Distinct cis-regulatory elements from the Dlx1/Dlx2 locus mark different progenitor cell populations in the ganglionic eminences and different subtypes of adult cortical interneurons

Abstract

Distinct subtypes of cortical GABAergic interneurons provide inhibitory signals that are indispensable for neural network function. The Dlx homeobox genes have a central role in regulating their development and function. We have characterized the activity of three cis-regulatory sequences involved in forebrain expression of vertebrate Dlx genes: upstream regulatory element 2 (URE2), I12b, and I56i. The three regulatory elements display regional and temporal differences in their activities within the lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE), and caudal ganglionic eminence (CGE) and label distinct populations of tangentially migrating neurons at embryonic day 12.5 (E12.5) and E13.5. We provide evidence that the dorsomedial and ventral MGE are distinct sources of tangentially migrating neurons during midgestation. In the adult cortex, URE2 and I12b/I56i are differentially expressed in parvalbumin-, calretinin-, neuropeptide Y-, and neuronal nitric oxide synthase-positive interneurons; I12b and I56i were specifically active in somatostatin-, vasoactive intestinal peptide-, and calbindin-positive interneurons. These data suggest that interneuron subtypes use distinct combinations of Dlx1/Dlx2 enhancers from the time they are specified through adulthood.

Figure 1.

Conserved cis-acting regulatory elements in the Dlx1/Dlx2 locus. A, Schematic representation of the Dlx1/Dlx2 locus of four vertebrate species. Each Dlx gene comprises three exons, which are indicated in light blue boxes. Colored squares are conserved noncoding DNA sequences: I12b (blue), URE1 (yellow), and URE2 (red). B, Enhancer activity of the mouse URE2 sequence in an E11.5 mouse embryo. URE2 targets lacZ reporter gene expression to two domains in the forebrain, the subpallial telencephalon, and diencephalon, as well as other tissues. H, Hyoid arch (second arch); Hy, hypothalamus; L, limbs; PT, prethalamus (ventral thalamus); ST, subpallial telencephalon; St, somites. Scale bar, 1 mm.

Figure 2.

Comparative enhancer activities of URE2, I12b, and I56i in the subpallial telencephalon of transgenic mice. Coronal hemisections showing lacZ expression under the control of each enhancer at E11.5 (A–F) and E12.5 (G–L) in medial and caudal levels. A, D, G, J, URE2-lacZ; B, E, H, K, I12b-lacZ; C, F, I, L, I56i-lacZ. B, C, E, F, Insets, Higher-magnification pictures of regions shown in black dashed boxes; arrowheads in the insets mark the beginning of tangential migrations at the level of the dLGE at E11.5. G, H, I, Insets, Higher-magnification pictures of the red dashed boxes. H, I, K, L, Arrows 1 and 2 show the two streams of migratory cells emerging from the LGE at the level of the corticostriatal boundary (CSB), which coalesce into one stream within the cortex at E12.5: a superficial stream (stream 1) and a deep stream (stream 2). Tangential migration of URE2-lacZ-positive cells was not observed at these stages. III, Third ventricle; CP, cortical plate; Hi, hippocampus; Hy, hypothalamus; LV, lateral ventricle; PCx, piriform cortex; SCB, suprachiasmatic band; Th, thalamus; VP, ventral pallium; VZ, ventricular zone. Scale bar: (in A) A–L, 250 μm; B, C, E, F, insets, 125 μm; G, H, I, insets, 75 μm.

Figure 3.

Activities of three Dlx CREs in the MGE and in the cortex near the CSB at E12.5. A–F, Double immunohistochemistry showing the expression of I12b-AP in green (A, D), URE2-lacZ (B), or I56i-lacZ in red (E). C, F, Merged pictures of A, B and D, E, respectively. Compared with the dMGE, only few radial columns of cells in B express URE2-lacZ in the SVZ of vMGE (arrowheads), whereas others do not (arrows). In C, arrows and arrowheads show radial columns of double-labeled cells (yellow) and single-labeled ones (red or green), respectively. Insets in B and C are high-magnification pictures of the areas shown in white dashed boxes. G–L, Labeling of tangentially migrating cells in the cortex near the CSB of I12b-AP mice (G, J; green), URE2-lacZ mice (H; red), and I56i-lacZ mice (K; red) at E12.5. I, L, Merged pictures of G, H and J, K, respectively. As described in Figure 2, migratory cells in H, I, K, and L follow superficial (stream 1) and deep (stream 2) streams of migrations at E12.5 that will merge into one along the Mz of the CSB. H, There is little URE2-lacZ expression in tangentially migrating cells at this age. I, L, Single-labeled and double-labeled cells are indicated with arrows and arrowheads, respectively. Scale bar: (in A) A–L, 25 μm; B, C, insets, 15 μm; G–L, 12.5 μm. Symbols are the same as in Figure 2.

Figure 4.

Differential activities of the URE2, I12b, and I56i CREs in migrating cells. A–F, Reporter transgene expression was determined at the level of the CSB at E12.5 (A, C, E) or at the level of the DP at E13.5 (B, D, F). The number of positive cells was determined from two to three sections per mouse of the CSB or DP at the level of the MGE or CGE (total = average from 3 mice). When both immunohistochemical signals were detected for both antigens in the same cell, regardless of signal intensity, the cell was counted as being positive for both antigens. Values are presented as mean ± SD. G, Schematic representation of an E13 coronal hemisection of the mouse telencephalon showing the CSB, the DP, and the two streams (stream 1 and 2) of tangentially migrating cells toward the dorsal cortex. Symbols are the same as in Figure 2.

Figure 5.

Activities of three Dlx CREs in tangentially migrating cells derived from the MGE at E13.5. A–F, Double immunohistochemistry showing the expression of I12b-AP in green (A, D) or URE2-lacZ (B) or I56i-lacZ in red (E), in the DP. C, F, Merged pictures of A, B and D, E, respectively. Tangentially migrating cells follow the Mz and IZ of the DP at E13.5. G–L, Higher-magnification pictures of the boxes shown in A–F, respectively. I, L, Merged pictures of G, H and J, K, respectively. There are more migrating cells expressing URE2-lacZ mice at this age (B, H) compared with E12.5 (Fig. 3 H). Single-labeled and double-labeled cells in I and L are indicated with arrows and arrowheads, respectively. Most migrating cells in I12b-AP/I56i-lacZ mice are double labeled, whereas a large number of cells in I12b-AP/URE2-lacZ mice are single labeled. Scale bar: (in A) A–F, 25 μm; G–L, 12.5 μm. Symbols are the same as in Figure 2.

Figure 6.

The dMGE/mMGE and vMGE are both sources of tangentially migrating cells to the DP at E12.5. A, DiI labeling (arrows) of the dMGE/mMGE and vMGE. A, Inset, A picture taken after 72 h in culture. B–D, Robust migrations toward the cortex are derived from both subdivisions. E, I, The left vMGE was dissected and transplanted into a rostral level to replace the LGE (asterisk in E, I, and M). The left dMGE/mMGE and right vMGE in E and I as well as the transplanted vMGEs in M were labeled with DiI. E, I, M, Insets, Pictures taken after 48 h in culture. F, J, N, Fluorescent pictures of E, I, M, respectively. G, K, O and H, L, P are higher magnifications of the left and right hemispheres in E, I, M, respectively. Hi, Hippocampus. Robust tangential migration to the DP is observed from progenitors located in the dMGE/mMGE (G, K) and control vMGE (H, L) as well as transplanted vMGE (O, P). Scale bar: (in A) A, B, E, F, I, J, M, N, 100 μm; C, D, G, H, K, L, O, P, 50 μm. Symbols are the same as in Figure 2.

Figure 7.

Percentage of cortical interneurons, classified according to marker gene expression, showing activity of the URE2, I12b, and I56i enhancers. Coexpression is an average from 8 to 10 sections of the somatosensory and motor cortices per mouse at P35 (n = 4). When both immunohistochemical signals were detected for both antigens in the same cell, regardless of signal intensity, the cell was counted as being positive for both antigens.

Figure 8.

Coexpression of the SOM and PV interneuron markers with URE2-lacZ, I12b-lacZ, and I56i-lacZ in the mouse adult somatosensory cortex at P35. Cells expressing URE2-lacZ (A–D, M–P), I12b-lacZ (E–H, Q–T), or I56i-lacZ (I–L, U–X) are shown in red. Interneurons expressing SOM (A–L) or PV (M–X) are shown in green. D, H, L, P, T, X, Higher magnifications of boxes shown in C, G, K, O, S, W, respectively. O, P, Most PV+ interneurons express URE2-lacZ. C, D, In contrast, only a few SOM+ interneurons express URE2-lacZ. The I12b-lacZ and I56i-lacZ transgenes are expressed in a large number of PV+ as well as SOM+ interneurons. Double- and single-labeled interneurons are indicated with arrows and arrowheads, respectively. Scale bar: (in A) A–C, E–G, I–K, M–O, Q–S, U–W, 25 μm; D, H, L, P, T, X, 8.7 μm.

Figure 9.

Coexpression of the CR and VIP interneuron markers with URE2-lacZ, I12b-lacZ, and I56i-lacZ in the mouse adult somatosensory cortex at P35. Cells expressing URE2-lacZ (A–D, M–P), I12b-lacZ (E–H, Q–T), or I56i-lacZ (I–L, U–X) are shown in red. Interneurons expressing CR (A–L) or VIP (M–X) are shown in green. D, H, L, P, T, X, Higher magnifications of boxes shown in C, G, K, O, S, W, respectively. C, D, Most CR+ interneurons express URE2-lacZ. O, P, In contrast, few VIP+ interneurons express URE2-lacZ. The I12b-lacZ and I56i-lacZ transgenes are expressed in a large number of CR+ as well as VIP+ interneurons. Double- and single-labeled interneurons are indicated with arrows and arrowheads, respectively. Scale bar: (in A) A–C, E–G, I–K, M–O, Q–S, U–W, 25 μm; D, H, L, P, T, X, 8.7 μm.

Figure 10.

Proposed model for the origin of adult cortical interneurons subtypes in the embryonic mouse brain. A, B, Schematic representation of E12.5 coronal hemisections/sections at the level of the MGE (A) and the CGE (B). Enhancer activities of I12b (red) and URE2 (blue) in the MGE and CGE are depicted in the left hemispheres in A and B. Colored arrows in the right hemispheres in A and B indicate the proposed origin(s) of Dlx-progenitor cells that will give rise to various subtypes of cortical GABAergic interneurons. Dlx-progenitors that express URE2-lacZ and I12b-lacZ are represented by blue and red arrows, respectively. Progenitors that express both transgenes are shown in orange. Dlx-PV-progenitors are possibly derived from the mMGE whereas Dlx-SOM-progenitors could derive from the dMGE and/or the vMGE (see Discussion). Dlx-NPY-progenitors and Dlx-nNOS-progenitors are probably derived from the mMGE; some of these cells could also form subpopulations of Dlx-SOM-progenitors. In the CGE, Dlx-VIP-progenitors and Dlx-CR-progenitors are probably derived from the mCGE but could also derive from the dCGE (see Discussion). Symbols are the same as in Figure 2. Hi, Hippocampus; Th, thalamus; d, dorsal; m, medial; v, ventral.