Extended life-span conferred by cotransporter gene mutations in Drosophila.

Aging is genetically determined and environmentally modulated. In a study of longevity in the adult fruit fly, Drosophila melanogaster, we found that five independent P-element insertional mutations in a single gene resulted in a near doubling of the average adult life-span without a decline in fertility or physical activity. Sequence analysis revealed that the product of this gene, named Indy (for I'm not dead yet), is most closely related to a mammalian sodium dicarboxylate cotransporter-a membrane protein that transports Krebs cycle intermediates. Indy was most abundantly expressed in the fat body, midgut, and oenocytes: the principal sites of intermediary metabolism in the fly. Excision of the P element resulted in a reversion to normal life-span. These mutations may create a metabolic state that mimics caloric restriction, which has been shown to extend life-span.

Cu, Zn superoxide dismutase deficiency accelerates the time course of an age-related marker in Drosophila melanogaster.

In the oxidative stress hypothesis of aging the random accumulation of oxidative damage over time is postulated to cause aging. The pace at which oxidative damage accrues determines the rate of aging, but it is less clear how the accumulation of random damage could cause the stereotypic pattern of aging. It has been proposed that oxidative damage induces changes in gene expression, translating a random input of damage into a patterned output. In support of this we show that in adult Drosophila melanogaster, with a deficiency in the anti-oxidant enzyme Cu, Zn superoxide dismutase (Sod), an increase in oxidative stress, and a shortened life span, there is acceleration in the normal age-related temporal pattern of wingless gene expression. The acceleration in the temporal pattern of wingless gene expression is proportional to the shortened life span suggesting that the shortened life span of Sod deficient animals is due, not to an abnormal pathological process, but to an increase in the rate of aging.

Regulation of gene expression is preserved in aging Drosophila melanogaster.

Aging, and the deterioration of biological performance that characterizes it, are routinely assumed to be due to a progressive global loss of homeostasis and a general increase in dysregulation [1-4] . We tested this hypothesis directly by measuring age-specific variability in gene expression. Analysis of the transcriptional activity of six genes in various inbred lines of Drosophila melanogaster unexpectedly failed to show an increase in variability among individuals as they age and die. Although regulation of gene expression is a central feature of life, a global decline in the control of gene expression does not appear to be either a cause or a consequence of the process of aging.

Drosophila drop-dead mutations accelerate the time course of age-related markers.

Mutations of the drop-dead gene in Drosophila melanogaster lead to striking early death of the adult animal. At different times, after emergence from the pupa, individual flies begin to stagger and, shortly thereafter, die. Anatomical examination reveals gross neuropathological lesions in the brain. The life span of flies mutant for the drop-dead gene is four to five times shorter than for normal adults. That raises the question whether loss of the normal gene product might set into motion a series of events typical of the normal aging process. We used molecular markers, the expression patterns of which, in normal flies, change with age in a manner that correlates with life span. In the drop-dead mutant, there is an acceleration in the temporal pattern of expression of these age-related markers.

Spatial and temporal pattern of expression of the wingless and engrailed genes in the adult antenna is regulated by age-dependent mechanisms.

The spatial and temporal pattern of expression of enhancer trap lines reporting on the wingless (wg) and engrailed (en) genes was characterized in the adult antenna of Drosophila melanogaster. The time courses of expression seen for wg and en, although different from each other, reveal a complex well-controlled pattern of temporal expression, providing evidence that regulatory mechanisms are preserved throughout the life span of the adult fly. Altering the life span demonstrates that the temporal patterns of expression of both wg and en are linked to life span. These studies suggest that the expression of wg and en in the adult antenna is controlled by age-dependent mechanisms.

Timing of expression of a gene in the adult Drosophila is regulated by mechanisms independent of temperature and metabolic rate.

The examination of beta-galactosidase (beta-gal) expression in the third segment of the antenna of the 2216 enhancer trap line in Drosophila melanogaster reveals two distinct spatial and temporal regulatory patterns of expression during adult life. Type 1 expression is characterized by a decline in the level of beta-gal expression with increasing age. Starting from a maximal level of expression at the time of adult emergence, there is a decrease in the number of cells that express beta-gal so that by 40-50 days of adult life few cells express beta-gal. Varying the ambient temperature and using hyperactivity mutants (Hyperkinetic, Shaker) demonstrates that the rate of this decline is independent of temperature and metabolic rate. Type II expression is distinctly different in spatial distribution and temporal regulation from the first pattern. Type II expression is restricted in the antenna to a small (< 20-30) set of cells whose level of expression changes in a periodic manner with time. The regulation of this periodicity appears to be linked to ambient temperature.

The expression of a reporter protein, beta-galactosidase, is preserved during maturation and aging in some cells of the adult Drosophila melanogaster.

The effects of maturation and aging on cell stability and maintenance of protein expression have been examined in adult Drosophila melanogaster. Counting the number of cells present in the antenna of the adult fly revealed little loss in cell number with aging. Enhancer map-marked genes expressing beta-galactosidase (beta-gal) in the antenna and an Rh1 opsin reporter gene construct expressing beta-gal in the R1-6 photoreceptor cells of the compound eye revealed no alteration in spatial distribution or amount of beta-gal with aging. A heat shock-inducible promoter coupled to the expression of beta-gal, hsp70-lacZ, revealed that the rate and amount of induction of beta-gal after heat shock is preserved during aging but the rate of decay of beta-gal may be slightly delayed in older animals. These studies suggest that the ability to express a reporter protein, beta-galactosidase, is preserved in at least a subset of cells in the aging fly.

A molecular marker confirms that the rate of adult maturation is largely independent of the rate of pre-adult development in Drosophila melanogaster.

The separation of adult from pre-adult life seen with animals such as Drosophila melanogaster, which are holometabolous and undergo complete metamorphosis, provides the opportunity to examine the contribution of pre-adult rate of development on the rate of maturation and aging of the adult. Recent work has shown that when ambient temperature is used to alter the rate of development there is little effect on adult life span. From this work it has been concluded that the rate of aging is largely independent of the rate of pre-adult development. However, the techniques used to examine life span did not allow for the examination of the earliest events of adult life. Our experimental design used a molecular marker linked to life span as a sensitive measure of determining physiological age. In this way, we were able to evaluate the effect of pre-adult rate of development on the earliest events of adult life. Using ambient temperature to alter both the rate of development in the pre-adult and the rate of aging in the adult independently, we were able to show that it is the ambient temperature at which the adults are living that is the principle determinant of the rate of maturation and aging of the adult. Little effect was seen on the rate of adult maturation in response to an acceleration or a slowing down of the rate of pre-adult development as measured by our molecular marker. These data support the conclusions drawn by others who examined the effect of the rate of development on adult life expectancy. The timing mechanisms at work during pre-adult and adult life appear to be largely regulated separately. If there is such a thing as a physiological clock, it appears to be reset upon eclosion.

Regulation of gene expression is linked to life span in adult Drosophila.

Examination of gene expression and aging in adult Drosophila reveals that the expression of some genes is regulated by age-dependent mechanisms. Genetic mutations, Hyperkinetic and Shaker, which are known to shorten life span through an acceleration of the aging process, were used to study the expression of an enhancer trap marked gene. The temporal pattern of expression for such a marked gene shows scaling with respect to life span; it is altered in direct proportion to the life expectancy of the adult animal. This demonstrates that expression of this gene is controlled through mechanisms coupled to physiologic as opposed to chronologic age. Results provide direct evidence for linkage between the regulation of gene expression and life span and establish a model system for the genetic analysis of aging.

Changes in gene expression during post-eclosional development in the olfactory system of Drosophila melanogaster.

We have found that the expression of some genes in Drosophila melanogaster changes during the life of the adult fly. These changes can be illustrated by the use of enhancer trap lines which mark the expression of particular genes in the adult fly. Although the fly is considered able to perform most necessary adult functions within the first 72 h after eclosion from the pupal case, we find changes in expression over the first 10 days of life in the antennae of several of the genes we have examined. Some genes change by increasing from an initially low level of expression of the marked gene, while other lines, which we have termed 'late-onset' genes, show no expression of the marked gene until 4-5 days following eclosion. In contrast, some genes decrease their expression during the first 10 days of life. The changes in gene expression seen over the first 10 days of the fly's adult life provides molecular evidence of the many maturational changes occurring during the early life of the adult fly.

Temporal patterns of gene expression in the antenna of the adult Drosophila melanogaster.

The time course of gene expression in the adult fruit fly has been partially characterized by using enhancer trap and reporter gene constructs that mark 49 different genes. The relative intensity of the reporter protein in individual cells of the antennae was measured as a function of adult age. Most genes showed a graduated expression, and the intensity of expression had a reproducible and characteristic time course. Different genes displayed different temporal patterns of expression and more often than not the pattern of expression was complex. We found a number of genes having patterns that scaled with life span. In these cases the intensity of gene expression was found to be invariant with respect to biological time, when expressed as a fraction of the life span of the line. The scaling was observed even when life span was varied as much as threefold. Such scaling serves to (1) further demonstrate that deterministic mechanisms such as gene regulation act to generate the temporal patterns of expression seen during adult life, (2) indicate that control of these regulatory mechanisms is linked to life span, and (3) suggest mechanisms by which this control is accomplished. We have concluded that gene expression in the adult fly is often regulated in a fashion that allows for graduated expression over time, and that the regulation itself is changing throughout adult life according to some prescribed program or algorithm.

The large upstream control region of the Drosophila homeotic gene Ultrabithorax.

Ultrabithorax (Ubx) is a Drosophila homeotic gene that determines the segmental identities of parts of the thorax and abdomen. Appropriate Ubx transcription requires a long upstream control region (UCR) that is defined genetically by the bithoraxoid (bxd) and postbithorax (pbx) subfunction mutations. We have directly analyzed UCR functions by the examination of beta-galactosidase expression in flies containing Ubx-lacZ fusion genes. 35 kb of UCR DNA confers upon beta-galactosidase an expression pattern that closely parallels normal Ubx expression throughout development. In contrast, 22 kb of UCR DNA confers fewer features of normal Ubx expression, and with 5 kb of UCR DNA the expression pattern has no resemblance to Ubx expression except in the visceral mesoderm. We have also shown that bxd chromosome breakpoint mutants form a comparable 5' deletion series in which the severity of the effect on Ubx expression correlates with the amount of upstream DNA remaining in the mutant. In Ubx-lacZ fusions containing 22 kb of UCR DNA, and in comparable bxd mutants, there is a persistent pair-rule pattern of metameric expression in early development, demonstrating that there are distinct mechanisms with different sequence requirements for the initial activation of Ubx in different metameres. The correction of this pair-rule pattern later in embryogenesis shows that there are also distinct mechanisms for the activation of Ubx at different times during development.

A simple chemosensory response in Drosophila and the isolation of acj mutants in which it is affected.

Although the Drosophila visual system has been described extensively, little is known about its olfactory system. A major reason for this discrepancy has been the lack of simple, reliable means of measuring response to airborne chemicals. This paper describes a jump response elicited by exposing Drosophila to chemical vapors. This behavior provides the basis for a single-fly chemosensory assay. The behavior exhibits dose dependence and chemical specificity: it is stimulated by exposure to ethyl acetate, benzaldehyde, and propionic acid but not ethanol. Animals can respond repeatedly at short intervals to ethyl acetate and propionic acid. The response relies on the third antennal segments. To illustrate the use of this behavior in genetic analysis of chemosensory response, nine acj mutants defective in response are isolated (acj = abnormal chemosensory jump), and their responses to two chemicals are characterized. All of the acj mutants are normal in giant fiber system physiology, and two exhibit defects in visual system physiology.

Genetic analysis of olfactory behavior in Drosophila: a new screen yields the ota mutants.

A simple means of measuring Drosophila olfactory response is described, and the behavior which it measures is characterized. The assay was used to screen for X-linked mutants defective in olfactory function. Six ota mutants were isolated and characterized (ota = olfactory trap abnormal). Four of the mutants were found to be abnormal in another chemosensory behavior as well. Two of the mutant phenotypes extend to include another sensory system: they are defective in visual system physiology. All were normal, however, in a test of giant fiber system physiology. Two of the mutations are dominant, and the recessive mutations define two complementation groups. Mutations representing each complementation group, as well as one of the dominant mutations, were mapped. For the mutants with defective visual system physiology, the visual defects were shown to cosegregate with olfactory phenotypes.

Isolation and characterization of an olfactory mutant in Drosophila with a chemically specific defect.

A Drosophila mutant was isolated and shown to exhibit defective response to the chemical odorant benzaldehyde in two distinctly different behavioral assays. The defect exhibited chemical specificity: response to three other chemicals was normal. The mutant also showed abnormalities in pigmentation and fertility. Genetic mapping and complementation analysis provide evidence that the olfactory, pigmentation, and fertility defects arise as a result of a lesion at the pentagon locus. The specificity of the olfactory defect suggests the possibility that the mutation may define a molecule required in reception, transduction, or processing of a specific subset of chemical information in the olfactory system.

Segmental distribution of bithorax complex proteins during Drosophila development.

The Ubx and bxd transcription units comprise a single functional domain in the bithorax complex of Drosophila melanogaster. The segmental distributions and nuclear localization of proteins encoded by the Ubx unit have been determined by immunofluorescence staining with antibodies raised against a fusion protein containing Ubx coding sequences. Wild-type and mutant distributions are consistent with a model in which the protein-coding functions of the domain derive from the Ubx unit and are regulated by the bxd unit.

Cell recognition during neuronal development.

Insect embryos, with their relatively simple nervous systems, provide a model system with which to study the cellular and molecular mechanisms underlying cell recognition during neuronal development. Such an approach can take advantage of the accessible cells of the grasshopper embryo and the accessible genes of Drosophila. The growth cones of identified neurons express selective affinities for specific axonal surfaces; such specificities give rise to the stereotyped patterns of selective fasciculation common to both species. These and other results suggest that early in development cell lineage and cell interactions lead to the differential expression of cell recognition molecules on the surfaces of small subsets of embryonic neurons whose axons selectively fasciculate with one another. Monoclonal antibodies reveal surface molecules in the Drosophila embryo whose expression correlates with this prediction. It should now be possible to isolate the genes encoding these potential cell recognition molecules and to test their function through the use of molecular genetic approaches in Drosophila.

Recognition of myelin-associated glycoprotein by the monoclonal antibody HNK-1.

Myelin-associated glycoprotein (MAG) is a quantitatively minor component in both peripheral and central myelin sheaths that is thought to have a role in cell-cell interactions within the nervous system. We show here that a mouse monoclonal antibody, HNK-1, which is directed against human natural killer cells also recognizes an antigenic determinant of human central and peripheral nervous system white matter by immunoperoxidase staining of tissue sections. Immunoblot analysis of myelin proteins and purified extracted MAG indicates that the antigen recognized is MAG.