Figure 1.

Chondrogenic ES cell differentiation is induced by transient Notch1 signaling. (a) Conditional activation of the constitutive active Notch1 intracellular domain (NICD) using the tamoxifen-inducible system. In the absence of tamoxifen (OHT), Notch signaling is inactive in cells stably expressing a fusion protein consisting of NICD and the hormone-binding part of the tamoxifen-sensitive human estrogen receptor (ERT) because the fusion protein NERT is kept in the cytoplasm by binding of HSP90 to the ERT part of the protein. Upon addition of OHT, the NERT fusion protein translocates to the nucleus and transactivates Notch1 target genes by binding to the transcriptional repressor RBP-J and converting it to a transcriptional activator. An animated version of the inducible Notch1 system can be found in the Supplementary Material Video 1. (b) NERT ESCs were differentiated in vitro as EBs and Notch1 signaling was activated by the addition of OHT during 0–2 days, 2–5 days or continuously from day 2 during EB culture. EBs were analyzed for the formation of cartilage nodules by Alcian blue staining. Transient Notch1 activation induced chondrogenic differentiation at 25–34 days after plating of the 5-day-old EBs (5+25 to 5+34 days). In contrast, continuous Notch1 activation (2–5+34 days) completely inhibited chondrogenic differentiation. The error bars indicate S.E.M. from three independent experiments. Two-way ANOVA revealed highly significant interactions and variabilities with P-values <0.0001. Significant differences determined by Bonferroni's post-test in comparison with the uninduced (control) NERT EBs (black asterisk) and with the continuously OHT-induced NERT EBs (green asterisk) are ^**P<0.01, ^***P<0.001. (c) Immunostaining for collagen type II confirmed the results. Collagen type II-positive nodules were detected during in vitro differentiation of NERT ES cells after early Notch1 activation (0–2 days OHT, 2–5 days OHT) and in the control from 28 up to 41 days after EB plating (5+(28−41) days). In contrast, only collagen type II-positive fibers but no nodules appeared after continuous Notch1 activation in EBs from 2 days before plating up to 41 days after EB plating (2 days to 5+41 days). Bar=100 μm