How did we get here? It's a question both basic and profound. A child might give a guileless answer: First comes love, then comes marriage, then comes the baby in the baby carriage.
How did we get here? Each of us begins as a zygote, as the sum of egg and sperm, as a primal cell. Genetically complete and raring to go, this single cell contains the biological software necessary to create a human, written in three billion base pairs of DNA, twisting through 23 pairs of chromosomes.
Understanding exactly how this software moves an organism from zygote to adulthood is another matter. What switches genes on and off, directing them first to develop a primitive skin, a crude nervous system, and a chamberless, but beating, heart? And then to refine--to become eyes that can see...with lashes and brows! To become hands and fingers swirled with a print unique in all the world. To become a mind that not only controls basic functions like breath and heartbeat, but one that burns with the electricity of reason and imagination.
How did we get here? Embryologists, developmental biologists, fetal pathologists and others have worked for years to sketch in the outlines of development. But it's still pretty much a mystery, and like all the best mysteries, it defies easy resolution. Clues are elusive. Red herrings abound. Methods, mechanisms, and motives are hard to deduce. Solving it will take more than good detective work; scientific sleuths also need good hunches and good luck.
In detective novels, it's frequently the chance clue--the stray fingerprint, the contradictory statement, the forgotten letter--that cracks the case. So it is too in science, as ORNL's Waldy Generoso has come to experience. From a seemingly ordinary experiment, Generoso has stumbled upon extraordinary results, clues that might help crack the mystery of how we got here, evidence that might also help to explain how things can go wrong on the journey.
A senior scientist in the Biology Division, Generoso never intended to start thumbing through the pages of the whodunit of human development--he is a mouse researcher. Laboring away in ORNL's monolithic ``Mouse House,'' his work has focused on mutagenesis--including mutations in the genetic structures of mice. In 1987, under funding from the Department of Energy and the National Institute of Environmental Health Science, Generoso attempted yet another experiment in mutagenesis. He wanted to try to change the distribution of the genetic material contributed by the mother to a zygote--that primal cell. So he administered ethylene oxide (a mutagen) to female mice immediately after conception, before the zygote made its first division.
hen as he says, ``Something happened,'' something that altered the whole course of his research. Generoso stumbled upon a fresh trail, a series of clues that led him from the puzzle of genetics to the riddle of development. His experiment revealed an eerie parallel between birth defects in mice--defects induced in the zygote, at the first moment of life--and birth defects in humans. Previously, it had been assumed that such defects could only be induced later, when major organs and limbs were forming. Suddenly, that assumption was shattered.
Something happened. The mutagen did affect the zygotes--that was no surprise. It was how it affected them that was so startling. Many of the embryos died early--damage caused by the mutagen was too severe. Generoso had expected that. But other fetuses had died quite late--almost at the end of gestation--and had begun to develop, unfurling with deformities both unusual and inconsistent. Some had cleft palates. Some had neural tube or abdominal wall defects. Others had small brains or deformed limbs. others were hydropic, swollen with fluid.
Late fetal deaths. Unusual deformities. Generoso's curiosity was piqued, and he began to dig deeper. What had that mutagen wrought?
What it had not wrought was mutagenesis. Defective and dead fetuses abounded, but their chromosomes were unmarked. There were no trisomies-extra chromosomes. There was no evidence of genetic damage. What had that mutagen wrought?
``We looked high and low, at two-cell, four-cell, eight-cell stages, at 10-day-old fetuses with deformities, and we could not attribute the effect we are seeing to chromosomal damage. The chromosomes were fine'' he says. ``Point mutations--incremental changes in the DNA--were unlikely because chemicals and X-rays known to affect genes don't cause these deformities. It had to be something else.''
His boss, geneticist Liane Russell (renowned in her own right for discovering that male development in mammal embryos is triggered by the ``Y'' chromosome at conception), was also surprised. ``I was willing to bet Waldy $1,000 that these deformities were caused by trisomies, but they're not. There are no structural malformations in the chromosomes. The anomalies happen at too high a rate to be gene mutations, and they'd all have to be dominant traits.''
This all might have been just another quirky research conundrum--intellectually challenging, hut of minor significance--if not for the sharp eyes of Generoso's collaborator, a detective grounded in the anomalies of human development. Enter Joe Rutledge, pediatric pathologist and researcher at the University of Washington School of Medicine. In 1971, as a college student, he spent a summer working in Generoso's lab. In the early '80s, Generoso asked his former student to look at some interesting mouse malformations, and the collaboration began.
``It blossomed into a very nice symbiotic relationship,'' Rutledge says. ``My major academic interest is in human malformations, birth defects in children. That's my day-to-day life. I provide a backboard to bounce ideas off of in regard to humans, and he provides me with an avenue to see how we might follow these ideas in mice and relate them back to humans.'' Naturally, Rutledge was asked to look at the results of Generoso's latest experiment. He saw his day-today life. He saw the faces of malformed children redrawn in miniature, etched again and again on unborn mice. Cleft palates, limb deformities, neural tube defects, organs blossoming through gaping holes in abdominal walls--as a pathologist, Rutledge had seen them all before, in human form, but he had rarely been able to explain how they could happen.
t's a fact of biology that humans are reproductively inefficient--half of all pregnancies never come to term. Most fail early--before a woman even knows she is pregnant--and are reabsorbed by the body. Inefficiency breeds tragedy when a flawed pregnancy continues, only to dead-end in a miscarriage, in a stillbirth, occasionally in the live birth of a child with severe deformities. And most of these defects cannot be accounted for; something just goes wrong. Genetic flaws account for only 20%. Ten percent are caused by specific reagents--chemicals, drugs, or viruses. As for the rest, well, who knows?
When Rutledge pointed out the similarities between the mutagen-induced defects and random human defects, the tiny, pink mouse fetuses suddenly became much more than scientific curiosities. Their deformities seemed so much like those unexplainable human defects. Because they died late in gestation, they echoed all those unexplainable human miscarriages and stillbirths. Could there be a connection? Generoso's experiment seemed to point to something bigger, some answer out there in the mists, a piece not just of the puzzle of birth defects but also of the entire cosmic mystery of development.
Something happened. If Generoso could figure out what went wrong in these fetuses, could he then understand what happens when reproduction goes right? And if he could explain it in the mouse, could he extrapolate that explanation to humans! Perhaps. Scientists study mice because mice are a lot like humans--90% of our two genomes are the same. But mice are, genetically, more compact and efficient. (We humans have a lot of evolutionary detritus in our DNA stringing along between the meaningful bits--the genes.) And because mice reproduce and mature so quickly, scientists can study hundreds of generations in the time it would take to follow one human generation from conception to adulthood.
So it was back to the lab, on a quest for a hypothesis--a theory that might explain the ``something.'' Generoso revamped his original experiment and tweaked the variables--trying different mutagens, exposing the mice for longer and shorter periods after conception. By transferring treated zygotes into untreated surrogate mice mothers, it became clear that the zygote itself was short-circuiting; its development was not hampered by an inhospitable uterus.
As he noodled away at this problem, Generoso came to believe that even if the damage was not caused genetically, genes must still be at fault. He hypothesized that the mutagens were interfering with some crucial, early moments of gene expression, the process that turns genes on and off in the precise order required to build a being.
``When a zygote begins to divide, begins to become an embryo, its development must proceed according to a well-synchronized symphony of gene expression,'' Generoso explains. ``Not all genes can be expressed at the same time--the organism would die. A two-cell stage will not express the gene that it does not need. It will express only the genes it initially needs to kick off a cascade of other gene expressions. Then its function is finished and it switches off.
``It's a very precise coordination of gene expression. And we believe that if you mess up that finely tuned process, you may cause these types of developmental anomalies.''
irst things must come first, and the order of that cascade is crucial. After all, there's no point in making a heart if there are no vessels to pump blood through; there's no point in making an eve without a brain to send messages to. And in those early stages, when the single cell, the double or quadrupled cells, all have the incipient ability to become heart, eye--anything--much can go wrong.
To begin to prove this hypothesis. Generoso tried another experiment. He set out to induce defects with 5-azacytidine, a chemical that is known to affect gene expression selectively-- turning on some but not all, of the genes in the genome. Generoso treated conceptuses at the zygote stage and shortly after, when the first cell divisions were beginning. When the results were in, the similarities were startling; the new chemical produced the same defects. And not only did it induce defects, 5-azacytidine proved particularly effective when applied at the two-cell stage-that point where the cascade of gene expression would have begun independently.
What Generoso's research seems to be saving is that from the moment of conception, the ability of genes to send the right messages at the right time can be ruined. The imprinting code that tells genes when to fire might be rearranged. Cells might not be able to differentiate--to become muscle cells or nerve cells or blood cells. They might not be able to migrate--to make that movement which puts your nose squarely in the middle of your face and your feet on the ends of your legs. They might not be able to make vital morphogens like insulin.
``These processes are altered, or they're not there, or they're there at the wrong time. And this is what happens when you mess up that cascade,'' Generoso explains. ``All of these are very active areas of research right now in molecular biology. And all of this leads us to believe more and more that our hypothesis must he correct--it must be gene expression.''
Now it is time for Generoso to test that hypothesis as rigorously as he can. He plans to test more chemicals, to see if they induce the same defects. He'll soon be able to use a DNA library to go right into the genome, looking for disruption on a molecular level. He hopes in the process to discover new genes--the genes that help a zygote grow from a single cell to complex organism. He also hopes to help prevent the tragedy of unexplained birth defects.
Because, if the mechanism that starts that cascade is, indeed, so extremely sensitive right from the moment of conception--a moment few women are actually aware of--then it is crucial to know what substances might harm it. Generoso hopes his research can accomplish two things; first, provide a test to see which chemicals might cause these defects, and second, provide women with vital information to use when planning a pregnancy.
Just as the acne medication Accutane is not given to women who might be pregnant, just as X-rays are not performed on women who might be pregnant, so may other substances be added to the proscribed list as a result of this work. The first mutagen Generoso used, ethylene oxide, is, in fact, used in sterilization equipment in hospitals. Are health-care workers at risk? Perhaps. Generoso has been asked to testify as an expert witness in ethylene oxide-related suits; so far, he has refused, not wanting to compromise his scientific impartiality.
The question of how chemicals may affect early development is being aired, not in court, but among researchers in toxicology and researchers in early development. For eight months beginning in September 1992, toxicologists and teratologists are sitting down with molecular embryologists and with human embryologists in a series of workshops and a symposium sponsored by the National Institute of Environmental Health Science. These very different researchers have never before joined forces to work on mammalian development, and the unprecedented alliance is due in large part to Generoso's work. Such meetings had been considered for a long time, but Generoso's research ``was one of the initial things that stimulated the beginning of the workshop idea,'' says NIEH's Jack Bishop, who is coordinating the series.
Titled ``Molecular and Cellular Mechanisms of Early Mammalian Development,'' the series is ``putting all these people together in one room and letting hem talk about their work,'' Bishop says. After the two or so years it will take to conduct the meetings, a symposium will summarize the discussions and identify key areas of research in abnormal development--``which problem areas are approachable and solvable, and the general framework of methodologies we'll need to do that.''
The role played by toxic substances is important to Generoso; he is also working with the National Research Council on a series of meetings to try to assess what substances might put newly pregnant women at risk. But his colleagues believe his work has even larger implications. They are looking beyond toxicology to the larger issues of how did we get here?
``This will have implications on our understanding of early development,'' Jack Bishop says. ``One of the major stepping stones to understanding normal development is creating abnormal development. These two aspects go hand-in-hand.''
``The biggest contribution of this research may be n mechanistic terms,'' says Liane Russell. ``We are learning a lot about what gene expression is in the early embryo--what is and is not being expressed. By collaborating with Joe Rutledge, we'll be able to classify these abnormalities and relate them back to humans.''
``It makes sense to study it because it may relate to what we see in humans,'' Rutledge elaborates. ``Many of the abnormalities are exactly analogous, but are they caused by the same mechanism? That's the real question.''
ell, are they? The problem with hypotheses is that they are hypothetical, and Generoso is still seeking a theory that will completely explain his results. His latest experiment only added more twists to the plot: Attempting to define a crucial period of vulnerability for the zygote, he found that it was much more sensitive three days after conception. Between 60 to 73 hours after conception--long after the seemingly crucial period of one to six hours--it took less mutagen--only half as much--to cause these deformities. And the way the zygotes implanted themselves in the uterus was unusual, too.
When he heard of the startling new plot twist, Joe Rutledge flew out to Oak Ridge immediately to see for himself and to gather specimens for further study back in Seattle. Together, the two are striving to make sense of the latest results.
``We're still working toward a hypothesis,'' Generoso said. ``Why are the embryos more sensitive later? Why is there this change in the implantation sites? This is a whole other aspect that we're still trying to sort out. Things are moving and more puzzles are arising.''
It's the typical frustration of scientific research: Just when you think you've got it all figured out, some experimental bombshell sends you back to square one. Perplexed, puzzled, but undaunted, Generoso is pushing on, because he is convinced that he's on to something--something important. Perhaps he will find a way to understand how some of those unexplainable birth defects happen. Perhaps he can help develop simple tests for new drugs and chemicals, so that women planning a pregnancy know what sub- stances to avoid if there's even a chance they're about to conceive.
