Embryonic Research: It's More Than Just Cloning

As some researchers pursue cloning and stem cell work, attracting media attention along the way, others concentrate on embryonic research that will help produce healthier babies. From esoteric work on genetic control mechanisms to studies of fetal nutrition, human genetics, and the effects of toxic substances on the fetus, scientists are trying to formulate a fuller picture of what occurs in utero. For example, scientists like Dennis Thiele and colleagues in the biological chemistry department at the University of Michigan Medical School, are investigating the genetic cues in development. Courtesy of Cynthia BearerCynthia Bearer Thiele's team is studying processes affected by the cell membrane and have identified a gene essential for copper transport into the embryonic cell, Ctr1. "A lot of people don't realize that copper is important for the appropriate neuropeptides—neuropeptide Y and other neuropeptides," says Thiele. "Our connective tissue, our skin, and our tendons have to be mature, and it's a copper-dependent enzyme that does that." Mammals without this copper transporter die during gestation, he says. Using knockout mouse models, Thiele and colleagues found that embryos lacking both copies of the Ctr1 gene did not survive;1 their neural tubes did not close before they died, indicating neural development deficits. Normally, the copper transporter Ctr1 picks up copper ion in the plasma membrane. Research led by Jonathan Gitlin at the Washington University School of Medicine, St. Louis, shows that the copper is then handed off to metallochaperone ATOX1, which carries it from the cytoplasm to the cellular organelles, where it is used.2 In the future, this research may well affect dietary advice given to expectant women. "What it might suggest," says Thiele, "[is] that pregnant mothers are often prescribed iron supplements, and we know that the ability to utilize iron absolutely depends upon the appropriate amount of copper in the diet. Without copper, the iron transport machinery can't work." Thiele states that, while he is not advocating copper supplementation for pregnant women, "it's something to look at." Mom's Diet Really Matters Biochemist William Rees, of The Rowett Research Institute, Aberdeen, Scotland, and fellow researchers in the European Union (EU) are studying how the mother's diet affects fetal growth and childhood disease susceptibility, as well as the genetic mechanisms involved. Rees is looking at amino acid use in utero because, he notes in a recent paper,3 "Epidemiological studies of human populations show that poor growth in utero predisposes an individual to the later development of Type 2 (non-insulin-dependent) diabetes mellitus and hypertension in adulthood." The initial hypothesis was that a protein-deficient diet limited the fetus' growth because it decreased the amino acid supply, explains Rees. His team presumed that the lack of specific amino acids would lead to overexpression of certain genes. "In fact, these responses are not initiated by feeding the low protein diet, and gene expression is reduced rather than increased," Rees says. In studies on rats, researchers found that the mother can mobilize her body proteins to maintain the supply for most amino acids, but diets that disturb the metabolism of sulfur-containing amino acids, especially methionine, may have extensive negative effects. While protein synthesis is not affected, the transfer of methyl groups to methylate DNA is. "We are now looking at genes which control the cell cycle," says Rees. "Because these genes are also regulated by methylation, it is possible that the diets are leading to epigenetic programming of fetal development." To further elucidate the roles of maternal nutrition and genetic factors on fetal development, Rees and his colleagues are participating in an EU program called, "NUTRIX." This program will fund research on animal models of nutrition, a retrospective study on human fetal development and nutrition, and related studies. A particular concern is whether adult diseases, such as Type 2 diabetes and cardiovascular disease, are programmed in utero and whether they are related to the expectant mother's diet. "Once the outcomes are known," says Rees, "then there is the possibility to identify potential therapeutic interventions." Other researchers are studying environmental effects on the fetus, from toxins, such as heavy metals and pesticides, to drugs of abuse. Clinical psychologist Lynn T. Singer, Case Western Reserve University School of Medicine in Cleveland, is studying how lung diseases that occur in preterm babies (those born prior to the end of the 37th week) affect brain development. The smallest and youngest of such infants develop bronchopulmonary dysplasia; however, with improved medical care, these babies, who would otherwise die, survive. Moreover, about 20% are mentally retarded according to Singer. "There's a kind of lowering of overall intelligence in the group." Singer also studies how a mother's illicit drug use affects the fetus. Her group has found that in babies exposed to cocaine in utero, "the amount of cocaine exposure was related to the baby's head circumference, even after controlling for all other factors."4 Head circumference is a gross measure of brain growth. Singer and colleagues in the UK have recently received a National Institutes of Health grant to study the effects of the illegal drug methylenedioxymethamphetamine (MDMA or "ecstasy") on prenatal development. In Europe, MDMA has been used extensively during the past decade; and the effects have been studied. "The issue is, what recovery can you get from various fetal insults and what are the long-term outcomes, because if we can identify what the insults are, we may be able to change the environment," Singer says. Children facing such exposures, and who might develop neurological deficits, could benefit from physical therapy, extensive stimulation, and other environmental interventions. Improving Tests Neonatologist Cynthia Bearer, a colleague of Singer, is developing what Bearer hopes will be better tests for fetal toxic exposure: tests on meconium, which is the first fecal discharge from the infant after birth. Meconium begins to accumulate in the uterus from week 13, so it could conceivably supply an extensive track record of various fat-soluble environmental chemical exposures. Bearer explains that meconium is a better sampling source than other biological specimens. Hair samples are not good because neonates are usually hairless and, if they do have hair, mothers usually won't let it be collected. Urine, umbilical cord blood, and neonatal breath tests only indicate exposure to chemicals that turn over rapidly in the bloodstream. The umbilical cord itself has not been of much value in tests. There is heavy maternal contamination of the placenta, so it may not accurately reflect fetal exposure. Transdermal testing would only indicate exposure immediately before birth. Bearer, who has tested meconium for alcohol metabolites to gauge if expectant women were drinking heavily (14 drinks per week or more) and thus allowing their children to face the risk of fetal alcohol damage, says that this test may aid in identifying children suffering from alcohol damage years earlier than it is usually diagnosed, allowing for interventions that may aid these children and increase brain synaptic development before they reach school age. Couresty of Philip LandriganPhilip Landrigan Physician Philip Landrigan, director of the Center for Children's Health and the Environment at New York's Mt. Sinai School of Medicine, notes that in 2000, Congress appropriated funding for a longitudinal study, known in the vernacular as "the Pediatric Framingham Study," to look at the long-term effects of toxic chemical exposure on childhood development, primarily of the brain.5 "The plan would be to enroll mothers when they come into the [obstetrician]," says Landrigan. He expects that mothers would sign an informed consent form, give hair and blood samples, and, at childbirth, medical staff would take samples of umbilical cord blood. He also expects that the children would be evaluated periodically for neural and endocrine development, and would be followed for about 25 years. Landrigan's group is engaged in a pilot project for this study, and has enrolled more than 500 families over the last four years. Bearer sees a role for meconium sampling in the proposed longitudinal study. "My dream would be [that] meconium becomes the standard for newborn screening," says Bearer. "It's not invasive, you get a large sample, [and] you can probably do DNA from it." She is collaborating with the Centers for Disease Control and Prevention, which can conduct more sensitive tests on the meconium than her laboratory can, and is working with a group funded by the Environmental Protection Agency and the National Institute of Environmental Health Sciences to develop a meconium test for environmental pollutants. Myrna Watanabe is a freelance writer in Patterson N.Y. References 1. J. Lee et al., "Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development," Proceedings of the National Academy of Sciences (PNAS), 98[12]:6842-7, June 5, 2001. 2. I. Hamza et al., "The metallochaperone Atox1 plays a critical role in perinatal copper homeostasis," PNAS, 98[12]:6848-52, June 5, 2001. 3. W.D. Rees, "Manipulating the sulphur amino acid content of the early diet and its implications for long-term health," Proceedings of the Nutrition Society, (In press). 4. L.T. Singer et al., "Effects of cocaine/polydrug exposure and maternal psychological distress on infant birth outcomes," Neurotoxicology and Teratology, (In press). 5. P.J. Landrigan, "Children's environmental health: Lessons from the past and prospects for the future," Pediatric Clinics of North America, 48[5]:319-30, October 2001. For information about NUTRIX, visit www.biol.ucl.ac.be/nutrix/; for information about the EU's Research Framework V, which supports NUTRIX, visit www.cordis.lu/fp5/home.html. Learning About Membrane Trafficking Raffi Aroian, a molecular geneticist/developmental biologist at the University of California, San Diego studies the role of certain genes in early development of the nematode Caenorhabditis elegans. Active genes in the earliest developmental stages (at cleavage from the one- to two-celled stage) may determine whether the embryo will continue developing. Before the first cell division, the embryo begins to develop polarity. Afterwards, the two resulting cells differ in size. "The bigger, unequal cell makes most of the hypodermis and the nervous system and the small cell makes the mesoderm and endoderm," explains Aroian. Aroian's team wanted to find the genes that made this asymmetrical cleavage. They discovered that worm embryos lacking a gene for one of the actin-binding proteins, which they named pod-1 for "polarity and osmotic defective genes," did not survive. The researchers have since found additional pod genes1 that are related to membrane trafficking and cell permeability. For example, C. elegans produces tiny eggs covered with an impermeable shell. But eggshells with a pod defect are permeable. pod-deficient embryos also show "polarity defects that are independent of those permeability defects," says Aroian. Regarding these genes' connection to mammalian development, Aroian notes that related genes, called par (partitioning defects resulting in the asymmetrical development in the one-, two-, and four-celled embryonic stages), produce proteins in C. elegans' epithelial cells. par, which performs the same function in Drosophila and mammalian epithelial cells, are responsible for cell polarization. These genes may play a major role in membrane trafficking as do pod genes. "It's already been shown that the same pathway that polarizes the C. elegans embryo polarizes neuroblast cells in Drosophila and mammals," says Aroian. — Myrna E. Watanabe A. Tagawa et al., "pod-2 along with pod-1, defines a new class of genes required for polarity in the early Caenorhabditis elegans embryo," Developmental Biology 233:412-24, May 15, 2001.

Embryonic Research: It's More Than Just Cloning

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