A regulatory toolbox of MiniPromoters to drive selective expression in the brain

Abstract

The Pleiades Promoter Project integrates genomewide bioinformatics with large-scale knockin mouse production and histological examination of expression patterns to develop MiniPromoters and related tools designed to study and treat the brain by directed gene expression. Genes with brain expression patterns of interest are subjected to bioinformatic analysis to delineate candidate regulatory regions, which are then incorporated into a panel of compact human MiniPromoters to drive expression to brain regions and cell types of interest. Using single-copy, homologous-recombination "knockins" in embryonic stem cells, each MiniPromoter reporter is integrated immediately 5' of the Hprt locus in the mouse genome. MiniPromoter expression profiles are characterized in differentiation assays of the transgenic cells or in mouse brains following transgenic mouse production. Histological examination of adult brains, eyes, and spinal cords for reporter gene activity is coupled to costaining with cell-type-specific markers to define expression. The publicly available Pleiades MiniPromoter Project is a key resource to facilitate research on brain development and therapies.

Fig. 1.

In vitro neural differentiation for prescreening MiniP designs. (A and E) Ple53 and Ple88 are cloned upstream of EGFP or lacZ, respectively. (B and F) RT-PCR assays across seven time points of ESC neural differentiation for endogenous and reporter genes demonstrate appropriate temporal expression. (C and G) Immunochemistry or X-gal staining demonstrates appropriate spatial expression. [Scale bars, 100 μm (C), 200 μm (G).] (D and H) Germline knockin adult mouse brain sagittal sections analyzed by immunochemistry or X-gal staining confirms expression in the appropriate regions (i.e., olfactory bulb and rostral migratory stream for Ple53 and glia throughout the brain for Ple88).

Fig. 2.

Regulatory resolution score prioritizes genes for MiniP design. (A) Score distribution for 100 manually curated genes. The width of the boxes are proportional to the number of observations in the groups. The increases in scores from 1 to 4 and 5 are significant (P = 1.4e⁻⁰³ and P = 7e⁻⁰⁴, respectively; Wilcoxon test), as well as from 2 to 5 (P = 4.5e⁻⁰²; Wilcoxon test). (B) Score frequency of the selected 57 genes (black) compared with all other brain region selective genes (white). The dotted gray line shows that the proportion of MiniP genes relative to total increases with the score (linear regression; individual values are marked as gray boxes). (C) Regulatory resolution scores for the genes selected for MiniP design.

Fig. 3.

Refined designs of control genes validate the bioinformatics approach. We analyzed adult brain sagittal sections of four positive strains carrying MiniPs designed using our bioinformatics pipeline on control genes. For Ple49, sections of the adrenal gland were also analyzed. EGFP is detected using anti-GFP immunochemistry (brown) and lacZ is detected using X-gal histochemistry (counterstained with neutral red). AG, adrenal gland; Bs, brainstem; Cb, cerebellum; Ctx, cortex; Hyp, hypothalamus; LC, locus coeruleus; OB, olfactory bulb; RMS, rostral migratory stream. (A) Ple49-lacZ (DBH RRs) expression is enriched in the LC (but in cells that do not co-stain with anti-TH) and the AG. The last image shows costaining of X-gal (blue) with tyrosine hydroxylase (brown) in the AG. (B) Ple54-EGFP (DCX RRs) expression is observed in different regions of the brain with enrichment in the OB as seen on the whole brain image. The last image shows costaining of EGFP (green) with the endogenous Dcx protein (red) in the RMS. (C) Ple90-EGFP (GFAP RRs) is expressed in astrocytes throughout the brain. The last image shows costaining of EGFP (green) with the endogenous GFAP protein (red). (D) Ple111-EGFP (HCRT RRs) is specifically expressed in a few cells of the lateral hypothalamus. The last image shows costaining of EGFP (green) with the endogenous HCRT protein (red). (Scale bars, 100 μm.)

Fig. 4.

Novel MiniP expression patterns in the adult brain and retina. EGFP is detected using anti-GFP immunochemistry (brown) or EGFP/cre, detected using X-gal histochemistry (counterstained with neutral red). All sections are sagittal unless otherwise stated. Hip, hippocampus; MB, mid brain; Ret, retina; VTA, ventral tegmental area. (A) Ple67-EGFP (FEV RRs) expression is enriched in all raphe nuclei: from left to right, dorsal (coronal section), magnus (sagittal section), pallidus/obscurus (coronal section). (B) Ple103-EGFP/cre (HAP1 RRs) shows sporadic staining in various regions of the brain. (C) Ple151-EGFP (OLIG1 RRs) expresses throughout the brain in a puffy-like manner. The second detail image shows no costaining of EGFP (green) with neuronal marker NeuN (red). The last image shows costaining of EGFP (green) with the myelinating oligodendrocyte marker RIP (red). (D) Ple162-EGFP/cre (PITX3 RRs) is very specifically expressed in cells just dorsal to the VTA as well as the retina. The last image shows no costaining of X-gal (blue) with tyrosine hydroxylase (brown). (E) Ple167-EGFP/cre (POGZ RRs) is expressed in patches of cells across the brain. The last image shows costaining of X-gal (blue) with NeuN (brown). (F) Ple178-EGFP/cre (RGS16 RRs) is expressed in various regions of the brain. The last image shows a costaining of X-gal (blue) with NeuN (brown). (G) Ple185-EGFP (S100B RRs) expresses in Bergmann glia of the cerebellum and myelinated fibers in the cortex. The last image is a costaining of EGFP (green) with the endogenous S100B protein (red). (Scale bars, 100 μm, except C rightmost image, 50 μm.)

Fig. 5.

A view of selected novel MiniP driving lacZ assessed in 1-mm brain slices. At least two chimeric and/or germline brains were analyzed for each strain using X-gal staining of 1-mm brain slices and presented similar phenotypes. More detailed images can be found in Fig. S5 and at http://www.pleiades.org.

Fig. 6.

Analysis of gene regulation based on MiniP activity. (Top) Human genomic sequence around OLIG1 and OLIG2 together with the candidate RRs included in Ple148, Ple150, and Ple151. Comparisons of TFBS predictions between Ple151 sequences (8, 10, 11) and all others (–7, 9) identify EGR1 (e; red) and FOS (f; green) binding sites putatively responsible for Ple151-specific expression. The conservation plots were captured from the University of California Santa Cruz Genome Browser.