Yasuhiro Itoh

The Wnt/β-catenin pathway directs neuronal differentiation of cortical neural precursor cells

Neural precursor cells (NPCs) have the ability to self-renew and to give rise to neuronal and glial lineages. The fate decision of NPCs between proliferation and differentiation determines the number of differentiated cells and the size of each region of the brain. However, the signals that regulate the timing of neuronal differentiation remain unclear. Here, we show that Wnt signaling inhibits the self-renewal capacity of mouse cortical NPCs, and instructively promotes their neuronal differentiation. Overexpression of Wnt7a or of a stabilized form of β-catenin in mouse cortical NPC cultures induced neuronal differentiation even in the presence of Fgf2, a self-renewal-promoting factor in this system. Moreover, blockade of Wnt signaling led to inhibition of neuronal differentiation of cortical NPCs in vitro and in the developing mouse neocortex. Furthermore, theβ -catenin/TCF complex appears to directly regulate the promoter of neurogenin 1, a gene implicated in cortical neuronal differentiation. Importantly, stabilized β-catenin did not induce neuronal differentiation of cortical NPCs at earlier developmental stages, consistent with previous reports indicating self-renewal-promoting functions of Wnts in early NPCs. These findings may reveal broader and stage-specific physiological roles of Wnt signaling during neural development.

The Cyclin-dependent Kinase Inhibitors p57 and p27 Regulate Neuronal Migration in the Developing Mouse Neocortex

Neuronal precursors remain in the proliferative zone of the developing mammalian neocortex until after they have undergone neuronal differentiation and cell cycle arrest. The newborn neurons then migrate away from the proliferative zone and enter the cortical plate. The molecules that coordinate migration with neuronal differentiation have been unclear. We have proposed in this study that the cdk inhibitors p57 and p27 play a role in this coordination. We have found that p57 and p27 mRNA increase upon neuronal differentiation of neocortical neuroepithelial cells. Knockdown of p57 by RNA interference resulted in a significant delay in the migration of neurons that entered the cortical plate but did not affect neuronal differentiation. Knockdown of p27 also inhibits neuronal migration in the intermediate zone as well as in the cortical plate, as reported by others. We have also found that knockdown of p27 increases p57 mRNA levels. These results suggest that both p57 and p27 play essential roles in neuronal migration and may, in concert, coordinate the timing of neuronal differentiation, migration, and possibly cell cycle arrest in neocortical development.

α2-chimaerin controls neuronal migration and functioning of the cerebral cortex through CRMP-2

Disrupted cortical neuronal migration is associated with epileptic seizures and developmental delay. However, the molecular mechanism by which disruptions of early cortical development result in neurological symptoms is poorly understood. Here we report α2-chimaerin as a key regulator of cortical neuronal migration and function. In utero suppression of α2-chimaerin arrested neuronal migration at the multipolar stage, leading to accumulation of ectopic neurons in the subcortical region. Mice with such migration defects showed an imbalance between excitation and inhibition in local cortical circuitry and greater susceptibility to convulsant-induced seizures. We further show that α2-chimaerin regulates bipolar transition and neuronal migration through modulating the activity of CRMP-2, a microtubule-associated protein. These findings establish a new α2-chimaerin-dependent mechanism underlying neuronal migration and proper functioning of the cerebral cortex and provide insights into the pathogenesis of seizure-related neurodevelopmental disorders.

Scratch regulates neuronal migration onset via an epithelial-mesenchymal transition-like mechanism

During neocortical development, the neuroepithelial or neural precursor cells that commit to neuronal fate need to delaminate and start migration toward the pial surface. However, the mechanism that couples neuronal fate commitment to detachment from the neuroepithelium remains largely unknown. Here we show that Scratch1 and Scratch2, members of the Snail superfamily of transcription factors, are expressed upon neuronal fate commitment under the control of proneural genes and promote apical process detachment and radial migration in the developing mouse neocortex. Scratch-induced delamination from the apical surface was mediated by transcriptional repression of the adhesion molecule E-cadherin. These findings suggest that Scratch proteins constitute a molecular link between neuronal fate commitment and the onset of neuronal migration. On the basis of their similarity to proteins involved in the epithelial-mesenchymal transition (EMT), we propose that Scratch proteins mediate the conversion of neuroepithelial cells to migrating neurons or intermediate neuronal progenitors through an EMT-related mechanism.

Selective induction of neocortical GABAergic neurons by the PDK1-Akt pathway through activation of Mash1

Extracellular stimuli regulate neuronal differentiation and subtype specification during brain development, although the intracellular signaling pathways that mediate these processes remain largely unclear. We now show that the PDK1-Akt pathway regulates differentiation of telencephalic neural precursor cells (NPCs). Active Akt promotes differentiation of NPC into γ-aminobutyric acid-containing (GABAergic) but not glutamatergic neurons. Disruption of the Pdk1 gene or expression of dominant-negative forms of Akt suppresses insulin-like growth factor (IGF)-1 enhancement of NPC differentiation into neurons in vitro and production of neocortical GABAergic neurons in vivo. Furthermore, active Akt increased the protein levels and transactivation activity of Mash1, a proneural basic helix-loop-helix protein required for the generation of neocortical GABAergic neurons, and Mash1 was required for Akt-induced neuronal differentiation. These results have unveiled an unexpected role of the PDK1-Akt pathway: a key mediator of extracellular signals regulating the production of neocortical GABAergic neurons.

Application of long range j c-h resolved 2d spectroscopy (lrjr) in structural elucidation of natural products. The structure of oxirapentyn

Abstract The structure of a fungal metabolite, oxyrapentyn has been determined as shown in Fig. 7 by application of a new NMR technique, long range J C-H resolved 2D spectroscopy (LRJR).

Tcf3 represses Wnt-β-catenin signaling and maintains neural stem cell population during neocortical development.

During mouse neocortical development, the Wnt–β-catenin signaling pathway plays essential roles in various phenomena including neuronal differentiation and proliferation of neural precursor cells (NPCs). Production of the appropriate number of neurons without depletion of the NPC population requires precise regulation of the balance between differentiation and maintenance of NPCs. However, the mechanism that suppresses Wnt signaling to prevent premature neuronal differentiation of NPCs is poorly understood. We now show that the HMG box transcription factor Tcf3 (also known as Tcf7l1) contributes to this mechanism. Tcf3 is highly expressed in undifferentiated NPCs in the mouse neocortex, and its expression is reduced in intermediate neuronal progenitors (INPs) committed to the neuronal fate. We found Tcf3 to be a repressor of Wnt signaling in neocortical NPCs in a reporter gene assay. Tcf3 bound to the promoter of the proneural bHLH gene Neurogenin1 (Neurog1) and repressed its expression. Consistent with this, Tcf3 repressed neuronal differentiation and increased the self-renewal activity of NPCs. We also found that Wnt signal stimulation reduces the level of Tcf3, and increases those of Tcf1 (also known as Tcf7) and Lef1, positive mediators of Wnt signaling, in NPCs. Together, these results suggest that Tcf3 antagonizes Wnt signaling in NPCs, thereby maintaining their undifferentiated state in the neocortex and that Wnt signaling promotes the transition from Tcf3-mediated repression to Tcf1/Lef1-mediated enhancement of Wnt signaling, constituting a positive feedback loop that facilitates neuronal differentiation.

PDK1–Akt pathway regulates radial neuronal migration and microtubules in the developing mouse neocortex

Significance In the developing mammalian neocortex, neurons migrate a long distance from their birthplace to the positions where they form appropriate layers and networks, and dysregulation of this process has been implicated in brain malformation and neurological diseases. Given the fine correlation between temporal order of various sequentially generated neuronal cell types and their spatial distribution, migration speed needs to be tightly controlled to achieve correct neocortical layering, although the underlying mechanisms remain unclear. Here we show that the serine/threonine kinase Akt and its activator phosphoinositide-dependent protein kinase 1 (PDK1) regulate the speed of locomotion of mouse neocortical neurons through the cortical plate. Our data suggest that the PDK1–Akt axis regulates microtubule organization, in part by regulating the cytoplasmic dynein/dynactin complex, in migrating neurons. Neurons migrate a long radial distance by a process known as locomotion in the developing mammalian neocortex. During locomotion, immature neurons undergo saltatory movement along radial glia fibers. The molecular mechanisms that regulate the speed of locomotion are largely unknown. We now show that the serine/threonine kinase Akt and its activator phosphoinositide-dependent protein kinase 1 (PDK1) regulate the speed of locomotion of mouse neocortical neurons through the cortical plate. Inactivation of the PDK1–Akt pathway impaired the coordinated movement of the nucleus and centrosome, a microtubule-dependent process, during neuronal migration. Moreover, the PDK1–Akt pathway was found to control microtubules, likely by regulating the binding of accessory proteins including the dynactin subunit p150glued. Consistent with this notion, we found that PDK1 regulates the expression of cytoplasmic dynein intermediate chain and light intermediate chain at a posttranscriptional level in the developing neocortex. Our results thus reveal an essential role for the PDK1–Akt pathway in the regulation of a key step of neuronal migration.

Transcriptional coupling of neuronal fate commitment and the onset of migration.

During mammalian CNS development, when the neural precursor cells commit to the neuronal fate they must delaminate and migrate toward the pial surface in order to reach the appropriate final location. Thus, the coordination of delamination and fate commitment is important in creating the correct structure. Although previous studies have proposed that spindle orientation during mitosis plays a role in both delamination and fate commitment, thus coordinating these events, subsequent studies have challenged this model. Recent work has identified several transcriptional mechanisms associated with neurogenesis that inhibit cell adhesion of newborn neurons and intermediate neuronal progenitors, thereby triggering delamination and linking it with fate commitment.

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