Ecdysis sequences consist of concatenated motor programs that serve to first loosen, then cast off an insect's exoskeleton and finally to expand a new exoskeleton to accommodate further growth. Survival and growth thus depend upon successful execution of ecdysis sequences, and each such sequence must be tailored to the animal's particular developmental stage. The developmental stage that has been the focus of most of the research in my laboratory is that of the newly metamorphosed adult, where expansion of the adult exoskeleton includes expansion of the newly formed wings. Since wing expansion is only undertaken in suitable surroundings, the ecdysis sequence includes an environmentally-sensitive behavioral program that mediates the search for a favorable environment. A subsequent, hormonally-driven program serves to expand the recently developed wings of the newly emerged fly. Because wing expansion must be performed within several hours of emergence, the need for safety must be balanced by the imperative to expand and each individual fly must decide when (and under what environmental circumstances) to expand. Wing expansion thus provides a behavioral paradigm for studying decision-making, the most fundamental aspect of behavioral integration, and for understanding how hormonal and environmental factors act, individually and in concert, to recruit motor patterns to assemble behavioral sequences. Such understanding should, in turn, shed light on the deficits in behavioral organization that lie at the root of many mental disorders, including obsessive-compulsive disorder, schizophrenia, and bipolar disorder. Some of the challenges and promises of research on the molecular mechanisms of behavior as studied in model organisms can be found in a meeting review I co-authored this year that was published in the journal Molecular Pharmacology (Langenhan et al. (2015) Mol Pharmacol. 88:596-603.) My laboratory's approach to understanding how the Drosophila nervous system produces behavioral sequences crucially depends on genetic techniques that allow specific subsets of brain cells to be turned off or on in freely behaving animals. Making such tools is another principal goal of our research. A palette of tools that we introduced this year (Diao et al., 2015, Cell Reports 10, 14101421), which has driven much of our recent research, permits targeting of transgene expression to neurons that express a specific gene of interest. We call such tools Trojan exons by way of analogy to the Trojan horse: They are stealthily introduced into the gene of interest, and then release transcriptional effectors that render the cells susceptible to exploitation by other transgenes. They thus allow investigators to gain genetic access to cell types of interest and manipulate their function. Trojan exons have the virtue of being easy to use and completely modular, in that Trojan exons of different types can be inserted into the same gene. When used in combination with the large number of existing Drosophila strains bearing MiMIC transposons in so-called coding introns, Trojan exons truly represent plug-and-play cassettes for cell-type specific targeting. Our various Trojan exons, and the tools we created for easily generating Trojan exon fly lines, have been made available to the community through either the Bloomington Drosophila Stock Center or Addgene. My own laboratory has used the Trojan exon techniquetogether with tools made by a previously developed method (Diao & White (2012) Genetics 190: 11391144)to investigate the circuitry underlying the wing expansion decision at adult ecdysis. To do so, we have targeted neurons that express the receptor of a hormone called Bursicon. We earlier showed that two Bursicon-expressing neurons (i.e., the BSEG) are not only necessary for wing expansion, but that activating them is sufficient to force the decision to expand even in unfavorable environmental conditions (Luan et al. (2012) J Neurosci 32: 880889). A focus of recent research has been to identify the downstream targets of the BSEG--which must necessarily express the Bursicon receptorin an effort to understand how they do so. As will be reported in a manuscript now being prepared for publication, the BSEG and their targets surprisingly comprise part of a positive feedback loop that mediates the wing expansion decision. In other work, we have used the Trojan exon technique to expand our investigation of the ecdysis circuitry beyond the circuit that mediates wing expansion at the adult stage. It has long been known that the ecdysis sequences of all developmental stages are initiated by release of Ecdysis Triggering Hormone (ETH). This hormone is thought to activate central brain circuits and orchestrate the execution of the entire ecdysis sequence, but how it does so has remained largely unknown. Historically, this is due to the lack of methods for systematically identifying and characterizing the neurons targeted by ETH. We have now succeeded in doing so by targeting cells that express the ETH receptor (ETHR) using the Trojan exon technique. By fully exploiting the versatility of this technique, we have developed a comprehensive toolset for manipulating not only the entire complement of ETHR neurons, but many subsets of such neurons, including subsets that individually express one its two isoforms, ETHRA and ETHRB. In work that has recently been submitted for publication, we have shown that ETHRA and ETHRB serve functionally and developmentally distinct roles in ecdysis. In addition, we have identified several other subsets of ETHR-expressing neurons that mediate ecdysis behaviors or the physiological changes that support their execution. In summary, we have made substantial progress during the last year in elucidating the developmentally dynamic circuit in the Drosophila nervous system that supports ecdysis and, at the adult stage, mediates the behavioral decision governing wing expansion. At the same time, we have continued to develop tools that will support not only our own circuit mapping efforts, but also those of other members of the Drosophila research community. As we use these tools to extend and refine our analysis of the circuitry underlying ecdysis sequences at all developmental stages in the fly, our work should provide insight into the principles that govern the development and function of behavioral circuits in all organisms, including humans.
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