Restriction of developmental potential during divergence of the enteric and sympathetic neuronal lineages.

In the peripheral nervous system, enteric and sympathetic neurons develop from multipotent neural crest cells. While local environmental signals in the gut and in the region of the sympathetic ganglia play a role in the choice of cell fate, little is known about the mechanisms that underlie restriction to specific neuronal phenotypes. We investigated the divergence and restriction of the enteric and sympathetic neuronal lineages using immuno-isolated neural crest-derived cells from the gut and sympathetic ganglia. Analysis of neuronal and lineage-specific mRNAs and proteins indicated that neural crest-derived cells from the gut and sympathetic ganglia had initiated neuronal differentiation and phenotypic divergence by E14.5 in the rat. We investigated the developmental potential of these cells using expression of tyrosine hydroxylase as a marker for a sympathetic phenotype. Tyrosine hydroxylase expression was examined in neurons that developed from sympathetic and enteric neuroblasts under the following culture conditions: culture alone; coculture with gut monolayers to promote enteric differentiation; or coculture with dorsal aorta monolayers to promote noradrenergic differentiation. Both enteric and sympathetic neuroblasts displayed developmental plasticity at E14.5. Sympathetic neuroblasts downregulated tyrosine hydroxylase in response to signals from the gut environment and enteric neuroblasts increased expression of tyrosine hydroxylase when grown on dorsal aorta or in the absence of other cell types. Tracking of individual sympathetic cells displaying a neuronal morphology at the time of plating indicated that neuroblasts retained phenotypic plasticity even after initial neuronal differentiation had occurred. By E19.5 both enteric and sympathetic neuroblasts had undergone a significant loss of their developmental potential, with most neuroblasts retaining their lineage-specific phenotype in all environments tested. Together our data indicate that the developmental potential of enteric and sympathetic neuroblasts becomes restricted over time and that this restriction takes place not as a consequence of initial neuronal differentiation but during the period of neuronal maturation. Further, we have characterized a default pathway of adrenergic differentiation in the enteric nervous system and have defined a transient requirement for gut-derived factors in the maintenance of the enteric neuronal phenotype.     

Expression of ezrin in subventricular zone neural stem cells and their progeny in adult and developing mice

Ezrin is a member of the ezrin–radixin–moesin (ERM) family of proteins, which link the cytoskeleton and cell membrane. ERM proteins are involved in pivotal cellular functions including cell–matrix recognition, cell–cell communication, and cell motility. Several recent studies have shown that ERM proteins are expressed in specific cell types of the adult rostral migratory stream (RMS). In this study, we found that ERM proteins are expressed highly in the early postnatal RMS and the ventricular zone of embryonic cerebral cortex, suggesting that these proteins may be expressed by neural progenitors. Furthermore, whereas ezrin previously was found to be expressed exclusively by astrocytes of the adult RMS, we found that ezrin-expressing cells also expressed the markers for indicating neuroblasts in vivo and in vitro, and that ezrin expression by neuroblasts decreases progressively as neuroblasts migrate. Using in vitro differentiation of adult neural stem cells, we found that ezrin is expressed by neural stem cells and their progeny (neuroblasts and astrocytes), but not by oligodendrocytic progeny. Collectively our findings demonstrate that adult neural stem cells and neuroblasts express ezrin and that ezrin may be involved in intracellular actin remodeling.     

Insights into neural stem cell biology from flies

Drosophila neuroblasts are similar to mammalian neural stem cells in their ability to self-renew and to produce many different types of neurons and glial cells. In the past two decades, great advances have been made in understanding the molecular mechanisms underlying embryonic neuroblast formation, the establishment of cell polarity and the temporal regulation of cell fate. It is now a challenge to connect, at the molecular level, the different cell biological events underlying the transition from neural stem cell maintenance to differentiation. Progress has also been made in understanding the later stages of development, when neuroblasts become mitotically inactive, or quiescent, and are then reactivated postembryonically to generate the neurons that make up the adult nervous system. The ability to manipulate the steps leading from quiescence to proliferation and from proliferation to differentiation will have a major impact on the treatment of neurological injury and neurodegenerative disease.     

Clonal analysis of Drosophila embryonic neuroblasts: neural cell types, axon projections and muscle targets.

An experimental analysis of neurogenesis requires a detailed understanding of wild-type neural development. Recent DiI cell lineage studies have begun to elucidate the family of neurons and glia produced by each Drosophila embryonic neural precursor (neuroblast). Here we use DiI labeling to extend and clarify previous studies, but our analysis differs from previous studies in four major features: we analyze and compare lineages of every known embryonic neuroblast; we use an in vivo landmark (engrailed-GFP) to increase the accuracy of neuroblast identification; we use confocal fluorescence and Nomarski microscopy to collect three-dimensional data in living embryos simultaneously for each DiI-labeled clone, the engrailed-GFP landmark, and the entire CNS and muscle target field (Nomarski images); and finally, we analyze clones very late in embryonic development, which reveals novel cell types and axon/dendrite complexity. We identify the parental neuroblasts for all the cell types of the embryonic CNS: motoneurons, intersegmental interneurons, local interneurons, glia and neurosecretory cells (whose origins had never been determined). We identify muscle contacts for every thoracic and abdominal motoneuron at stage 17. We define the parental neuroblasts for neurons or glia expressing well-known molecular markers or neurotransmitters. We correlate Drosophila cell lineage data with information derived from other insects. In addition, we make the following novel conclusions: (1) neuroblasts at similar dorsoventral positions, but not anteroposterior positions, often generate similar cell lineages, and (2) neuroblasts at similar dorsoventral positions often produce the same motoneuron subtype: ventral neuroblasts typically generate motoneurons with dorsal muscle targets, while dorsal neuroblasts produce motoneurons with ventral muscle targets. Lineage data and movies can be found at http://www.biologists. com/Development/movies/dev8623.html http://www.neuro.uoregon. edu/doelab/lineages/     

Expression of a neurofilament protein by the precursors of a subpopulation of ventral spinal cord neurons

The expression of neurofilament proteins (NF-H, NF-M, and NF-L) in replicating neuroepithelial cells and postmitotic neuroblasts in the embryonic chick trunk neural tube was examined by immunohistochemistry. Anti-NF-M, in particular, resulted in bright staining of some mitotic cells, which were found to be strictly localized to a midventral and an extreme dorsal position in the neural tube. Those in the midventral position were observed with greatest frequency during Days 3 and 4 of incubation and became increasingly rare thereafter. During the same period of time, and in the same small ventral region, NF-M-positive interphase cells, presumably migrating postmitotic neuroblasts, were also present. In contrast, NF-L-positive mitotic cells were rarely seen. NF-L-positive migrating and differentiating neuroblasts were observed throughout the ventral half of the neural tube except in the midventral area containing NF-M-positive mitotic cells and NF-M-positive migrating neuroblasts. These results, together with known temporal and spatial patterns of neurogenesis in the spinal cord, suggest that the expression of NF-L and NF-M, in the form recognized by our antibodies, may not be initiated coordinately, or even in the same sequence, in different types of neuroblasts, and that only the immediate precursors of a specific subpopulation of ventral spinal cord neurons begin expressing NF-M in the terminal cell cycle. In addition, the NF-M-positive mitotic cells, when observed in anaphase and telophase, had NF-M-positive material associated with both emerging daughter cells and the migrating neuroblasts were frequently found in closely associated pairs, consistent with the suggestion that these precursor cells undergo a symmetrical terminal division to yield two daughter postmitotic neuroblasts.     

Age-dependent changes in the subcallosal zone neurogenesis of mice

There are several known neurogenic areas including subventricular zone and subgranular layer in the dentate gyrus of the hippocampus. Both germinal centers exhibit an age-dependent decline in cell proliferation and neurogenesis, which may be associated with age-related decline in brain function. We recently identified the subcallosal zone (SCZ) as a novel neural stem cell niche with a potential to spontaneously produce new neuroblasts. We examined whether SCZ neurogenesis is also regulated by the age of mice. The number of newly generated neuroblasts was reduced in the SCZ with age, and only marginal number of DCX-labeled neuroblasts was found in 6-month-old SCZ, which is most likely due to reduced proliferation of progenitor cells and loss of neural stem cells (NSCs). This age-dependent changes in the SCZ occurred earlier than that of other neurogenic brain regions. The neurosphere assay in vitro confirmed the depletion of NSCs within the SCZ of young adults. However, marked induction of neuroblast production in the SCZ was seen in 6-month-old mice after traumatic brain injury. Taken together, these results indicate that a rapid decline in SCZ neurogenesis in mice is due to depletion of NSCs and reduced capacity to produce neuroblasts.     

Forever young: death-defying neuroblasts

During development, many neural stem cells "age" as they sequentially generate distinct neuronal or glial cell types. In this issue, Maurange et al. (2008) now identify the temporal control factors in Drosophila neural stem cells (neuroblasts) that regulate the fate of stem cell progeny and signal the end of stem cell proliferation.     

Long-Term Live Cell Imaging and Automated 4D Analysis of Drosophila Neuroblast Lineages

The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.     

Defective neuroblast commitment in mutants of the achaete-scute complex and adjacent genes of D. melanogaster

Loss of function mutations in genes of the achaete-scute complex (ASC) or in the gene vnd of D. melanogaster result in neural hypoplasia. Two types of defects contribute to the development of the neural hypoplasic phenotype: a lower than normal proportion of neuroblasts delaminate from the neuroectoderm, and there is abundant cell death in the neural primordium during later stages. In addition, we found that increasing the copy number of ASC wild-type alleles leads to effects opposite to those caused by their deletion. All of these results indicate that the function of these genes is required for the commitment of neuroectodermal cells as neuroblasts and that the loss of these genetic functions causes the cells either to take on an epidermal fate or to die.     

Comparison of early nerve cord development in insects and vertebrates

It is widely held that the insect and vertebrate CNS evolved independently. This view is now challenged by the concept of dorsoventral axis inversion, which holds that ventral in insects corresponds to dorsal in vertebrates. Here, insect and vertebrate CNS development is compared involving embryological and molecular data. In insects and vertebrates, neurons differentiate towards the body cavity. At early stages of neurogenesis, neural progenitor cells are arranged in three longitudinal columns on either side of the midline, and NK-2/NK-2.2, ind/Gsh and msh/Msx homologs specify the medial, intermediate and lateral columns, respectively. Other pairs of regional specification genes are, however, expressed in transverse stripes in insects, and in longitudinal stripes in the vertebrates. There are differences in the regional distribution of cell types in the developing neuroectoderm. However, within a given neurogenic column in insects and vertebrates some of the emerging cell types are remarkably similar and may thus be phylogenetically old: NK-2/NK-2.2-expressing medial column neuroblasts give rise to interneurons that pioneer the medial longitudinal fascicles, and to motoneurons that exit via lateral nerve roots to then project peripherally. Lateral column neuroblasts produce, among other cell types, nerve root glia and peripheral glia. Midline precursors give rise to glial cells that enwrap outgrowing commissural axons. The midline glia also express netrin homologs to attract commissural axons from a distance.     

Cortical aPKC kinase activity distinguishes neural stem cells from progenitor cells by ensuring asymmetric segregation of Numb

During asymmetric stem cell division, polarization of the cell cortex targets fate determinants unequally into the sibling daughters, leading to regeneration of a stem cell and production of a progenitor cell with restricted developmental potential. In mitotic neural stem cells (neuroblasts) in fly larval brains, the antagonistic interaction between the polarity proteins Lethal (2) giant larvae (Lgl) and atypical Protein Kinase C (aPKC) ensures self-renewal of a daughter neuroblast and generation of a progenitor cell by regulating asymmetric segregation of fate determinants. In the absence of lgl function, elevated cortical aPKC kinase activity perturbs unequal partitioning of the fate determinants including Numb and induces supernumerary neuroblasts in larval brains. However, whether increased aPKC function triggers formation of excess neuroblasts by inactivating Numb remains controversial. To investigate how increased cortical aPKC function induces formation of excess neuroblasts, we analyzed the fate of cells in neuroblast lineage clones in lgl mutant brains. Surprisingly, our analyses revealed that neuroblasts in lgl mutant brains undergo asymmetric division to produce progenitor cells, which then revert back into neuroblasts. In lgl mutant brains, Numb remained localized in the cortex of mitotic neuroblasts and failed to segregate exclusively into the progenitor cell following completion of asymmetric division. These results led us to propose that elevated aPKC function in the cortex of mitotic neuroblasts reduces the function of Numb in the future progenitor cells. We identified that the acyl-CoA binding domain containing 3 protein (ACBD3) binding region is essential for asymmetric segregation of Numb in mitotic neuroblasts and suppression of the supernumerary neuroblast phenotype induced by increased aPKC function. The ACBD3 binding region of Numb harbors two aPKC phosphorylation sites, serines 48 and 52. Surprisingly, while the phosphorylation status at these two sites directly impinged on asymmetric segregation of Numb in mitotic neuroblasts, both the phosphomimetic and non-phosphorylatable forms of Numb suppressed formation of excess neuroblasts triggered by increased cortical aPKC function. Thus, we propose that precise regulation of cortical aPKC kinase activity distinguishes the sibling cell identity in part by ensuring asymmetric partitioning of Numb into the future progenitor cell where Numb maintains restricted potential independently of regulation by aPKC.     

Cyto-differentiation of the neuroblasts from the neural ectodermal cells in grasshopper,Chortophaga viridifasciata (De Geer) (Orthoptera: Acrididae) embryos

The first sign of neurogenesis in the embryo of grasshopper, Chortophaga viridifasciata (Orthoptera: Acrididae), is signaled by a partition of the ectodermal cells into non-neural ectodermal cells and neural eetodermal cells. The neuroblasts are differentiated from neural ectodermal cells. In the present study, we examined the pattern of mitotic activity in the developing embryo by tracing the incorporation of BrdU in S phase nuclei. The results indicate that the ectodermal cells in 6-day old embryos do not show any signs of differentiation. In 7-day old embryos, in which ectodermal cells become partitioned into 2 types, almost no neural ectodermal cells are incorporated with BrdU, whereas a constant incorporation is revealed in non-neural ectodermal cells. Among the mitotically quiescent neural ectodermal cells, which are arrested at the GI stage of the cell cycle, in 8-day old embryos, the neuroblasts are the first to resume their mitotic activity, while the other cells are then released from the mitotic quiescence. It seems that the mitotic quiescence may be an essential process to acquire a neural fate.     

Subventricular zone astrocytes are neural stem cells in the adult mammalian brain.

Neural stem cells reside in the subventricular zone (SVZ) of the adult mammalian brain. This germinal region, which continually generates new neurons destined for the olfactory bulb, is composed of four cell types: migrating neuroblasts, immature precursors, astrocytes, and ependymal cells. Here we show that SVZ astrocytes, and not ependymal cells, remain labeled with proliferation markers after long survivals in adult mice. After elimination of immature precursors and neuroblasts by an antimitotic treatment, SVZ astrocytes divide to generate immature precursors and neuroblasts. Furthermore, in untreated mice, SVZ astrocytes specifically infected with a retrovirus give rise to new neurons in the olfactory bulb. Finally, we show that SVZ astrocytes give rise to cells that grow into multipotent neurospheres in vitro. We conclude that SVZ astrocytes act as neural stem cells in both the normal and regenerating brain.