Steven Robinow

The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages

The locus elav (ella-vee) of Drosophila melanogaster, which is necessary for the proper development of the embryonic and adult nervous systems, has been characterized both genetically and molecularly. This locus has been shown to be transcribed exclusively within, and ubiquitously throughout, the developing nervous system during Hours 6 to 12 of embryogenesis. We present in situ RNA localization data which demonstrate that elav is expressed in the central nervous system as well as the peripheral nervous system of embryos, larvae, pupae, and adults. We also demonstrate that elav is not transcribed in embryonic or larval neuroblasts (the neuronal progenitor cells), or in at least one type of glial cell. These data provide evidence that the requirement for elav function is not limited to the 6- to 12-hr embryonic nervous system and the adult eye and developing optic lobe, but that its function is required for the development and continued maintenance of all neurons of the organism.

Cell-Autonomous Requirement of the USP/EcR-B Ecdysone Receptor for Mushroom Body Neuronal Remodeling in Drosophila

Neuronal process remodeling occurs widely in the construction of both invertebrate and vertebrate nervous systems. During Drosophila metamorphosis, gamma neurons of the mushroom bodies (MBs), the center for olfactory learning in insects, undergo pruning of larval-specific dendrites and axons followed by outgrowth of adult-specific processes. To elucidate the underlying molecular mechanisms, we conducted a genetic mosaic screen and identified one ultraspiracle (usp) allele defective in larval process pruning. Consistent with the notion that USP forms a heterodimer with the ecdysone receptor (EcR), we found that the EcR-B1 isoform is specifically expressed in the MB gamma neurons, and is required for the pruning of larval processes. Surprisingly, most identified primary EcR/USP targets are dispensable for MB neuronal remodeling. Our study demonstrates cell-autonomous roles for EcR/USP in controlling neuronal remodeling, potentially through novel downstream targets.

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.

Molecular analysis of the locus elav in Drosophila melanogaster: a gene whose embryonic expression is neural specific.

The embryonic lethal abnormal visual system (elav) locus in Drosophila melanogaster, a vital gene mapping within the 1B5‐1B9 region of the X‐chromosome has been cloned and analysed. Previous developmental analyses have shown that in addition to the embryonic requirement there is a post‐embryonic requirement for elav function in the cells of the visual system. A DNA segment containing elav+ function was defined through germ line transformation experiments. This region encodes three embryonic poly(A)+ RNAs and two adult transcripts which are preferentially expressed in the head. In situ hybridization experiments clearly demonstrate that the embryonic expression of elav is restricted to the nervous system.

Isoform specific control of gene activity in vivo by the Drosophila ecdysone receptor

The steroid hormone 20-hydroxyecdysone induces metamorphosis in insects. The receptor for the hormone is the ecdysone receptor, a heterodimer of two nuclear receptors, EcR and USP. In Drosophila the EcR gene encodes 3 isoforms (EcR-A, EcR-B1 and EcR-B2) that vary in their N-terminal region but not in their DNA binding and ligand binding domains. The stage and tissue specific distribution of the isoforms during metamorphosis suggests distinct functions for the different isoforms. By over-expressing the three isoforms in animals we present results supporting this hypothesis. We tested for the ability of the different isoforms to rescue the lack of dendritic pruning that is characteristic of mutants lacking both EcR-B1 and EcR-B2. By expressing the different isoforms specifically in the affected neurons, we found that both EcR-B isoforms were able to rescue the neuronal defect cell autonomously, but that EcR-A was less effective. We also analyzed the effect of over-expressing the isoforms in a wild-type background. We determined a sensitive period when high levels of either EcR-B isoform were lethal, indicating that the low levels of EcR-B at this time are crucial to ensure normal development. Over-expressing EcR-A in contrast had no detrimental effect. However, high levels of EcR-A expressed in the posterior compartment suppressed puparial tanning, and resulted in down-regulation of some of the tested target genes in the posterior compartment of the wing disc. EcR-B1 or EcR-B2 over-expression had little or no effect.

GENETIC AND HORMONAL REGULATION OF THE DEATH OF PEPTIDERGIC NEURONS IN THEDROSOPHILA CENTRAL NERVOUS SYSTEM

To understand the role apoptosis plays in nervous system development and to gain insight into the mechanisms by which steroid hormones regulate neuronal apoptosis, we investigated the death of a set of peptidergic neurons in the CNS of the fruitfly Drosophila melanogaster. Typically, apoptosis in Drosophila is induced by the expression of the genes reaper, grim, or head involution defective (hid). We provide genetic evidence that the death of these neurons requires reaper and grim gene function. Consistent with this genetic analysis, we demonstrate that these doomed neurons accumulate reaper and grim transcripts prior to the onset of apoptosis. These neurons also accumulate low levels of hid, although the genetic analysis suggests that hid may not play a major role in the induction of apoptosis in these neurons. We show that the death of these neurons is dependent upon the fall in the titer of the steroid hormone 20-hydroxyecdysone that occurs at the end of metamorphosis, and demonstrate that the accumulation of both reaper and grim transcripts is inhibited by this steroid hormone. These observations support the notion that 20E controls apoptosis by regulating the expression of genes that induce apoptosis.

The dMRP/CG6214 gene of Drosophila is evolutionarily and functionally related to the human multidrug resistance-associated protein family

ATP‐binding cassette (ABC) transporters are involved in the transport of substrates across biological membranes and are essential for many cellular processes. Of the fifty‐six Drosophila ABC transporter genes only white, brown, scarlet, E23 and Atet have been studied in detail. Phylogenetic analyses identify the Drosophila gene dMRP/CG6214 as an orthologue to the human multidrug‐resistance associated proteins MRP1, MRP2, MRP3 and MRP6. To study evolutionarily conserved roles of MRPs we have initiated a characterization of dMRP. In situ hybridization and Northern analysis indicate that dMRP is expressed throughout development and appears to be head enriched in adults. Functional studies indicate that DMRP is capable of transporting a known MRP1 substrate and establishes DMRP as a high capacity ATP‐dependent, vanadate‐sensitive organic anion transporter.

The unfulfilled/DHR51 gene of Drosophila melanogaster modulates wing expansion and fertility

This is the first functional analysis in Drosophila of unfulfilled (unf; DHR51), the NR2E3 nuclear receptor superfamily ortholog of C. elegans fax‐1 and human PNR. Both fax‐1 and PNR mutations disrupt developmental events in a limited number of neurons, resulting in behavioral or sensory deficits. An analysis of two independent unf alleles revealed that unf mutants are characterized by one of two phenotypes. A proportion of the mutants eclosed but failed to expand their wings and were poorly coordinated. The remainder completed wing expansion but displayed severely compromised fertility. Consistent with the restricted neural expression of fax‐1 and PNR, unf expression was detected in situ only in mushroom body neurons and a small number of other cells of the central nervous system (CNS). These data support the hypothesis that the wing expansion failure and the compromised fertility of unf mutants are the result of underlying neural defects. Developmental Dynamics 238:171–182, 2009.

Characterization of the regulatory elements controlling neuronal expression of the A-isoform of the ecdysone receptor gene of Drosophila melanogaster

During the development of the adult central nervous system (CNS) of the fruitfly Drosophila melanogaster, the A-isoform of the ecdysone receptor (EcR-A), a typical nuclear hormone receptor, is expressed at high levels in the Type II neurons, a set of neurons that die shortly after the emergence of the adult. To understand the role that transcriptional regulation of nuclear receptor genes plays in CNS development, we have dissected the region controlling the transcription of EcR-A by analyzing the ability of this region to drive the expression of reporter genes in transgenic animals. These analyses have demonstrated that the Type II neurons are a heterogeneous collection of neurons that utilize different regulatory elements to coordinate the expression of the same transcript.

unfulfilled Interacting Genes Display Branch-Specific Roles in the Development of Mushroom Body Axons in Drosophila melanogaster

The mushroom body (MB) of Drosophila melanogaster is an organized collection of interneurons that is required for learning and memory. Each of the three subtypes of MB neurons, γ, α´/β´, and α/β, branch at some point during their development, providing an excellent model in which to study the genetic regulation of axon branching. Given the sequential birth order and the unique patterning of MB neurons, it is likely that specific gene cascades are required for the different guidance events that form the characteristic lobes of the MB. The nuclear receptor UNFULFILLED (UNF), a transcription factor, is required for the differentiation of all MB neurons. We have developed and used a classical genetic suppressor screen that takes advantage of the fact that ectopic expression of unf causes lethality to identify candidate genes that act downstream of UNF. We hypothesized that reducing the copy number of unf-interacting genes will suppress the unf-induced lethality. We have identified 19 candidate genes that when mutated suppress the unf-induced lethality. To test whether candidate genes impact MB development, we performed a secondary phenotypic screen in which the morphologies of the MBs in animals heterozygous for unf and a specific candidate gene were analyzed. Medial MB lobes were thin, missing, or misguided dorsally in five double heterozygote combinations (;unf/+;axin/+, unf/+;Fps85D/+, ;unf/+;Tsc1/+, ;unf/+;Rheb/+, ;unf/+;msn/+). Dorsal MB lobes were missing in ;unf/+;DopR2/+ or misprojecting beyond the termination point in ;unf/+;Sytβ double heterozygotes. These data suggest that unf and unf-interacting genes play specific roles in axon development in a branch-specific manner.