Disruption of the anterior commissure in Olig2 deficient mice.

In the present study, we examined neural circuit formation in the forebrain of the Olig2 knockout (Olig2-KO) mouse model and found disruption of the anterior commissure at the late foetal stage. Axon bundles of the anterior commissure encountered the wall of the third ventricle and ceased axonal extension. L1-CAM immunohistochemistry showed that Olig2-KO mice lose decussation formation in the basal forebrain. DiI tracing revealed that the thin bundles of the anterior commissure axons crossed the midline but ceased further extension into the deep part of the contralateral side. Furthermore, some fractions of DiI-labelled axons were oriented dorsolaterally, which was not observed in the control mouse forebrain. The rostral part of the third ventricle was much wider in the Olig2-KO mice than in wild-type mice, which likely resulted in the delay of midline fusion and subsequent delay and malformation of the anterior commissure. We analysed gene expression alterations in the Olig2-KO mice using a public database and found multiple genes, which are related to axon guidance and epithelial-mesenchymal transition, showing subtle expression changes. These results suggest that Olig2 is essential for anterior commissure formation, likely by regulating multiple biological processes.

Conserved and divergent functions of Pax6 underlie species-specific neurogenic patterns in the developing amniote brain.

The evolution of unique organ structures is associated with changes in conserved developmental programs. However, characterizing the functional conservation and variation of homologous transcription factors (TFs) that dictate species-specific cellular dynamics has remained elusive. Here, we dissect shared and divergent functions of Pax6 during amniote brain development. Comparative functional analyses revealed that the neurogenic function of Pax6 is highly conserved in the developing mouse and chick pallium, whereas stage-specific binary functions of Pax6 in neurogenesis are unique to mouse neuronal progenitors, consistent with Pax6-dependent temporal regulation of Notch signaling. Furthermore, we identified that Pax6-dependent enhancer activity of Dbx1 is extensively conserved between mammals and chick, although Dbx1 expression in the developing pallium is highly divergent in these species. Our results suggest that spatiotemporal changes in Pax6-dependent regulatory programs contributed to species-specific neurogenic patterns in mammalian and avian lineages, which underlie the morphological divergence of the amniote pallial architectures.

The evolution of basal progenitors in the developing non-mammalian brain.

The amplification of distinct neural stem/progenitor cell subtypes during embryogenesis is essential for the intricate brain structures present in various vertebrate species. For example, in both mammals and birds, proliferative neuronal progenitors transiently appear on the basal side of the ventricular zone of the telencephalon (basal progenitors), where they contribute to the enlargement of the neocortex and its homologous structures. In placental mammals, this proliferative cell population can be subdivided into several groups that include Tbr2(+) intermediate progenitors and basal radial glial cells (bRGs). Here, we report that basal progenitors in the developing avian pallium show unique morphological and molecular characteristics that resemble the characteristics of bRGs, a progenitor population that is abundant in gyrencephalic mammalian neocortex. Manipulation of LGN (Leu-Gly-Asn repeat-enriched protein) and Cdk4/cyclin D1, both essential regulators of neural progenitor dynamics, revealed that basal progenitors and Tbr2(+) cells are distinct cell lineages in the developing avian telencephalon. Furthermore, we identified a small population of subapical mitotic cells in the developing brains of a wide variety of amniotes and amphibians. Our results suggest that unique progenitor subtypes are amplified in mammalian and avian lineages by modifying common mechanisms of neural stem/progenitor regulation during amniote brain evolution.

NG2 expression in NG2 glia is regulated by binding of SoxE and bHLH transcription factors to a Cspg4 intronic enhancer.

NG2 is a type 1 integral membrane glycoprotein encoded by the Cspg4 gene. It is expressed on glial progenitor cells known as NG2 glial cells or oligodendrocyte precursor cells that exist widely throughout the developing and mature central nervous system and vascular mural cells but not on mature oligodendrocytes, astrocytes, microglia, neurons, or neural stem cells. Hence NG2 is widely used as a marker for NG2 glia in the rodent and human. The regulatory elements of the mouse Cspg4 gene and its flanking sequences have been used successfully to target reporter and Cre recombinase to NG2 glia in transgenic mice when used in a large 200 kb bacterial artificial chromosome cassette containing the 38 kb Cspg4 gene in the center. Despite the tightly regulated cell type- and stage-specific expression of NG2 in the brain and spinal cord, the mechanisms that regulate its transcription have remained unknown. Here, we describe a 1.45 kb intronic enhancer of the mouse Cspg4 gene that directed transcription of EGFP reporter to NG2 glia but not to pericytes in vitro and in transgenic mice. The 1.45 kb enhancer contained binding sites for SoxE and basic helix-loop-helix transcription factors, and its enhancer activity was augmented cooperatively by these factors, whose respective binding elements were found in close proximity to each other. Mutations in these binding elements abrogated the enhancer activity when tested in the postnatal mouse brain.

The Future Vocation of Neural Stem Cells: Lineage Commitment in Brain Development and Evolution.

Understanding the fate commitment of neural stem cells is critical to identify the regulatory mechanisms in developing brains. Genetic lineage-tracing has provided a powerful strategy to unveil the heterogeneous nature of stem cells and their descendants. However, recent studies have reported controversial data regarding the heterogeneity of neural stem cells in the developing mouse neocortex, which prevents a decisive conclusion on this issue. Here, we review the progress that has been made using lineage-tracing analyses of the developing neocortex and discuss stem cell heterogeneity from the viewpoint of comparative and evolutionary biology.

Origin of Oligodendrocytes in the Vertebrate Optic Nerve: A Review.

One of the unsolved problems in the research field of oligodendrocyte (OL) development has been the site(s) of origin of optic nerve OLs and its precursor cells (OPCs). It is generally accepted that OLs in the optic nerve are derived from the brain, and thus optic nerve OLs are immigrant cells. We previously demonstrated the brain origin of optic nerve OPCs in chick embryos. However, the site of optic nerve OPC origin has not been examined experimentally in developing rodents for the past two decades. We have recently reported that optic nerve OPCs in mice arise in the preoptic area by E12.5 and gradually migrate caudally and enter the optic nerve. These OPCs give rise to myelinating OLs in the optic nerve in the postnatal or adult stages. Surprisingly, there are species differences with respect to the origin of optic nerve OPCs between chicks and mice. Here, we summarize the site of OPC origin in the optic nerve based on our own previous and recent results, and discuss possible mechanisms underlying these species differences.

Sulfatide species with various fatty acid chains in oligodendrocytes at different developmental stages determined by imaging mass spectrometry.

HSO3-3-galactosylceramide (Sulfatide) species comprise the major glycosphingolipid components of oligodendrocytes and myelin and play functional roles in the regulation of oligodendrocyte maturation and myelin formation. Although various sulfatide species contain different fatty acids, it is unclear how these sulfatide species affect oligodendrogenesis and myelination. The O4 monoclonal antibody reaction with sulfatide has been widely used as a useful marker for oligodendrocytes and myelin. However, sulfatide synthesis during the pro-oligodendroblast stage, where differentiation into the oligodendrocyte lineage has already occurred, has not been examined. Notably, this stage comprises O4-positive cells. In this study, we identified a sulfatide species from the pro-oligodendroblast-to-myelination stage by imaging mass spectrometry. The results demonstrated that short-chain sulfatides with 16 carbon non-hydroxylated fatty acids (C16) and 18 carbon non-hydroxylated fatty acids (C18) or 18 carbon hydroxylated fatty acids (C18-OH) existed in restricted regions of the early embryonic spinal cord, where pro-oligodendroblasts initially appear, and co-localized with Olig2-positive pro-oligodendroblasts. C18 and C18-OH sulfatides also existed in isolated pro-oligodendroblasts. C22-OH sulfatide became predominant later in oligodendrocyte development and the longer C24 sulfatide was predominant in the adult brain. Additionally, the presence of each sulfatide species in a different area of the adult brain was demonstrated by imaging mass spectrometry at an increased lateral resolution. These findings indicated that O4 recognized sulfatides with short-chain fatty acids in pro-oligodendroblasts. Moreover, the fatty acid chain of the sulfatide became longer as the oligodendrocyte matured. Therefore, individual sulfatide species may have unique roles in oligodendrocyte maturation and myelination. Read the Editorial Highlight for this article on page 356.

Development of the prethalamus is crucial for thalamocortical projection formation and is regulated by Olig2.

Thalamocortical axons (TCAs) pass through the prethalamus in the first step of their neural circuit formation. Although it has been supposed that the prethalamus is an intermediate target for thalamocortical projection formation, much less is known about the molecular mechanisms of this targeting. Here, we demonstrated the functional implications of the prethalamus in the formation of this neural circuit. We show that Olig2 transcription factor, which is expressed in the ventricular zone (VZ) of prosomere 3, regulates prethalamus formation, and loss of Olig2 results in reduced prethalamus size in early development, which is accompanied by expansion of the thalamic eminence (TE). Extension of TCAs is disorganized in the Olig2-KO dorsal thalamus, and initial elongation of TCAs is retarded in the Olig2-KO forebrain. Microarray analysis demonstrated upregulation of several axon guidance molecules, including Epha3 and Epha5, in the Olig2-KO basal forebrain. In situ hybridization showed that the prethalamus in the wild type excluded the expression of Epha3 and Epha5, whereas loss of Olig2 resulted in reduction of this Ephas-negative area and the corresponding expansion of the Ephas-positive TE. Dissociated cultures of thalamic progenitor cells demonstrated that substrate-bound EphA3 suppresses neurite extension from dorsal thalamic neurons. These results indicate that Olig2 is involved in correct formation of the prethalamus, which leads to exclusion of the EphA3-expressing region and is crucial for proper TCA formation. Our observation is the first report showing the molecular mechanisms underlying how the prethalamus acts on initial thalamocortical projection formation.

Computational wave dynamics for innovative design of coastal structures.

For innovative designs of coastal structures, Numerical Wave Flumes (NWFs), which are solvers of Navier-Stokes equation for free-surface flows, are key tools. In this article, various methods and techniques for NWFs are overviewed. In the former half, key techniques of NWFs, namely the interface capturing (MAC, VOF, C-CUP) and significance of NWFs in comparison with the conventional wave models are described. In the latter part of this article, recent improvements of the particle method are shown as one of cores of NWFs. Methods for attenuating unphysical pressure fluctuation and improving accuracy, such as CMPS method for momentum conservation, Higher-order Source of Poisson Pressure Equation (PPE), Higher-order Laplacian, Error-Compensating Source in PPE, and Gradient Correction for ensuring Taylor-series consistency, are reviewed briefly. Finally, the latest new frontier of the accurate particle method, including Dynamic Stabilization for providing minimum-required artificial repulsive force to improve stability of computation, and Space Potential Particle for describing the exact free-surface boundary condition, is described.

Evolution of mammalian Opn5 as a specialized UV-absorbing pigment by a single amino acid mutation.

Opn5 is one of the recently identified opsin groups that is responsible for nonvisual photoreception in animals. We previously showed that a chicken homolog of mammalian Opn5 (Opn5m) is a Gi-coupled UV sensor having molecular properties typical of bistable pigments. Here we demonstrated that mammalian Opn5m evolved to be a more specialized photosensor by losing one of the characteristics of bistable pigments, direct binding of all-trans-retinal. We first confirmed that Opn5m proteins in zebrafish, Xenopus tropicalis, mouse, and human are also UV-sensitive pigments. Then we found that only mammalian Opn5m proteins lack the ability to directly bind all-trans-retinal. Mutational analysis showed that these characteristics were acquired by a single amino acid replacement at position 168. By comparing the expression patterns of Opn5m between mammals and chicken, we found that, like chicken Opn5m, mammalian Opn5m was localized in the ganglion cell layer and inner nuclear layer of the retina. However, the mouse and primate (common marmoset) opsins were distributed not in the posterior hypothalamus (including the region along the third ventricle) where chicken Opn5m is localized, but in the preoptic hypothalamus. Interestingly, RPE65, an essential enzyme for forming 11-cis-retinal in the visual cycle is expressed near the preoptic hypothalamus of the mouse and common marmoset brain but not near the region of the chicken brain where chicken Opn5m is expressed. Therefore, mammalian Opn5m may work exclusively as a short wavelength sensor in the brain as well as in the retina with the assistance of an 11-cis-retinal-supplying system.

Glycogenolysis-Induced Astrocytic Serping1 Expression Regulates Neuroinflammatory Effects on Hippocampal neuron.

The bacterial pathogen, lipopolysaccharide (LPS), elicits microglial response and induces cytokine secretion that subsequently activates astrocytes. Recent findings have indicated that LPS-induced activation of postnatal glial cells has led to alterations in synapse formation in hippocampal and cortical neurons, thereby resulting in a prolonged increased risk for seizure or depression. Nevertheless, its mechanisms remain to be fully elucidated. Cellular metabolism has recently gained recognition as a critical regulatory mechanism for the activation of peripheral immune cells, as it supplies the requisite energy and metabolite for their activation. In the present study, we report that LPS did not change the expression of reported astrocyte-derived synaptogenic genes in the postnatal hippocampus; however, it induced upregulation of astrocytic complement component regulator Serping1 within the postnatal hippocampus. As a regulatory mechanism, activation of glycogen degradation (glycogenolysis) governs the expression of a subset of inflammatory-responsive genes including Serping1 through reactive oxygen species (ROS)-NF-κB axis. Our study further demonstrated that glycogenolysis is implicated in neurotoxic phenotypes of astrocytes, such as impaired neuronal synaptogenesis or cellular toxicity. These findings suggested that activation of glycogenolysis in postnatal astrocytes is an essential metabolic pathway for inducing responses in inflammatory astrocytes.

A Neanderthal/Denisovan GLI3 variant contributes to anatomical variations in mice.

Changes in genomic structures underlie phenotypic diversification in organisms. Amino acid-changing mutations affect pleiotropic functions of proteins, although little is known about how mutated proteins are adapted in existing developmental programs. Here we investigate the biological effects of a variant of the GLI3 transcription factor (GLI3R1537C) carried in Neanderthals and Denisovans, which are extinct hominins close to modern humans. R1537C does not compromise protein stability or GLI3 activator-dependent transcriptional activities. In contrast, R1537C affects the regulation of downstream target genes associated with developmental processes. Furthermore, genome-edited mice carrying the Neanderthal/Denisovan GLI3 mutation exhibited various alterations in skeletal morphology. Our data suggest that an extinct hominin-type GLI3 contributes to species-specific anatomical variations, which were tolerated by relaxed constraint in developmental programs during human evolution.

Genetically-defined lineage tracing of Nkx2.2-expressing cells in chick spinal cord.

In the spinal cord, generation of oligodendrocytes (OLs) is totally dependent on the presence of Olig2, a basic helix-loop-helix transcription factor. However, it also requires Nkx2.2 for its generation, whose expression follows the expression of Olig2. Although it is believed that oligodendrocytes originate from the pMN domain, Nkx2.2 is present in the p3 domain located ventral to the pMN domain. According to recent reports, it is possible that oligodendrocytes are directly derived from the p3 domain in addition to the pMN domain in the chick spinal cord. We examined this hypothesis in this paper. To analyze OL development in the spinal cord, chick embryos are widely used for genetic modification by electroporation or for transplantation experiments, because it is relatively easy to manipulate them compared with mouse embryos. However, genetic modification by electroporation is not appropriate for glial development analyses because glia proliferate vigorously before maturation. In order to overcome these problems, we established a novel method to permanently introduce exogenous gene into a specific cell type. We introduced the CAT1 gene, a murine retroviral receptor, by electroporation followed by injection of murine retrovirus. By using this method, we successfully transduced murine retrovirus into the chick neural tube. We analyzed cell lineage from the p3 domain by restricting CAT1 expression by Nkx2.2-enhancer and found that most of the labeled cells became OLs when the cells were labeled at cE4. Moreover, the labeled OLs were found throughout the white matter in the spinal cord including the most dorsal spinal cord. Thus p3 domain directly generates spinal cord OLs in the chick spinal cord.

STAT3 transcriptionally regulates the expression of genes related to glycogen metabolism in developing motor neurons.

Motor neurons in the spinal cord are essential for movement. During the embryonic period, developing motor neurons store glycogen to protect against hypoglycemic and hypoxic stress. However, the mechanisms by which glycogen metabolism is regulated in motor neurons remain unclear. We herein investigated the transcriptional regulation of genes related to glycogen metabolism in the developing spinal cord. We focused on the regulatory mechanism of glycogen synthase (Gys1) and glycogen phosphorylase brain isoform (PygB), which play central roles in glycogen metabolism, and found that the transcription factor STAT3 regulated the expression of Gys1 and PygB via cis-regulatory promoter sequences in the developing spinal cord. These results suggest that STAT3 is important for the regulation of glycogen metabolism during motor neuron development.

Cell Type-Specific Transcriptional Control of Gsk3ß in the Developing Mammalian Neocortex.

Temporal control of neurogenesis is central for the development and evolution of species-specific brain architectures. The balance between progenitor expansion and neuronal differentiation is tightly coordinated by cell-intrinsic and cell-extrinsic cues. Wnt signaling plays pivotal roles in the proliferation and differentiation of neural progenitors in a temporal manner. However, regulatory mechanisms that adjust intracellular signaling amplitudes according to cell fate progression remain to be elucidated. Here, we report the transcriptional controls of Gsk3ß, a critical regulator of Wnt signaling, in the developing mouse neocortex. Gsk3ß expression was higher in ventricular neural progenitors, while it gradually declined in differentiated neurons. We identified active cis-regulatory module (CRM) of Gsk3ß that responded to cell type-specific transcription factors, such as Sox2, Sox9, and Neurogenin2. Furthermore, we found extensive conservation of the CRM among mammals but not in non-mammalian amniotes. Our data suggest that a mammalian-specific CRM drives the cell type-specific activity of Gsk3ß to fine tune Wnt signaling, which contributes to the tight control of neurogenesis during neocortical development.

Temperature sensitivity of Notch signaling underlies species-specific developmental plasticity and robustness in amniote brains.

Ambient temperature significantly affects developmental timing in animals. The temperature sensitivity of embryogenesis is generally believed to be a consequence of the thermal dependency of cellular metabolism. However, the adaptive molecular mechanisms that respond to variations in temperature remain unclear. Here, we report species-specific thermal sensitivity of Notch signaling in the developing amniote brain. Transient hypothermic conditions increase canonical Notch activity and reduce neurogenesis in chick neural progenitors. Increased biosynthesis of phosphatidylethanolamine, a major glycerophospholipid components of the plasma membrane, mediates hypothermia-induced Notch activation. Furthermore, the species-specific thermal dependency of Notch signaling is associated with developmental robustness to altered Notch signaling. Our results reveal unique regulatory mechanisms for temperature-dependent neurogenic potentials that underlie developmental and evolutionary adaptations to a range of ambient temperatures in amniotes.

Ultrastructural characteristics of oligodendrocyte precursor cells in the early postnatal mouse optic nerve observed by serial block-face scanning electron microscopy.

Oligodendrocyte precursor cells (OPC) arise from restricted regions of the central nervous system (CNS) and differentiate into myelin-forming cells after migration, but their ultrastructural characteristics have not been fully elucidated. This study examined the three-dimensional ultrastructure of OPCs in comparison with other glial cells in the early postnatal optic nerve by serial block-face scanning electron microscopy. We examined 70 putative OPCs (pOPC) that were distinct from other glial cells according to established morphological criteria. The pOPCs were unipolar in shape with relatively few processes, and their Golgi apparatus were localized in the perinuclear region with a single cisterna. Astrocytes abundant in the optic nerve were distinct from pOPCs and had a greater number of processes and more complicated Golgi apparatus morphology. All pOPCs and astrocytes contained a pair of centrioles (basal bodies). Among them, 45% of pOPCs extended a short cilium, and 20% of pOPCs had centrioles accompanied by vesicles, whereas all astrocytes with basal bodies had cilia with invaginated ciliary pockets. These results suggest that the fine structures of pOPCs during the developing and immature stages may account for their distinct behavior. Additionally, the vesicular transport of the centrioles, along with a short cilium length, suggests active ciliogenesis in pOPCs.

Changes in Wnt-Dependent Neuronal Morphology Underlie the Anatomical Diversification of Neocortical Homologs in Amniotes.

The six-layered neocortex is a shared characteristic of all mammals, but not of non-mammalian species, and its formation requires an inside-out pattern of neuronal migration. The extant reptilian dorsal cortex is thought to represent an ancestral form of the neocortex, although how the reptilian three-layered cortex is formed is poorly understood. Here, we show unique patterns of lamination and neuronal migration in the developing reptilian cortex. While the multipolar-to-bipolar transition of migrating neurons is essential for mammalian cortical development, the reptilian cortex lacks bipolar-shaped migrating neurons, resulting in an outside-in pattern of cortical development. Furthermore, dynamic regulation of Wnt signal strengths contributes to neuronal morphological changes, which is conserved across species. Our data preclude the idea that the six-layered mammalian neocortex emerged by simple addition to the reptilian dorsal cortex but suggest that the acquisition of a novel neuronal morphology based on conserved developmental programs contributed to neocortical evolution.

Differential potentials of neural progenitors for the generation of neurons and non-neuronal cells in the developing amniote brain.

Mature mammalian brains consist of variety of neuronal and non-neuronal cell types, which are progressively generated from embryonic neural progenitors through the embryonic and postnatal periods. However, it remains unknown whether all embryonic progenitors equivalently contribute to multiple cell types, or individual neural progenitors have variable potentials to generate specific cell types in a stochastic manner. Here, we performed population-level tracing of mouse embryonic neural progenitors by using Tol2-mediated genome integration vectors. We identified that neural progenitors in early embryonic stages predominantly contribute to cortical or subcortical neurons than astrocytes, ependymal cells, and neuroblasts in the postnatal brain. Notably, neurons and astrocytes were cumulatively labeled by the increase of total labeled cells, suggesting constant neurogenic and gliogenic potentials of individual neural progenitors. On the contrary, numbers of labeled ependymal cell are more fluctuated, implicating intrinsic variability of progenitor potentials for ependymal cell generation. Differential progenitor potentials that contribute to neurons, astrocytes, and ependymal cells were also detected in the developing avian pallium. Our data suggest evolutionary conservations of coherent and variable potentials of neural progenitors that generate multiple cell types in the developing amniote brain.

Localization of phospho-tyrosine489-beta-adducin immunoreactivity in the hypothalamic tanycytes and its involvement in energy homeostasis.

beta-Adducin is a cytoskeletal protein that interacts with the actin filaments to suppress actin polymerization and facilitate actin-spectrin binding. We have previously shown that beta-adducin is phosphorylated by Fyn at tyrosine489 in the rat brain and bound to its Src-homology 2 domain. In the present study, we examined the immunohistochemical localization of the tyrosine489-phosphorylated form of beta-adducin (pY489-beta-adducin) in the rat brain. Among brain regions, highest immunoreactivity was located in the hypothalamic tanycytes that are of glial origin lining around the third cerebral ventricle. Their immunoreactive processes extended into the arcuate nucleus, ventromedial hypothalamus and the median eminence. In addition, the pY489-beta-adducin immunoreactivity in the tanycytes was enhanced after fasting for 36-48 h, being associated with a morphological change of the DARPP-32-immunoreactivity. Intraperitoneal injection of 2-deoxy-d-glucose also enhances pY489-beta-adducin immunoreactivity in the tanycytes, along with increased food intake. These results suggest that tyrosine phosphorylation of beta-adducin in the tanycytes is involved in hypothalamic regulation of food intake and energy homeostasis.