The wmN1 Enhancer Region of the Mouse Myelin Proteolipid Protein Gene (mPlp1) is Indispensable for Expression of an mPlp1-lacZ Transgene in Both the CNS and PNS

Abstract

The myelin proteolipid protein gene (PLP1) encodes the most abundant protein in CNS myelin. Expression of the gene must be strictly regulated, as evidenced by human X-linked leukodystrophies resulting from variations in PLP1 copy number, including elevated dosages as well as deletions. Recently, we showed that the wmN1 region in human PLP1 (hPLP1) intron 1 is required to promote high levels of an hPLP1-lacZ transgene in mice, using a Cre-lox approach. The current study tests whether loss of the wmN1 region from a related transgene containing mouse Plp1 (mPlp1) DNA produces similar results. In addition, we investigated the effects of loss of another region (ASE) in mPlp1 intron 1. Previous studies have shown that the ASE is required to promote high levels of mPlp1-lacZ expression by transfection analysis, but had no effect when removed from the native gene in mouse. Whether this is due to compensation by another regulatory element in mPlp1 that was not included in the mPlp1-lacZ constructs, or to differences in methodology, is unclear. Two transgenic mouse lines were generated that harbor mPLP(+)Z/FL. The parental transgene utilizes mPlp1 sequences (proximal 2.3 kb of 5'-flanking DNA to the first 37 bp of exon 2) to drive expression of a lacZ reporter cassette. Here we demonstrate that mPLP(+)Z/FL is expressed in oligodendrocytes, oligodendrocyte precursor cells, olfactory ensheathing cells and neurons in brain, and Schwann cells in sciatic nerve. Loss of the wmN1 region from the parental transgene abolished expression, whereas removal of the ASE had no effect.

Keywords: Cre/loxP and Flp/Frt recombination; Enhancer; Gene regulation; Myelin proteolipid protein gene (Plp1); Transgenic mouse; lacZ.

Fig. 1

Schematic of the parental mPLP(+)Z/FL transgene which utilizes mPlp1 DNA, spanning the proximal 2.3 kb of 5ʹ-flanking DNA to the first 37 bp of exon 2, to drive expression of a lacZ reporter cassette. Black boxes depict mPlp1 exon 1 and the beginning of exon 2; the sequence for exon 2 is unnumbered as the transgene is drawn to scale. Black lines represent mPlp1 5ʹ-flanking and intron 1 DNA. Frt and loxP sites were introduced at the designated restriction enzyme sites in mPlp1 intron 1 (only pertinent restriction enzyme sites are indicated). Frt sites circumscribe the ASE, while loxP sites flank the wmN1 enhancer region. Removal of a portion of mPlp1 intron 1 DNA from the parental transgene was achieved by mating mPLP(+)Z/FL transgenic mice with the relevant deleter strain (see Material and Methods for further details). Intron 1 positions 971–1264 are missing from mPLPΔASE, and positions 3636–4815 are missing from mPLPΔwmN1, based on numbering mPlp1 intron 1 from 1 to 8140 [4]

Fig. 2

β-gal activity in brain of mPLP(+)Z/FL transgenic mice at P2 and P21 as determined by X-gal staining. Mid-sagittal brain sections (20 μm) were stained with X-gal for 6 h (P2) or 1 h (P21). Line 781 expresses the parental transgene to a higher degree than Line 823 based on the intensity of X-gal staining. In fact, β-gal activity was apparent in Line 781 at P21 after only 5 min of staining with X-gal (Fig. S1.) However, the pattern was consistent between the two lines, as indicated by staining the external layer of olfactory bulb at P2, and white matter areas of brain at P21

Fig. 3

Colocalization of β-gal activity with immunofluorescence for cell type-specific markers in brain and sciatic nerve. Tissues were obtained from mPLP(+)Z/FL mice (Line 781) at P9 (cerebellum; sciatic nerve) or P2 (olfactory bulb; brain stem). Brain sections (16 μm) and intact sciatic nerve were stained with X-gal followed by immunofluorescence staining with the indicated cell type-specific markers (red color); the X-gal stained sciatic nerve was sectioned longitudinally (16 μm) prior to immunofluorescence. The bright-field microscopic images for X-gal staining shown in black and white were converted to a green color in ImageJ for application with the merged pictures. Boxes in some of the bright-field images represent the area magnified in the immunofluorescence analysis. Magnification of bright-field images: cerebellum, 2.5×; olfactory bulb, 10×; brain stem and sciatic nerve, 20×

Fig. 4

The wmN1 region, but not the ASE, is required for mPlp1-lacZ expression in brain. Mid-sagittal brain sections (20 μm) from mPLPΔASE and mPLPΔwmN1 mice were stained with X-gal for 6 h (P2) or 1 h (P21). Removal of the wmN1 region from the parental transgene with Line 823 resulted in a lack of X-gal staining (mPLPΔwmN1), but loss of the ASE had no effect; comparable staining for mPLPΔASE and mPLP(+)Z/FL (see Fig. 2)

Fig. 5

The wmN1 region is required for mPlp1-lacZ expression in the PNS. X-gal staining of intact sciatic nerve from mPLP(+)Z/FL and mPLPΔwmN1 mice (Line 823) at P9 of age. Notice the significant amount of blue staining emanating from the mPLP(+)Z/FL transgene, which disappears when the wmN1 region is removed (mPLPΔwmN1)