Targeting of the enhanced green fluorescent protein reporter to adrenergic cells in mice

Abstract

Adrenaline and noradrenaline are important neurotransmitter hormones that mediate physiological stress responses in adult mammals, and are essential for cardiovascular function during a critical period of embryonic/fetal development. In this study, we describe a novel mouse model system for identifying and characterizing adrenergic cells. Specifically, we generated a reporter mouse strain in which a nuclear-localized enhanced green fluorescent protein gene (nEGFP) was inserted into exon 1 of the gene encoding Phenylethanolamine n-methyltransferase (Pnmt), the enzyme responsible for production of adrenaline from noradrenaline. Our analysis demonstrates that this knock-in mutation effectively marks adrenergic cells in embryonic and adult mice. We see expression of nEGFP in Pnmt-expressing cells of the adrenal medulla in adult animals. We also note that nEGFP expression recapitulates the restricted expression of Pnmt in the embryonic heart. Finally, we show that nEGFP and Pnmt expressions are each induced in parallel during the in vitro differentiation of pluripotent mouse embryonic stem cells into beating cardiomyocytes. Thus, this new mouse genetic model should be useful for the identification and functional characterization of adrenergic cells in vitro and in vivo.

Fig. 1

Construction of knock-in Pnmt::nEGFP allele. a Schematic depiction of (i) wild-type, (ii) Pnmt-GFP-Neo, and (iii) Pnmt-GFP alleles. The rectangles represent the coding sequences and the thickened lines show the DNA sequences used to direct homologous recombination to introduce GFP and NeoR into the first exon of Pnmt. b Representative Southern blot analyses of genomic DNAs from G418-resistant ES clones. After digestion with XbaI and hybridization with the 5′ external probe described in a, correctly targeted chromosomes display an 8 kb band in addition to the 14 kb band characteristic of the wild-type chromosome. The 3′ external probe identifies 6 and 14 kb bands from the targeted and wild-type chromosomes, respectively. Thus, lane 1 is from a correctly targeted cell line, while lane 2 shows the pattern for a non-targeted wild-type cell. R1 EcoRI; S SacI; Xb XbaI

Fig. 2

nEGFP expression in mouse adrenal glands. a, b Low-magnification views of fluorescent imaging of adrenal gland sections from wild-type control and Pnmt^+/nEGFP mice, respectively. Scale bar 200 μm. c, d Higher magnification of same image. Scale bar 50 μm. Ctx cortex, Med medulla

Fig. 3

Identification of nEGFP and endogenous Pnmt in adrenal chromaffin cells. a, b Pnmt IF histochemical staining in adult mouse adrenal gland sections as visualized for red fluorescence (Texas Red filter). c, d EGFP expression in the same adrenal sections but visualized for green fluorescence (GFP filter). e, f Overlay of Pnmt and nEGFP staining for each section. a, c, e Lo-mag low-magnification: scale bar 200 μm. b, d, f Hi-Mag high-magnification: scale bar 30 μm

Fig. 4

Nuclear localization of EGFP expression in adrenal chromaffin cells. a Propidium iodide (PI) staining of nuclei in adrenal chromaffin cells from an adult mouse adrenal gland section. b EGFP expression in the same section. c Overlay of PI and EGFP staining. Yellow staining indicates regions of overlap. Scale bar 50 μm (Color figure online)

Fig. 5

Co-localization of nEGFP and anti-GFP IF in nuclei of mouse adrenal gland sections. Positively stained cells were only found in the adrenal medulla. a nEGFP expression (green). b Anti-GFP IF staining (red). c DAPI nuclear stain (blue). d Overlay of images obtained in a–c. Arrows indicate examples of cells that were positively stained for nEGFP, anti-GFP IF, and DAPI. Scale bar 30 μm. Images were collected from heterozygous Pnmt^+/nEGFP mice. Wild-type (Pnmt^+/+) control adrenal sections showed no nEGFP or anti-GFP staining (see Supplemental Fig. 1) (Color figure online)

Fig. 6

Comparison of nEGFP expression in heterozygous (Pnmt^+/nEGFP) and homozygous (Pnmt^nEGFP/nEGFP) mouse adrenal glands. a, b Representative adrenal gland sections with nEGFP expression from heterozygous (a) and homozygous (b) mice. c Quantitative analysis of average number of nEGFP+ cells per section for each genotype (p < 0.05, n = 5). Scale bar 100 μm

Fig. 7

Detection of nEGFP expression from Pnmt^nEGFP/nEGFP mouse hearts at E10.5. a Sagittal section of E10.5 mouse heart visualized in the green spectrum, with atrium (Atr) and ventricle (Vent) clearly distinguishable. Scale bar 100 μm. Although many cells appear to exhibit green fluorescence in this image, most of this fluorescence is due to background autofluorescence. This was determined by comparing fluorescent images in the green and red spectra. An example is shown in b and c, respectively. Note that the images shown in b, c represent an expanded view of the region of the E10.5 near the A-V junction (boxed region from a) representing green and red fluorescent images, respectively. The nEGFP+ cells (arrows) were positively identified with the green fluorescence filter showing nEGFP expression to be exclusively evident when visualizing green but not red fluorescence. In contrast, autofluorescence was observed similarly in green and red spectra. Scale bar 20 μm

Fig. 8

Induction of cardiac differentiation and activation of Pnmt and nEGFP expression in mESCs. a Endogenous mouse Pnmt gene expression as detected by RT-PCR before and after induction of cardiac differentiation in mESCs. This procedure takes (7 + n) days. b–g Images of nEGFP+ cells in 7 + 5d cultures of cardiac-differentiated mESCs as visualized in green fluorescence for nEGFP (b), red fluorescence for anti-GFP IF (c), blue fluorescence for DAPI (d), Overlay of b–d images (e), DIC (f), and overlay of b–d plus f (g). Arrow indicates the same positively stained mESC in a–g. Scale bar 10 μm