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. 2016 Oct 5;92(1):59-74.
doi: 10.1016/j.neuron.2016.09.027.

Subpallial Enhancer Transgenic Lines: A Data and Tool Resource to Study Transcriptional Regulation of GABAergic Cell Fate

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Free PMC article

Subpallial Enhancer Transgenic Lines: A Data and Tool Resource to Study Transcriptional Regulation of GABAergic Cell Fate

Shanni N Silberberg et al. Neuron. .
Free PMC article

Abstract

Elucidating the transcriptional circuitry controlling forebrain development requires an understanding of enhancer activity and regulation. We generated stable transgenic mouse lines that express CreERT2 and GFP from ten different enhancer elements with activity in distinct domains within the embryonic basal ganglia. We used these unique tools to generate a comprehensive regional fate map of the mouse subpallium, including sources for specific subtypes of amygdala neurons. We then focused on deciphering transcriptional mechanisms that control enhancer activity. Using machine-learning computations, in vivo chromosomal occupancy of 13 transcription factors that regulate subpallial patterning and differentiation and analysis of enhancer activity in Dlx1/2 and Lhx6 mutants, we elucidated novel molecular mechanisms that regulate region-specific enhancer activity in the developing brain. Thus, these subpallial enhancer transgenic lines are data and tool resources to study transcriptional regulation of GABAergic cell fate.

Figures

Figure 1
Figure 1
Regional enhancer activity of transgenic lines harboring ten different enhancers at E11.5. Enhancers are grouped according to their expression domain in the transient transgenic assay at E11.5: LGE, CGE, MGE and POA, or multiple subpallial domains. Each row of coronal sections are arranged rostral (left) to caudal (right). A–J. Vista enhancer-LacZ transient transgenics chosen to make stable lines. Cells expressing enhancer-driven-LacZ are blue following X-gal histochemistry. A’–J’. Stable transgenic enhancer lines harboring an Enhancer-Hsp68-CreERT2–IRES-GFP transgene. Immunofluorescence (IF) (599, 953, 1060, 799, 841, 692, 204, 192) or in situ RNA hybridization (ISH) (1538 and 1056) of enhancer driven GFP expression marks cells activating the enhancer at E11.5. A’’–J’’. Schemata representing activity of the stable enhancer lines (blue) in the ventricular zone, subventricular zone, and mantle zone of the E11.5 telencephalon. Scale bar 500µM. Abbreviations: Amygd, amygdala; AT: acroterminal domain; C, central part of the telencephalon; CGE, caudal ganglionic eminence; d, dorsal; Dg, diagonal; hp1, hypothalamic prosomere 1; hp2, hypothalamic prosomere 2; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; MP, medial pallium; OS, optic stalk; Pal, pallidal; PHy, peduncular hypothalamus; POA, preoptic domain; PSe, pallidal septum; PTh, prethalamus; Se, septum; St, striatum; Thy, terminal hypothalamus; v, ventral.
Figure 2
Figure 2
Fate mapping of ten enhancer lines (CreERT2 expressing) crossed to the Ai14 reporter. Tamoxifen administration at E10.5 induced Cre–dependent tdTomato expression in cells activating the enhancer within a ~24 hour period. Fate mapped cells at E17.5 (tdTomato+) shown in coronal sections arranged rostral (left) to caudal (right). See also figures S1 and S2. Scale bar 500µM. Abbreviations for all figures: AAD, anterior amygdaloid area, dorsal; AAV, anterior amygdaloid area, ventral; ac, anterior commissure; Acb, accumbens nucleus; ACo, anterior amygdalar cortical nucleus; al, ansa lenticularis; AOA, anterior olfactory areas; BEC, bed nucleus of the external capsule; BLA, basal lateral amygdala; BLP, basal lateral amygdala posterior; BM, basal medial nucleus of the amygdala; BST, bed nucleus stria terminalis; BSTA, amygdala area of the bed nucleus stria terminalis; BSTsc, subcommissural zone of the bed nucleus stria terminalis; Ce, central amygdala; CeA, central amygdala, anterior part; CeC, central amygdala, central part; CeM, central amygdala, medial part; CGE, caudal ganglionic eminence; CP, choroid plexus; CR, cajal retzius ; Cx, cortex; CxA, cortical amygdala transition; DB, diagonal band; DG, dentate gyrus; d, dorsal; GpE, globus pallidus externa; GPI, globus pallidus interna; hem, hem; Hi, hippocampus; hif, hippocampal fissure; Hyp, hypothalumus; IA, interculated amygdala; In, interneurons; Ise, intermediate septum; L, lateral amygdala; LGE, lateral ganglionic eminence; LH, lateral hypothalamus; lot, lateral olfactory tract; MA, medial amygdala region; MeAD, anterodorsal medial amygdala; MePD, posterodorsal medial amygdala; MePV, posteroventral medial amygdala; MGE, medial ganglionic eminence; Mpal, medial pallium; MSe, medial septum; NLOT, nucleus lateral olfactory tract; OB, olfactory bulb; OT, olfactory tubercle; PalSe, pallial septum; PCx, piriform cortex; ped, peduncle; PLCo, posterolateral cortical amygdala nucleus; POA, preoptic area; PTh, prethalmus; PThE, prethalamic eminence; Se, septum; SI, substantia innominata; TCA, thalamocortical axons; Th, thalmus; St, striatum; v, ventral; Vpal, ventral pallidum; VSt, ventral striatum.
Figure 3
Figure 3. Differential contribution of cells with activity of enhancers 1538 and 1056 to the globus pallidus
Fate mapping of enhancer lines 1056 (A–A’) or 1538 (B–B’) at P40 using a tamoxifen dose at E10.5. Fate-mapped cells (tdTomato+, red) co-labeled with parvalbumin (PV) immunofluorescence (green) in the rostral (A and B) and caudal (A’ and B’) globus pallidus (GP). B. Density of colabeled cells (tdTomato+ and PV+) in the rostral and caudal globus pallidus (cells/mm2): blue = 1056, purple = 1538, respectively. C. Rostral-Caudal ratio of PV+ cell density in the globus pallidus: tdTomato+ and PV+ (blue = 1056); tdTomato+ and PV+ (purple = 1538); PV+ (grey). ANOVA with Tukey’s multiple correction p (1056 vs. 1538) = 0.0387; p (1056 vs. PV) = 0.9641; p (1538 vs. PV) = 0.0291. See also figure S2. Graphs show mean ± standard deviation. Scale bar 500µM. Abbreviations: GP(c), globus pallidus, caudal part; GP(r), globus pallidus, rostral part; PTh, prethalmus; St, striatum.
Figure 4
Figure 4
Fate mapping at P40 of enhancer lines 1056 or 1538 using a tamoxifen dose at E10.5. A–B. Coronal sections of P40 somatosensory neocortex showing fate mapped cells (tdTomato+, red) from either 1056 (A–A’) or 1538 (B–B’) co-labeled with PV (A–B, green) or SST (A’–B’, green) by immunofluorescence. C. Fraction of tdTomato+ cells that co-localize with SST or PV out total tdTomato+ cells. Blue = 1056 tdTomato+; Purple = 1538 tdTomato+. D. Fraction of neocortical 1056 tdTomato+ cells that co-label with MGE (SST+ and PV+), CGE (Reelin+ (SST), VIP+) or oligodendrocyte (Olig2+) markers out of total neocortical 1056 tdTomato+ cells. E–F. Enhancer 799 activity or fate mapping in the P40 somatosensory cortex. Coronal sections showing either enhancer-799-driven GFP+ cells (green) co-stained with PV (E, blue) and SST (F, blue), or temporally integrated fate mapped tdTomato+ cells (red) co-stained with PV (E’, blue) or SST (F’, blue). G. Fraction of 799-activating GFP+ cells (green, n=3) or of temporally integrated fate mapped tdTomato+ cells (Red, n=3) that co-localize with SST and PV out of total GFP+ or total tdTomato+ respectively. H. Ratio of SST+ cells to PV+ cells in adult somatosensory cortex. Green = 799-activating GFP+ cells, Red = integrated fate mapped tdTomato+ cells, Black = all interneurons. Statistical differences between groups (799 GFP n=3, 799 tdTomato n=3, WT, n=3), were determined by One-Way ANOVA with Tukey’s posthoc test. p (799 GFP vs 799 tdTomato) = 0.0147; p (799 GFP vs WT) = 0.0074; p (799 tdTomato vs WT) = 0.7981. Graphs show mean ± standard deviation. Scale bars 500µM
Figure 5
Figure 5
Fate mapping of enhancer line 1060-CreERT2-IRES-GFP (GFP) (Cre induced tdTomato) to P35. A–C. Adult fate of cells activating 1060 at E11.5, E13.5, and E15.5. Cells activating enhancer 1060 at E11.5, E13.5 or E15.5 were labeled with tdTomato following a tamoxifen dose either at E10.5 (A), E12.5 (B), or E14.5 (C), and fate mapped to P35, shown in a rostrocaudal series of coronal hemisections. D–F Characterization of cortical fate mapped cells at P35 following tamoxifen administration at E14.5. Co-labeling of fate mapped cells (tdTomato+, red) from E15.5 by immunofluorescence with interneuron markers: REELIN (D, green), SST (D, blue), SP8 (E, green) and VIP (F, green). G. Layer distribution of cortical fate mapped cells. Fraction of tdTomato+ cells per cortical layer out of total cortical tdTomato+ cells. Black bar = co-label with REELIN, Grey bar = tdTomato only. See also figure S4 and S1C”. Graph shows mean ± standard deviation Scale bar 500µM. For abbreviations see Figure 2.
Figure 6
Figure 6
A. In vivo TF binding on ten enhancers used for stable transgenics from 16 ChIP-seq experiments. Denominator = number of experimental replicas; numerator = number of replicas with called peak over an enhancer. B. ChIP-seq enrichment over enhancer 799 in mm9 (chr6: 978714–9785886). Asterisks mark mutated binding sites in 799Δ2. Image generated using IGV software (REF). C–E. Comparison of wild type enhancer 799 activity at E13.5 (C, green, GFP) in the stable enhancer line crossed to two transcription factor null mutants: Dlx1/2−/− (D, green) and Lhx6PLAP/PLAP (E, green) exhibit loss of GFP expression in the subpallium and cortical interneurons, and GFP maintenance in indicated subpallial locations. Scale bar 500 µM. F. Luciferase assay to measure activity of wild type and mutant enhancer 799 in primary MGE culture. Fold-activation over control is shown for: grey = baseline control empty pGl4.23; orange = 799pGl4.23 fold activation over control; black = 799Δ2pGl4.23. Statistical differences between experimental groups (n=3 for each group) were determined with the Ratio paired t-test using p (799pGl4.23 vs. pGl4.23) = 0.0175; p (799pGl4.23 vs. 799ΔpGL4.23) = 0.0062; p (pGl4.23 vs 799ΔpGL4.23) = 0.7012. Graph shows mean ± standard deviation. Abbreviations: Cx (In), cortical interneurons; GP, globus pallidus; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; MZ, mantle zone; SVZ, subventricular zone: VPal, ventral pallidum; VZ, ventricular zone.
Figure 7
Figure 7
Transcription factor binding signatures discriminate subregional (LGE and MGE) and laminar (VZ, SVZ+MZ) activities of subpallial enhancers. See also Figure S5. A. Matrix showing the ChIP-Seq binding profile of MGE and LGE enhancers. X axis: enhancers with either LGE or MGE activity (laminar activity is also indicated). Y axis: TF ChIP-seq analyses. TF binding to an enhancer with MGE or LGE activity; Black and Red, respectively. Rows and columns were clustered using complete linkage and a correlation-based distance. B. Average ranking of the TF ChIP-Seq data sets among the 5 most discriminative for the 33 LGE and MGE classifiers trained within the leave-one-out cross-validation framework. The ranking is calculated based on the mean decrease in accuracy associated with each ChIP-Seq data set. C. Average ChIP-Seq profile for MGE and LGE enhancers shown as a heatmap. Y axis: “relative frequency” corresponds to the fraction of enhancers containing sites for the indicated TFs (right side). D. Matrix showing the ChIP-Seq binding profile of enhancers with VZ, SVZ+MZ, and MZ activity (LGE and MGE activity also indicated). Rows and columns were clustered using complete linkage and a correlation-based distance. E. Average ranking of the TF ChIP-Seq data sets among the 5 most discriminative for the 73 VZ, SVZ+MZ and MZ classifiers trained within the leave-one-out cross-validation framework. The ranking is calculated based on the mean decrease in accuracy associated with each ChIP-Seq data set. F. Average ChIP-Seq profile for VZ, SVZ+MZ, and MZ enhancers shown as a heatmap (see C). See also Figure S5.

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