European Journal of Neuroscience, Vol. 11, pp. 1577–1586, 1999 © European Neuroscience Association A novel P 0 glycoprotein transgene activates expression of lacZ in myelin-forming Schwann cells M. Laura Feltri, 1,2 Maurizio D’Antonio, 2 Angelo Quattrini, 1 Rita Numerato, 2 Marta Arona, 2 Stefano Previtali, 1,2 Shing-Yan Chiu, 3 Albee Messing 4 and Lawrence Wrabetz 1,2 1 Department of Neurology, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milano, Italy 2 DIBIT, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milano, Italy 3 Department of Physiology, Waisman Center, University of Wisconsin-Madison, Madison, WI 53706, USA 4 School of Medicine and Department of Pathobiological Sciences, Waisman Center, University of Wisconsin-Madison, Madison, WI 53706, USA Keywords: development, mice, peripheral nerve, transcription, Wallerian degeneration Abstract P 0 glycoprotein, the most abundant protein in peripheral nerve, is expressed specifically in the Schwann cell lineage. Upstream of the rat P 0 gene 1.1 kb of DNA can activate expression of cDNAs specifically in Schwann cells in transgenic mice. However, the expression of P 0 promoter-based transgenes has been inconsistent. As much as 9 kb of 5' flanking sequence fused to lacZ never yielded detectable levels of β-galactosidase in multiple lines of mice. We describe transgenic mice that express lacZ in peripheral nerve, using the complete mouse P 0 gene, including 6 kb of 5' flanking sequence, all exons and introns, and the natural polyadenylation signal. This vector activated lacZ expression specifically in cultured Schwann cells, and myelin-forming Schwann cells in four out of six transgenic lines. Transgene expression paralleled that of the endogenous P 0 gene, both during development and after Wallerian degeneration. lacZ expression was lower than endogenous P 0 expression, and was not detected in neural crest or Schwann cell precursors, where low levels of P 0 mRNA are present. However, when the same vector contained a small myc tag instead of the 3.2-kb lacZ insert, the resulting transgenic mRNA was expressed at levels comparable to endogenous P 0 mRNA. These data suggest that intragenic or 3' flanking sequences are necessary to generate the remarkable levels of endogenous P 0 gene expression. Introduction P 0 glycoprotein is an adhesion molecule that compacts the spiralling Schwann cell (SC) plasma membrane to form myelin in the peripheral nervous system. P 0 is indeed the most abundant protein of peripheral myelin, where it constitutes over 50% of total protein (Greenfield et al., 1973). P 0 is expressed by myelin-forming SC, cells that contact a single axon and deposit a myelin sheath around it; but not by non- myelin forming SC, cells that ensheath several axons without forming myelin. Both types of cells derive from neural crest cells that subsequently differentiate into SC precursors and embryonic SC (Jessen et al., 1994; Dong et al., 1995). Basal levels of P 0 mRNA are first seen in neural crest and remain in SC precursors and embryonic SC (Bhattacharyya et al., 1991; Baron et al., 1994; Zhang et al., 1995; Lee et al., 1997). The type of axon that a SC subsequently encounters will determine its phenotype: large axons induce differentiation into a myelin-forming SC, small axons – a non-myelin-forming SC. Myelin-forming SC greatly upregulates the expression of P 0 glyco- protein and other myelin-specific genes, so that after differentiation, P 0 mRNA accounts for at least 8% of the mRNA synthesized (Stahl et al., 1990). SC-specific induction of P 0 expression is thought to be mediated at the level of transcription (Lemke et al., 1988). In keeping with this, the P 0 promoter successfully directed expression of foreign genes Correspondence: M.L. Feltri, as above. E-mail: l.feltri@hsr.it Received 4 August 1998, revised 8 December 1998, accepted 9 December 1998 to myelin-forming SC. In vitro, 1.1 kb of P 0 promoter drove SC- specific expression of reporter genes (Lemke et al., 1988). Moreover, in transgenic mice, 1.1 kb of P 0 promoter successfully activated expression of toxins, hormones, oncogenes, transcription factors and enzymes specifically in SC (Messing et al., 1992, 1994; Weinstein et al., 1995; Nikitin et al., 1996). However, transgene expression with the 1.1 kb promoter has been inconsistent. Thus, phenotypes suggesting transgene expression have been obtained with potent molecules (e.g. diphtheria toxin), although transgenic mRNA expres- sion was low and was not measured as a direct proportion of endogenous P 0 mRNA (Messing et al., 1992). Also, more consistent expression was obtained when the transgene contained both exons and natural introns from the heterologous gene, as for growth hormone, SV40 T antigen, and connexin 32 (Messing et al., 1992, 1994; Bone et al., 1997). Instead, many attempts to express transgenes containing P 0 promoter fragments fused to cDNAs or reporter genes, even with heterologous introns (e.g. rabbit β-globin) included, have had only limited success, sometimes requiring many independent transgenic lines in order to obtain even low-level expression (Feltri et al., unpublished observations; Maycox et al., 1997). For example, P 0 promoter–lacZ transgenes have never been successfully expressed in SC, even when 9 kb of 5' flanking sequence was included (Table 1). Thus, we asked if the intragenic portion of the mouse P 0 gene (Mpz) contributes to high-level P 0 mRNA expression in SC. As we had no indication of where important regulatory elements may reside outside of the promoter, we reconstructed the complete Mpz, including