Abstract
How is the initial set of neurons correctly established during the development of the vertebrate central nervous system? In the embryo, glycine and GABA are depolarizing due the immature chloride gradient, which is only reversed to become hyperpolarizing later in post-natal development. We previously showed that glycine regulates neurogenesis via paracrine signaling that promotes calcium transients in neural stem cells (NSCs) and their differentiation into interneurons within the spinal cord of the zebrafish embryo. However, the subjacent molecular mechanisms are not yet understood. Our previous work suggests that early neuronal progenitors were not differentiating correctly in the developing spinal cord. As a result, we aimed at identifying the downstream molecular mechanisms involved specifically in NSCs during glycine-dependent embryonic neurogenesis. Using a gfap:GFP transgenic line, we successfully purified NSCs by fluorescence-activated cell sorting from whole zebrafish embryos and in embryos in which the glycine receptor was knocked down. The strength of this approach is that it focused on the NSC population while tackling the biological issue in an in vivo context in whole zebrafish embryos. After sequencing the transcriptome by RNA-sequencing, we analyzed the genes whose expression was changed upon disruption of glycine signaling and we confirmed the differential expression by independent RTqPCR assay. While over a thousand genes showed altered expression levels, through pathway analysis we identified 14 top candidate genes belonging to five different canonical signaling pathways (signaling by calcium, TGF-beta, sonic hedgehog, Wnt, and p53-related apoptosis) that are likely to mediate the promotion of neurogenesis by glycine.
Highlights
Neurogenesis is a crucial step in vertebrate development and it requires multiple and integrated signals throughout development to be achieved correctly (Urban and Guillemot, 2014; Mitrousis et al, 2015)
Since we aimed at studying the transcriptomic changes induced by glycine receptors (GlyRs) knockdown in neural stem cells (NSCs), we set up a protocol to purify this population of cells from whole zebrafish embryos
We aimed at identifying the molecular mechanisms involved in glycine-dependent neurogenesis during embryonic development, precisely in NSCs
Summary
Neurogenesis is a crucial step in vertebrate development and it requires multiple and integrated signals throughout development to be achieved correctly (Urban and Guillemot, 2014; Mitrousis et al, 2015). These neurotransmitters are mostly inhibitory in the adult neural system, they elicit an excitatory activity in the early embryo Contrary to their hyperpolarizing role in adult neurons, GABA and glycine result in a chloride-induced membrane depolarization in the embryo, which generates the earliest forms of electrical signaling in the immature nervous system. This switch in the electrical activity of these neurotransmitters has been correlated with the late expression of the neuron-specific and extrusive chloride/potassium co-transporter (KCC2), which decreases chloride load in mature neurons and underlies the hyperpolarisation of the cell when the glycine receptors (GlyRs) open. Mutations of glycine signaling, such as mutations in GlyRs (Pilorge et al, 2015) and KCC2 (Merner et al, 2015), have recently been implicated in autism, arguably impacting on neurogenesis
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