James W. Truman

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.

Ecdysone receptors and their biological actions.

Publisher Summary This chapter discusses the ecdysone receptors and their biological actions. It also summarizes the insect endocrinology and the roles of these steroids in the molting and metamorphosis. Natural hormones that lead to molting and metamorphosis are ecdysones. Molecules whose structures resemble those of the natural hormones are called ecdysteroids. The receptor for ecdysone is a member of the nuclear receptor superfamily that acts as a ligand-dependent transcription factor. The vertebrate steroid hormone receptors act as homodimers whereas the functional ecdysone receptor is always a heterodimer of receptor for ecdysone (EcR) with another member of the nuclear receptor (NR) superfamily, Ultraspiracle, the insect homolog of the vertebrate retinoid X receptor (RXR). Two new technologies promise to transform the environment for investigations of insect hormones, ecdysone receptor, and metamorphosis. The microarrays of expressed sequence tags are constructed (ESTs) and used hybridization to catalog changes in gene expression during metamorphosis. The first technological achievement is reviewed: the complete sequence of the Drosophila genome is obtained and is about to be released. It will be the first complete insect sequence and also the first genomic sequence from an organism that has served as a model for nuclear receptor endocrinology.

Refinement of Tools for Targeted Gene Expression in Drosophila

A wide variety of biological experiments rely on the ability to express an exogenous gene in a transgenic animal at a defined level and in a spatially and temporally controlled pattern. We describe major improvements of the methods available for achieving this objective in Drosophila melanogaster. We have systematically varied core promoters, UTRs, operator sequences, and transcriptional activating domains used to direct gene expression with the GAL4, LexA, and Split GAL4 transcription factors and the GAL80 transcriptional repressor. The use of site-specific integration allowed us to make quantitative comparisons between different constructs inserted at the same genomic location. We also characterized a set of PhiC31 integration sites for their ability to support transgene expression of both drivers and responders in the nervous system. The increased strength and reliability of these optimized reagents overcome many of the previous limitations of these methods and will facilitate genetic manipulations of greater complexity and sophistication.

The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster.

BACKGROUND The timely onset of metamorphosis in holometabolous insects depends on their reaching the appropriate size known as critical weight. Once critical weight is reached, juvenile hormone (JH) titers decline, resulting in the release of prothoracicotropic hormone (PTTH) at the next photoperiod gate and thereby inducing metamorphosis. How individuals determine when they have reached critical weight is unknown. We present evidence that in Drosophila, a component of the ring gland, the prothoracic gland (PG), assesses growth to determine when critical weight has been achieved. RESULTS We used the GAL4/UAS system to suppress or enhance growth by overexpressing PTEN or Dp110, respectively, in various components of the ring gland. Suppression of the growth of the PG and CA, but not of the CA alone, produced larger-than-normal larvae and adults. Suppression of only PG growth resulted in nonviable larvae, but larvae with enlarged PGs produced significantly smaller larvae and adults. Rearing larvae with enlarged PGs under constant light enhanced these effects, suggesting a role for photoperiod-gated PTTH secretion. These larvae are smaller, in part as a result of their repressed growth rates, a phenotype that could be rescued through nutritional supplementation (yeast paste). Most importantly, larvae with enlarged PGs overestimated size so that they initiated metamorphosis before surpassing the minimal viable weight necessary to survive pupation. CONCLUSIONS The PG acts as a size-assessing tissue by using insulin-dependent PG cell growth to determine when critical weight has been reached.

The origins of insect metamorphosis

Insect metamorphosis is a fascinating and highly successful biological adaptation, but there is much uncertainty as to how it evolved. Ancestral insect species did not undergo metamorphosis and there are still some existing species that lack metamorphosis or undergo only partial metamorphosis. Based on endocrine studies and morphological comparisons of the development of insect species with and without metamorphosis, a novel hypothesis for the evolution of metamorphosis is proposed. Changes in the endocrinology of development are central to this hypothesis. The three stages of the ancestral insect species—pronymph, nymph and adult—are proposed to be equivalent to the larva, pupa and adult stages of insects with complete metamorphosis. This proposal has general implications for insect developmental biology.

Local caspase activity directs engulfment of dendrites during pruning

Pruning is important for sculpting neural circuits, as it removes excessive or inaccurate projections. Here we show that the removal of sensory neuron dendrites during pruning in Drosophila melanogaster is directed by local caspase activity. Suppressing caspase activity prevented dendrite removal, whereas a global activation of caspases within a neuron caused cell death. A new genetically encoded caspase probe revealed that caspase activity is confined to the degenerating dendrites of pruning neurons.

Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta

The rate of metamorphosis in Manduca appears to be under continuous regulation by the circulating titer of the ecdysteroids. Ecdysteroids promote development during the first third of adult differentiation. We present here several lines of evidence indicating that the role of the ecdysteroids then changes to being inhibitory during the later stages of adult differentiation. Abdomen ligation, which precipitously reduces the levels of ecdysteroids in the abdomen, accelerates the rates of tissue development in this region. This acceleration can be counteracted by ecdysteroid injection or by implantation of prothoracic glands. Infusion of ecdysteroids into insects late in development results in a dose-dependent depression in the rate of subsequent development. The effectiveness of a given dosage of steroid is dependent on the developmental stage, with older animals being more affected. Last, the normal ecdysteroid titer declines in a stepwise fashion over the last 3 days of development and these steps are paralleled by a drop-off in the effectiveness of abdomen ligation over this same period. It is concluded that this effect of the ecdysteroids late in development provides a mechanism to ensure that the various tissues of the insect complete metamorphosis in a coordinated fashion.

Nitric Oxide and Cyclic GMP Regulate Retinal Patterning in the Optic Lobe of Drosophila

The photoreceptors of Drosophila express a nitric oxide-sensitive guanylate cyclase during the first half of metamorphosis, when postsynaptic elements in the optic lobe are being selected. Throughout this period, the optic lobes show NADPH-diaphorase activity and stain with an antibody to nitric oxide synthase (NOS). The NOS inhibitor L-NAME, the NO scavenger PTIO, the sGC inhibitor ODQ, and methylene blue, which inhibits NOS and guanylate cyclase, each caused the disorganization of retinal projections and extension of photoreceptor axons beyond their normal synaptic layers in vitro. The disruptive effects of L-NAME were prevented with the addition of 8-bromo-cGMP. These results suggest NO and cGMP act to stabilize retinal growth cones at the start of synaptic assembly.

Programmed cell death in the Drosophila CNS is ecdysone-regulated and coupled with a specific ecdysone receptor isoform

At adult emergence, the ventral CNS of Drosophila shows a group of approximately 300 neurons, which are unique in that they express 10-fold higher levels of the A isoform of the ecdysone receptor (EcR-A) than do other central neurons. This expression pattern is established early in metamorphosis and persists throughout the remainder of the pupal stage. Although these cells represent a heterogeneous group of neurons, they all share the same fate of undergoing rapid degeneration after the adult emerges from the pupal case. One prerequisite for this death is the decline of ecdysteroids at the end of metamorphosis. Treatment of flies with 20-hydroxyecdysone blocks the death of the cells, but only if given at least 3 hours before the normal time of degeneration. The correlation of a unique pattern of receptor isoform expression with a particular steroid-regulated fate suggests that variations in the pattern of receptor isoform expression may serve as important switches during development.

Disruption of a Behavioral Sequence by Targeted Death of Peptidergic Neurons in Drosophila

The neuropeptide eclosion hormone (EH) is a key regulator of insect ecdysis. We tested the role of the two EH-producing neurons in Drosophila by using an EH cell-specific enhancer to activate cell death genes reaper and head involution defective to ablate the EH cells. In the EH cell knockout flies, larval and adult ecdyses were disrupted, yet a third of the knockouts emerged as adults, demonstrating that EH has a significant but nonessential role in ecdysis. The EH cell knockouts had discrete behavioral deficits, including slow, uncoordinated eclosion and an insensitivity to ecdysis-triggering hormone. The knockouts lacked the lights-on eclosion response despite having a normal circadian eclosion rhythm. This study represents a novel approach to the dissection of neuropeptide regulation of a complex behavioral program.