Sexually dimorphic distribution of Prokr2 neurons revealed by the Prokr2-Cre mouse model

Abstract

Prokineticin receptor 2 (PROKR2) is predominantly expressed in the mammalian central nervous system. Loss-of-function mutations of PROKR2 in humans are associated with Kallmann syndrome due to the disruption of gonadotropin releasing hormone neuronal migration and deficient olfactory bulb morphogenesis. PROKR2 has been also implicated in the neuroendocrine control of GnRH neurons post-migration and other physiological systems. However, the brain circuitry and mechanisms associated with these actions have been difficult to investigate mainly due to the widespread distribution of Prokr2-expressing cells, and the lack of animal models and molecular tools. Here, we describe the generation, validation and characterization of a new mouse model that expresses Cre recombinase driven by the Prokr2 promoter, using CRISPR-Cas9 technology. Cre expression was visualized using reporter genes, tdTomato and GFP, in males and females. Expression of Cre-induced reporter genes was found in brain sites previously described to express Prokr2, e.g., the paraventricular and the suprachiasmatic nuclei, and the area postrema. The Prokr2-Cre mouse model was further validated by colocalization of Cre-induced GFP and Prokr2 mRNA. No disruption of Prokr2 expression, GnRH neuronal migration or fertility was observed. Comparative analysis of Prokr2-Cre expression in male and female brains revealed a sexually dimorphic distribution confirmed by in situ hybridization. In females, higher Cre activity was found in the medial preoptic area, ventromedial nucleus of the hypothalamus, arcuate nucleus, medial amygdala and lateral parabrachial nucleus. In males, Cre was higher in the amygdalo-hippocampal area. The sexually dimorphic pattern of Prokr2 expression indicates differential roles in reproductive function and, potentially, in other physiological systems.

Dissociated Pmch and Cre Expression in Lactating Pmch-Cre BAC Transgenic Mice

“…Mounted tissue was fixed in 4% paraformaldehyde in DEPC-treated 1× PBS for 20 min, dehydrated in increasing concentrations of ethanol, cleared of lipids using xylenes, and subsequently rehydrated in decreasing concentrations of ethanol. The slides were pretreated by microwaving in sodium citrate buffer (pH 6.0), then dehydrated again in increasing concentrations of ethanol, air-dried, and stored at −20°C ( Sibony et al, 1995 ; Frazão et al, 2013 ; Mohsen et al, 2017 ).…”

“…Sections were stored at −20 • C in RNAse-free cryoprotectant (20% glycerol, 30% ethylene glycol in DEPC-PBS). (Sibony et al, 1995;Frazão et al, 2013;Mohsen et al, 2017). The Pmch cDNA (template) was produced from mouse hypothalamic RNA and used to amplify a 478 bp sequence in the coding region of the Pmch gene (IDT, Inc.).…”

Principles of Genetic Engineering

“…Ideally, the insertion of reporter cDNAs in the genome results in single copy transgene insertions in defined loci in such a way that endogenous genes are not disrupted, and reporters are placed under the control of specific endogenous promoters [179]. The application of CRISPR/Cas9 technology to address this problem shows it can be used to achieve these goals [63,82,180]. The development of CRISPR/Cas9 base editing technology shows that it is possible to make single-nucleotide changes in the genome [181][182][183][184].…”

Twenty-Seven Tamoxifen-Inducible iCre-Driver Mouse Strains for Eye and Brain, Including Seventeen Carrying a New Inducible-First Constitutive-Ready Allele

“…Certainly, CRISPR has now made knocking-in cre at any mouse gene at least as fast and easy as docking 5′ of Hprt . Methods such as synthesized guide RNAs, short homology sequences, purified Cas9 protein, and direct introduction into mouse zygotes, have already enabled researchers to develop new constitutive and inducible cre-driver mice (Feng et al 2016; Hasegawa et al 2016; Ackermann et al 2017; Mohsen et al 2017; Daigle et al 2018). However, with this strategy, the possibility remains of generating a deleterious allele in the very gene whose normal expression is to be captured.…”