Generation of Cre-transgenic mice using Dlx1/Dlx2 enhancers and their characterization in GABAergic interneurons

Abstract

DLX1 and DLX2 transcription factors are necessary for forebrain GABAergic neuron differentiation, migration, and survival. We generated transgenic mice that express Cre-recombinase under the control of two ultra-conserved DNA elements near the Dlx1 and 2 locus termed I12b and URE2. We show that Cre-recombinase is active in a "Dlx-pattern" in the embryonic forebrain of transgenic mice. I12b-Cre is more active than URE2-Cre in the medial ganglionic eminences and its derivatives. Fate-mapping of EGFP+ cells in adult Cre;Z/EG animals demonstrated that GABAergic neurons, but not glia, are labeled. Most NPY+, nNOS+, parvalbumin+, and somatostatin+ cells are marked by I12b-Cre in the cortex and hippocampus, while 25-40% of these interneuron subtypes are labeled by URE2-Cre. Labeling of neurons generated between E12.5 to E15.5 indicated differences in birth-dates of EGFP+ cells that populate the olfactory bulb, hippocampus, and cortex. Finally, we provide the first in vivo evidence that both I12b and URE2 are direct targets of DLX2 and require Dlx1 and Dlx2 expression for proper activity.

Figure 1. Generation of I12b-Cre and URE2-Cre transgenic mice

(A) Schematic of the Dlx1 and Dlx2 genomic locus showing the location of the I12b and URE2 regulatory elements. (B) Top, Schematic of the I12b-Cre transgene; Bottom, Wholemount X-gal staining of an E10.5 I12b-Cre embryo crossed to the ROSA26-lacZ reporter (R26R). (C) Top, Schematic of the URE2-Cre transgene; Bottom, Wholemount X-gal staining of an E10.5 URE2-Cre embryo crossed to the ROSA26-lacZ reporter (R26R). ba1, first branchial arch; ba2, second branchial arch; di, diencephalon; drg, dorsal root ganglia; fl, forelimb; fnp, frontonasal prominence; hl, hindlimb; mp, minimal promoter; NLS, nuclear localization signal; oe, olfactory epithelium; pA, poly A sequence; tel, telencephalon; v, trigeminal ganglion; x, vagus ganglion. Scale bar = 1 mm.

Figure 2. EGFP labeling at E12.5 in I12b-Cre;Z/EG and URE2-Cre;Z/EG reveals strong forebrain expression

(A-B) Immunohistochemistry for EGFP on parasagittal sections through an E12.5 (A) I12b-Cre;Z/EG or (B) URE2-Cre;Z/EG animal. (C-D) Immunohistochemistry for EGFP on coronal sections through the E12.5 forebrain of (C) I12b-Cre;Z/EG or (D) URE2-Cre;Z/EG mice. EGFP expression is present in the CGE in I12b-Cre;Z/EG but not URE2-Cre;Z/EG. Also note that tangentially migrating cells (arrowhead) are readily detected in I12b-Cre;Z/EG forebrain. cge, caudal ganglionic eminence; drg, dorsal root ganglion; ge, ganglionic eminences; hyp, hypothalamus; lge, lateral ganglionic eminence; mge, medial ganglionic eminence; ob, olfactory bulb; oe, olfactory epithelium; poa, preoptic area; pth, prethalamus; s, skin; svz, subventricular zones; v, trigeminal ganglion. Scale bar in A = 1mm and in C = 500 μm.

Figure 3. Comparison of EGFP expression between E15.5 I12b-Cre;Z/EG and URE2-Cre;Z/EG mice highlights similarities and differences in forebrain expression

(A-A′, B-B′) Rostral to caudal coronal sections through E15.5 I12b-Cre;Z/EG (A-A′) and URE2-Cre;Z/EG (B-B′) were labeled by immunohistochemistry for EGFP. Note the reduced expression in specific ventral forebrain regions (asterisks) in URE2-Cre;Z/EG embryos. acb, accumbens; ah, anterior hypothalamus; arc, arcuate nucleus; cb, cerebellum; cpu, caudate-putamen; dmh, dorsomedial hypothalamus; fr ctx, frontal cortex; gp, globus pallidus; lh, lateral hypothalamus; m amy, medial amygala; ob, olfactory bulb; ol, inferior olive; on, olfactory nerve layer; pt, pretectal nucleus; pa, paraventricular hypothalamic nucleus; poa, preoptic area; rt, thalamic reticular nucleus; sep, septum; sf, superior fiber bundle; sm, stria medullaris of thalamus; svz, subventricular zone; Tg, tegmental nucleus; tu, olfactory tubercle; vp, ventral pallidum; zi, zona incerta. Scale bars = 500 μm.

Figure 4. Activity of I12b and URE2 revealed by EGFP immunohistochemistry of sections through newborn I12b-Cre;Z/EG and URE2-Cre;Z/EG mice

(A-A″, B-B″) Rostral to caudal coronal sections through P0 I12b-Cre;Z/EG (A-A″) and URE2-Cre;Z/EG (B-B″) mice were labeled for EGFP. Expression in URE2-Cre;Z/EG mice is lower in specific regions (asterisks) compared to I12b-Cre;Z/EG. acb, accumbens; amy, amygdala; atc, anterior hypothalamic area; cpu, caudate-putamen; fr, frontal cortex; gcl, granule cell layer; gl, glomerular layer; hip, hippocampus; hyp, hypothalamus; on, olfactory nerve layer; poa, preoptic area; rt, thalamic reticular nucleus; sep, septum; sn, substantia nigra; stm, stria terminalis; stn, subthalamic nucleus; tu, olfactory tubercle; vp, ventral pallidum; zi, zona incerta. Scale bars = 500 μm.

Figure 5. Characterization of EGFP expression in adult I12b-Cre;Z/EG and URE2-Cre;Z/EG forebrain

(A-B) EGFP+ cells were identified in coronal sections through adult I12b-Cre;Z/EG (A) or URE2-Cre;Z/EG (B) mice. The boxes denote similar regions of the cortex and hippocampus shown at higher magnification in (C) and (D). (C) EGFP+ cells in the adult somatosensory cortex of I12b-Cre;Z/EG (left) and URE2-Cre;Z/EG (right) mice populate cortical layers 1 to 6. I12b-Cre labels about 2.3× more cells than URE2-Cre. (D) EGFP+ cells are found throughout the adult hippocampus of I12b-Cre;Z/EG (top) and URE2-Cre;Z/EG (bottom) mice. I12b-Cre labels about 1.6× more cells than URE2-Cre. amy, amygdala; aob, accessory olfactory bulb; AudCtx, auditory cortex; cpu, caudate-putamen; DG, dentate gyrus; epl, external plexiform layer; gcl, granule cell layer; gl, glomerular layer; gp, globus pallidus; hip, hippocampus; hyp, hypothalamus; on, olfactory nerve layer; poa, preoptic area; Sb, subiculum; sep, septum; snr, substania nigra reticulata; SomCtx, somatosensory cortex; tu, olfactory tubercle; VisCtx, visual cortex; vp, ventral pallidum. Scale bar in A, B = 500 μm; C = 100 μm; D= 150 μm.

Figure 6. I12b-Cre and URE2-Cre label GABAergic neurons and not glia in the adult brain

(A-B) Double labeling for EGFP (green) and GABA (red) in the somatosensory cortex of I12b-Cre;Z/EG (A) and URE2-Cre;Z/EG (B) mice. (C) Graph of the percentage of GABA+ cells that are EGFP+ in the adult hippocampus (HIP) and somatosensory cortex (CTX). I12b-Cre;Z/EG in blue and URE2-Cre;Z/EG in red. Bars are mean ± SD (n=3). (D) Graph of the percentage of EGFP+ cells that are GABA+ in the adult hippocampus (HIP) and somatosensory cortex (CTX). I12b-Cre;Z/EG in blue and URE2-Cre;Z/EG in red. Bars are mean ± SD (n=3). (E-F, G-H, I-J) Double labeling for EGFP and the oligodendrocyte markers OLIG2 (E-F), OLIG1 (G-H), and SOX10 (I-J) in I12b-Cre;Z/EG (E,G,I) or URE2-Cre;Z/EG (F,H,J) cortex. (K-L) Double labeling for EGFP and the cholinergic neuron marker choline acetyltransferase (ChAT) in I12b-Cre;Z/EG (K) and URE2-Cre;Z/EG (L) striatum. No co-expression was detected. The inserts depict higher magnification images of the striatum (K′, L′). (M-N) Double labeling for EGFP and the astrocytic marker GFAP in I12b-Cre;Z/EG (M) or URE2-Cre;Z/EG (N) hippocampus. Scale bars = 100 μm.

Figure 7. DLX2 and CRE expression in embryonic and newborn I12b-Cre;Z/EG mice

(A - C) Coronal forebrain sections from I12b-Cre;Z/EG animals at E12.5 (A), E15.5 (B), and P0 (C) were double-labeled for DLX2 (red), CRE (green) and counterstained for cell nuclei with DAPI (blue). The boxed regions in (A-C) are shown at higher magnification in (A′-C′). In A′-C′, the boxed regions are shown at higher magnification in A″-A‴, B″-B″″, and C″-C″″. DLX2-only expression is shown in A″, B‴, and C‴, CRE-only expression is shown in A‴, B″″, and C″″, and both DLX2 and CRE expression is shown in A′, B″ and C″. In contrast to CRE expression, DLX2 expression is high in the ventricular zone (VZ). CRE and DLX2 are co-expressed within the subventricular zone (SVZ) at all ages examined. Scale bars in A, B, C = 500 μm.

Figure 8. Co-expression of I12b-Cre and URE2-Cre lineage cells with GABAergic interneuron subtypes in the adult somatosensory cortex

Representative images of double-labeling immunofluorescence with EGFP (green) and interneuron markers (red) in the adult somatosensory cortex of I12b-Cre;Z/EG (A-F) and URE2-Cre;Z/EG (G-L) mice. Quantification of co-expression is presented in Table 1. (A, G) Calbindin; (B, H) Calretinin; (C,I) nNOS; (D,J) NPY; (E,K) Parvalbumin; (F,L) Somatostatin. Scale bar= 150 μm.

Figure 9. CRE and EGFP expression in embryonic and adult I12b-Cre;Z/EG mice

(A-C) Immunofluorescence on E12.5 forebrain sections from I12b-Cre;Z/EG mice for CRE (red), EGFP (green), and DAPI (blue). Boxed regions in (A) are shown at higher magnification in (B) and (C). LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; POA, preoptic area. (B) Overlapping CRE and EGFP expression is detectable in tangentially migrating interneurons (C) CRE expression precedes EGFP expression within the SVZ of the MGE. (D-E) Immunofluorescence on E15.5 forebrain sections from I12b-Cre;Z/EG mice for CRE (red), EGFP (green), and DAPI (blue). Boxed regions in (D) is shown at higher magnification in (E). (E-E″) CRE and EGFP are co-expressed in tangentially migrating interneurons within the marginal zone (MZ) and between the intermediate zone (IZ) and subventricular zone (SVZ). (F-H) Immunofluorescence on adult forebrain sections from I12b-Cre;Z/EG mice for CRE (red), EGFP (green), and DAPI (blue) in the olfactory bulb (F-F′), cortex (G-G″), and subventricular zone (H). (F) CRE and EGFP expression within the adult olfactory bulb. Note that migrating neuroblasts in the SVZ express CRE, but not EGFP. In contrast, the granule cell layer (GCL), mitral cell layer (MCL), external plexiform layer (EPL) and glomerular layer (GL) contain cells that co-express EGFP and CRE. (G) CRE is expressed in a subset of EGFP+ cells within the adult somatosensory cortex. (H) CRE is expressed within neurogenic niche of SVZ cells of the lateral ventricles, but is not detectable within the striatum (Str). Scale bars in E, G = 100 μm; B, C, H = 200 μm; A, D, F = 500 μm.

Figure 10. Co-expression of I12b-Cre and URE2-Cre labeled cells with GABAergic interneuron subtypes in the adult hippocampus

Representative images of double-labeling immunofluourescence with EGFP (green) and interneuron markers (red) in the adult hippocampus of I12b-Cre;Z/EG (A-F) and URE2-Cre;Z/EG (G-L) mice. Quantification of co-expression is presented in Table 2. (A, G) Calbindin; (B, H) Calretinin; (C, I) nNOS; (D, J) NPY; (E, K) Parvalbumin; (F, L) Somatostatin. Scale bar= 200 μm.

Figure 11. Birthdate analysis of EGFP+ neurons in the olfactory bulb, hippocampus and cortex of I12b-Cre;Z/EG and URE2-Cre;Z/EG mice

(A-C) Pregnant I12b-Cre;Z/EG and URE2-Cre;Z/EG dams were treated with thymidine analogs CldU or IdU (collectively labeled as XdU) at gestational day 12.5, 13.5, 14.5, or 15.5. Pups were collected at birth and coronal sections through the olfactory bulb (A), neocortex (B) and hippocampus (C) were double labeled for EGFP (green) and XdU (red). A representative image of EGFP expression is shown in the first column and XdU-labeling of each treatment timepoint shown in the right four columns. (D) Quantification of the percentage of EGFP+ cells that co-expressed XdU at P0 in the olfactory bulb (left graph), neocortex (middle graph), and hippocampus (right graph). At least 3 coronal sections from 3-4 animals were analyzed. Each bar represents the mean and error bars are the SEM. * p < 0.04; ** p < 0.008. Scale bar in A, B = 100 μm; C = 200 μm.

Figure 12. DLX2 binds directly to I12b and URE2 in vivo and DLX1&2 function is necessary for I12b and URE2 activity

(A) Schematic of the Dlx1 and Dlx2 genomic locus showing the location of I12b and URE2 regulatory elements; the three putative DLX binding sites within URE2 are labeled (a-c). The core binding motif for DLX transcription factors is predicted to be TAATT. Dlx5/6i is a regulatory element in the intergenic region between Dlx5 and Dlx6 known to bind DLX1 and DLX2 in vivo. The DLX binding motif is present in Dlx5/6i, I12b, and URE2 enhancer elements (red italics). (B) Chromatin immunoprecipitation (ChIP) assays from E15.5 ganglion eminences using DLX2-specific antisera (aDLX2) shows that DLX2 binds directly to Dlx5/6i (top panel) and also to I12b (second row) and URE2 (third row) in vivo. Control assays without antibody or with DLX2 antisera followed by PCR to irrelevant genomic loci (Opsin promoter region or ERC9a, an enhancer region upstream of the progesterone receptor), were negative. L, 100 bp DNA ladder; gDNA, genomic DNA; -, no antibody. (C) I12b activity was greatly diminished in Dlx1&2 mutants (Dlx1&2-/-) with some notable exceptions (arrows). I12b-Cre;Z/EG animals were bred with Dlx1&2 mutant mice to generate Dlx1&2 null mutants that were transgenic for I12b-Cre and Z/EG. EGFP expression was visualized in coronal brain sections from control (top row) or Dlx1&2-/- mutants (bottom row) at P0. (D) URE2 activity is nearly abolished in Dlx1&2 mutants. URE2-Cre;Z/EG animals were bred with Dlx1&2 mutant mice to generate Dlx1&2 null mutants that were transgenic for URE2-Cre and the EGFP reporter. EGFP expression was visualized in coronal forebrain sections from control (top row) or DLX1&2-/- mutants (bottom row) at P0. Expression was lost throughout the brain except in a few cells within the glomerular layer of the olfactory bulb (arrows). acb, accumbens; cpu, caudate-putamen; ctx, cortex; gc, granule cell layer; gl, glomerular layer; hip, hippocampus; hyp, hypothalamus; on, olfactory nerve layer; pt, pretectal nucleus; sep, septum. Scale bars = 500 μm.