Genes induced in programmed cell death of neuronal PC12 cells and developing sympathetic neurons in vivo.

To identify primary response genes induced during early stages of neuronal programmed cell death (PCD), we screened by differential hybridization a subtracted cDNA library prepared from neuronal PC12 cells deprived of NGF for 6 hr in the presence of cycloheximide. Eight induced cDNA sequences were identified and designated message up-regulated during death (mud)-1-8. To determine which cloned sequences might be involved in neuronal PCD in vivo, expression of mud genes was analyzed in developing rat superior cervical ganglia (SCG) undergoing programmed cell death, using a combination of reverse Southern, reverse transcription polymerase chain reaction (RT-PCR), and in situ hybridization. Five sequences (mud-1, -3, -5/8, -6, and -7) are induced in SCG undergoing cell death in vivo, and induction of at least three of these (mud-3, -6, and -7) occurs in neurons. Partial sequence analysis reveals that mud-1 corresponds to annexin VI; mud-3 corresponds to rat PC3, mouse TIS21; mud-4 appears to be the rat homolog of human TAFII70; mud-5 and -8 are >85% identical members of the rodent gene family of B2-transcribed repeats; and mud-6 appears to be the rat homolog of human Ring 3 and Drosophila female sterile homeotic (fsh). Mud-2 and mud-7 encode novel sequences. These new candidate genes provide markers for early stages of neuronal PCD, are potentially involved in the cell death process, and serve to expand our view of cell death control in the developing nervous system.

Death in the balance.

Two major hypotheses concerning programmed cell death are that it is the end result of a gene expression pathway and that it is the cellular response to conflicting growth control signals. These ideas are examined, and their potential applicant to neuronal cell death during development is discussed. Since most mammalian genes involved in cell death have other functions, it is possible that a novel set of death genes does not exist in mammals. Instead, the genes identified may serve to link an initial stimulus to die with the cellular events that actually cause death, primarily by providing regulatory signals that direct the decision. The idea of cell death as a response to conflicting growth regulatory signals, initially derived from studies on cycling, non-neuronal cells, is applied to proliferating neuronal precursors and postmitotic neurons. How neuronal death during development might be the outcome of conflicting signals, and how retinoblastoma protein might negotiate "death by conflict" in different populations of neurons is discussed.

Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration.

Tissue plasminogen activator (tPA) mRNA was localized in the developing cerebellum and the potential role of tPA in migration of cerebellar granule cells was investigated. Proteolytic assays and Northern blots showed little variation in levels of tPA proteolytic activity or tPA mRNA expression in the developing cerebellum. The distribution of cerebellar tPA mRNA at different ages was visualized by in situ hybridization histochemistry. At postnatal day 7 (P7), most labeled cells were in the internal granule layer or developing white matter, and very few if any premigratory granule cells contained tPA mRNA. Although the molecular layer contained labeled cells at all ages, cell counts indicated that a greater percentage of cells in the molecular layer contained tPA mRNA during adulthood than during the period of granule cell migration. The most striking change in tPA mRNA expression was in Purkinje neurons, most of which began to express tPA mRNA between P7 and P14. The potential role of tPA in granule cell migration was investigated by performing migration assays in cerebellar slice explants in the presence or absence of protease inhibitors. The presence of inhibitors did not affect the distance that granule cells migrated. Data in the present study do not support a role for tPA in granule neuron migration; however, they do indicate that tPA is both spatially and temporally regulated during cerebellar development. Possible functions of tPA in the cerebellum are discussed.

PC12 cells overexpressing tissue plasminogen activator regenerate neurites to a greater extent and migrate faster than control cells in complex extracellular matrix.

PC12 cells were stably transfected with expression vectors containing rat tissue plasminogen activator (tPA) under control of either a cytomegalovirus or rous sarcoma virus promoter. Cell lines were characterized using protease assays, ELISAs, immunoblots, northern blots, and Southern blots. Control PC12 cells or cells containing vectors alone released about 1 pg tPA/cell/24 h, whereas cells stably transfected with a tPA cDNA released 2-5 pg tPA/cell/24 h. A strong correlation existed between the amount of tPA released and the ability of cells to degrade extracellular matrix. Experiments with protease inhibitors and antibodies against tPA and plasminogen indicated that degradation of matrix involved tPA-generated plasmin and that the amount of matrix degraded was dependent on the amount of tPA released. Cells expressing high levels of tPA migrated on a three-dimensional matrix about twice as fast as control cells and regenerated neurites within three-dimensional gels of Matrigel to a greater extent than control cells. Antibodies that inhibited tPA and plasminogen decreased migration and neurite regeneration, indicating that tPA was involved in both events, PC12 cells overexpressing tPA should provide a useful model system for investigating neural functions of tPA including its role in migration and regeneration.

Localization of tissue plasminogen activator mRNA in adult rat brain.

The distribution of tissue plasminogen activator (tPA) messenger RNA in rat brain was studied using in situ hybridization with 35S UTP-labeled RNA probes derived from a full-length tPA cDNA. Sense strand controls produced low, even backgrounds, with small elevations in the hippocampus. Full-length antisense probes produced strong signals over cerebral ventricular ependyma (including ependyma of the subcommissural organ), meninges, blood vessels, and Purkinje cell layer of the cerebellum, as well as strong signals over scattered cells throughout the brain. Some of these scattered labeled cells were large with lightly stained nuclei, while others were small with darkly stained nuclei. The large labeled cells, which were probably neurons, constituted 6% and 8% of cells in the brain stem and neocortex, respectively, and 100% of Purkinje cells. The small cells, which were present in all areas of the brain, constituted 3-11% of cells in individual brain areas.

Neuronal cell death: searching for the smoking gun.

During the past year,several model systems have been developed to identify cellular and biochemical events involved in neuronal cell death, to investigate the role of bcl-2 in cell survival, and to characterize the relationship between cell death and the cell cycle.

Probing the function of Drosophila melanogaster accessory glands by directed cell ablation.

The female Drosophila melanogaster fly undergoes behavioral changes after mating, including an increase in egg laying and an avoidance of remating. Accessory-gland products elicit these changes transiently when introduced into unmated female flies. We report here the generation and phenotype of flies that lack functional accessory-gland main cells as a consequence of genetically directed delivery of diphtheria toxin subunit A to those cells. Only main-cell secretions are essential for the short-term inhibition to remating; no other products of the genital tract can replace their function. Long-term inhibition to remating depends only on the storage of sperm in the female. Both sperm and main-cell secretions have roles in the increase of egg laying by the mated female. In addition to full-strength diphtheria toxin, we used low-activity toxins to kill only those cells that express toxin at high levels. These transgenic strains that express diphtheria toxins of different strengths in accessory-gland main cells will be useful in further defining the role of these cells.

A system for characterizing cellular and molecular events in programmed neuronal cell death.

A model system has been established in which PC12 cells are converted to neuronal-like cells that undergo transcription-dependent cell death following removal of NGF. Nineteen sublines of PC12 cells were tested to establish parameters for making cells dependent on NGF for survival. In most sublines, a relatively small percentage of cells become dependent on NGF for survival, and following removal of NGF, most of the cells begin proliferating in serum-containing medium. In several sublines, however, a significant percentage of cells die following removal of NGF. One of these sublines, PC6-3, can be grown under conditions in which 90% of the cells undergo transcription-dependent cell death following removal of NGF in either serum-free or serum-containing medium. Fourteen hours after removing NGF, 50% of the cells are committed to die, while initial morphological signs of cell death as determined by time-lapse videomicroscopy occur 2-6 hr later and include loss of neurites followed by a 1-3 hr period of active membrane "blebbing" and protrusions. Cell death can be blocked by the RNA synthesis inhibitor actinomycin D, the protein synthesis inhibitor cycloheximide, KCl, basic fibroblast growth factor, or dibutryl-cAMP, but not by epidermal growth factor, leupeptin, or the endonuclease inhibitor aurintricarboxylic acid (ATA). Removal of NGF activates an endonuclease that causes nucleosomal laddering of the DNA; however, endonuclease activity does not appear to be required for cell death. In agreement with previous studies (Batistatou and Greene, 1991; Rukenstein et al., 1991) demonstrating that naive PC12 cells undergo transcription-independent cell death when shifted into serum-free medium in the absence of growth factors, all cell lines tested except for one die when cultured in RPMI medium lacking growth factors. DNA fragmentation is a prominent feature of transcription-independent cell death, and death can be blocked with NGF, ATA, and dibutryl-cAMP but not with actinomycin D or KCl. The PC12 model system described here should be useful for identifying cell death genes and for characterizing cellular and molecular events in programmed neuronal cell death.

Structure, cell-specific expression, and mating-induced regulation of a Drosophila melanogaster male accessory gland gene.

The accessory gland of male insects is a secretory tissue of the genital tract made up of several distinct cell types. It secretes components of the ejaculatory fluid which have an important effect on the postmating behavior of the female. We have examined the sequence, structure, and expression of a gene, mst 316, expressed exclusively in the accessory glands of male Drosophila melanogaster. The mst 316 RNA encodes a small, basic protein of 52 amino acids that exhibits features common to precursors of secreted peptides, including a hydrophobic N-terminus. The tissue-specific expression of the mst 316 gene was studied using an mst 316--lacZ hybrid gene inserted into Drosophila by germ line transformation. The mst 316-lacZ fusion protein is expressed exclusively in the "main" cells of the accessory gland. It is first detected upon eclosion and exhibits a burst of synthesis in the first 3 days of adult life. The synthesis of the fusion protein is stimulated by mating, so that beta-galactosidase activity levels are two- to sixfold higher in males allowed to copulate with females compared to virgin male controls of the same age. The mating-stimulated synthesis of the mst 316-lacZ fusion protein, and by inference of the native gene product, appears to be due at least in part to increased transcript levels.

Sequences expressed sex-specifically in Drosophila melanogaster adults.

To obtain probes for sex-specific gene regulation during development in D. melanogaster, sequences expressed sex-specifically in adult flies were isolated by differential cDNA hybridization screens of a genomic library. Ten clones define new sex-specifically expressed genes. The remaining three isolates correspond to previously cloned genes encoding female-specific yolk proteins and chorion proteins. The pattern of expression of these genes in sex determination mutants and in germlineless flies, as well as their tissue specificities, permitted us to distinguish transcripts whose expression is dependent on correct sexual development of the soma or the germline. One of the female transcripts is expressed in nurse cells and oocytes. Five of the male-specific sequences are expressed in the testis during spermatogenesis: the remaining one is expressed in the soma. Experiments using a temperature-sensitive allele of tra-2 show that the presence of this male-specific transcript, found only in the adult paragonia, is not affected by temperature shift of X/X; tra-2ts2 adults. This is in contrast to yolk protein genes, which require tra-2 function in the adult for their expression in the female fat body.