Section: Rotations

Postdoctoral Position Available

Steven Reppert, M.D.

Academic Role: Professor

Faculty Appointment(s) In:
   Neurobiology

Other Affiliation(s):
   Interdisciplinary Graduate Program
   Program in Neuroscience

Potential Rotation Projects

Three-dimensional reconstructions of Monarch butterfly brains (under supervision of Postdoctoral Fellow:  Stanley Heinze)  See Below

In vitro reconstruction of the purple sea urchin (Strongylocentrotus purpuratus) circadian clock. (under supervision of Postdoctroral Fellow: Christine Merlin)  See Below

Project: Three-dimensional reconstructions of Monarch butterfly brains

With Postdoctoral Fellow: Stanley Heinze

Monarch butterflies are well known for their most spectacular fall migrations, during which they cover thousands of kilometers across unfamiliar terrain in order to reach their winter habitats in Mexico. For this task these animal use time compensated sky compass navigation. This allows them to calculate their traveling direction by using information derived from the changing position of the sun during the day. Not only the sun itself serves as compass cue, but also the sky polarization pattern, as has been shown by behavioral experiments (Reppert et al, 2004). The summer form of the Monarch butterfly, however, is non migratory and has been also shown to lack directional flight behavior (Zhu et al, 2009).

The neuronal basis of sky compass navigation and directional flight behavior is completely unknown in the Monarch butterfly and shall be addressed in this project. microarray analysis of brains has revealed that several genes are differentially expressed in brains of migrants and summer butterflies, suggesting that the neuronal machinery of the brain differs between the two forms (Zhu et al, in press). This is likely to result in changes of synaptic density in brain regions involved in migratory behavior. As it is known from other species, in particular the desert locust, that the central complex and the anterior optic tubercle are crucially involved in processing of sun compass cues, this project will focus on these two brain areas (Homberg, 2004; Pfeiffer et al, 2005; Heinze and Homberg, 2007; Pfeiffer and Homberg, 2007).

One way to estimate the number of synapses and fine branches within specific brain areas is a detailed volumetric analysis of these regions. Therefore digital, three-dimensional reconstructions of the central complex and the anterior optic tubercle will be carried across a population of butterflies, resulting in average shape and volume data of these regions (Huetteroth and Schachtner, 2005, Kurylas et al., 2008). The results will be eventually compared between summer butterflies and migrants in order to establish neuronal correlates of their different migratory states.

During the course of this two-month project, the complete dataset for summer butterflies is going to be obtained, providing the base for the comparison with the migratory form, which only occurs within a short period during fall. The student will learn to perform immunocytochemical brain labeling with antibodies against synaptic proteins and neurotransmitters. These fluorescent stainings will be analyzed with high resolution confocal microscopy, and the resulting image stacks will be used for three dimensional reconstructions with sophisti­cated graphics software (Amira5.1). Emphasis will be laid on obtaining a sound under­standing of insect brain neuroanatomy and developing the capabilities to independently pursue immunocytochemical studies of insect brains, starting with the dissection of brains up to the final statistical analysis of volumetric data.

References

Heinze S, Homberg U (2007) Maplike representation of celestial E-vector orientations in the brain of an insect. Science 315:995-997.

Homberg U (2004) In search of the sky compass in the insect brain. Naturwiss 91:199-208.

Huetteroth W, Schachtner J (2005) Standard three-dimensional glomeruli of the Manduca sexta antennal lobe: a tool to study both developmental and adult neuronal plasticity. Cell Tissue Res 319:513-24.

Kurylas AE, Rohlfing T, Krofczik S, Jenett A, Homberg U (2008) Standardized atlas of the brain of the desert locust, Schistocerca gregaria. Cell Tissue Res 333:125-145.

Pfeiffer K, Kinoshita M, Homberg U (2005) Polarization-sensitive and light-sensitive neurons in two parallel pathways passing through the anterior optic tubercle in the locust brain. J Neurophysiol 94:3903-3915.

Pfeiffer K, Homberg U (2007) Coding of azimuthal directions via time-compensated combination of celestial compass cues. Curr Biol 17:960-965.

Reppert SM, Zhu H, White RH (2004) Polarized light helps monarch butterflies navigate. Curr Biol 14:155-158.

Zhu H, Gegear RJ, Casselman A, Kanginakudru S, Reppert SM (2009) Defining behavioural and molecular differences between summer and migratory monarch butterflies. BMC Biology 7:14.

Project:  A rotation project is now open in the lab to work on the in vitro reconstruction of the purple sea urchin (Strongylocentrotus purpuratus) circadian clock.

Rotation Project under Supervision of Postdoctroral Fellow: Christine Merlin

Most of organisms possess an internal clock controlling physiological and behavioral outputs. The main gear at the core of the clock is a simple autoregulatory feedback loop involving a set of clock genes, in which positive elements drive the transcription of negative elements that rhythmically feed back to inhibit the action of the former with a time delay of about 24h.

Recent activities in our lab led to the discovery of a new type of clockwork mechanism in the monarch butterfly and yielded insights into the evolution of the circadian clock in insects (1-3). In order to extend our knowledge on the evolution of circadian clocks in the animal kingdom, we are interested to look at the clockwork mechanism in more ancient species. The sea urchin appears to be a suitable model for that purpose as it belongs to an evolutionary ancient group and its genome has been fully sequenced and annotated (4), therefore allowing the identification of its clock genes by database searches. Indeed, a preliminary study performed in the lab identified into the genome of S. purpuratus a set of clock genes that could potentially represent a new way to build a clock. To test this hypothesis, we are thus seeking to reconstruct this clock in vitro with the cloned clock genes identified.

The rotation student will be in charge of the cloning of the genes into appropriate expression vectors as well as the reconstruction of the transcriptional feedback loop in vitro using a luciferase reporter assay in Schneider 2 cells, in which the genes will be expressed transiently.

The student can expect to be trained in molecular biology, biochemistry, cell culture, and in vitro assays, in addition to be exposed to a rich scientific environment as exemplified by the diversity of projects developed in the lab (see our website).

Motivated students with strong interests in molecular approaches are encouraged to apply.

Zhu H., Yuan Q., Briscoe A.D., Froy O., Casselman A., and Reppert S.M. (2005) The two CRYs of the butterfly. Curr. Biol., 15:R953-R954.

Yuan Q., Metterville D., Briscoe A.D., and Reppert, S.M. (2007) Insect cryptochromes: Gene duplication and loss define diverse ways to construct insect circadian clocks. Mol. Biol. Evol., 24:948-955.

Zhu H., Sauman I., Yuan Q., Casselman A., Emery-Le M., Emery P., and Reppert S.M. (2008) Cryptochromes define a novel circadian clock mechanism in monarch butterflies that may underlie sun compass navigation. PLoS Biol., 6:e4.

The genome of the Sea Urchin Strogylocentrotus purpuratus. (2006) Sea Urchin Genome Sequencing Consortium. Science, 314:941-952.

Office: LRB 728 A-D Rm 728
Phone: 508-856-6148
E-mail: Steven.Reppert@umassmed.edu

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Postdoctoral Position Available A postdoctoral position is available to study in this laboratory. Contact Dr. Reppert for additional details.