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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.

Entomological protocols for aging blow fly (Diptera: Calliphoridae) larvae to estimate the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analysing fly larvae differ, most rely on light microscopy, genetic analysis or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically-important blow fly Chrysomya rufifacies using high-content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti-ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, non-bleaching, autofluorescence of larval posterior spiracles. High content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC.

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.

BackgroundThe mushroom bodies (MBs) ofDrosophilaare required for complex behaviors and consist of three types of neurons, γ, α'/β' and α/β. Previously, roles for transcription factors in MB neuronal differentiation have only been described for a subset of MB neurons. We are investigating the roles ofunfulfilled(unf;HR51, CG16801) in MB development.unfencodes a nuclear receptor that is orthologous to the nuclear receptors fasciculation of axons defective 1 (FAX-1) of the nematode and photoreceptor specific nuclear receptor (PNR) of mammals. Based on our previous observations thatunftranscripts accumulate in MB neurons at all developmental stages and the presence of axon pathfinding defects infax-1mutants, we hypothesized thatunfregulates MB axon growth and pathfinding.ResultsWe show thatunfmutants exhibit a range of highly penetrant axon stalling phenotypes affecting all neurons of the larval and adult MBs. Phenotypic analysis ofunfX1mutants revealed that α'/β' and α/β neurons initially project axons but stall prior to the formation of medial or dorsal MB lobes.unfZ0001mutants form medial lobes, although these axons fail to branch, which results in a failure to form the α or α' dorsal lobes. In either mutant background, γ neurons fail to develop larval-specific dorsal projections. These mutant γ neurons undergo normal pruning, but fail to re-extend axons medially during pupal development.unfRNAianimals displayed phenotypes similar to those seen inunfZ0001mutants. Unique asymmetrical phenotypes were observed inunfX1/unfZ0001compound heterozygotes. Expression ofUAS-unftransgenes in MB neurons rescues the larval and adultunfmutant phenotypes.ConclusionsThese data support the hypothesis thatunfplays a common role in the development of all types of MB neurons. Our data indicate thatunfis necessary for MB axon extension and branching and that the formation of dorsal collaterals is more sensitive to the loss ofunffunction than medial projections. The asymmetrical phenotypes observed in compound heterozygotes support the hypothesis that the earliest MB axons may serve as pioneers for the later-born MB neurons, providing evidence for pioneer MB axon guidance in post-embryonic development.

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.