A robust and high-throughput Cre reporting and characterization system for the whole mouse brain

Abstract

The Cre/lox system is widely used in mice to achieve cell-type-specific gene expression. However, a strong and universally responding system to express genes under Cre control is still lacking. We have generated a set of Cre reporter mice with strong, ubiquitous expression of fluorescent proteins of different spectra. The robust native fluorescence of these reporters enables direct visualization of fine dendritic structures and axonal projections of the labeled neurons, which is useful in mapping neuronal circuitry, imaging and tracking specific cell populations in vivo. Using these reporters and a high-throughput in situ hybridization platform, we are systematically profiling Cre-directed gene expression throughout the mouse brain in several Cre-driver lines, including new Cre lines targeting different cell types in the cortex. Our expression data are displayed in a public online database to help researchers assess the utility of various Cre-driver lines for cell-type-specific genetic manipulation.

Figure 1

Generation of the Cre-reporter lines. (a) Schematic diagram of the gene targeting strategy to insert the Cre reporter cassette into the Rosa26 locus, in the intron between endogenous exons 1 and 2. Upper right panel shows a partial Southern blot screen using HindIII digested genomic DNA and the probe indicated in the diagram. Full-length blot is presented in Supplementary Fig. 1 online. (b) Configurations of different Cre-reporter constructs inserted into the Rosa26 locus. The PGK-Neo marker in Ai9 ES clone was deleted by transfection of a PhiC31-expressing plasmid, generating the Ai14 ES clone. (c) Comparison of reporter expression in the cortex between Rosa26-EYFP and new reporter lines. Cre lines used are indicated above each panel. Scale bar, 100 μm. (d) Quantification of EYFP ISH signals in EIIa-Cre/Rosa-YFP, EIIa-Cre/Ai2 and EIIa-Cre/Ai3 mice, with whole brain sections as AOIs. Relative optical density was measured as the IOD ratio: IOD ratio = IOD / total AOI area. IOD = ΣOD(p), such that the optical density of individual expression object pixels are summed over the AOI. *** p < 0.001 (n = 3 sections per brain; Student’s t-test). (e) Quantitative RT-PCR of EYFP using 2 sets of primer pairs on the total RNA extracted from cerebellums of Pcp2-Cre/Rosa26-EYFP, Pcp2-Cre/Ai2 or Pcp2-Cre/Ai3 mice. ΔCp = Cp YFP – Cp Gapdh. Cp, number of real-time PCR cycles. Rosa-YFP, n = 4; Ai2, n = 8; Ai3, n = 12; Student’s t-test. *** p < 0.001. * p < 0.05. Values plotted are mean ± SEM.

Figure 2

Significantly enhanced fluorescent labeling in the new reporter lines. (a–b) Comparison of fluorescence in various reporter lines crossed to the same Cre-driver line: Pcp2-Cre in (a), and Chat-Cre in (b). IHC indicates immunostaining using anti-GFP. Unlike tdTomato fluorescence which was also visible in axonal projections, ZsGreen fluorescence was mostly confined to the cell bodies. Scale bar, 500 μm. (c) Quantification of fluorescence in single neurons of Pcp2-Cre/Rosa26-EYFP, Pcp2-Cre/Ai2 or Pcp2-Cre/Ai3 mice, on section images with 140-ms exposure time (no saturation). Three types of neurons were quantified, cerebellar Purkinje cells (cb, upper panel), cortical neurons (ctx, middle panel), and hippocampal CA1 pyramidal neurons (hpc, lower panel). Relative intensity = intensity of the object (cell) – intensity of the background. For each of Ai2 and Ai3, n = 30 randomly selected neurons from 3 sections per region. For Rosa-YFP, only n = 10 randomly selected neurons from one section was used in the Purkinje cell quantification, due to undetectability in the other two. *** p < 0.001. Values plotted are mean ± SEM. (d–h) Distinctive morphologies of various cell types labeled by tdTomato: (d) dentate granule cells; (e) a cortical interneuron; (f) Purkinje cells; (g) cerebellar Bergmann glia cells; (h) dendritic spines. (i) Corticothalamic projections from cortical layer 6 neurons in Ntsr1-Cre/Ai14 mice. (j) Corticothalamic projections in an Ai14 mouse with rAAV-Cre injected into the somatosensory cortex. (k) In vivo 2-photon imaging of tdTomato expressing neurons (red) and OGB-loaded neurons (green) in visual cortical layer 2/3 of an anesthetized Wfs1-Tg2-CreERT2/Ai9 mouse. Scale bar, 50 μm.

Figure 3

Informatics processing of the ISH characterization data. (a) Co-labeling of EYFP (green) with Slc6a3 (red) in the Slc6a3-Cre/Ai3 mouse by DFISH. In the VTA/substantia nigra region (left panel), EYFP was largely co-localized with Slc6a3 (>90% cells were co-labeled), demonstrating expected Cre-recombination in dopamine neurons. In the basal forebrain area (right panel), the cluster of EYFP-positive cells were Slc6a3-negative. Scale bar, 200 μm. (b) EYFP ISH data from a Slc6a3-Cre/Ai3 mouse were digitized and registered to the Allen Reference Atlas, which allows anatomical search and comparison. Two sections of registered EYFP ISH data are shown overlaid with anatomical partitioning. EYFP expression is present in both sections (white and red arrows).

Figure 4

New Cre lines and their differential recombination patterns in different cortical cell types. For each Cre line, both CISH of the reporter gene (tdTomato (Tom) or EYFP) and DFISH of the reporter (green) with Gad1 (red) are shown for the cortex only. CISH of some of the endogenous genes used to make Cre lines is also shown in the rightmost panel. The mice used are: (a) Wfs1-Tg2-CreERT2/Ai9 and Wfs1-Tg3-CreERT2/Ai9 with tamoxifen induction. (b) Scnn1a-Tg2-Cre/Ai9 and Scnn1a-Tg3-Cre/Ai9. (c) Six3-Cre/Ai9. (d) A930038C07Rik-Tg4-Cre/Ai9. (e) Ntsr1-Cre/Ai3. (f) 8030451F13Rik-Tg2-Cre/Ai9. (g) Camk2a-CreERT2/Ai14 without or with tamoxifen induction. (h) Erbb4-2A-CreERT2/Ai14 with tamoxifen induction, shown for both cortex and hippocampus to demonstrate the scattered population of interneurons. DFISH of Erbb4 (green) with tdTomato (red) shows the faithful but incomplete recapitulation of recombination in Erbb4 positive cells. DAPI staining is shown in blue. Scale bar, 100 μm.

Figure 5

Comparison of recombination patterns in two closely related knock-in Cre lines, Pvalb-ires-Cre and Pvalb-2A-Cre. (a–i) CISH comparison of tdTomato reporter in Pvalb-ires-Cre/Ai14 mouse (left panels: a, d, g), the endogenous Pvalb gene (middle panels: b, e, h), and tdTomato reporter in Pvalb-2A-Cre/Ai14 mouse (right panels, c, f, i) in 3 areas of the brain: cortex (top panels: a, b, c), hippocampus (middle panels: d, e, f) and thalamus (bottom panels: g, h, i). (j–k) DFISH comparison of tdTomato (green) with Gad1 (red) between Pvalb-ires-Cre (j) and Pvalb-2A-Cre (k). (l–m) DFISH comparison of Pvalb (green) with tdTomato (red) between Pvalb-ires-Cre (l) and Pvalb-2A-Cre (m). Scale bar, 200 μm.