Ayano Kawaguchi

Asymmetric production of surface-dividing and non-surface-dividing cortical progenitor cells

Mature neocortical layers all derive from the cortical plate (CP), a transient zone in the dorsal telencephalon into which young neurons are continuously delivered. To understand cytogenetic and histogenetic events that trigger the emergence of the CP, we have used a slice culture technique. Most divisions at the ventricular surface generated paired cycling daughters (P/P divisions) and the majority of the P/P divisions were asymmetric in daughter cell behavior; they frequently sent one daughter cell to a non-surface (NS) position, the subventricular zone (SVZ), within a single cell-cycle length while keeping the other mitotic daughter for division at the surface. The NS-dividing cells were mostly Hu+ and their daughters were also Hu+, suggesting their commitment to the neuronal lineage and supply of early neurons at a position much closer to their destiny than from the ventricular surface. The release of a cycling daughter cell to SVZ was achieved by collapse of the ventricular process of the cell, followed by its NS division. Neurogenin2 (Ngn2) was immunohistochemically detected in a certain cycling population during G1 phase and was further restricted during G2-M phases to the SVZ-directed population. Its retroviral introduction converted surface divisions to NS divisions. The asymmetric P/P division may therefore contribute to efficient neuron/progenitor segregation required for CP initiation through cell cycle-dependent and lineage-restricted expression of Ngn2.

In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus

Neurogenesis persists in the adult mammalian hippocampus. To identify and isolate neuronal progenitor cells of the adult human hippocampus, we transfected ventricular zone-free dissociates of surgically-excised dentate gyrus with DNA encoding humanized green fluorescent protein (hGFP), placed under the control of either the nestin enhancer (E/nestin) or the Tα1 tubulin promoter (P/Tα1), two regulatory regions that direct transcription in neural progenitor cells. The resultant P/Tα1:hGFP+ and E/nestin:enhanced (E)GFP+ cells expressed βIII-tubulin or microtubule-associated protein-2; many incorporated bromodeoxyuridine, indicating their genesis in vitro. Using fluorescence-activated cell sorting, the E/nestin:EGFP+ and P/Tα1:hGFP+ cells were isolated to near purity, and matured antigenically and physiologically as neurons. Thus, the adult human hippocampus contains mitotically competent neuronal progenitors that can be selectively extracted. The isolation of these cells may provide a cellular substrate for re-populating the damaged or degenerated adult hippocampus.

Nestin-EGFP transgenic mice: Visualization of the self-renewal and multipotency of CNS stem cells

We generated transgenic mice carrying enhanced green fluorescent protein (EGFP) under the control of the nestin second-intronic enhancer (E/nestin:EGFP). Flow cytometry followed by in vitro assays revealed that in situ EGFP expression in the embryonic brain correlated with the mitotic index, the cogeneration of both neurons and glia, and the frequency of neurosphere formation in vitro. High-level EGFP expressors derived from embryos included a distinct subpopulation of cells that were self-renewable and multipotent, criteria that define neural stem cells (NSCs). Such cells were largely absent among lower-level or non-EGFP expressors, thereby permitting us to enrich for NSCs using EGFP expression level. In adults, although E/nestin:EGFP-positive cells included the NSC population, the frequency of neurosphere formation did not correlate directly with the level of EGFP expression. However, moderately EGFP-expressing cells in adults gained EGFP intensity when they formed neurospheres, suggesting embryonic and adult NSCs exist in different microenvironments in vivo.

Promoter-Targeted Selection and Isolation of Neural Progenitor Cells From the Adult Human Ventricular Zone

Adult humans, like their nonhuman mammalian counterparts, harbor persistent neural progenitor cells in the forebrain ventricular lining. In the absence of adequate surface markers, however, these cells have proven difficult to isolate for study. We have previously identified and selected neural progenitor cells from both the fetal and adult rodent ventricular zone (VZ), by sorting forebrain cells transfected with plasmid DNA encoding the gene for green fluorescent protein driven by the early neuronal promoter for Tα1 tubulin (P/Tα1:hGFP). We have now extended this approach by purifying both P/Tα1:hGFP tubulin‐defined neuronal progenitors, as well as potentially less committed E/nestin:hGFP‐defined neural progenitor cells, from the adult human VZ. The ventricular wall of the temporal horn of the lateral ventricle was dissected from temporal lobes obtained from four adult patients undergoing therapeutic lobectomy. These samples were dissociated, and the cultured cells transduced with either P/Tα1:hGFP or E/nestin:EGFP plasmid DNA. A week later, the cells were redissociated, selected via fluorescence‐activated cell sorting (FACS) on the basis of neural promoter‐driven GFP expression, and replated. The majority of these cells expressed the early neuronal protein βIII‐tubulin upon FACS; within the week thereafter, most matured as morphologically evident neurons that coexpressed βIII‐tubulin and microtubule‐associated protein (MAP)‐2. Many of these neurons had incorporated bromodeoxyuridine in vitro in the days before FACS, indicating their mitogenesis in vitro. Thus, the use of fluorescent transgenes under the control of early neural promoters permits the enrichment of neuronal progenitor cells from the adult human ventricular zone. The specific acquisition, in both purity and number, of residual neural progenitor cells from the adult human brain may now permit hitherto unfeasible studies of both their biology and practical application. J. Neurosci. Res. 59:321–331, 2000

Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis

Cellular diversity of the brain is largely attributed to the spatial and temporal heterogeneity of progenitor cells. In mammalian cerebral development, it has been difficult to determine how heterogeneous the neural progenitor cells are, owing to dynamic changes in their nuclear position and gene expression. To address this issue, we systematically analyzed the cDNA profiles of a large number of single progenitor cells at the mid-embryonic stage in mouse. By cluster analysis and in situ hybridization, we have identified a set of genes that distinguishes between the apical and basal progenitors. Despite their relatively homogeneous global gene expression profiles, the apical progenitors exhibit highly variable expression patterns of Notch signaling components, raising the possibility that this causes the heterogeneous division patterns of these cells. Furthermore, we successfully captured the nascent state of basal progenitor cells. These cells are generated shortly after birth from the division of the apical progenitors, and show strong expression of the major Notch ligand delta-like 1, which soon fades away as the cells migrate in the ventricular zone. We also demonstrated that attenuation of Notch signals immediately induces differentiation of apical progenitors into nascent basal progenitors. Thus, a Notch-dependent feedback loop is likely to be in operation to maintain both progenitor populations.

Flow cytometric analysis of neural stem cells in the developing and adult mouse brain

Despite recent progress in the neural stem cell biology, their cellular characteristics have not been described well. We investigated various characteristics of neural stem cells (NSCs) in vivo during CNS development, using FACS to identify the NSCs. We first examined stage‐dependent changes in the physical parameters, using forward scatter (FSC) and side scatter (SSC) profiles, of NSCs from the developing striatum, where they appear to be active throughout the life of mammals. NSCs were divided into several fractions according to their FSC/SSC profile. With development, their number decreased in the FSChigh fractions but increased in the FSClow/SSChigh fraction, whereas NSCs were significantly concentrated in the fraction containing the largest cells (about 20 μm in diameter) at any stage, which were mostly the cells with the highest nestin‐enhancer activity. Furthermore, we demonstrated that, at all stages examined, the “side population” (SP), defined as the Hoechst 33342 low/negative fraction, which is known to be a stem cell‐enriched population in bone marrow, was also enriched for Notch1‐positive immature neural cells (about 60%) from the developing striatum. However, these immature SP cells were not detected in the large‐cell fraction, however, but were concentrated instead in the FSClow/mid fractions. FACS analysis showed that SP cells from adults were included to some extent in the CD24low/PNAlow fraction, where NSCs were greatly concentrated. Collectively, the characteristics of NSCs were not uniform and changed developmentally.

Direct isolation of committed neuronal progenitor cells from transgenic mice coexpressing spectrally distinct fluorescent proteins regulated by stage‐specific neural promoters

Many tissues arise from pluripotent stem cells through cell‐type specification and maturation. In the bone marrow, primitive stem cells generate all the different types of blood cells via the sequential differentiation of increasingly committed progenitor cells. Cell‐surface markers that clearly distinguish stem cells, restricted progenitors, and differentiated progeny have enabled researchers to isolate these cells and to study the regulatory mechanisms of hematopoiesis. Neuronal differentiation appears to involve similar mechanisms. However, neural progenitor cells that are restricted to a neuronal fate have not been characterized in vivo, because specific cell‐surface markers are not available. We have developed an alternative strategy to identify and isolate neuronal progenitor cells based on dual‐color fluorescent proteins. To identify and isolate directly progenitor cells from brain tissue without the need for either transfection or intervening cell culture, we established lines of transgenic mice bearing fluorescent transgenes regulated by neural promoters. One set of transgenic lines expressed enhanced yellow fluorescent protein (EYFP) in neuronal progenitor cells and neurons under the control of the Tα1 α‐tubulin promoter. Another line expressed enhanced green fluorescent protein (EGFP) in immature neural cells under the control of the enhancer/promoter elements of the nestin gene. By crossing these lines we obtained mice expressing both transgenes. To isolate neuronal progenitor cells directly from the developing brain, we used flow cytometry, selecting cells that expressed EGFP and EYFP simultaneously. We expect this strategy to provide valuable material with which to study the mechanisms of neurogenesis and to develop cell‐based therapies for neurological disorders. J. Neurosci. Res. 65:220–227, 2001.

Mechanisms that regulate the number of neurons during mouse neocortical development

Cortical development progresses through an early phase of progenitor expansion, a middle phase of neurogenesis, and a final phase of gliogenesis. During the middle phase, the neurogenic phase, the neocortical primordium balances the production of neurons against the maintenance of neural precursor cells (NPCs). The final number of neurons is determined by the duration of the neurogenic phase, the rate of NPC division, and the mode of NPC division, that is, whether a division gives rise to two NPCs, one NPC and one cell committed to the neuronal lineage, or two committed cells. We discuss here recent advances in understanding these key aspects that are fundamental for normal brain development.

Morphological asymmetry in dividing retinal progenitor cells

For the understanding of histogenetic events in the 3‐D retinal neuroepithelium, direct observation of the progenitor cells and their morphological changes is required. A slice culture method has been developed by which the behavior of single progenitor cells can be monitored. Although it has been believed that each retinal progenitor cell loses its basal process while it is in M phase, it is reported here that the process is retained throughout M phase and is inherited by one daughter cell, which can be a neuron or a progenitor cell. Daughter neurons used an inherited process for neuronal translocation and positioning. In divisions that produced two mitotic daughters, both of which subsequently divided to form four granddaughter cells, only one daughter cell inherited the original basal process while the other extended a new process. Interestingly, behavioral differences were often noted between such mitotic sisters in the trajectory of interkinetic nuclear movement, cell cycle length, and the composition of the granddaughter pair. Therefore, ‘symmetric’ (progenitor → progenitor + progenitor) divisions are in fact morphologically asymmetric, and the behavior of the mitotic daughters can often be asymmetric, indicating the necessity for studying possible associations between the process inheritance and the cell fate choice.

Lunatic fringe potentiates Notch signaling in the developing brain.

Notch signaling is essential for the self-renewal of mammalian neural progenitor cells. A variety of mechanisms modulate Notch signaling to balance the self-renewal and differentiation of progenitor cells. Fringe is a major Notch regulator and promotes or suppresses Notch signaling, depending on the Notch ligands. In the developing brain, Lunatic fringe (Lfng) is expressed in self-renewing progenitors, but its roles are unknown. In this study, in vivo mosaic analyses using in utero electroporation were developed to investigate the roles of Lfng in neural progenitor cells. We found that Lfng potentiates Notch signaling cell-autonomously. Its depletion did not affect the balance between neuronally committed cells and self-renewing progenitors, however, irrespective of the cell density of Lfng-depleted cells, and caused no obvious defects in brain development. In vivo overexpression experiments with Notch ligands suggest that Lfng strongly augments Notch signaling mediated by Delta-like 1 but not Jagged 1.