A distal upstream enhancer from the myelin basic protein gene regulates expression in myelin-forming schwann cells

Abstract

In peripheral nerves, large caliber axons are ensheathed by myelin-elaborating Schwann cells. Multiple lines of evidence demonstrate that expression of the genes encoding myelin structural proteins occurs in Schwann cells in response to axonal instructions. To gain further insight into the mechanisms controlling myelin gene expression, we used reporter constructs in transgenic mice to search for the DNA elements that regulate the myelin basic protein (MBP) gene. Through this in vivo investigation, we provide evidence for the participation of multiple, widely distributed, positive and negative elements in the overall control of MBP expression. Notably, all constructs bearing a 0.6 kb far-upstream sequence, designated Schwann cell enhancer 1 (SCE1), expressed at high levels in myelin-forming Schwann cells. In addition, robust targeting activity conferred by SCE1 was shown to be independent of other MBP 5' flanking sequence. These observations suggest that SCE1 will make available a powerful tool to drive transgene expression in myelinating Schwann cells and that a focused analysis of the SCE1 sequence will lead to the identification of transcription factor binding sites that positively regulate MBP expression.

Fig. 1.

MBP-promotedlacZ reporter constructs used to map Schwann cell enhancers and repressors in transgenic mice. We cloned 12 kb of MBP 5′ flanking sequence from a lambda DASH-129 mouse genomic library. Constructs incorporating various lengths of these MBP sequences were then used to derive transgenic mice, and transgene expression was assessed by the β-galactosidase histochemical assay. 5′ flanking sequences extending to −6, −7, −8.5, and −9 kb were ligated to thelacZ reporter gene, and the sequence lying between −9 and −8.5 kb was found to be required for Schwann cell expression. To evaluate potential effects of position and orientation, the slightly longer 0.6 kb fragment extending from −9.0 to −8.4 kb, designated SCE1, was ligated to constructs promoted by −3.1 or −6 kb MBP sequence. A second Schwann cell targeting activity was detected in the further upstream sequence (−12 to −9 kb), and because it also functions as an enhancer when ligated 3′ of the lacZ reporter, it was designated SCE2. The striped region in the −6 kbMBP-lacZ-5′(SCE1)3′ construct designates the pSK+ vector included in the injected construct.

Fig. 2.

β-galactosidase labeling in transgenic mice bearing MBP-promoted, SCE-containing constructs. In transgenic mice bearing MBP 5′ flanking sequences that include SCE1, both oligodendrocytes and Schwann cells label intensely with incubation times varying from minutes to hours. a,Lumbar spinal cord and attached roots from a 21-d-old −9 kb line 17 mouse. Inset, A 1-μm-thick cross section of L4 ventral root embedded in plastic subsequent to whole-mount labeling. Bluo-gal reaction product is associated with the majority of the myelin and Schwann cell cytoplasmic profiles. b, A 21-d-old mouse bearing a −6.0 kb MBP-lacZ construct with SCE2 ligated to the 3′ end of lacZ expresses β-galactosidase in both oligodendrocytes and Schwann cells.Inset, A 1-μm-thick cross section of whole-mount-stained L4 root demonstrates labeling of a subpopulation of myelin and Schwann cell profiles in this mosaic, primary transgenic mouse. c, Reporter constructs bearing the −6 kb MBP promoter target expression to oligodendrocytes but not Schwann cells. d, Teased fiber preparation from an SCE1-bearing transgenic mouse shows β-galactosidase reaction product accumulated in the cytoplasmic compartments of the Schwann cell, including the perinuclear cytoplasm (asterisk), Schmidt–Lantermann incisures (closed arrowhead), and paranodal loops (open arrowhead). e, A 12-μm-thick cryostat cross section from a sciatic nerve of a transgenic mouse bearing the SCE1-containing −9 kb promoter shows labeling on many myelin and Schwann cell profiles. Because the intra-Schwann cell distribution of the reaction product is not uniform along the internode (d), not all such profiles should be labeled. Scale bar: a–c, 3.1 mm; a, b, insets, 0.05 mm; c, 0.05 mm; d,0.17 mm; e, 0.05 mm.

Fig. 3.

SCE1 targets Schwann cell expression in the context of a heterologous promoter. a, SCE1 was ligated to a 0.3 kb hsp68 minimal promoter in either orientation, and each construct was used to derive transgenic mice. The sequence represented by the striped box in 3′(SCE1)5′-hsp68-lacZ is from the vector, pSK+.b, Dorsal view of a whole-mount histochemical β-galactosidase preparation from a 7-d-old SCE1-hsp68-lacZ (line 18) mouse showing the intense labeling of fibers in lumbar spinal roots but no expression in oligodendrocytes in the CNS. c, d, Cranial nerves and cervical spinal roots also demonstrate intense labeling, whereas neither oligodendrocytes nor other cell types label in the brain. Note that this specimen was a B6C3F1 derivative and was pigmented accounting for the black pigmentation, and not reaction product, observed in the eyes. Scale bar: b, 1.6 mm; c,d, 4.4 mm.

Fig. 4.

Developmental expression of transgenes bearing SCE1. Low-level transgene expression is detected in some regions of the PNS in an SCE1-hsp68-lacZ line 18 fetus at E15.5. Mixed nerves exiting the spinal column have detectable but unevenly deposited β-galactosidase reaction product (a), whereas the trigeminal nerve is more obviously and uniformly labeled (b). High-level expression, detectable in whole-mount preparations, initiates only in the postnatal period. c, Whole-mount preparation of a 2-d-old −9 kb MBP-lacZ mouse (line 17) shows labeling in ventral but not dorsal spinal roots, reflecting the relative developmental delay in the myelination program of dorsal roots. d, Electron micrograph prepared from whole-mount-labeled tissue reveals β-galactosidase reaction product deposited adjacent to developing myelin sheaths in the L4 ventral root. At this stage of development, whereas some ventral root fibers have initiated sheath formation, the unlabeled dorsal roots (data not shown) do not contain any myelin profiles. e, Whole-mount preparation of roots and dorsal root ganglia from a preweaning −9 kbMBP-lacZ transgenic mouse. Uniform and intense staining is observed in both dorsal and ventral roots, whereas neither the neurons nor non-Schwann cell glia in the dorsal root ganglia label. f, In 3-month-old −9 kb line 17MBP-lacZ transgenic mice, Schwann cells ensheathing motor axons in ventral roots no longer label, whereaslacZ expression continues, at readily detectable levels, in Schwann cells ensheathing sensory axons in dorsal roots (root modality was identified by the spinal cord insertion site). Scale bar:a, b, 0.18 mm; b, 0.18 mm;c, 1.14 mm; d, 4 μm; e,1.25 mm; f, 1.6 mm.

Fig. 5.

Sequence analysis reveals a Krox-20 site that is neither essential nor sufficient for Schwann cell expression.a, Sequence analysis reveals multiple potential regulatory elements within SCE1, and only a few are shown. Among these is a Krox-20 site [(−)GCGTGGGTG; Sham et al., 1993]. This site lies near the 3′ end of SCE1, and consequently, was included in the 8.5 kb promoted construct that expressed only in oligodendrocytes, demonstrating that it is not sufficient for conferring Schwann cell expression. b, To determine whether the Krox-20 site is essential for SCE1-mediated expression, a construct bearing a mutation within the Krox-20 site was generated [(−)GCGTGGGTG→GCGGTTTCG, δKrox-20-SCE1]. The mutated SCE1 was ligated to the 3′ end oflacZ driven by 6 kb of MBP promoter.c, A P30 transgenic mouse bearing the δKrox-20-SCE1 construct demonstrates high-level reporter gene expression in both Schwann cells and oligodendrocytes, indicating that the Krox-20 site is not essential for SCE1 targeting function. Scale bar: c, 4.4 mm.