Origins of oligodendrocytes in the cerebellum, whose development is controlled by the transcription factor, Sox9.

Development of oligodendrocytes, myelin-forming glia in the central nervous system (CNS), proceeds on a protracted schedule. Specification of oligodendrocyte progenitor cells (OPCs) begins early in development, whereas their terminal differentiation occurs at late embryonic and postnatal periods. However, for oligodendrocytes in the cerebellum, the developmental origins and the molecular machinery to control these distinct steps remain unclear. By in vivo fate mapping and immunohistochemical analyses, we obtained evidence that the majority of oligodendrocytes in the cerebellum originate from the Olig2-expressing neuroepithelial domain in the ventral rhombomere 1 (r1), while about 6% of cerebellar oligodendrocytes are produced in the cerebellar ventricular zone. Furthermore, to elucidate the molecular determinants that regulate their development, we analyzed mice in which the transcription factor Sox9 was specifically ablated from the cerebellum, ventral r1 and caudal midbrain by means of the Cre/loxP recombination system. This resulted in a delay in the birth of OPCs and subsequent developmental aberrations in these cells in the Sox9-deficient mice. In addition, we observed altered proliferation of OPCs, resulting in a decrease in oligodendrocyte numbers that accompanied an attenuation of the differentiation and an increased rate of apoptosis. Results from in vitro assays using oligodendrocyte-enriched cultures further supported our observations from in vivo experiments. These data suggest that Sox9 participates in the development of oligodendrocytes in the cerebellum, by regulating the timing of their generation, proliferation, differentiation and survival.

Temporal identity transition from Purkinje cell progenitors to GABAergic interneuron progenitors in the cerebellum.

In the cerebellum, all GABAergic neurons are generated from the Ptf1a-expressing ventricular zone (Ptf1a domain). However, the machinery to produce different types of GABAergic neurons remains elusive. Here we show temporal regulation of distinct GABAergic neuron progenitors in the cerebellum. Within the Ptf1a domain at early stages, we find two subpopulations; dorsally and ventrally located progenitors that express Olig2 and Gsx1, respectively. Lineage tracing reveals the former are exclusively Purkinje cell progenitors (PCPs) and the latter Pax2-positive interneuron progenitors (PIPs). As development proceeds, PCPs gradually become PIPs starting from ventral to dorsal. In gain- and loss-of-function mutants for Gsx1 and Olig1/2, we observe abnormal transitioning from PCPs to PIPs at inappropriate developmental stages. Our findings suggest that the temporal identity transition of cerebellar GABAergic neuron progenitors from PCPs to PIPs is negatively regulated by Olig2 and positively by Gsx1, and contributes to understanding temporal control of neuronal progenitor identities.

Inhibitory and excitatory subtypes of cochlear nucleus neurons are defined by distinct bHLH transcription factors, Ptf1a and Atoh1.

The cochlear nucleus (CN), which consists of dorsal and ventral cochlear nuclei (DCN and VCN), plays pivotal roles in processing and relaying auditory information to the brain. Although it contains various types of neurons, the origins of the distinct subtypes and their developmental molecular machinery are still elusive. Here we reveal that two basic helix-loop-helix transcription factors play crucial roles in specifying neuron subtypes in the CN. Pancreatic transcription factor 1a (Ptf1a) and atonal homolog 1 (Atoh1) were found to be expressed in discrete dorsolateral regions of the embryonic neuroepithelia of the middle hindbrain (rhombomeres 2-5). Genetic lineage tracing using mice that express Cre recombinase from the Ptf1a locus or under the control of the Atoh1 promoter revealed that inhibitory (GABAergic and glycinergic) or excitatory (glutamatergic) neurons of both DCN and VCN are derived from the Ptf1a- and Atoh1-expressing neuroepithelial regions, respectively. In the Ptf1a or Atoh1 null embryos, production of inhibitory or excitatory neurons, respectively, was severely inhibited in the CN. These findings suggest that inhibitory and excitatory subtypes of CN neurons are defined by Ptf1a and Atoh1, respectively and, furthermore, provide important insights into understanding the machinery of neuron subtype specification in the dorsal hindbrain.

The PI3K-Akt pathway promotes microtubule stabilization in migrating fibroblasts.

Directed cell migration is controlled by extracellular cues such as growth factors/chemokines and extracellular matrix. In a migrating cell, a subset of microtubules becomes stabilized, and this stabilization is implicated in the establishment and maintenance of cell polarity. It is still not fully understood, however, how extracellular cues regulate the dynamics of microtubules. Here we show that the PI3K-Akt signaling pathway plays a pivotal role in growth factor regulation of microtubule stability. Treatment of NIH 3T3 fibroblasts with platelet-derived growth factor (PDGF) increases the amount of stabilized microtubules, and this increase is abrogated by the addition of a PI3K inhibitor or by expression of a dominant-negative form of Akt (DN-Akt), but not by the addition of a MEK inhibitor. Expression of an active form of Akt slightly increases the bulk amount of stabilized microtubules. Stabilization of microtubules induced in edge cells in the wounded monolayer culture is also attenuated by the PI3K inhibitor treatment or by expression of DN-Akt. Given that Akt is activated at the leading edge of a migrating cell and plays an essential role in directed cell migration, these results reveal a novel mechanism linking extracellular cues to directed cell migration, namely Akt regulation of microtubule stability.

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.

Non-cell-autonomous action of STAT3 in maintenance of neural precursor cells in the mouse neocortex.

The transcription factor STAT3 promotes astrocytic differentiation of neural precursor cells (NPCs) during postnatal development of the mouse neocortex, but little has been known of the possible role of STAT3 in the embryonic neocortex. We now show that STAT3 is expressed in NPCs of the mouse embryonic neocortex and that the JAK-STAT3 signaling pathway plays an essential role in the maintenance of NPCs by fibroblast growth factor 2. Conditional deletion of the STAT3 gene in NPCs reduced their capacity to form neurospheres in vitro, as well as promoted neuronal differentiation both in vitro and in vivo. Furthermore, STAT3 was found to maintain NPCs in the undifferentiated state in a non-cell-autonomous manner. STAT3-dependent expression of the Notch ligand Delta-like1 (DLL1) appears to account for the non-cell-autonomous effect of STAT3 on NPC maintenance, as knockdown of DLL1 by RNA interference or inhibition of Notch activation with a gamma-secretase inhibitor abrogated the enhancement of neurosphere formation by STAT3. Our results reveal a previously unrecognized mechanism of interaction between the JAK-STAT3 and DLL1-Notch signaling pathways, as well as a pivotal role for this interaction in maintenance of NPCs during early neocortical development.

JNK antagonizes Akt-mediated survival signals by phosphorylating 14-3-3.

Life and death decisions are made by integrating a variety of apoptotic and survival signals in mammalian cells. Therefore, there is likely to be a common mechanism that integrates multiple signals adjudicating between the alternatives. In this study, we propose that 14-3-3 represents such an integration point. Several proapoptotic proteins commonly become associated with 14-3-3 upon phosphorylation by survival-mediating kinases such as Akt. We reported previously that cellular stresses induce c-Jun NH2-terminal kinase (JNK)-mediated 14-3-3zeta phosphorylation at Ser184 (Tsuruta, F., J. Sunayama, Y. Mori, S. Hattori, S. Shimizu, Y. Tsujimoto, K. Yoshioka, N. Masuyama, and Y. Gotoh. 2004. EMBO J. 23:1889-1899). Here, we show that phosphorylation of 14-3-3 by JNK releases the proapoptotic proteins Bad and FOXO3a from 14-3-3 and antagonizes the effects of Akt signaling. As a result of dissociation, Bad is dephosphorylated and translocates to the mitochondria, where it associates with Bcl-2/Bcl-x(L). Because Bad and FOXO3a share the 14-3-3-binding motif with other proapoptotic proteins, we propose that this JNK-mediated phosphorylation of 14-3-3 regulates these proapoptotic proteins in concert and makes cells more susceptible to apoptotic signals.

Xenopus ILK (integrin-linked kinase) is required for morphogenetic movements during gastrulation.

It has been suggested that ILK (integrin-linked kinase) participates in integrin- and growth factor-mediated signaling pathways and also functions as a scaffold protein at cell-extracellular matrix (ECM) adhesion sites. As the recently reported ILK knockout mice were found to die at the peri-implantation stage, the stage specific to mammals, little is known about the function of ILK in early developmental processes common to every vertebrate. To address this, we isolated a Xenopus ortholog of ILK (XeILK) and characterized its role in early Xenopus embryogenesis. XeILK was expressed constitutively and ubiquitously throughout the early embryogenesis. Depletion of XeILK with morpholino oligonucleotides (XeILK MO) caused severe defects in blastopore closure and axis elongation without affecting the mesodermal specification. Furthermore, XeILK MO was found to interfere with cell-cell and cell-ECM adhesions in dorsal marginal zone explants and to result in a significant loss of cell-ECM adhesions in activin-treated dissociated animal cap cells. These results thus indicate that XeILK plays an essential role in morphogenetic movements during gastrulation.

Notch promotes survival of neural precursor cells via mechanisms distinct from those regulating neurogenesis.

During development of the mammalian brain, many neural precursor cells (NPCs) undergo apoptosis. The regulation of such cell death, however, is poorly understood. We now show that the survival of mouse embryonic NPCs in vitro was increased by culture at a high cell density and that this effect was attributable to activation of Notch signaling. Expression of an active form of Notch1 thus markedly promoted NPC survival. Hes proteins, key effectors of Notch signaling in inhibition of neurogenesis, were not sufficient for the survival-promoting effect of Notch1. This effect of Notch1 required a region of the protein containing the RAM domain and was accompanied by up-regulation of the anti-apoptotic proteins Bcl-2 and Mcl-1. Moreover, knockdown of these proteins by RNA interference resulted in blockade of the Notch1-induced survival. These results reveal a new function of Notch, the promotion of NPC survival.

The Sox-2 regulatory regions display their activities in two distinct types of multipotent stem cells.

The Sox-2 gene is expressed in embryonic stem (ES) cells and neural stem cells. Two transcription enhancer regions, Sox-2 regulatory region 1 (SRR1) and SRR2, were described previously based on their activities in ES cells. Here, we demonstrate that these regulatory regions also exert their activities in neural stem cells. Moreover, our data reveal that, as in ES cells, both SRR1 and SRR2 show their activities rather specifically in multipotent neural stem or progenitor cells but cease to function in differentiated cells, such as postmitotic neurons. Systematic deletion and mutation analyses showed that the same or at least overlapping DNA elements of SRR2 are involved in its activity in both ES and neural stem or progenitor cells. Thus, SRR2 is the first example of an enhancer in which a single regulatory core sequence is involved in multipotent-state-specific expression in two different stem cells, i.e., ES and neural stem cells.

The Wnt/beta-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 beta-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, the beta-catenin/TCF complex appears to directly regulate the promoter of neurogenin 1, a gene implicated in cortical neuronal differentiation. Importantly, stabilized beta-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.

Hes binding to STAT3 mediates crosstalk between Notch and JAK-STAT signalling.

Although the Notch and JAK-STAT signalling pathways fulfill overlapping roles in growth and differentiation regulation, no coordination mechanism has been proposed to explain their relationship. Here we show that STAT3 is activated in the presence of active Notch, as well as the Notch effectors Hes1 and Hes5. Hes proteins associate with JAK2 and STAT3, and facilitate complex formation between JAK2 and STAT3, thus promoting STAT3 phosphorylation and activation. Furthermore, suppression of endogenous Hes1 expression reduces growth factor induction of STAT3 phosphorylation. STAT3 seems to be essential for maintenance of radial glial cells and differentiation of astrocytes by Notch in the developing central nervous system. These results suggest that direct protein-protein interactions coordinate cross-talk between the Notch-Hes and JAK-STAT pathways.

JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins.

Targeted gene disruption studies have established that the c-Jun NH2-terminal kinase (JNK) is required for the stress-induced release of mitochondrial cytochrome c and apoptosis, and that the Bax subfamily of Bcl-2-related proteins is essential for JNK-dependent apoptosis. However, the mechanism by which JNK regulates Bax has remained unsolved. Here we demonstrate that activated JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3, a cytoplasmic anchor of Bax. Phosphorylation of 14-3-3 led to dissociation of Bax from this protein. Expression of phosphorylation-defective mutants of 14-3-3 blocked JNK-induced Bax translocation to mitochondria, cytochrome c release and apoptosis. Collectively, these results have revealed a key mechanism of Bax regulation in stress-induced apoptosis.

Adenosine A2A receptor facilitates calcium-dependent protein secretion through the activation of protein kinase A and phosphatidylinositol-3 kinase in PC12 cells.

Adenosine modulates a variety of cellular functions including calcium-dependent exocytosis. Activation of adenosine A(2A) receptor (A(2A)-R) facilitates neurotransmitter release in some cell types, although the underlying mechanisms are not fully understood. In this study, we found that treatment of PC12 cells with the A(2A)-R agonist CGS21680 promotes calcium-evoked secretion of the fusion protein between neuropeptide Y and modified yellow fluorescence protein (NPY-Venus). CGS21680 treatment of PC12 cells transiently increased the phosphorylation of p38 and JNK MAP kinases and Akt, as well as that of ATF2 and CREB, reaching maximal levels at around 10-15 min of CGS21680 treatment. Importantly, pretreatment of PC12 cells with the PI3K inhibitor LY294002, together with the protein kinase A (PKA) inhibitor KT5720, significantly inhibited CGS21680 enhancement of calcium-dependent NPY-Venus release. Moreover, expression of a dominant-negative form of Akt and the PKA inhibitory polypeptide protein kinase inhibitor (PKI) co-operatively inhibited the facilitating effect of CGS21680 on secretion of NPY-Venus. These data suggest that the PI3K-Akt and PKA pathways play a critical role in A(2A)-R-mediated facilitation of calcium-dependent secretion. We also found that CGS21680 treatment promoted recruitment of the NPY-Venus-containing vesicles to the proximity of the plasma membrane at around 10-15 min of CGS21680 treatment, which may in part account for the facilitated secretion by A(2A)-R activation.

The phosphatidylinositol-3 kinase (PI3K)-Akt pathway suppresses neurite branch formation in NGF-treated PC12 cells.

BACKGROUND: Previous studies have shown that phosphatidylinositol-3 kinase (PI3K) plays an important role in NGF (nerve growth factor)-induced neurite elongation. However, the roles of the PI3K pathway in neurite branch formation were not fully understood. Also, it was not clear where the PI3K pathway is activated during branch formation. RESULTS: We found that the treatment of PC12 cells with the PI3K inhibitor LY294002 resulted in a marked increase in the number of neurite branch points, suggesting a suppressive role of PI3K in neurite branch formation. Expression of a constitutively active form of Akt, a downstream effector of PI3K, decreased the number of branch points, whereas that of a dominant-negative form of Akt increased it. In contrast, inhibition of neither Rac, mTOR nor GSK3, other effectors of PI3K, promoted branch formation. Importantly, the phosphorylated form of endogenous Akt was localized at the tips of growth cones, but devoid of small branches in NGF-treated PC12 cells. A GFP-fusion protein of the plekstrin-homology (PH) domain of Akt was also localized at the tips of growth cones. CONCLUSIONS: The PI3K-Akt pathway thus plays a key role in suppression of neurite branch formation in NGF-treated PC12 cells.

Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions.

Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms.

Essential role of the transcription factor Ets-2 in Xenopus early development.

The fibroblast growth factor (FGF)/MAPK pathway plays an important role in early Xenopus developmental processes, including mesoderm patterning. The activation of the MAPK pathway leads to induction of Xenopus Brachyury (Xbra), which regulates the transcription of downstream mesoderm-specific genes in mesoderm patterning. However, the link between the FGF/MAPK pathway and the induction of Xbra has not been fully understood. Here we present evidence suggesting that Ets-2 is involved in the induction of Xbra and thus in the development of posterior mesoderm during early embryonic development. Overexpression of Ets-2 caused posteriorized embryos and led to the induction of mesoderm in ectodermal explants. Expression of a dominant-negative form of Ets-2 or injection of antisense morpholino oligonucleotides against Ets-2 inhibited the formation of the trunk and tail structures. Overexpression of Ets-2 resulted in the induction of Xbra, and expression of the dominant-negative Ets-2 inhibited FGF- or constitutively active MEK-induced Xbra expression. Moreover, overexpression of Ets-2 up-regulated the transcription from Xbra promoter reporter gene constructs. Ets-2 bound to the Xbra promoter region in vitro. These results taken together indicate that Xenopus Ets-2 plays an essential role in mesoderm patterning, lying between the FGF/MAPK pathway and the Xbra transcription.

Akt enhances Mdm2-mediated ubiquitination and degradation of p53.

p53 plays a key role in DNA damage-induced apoptosis. Recent studies have reported that the phosphatidylinositol 3-OH-kinase-Akt pathway inhibits p53-mediated transcription and apoptosis, although the underlying mechanisms have yet to be determined. Mdm2, a ubiquitin ligase for p53, plays a central role in regulation of the stability of p53 and serves as a good substrate for Akt. In this study, we find that expression of Akt reduces the protein levels of p53, at least in part by enhancing the degradation of p53. Both Akt expression and serum treatment induced phosphorylation of Mdm2 at Ser186. Akt-mediated phosphorylation of Mdm2 at Ser186 had little effect on the subcellular localization of Mdm2. However, both Akt expression and serum treatment increased Mdm2 ubiquitination of p53. The serum-induced increase in p53 ubiquitination was blocked by LY294002, a phosphatidylinositol 3-OH-kinase inhibitor. Moreover, when Ser186 was replaced by Ala, Mdm2 became resistant to Akt enhancement of p53 ubiquitination and degradation. Collectively, these results suggest that Akt enhances the ubiquitination-promoting function of Mdm2 by phosphorylation of Ser186, which results in reduction of p53 protein. This study may shed light on the mechanisms by which Akt promotes survival, proliferation, and tumorigenesis.

The phosphatidylinositol 3-kinase (PI3K)-Akt pathway suppresses Bax translocation to mitochondria.

Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but redistributes to mitochondria in response to apoptotic stimuli, where it induces cytochrome c release. In this study, we show that the phosphatidylinositol 3-OH kinase (PI3K)-Akt pathway plays an important role in the regulation of Bax subcellular localization. We found that LY294002, a PI3K inhibitor, blocked the effects of serum to prevent Bax translocation to mitochondria and that expression of an active form of PI3K suppressed staurosporine-induced Bax translocation, suggesting that PI3K activity is essential for retaining Bax in the cytoplasm. In contrast, both U0126, a MEK inhibitor, and active MEK had little effect on Bax localization. In respect to downstream effectors of PI3K, we found that expression of active Akt, but not serum and glucocorticoid-induced protein kinase (SGK), suppressed staurosporine-induced translocation of Bax, whereas dominant negative Akt moderately promoted Bax translocation. Expression of Akt did not alter the levels of Bax, Bcl-2, Bcl-X(L), or phosphorylated JNK under the conditions used, suggesting that there were alternative mechanisms for Akt in the suppression of Bax translocation. Collectively, these results suggest that the PI3K-Akt pathway inhibits Bax translocation from cytoplasm to mitochondria and have revealed a novel mechanism by which the PI3K-Akt pathway promotes survival.

JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates.

Recent genetic studies in Drosophila identified a novel non-canonical Wnt pathway, the planar cell polarity (PCP) pathway, that signals via JNK to control epithelial cell polarity in Drosophila. Most recently, a pathway regulating convergent extension movements during gastrulation in vertebrate embryos has been shown to be a vertebrate equivalent of the PCP pathway. However, it is not known whether the JNK pathway functions in this non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. In addition, it is not known whether JNK is in fact activated by Wnt stimulation. Here we show that Wnt5a is capable of activating JNK in cultured cells, and present evidence that the JNK pathway mediates the action of Wnt5a to regulate convergent extension movements in Xenopus. Our results thus demonstrate that the non-canonical Wnt/JNK pathway is conserved in both vertebrate and invertebrate and define that JNK has an activity to regulate morphogenetic cell movements.