Mayumi Okamoto

Pioneering Axons Regulate Neuronal Polarization in the Developing Cerebral Cortex

The polarization of neurons, which mainly includes the differentiation of axons and dendrites, is regulated by cell-autonomous and non-cell-autonomous factors. In the developing central nervous system, neuronal development occurs in a heterogeneous environment that also comprises extracellular matrices, radial glial cells, and neurons. Although many cell-autonomous factors that affect neuronal polarization have been identified, the microenvironmental cues involved in neuronal polarization remain largely unknown. Here, we show that neuronal polarization occurs in a microenvironment in the lower intermediate zone, where the cell adhesion molecule transient axonal glycoprotein-1 (TAG-1) is expressed in cortical efferent axons. The immature neurites of multipolar cells closely contact TAG-1-positive axons and generate axons. Inhibition of TAG-1-mediated cell-to-cell interaction or its downstream kinase Lyn impairs neuronal polarization. These results show that the TAG-1-mediated cell-to-cell interaction between the unpolarized multipolar cells and the pioneering axons regulates the polarization of multipolar cells partly through Lyn kinase and Rac1.

Interkinetic nuclear migration generates and opposes ventricular-zone crowding: insight into tissue mechanics

The neuroepithelium (NE) or ventricular zone (VZ), from which multiple types of brain cells arise, is pseudostratified. In the NE/VZ, neural progenitor cells are elongated along the apicobasal axis, and their nuclei assume different apicobasal positions. These nuclei move in a cell cycle–dependent manner, i.e., apicalward during G2 phase and basalward during G1 phase, a process called interkinetic nuclear migration (INM). This review will summarize and discuss several topics: the nature of the INM exhibited by neural progenitor cells, the mechanical difficulties associated with INM in the developing cerebral cortex, the community-level mechanisms underlying collective and efficient INM, the impact on overall brain formation when NE/VZ is overcrowded due to loss of INM, and whether and how neural progenitor INM varies among mammalian species. These discussions will be based on recent findings obtained in live, three-dimensional specimens using quantitative and mechanical approaches. Experiments in which overcrowding was induced in mouse neocortical NE/VZ, as well as comparisons of neocortical INM between mice and ferrets, have revealed that the behavior of NE/VZ cells can be affected by cellular densification. A consideration of the physical aspects in the NE/VZ and the mechanical difficulties associated with high-degree pseudostratification (PS) is important for achieving a better understanding of neocortical development and evolution.

Cell-cycle-independent transitions in temporal identity of mammalian neural progenitor cells

During cerebral development, many types of neurons are sequentially generated by self-renewing progenitor cells called apical progenitors (APs). Temporal changes in AP identity are thought to be responsible for neuronal diversity; however, the mechanisms underlying such changes remain largely unknown. Here we perform single-cell transcriptome analysis of individual progenitors at different developmental stages, and identify a subset of genes whose expression changes over time but is independent of differentiation status. Surprisingly, the pattern of changes in the expression of such temporal-axis genes in APs is unaffected by cell-cycle arrest. Consistent with this, transient cell-cycle arrest of APs in vivo does not prevent descendant neurons from acquiring their correct laminar fates. Analysis of cultured APs reveals that transitions in AP gene expression are driven by both cell-intrinsic and -extrinsic mechanisms. These results suggest that the timing mechanisms controlling AP temporal identity function independently of cell-cycle progression and Notch activation mode.

Ferret-mouse differences in interkinetic nuclear migration and cellular densification in the neocortical ventricular zone.

The thick outer subventricular zone (OSVZ) is characteristic of the development of human neocortex. How this region originates from the ventricular zone (VZ) is largely unknown. Recently, we showed that over-proliferation-induced acute nuclear densification and thickening of the VZ in neocortical walls of mice, which lack an OSVZ, causes reactive delamination of undifferentiated progenitors and invasion by these cells of basal areas outside the VZ. In this study, we sought to determine how VZ cells behave in non-rodent animals that have an OSVZ. A comparison of mid-embryonic mice and ferrets revealed: (1) the VZ is thicker and more pseudostratified in ferrets. (2) The soma and nuclei of VZ cells were horizontally and apicobasally denser in ferrets. (3) Individual endfeet were also denser on the apical (ventricular) surface in ferrets. (4) In ferrets, apicalward nucleokinesis was less directional, whereas basalward nucleokinesis was more directional; consequently, the nuclear density in the periventricular space (within 16 μm of the apical surface) was smaller in ferrets than in mice, despite the nuclear densification seen basally in ferrets. These results suggest that species-specific differences in nucleokinesis strategies may have evolved in close association with the magnitudes and patterns of nuclear stratification in the VZ.

Neurogenin2‐d4Venus and Gadd45g‐d4Venus transgenic mice: Visualizing mitotic and migratory behaviors of cells committed to the neuronal lineage in the developing mammalian brain

To achieve highly sensitive and comprehensive assessment of the morphology and dynamics of cells committed to the neuronal lineage in mammalian brain primordia, we generated two transgenic mouse lines expressing a destabilized (d4) Venus controlled by regulatory elements of the Neurogenin2 (Neurog2) or Gadd45g gene. In mid‐embryonic neocortical walls, expression of Neurog2‐d4Venus mostly overlapped with that of Neurog2 protein, with a slightly (1 h) delayed onset. Although Neurog2‐d4Venus and Gadd45g‐d4Venus mice exhibited very similar labeling patterns in the ventricular zone (VZ), in Gadd45g‐d4Venus mice cells could be visualized in more basal areas containing fully differentiated neurons, where Neurog2‐d4Venus fluorescence was absent. Time‐lapse monitoring revealed that most d4Venus+ cells in the VZ had processes extending to the apical surface; many of these cells eventually retracted their apical process and migrated basally to the subventricular zone, where neurons, as well as the intermediate neurogenic progenitors that undergo terminal neuron‐producing division, could be live‐monitored by d4Venus fluorescence. Some d4Venus+ VZ cells instead underwent nuclear migration to the apical surface, where they divided to generate two d4Venus+ daughter cells, suggesting that the symmetric terminal division that gives rise to neuron pairs at the apical surface can be reliably live‐monitored. Similar lineage‐committed cells were observed in other developing neural regions including retina, spinal cord, and cerebellum, as well as in regions of the peripheral nervous system such as dorsal root ganglia. These mouse lines will be useful for elucidating the cellular and molecular mechanisms underlying development of the mammalian nervous system.

Synaptic transmission from subplate neurons controls radial migration of neocortical neurons

Transient instruction changes migration The brain neocortex is built by waves of neurons migrating from deep within the brain to the surface layers. Ohtaka-Maruyama et al. found that a layer of neurons that multipolar neurons encounter on their travels instructs the migrating neurons to change phenotype and direction (see the Perspective by Schinder and Lanuza). These subplate neurons form transient glutamatergic synapses with the immature migrants. This results in the migrating multipolar neurons becoming bipolar, more directed, and faster in their final migrations. Science, this issue p. 313; see also p. 265 In the developing mouse neocortex, subplate neurons form transient synapses on immature migrating multipolar neurons. The neocortex exhibits a six-layered structure that is formed by radial migration of excitatory neurons, for which the multipolar-to-bipolar transition of immature migrating multipolar neurons is required. Here, we report that subplate neurons, one of the first neuron types born in the neocortex, manage the multipolar-to-bipolar transition of migrating neurons. By histochemical, imaging, and microarray analyses on the mouse embryonic cortex, we found that subplate neurons extend neurites toward the ventricular side of the subplate and form transient glutamatergic synapses on the multipolar neurons just below the subplate. NMDAR (N-methyl-d-aspartate receptor)–mediated synaptic transmission from subplate neurons to multipolar neurons induces the multipolar-to-bipolar transition, leading to a change in migration mode from slow multipolar migration to faster radial glial-guided locomotion. Our data suggested that transient synapses formed on early immature neurons regulate radial migration.

Temporal change of gene expression pattern in neural progenitor cells during neocortical development

is essential for the development of mammalian pancreas. Several groups including us have recently reported that Ptf1a is expressed in the developing CNS, such as cerebellum, medulla, spinal cord and retina, and involved in specifying neuronal cell types. In the hypothalamus, there are various types of neurons. Hypothalamus is believed to control energy expenditure and body weight balance through food intake regulation. In addition, it acts as the highest center for autonomic nervous system and sexual function, and regulates circadian rhythm, homeostasis, emotion, instinctive behavior such as aggression and mating. Therefore, it is indispensable not only to maintain individual life but also to preserve species. Although descriptions on its physiological function have been accumulated, little is known about the developmental machinery of the hypothalamic neurons. During development, we found Ptf1a is expressed in a part of diencephalic neuroepithelium, that is, a source of hypothalamus. Using the Cre/loxP system, we identified -gal-labeled Ptf1a lineage cells in the ventromedial hypothalamus (VMH), caudal dorsomedial hypothalamus (cDMH), tuberal nucleus (TU), arcuate nucleus (ARC), and medial preoptic area (MPA), all of which are critical sites to regulate energy balance. Conditional knockout (CKO) of this gene in the developing hypothalamus (Ptf1aNkx2.1−/−) resulted in late-onset obesity. We are now precisely analyzing the phenotypes of the CKO mice from anatomical and physiological points of view.

Heterogeneity of neural progenitor cells during neocortical development as revealed by single cell gene expression profiles

dyskinesia. However, little is known about recovery and regeneration mechanism of optimal neural circuits developing by precise neural activities and how neural activities form the appropriate local neural circuits with transplanted cells correctly processing the information with endogenous neurons. Therefore we investigated whether Neurogenin2 and Mash-1 can differentiate transplanted neural stem cells, which are thought to regulate differentiation of excitatory and inhibitory neuron respectively. This approach will allow us to reveal the formation mechanism of local excitatory and inhibitory circuits in the cerebral cortex. Additionally this method could be helpful to care the patients suffered from neurological disorders.