Distinct mechanisms triggering glial differentiation in Drosophila thoracic and abdominal neuroblasts 6-4

Neurons and glia are produced in stereotyped patterns after neuroblast cell division during development of the Drosophila central nervous system. The first cell division of thoracic neuroblast 6-4 (NB6-4T) is asymmetric, giving rise to a glial precursor cell and a neuronal precursor cell. In contrast, abdominal NB6-4 (NB6-4A) divides symmetrically to produce two glial cells. To understand the relationship between cell division and glia-neuron cell fate determination, we examined and compared the effects of known cell division mutations on the NB6-4T and NB6-4A lineages. Based on observation of expression of glial fate determination and early glial differentiation genes, the onset of glial differentiation occurred in NB6-4A but not in NB6-4T when both cell cycle progression and cytokinesis were genetically arrested. On the other hand, glial differentiation started in both lineages when cytokinesis was blocked with intact cell cycle progression. These results showed that NB6-4T, but not NB6-4A, requires cell cycle progression for acquisition of glial fate, suggesting that distinct mechanisms trigger glial differentiation in the different lineages.     

Asymmetric cell division of thoracic neuroblast 6-4 to bifurcate glial and neuronal lineage in Drosophila

In the development of the Drosophila central nervous system, some of the neuroblasts designated as neuroglioblasts generate both glia and neurons. Little is known about how neuroglioblasts produce these different cell types. NB6-4 in the thoracic segment (NB6-4T) is a neuroglioblast, although the corresponding cell in the abdominal segment (NB6-4A) produces only glia. Here, we describe the cell divisions in the NB6-4T lineage, following changes in cell number and cell arrangement. We also examined successive changes in the expression of glial cells missing (gcm) mRNA and protein, activity of which is known to direct glial fate from the neuronal default state. The first cell division of NB6-4T occurred in the medial-lateral orientation, and was found to bifurcate the glial and neuronal lineage. After division, the medial daughter cell expressed GCM protein to produce three glial cells, while the lateral daughter cell with no GCM expression produced ganglion mother cells, secondary precursors of neurons. Although gcm mRNA was present evenly in the cytoplasm of NB6-4T before the first cell division, it became detected asymmetrically in the cell during mitosis and eventually only in the medial daughter cell. In contrast, NB6-4A showed a symmetrical distribution of gcm mRNA and GCM protein through division. Our observations suggest that mechanisms regulating gcm mRNA expression and its translation play an important role in glial and neuronal lineage bifurcation that results from asymmetric cell division.     

A critical role for cyclin E in cell fate determination in the central nervous system of Drosophila melanogaster

We have examined the process by which cell diversity is generated in neuroblast (NB) lineages in the central nervous system of Drosophila melanogaster. Thoracic NB6-4 (NB6-4t) generates both neurons and glial cells, whereas NB6-4a generates only glial cells in abdominal segments. This is attributed to an asymmetric first division of NB6-4t, localizing prospero (pros) and glial cell missing (gcm) only to the glial precursor cell, and a symmetric division of NB6-4a, where both daughter cells express pros and gcm. Here we show that the NB6-4t lineage represents the ground state, which does not require the input of any homeotic gene, whereas the NB6-4a lineage is specified by the homeotic genes abd-A and Abd-B. They specify the NB6-4a lineage by down-regulating levels of the G1 cyclin, DmCycE (CycE). CycE, which is asymmetrically expressed after the first division of NB6-4t, functions upstream of pros and gcm to specify the neuronal sublineage. Loss of CycE function causes homeotic transformation of NB6-4t to NB6-4a, whereas ectopic CycE induces reverse transformations. However, other components of the cell cycle seem to have a minor role in this process, suggesting a critical role for CycE in regulating cell fate in segment-specific neural lineages.     

The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells

In Drosophila, neurons and glial cells are produced by neural precursor cells called neuroblasts (NBs), which can be individually identified. Each NB generates a characteristic cell lineage specified by a precise spatiotemporal control of gene expression within the NB and its progeny. Here we show that the homeobox genes ladybird early and ladybird late are expressed in subsets of cells deriving from neuroblasts NB 5-3 and NB 5-6 and are essential for their correct development. Our analysis revealed that ladybird in Drosophila, like their vertebrate orthologous Lbx1 genes, play an important role in cell fate specification processes. Among those cells that express ladybird are NB 5-6-derived glial cells. In ladybird loss-of-function mutants, the NB 5-6-derived exit glial cells are absent while overexpression of these genes leads to supernumerary glial cells of this type. Furthermore, aberrant glial cell positioning and aberrant spacing of axonal fascicles in the nerve roots observed in embryos with altered ladybird function suggest that the ladybird genes might also control directed cell movements and cell-cell interactions within the developing Drosophila ventral nerve cord.     

Cell cycle independent role of Cyclin E during neural cell fate specification in Drosophila is mediated by its regulation of Prospero function

During development, neural progenitor cells or neuroblasts generate a great intra- and inter-segmental diversity of neuronal and glial cell types in the nervous system. In thoracic segments of the embryonic central nervous system of Drosophila, the neuroblast NB6-4t undergoes an asymmetric first division to generate a neuronal and a glial sublineage, while abdominal NB6-4a divides once symmetrically to generate only 2 glial cells. We had earlier reported a critical function for the G1 cyclin, CyclinE (CycE) in regulating asymmetric cell division in NB6-4t. Here we show that (i) this function of CycE is independent of its role in cell cycle regulation and (ii) the two functions are mediated by distinct domains at the protein level. Results presented here also suggest that CycE inhibits the function of Prospero and facilitates its cortical localization, which is critical for inducing stem cell behaviour, i.e. asymmetric cell division of NB6-4t. Furthermore our data imply that CycE is required for the maintenance of stem cell identity of most other neuroblasts.     

Sloppy paired acts as the downstream target of wingless in the Drosophila CNS and interaction between sloppy paired and gooseberry inhibits sloppy paired during neurogenesis

Wingless (Wg) and other Wnt proteins play a crucial role in a number of developmental decisions in a variety of organisms. In the ventral nerve cord of the Drosophila embryo, Wg is non-autonomously required for the formation and specification of a neuronal precursor cell, NB4-2. NB4-2 gives rise to a well-studied neuronal lineage, the RP2/sib lineage. While the various components of the Wg-signaling pathway are also required for generating NB4-2, the target gene(s) of this pathway in the signal-receiving cell is not known. In this paper, we show that sloppy paired 1 and sloppy paired 2 function as the downstream targets of the Wg signaling to generate the NB4-2 cell. Thus, while the loss-of-function mutations in wg and slp have the same NB4-2 formation and specification defects, these defects in wg mutants can be rescued by expressing slp genes from a heterologous promoter. That slp genes function downstream of the Wg signaling is also indicated by the result that expression of slp genes is lost from the neuroectoderm in wg mutants and that ectopic expression of wg induces ectopic expression of slp. Finally, previous results show that Gooseberry (Gsb) prevents Wg from specifying NB4-2 identity to the wg-expressing NB5-3. In this paper, we also show that gsb interacts with slp and prevents Slp from specifying NB4-2 identity. Overexpression of slp overcomes this antagonistic interaction and respecifies NB5-3 as NB4-2. This respecification, however, can be suppressed by a simultaneous overexpression of gsb at high levels. This mechanism appears to be responsible for specifying NB5-3 identity to a row 5 neuroblast and preventing Wg from specifying NB4-2 identity to that cell.     

Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression

During neural lineage progression, differences in daughter cell proliferation can generate different lineage topologies. This is apparent in the Drosophila neuroblast 5-6 lineage (NB5-6T), which undergoes a daughter cell proliferation switch from generating daughter cells that divide once to generating neurons directly. Simultaneously, neural lineages, e.g. NB5-6T, undergo temporal changes in competence, as evidenced by the generation of different neural subtypes at distinct time points. When daughter proliferation is altered against a backdrop of temporal competence changes, it may create an integrative mechanism for simultaneously controlling cell fate and number. Here, we identify two independent pathways, Prospero and Notch, which act in concert to control the different daughter cell proliferation modes in NB5-6T. Altering daughter cell proliferation and temporal progression, individually and simultaneously, results in predictable changes in cell fate and number. This demonstrates that different daughter cell proliferation modes can be integrated with temporal competence changes, and suggests a novel mechanism for coordinately controlling neuronal subtype numbers.     

Successive specification of Drosophila neuroblasts NB 6-4 and NB 7-3 depends on interaction of the segment polarity genes wingless, gooseberry and naked cuticle

The Drosophila central nervous system derives from neural precursor cells, the neuroblasts (NBs), which are born from the neuroectoderm by the process of delamination. Each NB has a unique identity, which is revealed by the production of a characteristic cell lineage and a specific set of molecular markers it expresses. These NBs delaminate at different but reproducible time points during neurogenesis (S1-S5) and it has been shown for early delaminating NBs (S1/S2) that their identities depend on positional information conferred by segment polarity genes and dorsoventral patterning genes. We have studied mechanisms leading to the fate specification of a set of late delaminating neuroblasts, NB 6-4 and NB 7-3, both of which arise from the engrailed (en) expression domain, with NB 6-4 delaminating first. In contrast to former reports, we did not find any evidence for a direct role of hedgehog in the process of NB 7-3 specification. Instead, we present evidence to show that the interplay of the segmentation genes naked cuticle (nkd) and gooseberry (gsb), both of which are targets of wingless (wg) activity, leads to differential commitment to NB 6-4 and NB 7-3 cell fate. In the absence of either nkd or gsb, one NB fate is replaced by the other. However, the temporal sequence of delamination is maintained, suggesting that formation and specification of these two NBs are under independent control.     

RanGAP‐mediated nucleocytoplasmic transport of Prospero regulates neural stem cell lifespan in Drosophila larval central brain

By the end of neurogenesis in Drosophila pupal brain neuroblasts (NBs), nuclear Prospero (Pros) triggers cell cycle exit and terminates NB lifespan. Here, we reveal that in larval brain NBs, an intrinsic mechanism facilitates import and export of Pros across the nuclear envelope via a Ran‐mediated nucleocytoplasmic transport system. In rangap mutants, the export of Pros from the nucleus to cytoplasm is impaired and the nucleocytoplasmic transport of Pros becomes one‐way traffic, causing an early accumulation of Pros in the nuclei of the larval central brain NBs. This nuclear Pros retention initiates NB cell cycle exit and leads to a premature decrease of total NB numbers. Our data indicate that RanGAP plays a crucial role in this intrinsic mechanism that controls NB lifespan during neurogenesis. Our study may provide insights into understanding the lifespan of neural stem cells during neurogenesis in other organisms.     

Basement membrane attachment is dispensable for radial glial cell fate and for proliferation, but affects positioning of neuronal subtypes

Radial glial cells have been shown to act as neuronal precursors in the developing cortex and to maintain their radial processes attached to the basement membrane (BM) during cell division. Here, we examined a potential role of direct signalling from the BM to radial glial cells in three mouse mutants where radial glia attachment to the BM is disrupted. This is the case if the nidogen-binding site of the laminin gamma1 chain is mutated, in the absence of alpha6 integrin or of perlecan, an essential BM component. Surprisingly, cortical radial glial cells lacking contact to the BM were not affected in their proliferation, interkinetic nuclear migration, orientation of cell division and neurogenesis. Only a small subset of precursors was located ectopically within the cortical parenchyma. Notably, however, neuronal subtype composition was severely disturbed at late developmental stages (E18) in the cortex of the laminin gamma1III4-/- mice. Thus, although BM attachment seems dispensable for precursor cells, an intact BM is required for adequate neuronal composition of the cerebral cortex.     

Survival of hippocampal and cortical neurons in a mixture of MEM+ and B27-supplemented neurobasal medium

Serum-free B-27 supplemented neurobasal (NB) and a 10% fetal bovine serum-supplemented Eagle's minimum essential medium (MEM+) are used to culture rat embryonic hippocampal neurons for different purposes. Although NB medium leads to enhanced cell survival, it contains biological antioxidants and is not suitable for the study of free radical damage and oxidation in cultured neurons. MEM+ without additional antioxidants has been used widely in the study of free radical damage and oxidation, although it does not support optimum neuronal survival in culture. Serum in MEM+ leads to enhanced cell survival but also promotes glial cell proliferation. In this study, we used a new combination medium (NM-2) that consists of both NB and MEM+ for growing primary hippocampal and cortical neuronal cultures. NM-2 enhanced neuronal survival 78.9% for dissociated neurons at a density of 50 cells/mm(2) and 83.1% for 100 cells/mm(2), while decreasing glial cell proliferation to 2-3% and completely inhibiting oligodendrocytes. The NM-2 minimized the effectiveness of antioxidants in the medium to the neurotoxin 4-hydroxynonenal. It also decreased neuronal clumping and provided a more even distribution of neurons. Neurons survived for 4 weeks in NM-2 without changing the original medium. NM-2 provides a good environment for studies of free radical damage and oxidation of neurons. The combination incorporates the best of both NB and MEM+ that results in high neuron survival rate, low glial cell proliferation, reduced antioxidant level, and provides relatively pure cultures of hippocampal and cortical neurons.     

Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage

The developing central nervous system of vertebrates contains an abundant cell type designated radial glial cells. These cells are known as guiding cables for migrating neurons, while their role as precursor cells is less clear. Since radial glial cells express a variety of astroglial characteristics and differentiate as astrocytes after completing their guidance function, they have been considered as part of the glial lineage. Using fluorescence-activated cell sorting, we show here that radial glial cells also are neuronal precursors and only later, after neurogenesis, do they shift towards an exclusive generation of astrocytes. These results thus demonstrate a novel function for radial glial cells, namely their ability to generate two major cell types found in the nervous system, neurons and astrocytes.     

Early events in insect neurogenesis. II. The role of cell interactions and cell lineage in the determination of neuronal precursor cells

The insect central nervous system (CNS) is composed of a brain and a chain of segmental ganglia; each hemiganglion contains about 1000 individually identifiable neurons. How is the enormous neuronal diversity and specificity generated? Neurons of a hemiganglion largely arise during embryogenesis from a stereotyped pattern of individually identified neuronal precursor cells, called neuroblasts (NBs). The transition from ectoderm to individual neurons thus involves two major steps: first, an undifferentiated ectodermal cell sheet produces the stereotyped pattern of 30 NBs per hemisegment; second, each of these NBs contributes a specific family of neuronal progeny to the developing CNS. We have used a laser microbeam to ablate individual cells in the grasshopper embryo in order to study the initial events of neuronal determination. In particular, how does a layer of apparently equivalent ectodermal cells produce a highly stereotyped pattern of unique NBs? Our results suggest the following mechanism for NB determination. (1) Cell interactions between the approximately 150 equivalent ectodermal cells of a hemisegment allow 30 cells to enlarge into NBs. (2) As these young NBs enlarge they inhibit adjacent ectodermal cells from becoming NBs; the adjacent cells then either differentiate into nonneuronal support cells or die. (3) Each NB is assigned a unique identity due to its position of enlargement within the neuroepithelium. (4) The NB then generates its characteristic family of neurons by an invariant cell lineage. Development of the insect CNS depends on cell interactions and positional cues to create a pattern of NBs, and then on cell lineage to restrict the fate of the NB progeny.     

L-myc and N-myc influence lineage determination in the central nervous system.

The N-myc and the L-myc proto-oncogenes are expressed during embryonal development mainly in the developing brain. Studies of their expression in single neuroepithelial cells revealed that neural precursors not yet committed to the glial or the neuronal lineage expressed both genes, but after lineage commitment they expressed either N-myc or L-myc. Moreover, enforced expression of L-myc in the neural precursor cell line 2.3D caused neuronal differentiation, while the expression of N-myc promoted glial differentiation. These results indicate that L-myc and N-myc play critical roles in lineage determination for the central nervous system.     

A novel mode of asymmetric division identifies the fly neuroglioblast 6-4T.

Asymmetric cell divisions and segregation of fate determinants are crucial events in the generation of cell diversity. Fly neuroblasts, the precursors that self-reproduce and generate neurons, represent a clear example of asymmetrically dividing cells. Less is known about how neurons and glial cells are generated by multipotent precursors. Flies provide the ideal model system to study this process. Indeed, neuroglioblasts (NGBs) can be specifically identified and have been shown to require the glide/gcm fate determinant to produce glial cells, which otherwise would become neurons. Here, we follow the division of a specific NGB (NGB6-4T), which produces a neuroblast (NB) and a glioblast (GB). We show that, to generate the glioblast, glide/gcm RNA becomes progressively unequally distributed during NGB division and preferentially segregates. Subsequently, a GB-specific factor is required to maintain glide/gcm expression. Both processes are necessary for gliogenesis, showing that the glial vs. neuronal fate choice is a two-step process. This feature, together with glide/gcm subcellular RNA distribution and the behavior of the NGB mitotic apparatus identify a novel type of division generating cell diversity.     

Distribution and Development of Peripheral Glial Cells in the Human Fetal Cochlea

The adult human cochlea contains various types of peripheral glial cells that envelop or myelinate the three different domains of the spiral ganglion neurons: the central processes in the cochlear nerve, the cell bodies in the spiral ganglia, and the peripheral processes in the osseous spiral lamina. Little is known about the distribution, lineage separation and maturation of these peripheral glial cells in the human fetal cochlea. In the current study, we observed peripheral glial cells expressing SOX10, SOX9 and S100B as early as 9 weeks of gestation (W9) in all three neuronal domains. We propose that these cells are the common precursor to both mature Schwann cells and satellite glial cells. Additionally, the peripheral glial cells located along the peripheral processes expressed NGFR, indicating a phenotype distinct from the peripheral glial cells located along the central processes. From W12, the spiral ganglion was gradually populated by satellite glial cells in a spatiotemporal gradient. In the cochlear nerve, radial sorting was accomplished by W22 and myelination started prior to myelination of the peripheral processes. The developmental dynamics of the peripheral glial cells in the human fetal cochlea is in support of a neural crest origin. Our study provides the first overview of the distribution and maturation of peripheral glial cells in the human fetal cochlea from W9 to W22.