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Steven Reppert, M.D.Academic Role: Professor
Faculty Appointment(s) In:
Other Affiliation(s):
In an integrated set of studies, our laboratory is using anatomical, cellular, molecular, electrophysiological, genetic and behavioral approaches to more fully understand the biological basis of monarch butterfly (Danaus plexippus) migration, with a focus on the butterfly's navigational abilities and its ancestral circadian clock. Active projects include: Monarch Circadian Clockwork Rationale. Paradigm shifting studies in the monarch butterfly have enhanced our view of the evolution and function of CRYPTOCHROME (CRY) proteins in animals (Zhu et al., 2008). The butterfly's clock mechanism posits two CRY proteins as critical for circadian timing, giving rise to a NOVEL clockwork (Fig. 1A). Thus, in the same species, the two functionally distinct CRYs can be analyzed. In monarchs, a Drosophila-like CRY, designated CRY1, functions as a likely circadian photoreceptor, while a vertebrate-like CRY, designated CRY2, appears to function as the major transcriptional repressor of the clockwork transcriptional feedback loop. The analysis of distinct CRY function has been further aided by a monarch cell line (DpN1 cells), which expresses a light-driven clock, in which both monarch CRY1 and CRY2 function; no other light-sensitive insect cell line has been described. Our continued studies will expand our knowledge of the unique properties of the CRY proteins by further defining distinct CRY mechanisms of action in lepidopterans (butterflies and moths) using molecular, biochemical, genetic, and behavioral approaches. Because of the parallel, complementary nature of circadian clock discoveries between flies and mammals, which we believe can now be extended to lepidopterans, our overriding hypothesis is that further analysis of the two families of animal CRY proteins that are represented in insects will advance our fundamental understanding of animal clock mechanisms. Employ an RNAi screen to discover novel components of the insect CRY1 light-signaling pathway. We postulate that novel components of the CRY1-dependent circadian input pathway remain to be elucidated. The light-driven clock in DpN1 cells, along with a recently constructed monarch expressed sequence tag library (see below), allow for the development of an unbiased, high-throughput RNAi screen to discover novel components of light-induced CRY1 signaling that allow the molecule to function as a circadian photoreceptor. Mechanistic details of novel component function will be further analyzed in DpN1 cells. Candidate components will be characterized in Drosophila strains harboring loss-of-function mutations of the fly homologs. Use a proteomics approach to identify novel interactors within the insect CRY2/CLK:CYC transcriptional complex. We hypothesize that yet to be discovered insect CRY2 interactive proteins are essential for its role as a major transcriptional repressor of the clockwork transcriptional feedback loop. With DpN1 cells, we will use tandem affinity purification and coimmunoprecipitation to identify additional CRY2-, CLOCK-, and CYCLE-interacting proteins. Finding novel interactors may help define the transcriptional repressive role of CRY not only in insects, but also in mammals, in which the mechanism of CRY repression is not well understood. Monarch Navigation Rationale. During their fall migration, monarch butterflies travel distances approaching 4000 km. The remarkable navigational abilities of monarch butterflies are part of a genetic program that is initiated in migrations. We believe that the centerpiece of the navigational process is time-compensated sun compass orientation. The ability of migrants to successfully navigate to their overwintering sites in central Mexico requires that the underlying genetically program is constantly being recalibrated by environmental factors. Time-compensated sun compass orientation. We continue to use a flight simulator to examine this important aspect of monarch navigation. We are determining whether a time-compensated sun compass is used in the spring by migrants on the way back from Mexico to the Southern US and its utilization in subsequent summer generations that travel from the Southern US to the Northern limits of their habitat. We are also examining whether the use and orientation direction of the sun compass is influence by daylength in monarchs. Central complex. Based on studies in locusts and crickets, it appears that the sun compass resides in the central complex. The central complex is a midline structure consisting of the dorsally positioned protocerebral bridge and the more ventrally situated central body, which has upper and lower subdivisions. Our recent finding of a clock connection with the central complex (via a CRY2-positive pathway) in monarch butterflies represents a major advance (Fig. 1B). We are defining in more detail with confocal microscopy the anatomy of the central complex in monarch butterflies. Three-dimensional reconstruction can be used to determine where the CRY2 pathway originates. It will also be important to determine where the CRY2 pathway ends and whether it actually functionally communicates with the central complex. Our ultimate goal is to selectively manipulate gene expression in the central complex (and other relevant brain regions) to understand the molecular logic behind the neuronal signaling pathways that have been identified and how they ultimately contribute to time-compensated flight orientation. Magnetoreception. As migrants "funnel" into Texas in October, do they switch navigation strategies - the so-called "beacon effect"? Could this be geomagnetic in whole or part? We have developed an assay system in which light-dependent magnetoreception can be monitored and its molecular underpinnings deciphered (See Gegear et al., 2008). Social interactions. Migratory monarch butterflies are gregarious, while summer butterflies are not. Migrants spend their nights in roosts along the migration flyway. They migrate in large swarms, which increase in size the closer they get to Mexico. Do social interactions among butterflies influence their navigation. Flight simulator experiments could help determine whether social interactions actually influence time-compensated sun compass orientation mechanisms. We are also investigating whether pheromones are important. Real-time monitoring of navigation. We are developing tools for monitoring free-flying monarchs over long distances. Monarch Migration Genes Rationale. To fully understand migration, we need to determine the gene expression patterns that define the migratory "state". Transcriptional profiling. We continue to examine the differential gene expression patterns between migratory and summer butterflies. The idea is that a group of genes or a specific pattern of gene expression will define migratory butterflies in molecular terms. We also continue to examine the specific genes regulated by juvenile hormone, a key regulator of migration whose reduction is responsible for the curtailment of reproductive behavior and increased longevity in migrants. miRNAs. MicroRNAs (miRNAs) regulate gene expression by inhibiting translation, and each miRNA can regulate a complement of proteins. In other systems, miRNAs are involved in epigenetic developmental events. We are therefore addressing the possibility that miRNAs may be involved in initiating/mediating the migratory state in monarch butterflies. Accessing the Monarch Genome Rationale. Accessing the monarch genome is essential for moving the clockwork, navigation and migration issues into contemporary biology. Such access is necessary for the monarch butterfly to become a model organism to study circadian clock and migration mechanisms. Develop a targeted gene inactivation strategy in lepidopterans. In collaboration with Dr. Scot Wolfe in the Program in Gene Function & Expression, we are developing a novel gene targeting approach that uses a zinc finger nuclease (ZFN) strategy in the silkworm Bombyx mori, a genetically tractable species, to define the essential nature of CRY2 for clockwork function in lepidopterans. The effects of such gene targeting on two circadian behaviors, the timing of egg hatching and adult eclosion, will be monitored, as well as the effects on the molecular clock mechanism itself. Once developed, the ZFN strategy will be a powerful tool for targeting additional clock genes in Bombyx and ultimately targeting genes in monarchs. The method can also be used to knock-in reporters into clock gene loci. Develop artificial diet for monarchs. An artificial diet for consistently rearing monarchs from egg to adults is essential for the utilization of genetic approaches. We need to be able to maintain monarch "lines" in the laboratory.
Rationale. Developing more comprehensive genomic resources for the monarch butterfly is a fundamental step to fully utilize this system to address important issues in contemporary biology (e.g., sun compass orientation, and circadian clock mechanisms). Monarch brain expressed sequence tag library (http://titan.biotec.uiuc.edu/butterfly/) Sequence the monarch genome. We are in the process of sequencing the entire monarch butterfly genome. The monarch butterfly has the smallest genome of the lepidopterans examined (based on 59 different species;(Zhu et al., 2008) and is more similar in size to that of the mosquito, which is predicted to contain 13,683 genes. We have undertaken this project for transcriptional profiling studies of migratory and non-migratory butterflies, and for gathering more comparative genomic data (no other butterfly genome has been sequenced). See document: Why Sequence the Monarch Genome The sequencing of the monarch butterfly genome was performed initially with SymBio Corporation (www.sym-bio.com). As part of SymBio Corporation's support, a preliminary project was designed and performed. The goals of the preliminary project were to provide proof of principle for moving to a complete genome project by generating high quality random libraries, sequencing by a hybrid approach involving both standard Sanger and 454 Flx reads, and assembling the data and performing bioinformatic analysis, including ORF finding and BLAST analyses. The preliminary project consisted of three phases. Phase 1 generated two subclone plasmid libraries and produced 34,000 Sanger sequence reads to a 0.07X genome depth coverage. Phase 2 added >60,000 Sanger reads and a 454 Flx sequencing run producing 0.5X genome coverage. Phase 3 added Sanger reads to >100,000 and an additional 454 Flx run to produce 1.0X genome coverage. The data determined the baseline jumping off point for completing the genome by further sequencing (to at least 13X depth). Additional sequencing and annotation are ongoing.
Office: LRB 728 A-D Rm 728
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Molecular Neuroethology
Lazare Research Bldg., 364 Plantation Street Worcester, MA 01605-2324