Lineage-specific gene expression in CNC derivatives in vivo and in vitro. (a) Our strategy for conditional expression of a target gene using the dual-level expression system. Tissue or lineage specificity is provided by Cre-mediated excision of the loxP-flanked STOP sequence, and further temporal activation is controlled by Dox administration. The expression of rtTA in this system becomes independent of regulation by the tissue-specific promoter (TSP) after the excision of the loxP-flanked STOP sequence. The R26STOPrtTA gene activated by Cre remains active in all of the daughter cells even after Cre expression has been turned off. (b) E11.5 Wnt1-Cre/R26STOPrtTA/TRE-lacZ triple transgenic embryos, induced with Dox for 5 days (embryo, Upper Left) or without Dox induction (other three embryos) (β-gal staining for 2 h). (c) Enlargement of Dox-induced embryo from a, showing expression in derivatives of the Wnt1-expressing neural crest cells, including maxilla, mandible, cranial nerves, skull, eyes, skin, and salivary glands. (d-i) Sections of the β-gal-stained E11.5 embryo showing targeted gene expression in neural crest derivatives, including the lateral (LNP) and medial (MNP) nasal processes (d), the mandibular (Mn) and maxillary (Mx) components of branchial arch (e and f), the optic cup (f, Oc), the trigeminal (g, TG) and dorsal root ganglia (h, DRG), the dorsal neural tube (h, NT), the united ganglion of XI and X nerves (black arrowhead), the inferior ganglion of XI nerve (black arrow), and the branches of VII nerve (white arrowhead) (i). β-Gal staining of a triple transgenic E16.5 embryo (k and l) and 21-day-old mouse (n) showed that conditional gene expression could be manipulated in the CNC-derived skeletal elements of the developing mouse skull vault. Control mice without Dox induction revealed no expression of lacZ (j and m). f, frontal bone; ip, interparietal bone; n, nasal bone; nc, nasal cartilage; p, parietal bone. Sections of the β-gal-stained E16.5 embryos revealed that the lacZ reporter is expressed in the cranial sutures (o, arrow) and salivary glands (p). The neural crest-derived osteoblasts isolated from nasal and frontal bones were cultured in medium containing (r) or lacking (q) Dox for 16 h and analyzed by β-gal staining (at 37°C for 1 h) for inducible expression of the target gene. Absolutely no activation of the TRE-lacZ reporter (0%) was observed in cells without Dox. In contrast, 95% of the neural crest-derived osteoblasts exhibited nuclear β-gal staining upon Dox treatment. A similar expression pattern was observed in calvarial osteoblasts isolated from the newborn carrying Wnt1-Cre/R26RlacZ transgenes. No lacZ expression was detected in osteoblasts isolated from the parietal bone (s), whereas ≈95% of the cells were positive for β-gal staining (at 37°C for 2 h) in the nasal/frontal bone-derived osteoblasts (t). [Scale bars: 3 mm (b, m, and n); 1 mm (c); 100 μm (d-g and i); 200 μm (h, and o-t); and 2 mm (j-l).]