Transcriptomics analysis of developing Bergmann glia in anterior and posterior lobules

Bergmann glia (BG) comprises specialized glial cells of the cerebellum with cell bodies situated in the Purkinje cell layer and radial fibers passing through the molecular layer. In the adult, BG is an integral part of the cerebellar circuitry, modulating extracellularion homeostasis, synapse stability, plasticity, neuronal metabolism and neuroprotection. During early cerebellar development, BG is responsible for migration and correct layering of neuronal granule cell precursors (GC). Cerebellar neurons form highly organized zonal circuits and the neurons in each zone are normally associated with glial cells, and there are indications that also BG follows the spatial and temporal patterning of each functional zone. The molecular mechanisms regulating BG function are not completely clear. In addition, neither key transcriptional programs nor epigenetic modifications have been studied in depth in BG. More accurate and deeper omics data analysis, especially with purified BG samples are therefore necessary. To study the spatial patterning of BG's zones, we investigated the whole transcriptome profile of isolated BG cells from anterior (I-V) and posterior lobules (VI-IX) of the developing cerebellum (postnatal day P10), during the peak of GC migration along glial fibers. Cerebellar vermis from C57BL/6J wild-type mice were isolated from vibratome slices and the anterior lobules (A-L) were anatomically separated from posterior lobules (P-L) with an incision at the primary fissure. Lobules were cut in small pieces and dissociated by enzymatic digestion, followed by mechanical disaggregation to obtain mono- dispersed cell populations. BG cell populations from A-L or P-L were isolated by FACS sorting, based on the expression of a specific BG's cell membrane marker. Total RNA was prepared from the sorted samples for RNA-seq analysis and subsequent validation by RT-qPCR assays. RNA-seq reads were analyzed by DESeq2 R package to identify differentially expressed genes between P-L and A-L BG cells. Gene set enrichment analysis (GSEA) allowed the identification of biological processes and functional pathways affected by the deregulated genes. We present original results on 522 genes that were differentially expressed between P-L and A-L lobules (181 up-regulated, 341 down-regulated) and were functionally classified based on Gene Ontology-Biological Process (GO-BP) database.