Differentiation of a Schwann cell line expressing reporter genes in the presence and absence of nerve axons

Abstract

The formation of the myelin sheath in peripheral nerves requires a complex sequence of morphological interactions between myelincompetent Schwann cells and the nerve axon and a concomitant switch in Schwann cell gene expression patterns from the immature to the mature myelin-forming phenotype [l]. The degree of interdependence between these two phenomena is not yet understood. To explore links between molecular and cellular events in myelination and demyelination we are expressing reporter genes in clonallyderived Schwann cells in order to study gene activity in relation to axon ensheathment in neuron coculture.The SCL4.lk7 immortal diploid Schwann cell strain 121 can undergo postmitotic differentiation in culture either in the presence or the absence of neurons. Rapidly growing cells were transfected using CaZPO4 coprecipitation with plasmid pffiFP-C I containing the cytomegalovirus immediate early (CMVlE) promoter reporting green fluorescent protein (GFP) to allow repeated observation of Schwann cells either in monoculture or coculture systems. Following subculture onto monolayers of embryonic rat dorsal root ganglion neurons under growtharrestdmyelin-forming conditions (Human Placental Serumsupplemented medium preconditioned against growth-arrested cultures), SCL 4.IF7 cells rapidly ensheathed and myelinated nerve axons. The constitutively active promoter was used to determine the response time of the reporter gene and the duration of persistence of its expression in coculture systems enabling myelin-fomtion. We also investigated the expression of a neural cell type-specific promoter sequence, the distal portion of the human neurofilament Lprotein (hNF-L) promoter [3], which becomes active in Schwann cells which are induced to demyelinate [4] but is suppressed in myelinatingSchwann cells. The NF-L promoter sequence was inserted between BamHl and Psrl sites of plasmid pEGFP-1 containing the GFP coding region. The plasmid (pEGFP-NFL) was transfected into SCU. 1/F7 cells as before. Cells transfected with pEGF-CI expressed GFP during proliferation (Fig 1A) and after growth arrest. Cells also expressed GFP when transfected during or after growth-arrest (Fig. IB). When under control of the distal hNF-L promoter sequence, constitutive activity was seen in SCIA.lF7 cells adopting process-bearing morphology during early differentiation [2], however when transfected cells were allowed to differentiate further, expression was reduced in some cells and was absent in others (Fig 1C.D). pEGF-CI expression in SCL4.1/F7 was stable following passage. and transfer to coculture and GFP expression could be visualized repetitively in single cells contacting axons up to two weeks after transfection (Fig lE,F). An alternative transduction method was developed using replicationdefective adenovirus (RAd35). GFP or P-galactosidase structural genes were inserted into the Nhel site of plasmid pAL119, which provides adenovirus flanking sequences. Recombined adenoviruses are generated following co-tnlnsfection of the transfer vector with t...