Cre-mediated recombination in the Wnt1-expressing neural precursors is induced by tetracycline-dependent activation. (A) A diagram illustrates our strategy for spatiotemporal specific-recombination in the Wnt1-expressing cells. Expression of rtTA in the Wnt1-expressing cells permits the Dox-inducible activation of TRE-Cre. The Cre-mediated recombination, which deletes DNA sequences flanked by two loxP sites, occurs in the Wnt1-expressing neural precursors, thereby achieving either gene expression or ablation. Note that the conditional allele, containing the loxP site flanking sequence, could be a gene specific floxed allele (for conditional gene ablation) or an allele permitting target gene expression upon Cre-mediated recombination (for conditional gene expression). (B, C) Embryos carrying Wnt1-rtTA and TRE-Cre transgenes (double transgenics, right; control: carrying Wnt1-rtTA, left) enable conditional excision of the sequence flanked by two loxP sites upon Dox treatment, leading to the expression of a lacZ reporter (R26RlacZ). The expression of Cre is stimulated by Dox at E6.5 (D–F), E9.5 (I–K) and E10.5 (L–N), followed by harvesting of embryos at E13.5. Scale bars, 2 mm (B, C); 1 mm (D–N).

Inducible gene expression is targeted to the Wnt1-expressing cells and their derivatives using Wnt1-rtTA and TRE-Cre transgenes. Sections of the β-gal stained embryo shown in Figure 2D reveal that the target gene expression is stimulated in the Wnt1-expressing precursors and their derivatives, including those in hindbrain (A, D), midbrain (B, C), neural tube (E), trigeminal nerve (F) and ganglion (G), dorsal root ganglion (H), exoccipital bone and vagus nerve (I), nasal septum (J), nasal capsule (K), ear (M, N), eye (L), molar (O), follicle (P), thymic rudiment (Q), heart (R). CP, choroid plexus; co, cochlea; di, diencephalon; DRG, dorsal root ganglion; e, ear; el, eyelid; EB, exoccipital bone; F, follicle; met, metencephalon; my, myelencephalon; M, molar; MEO, middle ear ossicle; NC, nasal cavity; NS, nasal septum; NT, neural tube; OM, ocular muscle; ON, optic nerve; RA, right atrium; T, thymic rudiment; TG, trigeminal ganglion; TN, trigeminal nerve; VN, vagus nerve. Scale bars, 1 mm (A); 300 μm (B–D, G, J, K); 150 μm (E, F, H, I, L, R); 200 μm (M, N); 75 μm (O–Q).

Schematic representation illustrates the use of transgenic systems for manipulating gene activity in neural precursors and CNC cells. (A) Neural crest cells arise at the lateral margin of neural fold at the boundary between surface (SE) and neural (NE) ectoderms. They migrate ventrolaterally to populate the head and neck regions upon closure of the neural tube. (B) The Wnt1-rtTA strain expresses rtTA only in the pre-migratory precursors (red cells) in dorsal midline because the Wnt1 regulatory element is not active after emigration of these cells (blue cells). (C) The Cre-mediated recombination, permitting deletion of the loxP-flanked STOP sequence, activates the expression of rtTA under control of ROSA26 promoter in both neural crest precursors and their derivatives in the neural tube and craniofacial primordium, respectively. After the Cre-mediated expression, rtTA is constitutively expressed in the Wnt1-expressing cells and their derivatives, independent of the activity of the Wnt1 regulatory elements. (D) The inducible expression domain of Cre in the Wnt1-rtTA/TRE-Cre system varies by the timing of Dox administration. The presence of Dox prior to the migration of neural crest precursors enables the Cre-mediated recombination to occur in both the dorsal midline and craniofacial regions (Stage 1). When Dox is administered during the migration of CNC, only the cells leaving late will be activated (Stage 2). Cre would not be present in the derivatives of CNC if Dox is added after they have emigrated (Stage 3). Pre-migratory and post-migratory cells are shown in red and blue, respectively. Cells permitting the manipulation of gene activity are highlighted in green.

Wnt1-rtTA mouse strain permits targeted gene expression in Wnt1-expressing neural precursors. (A) A schematic representation of the tetracycline-dependent gene activation by Wnt1-rtTA mouse strain. The Wnt1 promoter (P) and enhancer (E) direct the expression of rtTA in the Wnt1-expressing precursors. The presence of Dox induces the binding of rtTA to TRE, activating the reporter (lacZ or GFP) expression. (B) Embryos carrying Wnt1-rtTA and TRE-lacZ transgenes (double transgenics, right; control, left) enable conditional expression of the lacZ gene in the Wnt1-expressing cells of developing brain and neural tube. Embryos were recovered at E12.5 and analyzed by β-gal staining in whole mounts (B–H) and sections (M–T). The lacZ reporter is highly stimulated in the dorsal midline of developing neural tube and mid/hind brain upon Dox induction (B–D, M–T). Inducible expression of the lacZ reporter is detected in the double transgenic embryos with 7 days (E, G) or 17 hours (F, H) of Dox treatment. (I–L) Whole mount fluorescent analysis revealed an inducible expression of the GFP reporter in double transgenic embryos for Wnt1-rtTA and TRE-H2BGFP at E13.5. The GFP reporter is expressed in the double transgenic embryos with 7 days (I, J), 17 hours (K) or 12 hours (L) of Dox treatment. Enlargements of the insets in panels B and M are shown in C, D, N, O, P. Insets exhibit enlargement of the stained regions (R–T). CP, choroid plexus; di, diencephalon; Hy, hypothalamus; met, metencephalon; my, myelencephalon; NT, neural tube; Th, thalamus. Scale bars, 2 mm (B, C, I); 1 mm (D, E–H, J–L); 0.5 mm (M, R–T); 0.1 mm (N–Q).

Neural crest lineage-specific gene activation in an inducible fashion. (A) A scheme represents a strategy for manipulating gene expression in neural crest lineage. Neural crest 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 the Wnt1-expressing cells is controlled by the Cre-mediated excision of the loxP-flanked STOP sequence. The rtTA gene, expressed in all Wnt1-expressing cells and their descendants, enables inducible gene expression by the tetracycline-dependent activation system. (B) Whole mount GFP analysis of the E12.5 control (R26STOPrtTA; TRE-H2BGFP double transgenics, left) and Wnt1-Cre; R26STOPrtTA; TRE-H2BGFP triple transgenic embryos (right), with Dox treatment. (C–E) Inducible expression of the H2BGFP reporter in the triple transgenic embryos with varying lengths of Dox treatment (C, 7d; D, 17h; E, 10h). (F) Enlargement of the Dox-induced embryo from panel B, showing targeted gene expression in the Wnt1-expressing neural precursors (mid/hind brain) and their derivatives/neural crest cells (craniofacial regions). (G) Dorsal view of embryos exhibits the GFP expression in the developing neural tube of the triple transgenic animal (upper), but not the control (R26STOPrtTA; TRE-H2BGFP double transgenics, lower). (H) Enlargement of the GFP positive embryo displays target gene expression in the neural tube (arrowheads) and neural crest derivatives, dorsal root ganglions (arrows). (I–N) Sections of the GFP positive E13.5 embryos show conditional gene expression in developing neural tube as well as neural crest derivatives, facial processes. The reporter is highly expressed in developing mid/hind brain, including mesencephalon (mes), myelencephalon (my), diencephalon (di) and cerebellum (cb), as well as neural crest derived craniofacial mesenchyme, including maxillary (mx) and mandibular (mn) prominences. Enlargements of the inset in panels I, K and M are shown in J, L and N. An inset displays enlargement of the dorsal root ganglion (DRG) regions in panel K. (O) Whole mount and (P) GFP analysis reveals targeted expression of the H2BGFP reporter in the neural crest-derived craniofacial region of the triple transgenic newborn (right), but not the control (left), with Dox treatment. (Q) GFP analysis of the skull indicates the transgenic expression in the skeletal elements, including the nasal cartilage (nc), nasal bone (n), frontal bone (f) and interparietal (ip) bone, but not in mesoderm-derived parietal bone (p). Scale bars, 2 mm (B, G, O–Q); 1 mm (F, H, I, K, M); 0.5 mm (J, L, N).