Corrigendum: The Dietary Flavonoid, Luteolin, Negatively Affects Neuronal Differentiation.

[This corrects the article DOI: 10.3389/fnmol.2019.00041.].

Glycine Regulates Neural Stem Cell Proliferation During Development via Lnx1-Dependent Notch Signaling.

During development of the zebrafish embryo, glycine signaling promotes the differentiation of neural stem cells (NSCs). We found that glycine signaling suppresses the expression of Ligand of Numb X1 (lnx1, Ligand of numb protein-x1), a gene of unknown function during NSC differentiation that is selectively expressed in the embryonic central nervous system (CNS). As a consequence, Numb levels were stabilized and Notch activity (measured as her4.1 expression) was reduced, promoting NSC differentiation. These consequent actions were blocked by knockdown of lnx1. In contrast, lnx1 overexpression increased NSC proliferation and led to defects of neural tube closure at the early stages of development. Thus, our data provide evidence that glycine/lnx1 signaling modulates NSC proliferation by regulation of Notch signaling.

The Dietary Flavonoid, Luteolin, Negatively Affects Neuronal Differentiation.

Luteolin, a polyphenolic plant flavonoid, has been attributed with numerous beneficial properties like anti-cancer, antioxidant, and anti-inflammatory action. Luteolin has been reported earlier to be neuroprotective in models of spinal cord injury and traumatic brain injury and also induces neurite outgrowth in PC12 cells. However, the effect of luteolin on early differentiation, which might be important for its beneficial effects, is unknown. In this report, we show that luteolin negatively affects early differentiation of embryonic stem cells, hampering the formation of embryoid bodies. At later stages of differentiation, luteolin specifically inhibits neuronal differentiation, where the expression of early neuronal markers is suppressed, whereas luteolin treatment does not inhibit expression of meso- and endodermal markers. Further, in a developing zebrafish model, luteolin treatment leads to fewer numbers of mitotic cells in the brain. These specific effects of luteolin on neuronal differentiation could possibly be due to its ability to inhibit the lysine acetyltransferase, p300, since the structurally closely related p300 non-inhibitor flavonoid, apigenin, does not inhibit neuronal differentiation. These results show that luteolin perturbs neuronal differentiation of embryonic stem cells.

Glycine Promotes the Survival of a Subpopulation of Neural Stem Cells.

Glycine is mainly known as an inhibitory neurotransmitter in adult mature neurons, regulating neuronal network activity in the central nervous system. In contrast, during embryogenesis glycine can act as an excitatory neurotransmitter and generates the first electrical signal in immature neurons. The roles and functional significance of this excitatory glycinergic activity during neurodevelopment are still unclear. Using the zebrafish embryo as a model, we previously showed that glycine regulates proliferation and differentiation of neural stem cells (NSCs) to interneurons. Moreover, we identified that glycine signaling in NSCs is associated with several common developmental pathways and surprisingly also the p53-related apoptosis. Here we investigated how glycine signaling regulates NSC survival. First, we showed by two approaches, acridine orange staining and active caspase 3 immunostaining that defects in glycine signaling induce an early and transient cell death, which was suppressed by knockdown of p53. Then, we developed an NSC transplantation strategy to directly assess NSC-autonomous development upon perturbing glycine signaling. In vivo time-lapse imaging showed that disruption of glycine signaling disturbed the normal NSC interkinetic nuclear migration, leading to cell cycle arrest and apoptosis. Finally, we analyzed two main subpopulations of NSCs, expressing either nestin or GFAP, by in situ labeling and in transgenic lines expressing GFP in either population. We found that disruption of glycine signaling induced a drastic and selective loss of nestin-positive (nestin+) NSCs, which was only partially rescued upon p53 knockdown. Taken together, our findings support a role of glycine signaling in promoting survival of the nestin+ NSC subpopulation early during development.

Transcriptomic Analysis of Purified Embryonic Neural Stem Cells from Zebrafish Embryos Reveals Signaling Pathways Involved in Glycine-Dependent Neurogenesis.

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.

Analysis of NUAK1 and NUAK2 expression during early chick development reveals specific patterns in the developing head.

Several human diseases are associated with the NUAK1 and NUAK2 genes. These genes encode kinases, members of the AMPK-related kinases (ARK) gene family. Both NUAK1 and NUAK2 are known targets of the serine threonine kinase LKB1, a tumor suppressor involved in regulating cell polarity. While much is known about their functions in disease, their expression pattern in normal development has not been extensively studied. Here, we present the expression patterns for NUAK1 and NUAK2 in the chick during early-stage embryogenesis, until day 3 (Hamburger and Hamilton stage HH20). Several embryonic structures, in particular the nascent head, showed distinct expression levels. NUAK1 expression was first detected at stage HH6 in the rostral neural folds. It was then expressed (HH7-11) throughout the encephalalon, predominantly in the telencephalon and mesencephalon. NUAK1 expression was also detected in the splanchnic endoderm area at HH8-10, and in the vitellin vein derived from this area, but not in the heart. NUAK2 expression was first detected at stage HH6 in the neural folds. It was then found throughout the encephalon at stage HH20. Particular attention was paid in this study to the dorsal ectoderm at stages HH7 and HH8, where a local deficit or accumulation of NUAK2 mRNA were found to correlate with the direction of curvature of the neural plate. This is the first description of NUAK1 and NUAK2 expression patterns in the chick during early development; it reveals non-identical expression profiles for both genes in neural development.