Neurotransmitter signalling to oligodendrocytes

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Gene Editing in Rat Embryonic Stem Cells to Produce In Vitro Models and In Vivo Reporters

Rat embryonic stem cells (ESCs) offer the potential for sophisticated genome engineering in this valuable biomedical model species. However, germline transmission has been rare following conventional homologous recombination and clonal selection. Here, we used the CRISPR/Cas9 system to target genomic mutations and insertions. We first evaluated utility for directed mutagenesis and recovered clones with biallelic deletions in Lef1. Mutant cells exhibited reduced sensitivity to glycogen synthase kinase 3 inhibition during self-renewal. We then generated a non-disruptive knockin of dsRed at the Sox10 locus. Two clones produced germline chimeras. Comparative expression of dsRed and SOX10 validated the fidelity of the reporter. To illustrate utility, live imaging of dsRed in neonatal brain slices was employed to visualize oligodendrocyte lineage cells for patch-clamp recording. Overall, these results show that CRISPR/Cas9 gene editing technology in germline-competent rat ESCs is enabling for in vitro studies and for generating genetically modified rats

Signal requirement for cortical potential of transplantable human neuroepithelial stem cells.

The cerebral cortex develops from dorsal forebrain neuroepithelial progenitor cells. Following the initial expansion of the progenitor cell pool, these cells generate neurons of all the cortical layers and then astrocytes and oligodendrocytes. Yet, the regulatory pathways that control the expansion and maintenance of the progenitor cell pool are currently unknown. Here we define six basic pathway components that regulate proliferation of cortically specified human neuroepithelial stem cells (cNESCs) in vitro without the loss of cerebral cortex developmental potential. We show that activation of FGF and inhibition of BMP and ACTIVIN A signalling are required for long-term cNESC proliferation. We also demonstrate that cNESCs preserve dorsal telencephalon-specific potential when GSK3, AKT and nuclear CATENIN-β1 activity are low. Remarkably, regulation of these six pathway components supports the clonal expansion of cNESCs. Moreover, cNESCs differentiate into lower- and upper-layer cortical neurons in vitro and in vivo. The identification of mechanisms that drive the neuroepithelial stem cell self-renewal and differentiation and preserve this potential in vitro is key to developing regenerative and cell-based therapeutic approaches to treat neurological conditions

Nutritional regulation of oligodendrocyte differentiation regulates perineuronal net remodeling in the median eminence.

The mediobasal hypothalamus (arcuate nucleus, ARH and median eminence, ME) is a key nutrient sensing site for the production of the complex homeostatic feedback responses required for the maintenance of energy balance. Here we show that refeeding after an overnight fast rapidly triggers proliferation and differentiation of oligodendrocyte progenitors, leading to the production of new oligodendrocytes in the ME specifically. During this nutritional paradigm, ME perineuronal nets (PNN), emerging regulators of ARH metabolic functions, are rapidly remodelled, and this process requires myelin regulatory factor (Myrf) in oligodendrocyte progenitors. In genetically obese ob/ob mice, nutritional regulations of ME oligodendrocyte differentiation and PNN remodelling are blunted, and enzymatic digestion of local PNN increases food intake and weight gain. We conclude that MBH PNN are required for the maintenance of energy balance in lean mice and are remodelled in the adult ME via the nutritional control of oligodendrocyte differentiation