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

Abstract

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.

Dynamic organelle localization and cytoskeletal reorganization during preimplantation mouse embryo development revealed by live imaging of genetically encoded fluorescent fusion proteins

“…These reporter mouse lines could circumvent common problems in other transgenic reporter mice as mentioned above. Indeed, our R26 reporter mouse lines have proven very useful for live cell imaging of not only early embryos (Abe et al, ; Ichikawa et al, ; Kawaue et al, ; Ke et al, ; Lau et al, ; Namba et al, ; Nonomura et al, ; Okamoto et al, ; Shimozawa et al, ; Shioi et al, ), but also several of adult tissue types and primary cultured cells obtained from these mice (Ito et al, ; Jin et al, ; Shinomura et al, ; Son et al, ; Susaki et al, ; Tainaka et al, ; Yokose et al, ).…”

Differences in the Mechanical Properties of the Developing Cerebral Cortical Proliferative Zone between Mice and Ferrets at both the Tissue and Single-Cell Levels

“…Slices were imaged using an Olympus IX71 [4x (Figure 3A) or 20x (Figure 3B)] equipped with an Orca ER camera (Hamamatsu Photonics). To measure the volume of each dissociated cell (Figure 6), cells were plated on polyethyleneimine-coated glass-bottomed 35 mm dishes (Iwaki) at a density of 5 × 10 4 / cm 2 , and then stained with the styryl dye FM4-64 (Thermo Fisher Scientific) at a concentration of 5 μg/ml for 30 min (Kawaue et al, 2014). …”

“…For confocal imaging of all VZ cells in the subapical space (Figure 8), processed cerebral hemispheric walls (500 × 500 μm) were stained with 5 μg/ml FM4-64 for 30 min (Kawaue et al, 2014) before being mounted on polystyrene cell culture dish (Corning) using AteloCell IAC-30 collagen gel (Koken). Live confocal images were taken with a BX51W1 microscope (Olympus) equipped with a CSU-X1 laser scanning confocal unit (Yokogawa), a 100x objective lens (LUMPLFL 100XW, Olympus), an iXon+ EMCCD camera (Andor), and an on-stage culture chamber (Tokai Hit) filled with 45% N 2 , 40% O 2 , and 5% CO 2 .…”

Radial Glial Fibers Promote Neuronal Migration and Functional Recovery after Neonatal Brain Injury

“…The following transgenic mouse lines were used: R26-tdTomato mice (Stock No. 7914, the Jackson Laboratory) provided by Dr. Masahiro Yamaguchi (Kochi Medical School, Japan), Neurog2-d4Venus mice (Kawaue et al, 2014), NSE-DTA mice (Imayoshi et al, 2008;Kobayakawa et al, 2007) provided by Dr. Shigeyoshi Itohara (RIKEN, Japan), and Dcx-EGFP mice (Gong et al, 2003) provided by the Mutant Mouse Research Resource Center (MMRRC. RRID: MMRRC_000244-MU).…”