Figure 1.
(A) Targeting strategy for generating mice in which tamoxifen-inducible Cre is expressed from the endogenous Rax locus (Rax-CreERT2). The targeting vector containing a tamoxifen-inducible Cre (CreERT2) and pGK-neo cassette flanked with Frt sites was inserted six nucleotides downstream from the start of the initiator codon of the endogenous Rax locus. After homologous recombination, the correct 3′ integration was tested in embryonic stem cells (ES) by PCR using a forward primer (P2) inside the second intron of Rax gene and a reverse primer (P1) in the neo cassette generating a 3 kb fragment. The Rax-CreERT2-neo mice were later mated with FLPeR mice to remove the neo cassette. Frt, Flp recombinase targets. (B) Correct insertion was confirmed by Southern blot using a 5′ probe which hybridized upstream of the long arm of double digested (AfIII and SexAI) DNA from ES cells. Duplicate samples of heterozygous and wildtype mice are shown. (C) Schema for Rax-regulated Cre-mediated Tdtomato expression induced by 4-OHT.
Figure 2.
Tdtomato reporter expression is seen in neural progenitors of the ventral forebrain and retina in Rax-CreERT2;Ai9 mice following 4-OHT induction at E8.5.
(A) Diagram of experimental schematic. E8.5 timed pregnant mice received 1.5 mg of 4-OHT through oral gavage and were sacrificed at E10.5. (B) Anatomical structure of sagittal section. lv, lateral ventricle; di, diencephalon; 3v, third ventricle; PTh, prethalamus; CPa, caudal paraventricular nucleus area; Tu, tuberal region; Ahy, anterior hypothalamus. (C) Coronal sections corresponding to the line in (B). (D and E) Sagittal sections of Rax-CreERT2;Ai9 mice. vfb, ventral forebrain; mv, mesencephalic vesicle; 4v, fourth ventricle. (F and G) High power images of (C) and (E) respectively. (H) Coronal view of retina. OC, optic cup; LP, lens pit. Tdtomato signal was boosted with anti-DsRed immunolabeling. Red fluorescence exhibited mosaic pattern limited to ventral regions of the telencephalon and diencephalon (C–G), and as robust expression was detected in neuroretina (H). Sections were counterstained with DAPI (blue). Scale bars: 100 µm (C, F–H), 200 µm (D, E).
Table 1.
Summary of 4-OHT experiments described in the study.
Figure 3.
Tdtomato reporter expression is seen in neural progenitors of the ventral forebrain and retina in Rax-CreERT2;Ai9 mice following 4-OHT induction at E10.5.
E10.5 pregnant mice received 1.5-OHT through oral gavage and were sacrificed at E12.5. (A–C) Coronal views of the anterior, medial and posterior hypothalamus, respectively. Tdtomato positive cells, visualized by anti-DsRed immunolabeling, co-express the neuronal markers NeuN and Hu (A, dashed square), as well as the neural progenitor marker vimentin (Vim) (B). A region of ventral hypothalamic neuroepithelium by the dashed square is enlarged to show the colocalization with neuronal markers in more detail (A, bottom left panel). A white asterisk indicates the dorsal hypothalamic domain of Tdtomato expression. (D and E) Coronal view of posterior pituitary and retina. Arrowheads indicate coexpression of Tdtomato with vimentin in optic nerve (ON), which contains axons of retinal ganglion cells (RGCs), an early-born cell type. 3V, third ventricle; PP, posterior pituitary; NR, neural retina; L, lens. Sections were counterstained with DAPI (blue). Scale bars: 100 µm.
Figure 4.
Tdtomato reporter expression is restricted to hypothalamus and retina in Rax-CreERT2;Ai9 mice following 4-OHT induction at E12.5.
E12.5 pregnant mice received 1.5-OHT orally through gavage and were sacrificed at E14.5. (A) Sagittal view represents the specific induction of reporter gene within the ventral hypothalamic region. LV, lateral ventricle; Th, thalamus; Hy, hypothalamus; PP, posterior pituitary; 4V, fourth ventricle; ON, optic nerve; DAhy, dorsal anterior hypothalamus. (B–D) Coronal views of the anterior, medial and posterior hypothalamus, respectively. Tdtomato expression was higher in the medial hypothalamus than either anterior or posterior and overlapped with vimentin signal along the third ventricle (3V) (C). (E) Coronal section of the retina. The weak red fluorescence observed in the hyloid vessel and in periocular tissues represents background staining, with inset showing a wildtype control eye for comparison. Red fluorescence was detected in the RGCs and their axonal projection toward the optic nerve (ON). NR, neural retina; L, lens. Sections were counterstained with DAPI (blue). Scale bars: 500 µm (A), 200 µm (B–E).
Figure 5.
Tdtomato reporter expression is restricted to radial glia-like cells in hypothalamus, posterior pituitary, and specific retinal cell subtypes following 4-OHT induction at E16.5.
E14.5 pregnant mice received 1.5-OHT orally through gavage and were sacrificed at E18.5. (A–D) Coronal views of the anterior, medial, posterior hypothalamus and retina, respectively. Reporter expression was highly limited to the radial glial cells labeled with vimentin along the third ventricular wall (A–C and E, white arrows). 3V, third ventricle; ME, median eminence; PP, posterior pituitary; NR, neural retina; ONH, optic nerve head; L, lens. (E–G) High power confocal images of hypothalamus (E) and central retina (F,G). A small number of parenchymal cells were colocalized with vimentin (E, white arrows), GFAP (F, white arrows) and Olig2 (G, white arrows) in hypothalamus (Hy). A small set of Tdtomato-positive cells in retina were colocalized with GFAP (I) and Olig2 (J) in GCL and ONBL respectively, but not with NF165 (H). IL, intermediate lobe; ONBL, outer neuroblastic layer; INBL, inner neuroblastic layer; GCL, ganglion cell layer. Sections were counterstained with DAPI (blue). Scale bars: 200 µm.
Figure 6.
Tdtomato reporter expression in hypothalamus following postnatal 4-OHT induction in Rax-CreERT2;Ai9 mice.
Rax-CreERT2;Ai9 mice received a single dose of 4-OHT (1.0 mg,1.5 mg and 2 mg, respectively) by intraperiotoneal injection (i.p) at three different time points, P7 (A–C), P28 (D–F) and P50(G–I), and were analyzed 2(A–C) or 3 (D–I) days following injection. (J) Representative confocal tile scan images of triple immunolabeling shows co-expression of DsRed, Sox2, and vimentin in the hypothalamic tanycytes along the third ventricular wall. AH, anterior hypothalamus; 3V, third ventricle; ArcN, arcuate nucleus; DMH, dorsomedial hypothalamus; VMH, ventromedial hypothalamus; ME, median eminence; PH, posterior hypothalamus; PMv, ventral premammillary nucleus. Sections were counterstained with DAPI (blue). Scale bars: 200 µm. (K) Efficiency of reporter induction in hypothalamic tanycytes in the indicated third ventricular region following a single 4-OHT injection at P7 and at P28, quantifying the data shown in 6A–C and 6D–F. A total of 2226 DsRed-positive and 7629 DAPI-positive cells were counted for this analysis, which was obtained by analyzing a total of 4 sections each from 4 Rax-CreERT2;Ai9 mice for each condition tested. Standard deviation is shown. Two-way ANOVA analysis did not reveal any significant (p<0.05) differences in efficiency of reporter induction among tanycytes in any of the four different zones indicated.
Figure 7.
Specificity and efficiency of induction of TdTomato reporter in hypothalamus following postnatal 4-OHT induction in Rax-CreERT2;Ai9 mice.
(A–C) Comparison of hypothalamic reporter expression between uninduced oil-treated control (right panels) and 4-OHT induced mice (left panels). P18 Rax-CreERT2;Ai9 mice received i.p. injections daily with either oil vehicle or 4-OHT (0.3 mg at P18–20 and 1.0 mg at P21–22) for 5 days and were analyzed at P23. Control mouse brains did not have morphological defects and did not show any ectopic reporter expression in the anterior (A), medial (B) or posterior hypothalamus (C). (D–E) Efficiency of induction with a single dose (1.0 mg of 4-OHT) at P4 (D) or three daily doses (0.3 mg of 4-OHT) each at P5, P7, and P11 (E). Multiple injections resulted in enhanced induction compared to single injection. Sections were counterstained with DAPI (blue). Scale bars: 200 µm (F).
Figure 8.
Tdtomato reporter expression in retina following postnatal 4-OHT induction in Rax-CreERT2;Ai9 mice.
Rax-CreERT2;Ai9 mice received 4-OHT (1.0 mg,1.5 mg and 2 mg, respectively) i.p at three different time points, P7 (A,F), P28 (B,D,E,G), and P50(C,H), and their neural retinas (A–E) and ciliary margins (F–G) were analyzed after 2(A,F) or 3 (B–E, G–H) days. A single i.p. injection successfully induced reporter expression in radial Müller glia in all three different postnatal stages tested, particularly at P31. Tdtomato co-labeled with the Müller glia markers GS (D) and P27 (E). Interestingly, reporter expression was also observed in the non-pigmented zone of the ciliary margin at all indicated postnatal stages (F–H). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; PE, pigmented epithelium; CB, ciliary body. Sections were counterstained with DAPI (blue). Scale bars: 200 µm.
Figure 9.
Tdtomato reporter expression in posterior pituitary and cerebellum following postnatal 4-OHT induction in Rax-CreERT2;Ai9 mice.
(A–H) Rax-CreERT2;Ai9 double heterozygote mice received 1.5 mg of 4-OHT at P28 and their pituitary glands were analyzed 3 days later. Coronal (A) and sagittal (B) sections stained with DsRed. Reporter expression was restricted in only pars nervosa (PN). PI, pars intermediate; PD, pars distalis. (C–F and G–H) High power confocal images of (A) and (B), respectively. A subset of Tdtomato-positive cells colocalized with vimentin (C, F), but not with GFAP or NF165. (I) Rax-CreERT2;Ai9 double heterozygote mice received 0.3 mg of 4-OHT three times at P5, P7, and P9, and their cerebellums were analyzed at P15. Reporter-positive cells were prominently robust in the granular layer (GL). (J) Rax mRNA in situ hybridization signal in cerebellum at P4.5 (data obtained from the Allen Brain Atlas), with the flocculus indicated by a black arrowhead. Sections were counterstained with DAPI (blue). ML, molecular layer. Scale bars: 200 µm (A–H), 400 µm (J).