Olfactory ensheathing cells (OECs), the glia of the olfactory nerve, are promising candidates for patient-specific cell-mediated repair of both peripheral nerves and the spinal cord. The recent discovery that OECs originate from the neural crest, rather than the olfactory epithelium as previously thought, potentially means that homogeneous populations of OECs for repair could be expanded in culture from neural crest stem cells persisting in the patient's own skin and hair follicles. The first step towards this long-term goal is to understand the molecular mechanisms underlying neural crest differentiation into OECs, as opposed to Schwann cells (the glia of all other peripheral nerves), which are less effective in spinal cord repair. To identify transcription factors and signalling pathways that might be involved in OEC versus Schwann cell differentiation, I took an unbiased transcriptome profiling approach. Taking advantage of Sox10 expression throughout both OEC and Schwann cell development, I used laser-capture microdissection on cryosections of mouse embryos carrying a Sox10:H2BVenus transgene, to isolate OEC subpopulations (olfactory mucosal OECs, from the olfactory nerve, and olfactory nerve layer OECs, from the olfactory nerve layer surrounding the olfactory bulb) at different stages of development, and Schwann cells from trigeminal nerve branches on the same sections, for RNA-seq and cross-wise comparison of transcriptomes. Validation of candidate genes by in situ hybridisation revealed some contamination with adjacent cells from mesenchyme, olfactory epithelium or olfactory bulb, but also identified the expression in developing OECs of various genes previously reported to be expressed in adult OECs, and of over 20 genes previously unknown in OECs. Some of these genes are expressed by OECs but not Schwann cells; some are expressed by olfactory nerve layer OECs but not olfactory mucosal OECs, while some are expressed by olfactory mucosal OECs and Schwann cells but not olfactory nerve layer OECs. For a subset of the genes, I was also able to analyse OEC differentiation in mouse mutants. I also collected transcriptome data from neural crest-derived cells that persist on the olfactory nerve in Sox10-null embryos (in which neural crest-derived cells colonise the olfactory nerve, but normal OEC differentiation is disrupted). Comparison with wild-type OEC transcriptome data from the same embryonic stage identified genes whose expression is likely either downregulated or up-regulated in the absence of Sox10, supporting a role in normal OEC differentiation. Overall, these various transcriptomic comparisons (between OECs at different developmental stages, different OEC subpopulations, OECs versus Schwann cells, and OECs versus Sox10-null neural crest-derived cells on the olfactory nerve) have identified multiple transcription factor and signalling pathway genes, amongst others, that are expressed during OEC development in vivo (including some specific to different OEC subpopulations) and that may be important for OEC differentiation. Furthermore, some of these genes are not expressed by embryonic Schwann cells. This work provides a foundation for understanding how to promote OEC rather than Schwann cell differentiation from neural crest stem cells in culture, with the potential for clinical application in the future.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:767758 |
| Date | January 2019 |
| Creators | Perera, Surangi Nalika |
| Contributors | Baker, Clare V. H. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/288787 |
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