A novel role for gamma-secretase in the formation of primitive streak-like intermediates from ES cells in culture.
gamma-Secretase is a membrane-associated protease with multiple intracellular targets, a number of which have been shown to influence embryonic development and embryonic stem (ES) cell differentiation. This paper describes the use of the gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) to evaluate the role of gamma-secretase in the differentiation of pluripotent stem cells to the germ lineages. The addition of DAPT did not prevent the formation of primitive ectoderm-like cells from ES cells in culture. In contrast, the addition of DAPT during primitive ectoderm-like cell differentiation interfered with the ability of both serum and BMP4 to induce a primitive streak-like intermediate and resulted in the preferential formation of neurectoderm. Similarly, DAPT reduced the formation of primitive streak-like intermediates from differentiating human ES cells; the culture conditions used resulted in a population enriched in human surface ectoderm. These data suggest that gamma-secretase may form part of the general pathway by which mesoderm is specified within the primitive streak. The addition of an E-cadherin neutralizing antibody was able to partially reverse the effect of DAPT, suggesting that DAPT may be preventing the formation of primitive streak-like intermediates and promoting neurectoderm differentiation by stabilizing E-cadherin and preventing its proteolysis.
Hughes
James N
JN
School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South Australia, 5005, Australia.
Dodge
Natasha
N
Rathjen
Peter D
PD
Rathjen
Joy
J
D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004151:Dipeptides / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D015398:Signal Transduction
C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D015820:Cadherins; D004151:Dipeptides; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; D011480:Protease Inhibitors; D053829:Amyloid Precursor Protein Secretases