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/     

ming is expressed in neuroblast sublineages and regulates gene expression in the Drosophila central nervous system.

Cell diversity in the Drosophila central nervous system (CNS) is primarily generated by the invariant lineage of neural precursors called neuroblasts. We used an enhancer trap screen to identify the ming gene, which is transiently expressed in a subset of neuroblasts at reproducible points in their cell lineage (i.e. in neuroblast 'sublineages'), suggesting that neuroblast identity can be altered during its cell lineage. ming encodes a predicted zinc finger protein and loss of ming function results in precise alterations in CNS gene expression, defects in axonogenesis and embryonic lethality. We propose that ming controls cell fate within neuroblast cell lineages.     

Polycomb group genes are required for neural stem cell survival in postembryonic neurogenesis of Drosophila

Genes of the Polycomb group (PcG) are part of a cellular memory system that maintains appropriate inactive states of Hox gene expression in Drosophila. Here, we investigate the role of PcG genes in postembryonic development of the Drosophila CNS. We use mosaic-based MARCM techniques to analyze the role of these genes in the persistent larval neuroblasts and progeny of the central brain and thoracic ganglia. We find that proliferation in postembryonic neuroblast clones is dramatically reduced in the absence of Polycomb, Sex combs extra, Sex combs on midleg, Enhancer of zeste or Suppressor of zeste 12. The proliferation defects in these PcG mutants are due to the loss of neuroblasts by apoptosis in the mutant clones. Mutation of PcG genes in postembryonic lineages results in the ectopic expression of posterior Hox genes, and experimentally induced misexpression of posterior Hox genes, which in the wild type causes neuroblast death, mimics the PcG loss-of-function phenotype. Significantly, full restoration of wild-type-like properties in the PcG mutant lineages is achieved by blocking apoptosis in the neuroblast clones. These findings indicate that loss of PcG genes leads to aberrant derepression of posterior Hox gene expression in postembryonic neuroblasts, which causes neuroblast death and termination of proliferation in the mutant clones. Our findings demonstrate that PcG genes are essential for normal neuroblast survival in the postembryonic CNS of Drosophila. Moreover, together with data on mammalian PcG genes, they imply that repression of aberrant reactivation of Hox genes may be a general and evolutionarily conserved role for PcG genes in CNS development.     

Cell division cycle of cultured neural precursor cells from Drosophila

In Drosophila neuroblast cells, which give rise to the embryonic nervous system, undergo a limited number of asymmetric cell divisions. These cell lineages result in the formation of clusters of neurons when neuroblasts are isolated and cultured. A significant proportion of these neural cell clusters (NCC) arise from individual precursor cells. The formation of NCC containing more than two neurons is repressed when DNA synthesis is inhibited. Cell division during NCC development was examined by [3H]thymidine autoradiography. The pattern of DNA synthesis by neural cells was that expected based on observations in situ. The pattern in individual NCC was consistent with single precursor origins for more than 80% of NCC, under our conditions of culture. Based on this, we show that the largest neural precursors at gastrulation undergo the most cell divisions in culture. The neuroblast cell division cycle averages approximately 1.5 hr, and is similar to that of blastoderm cells.     

Molecular markers for identified neuroblasts and ganglion mother cells in the Drosophila central nervous system.

The first step in generating cellular diversity in the Drosophila central nervous system is the formation of a segmentally reiterated array of neural precursor cells, called neuroblasts. Subsequently, each neuroblast goes through an invariant cell lineage to generate neurons and/or glia. Using molecular lineage markers, I show that (1) each neuroblast forms at a stereotyped time and position; (2) the neuroblast pattern is indistinguishable between thoracic and abdominal segments; (3) the development of individual neuroblasts can be followed throughout early neurogenesis; (4) gene expression in a neuroblast can be reproducibly modulated during its cell lineage; (5) identified ganglion mother cells form at stereotyped times and positions; and (6) the cell lineage of four well-characterized neurons can be traced back to two identified neuroblasts. These results set the stage for investigating neuroblast specification and the mechanisms controlling neuroblast cell lineages.     

Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts

Cell polarity must be integrated with tissue polarity for proper development. The Drosophila embryonic central nervous system (CNS) is a highly polarized tissue; neuroblasts occupy the most apical layer of cells within the CNS, and lie just basal to the neural epithelium. Neuroblasts are the CNS progenitor cells and undergo multiple rounds of asymmetric cell division, ;budding off' smaller daughter cells (GMCs) from the side opposite the epithelium, thereby positioning neuronal/glial progeny towards the embryo interior. It is unknown whether this highly stereotypical orientation of neuroblast divisions is controlled by an intrinsic cue (e.g. cortical mark) or an extrinsic cue (e.g. cell-cell signal). Using live imaging and in vitro culture, we find that neuroblasts in contact with epithelial cells always ;bud off' GMCs in the same direction, opposite from the epithelia-neuroblast contact site, identical to what is observed in vivo. By contrast, isolated neuroblasts 'bud off' GMCs at random positions. Imaging of centrosome/spindle dynamics and cortical polarity shows that in neuroblasts contacting epithelial cells, centrosomes remained anchored and cortical polarity proteins localize at the same epithelia-neuroblast contact site over subsequent cell cycles. In isolated neuroblasts, centrosomes drifted between cell cycles and cortical polarity proteins showed a delay in polarization and random positioning. We conclude that embryonic neuroblasts require an extrinsic signal from the overlying epithelium to anchor the centrosome/centrosome pair at the site of epithelial-neuroblast contact and for proper temporal and spatial localization of cortical Par proteins. This ensures the proper coordination between neuroblast cell polarity and CNS tissue polarity.     

Antagonistic roles for Ultrabithorax and Antennapedia in regulating segment-specific apoptosis of differentiated motoneurons in the Drosophila embryonic central nervous system

The generation of morphological diversity among segmental units of the nervous system is crucial for correct matching of neurons with their targets and for formation of functional neuromuscular networks. However, the mechanisms leading to segment diversity remain largely unknown. We report here that the Hox genes Ultrabithorax (Ubx) and Antennapedia (Antp) regulate segment-specific survival of differentiated motoneurons in the ventral nerve cord of Drosophila embryos. We show that Ubx is required to activate segment-specific apoptosis in these cells, and that their survival depends on Antp. Expression of the Ubx protein is strongly upregulated in the motoneurons shortly before they undergo apoptosis, and our results indicate that this late upregulation is required to activate reaper-dependent cell death. We further demonstrate that Ubx executes this role by counteracting the function of Antp in promoting cell survival. Thus, two Hox genes contribute to segment patterning and diversity in the embryonic CNS by carrying out opposing roles in the survival of specific differentiated motoneurons.     

Segment-specific muscle degeneration is triggered directly by a steroid hormone during insect metamorphosis

During metamorphosis of the hawkmoth, Manduca sexta, some larval muscles degenerate while others are respecified for new functions. In larvae, accessory planta retractor muscles (APRMs) are present in abdominal segments 1 to 6 (A1 to A6). APRMs serve as proleg retractors in A3 to A6 and body wall muscles in A1 and A2. At pupation, all APRMs degenerate except those in A2 and A3, which are respecified to circulate hemolymph in pupae. The motoneurons that innervate APRMs, the APRs, likewise undergo segment-specific programmed cell death (PCD), as a direct, cell-autonomous response to the prepupal peak of ecdysteroids. The segment-specific patterns of APR and APRM death differ. The present study tested the hypothesis that APRM death is a direct, cell-autonomous response to the prepupal peak of ecdysteroids. Prevention of the prepupal peak prevented APRM degeneration, and replacement of the peak by infusion of 20-hydroxyecdysone restored the correct segment-specific pattern of APRM degeneration. Surgical denervation of APRMs did not perturb their segment-specific degeneration at pupation, indicating that signals from APRs are not required for the muscles' segment-specific responses to ecdysteroids. The possibility that instructive signals originate from APRMs' epidermal attachment points was tested by treating the epidermis with a juvenile hormone analog to prevent pupal development. This manipulation likewise did not alter APRM fate. We conclude that both the muscles and motoneurons in this motor system respond directly and cell-autonomously to prepupal ecdysteroids to produce a segment-specific pattern of PCD that is matched to the functional requirements of the pupal body.     

Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster

Neurogenesis in the ventral CNS of Drosophila was studied using staining with toluidine blue and birth dating of cells monitored by incorporation of bromodeoxyuridine into DNA. The ventral CNS of the larva contains sets of neuronal stem cells (neuroblasts) which are thought to be persistent embryonic neuroblasts. Each thoracic neuromere has at least 47 of these stem cells whereas most abdominal neuromeres possess only 6. They occur in stereotyped locations so that the same neuroblast can be followed from animal to animal. The thoracic neuroblasts begin enlarging at 18-26 hr of larval life, DNA synthesis commences by 31-36 hr, and the first mitoses occur shortly thereafter. Mitotic activity continues through the remainder of larval life with the neuroblasts showing a minimum cell cycle time of less than 55 min during the late third larval instar. By 12 hr after pupariation each neuroblast has produced approximately 100 progeny which are collected with it into a discrete packet. The progeny accumulate in an immature, arrested state and only finish their differentiation into mature neurons with the onset of metamorphosis. Most of the abdominal neuroblasts differ from their thoracic counterparts in their minimum cell cycle time (less than 2 hr) and the duration of proliferation (from about 50 to 90 hr of larval life). Neurons produced during the larval stage account for more than 90% of the cells found in the ventral CNS of the adult.     

Target muscles and sensory afferents do not influence steroid-regulated,segment-specific death of identified motoneurons in Manduca sexta

Programmed cell death plays a critical role in sculpting the nervous system during embryonic development. In holometabolous insects, cell death also plays an important role in the reorganization of the nervous system during metamorphosis. In Manduca sexta, cell death and the factors that regulate it can be studied at the level of individually identified neurons. The accessory planta retractor (APR) motoneurons undergo segment-specific death during the larval-pupal transformation. APRs in abdominal segments 1, 5, and 6 die at pupation; those in abdominal segments 2, 3, and 4 survive until adulthood. Juvenile hormone and ecdysteroids regulate the metamorphic restructuring of the nervous system, but the factors that determine which APRs will live and which will die are not known. The present study assessed the possible importance of cell-cell interactions in determining APR survival at pupation by removing APR's target muscle or mechanosensory input early in the final larval instar, prior to the hormonal cues that trigger the larval-pupal transformation. The motoneurons showed their normal, segment-specific pattern of death in nearly all cases. These results suggest that target muscles and sensory input play little or no role in determining the segment-specific pattern of APR survival at pupation. © 1996 John Wiley & Sons, Inc.     

Coordinated expression of cell death genes regulates neuroblast apoptosis

Properly regulated apoptosis in the developing central nervous system is crucial for normal morphogenesis and homeostasis. In Drosophila, a subset of neural stem cells, or neuroblasts, undergo apoptosis during embryogenesis. Of the 30 neuroblasts initially present in each abdominal hemisegment of the embryonic ventral nerve cord, only three survive into larval life, and these undergo apoptosis in the larvae. Here, we use loss-of-function analysis to demonstrate that neuroblast apoptosis during embryogenesis requires the coordinated expression of the cell death genes grim and reaper, and possibly sickle. These genes are clustered in a 140 kb region of the third chromosome and show overlapping patterns of expression. We show that expression of grim, reaper and sickle in embryonic neuroblasts is controlled by a common regulatory region located between reaper and grim. In the absence of grim and reaper, many neuroblasts survive the embryonic period of cell death and the ventral nerve cord becomes massively hypertrophic. Deletion of grim alone blocks the death of neuroblasts in the larvae. The overlapping activity of these multiple cell death genes suggests that the coordinated regulation of their expression provides flexibility in this crucial developmental process.     

Patterns of dye coupling involving serotonergic neurons provide insights into the cellular organization of a central complex lineage of the embryonic grasshopper Schistocerca gregaria

All eight neuroblasts from the pars intercerebralis of one protocerebral hemisphere whose progeny contribute fibers to the central complex in the embryonic brain of the grasshopper Schistocerca gregaria generate serotonergic cells at stereotypic locations in their lineages. The pattern of dye coupling involving these neuroblasts and their progeny was investigated during embryogenesis by injecting fluorescent dye intracellularly into the neuroblast and/or its progeny in brain slices. The tissue was then processed for anti-serotonin immunohistochemistry. A representative lineage, that of neuroblast 1-3, was selected for detailed study. Stereotypic patterns of dye coupling were observed between progeny of the lineage throughout embryogenesis. Dye injected into the soma of a serotonergic cell consistently spread to a cluster of between five and eight neighboring non-serotonergic cells, but never to other serotonergic cells. Dye injected into a non-serotonergic cell from such a cluster spread to other non-serotonergic cells of the cluster, and to the immediate serotonergic cell, but never to further serotonergic cells. Serotonergic cells tested from different locations within the lineage repeat this pattern of dye coupling. All dye coupling was blocked on addition of an established gap junctional blocker (n-heptanol) to the bathing medium. The lack of coupling among serotonergic cells in the lineage suggests that each, along with its associated cluster of dye-coupled non-serotonergic cells, represents an independent communicating pathway (labeled line) to the developing central complex neuropil. The serotonergic cell may function as the coordinating element in such a projection system.     

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.     

Role of caspases and mitochondria in the steroid-induced programmed cell death of a motoneuron during metamorphosis

Accessory planta retractor (APR) motoneurons of the hawk moth, Manduca sexta, undergo a segment-specific pattern of programmed cell death (PCD) 24 to 48 h after pupal ecdysis (PE). Cell culture experiments show that the PCD of APRs in abdominal segment 6 [APR(6)s] is a cell-autonomous response to the steroid hormone 20-hydroxyecdysone (20E) and involves mitochondrial demise and cell shrinkage. Twenty-four hours before PE, at stage W3-noon, APR(6)s require further 20E exposure and protein synthesis (as tested with cycloheximide) to undergo PCD, and death can be blocked by a broad-spectrum caspase inhibitor. By PE, death is 20E- and protein synthesis-independent and the caspase inhibitor blocks cell shrinkage but not loss of mitochondrial function. Thus, the commitment to mitochondrial demise precedes the commitment to execution events. The phenotype of necrotic cell death induced by a mitochondrial electron transfer inhibitor differs unambiguously from 20E-induced PCD. By inducing PCD pharmacologically, the readiness of APR(6)s to execute PCD was found to increase during the final larval instar. These data suggest that the 20E-induced PCD of APR(6)s includes a premitochondrial phase which includes 20E-induced synthetic events and apical caspase activity, a mitochondrial phase which culminates in loss of mitochondrial function, and a postmitochondrial phase during which effector caspases are activated and APR(6) is destroyed.     

Developmental change in the steroid hormone signal for cell-autonomous, segment-specific programmed cell death of a motoneuron.

During metamorphosis of the hawkmoth, Manduca sexta, accessory planta retractor (APR) motoneurons undergo a segment-specific pattern of programmed cell death (PCD): e.g., APRs from abdominal segment six [APR(6)s] die at pupation in direct response to the prepupal rise in 20-hydroxyecdysone (20E), whereas APR(4)s survive through the pupal stage and die at eclosion (adult emergence). The hypothesis that the death of APR(4)s is triggered by the decline in 20E at eclosion was supported by findings that injection of 20E into developing pupae to delay the fall in 20E delayed APR(4) death. Furthermore, abdomen isolation to advance the fall in 20E caused precocious APR(4) death. In other experiments, APR(4)s were placed in primary cell culture 4 days before eclosion in medium containing 1 microg/ml 20E. A switch to hormone-free medium induced PCD in a significant proportion of APR(4)s, compared to APR(4)s that remained in 20E. Process fragmentation was a reliable early indicator of PCD. These results show that a decline in 20E triggers cell-autonomous PCD of APR(4)s, in contrast to the rise in 20E that triggers cell-autonomous PCD of APR(6)s. Thus, the PCD of homologous motoneurons in different body segments at different developmental times is triggered by different steroid hormone signals.     

Dap160/intersectin binds and activates aPKC to regulate cell polarity and cell cycle progression

The atypical protein kinase C (aPKC) is required for cell polarization of many cell types, and is upregulated in several human tumors. Despite its importance in cell polarity and growth control, relatively little is known about how aPKC activity is regulated. Here, we use a biochemical approach to identify Dynamin-associated protein 160 (Dap160; related to mammalian intersectin) as an aPKC-interacting protein in Drosophila. We show that Dap160 directly interacts with aPKC, stimulates aPKC activity in vitro and colocalizes with aPKC at the apical cortex of embryonic neuroblasts. In dap160 mutants, aPKC is delocalized from the neuroblast apical cortex and has reduced activity, based on its inability to displace known target proteins from the basal cortex. Both dap160 and aPKC mutants have fewer proliferating neuroblasts and a prolonged neuroblast cell cycle. We conclude that Dap160 positively regulates aPKC activity and localization to promote neuroblast cell polarity and cell cycle progression.