Dorsal telencephalon-specific expression of Cre recombinase in PAC transgenic mice

© 2004 Wiley-Liss, Inc.

genesis 38:130 –138 (2004)

TECHNOLOGY REPORT

Dorsal Telencephalon-Specific Expression of Cre Recombinase in PAC Transgenic Mice Takuji Iwasato,1,2* Ryochi Nomura,2 Reiko Ando,2,4 Toshio Ikeda,2 Mika Tanaka,2,3 and Shigeyoshi Itohara2* 1

PRESTO, Japan Science and Technology Agency, Saitama, Japan Laboratory for Behavioral Genetics, Brain Science Institute (BSI), Riken Saitama, Japan 3 Research Resources Center, Brain Science Institute (BSI), Riken Saitama, Japan 4 Brain Science and Life Technology Research Foundation, Tokyo, Japan 2

Received 5 August 2003; Accepted 3 November 2003

Summary: The ability to restrict gene expression or disruption to specific regions of the brain would enhance understanding of the molecular basis for brain development and function. For this purpose, brain region-restricted promoters are essential. Here we report the isolation of a DNA fragment containing the Emx1 gene promoter, which is responsible for dorsal telencephalon-specific expression. The Cre recombinase gene was inserted into a mouse PAC (P1-derived artificial chromosome) Emx1-locus clone (PAC-Emx1#1 clone) and utilized to generate three transgenic mouse lines. In all three lines, especially Tg3, Cre-mediated recombination was highly restricted to Emx1-expressing cell lineages, from embryonic stages to adulthood. Immunohistochemical analyses showed that Cre protein is expressed in the dorsal telencephalon in all three lines in adulthood. Thus, the PAC-Emx1#1 clone contains essentially all regulatory elements necessary for Emx1 gene expression. Our results suggest that Emx1-Cre Tg3 mice and the PAC-Emx1#1 clone constitute powerful tools for dorsal telencephalon-specific gene manipulation. genesis 38:130 –138, 2004. © 2004 Wiley-Liss, Inc. Key words: P1-derived artificial chromosome; Cre/loxP recombination; Emx1 promoter; central nervous system

The cerebral cortex is a region of the brain that is important for higher brain functions. Its development and function depend on its connectivity with other regions of the brain (Iwasato et al., 1997; Schlaggar and O’Leary, 1991; Sharma et al., 2000). Therefore, the ability to manipulate genes in a cerebral cortex-restricted manner may be necessary to dissect out the molecular and cellular mechanisms running in the cerebral cortex during cortical development and operation from those running in other brain regions, including the striatum, thalamus, and brainstem. Transgenic mice are a powerful model system for studying the molecular mechanisms of brain function and development specific to particular regions of the brain and/or cell-types (Huang et al., 1999; Ichise et al.,

2000). Proper study of these mechanisms requires the use of region- and/or cell type-specific promoters, such as the CaMKII promoter specific to excitatory neurons in the postnatal forebrain (Mayford et al., 1995) and the L7 promoter specific to the cerebellar Purkinje neurons (Oberdick et al., 1990). Since no promoter has been available for the dorsal telencephalon, however, the ability to utilize transgenic mice to study the development or function of this region of the brain has been limited. The promoter of Emx1, a homeobox gene, is an ideal candidate for the purpose, because its expression is restricted to the dorsal telencephalon from embryonic stages to adulthood (Gulisano et al., 1996; Simeone et al., 1992). To isolate a DNA fragment containing transcriptional regulatory elements for the Emx1 gene expression, we examined a mouse PAC Emx1-locus clone (PACEmx1#1) (Fig. 1). The Cre recombinase gene was inserted into the PAC-Emx1#1 clone and utilized to generate three transgenic mouse lines (Tg1, Tg2, and Tg3). Cre recombinase is a unique reporter of promoter activity. Using Cre/loxP recombination reporters, such as CAG-CAT-Z (Sakai and Miyazaki, 1997) and R26R mice (Soriano, 1999), cell lineages in which the promoter has been activated transiently or permanently can be detected. In addition, immunostaining with an anti-Cre antibody can detect cells in which the promoter is active

* Correspondence to: Takuji Iwasato, BSI, Riken, 2-1 Hirosawa Wako-shi, Saitama 351-0198, Japan. E-mail: [email protected] or Shigeyoshi Itohara, BSI, Riken, 2-1 Hirosawa Wako-shi, Saitama 351-0198, Japan. E-mail: [email protected] Contract grant sponsors: Grant-in-Aid for Scientific Research-C, Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Priority Areas-A, Ministry of Education, Culture, Sports, Science, and Technology (to T.I.).

DOI: 10.1002/gene.20009

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FIG. 1. PAC modification and generation of Emx1-Cre transgenic mice. A: The transgenic construct. A nuclear localization signal (NLS)-Cre-poly (A) signal (pA) cassette was inserted, in the sense orientation, immediately 5⬘ to the Emx1 translational initiation site (ATG) into the PAC-Emx1#1 clone. B: Restriction enzyme digestions of PAC-Emx1#1 and two independent transgenic constructs (PAC-Cre-A and -B). Arrowheads show bands whose sizes were expected to be shifted by homologous recombination in bacteria. Other bands of PACEmx1-Cre#1, PAC-Cre-A, and PAC-Cre-B had similar sizes, suggesting no major deletion during homologous recombination in the bacteria. C: Pulsed-field gel electrophoresis showing that the linearized transgenic construct (PAC-Cre-A) was intact just prior to injection into pronuclei.

at particular developmental stages (Nakazawa et al., 2002). To characterize the patterns of Cre/loxP recombination (the promoter activity in cell lineages), we crossed each of our three transgenic lines with CAG-CAT-Z recombination-reporter mice to obtain double transgenic (Emx1-Cre Tg/CAG-CAT-Z) mice. Brains of adult mice (up to 18-month-old) were removed and thick (400 ␮m) coronal sections from the olfactory bulb to the anterior spinal cord were stained for ␤-galactosidase activity (Figs. 2, 3). This strategy allowed us to examine all brain regions for Cre-mediated recombination with high sensitivity. We compared the staining intensity in Tg/CAGCAT-Z mice with that in CAG-⌬-Z-positive control mice (see Materials and Methods). We detected dense LacZ staining, indicative of many cells with Cre-mediated recombination, in the olfactory bulb, neocortex, piriform cortex, hippocampus, and amygdala in each of Tg3/ CAG-CAT-Z mice (n ⫽ 4). In contrast, there were no LacZ-positive cells in other regions of the brain, including the striatum, thalamus, hypothalamus, brainstem, cerebellum, and spinal cord. Tg1/CAG-CAT-Z (n ⫽ 4)

and Tg2/CAG-CAT-Z (n ⫽ 2) mice showed staining patterns similar to those of Tg3/CAG-CAT-Z mice, although several differences were also observed. To determine whether there are developmental changes in recombination patterns, we examined Tg/ CAG-CAT-Z mice at postnatal days (P)7– 8 by X-gal staining of coronal or parasagittal slices (400 ␮m-thick) (data not shown). At this stage of development all Tg1/CAGCAT-Z (n ⫽ 1), Tg2/CAG-CAT-Z (n ⫽ 5), and Tg3/CAGCAT-Z (n ⫽ 4) mice showed strong staining in the dorsal telencephalon. In the Tg1/CAG-CAT-Z brain, we observed staining in the inferior colliculus. In the Tg2/CAGCAT-Z brain, staining of the olfactory bulb was weak and staining of the thalamus and cerebellum was not detected yet. Using whole-mount X-gal staining, we examined Tg3/CAG-CAT-Z embryos at embryonic days (E)8.5–17 (n ⫽ 14) and found that Cre-mediated recombination in the dorsal telencephalon was first detectable at E10 (Fig. 4). These results were similar to those for onset of endogenous Emx1 expression (Simeone et al., 1992).

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FIG. 2. Dorsal telencephalon-specific Cre-mediated recombination in transgenic mouse brains. Cre-mediated recombination was assayed by X-gal staining of 400-␮m-thick coronal sections of adult Tg1/CAG-CAT-Z (Tg1), Tg2/CAG-CAT-Z (Tg2), Tg3/CAG-CAT-Z (Tg3), knockin/CAG-CAT-Z (KI), CAG-⌬-Z, and CAG-CAT-Z mice. A–C: All Tg1, Tg2, and Tg3 mice showed dense staining, indicative of recombination, in the neocortex (cx), piriform cortex (pir), and hippocampus (hip). Tg1 and Tg3 amygdala were stained in some compartments, such as the basolateral complex (BL), while others, such as the MePD nuclei, were not stained. In Tg2, the amygdala showed much weaker staining than the other two transgenic lines, whereas there were a few and some positive cells in the thalamus (th) (arrowhead) and cerebellar granule cell layer (data not shown), respectively. D: In KI mice, there were some positive cells in the thalamus, cerebellum, brainstem, etc. (data not shown). E: In CAG-⌬-Z mice (positive control, PC), all areas of the brain were LacZ-positive. F: In CAG-CAT-Z (negative control, NC) mice, there was no staining in any area of the brain. str, striatum; ht, hypothalamus; fi, fimbria. Scale bar ⫽ 2 mm.

To determine the recombination specificities outside the brain, we dissected various body parts from Tg3/ CAG-CAT-Z mice (n ⫽ 2) at P7 and stained them for X-gal (data not shown). We detected various staining intensities in the thymus, stomach, testis, and some regions of the skin, including the limbs and tail. In contrast, there was no staining of the heart, lung, liver, spleen, muscle, or bladder. In CAG-⌬-Z-positive control mice at P7, all of these organs were stained, suggesting that CAG-CAT-Z reporter system is suitable for detecting recombination in these tissues. We further found that some (not all) cells in germline had Cre-mediated recombination in Tg3 male (data not shown). Recombination outside the brain was also detected in Emx1-Cre knockin (KI) mice (Iwasato et al., 2000), although these animals were not analyzed extensively. To characterize the recombination patterns at the cellular level, thin (8 –10 ␮m) coronal brain slices of Tg1/

CAG-CAT-Z (n ⫽ 6), Tg2/CAG-CAT-Z (n ⫽ 7), and Tg3/ CAG-CAT-Z (n ⫽ 6) mice (P7 to 13-month-old) were analyzed. X-gal/anti-GABA-antibody double and X-gal/anti-GABA-antibody/Nissl triple staining revealed that, in the neocortex and hippocampus of all three lines of mice, most GABA-positive (inhibitory) neurons were LacZ-negative, whereas most GABA-negative (excitatory) neurons were LacZ-positive (Fig. 5). Thus, throughout the life span of all three lines Cre-mediated recombination is specific to excitatory neurons. These results are consistent with those of Emx1-Cre KI mice (Fig. 5D) (Gorski et al., 2002; Iwasato et al., 2000), as well as with the cell type specificities of endogenous Emx1 expression (Chan et al., 2001). To determine whether Cre-mediated recombination occurred in astrocytes, we stained brain slices of Tg1/ CAG-CAT-Z and Tg3/CAG-CAT-Z mice with X-gal and an anti-GFAP antibody. In both transgenic lines, most GFAP-

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FIG. 3. X-gal staining of coronal sections (400-␮m-thick) of Tg3/CAG-CAT-Z (18-month-old) (A), and Tg1/CAG-CAT-Z (14-month-old) (B) mice. In Tg1/CAG-CAT-Z mice, staining was detected in the mammillary nuclei (mn) of the hypothalamus, in the pontine nuclei (pn), and in the inferior colliculus (ic). Ob, olfactory bulb; str, striatum; sp, septum; cx, neocortex; pir, piriform cortex; hip, hippocampus; am, amygdala; cc, corpus callosum; th, thalamus; cb, cerebellum; bs, brainstem; sc, spinal cord. Scale bar ⫽ 2 mm.

positive astrocytes in the dentate gyrus (DG) molecular layer and in neocortex layer I were LacZ-positive (data not shown). Thus, in addition to excitatory neurons, astrocytes (GFAP-positive cells) showed Cre-mediated recombination in the dorsal telencephalon of Emx1-Cre transgenic mice. This finding is consistent with the re-

sults with our (data not shown) and others’ (Gorski et al., 2002) Emx1-Cre KI mice. Furthermore, we utilized R26R (Soriano, 1999), another recombination-reporter mouse line, to determine the reproducibility of the tight regulation of Cre-mediated recombination observed in Tg3 mice. In Tg3/R26R

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FIG. 4. Whole-mount X-gal staining of Tg3/CAG-CAT-Z (A–C) and Tg3/R26R (D) embryos at E8.5 (A), E9.5 (B), E10 (C), and E12.5 (D). Cre-mediated recombination in the dorsal telencephalon (arrowheads) was first detectable at E10. Recombination was also detected in part of the nonneural ectoderm (arrows). Scale bars ⫽ 0.5 mm.

mice (n ⫽ 3; 2– 4-month-old), Cre-mediated recombination in the brain was restricted to the cerebral cortex, hippocampus, olfactory bulb, and amygdala (Fig. 6), a result consistent with those observed in Tg3/CAG-CAT-Z mice. In addition, most of the LacZ-negative neurons in the cerebral cortex and hippocampus were GABA-positive inhibitory neurons. With whole-mount X-gal staining of E12.5 Tg3/R26R embryos (n ⫽ 2), we found that Cre-mediated recombination occurred at early embryonic stages in R26R allele as in CAG-CAT-Z allele (Fig. 4). Finally, we assayed Cre protein expression in the adult dorsal telencephalon of Emx1-Cre Tg1, Tg2, Tg3, and KI mice (Fig. 7). Using an anti-Cre antibody, we found that all cortical layers (II–VI) and the piriform cortex were stained densely and uniformly in Tg1 mice (n ⫽ 3, 4.5–10-month-old). In the hippocampus, granule cells in the DG were strongly stained, whereas CA1 and CA3 pyramidal cells were stained relatively weakly. In contrast, the oriens layers, stratum radiatum, and DG molecular layer in the hippocampus had no Cre-positive cells. In the amygdala, the basolateral complex showed high levels of expression, while no expression was detected in the MePD nuclei. These patterns were almost identical to those in homozygous (n ⫽ 8, 1– 8-month-old) and

heterozygous (n ⫽ 3, 3–5-month-old) KI mice. In Tg2 (n ⫽ 2, 4.5- and 7.5-month-old) and Tg3 (n ⫽ 2, 4.5- and 7-month-old) mice, we observed overall expression patterns similar to those in Tg1 mice. However, Cre expression in the ventral cerebral cortex and amygdala was much weaker in Tg2 than in Tg1, Tg3, and KI mice. In the Tg2 and Tg3 neocortex, Cre expression was detected in all layers (II–VI), but expression levels were not even. Weak Cre protein expression in some layers of Tg2 and Tg3 cortex might be sufficient for Cre function, because Cre-mediated recombination was detected uniformly in cortical layers even in Tg2 and Tg3 mice (Figs. 2, 3). Alternatively, transient Cre expression in earlier developmental stages in these two lines may make uniform Cre-mediated recombination in cortical layers. Thus, although there were slight differences among the transgenic and KI lines, in all lines the patterns of Cre protein expression as a whole were similar to that of endogenous Emx1 gene expression (Gulisano et al., 1996; Simeone et al., 1992). Taken together, our data demonstrate that the 135-kb PAC-Emx1#1 fragment contains virtually all essential transcriptional-regulatory elements for dorsal telencephalon-specific Emx1 gene expression. Transgenic mouse lines generated by others (Guo et al., 2000c) using a short (11 kb) Emx1 promoter had transgene expression patterns completely different from those of endogenous Emx1 gene. Therefore, regulatory elements for Emx1 gene expression appear to be widespread within the 135-kb PAC-Emx1#1 fragment. Our three transgenic lines, especially Tg3, demonstrated extremely strict region- and cell type-specificity in Cre-mediated recombination from embryonic to aged stages. In Tg3 mice, these rigid specificities were observed at two distinct target loxP loci of CAG-CAT-Z and R26R mice. Therefore, using Tg3 mice may enable manipulation of any target gene in a similarly strict dorsal telencephalon-specific manner. We previously generated Emx1-Cre KI mice and showed that Cre-mediated recombination occurs only in the dorsal telencephalon between stages E11.5 and P7 (Iwasato et al., 2000). At later developmental stages, such as 3-month-old, however, the CAG-CAT-Z reporter system detected Cre-mediated recombination in parts of the thalamus, brainstem, and cerebellum (Fig. 2D, data not shown). In Emx1-Cre KI mice, the pgk promoter inserted in the Emx1 locus appears to have caused ectopic Cre-mediated recombination in the brain. This ectopic recombination disappeared completely in an Emx1-Cre KI variant in which the pgk-neo selection marker gene had been deleted (data not shown). Thus, Tg3 line as well as the Emx1-Cre KI variant has an advantage in analyzing adult brain function. Tg3 mice have additional advantages over KI mice. In most Emx1-Cre KI mouse lines (Guo et al., 2000b; Iwasato et al., 2000), one out of two endogenous Emx1 genes is disrupted by insertion of the Cre gene. Because a half dosage of Emx1 expression might be insufficient for some mechanisms (Guo et al., 2000a; Qiu et al.,

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FIG. 5. Cre-mediated recombination is restricted to excitatory neurons in the cerebral cortex of Emx1-Cre transgenic and KI mice. X-gal/anti-GABA-antibody/Nissl triple staining of the adult barrel cortex (10-␮m-thick) of Tg1/CAG-CAT-Z (A), Tg2/CAG-CAT-Z (B), Tg3/CAG-CAT-Z (C), KI/CAG-CAT-Z (D), and CAG-⌬-Z positive control (E) mice. A–D: GABA-negative (excitatory) neurons (purple) had blue particles, indicative of Cre-mediated recombination, while GABA-containing (inhibitory) neurons (brown, arrows) had no blue particles. E: LacZ-staining of both GABA-positive (arrows) and GABA-negative neurons in CAG-⌬-Z mice, confirming that the CAG-CAT-Z reporter system can detect recombination in both excitatory and inhibitory neurons. Scale bar ⫽ 100 ␮m.

1996; Yoshida et al., 1997), Tg3 mice may provide a more suitable model for studying the functions of molecules that may be closely related to Emx1 function. In addition, KI mice were generated in a 129 genetic background (Guo et al., 2000b; Iwasato et al., 2000), whereas Tg3 mice were generated in a C57BL/6 genetic background. Pure C57BL/6 genetic background is more advantageous in many fields of research, including learning and memory. In summary, using the PAC-Emx1#1 fragment, we have generated transgenic mouse lines (especially Tg3) that demonstrate lifelong expression of Cre recombinase in a pattern highly specific to the dorsal telencephalon. Emx1-Cre Tg3 mice may enable the manipulation of any target gene in a manner restricted to the dorsal telencephalon. Furthermore, the PAC-Emx1#1 fragment will allow us to express any gene of interest in the dorsal telencephalon by the transgenic mouse approach. MATERIALS AND METHODS PAC Modification An RPCI-21 PAC mouse genomic library (Roswell Park Cancer Institute, Buffalo, NY) was screened with an

Emx1 probe and several positive clones were analyzed by restriction enzyme digestion, pulsed-field gel electrophoresis (FIGE Mapper: Bio-Rad, Hercules, CA), and Southern hybridization. We chose one clone (PACEmx1#1), which contained about 95 kb upstream and 40 kb downstream of the NotI site immediately 5⬘ to the Emx1 translational initiation site. The NLS-Cre-poly(A) cassette was inserted into the NotI site in the PACEmx1#1 clone by homologous recombination in bacteria (Yang et al., 1997). The first and second homologous recombination events were confirmed by Southern blot hybridization. Generation of Transgenic Founder Mice Two independent homologous recombinants (PACCre-A and B) were digested with NotI. The PAC-Cre-A fragment was separated from the vector sequence by pulsed-field gel electrophoresis in 1% SeaPlaque agarose (BMA, Portland, ME) in TAE buffer and purified by ␤-agarase digestion and with Ultra Free-MC UFC3LTK25 (Millipore, Billerica, MA). The PAC-Cre-B fragment was separated by electrophoresis in 0.5% SeaPlaque agarose in TAE and purified by extraction with phenol (three times), phenol/chloroform, and chloroform, followed by

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FIG. 6. Dorsal telencephalon-specific Cre-mediated recombination patterns in R26R reporter mice. A–F: X-gal/Nissl double staining of coronal sections (10-␮m-thick) of adult Tg3/R26R mice. G: X-gal/anti-GABA-antibody/Nissl triple staining of the barrel cortex (10-␮m-thick) of adult Tg3/R26R mice. Most LacZ-negative cells were GABAergic (brown, arrows). Cx, neocortex; Str, striatum; Am, amygdala; Th, thalamus; Hip, hippocampus; IC, inferior colliculus; Cb, cerebellum; BS, brainstem. Scale bar ⫽ 2 mm (A–F) and 62.5 ␮m (G).

ethanol precipitation. Microinjection of the PAC-Cre-A fragment (1 ng/␮l) into the pronuclei of zygotes of C57BL/6 inbred mice generated two transgenic mouse lines (Tg1 and Tg2), whereas microinjection of the PACCre-B fragment (2 ng/␮l) generated one line (Tg3). Southern blot hybridization using a Cre probe revealed that Tg1 and Tg2 mice each have 2–3 copies of the transgene, whereas Tg3 mice have 7–9 copies of this transgene (data not shown). Transgenic mice were genotyped by Southern hybridization and/or PCR with the Cre primers (5⬘-ACCTGATGGACATGTTCAGGGATCG-3⬘ and 5⬘-TCCGGTTATTCAACTTGCACCATGC-3⬘; product size, 108 bp). The experimental procedures and housing conditions for animals were approved by the Institute’s Animal Experimental Committee and all animals were cared for and treated humanely in accordance with the Institutional Guidelines for Experiments using Animals. Histology Whole embryos and the brains of young (⬍P8) mice were fixed in 10% formalin in 0.1M sodium phosphate buffer (PB) (pH 7.4) for 30 min on ice. Adult mice were perfused intracardially with cold fixative. Brains were embedded in 2% agarose in 0.1M PB and cut into 400␮m-thick sections with a Micro-slicer (Dosaka, Kyoto,

Japan). Whole embryos and brain slices were stained overnight (CAG-CAT-Z mice) or for 6 h (R26R mice) in X-gal solution (5 mM K3FeCN6, 5 mM K4FeCN6, 2 mM MgCl2, 0.02% NP-40, 0.01% Na-deoxycholate, 1 mg/ml X-gal in 0.1M PB) at 37°C. In another set of experiments, mice were perfused and the brains were postfixed for 4 h with 4% paraformaldehyde (PFA) in 0.1M PB and equilibrated in 30% sucrose overnight at 4°C. Eight or 10 ␮m-thick sections were cut in a cryostat and mounted onto slides. Slides were stained with X-gal overnight (CAG-CAT-Z mice) or for 30 min (R26R mice) at 37°C. The tissue sections were subsequently incubated with antibodies against GABA (Sigma, St. Louis, MO; A2052, 1:2,000) or GFAP (Progen GF12.24, 1:500). CAG-⌬-Z mice were generated by crossing CAG-CAT-Z mice with “deleter” mice to remove the “loxP-CAT-loxP” fragment in the germline (Iwasato et al., 2000). Cre immunohistochemistry was performed as described previously (Nakazawa et al., 2002) with some modifications. Briefly, 50-␮m-thick coronal sections were permeabilized by incubation in 50% ethanol/PBS for 30 min and then incubated in 3% hydrogen peroxide in PBS for 10 min to inactivate endogenous peroxidases. Sections were preincubated in 10% normal goat serum (NGS) in TNB buffer (0.1M Tris 7.5, 0.15M NaCl, 0.5% blocking reagent (TSA kit; NEN, Boston, MA)) for 30 min

FIG. 7. Dorsal-telencephalon-specific Cre protein expression in three lines of transgenic mice. Coronal sections (50-␮m-thick) of Tg1 (7-month-old), Tg2 (7.5-month-old), Tg3 (7-month-old), homozygous KI (4-month-old), and wildtype (WT) mice were stained with an anti-Cre antibody. Cre protein expression was observed in layers II–VI of the neocortex in all Tg1, Tg2, Tg3, and KI mice, although there were slight differences in expression patterns. In the hippocampus, there was higher expression in the dentate gyrus (DG) than in the CA1 and CA3 in all four transgenic lines. In the amygdala, Tg1, Tg3, and KI mice had strong expression in the basolateral complex (BL) and basomedial nucleus (BM), but no expression in the MePD nucleus; whereas Cre expression in the Tg2 amygdala was weak. Or, oriens layer; rad, stratum radiatum; mol, molecular layer; pir, piriform cortex. Scale bar ⫽ 250 ␮m (left and middle columns), 500 ␮m (right column).

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and incubated in anti-Cre antibody (BABCO, 1:2,000) in TNB buffer in a cold room overnight. After incubation in biotinylated goat antirabbit IgG (H⫹L) (1:200)/3% NGS in TNB buffer at room temperature for 1 h, sections were incubated in StrABC/HRP (Vectastain Elite PK-6101, Vector, Burlingame, CA) for 1 h at room temperature and subsequently in Biotinylated Tyramide Amplification Reagent (1:50 in 1X Amplification diluent, TSA kit) for 10 min at room temperature. After incubation in SA-HPR (1:100 in TNB: TSA kit) for 30 min at room temperature, signals were detected by 5-min treatment in DAB (SK4100, Vector). ACKNOWLEDGMENTS We thank Dr. J.-I. Miyazaki for CAG-CAT-Z mice; Dr. P. Soriano for R26R mice; Drs. X.W. Yang and N. Heintz for the pSv.RecA vector; Drs. K. Nakazawa and M. Takemura for protocols of Cre and GFAP immunohistochemistry, respectively; Ms. N. Yoshida, Ms. Y. Onodera, Mr. Y. Taguchi, and Ms. M. Terasawa for technical assistance; Drs. H. Kanki and S. Nishimura for helpful discussion and/or comments on the manuscript; and the Research Resources Center of BSI for help in mouse care. LITERATURE CITED Chan CH, Godinho LN, Thomaidou D, Tan SS, Gulisano M, Parnavelas JG. 2001. Emx1 is a marker for pyramidal neurons of the cerebral cortex. Cereb Cortex 11:1191–1198. Gorski JA, Talley T, Qiu M, Puelles L, Rubenstein JL, Jones KR. 2002. Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-expressing lineage. J Neurosci 22: 6309 – 6314. Gulisano M, Broccoli V, Pardini C, Boncinelli E. 1996. Emx1 and Emx2 show different patterns of expression during proliferation and differentiation of the developing cerebral cortex in the mouse. Eur J Neurosci 8:1037–1050. Guo H, Christoff JM, Campos VE, Jin XL, Li Y. 2000a. Normal corpus callosum in Emx1 mutant mice with C57BL/6 background. Biochem Biophys Res Commun 276:649 – 653. Guo H, Hong S, Jin XL, Chen RS, Avasthi PP, Tu YT, Ivanco TL, Li Y. 2000b. Specificity and efficiency of Cre-mediated recombination in Emx1-Cre knock-in mice. Biochem Biophys Res Commun 273: 661– 665. Guo H, Mao C, Jin XL, Wang H, Tu YT, Avasthi PP, Li Y. 2000c. Cre-mediated cerebellum- and hippocampus-restricted gene mu-

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