Abstract
Definitive mesoderm arises from a bipotent mesendodermal population, and to study processes controlling its development at this stage, embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in beta-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context, we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively, EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation, EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition, the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context.
Highlights
Definitive mesoderm and endoderm both arise from the primitive streak during gastrulation
Blood islands consist of primitive hematopoietic blood cells surrounded by a layer of endothelial cells and support primitive hematopoiesis between embryonic days 7 and 14.5
When comparing the SHBϩ/Ϫ clone with the control and the SHBϪ/Ϫ clones on day 12 of differentiation, it was observed that tion of SHB, we performed targeted inactivation of embryonic stem (ES) cells the loss of one SHB allele resulted in a response that was very using the loxP-SHB construct (Fig. 1A)
Summary
Definitive mesoderm and endoderm both arise from the primitive streak during gastrulation. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. Expression of Mesodermal Markers in SHBϪ/Ϫ EBs—Previous studies have suggested an involvement of SHB in vascular formation [27].
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