"We know surprisingly little at the cellular and molecular level about how embryos cope with their environments, yet the embryo is the most fragile stage in the life cycle, and its survival is critical for sustaining the life of a species," said David Epel, a professor of biology at Stanford's Hopkins Marine Station. Epel is co-organizer of the AAAS session with James Clegg, a professor of biology at the University of California-Davis' Bodega Marine Laboratory.
Studies of embryos under stress may shed light on a range of biological questions, including how drug resistance evolves, which species are most likely to be vulnerable to global climate change, and how ultraviolet light from the sun may contribute to the apparent worldwide decline in frog populations. Among the participants in the symposium were Clegg, who presented an overview of the topic; Epel, who described coping strategies that embryos use to repel toxins, and Susan Brawley of the University of Maine, who described strategies that marine algae use for reproductive success. Susan Lindquist of the University of Chicago and Gretchen Hofmann, of the University of New Mexico-Albuquerque, both discussed how embryos balance the good and bad effect of proteins that protect from heat shock. Roger Pedersen of the University of California-San Francisco and Joseph M. Kiesecker of Yale described the influence of DNA repair mechanisms in protecting embryos from damage during development.
All organisms have built-in mechanisms to survive insults from their environments, but in embryos these mechanisms often are different than in adults, Epel said. "One reason is that the embryo has not developed the adult's mechanisms for coping. It is adapted to a particular niche and to a particular task - rapid cell division and development. One purpose of this symposium is to look at how the rules and principles for handling stress appear to be different for embryos than for adults."
Spitting out toxins
Epel's own research illustrates a coping mechanism that may be useful for an adult but is essential to its embryo, at least if the species is like the fat innkeeper worm, which survives in coastal mud flats, resisting natural and man-made toxins washed off the land by streams. "Adult organisms often defend against toxins by taking the substance into their cells and transforming it," Epel said. During embryogenesis, contact with such toxins could damage DNA, proteins or cell membranes, and thus interfere with the embryo's development. So the innkeeper worm1s embryo detects toxins as soon as they touch a cell wall, and spits them out of the cell.
To do so, Epel found, the embryo relies on a protein woven into the walls of some of its cells that forces toxins out. The protein is called an MDR transporter pump - "MDR" stands for "multi-drug resistance," because these proteins were first discovered by cancer researchers trying to find out how malignant tumors manage to eject certain chemotherapy drugs. Recently, Epel and others have found MDR pumps in many organisms, from bacteria to mammals. This defense against toxins almost certainly has been around for millions of years. Unfortunately it's now one of the mechanisms that cancers use to resist treatment, and that disease-causing organisms use to resist antibiotics.
Epel has identified the MDR transporter as one mechanism that an adult fat innkeeper worm uses to survive in some of the most polluted mud flats along the Pacific coast. At the AAAS meeting, he presented data showing that the worm's embryos use MDR transporters as well, as a first line of defense against even tiny amounts of toxins. For the embryos, there is a tradeoff, however: The transporter requires a significant amount of the cell's chemical energy, or ATP, to do its work. Even though it succeeds in fending off toxins, the embryo may pay a price in delayed development.
(For more information on Epel1s discoveries about MDR transporters in mussels and worms, see Stanford Report, March 18, 1997, available on the Web at http://www-leland.stnaford.edu/dept/news/relaged/970319epel.html.)
Surviving heat shock
That tension between the benefits and the costs of a defense mechanism is even more starkly defined in the case of heat shock proteins, protective substances that flood the cells of certain organisms at the first signal that they will be exposed to damaging high temperatures. The cost of this protection comes when temperatures return to normal, and the heat shock proteins severely restrict growth.
"Balancing the beneficial effects of heat shock proteins on stress tolerance and the detrimental effects to growth is particularly crucial in early embryos, which are characterized by rapid rates of growth and cell division," said Lindquist, professor of molecular genetics and cell biology at the University of Chicago, and a fellow of the Howard Hughes Medical Institute.
Both Lindquist and Hofmann presented research on heat shock proteins at the symposium. Hofmann is an assistant professor of biology at the University of New Mexico-Albuquerque. Her research highlights the tradeoffs for embryos. She studies survival strategies in the intertidal zone, where temperatures in a single day range from the chill of ocean waves to the sun-baked heat of a tidepool.
She found that the embryos of snails, laid in gelatinous masses in tidepools, began expressing heat shock proteins 30 hours or more into their development; at that point, the proteins offered some protection from temperatures that rose as high as 88 degrees Fahrenheit. For the first day of life, however, the early embryos had to depend on the good fortune of a cool day to protect them from heat shock.
"There is a strategic tension between the benefit of heat shock proteins as protection from environmental heat stress, the biological consequences of heat shock protein expression in early embryos, and the unpredictability of temperature stress in the intertidal zone," Hofmann said.
She said the embryos may have other protective factors as well that scientists haven't discovered yet; or the actions of their parents may offer some protection. "Perhaps the female snail can sense when to lay the eggs so as to reduce mortality of her progeny," Hofmann said.
Lindquist's research has uncovered two unexpected ways that embryos restrict heat shock proteins. Her lab studies the fruit fly Drosophila, which has a dramatic response to high temperatures - within an hour of exposure to heat, the fly1s cells are flooded with a thousandfold increase in the heat shock protein Hsp70.
That is what happens in most stages of the fly's life cycle, including late embryos, larvae and adults. However, built into the normal steps for development of Drosophila's early embryo is a specific mechanism to prevent expression of Hsp70. This preventer mechanism remains in place only through the most rapid phase of cell division, when the embryo's healthy development would be disrupted by the presence of the heat shock protein.
In a later stage of development, the embryo expresses heat shock protein when temperatures rise. But as soon as the heat goes down to normal, the embryo gathers up the Hsp70 proteins and sequesters them in tiny granules within its cells. Most organisms and cell types use a different method, proteolysis, to dissolve unwanted proteins and get rid of them, Lindquist said. The fly embryo probably resorts to sequestration in the interests of speed, since it can not go about its business of growing and developing until Hsp70 is gone.
"The organism needs to inactivate one of the most abundant proteins in the cell in a highly selective manner in just a few minutes," Lindquist said. "This step ensures that Hsp70 will be inactivated as soon as it is no longer needed." UV light, DNA damage and missing frogs
Worldwide, scientists have noticed a decline in many species of amphibians. Several apparently have become extinct, yet no single cause for the losses has been identified. Kiesecker, a postdoctoral fellow in biodiversity at Yale, and Andrew Blaustein, professor of zoology at Oregon State University, have identified some contributing factors that could apply to amphibians in many locations if their embryos are exposed to extra ultraviolet light from the sun, thanks to a thinning ozone layer.
When frog eggs are exposed to ultraviolet-B radiation, the light damages the embryos1 DNA. The damage can cause mutations and can kill embryonic cells, but the embryo has a well-developed rescue mechanism, based on the work of a DNA repair enzyme called photolyase. Frog eggs from species with the greatest photolyase activity are the ones most likely to survive a blast of UV-B light, Kiesecker and Blaustein found. However, the researchers also have identified a fungus that appears to act synergistically with UV-B to kill developing frog eggs. DNA damage may make the eggs more susceptible to the fungus.
Mammals also have DNA repair mechanisms; even in the protection of the womb, damage can occur during embryonic development. In the AAAS session, Pedersen, research director of the reproductive genetics unit at the University of California-San Francisco reported on the role of one type of DNA repair - base excision repair - in protecting mouse embryos.
"In our research we are finding that base excision repair is essential for life even at the earliest stages of intrauterine embryonic development of the mouse," Pedersen said. "We are investigating the response of embryos to injury that occurs within the womb, and the mechanisms that embryos use to cope with the damage, or to initiate a 'self-destruct' program if the damage cannot be repaired."
Timing and seaweed success
Epel1s fascination with the coping strategies of embryos began years ago, with pioneering studies that show how they begin; he worked with sea urchin eggs to demonstrate how a successful meeting between sperm and egg triggers the egg to become an embryo. To wrap up the symposium, he invited Brawley, a professor of marine biology at the University of Maine, who may have answered one of the longstanding mysteries about organisms like sea urchins, corals and seaweed, that depend on the vagaries of current and tide to get sperm and egg together close enough for fertilization to begin.
Previous studies assumed that these organisms release huge numbers of reproductive cells, or gametes, when water is turbulent - good for distribution, but with a fertilization success rate likely to be 1 percent or less. Brawley and her research team studied Fucus, a brown seaweed that lives around the world. In laboratory experiments, they showed that Fucus waits to release its gametes when the water is calm and salinity is high - two conditions likely to strongly increase the success rate of external fertilization.
Looking further, they found that the seaweed waits for chemical signals, a high level of salt and a low level of carbon dioxide dissolved in the seawater, as indicators that conditions are right for romance. Brawley gives credit to her former postdoctoral fellow, Gareth Pearson, for demonstrating the low carbon hypothesis, and to former graduate student Ester Serrao, who did what Brawley calls the "clencher" work - showing that, contrary to earlier assumptions, high water motion inhibits gamete release.
Brawley's lab now is working on the details of what happens inside the seaweed to inhibit or trigger gamete release. They also are studying how the embryos of Fucus fare once fertilization is successful. They have found that the embryos at first are resistant to the stresses of tidepool living, such as daily shifts from low to high temperatures and from wet to dry conditions. At the age of one to two days, they become sensitive to these stresses, then become resistant again. Also, former graduate student Rui Li has shown that the temperatures experienced by adult algae affect the heat tolerance of their embryonic offspring.
(For a press release from the University of Maine describing Brawley1s research, see 3When to Reproduce? It1s all in the timing,2 available on Eurekalert! or on the UMaine website at http://www.ume.maine.edu/~paffairs/releases/releases.html.)