David Epel, PhD - US grants

The Hopkins Marine Station (HMS) of Stanford University was founded in 1892 and has been in continuous operation as a year- round facility with resident faculty since 1917. It is located in Pacific Grove, California, 90 miles from the Stanford campus. Its site consists of 11 acres with one mile of shoreline that preserves one of the richest pieces of intertidal environment on the California coast. The Station has seven resident faculty and six other faculty members from the main campus also maintain labs at HMS. A major new effort at HMS, to be supported by erection of a large new building currently in the planning stage, is the application of modern molecular techniques to address fundamental questions in marine biology and biological oceanography. The resident and non-resident faculty of HMS are leaders in this new research frontier. This award will purchase some essential equipment (ultracentrifuges, gel boxes, microscopes, etc.) for use in marine biotechnological research. Stanford will commit at least $700,000 in matching funds to ensure that this exciting new adventure succeeds. Moreover, Stanford has earmarked substantial funds in future years for new faculty positions, set-up funds, laboratory renovations and technical support for the facilities at HMS.

The objective of this proposal is to provide travel costs for three speakers to participate in a symposium on Fertilization to be held as a part of the 5th International Congress of Invertebrate Reproduction in Nagoya, Japan, July 23-28, 1989. The speakers in this session will describe current work on echinoderms, echiuroids and ascidians covering frontier research on biochemistry of egg envelope formation, molecular basis of sperm-egg binding, receptors for egg activation, transduction of ionic signals into metabolic responses and the enzymology of sperm passage through the egg coat. %%% This Congress is unique in looking at reproduction in the invertebrate phyla and brings together researchers from around the world. The opportunity provided will lead to important dissemination of information and to further research insights about fertilization in particular and development in general in important invertebrate model systems.

Dr. Epel is seeking support under the Small Grants for Exploratory Research for assistance in moving his lab into a new research direction - the cellular mechanisms by which embryos of aquatic organisms resist deleterious effects of xenobiotics, both natural and anthropogenic. He will focus on embryos since this is a particularly sensitive phase of the organism's life history. This new work combines his long term work and interest in cell and developmental biology with a new interest and concern about the effects of man-introduced chemicals into the marine environment. Besides providing new insights about embryonic defense mechanisms, this work will also yield new and unexpected insights on the normal functioning of cells in both the embryonic and adult phases of the organisms's life history. It will test the possibility that the multi-drug resistance or P-glycoproteins transport system, whose role has been primarily assumed to be of medical importance in terms of transporting hydrophobic drugs out of cells, is normally (i.e., in the historical or evolutionary sense) utilized for removal or detoxification of xenobiotics/pollutants. This idea, if correct, would provide a novel insight into how organisms, and in particular aquatic organisms, can resist the effects of man-made and natural noxious compounds. This work could also provide a "natural history" or evolutionary basis for why these proteins are present and their role in the normal life history of the organism.

The long term goal of this work is to understand regulation of enzyme activity upon fertilization, using sea urchin oocytes as a model system for studying activation. The proposed research will investigate an apparent activation of enzymes at fertilization, particularly glucose-6-phosphate dehydrogenase (G6PDH). This enzyme change is not seen in cell homogenates but is seen in vitro as a binding change, in permeabilized cells as an activity change and in vivo as an activity change. Comprehension of this phenomena will lead to greater understanding of egg activation at fertilization in particular and could be applicable to other cases of signal transduction, e.g., the action of growth factors on cells, in general. The objectives are to (1) identify the structural elements associated with G6PDH using crosslinking reagents or affinity chromatography, (2) characterize these proteins and their cDNAs, (3) use partial amino acid sequences to prepare antibodies and study the cytological locale of these proteins and any changes in locale and (4) also use the antibodies to examine any chemical modifications as assessed with Western blots. The research will also ascertain (5) whether the dissociation might be affected by small molecules produced or destroyed at fertilization and as assessed by enriching the permeabilization medium for endogenous small molecules. An ancillary but related goal is to extend new technologies for measuring enzyme activity in vivo by using caged substrates to ascertain (6) whether amino acid catabolism is an important energy source for development. All oocytes, even those of mammals, have yolk but the role of this yolk in supporting early development is not yet appreciated. This work with caged substrates will extend this technology and also provide new insights into the energetics of development.

9321532 EPEL Current understanding of the defense mechanisms utilized by early embryos to evade environmental threats is rudimentary in comparison to our knowledge of multicellular defense systems, such as the immune system, employed by adult life stages. Strong ecological selectiv pressures for biological devices to resist pathogens and predators must exist in embryos to assure survival of these critical primary stages in oceanic life cycles. The egg capsule of the Pacificc squid, Loligo opalescens, provides a model system to study such embryo protection processes. These squid deposit their eggs in large communal aggregates in shallow coastal waters such as in Monterey Bay. During their month-long gestation period these embryos endure little or no animal, fungal, or microbial predations. Initial studies revealed the presence of several distinct bacterial symbionts specifically associated with the egg capsule sheath. These bacteria apparently are secreted from the accessory nidamental gland connected to the squid ovary, which also contains symbioticbacteria. This research project aims to identify these microbes and assess their contribution to the protection of the squid embryos in the wild. By integrating prokaryotic genetics and molecular biology with more classical approaches to ocean ecology, this research will address questions concerning both marine embryo defense mechanisms and potential therapeutic agents for use in aquaculture and medicine. Additionally, the molecular devices responsible for the mutualistic relationship between marine bacteria and the eukaryotic host that will be studied may represent a global phenomenon with implications for a wide spectrum of disciplines including immunology, cell biology, and infectious disease. ***

The long term objective of this work is the enhancement of the sea urchin gamete system as a model for cell and developmental studies. The proposed research will develop procedures to store ovulated eggs in vitro for extended periods of time, from two to four weeks or longer, and still maintain fertilizability and normal development. This would permit experiments that depend on preloading eggs with isotopes to equilibrium, allow expression of injected mRNA and permit multiple experiments on the same batch of eggs. Finally, having long-lived eggs will allow development of methods to ship eggs (as opposed to shipping hard-to-maintain adults), which would expand the pool of researchers using this resource. Sea urchin eggs are normally stored in the ovary for many months. Our preliminary results have demonstrated a significant extension of the lifetime of ovulated eggs by culture under axenic conditions. Because eggs remain viable for many months in the ovary, our experimental approach will involve mimicking the ovarian conditions. The proposed research will examine such parameters as low O2 tension, high CO2, provision of nutrients and antioxidants. When the optimal conditions have been determined, the procedure will be scaled to handle large amounts of egg material. Investigations using the easily studied sea urchin gametes have provided important insights into fertilization, cell division and regulation of gene activity through transcription factors. These findings have led to discovery of similar phenomena in vertebrates such as molecules that control call division (the cyclins), alternate means of calcium release (cyclic ADP ribose) and intracellular pH regulation (Na+-H+ exchangers) to name a few. Enhancement of this resource will expand research on these forms with a resultant increase in our knowledge of general cell and developmental phenomena applicable to all animals.

9553566 Epel This project is to provide high school biology laboratory exercises to teachers over the Internet. New mult-disciplinary protocols in biology and cell biology are to be developed, disseminated as supplementary materials and maintained on the World Wide Web and in other forms. The project is a model of how research laboratories can efficiently share expertise with the nation's educators. Concepts of scientific method and experimentation is to be taught through modules featuring sea urchin gametes, illustrating fertilization, cell physiology, cell division, embryonic development, biochemistry, speciation, and the interaction between organisms and their environment. Teacher support is to be by way of interactive tutorials and Web pages over the Internet.

Epel 96-04354 This one year grant will fund a pilot project aimed at understanding how embryos defend themselves against environmental toxins. Dr. Epel hypothesizes that the P-glycoprotein drug transporters provide this protection, acting as a first line of defense to keep toxins out of the cell. It is present in embryos and adults of a mud-dwelling worm, Urechis caupo, and its activity protects its embryos against added test toxins. This protection might account for the ability of Urechis embryos to develop normally in pore water (the supernatant sea water from the mud flat sediments). The embryos of starfish and sea urchins do not possess this transporter and their embryos cannot develop in pore water from the mud flats. If this drug transporter in fact protects embryos, then expression of the transporter in the embryos of the sea urchin or starfish should confer protection against environmental toxins. The major goal of this one year pilot study is to express this transporter, using the gene cloned from the mouse or nematode worm, in starfish or sea urchins oocytes and to determine if the resultant embryos will develop. If so, a large number of questions can then be asked and answered about the role of this transporter in protection of embryos and also about protection against toxins in the marine environment in general. This transporter is not ubiquitous in the marine environment, and our limited survey indicates its presence in filter and suspension feeders but not in sea urchins or starfish. A second question we wish to address in this pilot study relates to the distribution of this transporter. It is present in an echiuroid worm which lives in sediments. Is having this transporter a general attribute of being an echiuroid? Or is it something restricted to animals whose lifestyle exposes them to toxins (such as sediment dwelling or filter feeding)? To provide an answer to this question, a minor component of this pilot study will determine whether the transport protein is ex pressed or over expressed in members of this group that live in clean habitats or have different lifestyles. Providing answers to these two questions, especially information about the expression of the cloned transporter gene in echinoderm embryos, will indicate will indicate the feasibility of research approaches using expressed genes in embyros. These approaches, if feasible, would provide rigorous information on the biology of embryo defense mechanisms in general and the role of this drug transporter in particular

DESCRIPTION (adapted from the applicant's abstract): The goal of this study is to determine the roles of nitric oxide (NO) and its possible relationship with the intracellular Ca2+ transients in the processes of oocyte activation and cell cycle. The possible role of NO in regulating the egg's ion channels (as part of polyspermy block) and sperm physiology (such as motility and the acrosome reaction) will also be investigated.

0216458
Luthy
This proposal will create a modern analytical facility at Stanford University capable of accurately measuring emerging environmental contaminants at trace levels in aquatic systems. By building on the University's new campus-wide Environmental Initiative, this proposal represents a strategic opportunity to significantly impact environmental research and education at Stanford University among faculty and students in chemistry, biology, geological and environmental sciences, and environmental engineering. The proposed emerging contaminant analytical facility will create interdisciplinary research opportunities and promote the teaching on the fate, transport, and effects of emerging contaminants in aquatic systems.

Project Summary for IBN-0446384
Efflux Transporters in Sea Urchin Development


Efflux transporters in embryos can have both housekeeping and developmental roles. The housekeeping role can protect the embryo from external toxicants during embryogenesis. The developmental role can pump endogenous signaling molecules out of cells to influence adjacent cells or to feedback on the cell itself in an autocrine fashion. Preliminary work reveals members of the mrp family of ABC efflux transporters are dormant in the unfertilized sea urchin egg and become active as part of the response to fertilization. The increased activity appears to result from movement of vesicles containing these transporters to the egg surface after fertilization and insertion of the proteins into the membrane. Proposed experiments will prove or disprove the vesicle movement hypothesis using antibodies to follow any changes in the location of the transport proteins.
Preliminary results suggest several developmental roles. One is a role in forwarding cell division during the cleavage period. The proposed research will examine specificity of the effect by injection of antibodies to the active site of the transporter. If indicated as a specific effect a search will be made for the active substance effluxed by the transporter. A plausible endogenous substrate are leukotrienes and levels of these compounds will be measured during cleavage and later development.
The other transporter function is a role in signaling in primary mesenchyme cell differentiation, perhaps in the cross-talk between ectoderm and underlying mesenchyme cells. The proposed studies will describe the expression of new transporters during development, localize them by in situ hybridization and assess their role using morpholinos to specifically prevent new transport expression.
This work also broadens the view of efflux transporters, which has in the past been primarily centered on their medical relevance in terms of evading drug therapy during treatment against cancer. The proposed research looks at these ubiquitous molecules in new ways; in terms of a fertilization response leading to re-organization of the plasma membrane, in terms of a role in normal cell functions such as mitosis and in terms of a signaling role for integrating morphogenetic responses.
The proposed research also has broad impact on education, as many of these experimental results can be integrated into laboratory exercises used in introductory biology labs at Stanford and in high school and college exercises across the country. To facilitate this, exercises suggested by this research will be disseminated on the widely used (last year received 4 million hits) Sea Urchin Embryology web site (http://www.stanford.edu/group/Urchin/ ).This combination of environmental work with cell and developmental biology also attracts numerous undergraduate students to the P.I's lab (often from under-represented groups); these students often receive University recognition for outstanding undergraduate research and many of these students later go on to graduate study in developmental biology and environmental sciences

This project uses sea urchin embryos to provide a curriculum module for inquiry-based high school biology. The curriculum is provided via a new open access website, addresses several of the National Science Content Standards and provides a range of activities suitable for all levels of high school biology. The curriculum, through the website, will also provide instructional support materials such as video demonstrations, animations, time lapse videos and image galleries relevant to each exercise. Also included will be substantial material for professional development, including descriptions relating each activity to the national content standards, discussion of major ideas and concepts and links to additional pertinent resources. For each lab concept, five types of activities will be developed: classroom wet labs, virtual wet labs, web-based bioinformatics labs, data manipulation activities and activities for accessing the primary scientific literature. Each activity provides an independent lesson and teachers will be able to create a custom module for one to six weeks of biology curriculum in Grades 9-12.