Retracing Schwann cell developmental transitions in embryonic dissociated DRG/Schwann cell cocultures in mice

Abstract

Embryonic Dissociated Dorsal Root Ganglia cultures are often used to investigate the role of novel molecular pathways or drugs in Schwann cell development and myelination. These cultures largely recapitulate the order of cellular and molecular events that occur in Schwann cells of embryonic nerves. However, the timing of Schwann cell developmental transitions, notably the transition from Schwann Cell Precursors to immature Schwann cells, has not been estimated so far in this culture system. In this study, we use RTqPCR to determine the expression profiles of Schwann cell developmental genes during the first week of culture. We first identified stable reference genes that show minimal variation across different experimental time points. Consequently, we normalized the mRNA profiles of Schwann cell developmental genes using the best internal reference genes. We then compared our data to the expression profiles of these genes in developing spinal nerves elaborated in numerous high-throughput and lineage tracing studies. This comparison helped in identifying that Schwann Cell Precursors transition into immature Schwann Cells between the 5th and 7th day in vitro. In effect, our data allows for a better understanding and interpretation of DRG culture experiments especially in studies that aim to elucidate the role of a novel gene in Schwann Cell development and myelination.

Mineralocorticoid receptor knockout in Schwann cells alters myelin sheath thickness

“…Aldosterone, spironolactone, dexamethasone and RU486 (all from Roche) were diluted in ethanol, and added at 1/10,000 final dilution each in culture medium. Primary SC cultures derived from E13.5 mouse dorsal root ganglia (pregnant C57BL/6 dams from Janvier Labs, Le Genest-Saint-Isle, France) that were dissociated in trypsin, plated and grown as previously described (Sundaram et al 2021). Confluent cultures were used to seed 12 well plates at 50,000 cells/wells and treatments were performed as described with MSC80 cells.…”

“…Twelve µm thick sections were cut with a cryostat and disposed on 3-(triethoxysily)propylamine (TESPA)-treated slides. Section were then permeabilized with 0.2% triton X100, 0.1% tween 20, blocked with 2% BSA and 5% donkey serum in PBS tween 0.1%, incubated at 4°C overnight with primary antibodies (rabbit anti-MR Thermofisher PA-95019, 1/100 and goat anti-sox10 R&D AF 2864 1/200 as previously validated (Sundaram et al 2021)) then incubated with secondary antibodies (donkey antirabbit Alexa 488 1/2,000 and donkey anti-goat Cy3 1/500, Jackson IR). Nuclei were labeled with Hoescht, slides were mounted in coverslips with fluoromount-G (Thermofisher) and dry overnight.…”

“…1E). This preparation method allows to obtain a very pure SC culture with low fibroblast contaminations compared to SC cultures isolated from sciatic nerve (Sundaram et al 2021). Indeed, only few cells are negative for Sox10 as visualized in the merged panel (Fig.…”