Tg/Tg [21] to generate the Actb −/− R26+hGata2 EpoR-iCre/+ rescue mice. Genotyping was done by fluorescent microscopy followed by PCR.…”

BMP-mediated specification of the erythroid lineage suppresses endothelial development in blood island precursors

“…VBI explants expressed high levels of erythroid differentiation markers relative to VP explants (supplemental Figure 3C), consistent with their known lineage potential, and the presence of VEGF down-regulated erythroid marker expression, in agreement with previous studies. 24,25 In contrast, analysis of a range of EC differentiation markers showed no appreciable enrichment in the VBI explants relative to the negative control VP explants (supplemental Figure 3D). The presence of active VEGF did not result in up-regulation of endothelial marker expression.…”