Gene expression analysis at three different stages of myelination in dorsal root ganglion (DRG) explant cultures isolated from E13.5 C57BL/6J mouse embryos.. Mus musculus

Myelin sheath formation in the peripheral nervous system is a complex event resulting from spatially and temporally regulated reciprocal interactions between the neuron and myelin-forming Schwann cells. Although many of the components of the sheath have been identified and well-characterized, our knowledge of the dynamic cellular and molecular processes and of the protein functional networks that participate in its formation is still weak. This paper describes a robust approach, combining transcriptomics, proteomics and immunocytochemistry analyses of myelinating dorsal root ganglion cultures derived from C57BL/6J mice, which can be exploited to identify protein networks, and their constituent modules, involved in peripheral nerve myelin formation. The approach allows distinguishing clear, reproducible and predictable differences between the structural properties of the cultures and their genome-wide and proteomic expression profiles at different stages of the myelination process. The expression profiles of selected neuronal and Schwann cell genes and proteins in the cultures reflect those observed in vivo, and the structural and ultrastructural properties and myelination schedule of the cultures closely resemble those observed in peripheral nerves in situ. The approach provides a unique and powerful tool to study comprehensive transcriptome and proteomic changes taking place simultaneously in Schwann cells and neurons during PNS myelination. Overall design: We performed both biological and technical replicates. Biological replicates represent different DRG batches (M1-M3), collected at different times from different pools of embryos. Technical replicates are samples from same DRG batch (R1 and R2). This allows comparison of both the inter-batch and intra-batch variations and provides a fair estimation of the reproducibility and robustness of the methodology. DRG explant cultures representing biological replicates that were collected 0 days (N = 3), 10 days (N = 3) and 22 days (N = 3) after switching to myelination medium, and two technical replicates (n = 2) for each sample (i.e. a total of 6 replicates for each time point, altogether 18 samples), were processed for gene expression analysis.