Neural Stem Cell Research

One of the most notable features in the evolution of the neocortex is the increase in neuron number that reaches its peak in the human brain. Although the laminar organization of the cortex is relatively similar in all mammals, an expansion in cortical surface area underlies the transformation from smooth cortex to the highly folded primate neocortex, and the associated alteration of cortical architecture that is the substrate for the 'higher' cortical functions that distinguish human from other species. This transition underscores the importance of understanding the process of neurogenesis in the developing neocortex.

Recent studies have identified two subtypes of neuronal progenitor cell in the developing rodent embryonic neocortex: radial glia (RG) and intermediate progenitors (IP). Neuroepithelial cells located in the apical-most region, the ventricular zone, transform to RG cells at the onset of neurogenesis. In addition to their well characterized function as a scaffold supporting neuronal migration, RG constitute the main population of neural progenitor cells in the developing mammalian neocortex.

An evolutionary increase in size and functional complexity of the cerebral cortex has culminated in the modern human brain, which diverged from a rodent lineage ~100 million years ago. Recent studies suggest that the development of oRG cells and their transit-amplifying daughter cells (that is, intermediate progenitor¡Vlike cells) may be the cellular mechanism underlying expansion in primate corticogenesis. Recently in the fetal human cortical tissue, a new subtype of neural progenitor cells, termed the oRG (outer subventricular zone-RG) cells, with radial glia¡Vlike morphology but lacking apical processes was discovered. oRG cells can self-renew and produce neuronal precursors. It has been suggested that the outer subventricular zone (OSVZ) may be a primate-specific feature and a hallmark of primate corticogenesis. Although the radial glia cells and intermediate progenitor cells of the ventricular zone and SVZ, respectively, are responsible for generating most cortical neurons in rodent, extra sites of progenitor cell activity have been suggested, which prompted us to ask whether oRG-like cells exist in the developing mouse neocortex.

To address these issues, we investigated whether progenitor cells resembling oRG cells exist in the rodent brain during periods of neocortical neurogenesis. We found cells in the superficial region of the subventricular zone (SVZ) in the developing mouse cortex that morphologically resembled oRG cells. Time-lapse imaging revealed that these cells underwent "mitotic somal translocation" and asymmetric division in which one daughter cell inherited the basal process. Our long-term imaging revealed that oRG cells were generated directly from radial glia cells and that they produced neurons directly, without an intervening intermediate progenitor cell. Furthermore, we found that during interphase, the centrosome moved into the basal process to maintain polarity before mitotic somal translocation. These results suggest that oRG cells are not a specialization of a larger brain with greater cortical area. Instead, oRG-like cells are probably present in all mammals, and an evolutionary increase in the number of oRG cells likely amplified neuronal production and contributed to cortical expansion.

Cell division of an ORG progenitor

Nature Neuroscience features the finding of the new subtype of neural progenitor

Neocortical neurons are born in the germinal zone of the developing mammalian brain and migrate over substantial distances to the forming cortical layers. The mechanisms that are involved in the initial stages of neocortical neurogenesis are not well understood. Neuroepithelial cells, referred to as radial glial progenitor cells (RGPCs) as the neocortex thickens, divide rapidly to expand their pool and undergo asymmetric divisions to generate most cortical pyramidal neurons and glia. Each progenitor spans the entire thickness of the neural tube and developing neocortex and shows an unusual behavior termed interkinetic nuclear migration (INM). After mitosis, which occurs exclusively at the ventricular surface, the nuclei ascend to the upper region of the ventricular zone, where they undergo S phase, and then descend back to the ventricular surface. This behavior is seen in most neuroepithelial cells in the CNS and in some polarized non-neuronal cells.

Although INM was described in the early part of the twentieth century, little was known until recently about its biological significance, its role in neurogenesis and its underlying mechanism. We carried out a detailed analysis of nuclear migration and microtubule organization in RGPCs and evaluated the contributions of microtubule- and actin-based motors to INM. We found that dynein was required for apical, but not basal, migration, which instead required an unconventional kinesin. Nuclear movement was independent of centrosome behavior and occurred along an array of uniformly oriented microtubules that span the entire length of the progenitor cell. Unlike others, we did not find any effect of inhibition of myosin II in our system. These results lead to a model in which INM is powered by oppositely directed microtubule motors that are regulated in a cell cycle¡Vdependent manner.

Behavior of a neural progenitor (RG) cell (green) and its centrosome (magenta)

Radial glia cells constitute a major population of neural progenitor cells that occupy the proliferative VZ in the developing mammalian neocortex. In addition to their well-characterized function as a scaffold in supporting neuronal migration, radial glia cells display interkinetic nuclear oscillation and proliferate extensively at the luminal surface of the VZ. During the peak phase of neurogenesis, they predominantly undergo asymmetric division to self-renew while simultaneously giving rise either directly to a neuron, or to an intermediate progenitor cell which subsequently divides symmetrically to produce neurons. Whereas differentiating progeny progressively migrate away from the VZ to form the cortical plate (CP)¡Xthe future neocortex¡Xrenewing radial glia progenitors remain in the VZ for subsequent divisions. The distinct migratory behaviour of radial glia progenitors and their differentiating progeny is fundamental to the proper development of the mammalian neocortex; however, little is known about the basis of these behavioural differences.

Centrosomes, the main microtubule-organizing centres in animal cells, have an important role in many cell processes, particularly during cell division10 and cell migration. All normal animal cells initially inherit one centrosome, consisting of a pair of centrioles surrounded by an amorphous pericentriolar material. The two centrioles differ in their structure and function. The older ¡¥mother¡¦ centriole, which is formed at least one-and-a-half generations earlier, possesses appendages/satellites that bear specific proteins, such as cenexin (also known as Odf2) and ninein, and anchor microtubules and support ciliogenesis. In contrast, the younger ¡¥daughter¡¦ centriole, which is formed during the preceding S phase, lacks these structures. Full acquisition of appendages/satellites by the daughter centriole is not achieved until at least one-and-a-half cell cycles later. During each cell cycle, the centrosome replicates once in a semi-conservative manner, resulting in the formation of two centrosomes: one of which retains the original old mother centriole (that is, the mother centrosome) while the other receives the new mother centriole (that is, the daughter centrosome). This intrinsic asymmetry in the centrosome has recently been demonstrated to be important for proper spindle orientation during the division of male germline stem cells and neuroblasts in Drosophila, although female germline stem cells appear to divide normally in the absence of centrioles/centrosomes. These studies indicate a critical role for the differential behaviour of centrosomes with differently aged mother centrioles in asymmetric division of the progenitor/stem cells, although it remains unclear whether proper behaviour and development of the progenitor/stem cells and their differentiating daughter cells depend on centrosome asymmetry.

Asymmetric division of radial glia progenitors accounts for nearly all neurogenesis in the developing mammalian neocortex. Three out of four autosomal recessive primary microcephaly (MCPH) genes identified so far encode centrosomal components, suggesting that proper neocortical neurogenesis and development entail a tight regulation of the centrosome, which is poorly understood. In collaboration with Dr. Songhai Shi's lab, we investigated centrosome regulation during the peak phase of mammalian neocortical neurogenesis. We show that asymmetric centrosome inheritance regulates the differential behaviour of renewing progenitors and their differentiating progeny in the embryonic mouse neocortex. Centrosome duplication in dividing radial glia progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glia progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results indicate that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glia progenitors in the developing mammalian neocortex.

Nature magzine features the story of asymmetric centrosome inheritance in nerual stem cell division

Asymmetric inheritance of the mother (red/green) and daughter (green) centrosomes