Fig. 6.

Ectopic induction of pluripotency factors in the PS/tail bud. a Diagram showing the components of the Dox-inducible pluripotency factor expression cassette targeted to the Hprt locus. Numbers indicate Hprt exons. TetOP, tet operator sequences; P, promoter. b Venus expression in the tail bud of an E10.5 wt a Tps/tb-Oct-Nanog-Venus chimeric embryo treated with Dox at E8.5. c Nanog and Venus expression in the tail bud of an E10.5 wt a Tps/tb-Oct-Nanog-Venus chimeric embryo treated with Dox at E8.5 following immunostaining with a anti-Nanog antibody. d Experimental strategy employed for the derivation of tail bud EpiSC explants from Tps/tb-Oct-Nanog-Venus chimeric embryos. e qPCR expression analysis of Oct4, Nanog and endogenous Nanog expression in Dox-treated, untreated and wild type (wt) EpiSC explants as well as ES cells. For detection of endogenous Nanog expression a set of primers complementary to the Nanog 5’UTR region were employed as previously described [34]. Expression is shown as relative to TBP. Error bars represent s.d. f Morphology of Tps/tb-Oct-Nanog-Venus chimeric embryos and P7 pups after continuous Dox treatment starting at E8.5. A wild type embryo and an untreated chimeric pup are also shown as controls. The arrowhead in the E14.5 embryo image indicates exencephaly. Far right: skeletal preps of a wild type and a chimeric P7 pup. The arrowhead indicates a kink in the tail of the chimeric embryo. g Summary of the phenotypes observed in Tps/tb-Oct-Nanog-Venus chimeric embryos after continuous Dox treatment starting at E8.5. These correspond to each of the chimeric embryos recorded in the second column of the table with the exception of the E10.5 chimeras where the same 4/6 chimeric embryos exhibited the phenotypes noted in the panel and the remaining two chimeric embryos appeared normal. The E9.5 chimeric embryo exhibited endogenous Nanog reactivation following explantation in EpiSC conditions. In all cases Doxycycline administration started at E8.5