Differential CRH expression level determines efficiency of Cre- and Flp-dependent recombination

Abstract

Corticotropin-releasing hormone expressing (CRH⁺) neurons are distributed throughout the brain and play a crucial role in shaping the stress responses. Mouse models expressing site-specific recombinases (SSRs) or reporter genes are important tools providing genetic access to defined cell types and have been widely used to address CRH⁺ neurons and connected brain circuits. Here, we investigated a recently generated CRH-FlpO driver line expanding the CRH system-related tool box. We directly compared it to a previously established and widely used CRH-Cre line with respect to the FlpO expression pattern and recombination efficiency. In the brain, FlpO mRNA distribution fully recapitulates the expression pattern of endogenous Crh. Combining both Crh locus driven SSRs driver lines with appropriate reporters revealed an overall coherence of respective spatial patterns of reporter gene activation validating CRH-FlpO mice as a valuable tool complementing existing CRH-Cre and reporter lines. However, a substantially lower number of reporter-expressing neurons was discerned in CRH-FlpO mice. Using an additional CRH reporter mouse line (CRH-Venus) and a mouse line allowing for conversion of Cre into FlpO activity (CAG-LSL-FlpO) in combination with intersectional and subtractive mouse genetic approaches, we were able to demonstrate that the reduced number of tdTomato reporter expressing CRH⁺ neurons can be ascribed to the lower recombination efficiency of FlpO compared to Cre recombinase. This discrepancy particularly manifests under conditions of low CRH expression and can be overcome by utilizing homozygous CRH-FlpO mice. These findings have direct experimental implications which have to be carefully considered when targeting CRH⁺ neurons using CRH-FlpO mice. However, the lower FlpO-dependent recombination efficiency also entails advantages as it provides a broader dynamic range of expression allowing for the visualization of cells showing stress-induced CRH expression which is not detectable in highly sensitive CRH-Cre mice as Cre-mediated recombination has largely been completed in all cells generally possessing the capacity to express CRH. These findings underscore the importance of a comprehensive evaluation of novel SSR driver lines prior to their application.

Keywords: CRF; CRH; Cre; Flp; corticotropin-releasing hormone; reporter; stress.

Figure 1

FlpO expression pattern in CRH-FlpO mice recapitulates endogenous CRH expression. (A) Scheme illustrating the generation of CRH-FlpO mice by homologous recombination-mediated gene targeting. (B) In situ hybridization (ISH) on coronal brain sections of heterozygous (Crh^+/FlpO) (n = 3) and wild-type (Crh^+/+) CRH-FlpO (n = 3) mice using FlpO- and Crh-specific riboprobes showing comparable expression patterns, e.g., the in piriform cortex (Pir), lateral part of the interstitial nucleus of the anterior commissure (IPACL), bed nucleus of the stria terminalis (BNST), central amygdala (CeA) and paraventricular nucleus of the hypothalamus (PVN). I, internal ribosomal entry site; loxP, loxP2272 Neo, neomycine resistance gene. Scale bar, 1,000 μm.

Figure 2

Comparison of the distribution of tdTomato⁺ cells and fibers in the brain of CRH-Cre::Ai9 and CRH-FlpO::Ai65F mice. (A,B) Schematic illustration combining photomicrographs of CRH-Cre::Ai9 (left, n = 5) and CRH-FlpO::Ai65F (right, n = 4) reporter mice. (C) Distribution of tdTomato⁺ cells and fibers in CRH-Cre::Ai9 and CRH-FlpO::Ai65F mice. ac, anterior commissure; BAR, Barrington’s nucleus; BNST, bed nucleus of the stria terminalis; CeA, central amygdala; I, internal ribosomal entry site; icp, inferior cerebellar peduncle; IO, inferior olivary nucleus; IPACL, lateral part of the interstitial nucleus of the anterior commissure; ME, median eminence; MGM, medial division of the medial geniculate; OB, olfactory bulb; Pir, piriform cortex; PVN, paraventricular nucleus of the hypothalamus; SN, substantia nigra. WRPE, woodchuck hepatitis virus posttranscriptional regulatory element. Scale bar, 1,000 μm.

Figure 3

Reporter mice reveal diverging FlpO- and Cre-mediated recombination efficiency. (A) Schematic illustration of CRH-Cre::CRH-FlpO::Ai65 reporter mice. (B) Comparison of the distribution of tdTomato⁺ cells and fibers in regions of interest in CRH-Cre::Ai9 (n = 5), CRH-Cre::CRH-FlpO::Ai65 (n = 4) and CRH-FlpO::Ai65F (n = 4) mice. (C) Schematic illustration of CRH-Cre::CRH-FlpO::Ai9 reporter mice. (D) Distribution of tdTomato⁺ cells and fibers in regions of interest in CRH-Cre::CRH-FlpO::Ai9 mice (n = 6). BNST, bed nucleus of the stria terminalis; CeA, central amygdala; I, internal ribosomal entry site; CTX, cortex; IPACL, lateral part of the interstitial nucleus of the anterior commissure; ME, median eminence; pA, BGH polyA signal; PVN, paraventricular nucleus of the hypothalamus; WRPE, woodchuck hepatitis virus posttranscriptional regulatory element. Scale bars, 200 μm.

Figure 4

Cre recombinase possesses high recombination efficiency, even at low expression levels. (A) Schematic illustration of CRH-Venus reporter mice. (B) Visualization of Venus⁺ cells with or without GFP antibody staining. Scale bar, 100 μm. (C) Comparison of the distribution of tdTomato⁺ and Venus⁺ cells and fibers in regions of interest in CRH-Venus::CRH-Cre::Ai9 mice (n = 3). Scale bar, 200 μm. (D) Comparison of the distribution of tdTomato⁺ and Venus⁺ cells and fibers in regions of interest in CRH-Venus::CRH-FlpO::Ai65F mice (n = 6). Scale bar, 200 μm. (E) Distribution of Venus⁺ cells and fibers in regions of interest in homozygous CRH-Venus mice (n = 3). Scale bar, 200 μm. BNST, bed nucleus of the stria terminalis; CeA, central amygdala; CTX, cortex; IPACL, lateral part of the interstitial nucleus of the anterior commissure; ME, median eminence; PVN, paraventricular nucleus of the hypothalamus.

Figure 5

FlpO recombination efficiency depends on strength of driving promoter. (A,B) Schematic illustration of CRH-Cre::CAG-LSL-FlpO::Ai65 and CRH-Cre::CAG-LSL-FlpO::Ai9 reporter mice. (C) Distribution of tdTomato⁺ cells and fibers in regions of interest in CRH-Cre::CAG-LSL-FlpO::Ai65 (n = 3) and CRH-Cre::CAG-LSL-FlpO::Ai9 (n = 7) mice. (D) Distribution of tdTomato⁺ cells and fibers in regions of interest in homozygous CRH-FlpO::Ai65F mice (n = 4) and heterozygous CRH-FlpO::Ai65F mice (n = 4). BNST, bed nucleus of the stria terminalis; CeA, central amygdala; CTX, cortex; I, internal ribosomal entry site; IPACL, lateral part of the interstitial nucleus of the anterior commissure; ME, median eminence; pA, BGH polyA signal; PVN, paraventricular nucleus of the hypothalamus. Scale bars, 200 μm.

Figure 6

FlpO recombination efficiency is enhanced by acute restraint stress. Comparison of distribution of tdTomato⁺ cells and fibers in regions of interest in stressed and unstressed CRH-FlpO::Ai65F mice (each n = 5). BNST, bed nucleus of the stria terminalis; CeA, central amygdala; CTX, cortex; I, internal ribosomal entry site; IPACL, lateral part of the interstitial nucleus of the anterior commissure; ME, median eminence; PVN, paraventricular nucleus of the hypothalamus. Scale bar, 200 μm.