Motoshi Nagao

Combinatorial Roles of Olig2 and Neurogenin2 in the Coordinated Induction of Pan-Neuronal and Subtype-Specific Properties of Motoneurons

Distinct classes of neurons are generated at defined times and positions during development of the nervous system. It remains elusive how specification of neuronal identity coordinates with acquisition of pan-neuronal properties. Here we show that basic helix-loop-helix (bHLH) transcription factors Olig2 and Neurogenin2 (Ngn2) play vital roles in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Olig2 and Ngn2 are specifically coexpressed in motoneuron progenitors. Misexpression studies in chick demonstrate the specific, combinatorial actions of Olig2 and Ngn2 in motoneuron generation. Our results further revealed crossregulatory interactions between bHLH and homeodomain transcription factors in the specification of motoneurons. We suggest that distinct classes of transcription factors collaborate to generate motoneurons in the ventral neural tube.

Combinatorial actions of patterning and HLH transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing spinal cord

During development, the three major neural cell lineages, neurons, oligodendrocytes and astrocytes, differentiate in specific temporal orders at topologically defined positions. How the timing and position of their generation are coordinately regulated remains poorly understood. Here, we provide evidence that the transcription factors Pax6, Olig2 and Nkx2.2 (Nkx2-2), which define the positional identity of multipotent progenitors early in development, also play crucial roles in controlling the timing of neurogenesis and gliogenesis in the developing ventral spinal cord. We show that each of these factors has a unique ability to either enhance or inhibit the activities of the proneural helix-loop-helix (HLH) factors Ngn1 (Neurog1), Ngn2 (Neurog2), Ngn3 (Neurog3) and Mash1 (Ascl1), and the inhibitory HLH factors Id1 and Hes1, thereby regulating both the timing of differentiation of multipotent progenitors and their fate. Consistent with this, dynamic changes in their co-expression pattern in vivo are closely correlated to stage- and domain-specific generation of three neural cell lineages. We also show that genetic manipulations of their temporal expression patterns in mice alter the timing of differentiation of neurons and glia. We propose a molecular code model whereby the combinatorial actions of two classes of transcription factors coordinately regulate the domain-specific temporal sequence of neurogenesis and gliogenesis in the developing spinal cord.

Cross Talk between Notch and Growth Factor/Cytokine Signaling Pathways in Neural Stem Cells

ABSTRACT Precise control of proliferation and differentiation of multipotent neural stem cells (NSCs) is crucial for proper development of the nervous system. Although signaling through the cell surface receptor Notch has been implicated in many aspects of neural development, its role in NSCs remains elusive. Here we examined how the Notch pathway cross talks with signaling for growth factors and cytokines in controlling the self-renewal and differentiation of NSCs. Both Notch and growth factors were required for active proliferation of NSCs, but each of these signals was sufficient and independent of the other to inhibit differentiation of neurons and glia. Moreover, Notch signals could support the clonal self-renewing growth of NSCs in the absence of growth factors. This growth factor-independent action of Notch involved the regulation of the cell cycle and cell-cell interactions. During differentiation of NSCs, Notch signals promoted the generation of astrocytes in collaboration with ciliary neurotrophic factor and growth factors. Their cooperative actions were likely through synergistic phosphorylation of signal transducer and activator of transcription 3 on tyrosine at position 705 and serine at position 727. Our data suggest that distinct intracellular signaling pathways operate downstream of Notch for the self-renewal of NSCs and stimulation of astrogenesis.

Ascl1 is required for oligodendrocyte development in the spinal cord

Development of oligodendrocytes, myelin-forming glia in the central nervous system (CNS), proceeds on a protracted schedule. Specification of oligodendrocyte progenitors (OLPs) begins early in development, whereas their terminal differentiation occurs at late embryonic and postnatal periods. How these distinct steps are controlled remains unclear. Our previous study demonstrated an important role of the helix-loop-helix (HLH) transcription factor Ascl1 in early generation of OLPs in the developing spinal cord. Here, we show that Ascl1 is also involved in terminal differentiation of oligodendrocytes late in development. Ascl1-/- mutant mice showed a deficiency in differentiation of myelin-expressing oligodendrocytes at birth. In vitro culture studies demonstrate that the induction and maintenance of co-expression of Olig2 and Nkx2-2 in OLPs, and thyroid hormone-responsive induction of myelin proteins are impaired in Ascl1-/- mutants. Gain-of-function studies further showed that Ascl1 collaborates with Olig2 and Nkx2-2 in promoting differentiation of OLPs into oligodendrocytes in vitro. Overexpression of Ascl1, Olig2 and Nkx2-2 alone stimulated the specification of OLPs, but the combinatorial action of Ascl1 and Olig2 or Nkx2-2 was required for further promoting their differentiation into oligodendrocytes. Thus, Ascl1 regulates multiple aspects of oligodendrocyte development in the spinal cord.

Homeobox genes Gsx1 and Gsx2 differentially regulate telencephalic progenitor maturation

Homeobox genes Gsx1 and Gsx2 (formerly Gsh1 and Gsh2) are among the earliest transcription factors expressed in neuronal progenitors of the lateral ganglionic eminence (LGE) in the ventral telencephalon. Gsx2 is required for the early specification of LGE progenitor cells and recently has been shown to specify different LGE neuronal subtypes at distinct time points. In Gsx2 mutants, Gsx1 compensates, at least in part, for the loss of Gsx2 in the specification of LGE neuronal subtypes. Because no specific phenotype has been described in Gsx1 mutants, it is unclear what role this factor plays in the development of the ventral telencephalon. Here, we used a gain-of-function approach to express either Gsx1 or Gsx2 throughout the telencephalon and found that Gsx1 functions similarly to Gsx2 in the specification of LGE identity. However, our results show that Gsx1 and Gsx2 differentially regulate the maturation of LGE progenitors. Specifically, Gsx2 maintains LGE progenitors in an undifferentiated state, whereas Gsx1 promotes progenitor maturation and the acquisition of neuronal phenotypes, at least in part, through the down-regulation of Gsx2. These unique results indicate that the two closely related Gsx genes similarly regulate LGE patterning but oppositely control the balance between proliferation and differentiation in the neuronal progenitor pool.

Zbtb20 promotes astrocytogenesis during neocortical development

Multipotent neural precursor cells (NPCs) generate astrocytes at late stages of mammalian neocortical development. Many signalling pathways that regulate astrocytogenesis directly induce the expression of GFAP, a marker of terminally differentiated astrocytes. However, astrocyte specification occurs before GFAP expression and essential factors for the specification step have remained elusive. Here we show that Zbtb20 regulates astrocyte specification in the mouse neocortex. Zbtb20 is highly expressed in late-stage NPCs and their astrocytic progeny. Overexpression and knockdown of Zbtb20 promote and suppress astrocytogenesis, respectively, although Zbtb20 does not directly activate the Gfap promoter. Astrocyte induction by Zbtb20 is suppressed by knockdown of Sox9 or NFIA. Furthermore, in the astrocyte lineage, Zbtb20 directly represses the expression of Brn2, which encodes a protein necessary for upper-layer neuron specification. Zbtb20 is thus a key determinant of astrocytogenesis, in which it collaborates with Sox9 and NFIA, and acts in part through direct repression of Brn2 expression.

Paxillin is the target of c-Jun N-terminal kinase in Schwann cells and regulates migration.

During development of the peripheral nervous system (PNS), Schwann cells migrate along axons, wrapping individual axons to form a myelin sheath. This process is all mediated by the intercellular signaling between neurons and Schwann cells. As yet, little is known about the intracellular signaling mechanisms controlling these morphological changes including Schwann cell migration. We previously showed that c-Jun N-terminal kinase (JNK) plays a key role in Schwann cell migration before the initiation of myelination. Here we show that JNK, acting through phosphorylation of the cytoskeletal protein paxillin, regulates Schwann cell migration and that it mediates dorsal root ganglion (DRG) neuronal conditioned medium (CM). Phosphorylation of paxillin at the Ser-178 position, the JNK phosphorylation site, is observed following stimulation with neuronal CM. Phosphorylation is also detected as a result of stimulation with each of growth factors contained in neuronal CM. Knockdown of paxillin with the specific small interfering RNA (siRNA) inhibits migration. The reintroduction of paxillin reverses siRNA-mediated inhibition of migration, whereas paxillin harboring the Ser-178-to-Ala mutation fails to reverse it. In addition, while JNK binds to the N-terminal region (called LD1), the deletion of LD1 blocks migration. Together, JNK binds and phosphorylates paxillin to regulate Schwann cell migration, illustrating that paxillin provides one of the convergent points of intracellular signaling pathways controlling Schwann cell migration.

High Mobility Group Nucleosome‐Binding Family Proteins Promote Astrocyte Differentiation of Neural Precursor Cells

Astrocytes are the most abundant cell type in the mammalian brain and are important for the functions of the central nervous system. Although previous studies have shown that the STAT signaling pathway or its regulators promote the generation of astrocytes from multipotent neural precursor cells (NPCs) in the developing mammalian brain, the molecular mechanisms that regulate the astrocytic fate decision have still remained largely unclear. Here, we show that the high mobility group nucleosome‐binding (HMGN) family proteins, HMGN1, 2, and 3, promote astrocyte differentiation of NPCs during brain development. HMGN proteins were expressed in NPCs, Sox9+ glial progenitors, and GFAP+ astrocytes in perinatal and adult brains. Forced expression of either HMGN1, 2, or 3 in NPCs in cultures or in the late embryonic neocortex increased the generation of astrocytes at the expense of neurons. Conversely, knockdown of either HMGN1, 2, or 3 in NPCs suppressed astrocyte differentiation and promoted neuronal differentiation. Importantly, overexpression of HMGN proteins did not induce the phosphorylation of STAT3 or activate STAT reporter genes. In addition, HMGN family proteins did not enhance DNA demethylation and acetylation of histone H3 around the STAT‐binding site of the gfap promoter. Moreover, knockdown of HMGN family proteins significantly reduced astrocyte differentiation induced by gliogenic signal ciliary neurotrophic factor, which activates the JAK‐STAT pathway. Therefore, we propose that HMGN family proteins are novel chromatin regulatory factors that control astrocyte fate decision/differentiation in parallel with or downstream of the JAK‐STAT pathway through modulation of the responsiveness to gliogenic signals. Stem Cells 2014;32:2983–2997

Oligodendrocyte Progenitor Cells Directly Utilize Lactate for Promoting Cell Cycling and Differentiation

Oligodendrocyte progenitor cells (OPCs) undergo marked morphological changes to become mature oligodendrocytes, but the metabolic resources for this process have not been fully elucidated. Although lactate, a metabolic derivative of glycogen, has been reported to be consumed in oligodendrocytes as a metabolite, and to ameliorate hypomyelination induced by low glucose conditions, it is not clear about the direct contribution of lactate to cell cycling and differentiation of OPCs, and the source of lactate for remyelination. Therefore, we evaluated the effect of 1,4‐dideoxy‐1,4‐imino‐d‐arabinitol (DAB), an inhibitor of the glycogen catabolic enzyme glycogen phosphorylase, in a mouse cuprizone model. Cuprizone induced demyelination in the corpus callosum and remyelination occurred after cuprizone treatment ceased. This remyelination was inhibited by the administration of DAB. To further examine whether lactate affects proliferation or differentiation of OPCs, we cultured mouse primary OPC‐rich cells and analyzed the effect of lactate. Lactate rescued the slowed cell cycling induced by 0.4 mM glucose, as assessed by the BrdU‐positive cell ratio. Lactate also promoted OPC differentiation detected by monitoring the mature oligodendrocyte marker myelin basic protein, in the presence of both 36.6 mM and 0.4 mM glucose. Furthermore, these lactate‐mediated effects were suppressed by the reported monocarboxylate transporter inhibitor, α‐cyano‐4‐hydroxy‐cinnamate. These results suggest that lactate directly promotes the cell cycling rate and differentiation of OPCs, and that glycogen, one of the sources of lactate, contributes to remyelination in vivo. J. Cell. Physiol. 232: 986–995, 2017.

Chd7 collaborates with Sox2 to regulate activation of oligodendrocyte precursor cells after spinal cord injury

Oligodendrocyte precursor cells (OPCs) act as a reservoir of new oligodendrocytes (OLs) in homeostatic and pathological conditions. OPCs are activated in response to injury to generate myelinating OLs, but the underlying mechanisms remain poorly understood. Here, we show that chromodomain helicase DNA binding protein 7 (Chd7) regulates OPC activation after spinal cord injury (SCI). Chd7 is expressed in OPCs in the adult spinal cord and its expression is upregulated with a concomitant increase in Sox2 expression after SCI. OPC-specific ablation of Chd7 in injured mice leads to reduced OPC proliferation, the loss of OPC identity, and impaired OPC differentiation. Ablation of Chd7 or Sox2 in cultured OPCs shows similar phenotypes to those observed in Chd7 knock-out mice. Chd7 and Sox2 form a complex in OPCs and bind to the promoters or enhancers of the regulator of cell cycle (Rgcc) and protein kinase Cθ (PKCθ) genes, thereby inducing their expression. The expression of Rgcc and PKCθ is reduced in the OPCs of the injured Chd7 knock-out mice. In cultured OPCs, overexpression and knock-down of Rgcc or PKCθ promote and suppress OPC proliferation, respectively. Furthermore, overexpression of both Rgcc and PKCθ rescues the Chd7 deletion phenotypes. Chd7 is thus a key regulator of OPC activation, in which it cooperates with Sox2 and acts via direct induction of Rgcc and PKCθ expression. SIGNIFICANCE STATEMENT Spinal cord injury (SCI) leads to oligodendrocyte (OL) loss and demyelination, along with neuronal death, resulting in impairment of motor or sensory functions. Oligodendrocyte precursor cells (OPCs) activated in response to injury are potential sources of OL replacement and are thought to contribute to remyelination and functional recovery after SCI. However, the molecular mechanisms underlying OPC activation, especially its epigenetic regulation, remain largely unclear. We demonstrate here that the chromatin remodeler chromodomain helicase DNA binding protein 7 (Chd7) regulates the proliferation and identity of OPCs after SCI. We have further identified regulator of cell cycle (Rgcc) and protein kinase Cθ (PKCθ) as novel targets of Chd7 for OPC activation.