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Steven Reppert, M.D.Academic Role: Professor
Faculty Appointment(s) In:
Other Affiliation(s):
NEW: NIH ARRP funding of monarch genome
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. 1). 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. Public health relevance: The monarch butterfly clock mechanism affords unique opportunities to further our understanding of fundamental clock mechanisms in animals. Defining the molecular and biochemical basis of circadian timing in animals has important implications. In terms of fundamental brain mechanisms, the circadian system is among the most tractable models for providing a complete understanding of the cellular and molecular events connecting genes to behavior. Thorough dissection of the genetic basis of circadian behavior may help decipher this connection for more complex behaviors. There are also important biomedical implications: Understanding the molecular clock has already increased our knowledge of how clock gene mutations contribute to disorders of the timing of sleep and could increase our knowledge of how clock gene mutations contribute to psychopathology (e.g., major depression and seasonal affective disorder). Likewise, such understanding should lead to new strategies for pharmacological manipulation of the human clock to improve the treatment of jet lag and shift-work ailments, and of clock-related sleep and psychiatric disorders. 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. Brain clocks and circuits. Using a strategy that relied on the coexpression of PER, TIM, and CRY1, four cells in the dorsolateral region of monarch butterfly brain (the pars lateralis, PL) were identified as the putative location of circadian clocks (Sauman et al., 2005) (Fig. 2). Further study showed that CRY2 is also expressed in those cells, in which it cycles in and out of the nucleus at the appropriate times to regulate the clockwork feedback loop (Zhu et al., 2008). Clock proteins are also expressed in large neurosecretory cells in the pars intercerebralis (PI); these cells may be part of a circadian network that contributes to circadian behaviors. Importantly, the CRY proteins mark neural pathways that may be relevant for migration and circadian behaviors. CRY1-positive fibers connect the PL clock cells to axons from polarization-sensitive photoreceptors in the dorsolateral medulla. CRY1-positive fibers also connect the PL to the PI that may be critical for the photoperiodic regulation of reproductive diapause and the initiation of the migratory state. A CRY2 –staining neural pathway may connect the PL clocks to the central complex, ultimately regulating circadian behaviors (e.g., daily flight activity, metabolic rhythms, and the sleep-wake cycle). Antennal clocks. It has been assumed that the circadian clock that provides time compensation for time-compensated sun compass orientation resides in the brain (specifically the PL), although this assumption has never been examined directly. We recently discovered that the antennae are necessary for proper time-compensated sun compass orientation in migratory monarch butterflies, that antennal clocks exist in monarchs, and that they likely provide the primary timing mechanism for sun compass orientation (Merlin et al., 2009). These unexpected findings pose a novel function for the antennae and open a new line of investigation into clock-compass connections that may extend widely to other insects that use this orientation mechanism. They also suggest the existence of a crucial but hitherto unknown neural circuit between the antennae and the central complex system (Fig. 3) that is now under investigation. 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 protocerebral bridge, the central body, which has upper and lower subdivisions, and the noduli. We are defining in more detail with confocal microscopy and three-dimensional reconstruction the anatomy of the central complex in the monarch butterfly. Input, intermediate and output neurons to the central complex are being defined and electrophysiological recordings of relevant neurons that process skylight information has begun. Our ultimate goal is to define how information about time and space are integrated in the brain of migratory butterflies, allowing them to maintain a southerly flight bearing over the course of the day. Elucidating the neural pathway connecting antennal clocks to the central complex looms large in this context. 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 recently showed that increasing juvenile hormone activity to induce summer-like reproductive development in normally juvenile hormone-deficient fall migrants does not alter directional flight behavior or its time-compensated orientation, as monitored in a flight simulator. Reproductive summer butterflies, in contrast, uniformly fail to exhibit directional, oriented flight. To define molecular correlates of behavioral state, we used microarray analysis of 9417 unique cDNA sequences (see Monarch brain expressed sequence tag library, below). Gene expression profiles reveal a suite of 40 genes whose differential expression in brain correlates with oriented flight behavior in individual migrants, independent of juvenile hormone activity, thereby molecularly separating fall migrants from summer butterflies. We also identified 23 juvenile hormone-dependent genes in brain, which separate reproductive from non-reproductive monarchs; genes involved in longevity, fatty acid metabolism, and innate immunity are upregulated in non-reproductive (juvenile-hormone deficient) migrants. Our results link key behavioral traits with gene expression profiles in brain that differentiate migratory from summer butterflies and thus show that seasonal changes in genomic function help define the migratory state. See Zhu et al., 2009. 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. Such a diet is being established by Orley (Chip) Taylor at Monarch Watch.
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/) The monarch genome. We have received NIH ARRP funds to generate a high depth sequence of the monarch butterfly genome using a combination of state-of-the-art "next generation" sequencing technologies, and then appropriately assemble, annotate and interpret the genome. To fully develop the monarch butterfly as a model organism to study circadian clock mechanisms and the associated molecular mechanisms of sun compass navigation used during migration, a sequenced and fully annotated genome is needed. The sequencing and annotation project is being performed in collaboration with Jeffrey L. Boore of the University of California Berkeley and CEO of Genome Project Solutions, Inc. through a consortium agreement for bioinformatics support. Genome Project Solutions (http://genomeprojectsolutions.com/) is a service provider of genome-level bioinformatics and developer of a new suite of genome analysis tools. A public database will be available to access all sequence and annotation information, as it becomes available.
Office: LRB 728 A-D Rm 728
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Molecular Neuroethology
Lazare Research Bldg, 364 Plantation Street, Rm. 708 Worcester, MA 01605-2324