Supplementary Figure 2: Crh-ires-Cre-driven reporter expression faithfully reproduces native Crh expression patterns. | Nature Neuroscience

Supplementary Figure 2: Crh-ires-Cre-driven reporter expression faithfully reproduces native Crh expression patterns.

From: Chronic CRH depletion from GABAergic, long-range projection neurons in the extended amygdala reduces dopamine release and increases anxiety

Supplementary Figure 2

Crh-ires-Cre mice were generated by inserting an ires-Cre expression cassette immediately after the translational STOP codon in the 3’ UTR of the endogenous Crh gene21. As a result, Cre expression is regulated by endogenous Crh promoter/enhancer elements, without compromising Crh expression. (a) Left row depicts the endogenous Crh mRNA expression pattern in adult wild-type mice (C57BL/6J) determined by ISH. Right row shows representative coronal sections of Crh-ires-Cre mice crossed with Ai9 reporter mice (R26::loxP-STOP-loxP-tdTomato). tdTomato fluorescence (red) mirrors the native Crh mRNA expression pattern. Regions of interest are highlighted with arrowheads or dashed lines. Representative images of three independent experiments. (b) High specificity of Cre recombinase expression in CRH neurons was additionally confirmed by double ISHs against endogenous Crh (white) and Crh-ires-Cre-driven tdTomato (red). (c) Quantification of double positive cells in (b); n = 3 mice, 2-3 sections/mouse. Most tdTomato-expressing neurons co-expressed Crh, and likewise the majority of Crh-positive neurons also expressed tdTomato (arrowheads). Upon Cre/loxP recombination-mediated removal of the transcriptional STOP cassette from the R26CAG::loxP-STOP-loxP-tdTomato allele, tdTomato-reporter expression is driven by a CAG promoter and continuously detected in all neurons that have expressed CRH at any given time point within their lineage. Thus, tdTomato-positive neurons that lack Crh expression (arrows) could represent cells that expressed CRH during the course of development, but ceased to do so in adulthood. The presence of Crh-positive, but tdTomato-negative cells (dashed arrows), could be explained by lack of activity of the CAG promoter (that drives tdTomato expression) in those particular cells. Abbreviations: anterior dorsal BNST (adBNST), anterior ventral BNST (avBNST), central nucleus of the amygdala (CeA), cortex (Ctx), hippocampus (Hip), piriform cortex (Pir), paraventricular nucleus of the hypothalamus (PVN). Error bars represent s.e.m.

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