The SHB adapter protein is required for normal maturation of mesoderm during in vitro differentiation of embryonic stem cells.
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.
Kriz
Vitezslav
V
Department of Medical Cell Biology, Uppsala University, Uppsala 75123, Sweden.
Agren
Nina
N
Lindholm
Cecilia K
CK
Lenell
Samuel
S
Saldeen
Johan
J
Mares
Jaroslav
J
Welsh
Michael
M
D000818:Animals; D001808:Blood Vessels / Q000378:metabolism / Q000473:pathology; D002454:Cell Differentiation; D003114:Colony-Forming Units Assay; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000166:cytology* / Q000378:metabolism; D005326:Fetal Proteins / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D018390:Gene Targeting; D006410:Hematopoiesis; D007124:Immunoenzyme Techniques; D019426:Integrases; D008648:Mesoderm / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020825:T-Box Domain Proteins / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D000098908:Brachyury Protein
D005326:Fetal Proteins; D019426:Integrases; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; D020825:T-Box Domain Proteins; D040301:Vascular Endothelial Growth Factor Receptor-2; D000098908:Brachyury Protein; C085634:Shb protein, mouse; C045073:Cre recombinase