grim promotes programmed cell death of Drosophila microchaete glial cells

The Inhibitor of apoptosis (IAP) antagonists Reaper (Rpr), Grim and Hid are central regulators of developmental apoptosis in Drosophila. Ectopic expression of each is sufficient to trigger apoptosis, and hid and rpr have been shown to be important for programmed cell death (PCD). To investigate the role for grim in PCD, a grim null mutant was generated. grim was not a key proapoptotic gene for embryonic PCD, confirming that grim cooperates with rpr and hid in embryogenesis. In contrast, PCD of glial cells in the microchaete lineage required grim, identifying a death process dependent upon endogenous grim. Grim associates with mitochondria and has been shown to activate a mitochondrial death pathway distinct from IAP antagonization; therefore, the Drosophila bcl-2 genes buffy and debcl were investigated for genetic interaction with grim. Loss of buffy led to microchaete glial cell survival and suppressed death in the eye induced by ectopic Grim. This is the first example of a developmental PCD process influenced by buffy, and places buffy in a proapoptotic role. PCD of microchaete glial cells represents an exceptional opportunity to study the mitochondrial proapoptotic process induced by Grim.     

A glial cell arises from an additional division within the mechanosensory lineage during development of the microchaete on the Drosophila notum.

We have used different cell markers to trace the development of the sensory cells of the thoracic microchaete. Our results dictate a revision in the currently accepted model for cell lineage within the mechanosensory bristle. The sensory organ progenitor divides to form two secondary progenitors: PIIa and PIIb. PIIb divides first to give rise to a tertiary progenitor-PIII and a glial cell. This is followed by division of PIIa to form the shaft and socket cells as described before. PIII expresses high levels of Elav and low levels of Prospero and divides to produce neuron and sheath. Its sibling cell expresses low Elav and high Prospero and is recognized by the glial marker, Repo. This cell migrates away from the other cells of the lineage following differentiation. The proposed modification in lineage has important implications for previous studies on sibling cell fate choice and cell fate specification in sensory systems.     

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.     

The regulation of apoptosis by Numb/Notch signaling in the serotonin lineage of Drosophila

Apoptosis is prevalent during development of the central nervous system (CNS), yet very little is known about the signals that specify an apoptotic cell fate. In this paper, we examine the role of Numb/Notch signaling in the development of the serotonin lineage of Drosophila and show that it is necessary for regulating apoptosis. Our results indicate that when Numb inhibits Notch signaling, cells undergo neuronal differentiation, whereas cells that maintain Notch signaling initiate apoptosis. The apoptosis inhibitor p35 can counteract Notch-mediated apoptosis and rescue cells within the serotonin lineage that normally undergo apoptosis. Furthermore, we observe tumor-like overproliferation of cells in the CNS when Notch signaling is reduced. These data suggest that the distribution of Numb during terminal mitotic divisions of the CNS can distinguish between a neuronal cell fate and programmed cell death.     

An epithelial niche in the Drosophila ovary undergoes long-range stem cell replacement

Adult epithelial stem cells are thought to reside in specific niches, where they are maintained by adhesion to stromal cells and by intercellular signals. In niches that harbor multiple adjacent stem cells, such as those maintaining Drosophila germ cells, lost stem cells are replaced by division of neighboring stem cells or reversion of transit cells. We have characterized the Drosophila follicle stem cell (FSC) niche as a model of the epithelial niche to learn whether nonneighboring cells can also generate stem cell replacements. Exactly two stroma-free FSC niches holding single FSCs are located in fixed locations on opposite edges of the Drosophila ovariole. FSC daughters regularly migrate across the width of the ovariole to the other niche before proliferating and contributing to the follicle cell monolayer. Crossmigrating FSC daughters compete with the resident FSC for niche occupancy and are the source of replacement FSCs. The ability of stem cell daughters to target a distant niche and displace its resident stem cell suggests that precancerous mutations might spread from niche to niche within stem cell-based tissues.     

The Drosophila transmembrane protein Fear-of-intimacy controls glial cell migration

Development of complex organs depends on intensive cell-cell interactions, which help coordinate movements of many cell types. In a genetic screen aimed to identify genes controlling midline glia migration in the Drosophila nervous system, we have identified mutations in the gene kastchen. Here we show that during embryogenesis kastchen is also required for the normal migration of longitudinal and peripheral glial cells. During larval development, kastchen non-cell autonomously affects the migration of the subretinal glia into the eye disc. During embryonic development, kastchen not only affects glial cell migration but also controls the migration of muscle cells toward their attachment sites. In all cases, kastchen apparently functions in terminating or restricting cell migration. We identified the molecular nature of the gene by performing transgenic rescue experiments and by sequence analysis of mutant alleles. Kastchen corresponds to the recently described gene fear-of-intimacy (foi) that was identified in screen for genes affecting germ cell migration, suggesting that Foi-Kastchen is more generally involved in regulating cell migration. It encodes a member of an eight-transmembrane domain protein family of putative Zinc transporters or proteases. We determined the topology of the Foi protein by using antisera against luminal and intracellular domains of the protein and provide evidence that it does not act as a Zinc transporter. Genetic evidence suggests that one of the functions of foi may be associated with hedgehog signaling.     

黑腹果蝇细胞谱系分析方法进展

对动物体内单个细胞的谱系进行分析有助于追踪其在发育过程中的作用,但是体内各种组织都是由很多形态、结构、功能各不相同的细胞构成的复杂系统,这种复杂性严重阻碍了对单个细胞的研究。嵌合克隆技术(Mosaic technique)和标记技术(Labeling technique)的出现为这一研究提供了强有力的手段。文章介绍了近几年来黑腹果蝇(Drosophila melanogaster)研究中常用的7种嵌合克隆标记方法,包括FRT介导的有丝分裂重组(FRT-mediated mitotic recombination)、MARCM(Mosaic analysis with a repressible cell marker)、TSG(Twin spotgenerator)、Twin-spot MARCM、Q-MARCM(Q system-based MARCM)、Coupled MARCM和G-TRACE(Gal4technique for real-time and clonal expression)技术,详述了这些技术的原理及应用,并对不同技术进行了对比。运用这些技术研究者可以从单细胞水平进行遗传学标记和操作,特别是在神经系统等复杂系统中追踪单个细胞的发育过程。果蝇中的这些技术也将为其他模式生物追踪细胞谱系提供参考。     

Heparin is a unique marker of progenitors in the glial cell lineage.

The oligodendrocyte-type-2 astrocyte progenitor cells (precursors of oligodendrocytes and type-2 astrocytes) are an excellent system in which to study differentiation as they can be manipulated in vitro. Maintenance of oligodendrocyte-type-2 astrocyte progenitor cells requires basic fibroblast growth factor, a growth factor whose action normally depends on a heparan sulfate coreceptor. Biochemical analysis revealed a most surprising result: that the oligodendrocyte-type-2 astrocyte progenitors did not synthesize heparan sulfate, the near ubiquitous N-sulfated cell surface polysaccharide, but the chemically related heparin in a form that was almost completely N- and O-sulfated. The heparin was detected in the pericellular fraction of the cells and the culture medium. In contrast the differentiated glial subpopulations (oligodendrocytes and type-2 astrocytes) synthesized typical heparan sulfate but with distinctive fine structural features for each cell type. Thus heparin is a unique differentiation marker in the glial lineage. Previously heparin has been found only in a subset of mature mast cells called the connective tissue mast cells. Its presence within the developing nervous system on a precise population of progenitors may confer specific and essential recognition properties on those cells in relation to binding soluble growth and/or differentiation factors and the extracellular matrix.     

Apoptosis-mediated cell death within the ovarian polar cell lineage of Drosophila melanogaster

Polar cells have been described as pairs of specific follicular cells present at each pole of Drosophila egg chambers. They are required at different stages of oogenesis for egg chamber formation and establishment of both the anteroposterior and planar polarities of the follicular epithelium. We show that definition of polar cell pairs is a progressive process since early stage egg chambers contain a cluster of several polar cell marker-expressing cells at each pole, while as of stage 5, they contain invariantly two pairs of such cells. Using cell lineage analysis, we demonstrate that these pre-polar cell clusters have a polyclonal origin and derive specifically from the polar cell lineage, rather than from that giving rise to follicular cells. In addition, selection of two polar cells from groups of pre-polar cells occurs via an apoptosis-dependent mechanism and is required for correct patterning of the anterior follicular epithelium of vitellogenic egg chambers.     

Cell lineage and cell fate specification in the embryonic CNS of Drosophila

The Drosophila CNS derives from a population of neural stem cells, called neuroblasts (NBs), which delaminate individually from the neurogenic region of the ectoderm. In the embryonic ventral nerve cord each NB can be uniquely identified and gives rise to a specific lineage consisting of neurons and/or glial cells. This 'NB identity' is dependent on the position of the progenitor cells in the neuroectoderm before delamination. The positional information is provided by the products of segment polarity and dorsoventral (D/V) patterning genes. Subsequently, 'cell fate genes' like huckebein (hkb) and eagle (eg) contribute to the generation of specific NB lineages. These genes act downstream of segment polarity and D/V patterning genes and regulate different processes such as the generation of glial cells and the determination of serotonergic neurons.     

The Drosophila proteasome undergoes changes in its subunit pattern during development

The two-dimensional electrophoretic protein subunit pattern of the proteasome, which is a mulifunctional non-lysosomal proteinase, was analyzed throughout the development of Drosophila melanogaster. The experiments show that the proteasome is already present in early embryos and its characteristic gross morphology as judged by the outer diameter of 12 nm and the inner depression of 3 nm remains unaltered. The electrophoretic analysis of the enzyme subunits demonstrates that the proteasome undergoes, dependent on development, alterations in its protein composition. The most simple subunit pattern is observed in Schneider's S-3 tissue culture cells and early embryos while with ongoing fly development the subunit pattern of the proteasome becomes increasingly complex. 32P-Labeling and immunoblotting experiments indicate that post-translational modification of the subunits must in part be responsible for the development-dependent diversification of the subunit pattern. Our data raise the possibility that the in vivo proteolytic activity and the in vivo substrate specificity of the proteasome may be regulated by modification of its subunit composition during fly development.     

Two types of asymmetric divisions in the Drosophila sensory organ precursor cell lineage

Asymmetric partitioning of cell-fate determinants during development requires coordinating the positioning of these determinants with orientation of the mitotic spindle. In the Drosophila peripheral nervous system, sensory organ progenitor cells (SOPs) undergo several rounds of division to produce five cells that give rise to a complete sensory organ. Here we have observed the asymmetric divisions that give rise to these cells in the developing pupae using green fluorescent protein fusion proteins. We find that spindle orientation and determinant localization are tightly coordinated at each division. Furthermore, we find that two types of asymmetric divisions exist within the sensory organ precursor cell lineage: the anterior-posterior pI cell-type division, where the spindle remains symmetric throughout mitosis, and the strikingly neuroblast-like apical-basal division of the pIIb cell, where the spindle exhibits a strong asymmetry at anaphase. In both these divisions, the spindle reorientates to position itself perpendicular to the region of the cortex containing the determinant. On the basis of these observations, we propose that two distinct mechanisms for controlling asymmetric cell divisions occur within the same lineage in the developing peripheral nervous system in Drosophila.     

Drosophila E-cadherin regulates the orientation of asymmetric cell division in the sensory organ lineage

BACKGROUND: Generation of cell-fate diversity in Metazoan depends in part on asymmetric cell divisions in which cell-fate determinants are asymmetrically distributed in the mother cell and unequally partitioned between daughter cells. The polarization of the mother cell is a prerequisite to the unequal segregation of cell-fate determinants. In the Drosophila bristle lineage, two distinct mechanisms are known to define the axis of polarity of the pI and pIIb cells. Frizzled (Fz) signaling regulates the planar orientation of the pI division, while Inscuteable (Insc) directs the apical-basal polarity of the pIIb cell. The orientation of the asymmetric division of the pIIa cell is identical to the one of its mother cell, the pI cell, but, in contrast, is regulated by an unknown Insc- and Fz-independent mechanism. RESULTS: DE-Cadherin-Catenin complexes are shown to localize at the cell contact between the two cells born from the asymmetric division of the pI cell. The mitotic spindle of the dividing pIIa cell rotates to line up with asymmetrically localized DE-Cadherin-Catenin complexes. While a complete loss of DE-Cadherin function disrupts the apical-basal polarity of the epithelium, both a partial loss of DE-Cadherin function and expression of a dominant-negative form of DE-Cadherin affect the orientation of the pIIa division. Furthermore, expression of dominant-negative DE-Cadherin also affects the position of Partner of Inscuteable (Pins) and Bazooka, two asymmetrically localized proteins known to regulate cell polarity. These results show that asymmetrically distributed Cad regulates the orientation of asymmetric cell division. CONCLUSIONS: We describe a novel mechanism involving a specialized Cad-containing cortical region by which a daughter cell divides with the same orientation as its mother cell.     

The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons

Neuron-glia communication is central to all nervous system responses to trauma, yet neural injury signaling pathways remain poorly understood. Here we explore cellular and molecular aspects of neural injury signaling in Drosophila. We show that transected Drosophila axons undergo injury-induced degeneration that is morphologically similar to Wallerian degeneration in mammals and can be suppressed by the neuroprotective mouse Wlds protein. Axonal injury elicits potent morphological and molecular responses from Drosophila glia: glia upregulate expression of the engulfment receptor Draper, undergo dramatic changes in morphology, and rapidly recruit cellular processes toward severed axons. In draper mutants, glia fail to respond morphologically to axon injury, and severed axons are not cleared from the CNS. Thus Draper appears to act as a glial receptor for severed axon-derived molecular cues that drive recruitment of glial processes to injured axons for engulfment.     

The secreted cell signal Folded Gastrulation regulates glial morphogenesis and axon guidance in Drosophila

During gastrulation in Drosophila, ventral cells change shape, undergoing synchronous apical constriction, to create the ventral furrow (VF). This process is affected in mutant embryos lacking zygotic function of the folded gastrulation (fog) gene, which encodes a putative secreted protein. Fog is an essential autocrine signal that induces cytoskeletal changes in invaginating VF cells. Here we show that Fog is also required for nervous system development. Fog is expressed by longitudinal glia in the central nervous system (CNS), and reducing its expression in glia causes defects in process extension and axon ensheathment. Glial Fog overexpression produces a disorganized glial lattice. Fog has a distinct set of functions in CNS neurons. Our data show that reduction or overexpression of Fog in these neurons produces axon guidance phenotypes. Interestingly, these phenotypes closely resemble those seen in embryos with altered expression of the receptor tyrosine phosphatase PTP52F. We conducted epistasis experiments to define the genetic relationships between Fog and PTP52F, and the results suggest that PTP52F is a downstream component of the Fog signaling pathway in CNS neurons. We also found that Ptp52F mutants have early VF phenotypes like those seen in fog mutants.