Figure 2

Notch signaling is active in neuroectodermal spheres derived from human embryonic stem cells. (A) Transcriptional expression of Notch signaling members in neuroectodermal spheres (NESs). All components functionally related with Notch signaling pathway were up-regulated in the NES samples. NOTCH4 was not expressed in all samples (data not shown). HEY1 and HEY2 were not preferentially expressed in the NESs, because they are regulated by Notch signaling but are associated with vascular development [30]. GAPDH, loading control. (B) Western-blot analyses of Notch signaling molecules in the NESs. All proteins of the Notch signaling pathway examined were expressed in hESCs, EBs and NESs but all were most abundant in the NES protein extracts. NICD, NOTCH Intracellular Domain (or cleaved Notch1). α-TUBULIN, loading control. (C) Immunostaining of the rosettes for the Notch ligands (JAG1 and DLL1) and receptor (NOTCH1). Rosettes structures derived from hESC-derived NESs were visualized using the rosette marker N-cadherin (N-CAD) and shown to locate to the inner rims of the rosettes. Both JAG1 (a and c) and DLL1 (b and d) transmembrane ligands are localized in the regions where N-cadherin signals exist. Anti-NOTCH1 antibody, which recognizes both NICD and C-terminal cytoplasmic domain of membrane-bound NOTCH1 receptor simultaneously, generates a rather diffused signal (c and d); nevertheless, its location at the luminal side of rosette is evident. Scale bars, 100 μm.