Contemporary Approaches to Endocrine Signaling

(earlier name: New Millennium Approaches to Comparative Endocrinology)

Symposium organized by Sunny Boyd, Miles Orchinik, and Juli Wade

Unique model organisms used by comparative scientists provide insight into biological processes from molecular to evolutionary levels. However, technological innovations often arise from work on more traditional model organisms, such as rats, mice and fruit flies. Timely application of state-of-the-art methods to comparative organisms can significantly advance progress in these model systems, as well as point to new directions for research across animals. Therefore, this symposium is designed to begin to bridge the technological gap across model systems by inviting speakers who are already applying exciting new methods in a diverse array of organisms.

Preliminary Schedule

Day One

8:00-8:20 Welcome and Introduction to the Symposium

8:20-9:00 Dr. Russ Fernald: Social regulation of the brain: New ways to discover the roles of status, sex and size

9:00-9:40 Dr. Laura Carruth: Sex differences in the songbird brain: using molecular tools to investigate brain sexual differentiation in a comparative model.

9:40-10:20 Coffee

10:20-11:00 Dr. Simon Evans: Microarrays and Brain Research

11:00-11:40 Dr. Christina Grozinger: Microarray analysis of pheromone-mediated gene expression in the honey bee brain.

11:40-1:20 Lunch

1:20-2:00 Dr. Rick Goetz: The “ups” and “downs” in using subtractive cloning approaches to isolate regulated genes

2:00-2:40 Dr. Stuart Tobet: Viewing cell movements in the developing neuroendocrine brain

2:40-3:20 Coffee

3:20-4:00 Dr. Rob Grainger: Xenopus tropicalis, a new model for vertebrate developmental genetics.

Day Two

8:00-9:40 Microarray Workshop; led by Dr. Simon Evans

9:40-10:20 Coffee

10:20-12:00 Protein Interactions Workshop; led by Biacore, Inc.

12:00-1:20 Lunch

1:20-5:00 Contributed oral presentations (plus a coffee break)

Abstracts

Dr. Laura Carruth, Department of Biology, Georgia State University
Sex differences in the songbird brain: using molecular tools to investigate brain sexual differentiation in a comparative model.

Early in development, male and female brains diverge in their patterns of growth and differentiation, especially in brain regions involved in the control of behavior. The classical model of brain sexual differentiation states that testosterone is aromatized in the brain into estrogen that then acts to either initiate male neural development or inhibit female neural development. One vertebrate model that is ideal for studying sexual differentiation is the Australian zebra finch (Taeniopygia guttata). Male zebra finches sing a courtship song while females do not. This distinct behavioral dimorphism is paralleled by large morphological se differences in the neural circuit for song learning and production. The differentiation of these morphological sex differences has previously been thought to be under hormonal control, but despite extensive research, sexual differentiation of the avian song system does not appear to completely follow this model. We are now examining different factors or mechanisms that may influence early song system development. We identified early markers of sexual differentiation (genes that might or might not respond to hormone treatment) using suppression subtractive hybridization on mRNA from hatchling male or female zebra finch telencephalon in order to isolate cDNAs representing genes that are expressed at a higher level in one sex or the other. Plasmid libraries were constructed of cDNAs that are enriched in male or female brain. We are characterizing these cDNAs and confirming their differential expression using mRNA dot blots, northern analysis, and in situ hybridization. Some of the cDNAs correspond to recognized avian genes, some are sex chromosomal genes, while several clones are novel.

Dr. Simon Evans, Mental Health Institute, University of Michigan
Promises and pitfalls of microarray technology applied to neuroscience research.
Neuroscience presents a significant challenge to DNA microarray technology because of high tissue complexity, low abundance transcripts and minor but biologically significant changes in gene expression levels. To examine the current utility of DNA microarrays for neuroscience applications we have used various experimental paradigms. First, with respect to sensitivity we have utilized existing data from hippocampal serial analysis of gene expression (SAGE) studies in combination with hippocampal microarray data to evaluate the limits of reliable detection of microarrays in complex brain tissue. Second, to evaluate the performance of microarrays in detecting expression differences in brain tissue we examined hypothalamic transcriptional profiles of adrenalectomized (ADX) rats. Because this model has been used extensively in the study of stress there exists a large literature regarding the effect of ADX on gene expression changes in transcripts of varying abundance, which can provide predictive value for the microarray results. Finally, we have examined data from replicate experiments to estimate both random and systematic error within microarray studies, which are plagued with high false positive rates because of the large number of observations in microarray experiments.

Dr. Russ Fernald, Psychology Department, Stanford University
Social regulation of the brain: status, sex and size.
It is self evident that the brain controls behavior but can behavior also ‘control’ the brain? Recent evidence has revealed that social behavior can cause changes in certain brain structures of adult animals. Such alterations can be dramatic, reversible and are typically related to reproductive behavior. How does behavior sculpt the brain and how are these changes controlled? Our studies link molecular events with organismal behavior by using a model system in which social behaviors regulate reproduction. We have shown that a variety of neural and endocrine changes result from changes in social status. Surprisingly, we have also demonstrated that body growth rate is also regulated by social status and immediate social history. Discovering how social information is transduced into physiological processes via cellular and molecular changes presents a major challenge. Using a range of techniques from behavioral observations to real-time PCR and gene chip technology, we are trying to discover how changes in behavioral status change the brain.

Dr. Rick Goetz, Marine Resources Center, Marine Biological Lab, Woods Hole
The “ups” and “downs” in using subtractive cloning techniques to isolate regulated genes.
Over the last decade, subtractive cloning approaches have been used extensively to isolate genes that are up- or down-regulated under various conditions. These techniques have provided the foundation for many subsequent studies concerning gene function and regulation and, as such, have been valuable tools for many biological fields. Over the past 10 years, we have used four different subtractive cloning approaches to isolate genes in fish that are regulated in relation to hormonal stimulation or the stage of ovarian maturation. These include conventional cDNA probe subtraction followed by library screening; differential display PCR; suppression subtraction hybridization; and chemical crosslinking subtraction. We continue to use these techniques for the isolation of new genes involved in physiological processes in fish and bivalve molluscs. Examples that illustrate our use of different subtractive cloning techniques will be described, including advantages and disadvantages of each. In addition, the use of ancillary methods (e.g., “Reverse Northerns”) to facilitate the use of these subtractive approaches are discussed.

Dr. Robert Grainger, Department of Biology, University of Virginia
Xenopus tropicalis, a new model for vertebrate developmental genetics.
The pipid frog Xenopus laevis has been among the most productive model systems for vertebrate experimental embryology. However, to determine whether a newly-identified activity is actually required for a given process, it is necessary to subtract a given gene function. In complex developmental systems, tools provided by genetics- for example, the phenotype of a null mutant- provide the strongest proof of participation. Further impetus for developing an amphibian genetic model derives from a recent breakthrough which allows transgenic frogs to be produced cheaply, efficiently, and in large numbers. Transgenesis-related genetic approaches, combined with the frog’s embryological advantages and the low cost of husbandry, should permit the dissection of many basic developmental processes quickly and inexpensively. However, Xenopus workers currently lack genetic tools for dissecting complex biological pathways. A frog which is better suited to genetic approaches is Xenopus (Silurana) tropicalis. X. tropicalis has a much shorter generation time (3-4 months), and a smaller diploid genome. The embryological techniques and molecular assays which have been described for X. laevis are readily applied to X. tropicalis, but may be supported by multigeneration genetic analyses. Using mutants or transgenic animals in highly developed tissue transplantation regimes will facilitate analysis of individual animals containing tissues of more than one genotype. Such genetic mosaic analyses have been very useful in studies of Drosophila embryogenesis, but are technically challenging in extant vertebrate models.

Dr. Christina Grozinger, Beckman Institute, University of Illinois
Microarray analysis of pheromone-mediated gene expression in the honey bee brain.
Pheromones regulate a wide variety of behaviors in both vertebrate and invertebrates. Honey bees are an established model organism to study pheromones: their highly complex society is predominantly regulated by chemical communication, and many of the components of different honey bee pheromones have been identified. Using recently developed honey bee cDNA microarrays, we have begun to analyze the molecular mechanisms of pheromonal regulation of honey bee behavior. Here we report on brain transcriptional responses to queen mandibular pheromone (QMP). QMP is a blend of five identified chemicals that has potent and diverse influences, with effects on brain structure, hormone systems, and several different aspects of behavior including queen rearing, foraging, and learning. We analyzed changes in gene expression in the brain caused by exposure to QMP in both one-day old and 8-9 day-old adult bees using microarrays, in both lab and field experiments. Several sets of co-regulated genes were identified, and the time course of their expression determined. This study will allow us to hypothesize the changes in brain architecture, neurochemical and hormonal state that lead to the dramatic alterations in behavior observed upon stimulation with pheromone.

Dr. Stuart Tobet, Department of Physiology, Univ. Of Massachusetts Medical School
Viewing cell movements in the developing neuroendocrine brain.
Many studies suggest that migratory guidance cues within the developing brain are diverse across many regions. To better understand the early development and differentiation of select brain regions; an in vitro method was developed using selected strains of embryonic mice. In particular, organotypic slices are used to test factors that influence the development of brain nuclei or layers. 250µm thick slices cut on a vibrating microtome are prepared and maintained in vitro for 0-3 days. Nissl stain analyses often show a uniform distribution of cells in the regions of interest on the day of plating (embryonic days 12-15). After 3 days in vitro, cellular aggregation suggesting nuclear or layer formation has occurred in several regions. Nuclear or layer formation in vitro suggests that key factors reside locally within the plane of the slices. Video microscopy studies are used to follow the migration of fluorescently-labeled cells in brain slices from mice maintained in serum-free media for 1 to 3 days. Transgenic mice with selective promoter driven expression of fluorescent proteins allows us to view specific cell types (e.g., neurons expressing gonadotropin releasing hormone). The accessibility of an in vitro system that provides for relatively normal brain development over key brief windows of time allows for the significant testing of important mechanisms.