Makoto Masaoka

Periventricular notch activation and asymmetric Ngn2 and Tbr2 expression in pair-generated neocortical daughter cells.

To understand the cellular and molecular mechanisms regulating cytogenesis within the neocortical ventricular zone, we examined at high resolution the spatiotemporal expression patterns of Ngn2 and Tbr2. Individually DiI-labeled daughter cells were tracked from their birth in slice cultures and immunostained for Ngn2 and Tbr2. Both proteins were initially absent from daughter cells during the first 2 h. Ngn2 expression was then initiated asymmetrically in one of the daughter cells, with a bias towards the apical cell, followed by a similar pattern of expression for Tbr2, which we found to be a direct target of Ngn2. How this asymmetric Ngn2 expression is achieved is unclear, but gamma-secretase inhibition at the birth of daughter cells resulted in premature Ngn2 expression, suggesting that Notch signaling in nascent daughter cells suppresses a Ngn2-Tbr2 cascade. Many of the nascent cells exhibited pin-like morphology with a short ventricular process, suggesting periventricular presentation of Notch ligands to these cells.

Dynamics of Centrosome Translocation and Microtubule Organization in Neocortical Neurons during Distinct Modes of Polarization

Neuronal migration and process formation require cytoskeletal organization and remodeling. Recent studies suggest that centrosome translocation is involved in initial axon outgrowth, while the role of centrosomal positioning is not clear. Here, we examine relations between centrosomal positioning, axonogenesis, and microtubule (MT) polarization in multipolar and bipolar neocortical neurons. We monitored dynamic movements of centrosomes and MT plus ends in migratory neurons in embryonic mouse cerebral slices. In locomoting bipolar neurons, the centrosome oriented toward the pia-directed leading process. Bipolar neurons displayed dense MT plus end dynamics in leading processes, while trailing processes showed clear bidirectional MTs. In migrating multipolar neurons, new processes emerged irrespective of centrosome localization, followed by centrosome reorientations toward the dominant process. Anterograde movements of MT plus ends occurred in growing processes and retrograde movements were observed after retraction of the distal tip. In multipolar neurons, axon formed by tangential extension of a dominant process and the centrosome oriented toward the growing axon, while in locomoting neurons, an axon formed opposite to the direction of migration and the centrosome localized to the base of the leading process. Our data suggest that MT organization may alter centrosomal localization and that centrosomal positioning does not necessarily direct process formation.

Survey of the Morphogenetic Dynamics of the Ventricular Surface of the Developing Mouse Neocortex

To understand the morphogenetic dynamics of the inner surface of the embryonic pallial (neocortical) wall, we immunohistochemically surveyed the cellular endfeet facing the lateral ventricle and found that the average endfoot area was minimal at embryonic day (E)12 in mice. This endfoot narrowing at E12 may represent a change in the mode of cell production at the surface from a purely proliferative mode that retains all daughter cells to a more differentiation‐directed mode that allows some daughter cells to leave the surface. The apices of cells undergoing mitosis were 1.5–3.9 times larger than the overall cell apices and 6.7–8.7 times smaller than the cross‐sectional area of mitotic somata. En face time‐lapse monitoring of each endfoot permitted observation of its cell cycle‐dependent size changes, division, and relationships with neighboring endfeet. Planar divisions oriented along the lateral–medial axis were less abundant than those oriented along the rostral–caudal axis at E10 and E11, but basal body distribution in each endfoot was random. Developmental Dynamics 236:3061–3070, 2007.

Microtubule dynamics during migration and process formation in neocortical pyramidal neurons in situ

glial cell feeders. The interneurons dissociated from embryonic cortex show more robust migration, compared to those from postnatal cortex. Embryonic interneurons, when plated on the glial feeder cells that had been cultured for two weeks, lose motility during four days in culture indicating intrinsic regulation of their motility. This was supported by an independent set of experiments in which the motility of interneurons in cortical explants labeled by in utero electroporation at E12.5 was lower than those labeled at E15.5 when observed at E18.5. The motility is down-regulated when plated on postnatal cortical neuron feeder layers. Interestingly, when embryonic cortical neurons are co-cultured with postnatal cortical cells, which are physically separated from embryonic neurons by the Millicell membrane, migration of embryonic interneuron is attenuated. These results suggest that termination of cortical interneuron migration is achieved by a synergistic action of intrinsic mechanism and environmental factors.

Behaviors of nascent Purkinje cells in the embryonic cerebellum

DHEAS, one of the most abundant steroids in mammalian brains, exerts many effects on memory and synaptic plasticity; e.g., DHEAS increased the length of MAP2+ neurites in cultures of dissociated mouse neocortical neurons. However, the effects of DHEAS on the dendritic structure in organotypic slice cultures have not been examined. The aim of this study is to examine the action of DHEAS to the dendritic structure of dentate granule neurons in hippocampal organotypic slice cultures prepared from neonatal GFP rats. The density of dendrites in the molecular layer was increased by DHEAS (10–100 nM for 3 days). DiI labeling of single granule cells also showed that DHEAS increased the length of neurites and the number of neurite branches.

20-P018 Live imaging the cortical lamination in slice culture of embryonic mouse cerebrum

tem to observe mesoderm cell migration in chicken embryo using live imaging. In early-stage chicken embryo, mesoderm cells move away from the primitive streak and follow stereotyped migratory route according to their dorsal-ventral fate. Immunohistochemical studies and live cell imaging with fluorescent labeled nucleus and Golgi complex showed a consistent positioning of Golgi complex at the rear end of migrating mesoderm cells, in contrast to what has been reported in cultured cells. Prior to mesoderm cell ingression, Golgi complex in epiblast cells is always located apical to the nucleus. These results suggest that newly formed mesoderm cells, although lost the apical/basal polarity, have inherited this ‘‘basal forward, apical behind’’ organization. We will also provide evidence that the Golgi complex and the microtubule-organizing center (MTOC) positions are related to microtubules organization during mesoderm cell migration. Similar to Golgi complex, abundant microtubules are detected at the apical side. After ingression, microtubules can be seen with GFP-EB1 fusion protein, to project radially and dynamically from MTOC toward the cell periphery. MTOC is also located at the rear end of migrating mesoderm cells as well as Golgi complex.