Large-scale animal model study uncovers altered brain pH and lactate levels as a transdiagnostic endophenotype of neuropsychiatric disorders involving cognitive impairment.

Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer's disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

Organization of self-advantageous niche by neural stem/progenitor cells during development via autocrine VEGF-A under hypoxia.

BACKGROUND: Tissue stem cells are confined within a special microenvironment called niche. Stem cells in such a niche are supplied with nutrients and contacted by other cells to maintain their characters and also to keep or expand their population size. Besides, oxygen concentration is a key factor for stem cell niche. Adult neural stem/progenitor cells (NSPCs) are known to reside in a hypoxic niche. Oxygen concentration levels are lower in fetal organs including brain than maternal organs. However, how fetal NSPCs adapt to the hypoxic environment during brain development, particularly before pial and periventricular vessels start to invade the telencephalon, has not fully been elucidated. METHODS: NSPCs were prepared from cerebral cortices of embryonic day (E) 11.5 or E14.5 mouse embryos and were enriched by 4-day incubation with FGF2. To evaluate NSPC numbers, neurosphere formation assay was performed. Sparsely plated NSPCs were cultured to form neurospheres under the hypoxic (1% O2) or normoxic condition. VEGF-A secreted from NSPCs in the culture medium was measured by ELISA. VEGF-A expression and Hif-1a in the developing brain was investigated by in situ hybridization and immunohistochemistry. RESULTS: Here we show that neurosphere formation of embryonic NSPCs is dramatically increased under hypoxia compared to normoxia. Vegf-A gene expression and its protein secretion were both up-regulated in the NSPCs under hypoxia. Either recombinant VEGF-A or conditioned medium of the hypoxic NSPC culture enhanced the neurosphere forming ability of normoxic NSPCs, which was attenuated by a VEGF-A signaling inhibitor. Furthermore, in the developing brain, VEGF-A was strongly expressed in the VZ where NSPCs are confined. CONCLUSIONS: We show that NSPCs secret VEGF-A in an autocrine fashion to efficiently maintain themselves under hypoxic developmental environment. Our results suggest that NSPCs have adaptive potential to respond to hypoxia to organize self-advantageous niche involving VEGF-A when the vascular system is immature.

A Synthetic Polymer Scaffold Reveals the Self-Maintenance Strategies of Rat Glioma Stem Cells by Organization of the Advantageous Niche.

Cancer stem cells (CSCs) are believed to be maintained within a microenvironmental niche. Here we used polymer microarrays for the rapid and efficient identification of glioma CSC (GSC) niche mimicries and identified a urethane-based synthetic polymer, upon which two groups of niche components, namely extracellular matrices (ECMs) and iron are revealed. In cultures, side population (SP) cells, defined as GSCs in the rat C6 glioma cell line, are more efficiently sustained in the presence of their differentiated progenies expressing higher levels of ECMs and transferrin, while in xenografts, ECMs are supplied by the vascular endothelial cells (VECs), including SP cell-derived ones with distinctively greater ability to retain xenobiotics than host VECs. Iron is stored in tumor infiltrating host macrophages (Mφs), whose protumoral activity is potently enhanced by SP cell-secreted soluble factor(s). Finally, coexpression of ECM-, iron-, and Mφ-related genes is found to be predictive of glioma patients' outcome. Our polymer-based approach reveals the intrinsic capacities of GSCs, to adapt the environment to organize a self-advantageous microenvironment niche, for their maintenance and expansion, which redefines the current concept of anti-CSC niche therapy and has the potential to accelerate cancer therapy development. Stem Cells 2016;34:1151-1162.

Increase in GFAP-positive astrocytes in histone demethylase GASC1/KDM4C/JMJD2C hypomorphic mutant mice.

GASC1, also known as KDM4C/JMJD2C, is a histone demethylase for histone H3 lysine 9 (H3K9) and H3K36. In this study, we observed an increase of GFAP-positive astrocytes in the brain of Gasc1 hypomorphic mutant mice at 2-3 months of age, but not at postnatal day 14 and day 30 by immunohistochemistry. Increases of GFAP-positive astrocytes were widely observed in the forebrain and prominent in such regions as cerebral cortex, caudate putamen, amygdala and diencephalon, but not obvious in hippocampus. Taken together with our observations to be published elsewhere that Gasc1 hypomorphic mutant mice exhibit abnormal behaviors including hyperactivity, persistence and many types of learning and memory deficits and abnormal synaptic functions such as prolonged long-term potentiation, the increase in GFAP-positive astrocytes may help understand their phenotypes, because astrocytes are known to affect synaptic plasticity.

A growth-promoting signaling component cyclin D1 in neural stem cells has antiastrogliogenic function to execute self-renewal.

Self-renewing proliferation of neural stem cells (NSCs) is intimately linked to the inhibition of neuronal and glial differentiation, however, their molecular linkage has been poorly understood. We have proposed a model previously explaining partly this linkage, in which fibroblast growth factor 2 (FGF2) and Wnt signals cooperate to promote NSC self-renewal via ß-catenin accumulation, which leads to the promotion of proliferation by lymphoid enhancer factor (LEF)/T-cell factor (TCF)-mediated cyclin D1 expression and at the same time to the inhibition of neuronal differentiation by ß-catenin-mediated potentiation of Notch signaling. To fully understand the mechanisms underlying NSC self-renewal, it needs to be clarified how these growth factor signals inhibit glial differentiation as well. Here, we demonstrate that cyclin D1, a NSC growth promoting signaling component and also a common component of FGF2 and Wnt signaling pathways, inhibits astroglial differentiation of NSCs. Interestingly, this effect of cyclin D1 is mediated even though its cell cycle progression activity is blocked. Forced downregulation of cyclin D1 enhances astrogliogenesis of NSCs in culture and in vivo. We further demonstrate that cyclin D1 binds to STAT3, a transcription factor downstream of astrogliogenic cytokines, and suppresses its transcriptional activity on the glial fibrillary acidic protein (Gfap) gene. Taken together with our previous finding, we provide a novel molecular mechanism for NSC self-renewal in which growth promoting signaling components activated by FGF2 and Wnts inhibit neuronal and glial differentiation.

Wnt3a promotes hippocampal neurogenesis by shortening cell cycle duration of neural progenitor cells.

The effects of Wnt signaling on neural progenitor cells have been controversial. Activation of the canonical Wnt signaling pathway either promotes neural progenitor cell proliferation or accelerates their differentiation into postmitotic neurons. This study demonstrates that activation of the Wnt signaling pathway by itself induces neural progenitor cell proliferation but does not directly affect neuronal differentiation processes. To investigate whether Wnt signaling promotes expansion and/or differentiation of neural progenitor cells in the developing hippocampus, we prepared primary mouse hippocampal progenitors and treated them with Wnt3a in a chemically defined culture medium. Wnt3a increased the total number of cells, including the numbers of Ki67(+) proliferating cells and Tuj1(+) differentiated neurons. This result verified that Wnt3a promoted neural progenitor cell proliferation. Meanwhile, Wnt3a did not appear to actively enhance the neuronal differentiation process itself, because (1) the ratio of Tuj1(+) cells to the total cells, and (2) the ratio of BrdU(+) Tuj1(+) cells to the total BrdU(+) cells, were both comparable between cultures with or without Wnt3a. Indeed, Wnt3a caused no significant change in either cell survival or the proportion of symmetric and asymmetric cell divisions that directly affected neuron production. We finally demonstrated that the Wnt3a treatment simply shortened cell cycle duration of neural progenitor cells by 2.9 h. The accelerated cell cycle progression without affecting the ratio of symmetric/asymmetric cell divisions explains how Wnt signaling per se leads to the expansion of both proliferative cell population and differentiated neuronal cell population.

Involvement of the Hipk family in regulation of eyeball size, lens formation and retinal morphogenesis.

Members of the homeodomain-interacting protein kinase (HIPK) family are involved in various intracellular regulatory mechanisms. The present study focused on clarifying the functions of HIPK family members in ocular organization during late embryogenesis. HIPK1 and HIPK2 were expressed in the inner retina during late embryogenesis. Hipk1(+/-)Hipk2(-/-) mice had a greater frequency of small eyes with a lens deficiency and abnormally laminated and thickened retinas than did wild-type littermates. These data indicate that Hipk1 and Hipk2 are involved in regulation of eye size, lens formation and retinal lamination during late embryogenesis.

Stabilized beta-catenin functions through TCF/LEF proteins and the Notch/RBP-Jkappa complex to promote proliferation and suppress differentiation of neural precursor cells.

The proliferation and differentiation of neural precursor cells are mutually exclusive during brain development. Despite its importance for precursor cell self renewal, the molecular linkage between these two events has remained unclear. Fibroblast growth factor 2 (FGF2) promotes neural precursor cell proliferation and concurrently inhibits their differentiation, suggesting a cross talk between proliferation and differentiation signaling pathways downstream of the FGF receptor. We demonstrate that FGF2 signaling through phosphatidylinositol 3 kinase activation inactivates glycogen synthase kinase 3beta (GSK3beta) and leads to the accumulation of beta-catenin in a manner different from that in the Wnt canonical pathway. The nuclear accumulated beta-catenin leads to cell proliferation by activating LEF/TCF transcription factors and concurrently inhibits neuronal differentiation by potentiating the Notch1-RBP-Jkappa signaling pathway. beta-Catenin and the Notch1 intracellular domain form a molecular complex with the promoter region of the antineurogenic hes1 gene, allowing its expression. This signaling interplay is especially essential for neural stem cell maintenance, since the misexpression of dominant-active GSK3beta completely inhibits the self renewal of neurosphere-forming stem cells and prompts their neuronal differentiation. Thus, the GSK3beta/beta-catenin signaling axis regulated by FGF and Wnt signals plays a pivotal role in the maintenance of neural stem/precursor cells by linking the cell proliferation to the inhibition of differentiation.

Potentiation of astrogliogenesis by STAT3-mediated activation of bone morphogenetic protein-Smad signaling in neural stem cells.

Astrocytes play important roles in brain development and injury response. Transcription factors STAT3 and Smad1, activated by leukemia inhibitory factor (LIF) and bone morphogenetic protein 2 (BMP2), respectively, form a complex with the coactivator p300 to synergistically induce astrocytes from neuroepithelial cells (NECs) (K. Nakashima, M. Yanagisawa, H. Arakawa, N. Kimura, T. Hisatsune, M. Kawabata, K. Miyazono, and T. Taga, Science 284:479-482, 1999). However, the mechanisms that govern astrogliogenesis during the determination of the fate of neural stem cells remain elusive. Here we found that LIF induces expression of BMP2 via STAT3 activation and leads to the consequent activation of Smad1 to efficiently promote astrogliogenic differentiation of NECs. The BMP antagonist Noggin abrogated LIF-induced Smad1 activation and astrogliogenesis by inhibiting BMPs produced by NECs. NECs deficient in suppressor of cytokine signaling 3 (SOCS3), a negative regulator of STAT3, readily differentiated into astrocytes upon activation by LIF not only due to sustained activation of STAT3 but also because of the consequent activation of Smad1. Our study suggests a novel LIF-triggered positive regulatory loop that enhances astrogliogenesis.

Involvement of the Olig2 transcription factor in cholinergic neuron development of the basal forebrain.

Cholinergic neurons, which express choline acetyltransferase (ChAT), are a major neuron subset generated in the basal forebrain. Areas presumed to be sites of origin of cholinergic neurons are roughly demarcated by expression of Olig2, a basic helix-loop-helix transcription factor, which includes the medial ganglionic eminence, septal area, and anterior entopeduncular/preoptic area. In the present study, we examined the involvement of Olig2 in cholinergic differentiation. When the Olig2-expressing cells at E12.5 were permanently modified to express the lacZ or EGFP gene by tamoxifen-induced Cre-mediated recombination, the cells marked by reporter gene expression were widely distributed in the basal forebrain by E18.5, some of which expressed neuronal markers. We showed that a small number of cells were double-positive for ChAT and X-gal or EGFP in almost all cases. In addition, the number of ChAT+ cells was reduced to 60% in the Olig2 knockout mouse basal forebrain. No evidence of elevated apoptosis or reduced proliferation was observed in the knockout mouse forebrain. The present study provides the first direct evidence for involvement of the Olig2 gene in cholinergic differentiation in the basal forebrain.

Basic fibroblast growth factor endows dorsal telencephalic neural progenitors with the ability to differentiate into oligodendrocytes but not gamma-aminobutyric acidergic neurons.

Basic fibroblast growth factor (bFGF) is commonly used to enrich and maintain neural stem cells in vitro. Olig2 is an essential transcription factor for oligodendrocyte lineage specification and is expressed predominantly in ventral neuroepithelial cells in the medial and lateral ganglionic eminence (GE), where oligodendrocyte progenitors originate. Here we report significant induction of Olig2 expression in dorsal neuroepithelium-derived cells cultured in the presence of bFGF, in which Olig2-expressing cells were initially negligible. Among Olig2-expressing cells appearing after a 5-day treatment with bFGF, 99.8% coexpressed nestin. There was no significant difference in proliferation or apoptosis in dorsal and ventral neuroepithelial cultures in the presence of bFGF, suggesting that bFGF induces ectopic expression of Olig2 in dorsal "cortical" neuroepithelial cells. Similarly, expression of Mash1, another ventral neuroepithelial cell marker gene, was also induced in cultured dorsal neuroepithelial cells in the presence of bFGF. Conversely, in this culture, expression of dorsal neuroepithelial cell markers, such as Neurogenin1, Neurogenin2, Pax6, and Emx2, was down-regulated. These results suggested a possible ventralizing activity of bFGF. In fact, bFGF-treated dorsal neuroepithelial cells acquired the potential to generate O4-positive oligodendrocytes with efficacy comparable to that observed with GE-derived cells. In marked contrast, bFGF did not enable dorsal neuroepithelial cells to generate gamma-aminobutyric acid (GABA) neurons, which normally develop only from GE in vivo. Thus, bFGF endows dorsal telencephalic neural progenitors with the ability to differentiate into oligodendrocytes but not GABAergic neurons, suggesting the presence of different mechanisms governing specification of dorsoventral cell identities of neuronal and glial cell lineages.

Direct evidence that ventral forebrain cells migrate to the cortex and contribute to the generation of cortical myelinating oligodendrocytes.

Cortical neuroepithelial cells generate neurons, astrocytes, and oligodendrocytes (OLs) in vitro. However, whether cortical OLs are derived from the cortical neuroepithelium or migrate from the ventral forebrain is under severe debate yet. This is due to the fact that OL progenitor cells (OPCs), as marked by the expression of PDGFRalpha or NG2, are generated at around embryonic day (E) 11 or 12 in the mouse ganglionic eminences, but the myelinating OLs appear during the second week postnatally in the cortex. There has been no labeling method for long-term glial cell-lineage tracing. Thus, we developed a new strategy: plasmid DNA encoding Cre recombinase was introduced into the Cre/loxP reporter forebrain in ventral- or dorsal-specific manner by in utero DNA electroporation. The reporter gfp gene is expressed permanently owing to the chromosomal DNA recombination. The GFP-labeled myelinating OLs were detected in the adult cortex when electroporation was targeted to the ventral neuroepithelium, demonstrating at least some of the myelinating OLs are derived from the ventral forebrain. However, when electroporation was targeted to the dorsal, we could not find GFP-labeled myelinating OLs. This suggests that the progenitors of cortical OPCs are absent or located at restricted regions in the dorsal forebrain (cortex) at E12.

Activation of canonical Wnt pathway promotes proliferation of retinal stem cells derived from adult mouse ciliary margin.

Adult retinal stem cells represent a possible cell source for the treatment of retinal degeneration. However, only a small number of stem cells reside in the ciliary margin. The present study aimed to promote the proliferation of adult retinal stem cells via the Wnt signaling pathway. Ciliary margin cells from 8-week-old mice were dissociated and cultured to allow sphere colony formation. Wnt3a, a glycogen synthase kinase (GSK) 3 inhibitor, fibroblast growth factor (FGF) 2, and a FGF receptor inhibitor were then applied in the culture media. The primary spheres were dissociated to prepare either monolayer or secondary sphere cultures. Wnt3a increased the size of the primary spheres and the number of Ki-67-positive proliferating cells in monolayer culture. The Wnt3a-treated primary sphere cells were capable of self-renewal and gave rise to fourfold the number of secondary spheres compared with nontreated sphere cells. These cells also retained their multilineage potential to express several retinal markers under differentiating culture conditions. The Wnt3a-treated cells showed nuclear accumulation of beta-catenin, and a GSK3 inhibitor, SB216763, mimicked the mitogenic activity of Wnt3a. The proliferative effect of SB216763 was attenuated by an FGF receptor inhibitor but was enhanced by FGF2, with Ki-67-positive cells reaching over 70% of the total cells. Wnt3a and SB216763 promoted the proliferation of retinal stem cells, and this was partly dependent on FGF2 signaling. A combination of Wnt and FGF signaling may provide a therapeutic strategy for in vitro expansion or in vivo activation of adult retinal stem cells.

Fetal pituitary gonadotropin as an initial target of dioxin in its impairment of cholesterol transportation and steroidogenesis in rats.

Reproductive and developmental disorders are the most sensitive toxic effects caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is thought to produce many, if not all, of these toxic effects by impairing steroidogenesis and/or steroid action during the prenatal or early postnatal stages. However, the mechanism of the antisex steroid effect of TCDD is not well understood. This study revealed that steroidogenic acute-regulatory protein (StAR), a key transporter of cholesterol for steroidogenesis, in the testes of fetal rats are down-regulated by maternal exposure to TCDD. It was also shown that many mRNAs of steroidogenetic enzymes, including cytochromes P450 11A1, 17, and 11B1 and 3beta-hydroxysteroid dehydrogenase, are reduced in fetuses of TCDD-treated dams in a testis-specific manner. The same was also observed for the expression of estrogen-alpha receptors and androgen receptors. Whereas StAR expression was not affected by TCDD in cultured fetal testis, the fetal serum content of LH, a pituitary regulator of StAR, was significantly reduced by TCDD. In agreement with this, pituitary expression of LHbeta subunit mRNA in fetuses was reduced by maternal exposure to TCDD, whereas the alpha-subunit remained unchanged. The reduction in LHbeta is suggested to occur by a mechanism different from the reduction in the GnRH level. Direct supply of exogenous gonadotropin to TCDD-exposed fetuses completely abolished the reduction of StAR expression. Taken together, these results demonstrate that TCDD impairs steroidogenesis in the fetus by targeting pituitary gonadotropins.

Wnt signaling controls the timing of oligodendrocyte development in the spinal cord.

During spinal cord development, oligodendrocytes are generated from a restricted region of the ventral ventricular zone and then spread out into the entire spinal cord. These events are controlled by graded inductive and repressive signals derived from a local organizing center. Sonic hedgehog was identified as an essential ventral factor for oligodendrocyte lineage specification, whereas the dorsal cue was less clear. In this study, Wnt proteins were identified as the dorsal factors that directly inhibit oligodendrocyte development. Wnt signaling through a canonical beta-catenin pathway prevents its differentiation from progenitor to an immature state. Addition of rmFz-8/Fc, a Wnt antagonist, increased the number of immature oligodendrocytes in the spinal cord explant culture, demonstrating that endogenous Wnt signaling controls oligodendrocyte development.

Control of axonophilic migration of oligodendrocyte precursor cells by Eph-ephrin interaction.

The migration of oligodendrocyte precursor cells (OPCs) is modulated by secreted molecules in their environment and by cell-cell and matrix-cell interactions. Here, we ask whether membrane-anchored guidance cues, such as the ephrin ligands and their Eph receptors, participate in the control of OPC migration in the optic nerve. We postulate that EphA and EphB receptors, which are expressed on axons of retinal ganglion cells, interact with ephrins on the surface of OPCs. We show the expression of ephrinA5, ephrinB2 and ephrinB3 in the migrating OPCs of the optic nerve as well as in the diencephalic sites from where they originate. In addition, we demonstrate that coated EphB2-Fc receptors, which are specific for ephrinB2/B3 ligands, induce dramatic changes in the contact and migratory properties of OPCs, indicating that axonal EphB receptors activate ephrinB signaling in OPCs.Based on these findings, we propose that OPCs are characterized by an ephrin code, and that Eph-ephrin interactions between axons and OPCs control the distribution of OPCs in the optic axonal tracts, and the progress and arrest of their migration.

Evidence for a second wave of oligodendrogenesis in the postnatal cerebral cortex of the mouse.

The existing view is that cortical oligodendrocytes (OLs) in rodents are born from the cortical subventricular zone (SVZ) after birth, but recent data suggest that many forebrain oligodendrocyte progenitor cells (OPCs) are specified much earlier (between E9.5 and E13.5 in the mouse) in the ventricular zone of the ventral forebrain under the control of sonic hedgehog (Shh) and migrate into the cortex afterward. We examined expression of specific early OL markers (PDGFRalpha, PLP/DM20, Olig2, and NG2) in the developing forebrain to clarify this issue. We propose that OPCs colonize the developing cortex in two temporally distinct waves. The gray matter is at least partially populated by a first wave of OPCs that arises in the medial ganglionic eminence and the entopeduncular area and spreads into the cortex via the developing cortical plate. The cerebral cortex benefits from the second wave of OPCs coming from residential SVZ. In the second wave, there might be two different types of precursor cells: PLP/DM20(+) cells populating only inner layers and PDGFRalpha(+) cells, which might eventually myelinate the outer regions as well.

Dysregulation of axonal sodium channel isoforms after adult-onset chronic demyelination.

Demyelination results in conduction block through changes in passive cable properties of an axon and in the expression and localization of axonal ion channels. We show here that adult-onset chronic demyelination, such as occurs in demyelinating disorders and after nerve injury, alters the complement of axonal voltage-dependent Na+ (Nav) channel isoforms and their localization. As a model, we used heterozygous transgenic mice with two extra copies of the proteolipid protein gene (Plp/-). Retinal ganglion cell axons in these mice myelinate normally, with young Plp/- and wild-type mice expressing Nav1.2 at low levels, whereas Nav1.6 is clustered in high densities at nodes of Ranvier. At 7 months of age, however, Plp/- mice exhibit severe demyelination and oligodendrocyte cell death, leading to a profound reduction in Nav1.6 clusters, loss of the paranodal axoglial apparatus, and a marked increase in Nav1.2. We conclude that myelin is crucial not only for node of Ranvier formation, but also to actively maintain the proper localization and complement of distinct axonal Nav channel isoforms throughout life. The altered Nav channel isoform localization and complement induced by demyelination may contribute to the pathophysiology of demyelinating disorders and nerve injury.

[Regulation of development of oligodendrocyte].

Oligodendrocyte (OL) progenitors in the mouse spinal cord are generated from a strictly restricted region in the ventricular zone of the ventral cord as early as on embryonic day 11 (E11). We previously reported that one of the factors that restrict this ventral specific appearance of OLs is an inhibitory factor secreted from the dorsal spinal cord, in addition to well-known stimulatory ventral factors such as sonic hedgehog. We characterized the developmental change of the inhibitory activities. They were very strong at E11, gradually reduced, and disappeared by E14. This pattern seemed to be well correlated with the developmental profile of Wnt3a expression at/near the roof plate. A conditioned medium of L cells that stably express Wnt3a showed significant reduction of O4 positive OLs in the ventral spinal cord explants, indicating that Wnt3a is one of the dorsal factors that inhibit OL development. Addition of Wnt3a supernatant to CG4 cells, an OL progenitor strain, and to the dissociated primary cultured cells suggested that Wnt3a directly acts on OL lineage cells and inhibits a differentiation step from OL progenitor to O4-positive stage. Thus, Wnt3a may directly control the timing of OL differentiation and the motility of OL lineage cells. A population of myelinating OLS in the dorsal area of telencephalon was further demonstrated to be ventral origin by the newly established cell marking system using in utero DNA electroporation.