Upregulation of Mitimere and Nubbin acts through cyclin E to confer self-renewing asymmetric division potential to neural precursor cells

In the Drosophila CNS, neuroblasts undergo self-renewing asymmetric divisions, whereas their progeny, ganglion mother cells (GMCs), divide asymmetrically to generate terminal postmitotic neurons. It is not known whether GMCs have the potential to undergo self-renewing asymmetric divisions. It is also not known how precursor cells undergo self-renewing asymmetric divisions. Here, we report that maintaining high levels of Mitimere or Nubbin, two POU proteins, in a GMC causes it to undergo self-renewing asymmetric divisions. These asymmetric divisions are due to upregulation of Cyclin E in late GMC and its unequal distribution between two daughter cells. GMCs in an embryo overexpressing Cyclin E, or in an embryo mutant for archipelago, also undergo self-renewing asymmetric divisions. Although the GMC self-renewal is independent of inscuteable and numb, the fate of the differentiating daughter is inscuteable and numb-dependent. Our results reveal that regulation of Cyclin E levels, and asymmetric distribution of Cyclin E and other determinants, confer self-renewing asymmetric division potential to precursor cells, and thus define a pathway that regulates such divisions. These results add to our understanding of maintenance and loss of pluripotential stem cell identity.     

Identification of Drosophila type II neuroblast lineages containing transit amplifying ganglion mother cells

Mammalian neural stem cells generate transit amplifying progenitors that expand the neuronal population, but these type of progenitors have not been studied in Drosophila. The Drosophila larval brain contains approximately 100 neural stem cells (neuroblasts) per brain lobe, which are thought to bud off smaller ganglion mother cells (GMCs) that each produce two post-mitotic neurons. Here, we use molecular markers and clonal analysis to identify a novel neuroblast cell lineage containing "transit amplifying GMCs" (TA-GMCs). TA-GMCs differ from canonical GMCs in several ways: each TA-GMC has nuclear Deadpan, cytoplasmic Prospero, forms Prospero crescents at mitosis, and generates up to 10 neurons; canonical GMCs lack Deadpan, have nuclear Prospero, lack Prospero crescents at mitosis, and generate two neurons. We conclude that there are at least two types of neuroblast lineages: a Type I lineage where GMCs generate two neurons, and a type II lineage where TA-GMCs have longer lineages. Type II lineages allow more neurons to be produced faster than Type I lineages, which may be advantageous in a rapidly developing organism like Drosophila.     

Neuronal determination during embryonic development of the grasshopper nervous system

We have examined the roles of cell lineage and interactions in the determination of individual identified neurons in the grasshopper embryo by selective ablations of individual cells and/or their neighbors at successive stages following their birth. The neurons in the grasshopper central nervous system (CNS) are produced by two types of identifiable neuronal precursor cells: neuroblasts (NBs), which generate most of the neurons, and midline precursors (MPs), which generate only a few. NBs divide asymmetrically in a stem cell fashion to generate a chain of ganglion mother cells (GMCs) which then divide once more symmetrically to produce pairs of sibling neurons: MPs cleave once to generate a single pair of sibling neurons. We analyzed the determination of (1) the pair of sibling progeny produced by midline precursor 3 (MP3) and the determination of (2) the pair of sibling progeny produced by the first GMC from neuroblast 1-1 (NB 1-1); in each case the siblings normally differentiate into morphologically distinct neurons. Our results indicate that both pairs of neuronal progeny (1) are born equivalent, (2) become determined by cell interactions early in their development before axonogenesis, and (3) demonstrate a hierarchy of fates with one fate dominant over the other. These results suggest a common pattern of neuronal determination in the grasshopper and possibly all insect embryos.     

Slit signaling promotes the terminal asymmetric division of neural precursor cells in the Drosophila CNS.

The bipotential Ganglion Mother Cells, or GMCs, in the Drosophila CNS asymmetrically divide to generate two distinct post-mitotic neurons. Here, we show that the midline repellent Slit (Sli), via its receptor Roundabout (Robo), promotes the terminal asymmetric division of GMCs. In GMC-1 of the RP2/sib lineage, Slit promotes asymmetric division by down regulating two POU proteins, Nubbin and Mitimere. The down regulation of these proteins allows the asymmetric localization of Inscuteable, leading to the asymmetric division of GMC-1. Consistent with this, over-expression of these POU genes in a late GMC-1 causes mis-localization of Insc and symmetric division of GMC-1 to generate two RP2s. Similarly, increasing the dosage of the two POU genes in sli mutant background enhances the penetrance of the RP2 lineage defects whereas reducing the dosage of the two genes reduces the penetrance of the phenotype. These results tie a cell-non-autonomous signaling pathway to the asymmetric division of precursor cells during neurogenesis.     

Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal

An important question in stem cell biology is how a cell decides to self-renew or differentiate. Drosophila neuroblasts divide asymmetrically to self-renew and generate differentiating progeny called GMCs. Here, we report that the Brain tumor (Brat) translation repressor is partitioned into GMCs via direct interaction with the Miranda scaffolding protein. In brat mutants, another Miranda cargo protein (Prospero) is not partitioned into GMCs, GMCs fail to downregulate neuroblast gene expression, and there is a massive increase in neuroblast numbers. Single neuroblast clones lacking Prospero have a similar phenotype. We conclude that Brat suppresses neuroblast stem cell self-renewal and promotes neuronal differentiation.     

Patterns of cell division and expression of asymmetric cell fate determinants in postembryonic neuroblast lineages of Drosophila

We have studied the division of postembryonic neuroblasts (Nbs) in the outer proliferation center (OPC) and central brain anlagen of Drosophila. We focused our attention on three aspects of these processes: the pattern of cellular division, the topological orientation of those divisions, and the expression of asymmetric cell fate determinants. Although larval Nbs are of embryonic origin, our results indicate that their properties appear to be modified during development. Several conclusions can be summarized: (i) In early larvae, Nbs divide symmetrically to give rise to two Nbs while in the late larval brain most Nbs divide asymmetrically to bud off an intermediate ganglion mother cell (GMC) that very rapidly divides into two ganglion cells (GC). (ii) Symmetric and asymmetric divisions of OPC Nbs show tangential and radial orientations, respectively. (iii) This change in the pattern of division correlates with the expression of inscuteable, which is apically localized only in asymmetric divisions. (iv) The spindle of asymmetrically dividing Nb is always oriented on an apical-basal axis. (v) Prospero does not colocalize with Miranda in the cortical crescent of mitotic Nbs. (vi) Prospero is transiently expressed in one of the two sibling GCs generated by the division of GMCs. The implications of these results on cell fate specification and differentiation of adult brain neurons are discussed.     

Prospero maintains the mitotic potential of glial precursors enabling them to respond to neurons

During central nervous system development, glial cells need to be in the correct number and location, at the correct time, to enable axon guidance and neuropile formation. Repair of the injured or diseased central nervous system will require the manipulation of glial precursors, so that the number of glial cells is adjusted to that of neurons, enabling axonal tracts to be rebuilt, remyelinated and functional. Unfortunately, the molecular mechanisms controlling glial precursor proliferative potential are unknown. We show here that glial proliferation is regulated by interactions with axons and that the Drosophila gene prospero is required to maintain the mitotic potential of glia. During growth cone guidance, Prospero positively regulates cycE promoting cell proliferation. Neuronal Vein activates the MAPKinase signalling pathway in the glia with highest Prospero levels, coupling axon extension with glial proliferation. Later on, Prospero maintains glial precursors in an undifferentiated state by activating Notch and antagonising the p27/p21 homologue Dacapo. This enables prospero-expressing cells alone to divide further upon elimination of neurons and to adjust glial number to axons during development.     

Ultrastructure of stomatal development in Arabidopsis (Brassicaceae) leaves

Stomatal development was studied in wild-type Arabidopsis leaves using light and electron microscopy. Development involves three successive types of stomatal precursor cells: meristemoid mother cells, meristemoids, and guard mother cells (GMCs). The first two types divide asymmetrically, whereas GMCs divide symmetrically. Analysis of cell wall patterns indicates that meristemoids can divide asymmetrically a variable number of times. Before meristemoid division, the nucleus and a preprophase band of microtubules become located on one side of the cell, and the vacuole on the other. Meristemoids are often triangular in shape and have evenly thickened walls. GMCs can be detected by their roughly oval shape, increased starch accumulation, and wall thickenings on opposite ends of the cells. Because these features are also found in developing stomata, stomatal differentiation begins in GMCs. The wall thickenings mark the division site in the GMC since they overlie a preprophase band of microtubules and occur where the cell plate fuses with the parent cell wall. Stomatal differentiation in Arabidopsis resembles that of other genera with kidney-shaped guard cells. This identification of stages in stomatal development in wild-type Arabidopsis provides a foundation for the analysis of relevant genes and of mutants defective in stomatal patterning, cell specification, and differentiation.     

Progenitor properties of symmetrically dividing Drosophila neuroblasts during embryonic and larval development

Asymmetric cell division generates two daughter cells of differential gene expression and/or cell shape. Drosophila neuroblasts undergo typical asymmetric divisions with regard to both features; this is achieved by asymmetric segregation of cell fate determinants (such as Prospero) and also by asymmetric spindle formation. The loss of genes involved in these individual asymmetric processes has revealed the roles of each asymmetric feature in neurogenesis, yet little is known about the fate of the neuroblast progeny when asymmetric processes are blocked and the cells divide symmetrically. We genetically created such neuroblasts, and found that in embryos, they were initially mitotic and then gradually differentiated into neurons, frequently forming a clone of cells homogeneous in temporal identity. By contrast, larval neuroblasts with the same genotype continued to proliferate without differentiation. Our results indicate that asymmetric divisions govern lineage length and progeny fate, consequently generating neural diversity, while the progeny fate of symmetrically dividing neuroblasts depends on developmental stages, presumably reflecting differential activities of Prospero in the nucleus.     

From epithelium to neuroblasts to neurons: the role of cell interactions and cell lineage during insect neurogenesis

The grasshopper central nervous system is composed of a brain and a chain of segmental ganglia. Each hemiganglion contains about 1000 neurons, most of which can be individually identified by their unique morphology and synaptic connectivity. Shortly after gastrulation the ventral ectoderm becomes a neurogenic region. In each hemisegment, ca. 150 neurogenic ectodermal cells (nECs) give rise to a stereotyped pattern of 30 identified neuroblasts (NBs, neuronal stem cells); the remaining nECs become various non-neuronal cells or die. The 30 NBs then give rise to about 1000 neurons as each NB initiates an invariant lineage, generating a stereotyped chain of ganglion mother cells (GMCs), each of which in turn divides once to generate two identified neurons. We have used a laser microbeam or microelectrode to ablate individual cells in ovo and in vitro at various stages of embryogenesis to study how neuronal diversity and specificity are generated during development. Our results suggest that cell interactions between ca. 150 equivalent nECs allow 30 cells to enlarge into NBs, the dominant fate in a hierarchy; the NBs inhibit adjacent nECs and thus cause them to differentiate into various non-neuronal cells; each NB is assigned its unique identity according to its position of enlargement within the neurogenic epithelium; each NB then generates its characteristic chain of GMCs by an invariant cell lineage; and each GMC generates a pair of equivalent progeny, the fate of each individual neuron being determined by both its GMC of origin and interactions with its sibling.     

Drosophila homologs of mammalian TNF/TNFR-related molecules regulate segregation of Miranda/Prospero in neuroblasts

During neuroblast (NB) divisions, cell fate determinants Prospero (Pros) and Numb, together with their adaptor proteins Miranda (Mira) and Partner of Numb, localize to the basal cell cortex at metaphase and segregate exclusively to the future ganglion mother cells (GMCs) at telophase. In inscuteable mutant NBs, these basal proteins are mislocalized during metaphase. However, during anaphase/telophase, these mutant NBs can partially correct these earlier localization defects and redistribute cell fate determinants as crescents to the region where the future GMC "buds" off. This compensatory mechanism has been referred to as "telophase rescue". We demonstrate that the Drosophila homolog of the mammalian tumor-necrosis factor (TNF) receptor-associated factor (DTRAF1) and Eiger (Egr), the homolog of the mammalian TNF, are required for telophase rescue of Mira/Pros. DTRAF1 localizes as an apical crescent in metaphase NBs and this apical localization requires Bazooka (Baz) and Egr. The Mira/Pros telophase rescue seen in inscuteable mutant NBs requires DTRAF1. Our data suggest that DTRAF1 binds to Baz and acts downstream of Egr in the Mira/Pros telophase rescue pathway.     

Two distinct mechanisms segregate Prospero in the longitudinal glia underlying the timing of interactions with axons

Prospero is required in dividing longitudinal glia (LG) during axon guidance; initially to enable glial division in response to neuronal contact, and subsequently to maintain glial precursors in a quiescent state with mitotic potential. Only Prospero-positive LG respond to neuronal ablation by over-proliferating, mimicking a glial-repair response. Prospero is distributed unequally through the progeny cells of the longitudinal glioblast lineage. Just before axon contact the concentration of Prospero is higher in two of the four progeny cells, and after axon guidance Prospero is present only in six out of ten progeny LG. Here we ask how Prospero is distributed unequally in these two distinct phases. We show that before neuronal contact, longitudinal glioblasts undergo invaginating divisions, perpendicular to the ectodermal layer. Miranda is required to segregate Prospero asymmetrically up to the four glial-progeny stage. After neuronal contact, Prospero is present in only the LG that activate Notch signalling in response to Serrate provided by commissural axons, and Numb is restricted to the glia that do not contain Prospero. As a result of this dual regulation of Prospero deployment, glia are coupled to the formation and maintenance of axonal trajectories.     

Cell cycling and DNA replication in a mutant blocked in cell division in the nematode Caenorhabditis elegans

The postembryonic development of the nematode Caenorhabditis elegans has been described at the level of individual cell lineages. A mutant of postembryonic development, lin-5 II, causes a failure of postembryonic nuclear and cell divisions. Mitosis in living animals is seen by light microscopy to proceed through prophase and nuclear envelope breakdown, but an abnormal-looking metaphase plate forms in the mutant, after which the interphase nuclear morphology reappears until the next attempted round of division. The precursor cells which give rise to the ventral nerve cord have been studied in lin-5. In the wild type these cells divide asymmetrically to give six descendants (one hypodermal cell and five neurons). In the mutant these precursors accumulate approximately six times the diploid quantity of DNA within a single nucleus, while attempting mitosis up to three times. These polyploid cells display characteristics of the cells they would have produced ordinarily.