The Visible Embryo News Archive

Transcription Factors May Dictate Differences Between Individual People

Researchers are only beginning to understand how individual variation in gene regulation can have a lasting impact on one’s health and susceptibility to certain diseases. Now, an ambitious survey of the human genome has identified differences in the binding of master regulators called transcription factors to DNA that affect how genes are expressed in different people.

The study, published in the March 18, 2010, issue of Science, looked at two common transcription factors. HHMI medical research fellow Maya Kasowski and her colleagues in the laboratory of molecular biologist Michael Snyder at Yale University conducted the work with Jan Korbel at the European Molecular Biology Laboratory. Snyder has since joined the faculty at Stanford University.

Transcription factors account for as much as 10 percent of the coding genome in humans and other organisms. When activated, transcription factors switch on or off hundreds or thousands of genes, a cascade that programs cells to grow or divide.

“The activity of transcription factors determines what a cell is doing at any given moment,” says Kasowski, who was a medical student at Yale when she received her HHMI medical research fellowship. She has since decided to pursue an M.D./Ph.D. degree.

Despite their large numbers and critical role, until now, no one had looked at whether there was any variability in the targets of transcription factors from one person to the next. The current study found a “number of differences between individuals” in the binding sites of two transcription factors, Snyder says.

Transcription factors bind to the human genome within areas of the genome still viewed as a black box - vast stretches of DNA sequence between known genes. Gradually, biologists have found that much of this DNA performs a vital function - helping turn genes on and off in specific situations. Some of the regulatory regions, known as binding regions, serve as handholds for transcription factors.

“We know there are differences in gene expression between people." says Dr. Kasowski. Understanding the differences in how genes are regulated could help us understand human diversity. But identifying the regulatory DNA that controls expression is much more difficult than looking for differences in the regions of the genome that code for genes.”

For the current study, Kasowski, Snyder, and their colleagues examined two important transcription factors: RNA polymerase II and NFkappaB. RNA polymerase II, which is active in all cells, transcribes DNA into RNA. NFkappaB is activated by stress, plays a key role in immune responses to infections, and has been implicated in several diseases, including cancer.

The team mapped every binding region for these two factors inside the genomes of 10 individuals. To do so, they deployed a new technology that uses chemicals to freeze transcription factors as they bind to the genome. The scientists then sequenced the segment of DNA to which the transcription factor bound. After the team combined the data from all 10 individuals, they found around 19,000 binding regions for RNA polymerase II and another 15,500 binding regions for NFkappaB.

They discovered that the number of transcription factors binding at the different sites often varied near different genes, which in many cases influenced how much of the gene was expressed.

Variation in transcription factor binding can help explain why one person may make more of a certain gene product than another, Snyder says. Among any two individuals, the team found that 25 percent of the RNA polymerase II binding regions varied in time or frequency, as did 7.5 percent of the NFkappaB binding regions.

Closer examination of these variable binding regions showed that single-letter differences in the genome - called SNPs - accounted for some of the difference in transcription factor binding. To be exact, in some individuals, a single letter change at a certain binding region influenced the likelihood that the transcription factor would or would not bind there. “We found that differences in DNA sequence contributed to differences in transcription factor binding,” Kasowski says. “The more SNPs we found in a particular binding region, the more variation in binding we saw.”

Larger differences in the genome, called structural variation, also accounted for a number of the differences in transcription factor binding. Structural variation happens when large segments of the genome are deleted, duplicated, or inverted. It varies widely among humans, and the role of such variability in human biology is not well understood.

But the new study shows that SNPs and structural variation can either increase or decrease transcription factor binding, and subsequently, the amount of a protein that gets made from a particular gene.

“We found that about one third of the differences in binding was caused by SNPs and structural variation,” Snyder says. “This is the first time anyone has shown that SNPs and structural variation affect large number of regulatory elements that control gene expression. Normally, people look at differences in the gene themselves rather than in the regulatory regions, because they are difficult to identify.”

The study also reports differences in binding of RNA polymerase II and NFkappaB near genes implicated in many major diseases, including type 1 diabetes, lupus, chronic lymphatic leukemia, schizophrenia, asthma, Crohn’s disease, and rheumatoid arthritis. “Variation in the regulation of genes might eventually help account for some of the varying susceptibility to diseases we see in the population,” Kasowski says.

In addition to looking at humans, Kasowski, Snyder and their colleagues looked at transcription factor binding for a single chimpanzee. The study shows that 32 percent of RNA polymerase II binding regions differed between the humans in the study and the chimp.

Snyder says that he included the chimp out of curiosity to see how transcription factor binding might account for differences between ourselves and our closest genetic cousin. But the 32 percent difference between chimps and humans was not that much larger than the 25 percent difference in RNA polymerase II binding found among two individuals.

Still, Snyder says that the study opens a new genomic frontier for biologists. “Only about two percent of our DNA codes for genes,” he says. “Studying the rest of the genome, including gene regulation and transcription factors, is the next wave in understanding human variation.”

Stem Cells Used to Model Infant Birth Defect

Findings reveal why a longstanding treatment works, and suggest better approaches.

Hemangiomas - strawberry-like birthmarks that commonly develop in early infancy - are generally harmless, but up to 10 percent cause tissue distortion or destruction and sometimes obstruction of vision or breathing. Since the 1960s, problematic hemangiomas have been treated with corticosteroids such as dexamethasone or prednisone. But steroids have considerable side effects, don't always work, and their mechanism of action in hemangioma has remained a mystery.

Researchers at Children's Hospital Boston recently discovered that infantile hemangiomas originate from stem cells, and have used these stem cells to better understand this tumor in the laboratory. In the March 18 issue of The New England Journal of Medicine, they show that steroids target hemangioma stem cells specifically, reveal their mechanism of their action and suggest other possible ways to halt and shrink hemangiomas.

Hemangiomas, affecting 4 to 10 percent of infants, are noncancerous tumors consisting of a tangled mass of blood vessels. Previously, it was assumed that steroids act on endothelial cells, which make up about 30 percent of cells in the tumor. The new research, led by dermatologist Shoshana Greenberger, MD, PhD, working in the lab of Joyce Bischoff, PhD, in Children's Vascular Biology Program, shows that steroids interfere with a much rarer and more primitive cell type - hemangioma stem cells.

Steroids target hemangioma stem cells, not the endothelial cells that are the major cell type in blood vessels. When pre-treated with steroids and suspended in a gel, hemangioma stem cells formed no blood cells when injected into mice (left). When the progenitors of endothelial cells were treated in the same way, there was no effect; blood vessels formed just as they do in cells not treated with steroids (right).

Greenberger and Bischoff further showed that steroids work by inhibiting hemangioma stem cells' ability to stimulate blood vessel growth, and that they do so by shutting down production of a specific factor called vascular endothelial growth factor (VEGF-A). VEGF is well known as a stimulator of angiogenesis (blood vessel growth) in cancer and age-related macular degeneration.

"We now have more therapies targeting VEGF, so our findings open the way to finding a more specific and safer therapy for hemangioma," says Greenberger.

Steroids usually result only in stabilization of hemangioma growth, and about 30 percent of hemangiomas don't respond to steroid treatment. Steroids also have side effects including facial swelling, hyperactivity, growth retardation and increased blood pressure. Although the effects on appearance may seem minor, research indicates that a baby's physical appearance can interfere with maternal bonding.

"My dream has always been to give a drug to stop hemangioma at its first appearance," says Children's plastic surgeon John Mulliken, MD, co-director of Children's Vascular Anomalies Center and a co-author on the study.

Greenberger, Bischoff and colleagues worked with hemangioma stem cells isolated from patient tissue samples provided by Mulliken, and showed that:

• When human hemangioma stem cells were pretreated with dexamethasone, then implanted in mice, the tumors that formed had far fewer blood vessels.
• Dexamethasone suppressed the stem cells' production of VEGF-A, but did not suppress VEGF-A production by endothelial cells from the same hemangioma.
• When VEGF-A production was suppressed in hemangioma stem cells using shRNA silencing, then implanted in the mice, there was an 89 percent reduction in vessel growth.
• VEGF-A was detected in actively growing hemangiomas, but not in regressing (involuting) hemangiomas.

This image demonstrates the presence of vascular endothelial growth facter (VEGF-A), shown in green, in an actively growing hemangioma -- and shows that it's not found in the endothelial cells, the major cell type in blood vessels, but in the interstitial space outside the blood vessels, so is presumably made by the hemangioma stem cells.

Earlier research in Bischoff's lab and that of Bjorn Olsen, MD, PhD, of the Harvard School of Dental Medicine, indicates that hemangiomas may result from an in utero mutation in a stem cell destined to become an endothelial cell, causing a disruption in the normally well-ordered process of blood vessel development.

Under a 2008 Translational Research Program grant from Children's, Bischoff's lab has been using hemangioma stem cells to test a library of existing medications that might specifically inhibit the proliferation of the hemangioma stem cells, and thereby limit growth of the hemangioma tumor.

"Steroids are inhibiting expression of a central regulator of blood vessel growth: VEGF-A," says Bischoff. "But we'd like to target the stem cell itself - stop its proliferation, prevent it from differentiating into unwanted blood vessels and, at the same time, eliminate the cellular source of VEGF-A."

The study was funded by the National Institutes of Health, the Translational Research Program at Children's Hospital Boston, a Harvard Skin Diseases Pilot Study Grant, Sheba Medical Center (Israel), and the John Butler Mulliken Foundation.

Citation: Greenberger S, Boscolo E, Adini I, Mulliken J and Bischoff J. Corticosteroid suppression of VEGF-A in infantile hemangioma-derived stem cells. N Engl J Med 2010 Mar 18; 362(11):30-38.

Pregnancy Helps Liver?

Pregnancy boosts the regenerative capacity of the liver in mice, a finding that may shed light on a process entirely separate from pregnancy -- aging, researchers report in a study published this week in Genes and Development.

The findings are "really unexpected," said Nikolai Timchenko, who studies liver regeneration and aging at Baylor College of Medicine in Houston, Texas.

He noted that the researchers identified a specific regeneration mechanism present only during pregnancy and harnessed the relevant pathway to boost liver regeneration in aging mice. "I think from a molecular point of view this is the major discovery of this work," he said.

"The loss of regenerative capacity of tissues with age is one of the main characteristics of aging," Timchenko, who didn't participate in the work, wrote in an email. Scientists have known for many years that old mice lose the regenerative capacity in their livers, but little about the molecular mechanisms underlying this process, he added. "Impaired liver regeneration and high rate of mortality after surgical resections in old patients are [the] two most significant problems which are discussed on each meeting of liver biology."

Yehudit Bergman, a molecular biologist studying epigenetics in embryonic stem cells at The Hebrew University of Jerusalem, and her colleagues were interested in studying tissue regenerative capacity. They began with an observation published five years ago that the regenerative capacity of muscle progenitor cells in an older animal can be boosted by parabiosis -- an experimental technique in which the older animal's circulatory system is surgically linked to a younger animal, allowing it to benefit from the younger animal's systemic environment.

Musing that pregnancy might be called a kind of parabiosis, the researchers tested the ability of the liver to regenerate after removing two thirds of it in older (10-12 month old) pregnant and nonpregnant mice. They observed a striking difference - the liver regenerated to only about half its original volume in nonpregnant animals, but it grew back to 96 percent its normal size in pregnant ones. Nine out of 19 older nonpregnant mice died after the surgery, compared to just two out of 22 of the pregnant group. In contrast, younger animals (3 months old) showed robust regeneration whether they were pregnant or not.

The researchers assumed this bounce-back was caused by liver cells proliferating during pregnancy, but a marker for dividing cells showed almost no proliferation in the liver between the second and third day after the surgery -- when proliferation is highest in nonpregnant animals. Rather than dividing, the cells that were present seemed to be growing larger. The results revealed that "in pregnancy the mechanism by which liver regeneration occurs is very different than the mechanism in nonpregnant mice," Bergman said.

They then linked this effect to a signaling pathway called Akt/mTORC1, which is known to play a role in cell growth. When they blocked the pathway, the cell growth they had observed was eliminated. Meanwhile, proliferation got a small boost, but not enough to spur significant regeneration.

"Once we knew that in pregnancy the model of regeneration is different, and we also knew that in aged mice liver regeneration is suffering, we thought, ok, maybe we can persuade aged mice to behave like pregnant mice," Bergman said. They tested the effect of a compound that activated the Akt/mTORC1 signaling pathway in old mice, between 18 and 24 months of age, and again removed two-thirds of their livers. None of the nine treated mice died after partial hepatectomy while four out of nine untreated mice died.

"I'm not sure how important this observation is, and whether it has clinical implications," said Douglas Schmucker, an expert in the liver and aging at the University of California, San Francisco, who was not involved in the work. Although he agreed that the observed effects were "pretty startling," he noted that it's not clear whether cell growth is equivalent to cell proliferation in how well regenerated liver will function. "Basically...are fatter, old cells better than thinner new cells?" he asked. Also, he said, if the effects are partly maternal, as the study suggests, it's not clear that men would be benefited by an approach based in this signaling pathway.

Bergman noted that in their experiments, the liver appeared to be working normally, but agreed that the findings are extremely preliminary. Her group is now working on pinning down the molecular mechanism of the effect. "There must be something in pregnancy that induces this pathway," she said. The researchers are also trying to determine whether pregnancy has a similar regenerative effect on other organs.

Influencing Stem-Cell Development Using Geometry

University of Chicago scientists have successfully used geometrically patterned surfaces to influence the development of stem cells.

The new approach is a departure from that of many stem-cell biologists, who focus instead on uncovering the role of proteins in controlling the fate of stem cells.

"The cells are seeing the same soluble proteins. In both cases it's the shape alone that's dictating whether they turn into fat or bone, and that hasn't been appreciated before," said Milan Mrksich, Professor in Chemistry and a Howard Hughes Medical Institute Investigator, who led the study.

"That's exciting because stem-cell therapies are of enormous interest right now, and a significant effort is ongoing to identify the laboratory conditions that can take a stem cell and push it into a specific lineage."

The UChicago team found that making cells assume a star shape promotes a tense cytoskeleton, which provides structural support for cells, while a flower shape promotes a looser cytoskeleton.

"On a flower shape you get the majority of cells turning to fat, and on a star shape you've got the majority of cells turning into bone," said Kris Kilian, a National Institutes of Health Fellow in Mrksich's research group.

The University of Chicago team published its findings in the March 1 Early Edition of the Proceedings of the National Academy of Sciences. Citation: "Geometric cues for directing the differentiation of mesenchymal stem cells," Proceedings of the National Academy of Sciences, March 1 Early Edition, by Kristopher A. Kilian, Branimir Bugarija, Bruce T. Lahn and Milan Mrksich.

Mrksich cautioned that the method is far from ready for use in the harvest of stem cells for therapeutic use, but it does signal a potentially promising direction for further study. Mrksich's research group has a long history of developing methods for patterning surfaces with chemistry to control the positions, sizes and shapes of cells in culture, and applying those patterned cells to drug-discovery assays, and studies of cell migration and cell adhesion.

Flame Retardants, PCBs & Pesticides Found in Blood of Young Girls

Banned chemicals - present in amounts higher than levels found in recent years in US adults - are turning up in the blood of young girls being studied in California and Ohio.

Researchers from Kaiser Permanente Northern California, UCSF and the California Department of Public Health (CDPH) led the study to measure blood levels of the chemicals. They assessed levels of PCBs, DDT and related pesticides, and flame retardants in nearly 600 girls, ages six to eight. These chemicals can act like hormones. Scientists and public health experts want to know how they might affect children as they develop through adolescence and into adulthood.

The main difference found between girls in Ohio and California was that the California girls had higher levels of all three classes of chemicals in their blood, on average.

The potentially hormonally active fire retardants measured in the study are called polybrominated diphenyl ethers (PBDEs). They have been banned in Europe, but they are still used in products sold in the United States to help meet fire safety standards. Products containing PBDEs include foam upholstery, textiles and home furnishings.

In the United States, PCBs were used in transformers and industrial applications until 1979, when their use was banned. DDT and related pesticides also are banned but persistent. In addition, these pesticides are still used on crops in Mexico.

The study identified additional differences in average blood levels of chemicals among the girls. Black girls had significantly higher levels of PBDEs in their blood in comparison to white girls. In general, levels of PCBs and pesticides were significantly lower among girls born to less educated mothers. Mexican-American girls had the highest levels of pesticides in their blood. Chemical levels also tended to be lower in girls who were obese.

The research report is in press and already available online in the scientific journal Environmental Research. The findings are among the first to be reported from a long-term, multi-site collaboration called the Breast Cancer and the Environment Research Centers (BCERCs). The project is funded primarily by the National Cancer Institute and the National Institute of Environmental Health Sciences.

The main exposures to these chemicals occur in utero or through breast feeding, according to the study authors, but additional exposures occur during childhood.

”This study demonstrates that measurable levels of chemicals that might interfere with normal hormonal pathways are present in the blood of girls during the time of breast development,” says Robert Hiatt, MD, PhD, director for population sciences for the UCSF Helen Diller Family Comprehensive Cancer Center and lead scientist for the Bay Area BCERC.

“The next step is to determine if we can detect any evidence that these chemicals have effects on pubertal development at the levels we have detected them.”

‘Cause for Concern’
According to study leader Lawrence Kushi, ScD, associate director for epidemiology at Kaiser Permanente Northern California, these findings indicate that exposures to these chemicals are widespread. “Although we don’t know what the health implications of these exposures are, the fact that they are found in almost all of our study population suggests some cause for concern,” Kushi says.

Childhood and especially adolescence are thought to represent a window of time when girls might be especially vulnerable to environmental exposures that can influence breast cancer risk in later years. This idea stems from lab studies of animals and population studies of women who had received radiation treatment when they were girls. Reproductive factors — including early age of first menstruation, late age of menopause and delayed childbearing — already are known to increase a woman’s risk for breast cancer.

That’s why cancer researchers are interested in identifying influences on reproductive development of young girls. When they entered the study, the girls had not yet begun to experience the hormone-driven changes that eventually lead to puberty and adolescence.

The procedure used in the study to prepare and analyze blood samples is the same one used by the Centers for Disease Control and Prevention (CDC), and the samples were analyzed by the same CDC laboratories. Every two years for the past decade, the CDC has conducted “biomonitoring” surveys to measure levels of chemicals in the blood and urine of people across the nation.

According to the study authors, “Our study provides data that was previously lacking on body burdens of hormonally active agents in young children from a large, racially diverse population using the most sensitive assays now available.”

The study was led by Gayle Windham, PhD, chief of the Epidemiology Surveillance Unit at the Environmental Health Investigations Branch of the CDPH, along with Kushi, and Hiatt. These three were joined in the research by additional collaborators from the University of Cincinnati, Cincinnati Children’s Hospital Medical Center, the US Centers for Disease Control and Prevention, and the CDPH.

Amniotic Fluid Cells More Efficiently Reprogrammed to Pluripotency Than Adult Cells

May Fill Need for Easily Reprogrammable Cell Type.

In a breakthrough that may help fill a critical need in stem cell research and patient care, researchers at Mount Sinai School of Medicine have demonstrated that skin cells found in human amniotic fluid can be efficiently “reprogrammed” to pluripotency, where they have characteristics similar to human embryonic stem cells that can develop into almost any type of cell in the human body.

The study is online now and will appear in print in the next issue of the journal Cellular Reprogramming (formerly Cloning and Stem Cells), to be published next month.

The Mount Sinai researchers found that when compared to cultured adult skin cells, the amniotic fluid skin cells formed stem cell colonies in about half the time and yielded nearly a 200 percent increase in number. Reprogramming fetal skin cells also cuts significantly the cost of generating patient-specific induced pluripotent stem cells when compared to reprogramming other cell types.

“There remains today a need in stem cell research for an easily reprogrammable cell type,” said the study’s lead author, Dr. Katalin Polgar, Assistant Professor of Medicine, Cardiology and Obstetrics, Gynecology and Reproductive Science, Mount Sinai School of Medicine. “Our study shows that reprogramming of cultured, terminally differentiated amniotic fluid cells results in pluripotent stem cells that are identical to human embryonic stem cells, and that it is much easier, faster and more efficient than reprogramming neonatal and adult cells.”

Amniotic fluid skin cells can be safely obtained from pregnant women undergoing amniocentesis at about 15 weeks of pregnancy as part of a diagnostic workup for chromosome aberrations and other genetic diseases. About 99 percent of cells found in amniotic fluid are terminally differentiated cells mostly from fetal skin, which are shed into the amniotic fluid as a fetus develops. Since these cells can be reprogrammed to pluripotency more efficiently than other cell types, they could be an important source for generating stem cells for basic research and future therapies and may be used to study and potentially cure fatal embryonic diseases with prenatal, perinatal gene therapy.

“We induced amniotic fluid skin cells to return from their final differentiated stage back to an undifferentiated stem cell stage from where they can develop into any cell type of the body,” said Dr. Polgar. “Amniotic fluid cells work much better than any other cell types when turning back their ‘internal clock.’

These cells can potentially be used as a model system in studying different regenerative therapies for diseases of the heart, liver, kidney, lung, pancreas, as well as for replacement of lost neurons in Alzheimer’s, Parkinson’s, even for cancer vaccines. They may also be used for future personalized stem cell banks. As the pluripotent stem cells induced from amniotic fluid skin cells are the patient’s own cells, there is no risk of immunorejection or teratocarcinoma formation.

“Additionally, stem cells reprogrammed from amniotic fluid skin cells could be used for drug discovery in disease models,” added Dr. Polgar. “Their potential use in toxicology models could reduce the need for experimental animals. Developing cell lines from individual amniotic fluid samples can accelerate the development of existing targets for different diseases. This all will bring new opportunities to explore innovative therapeutic models or targets in regenerative personalized medicine.”

The scientists were able to genetically reprogram the amniotic fluid skin cells using the four transcription factors (proteins that regulate the transcription of genes) OCT3/4, SOX2, KLF4, and c-MYC. After reprogramming, the cells were found to be identical to human embryonic stem cells in numerous ways, including for morphological and growth characteristics, antigenic stem cell markers, stem cell gene expression, and telomerase activity, in vitro and in vivo differentiation.

“These reprogrammed amniotic fluid cells are able to form, as embryonic stem cells can, three dimensional spheroid structures called ‘embryoid bodies.’ They also have the ability to self-renew themselves indefinitely. Pluripotent stem cells created from amniotic fluid cells shed from the fetal skin maintain all the potential of embryonic stem cells without using embryos, thereby eliminating ethical concerns associated with human embryonic stem cells obtained from preimplantation embryos,” Dr. Polgar said.

Brain Abnormalities Found in Children Exposed to Meth in Utero

Knowing the pattern of damage could help with diagnosis of affected children.

It has long been known that alcohol exposure is toxic to the developing fetus and can result in lifelong brain, cognitive and behavioral problems. Now, a new report out of UCLA shows that the effects of prenatal methamphetamine exposure - or worse, a combination of methamphetamine and alcohol - may be even more damaging.

Reporting in the March 17 issue of the Journal of Neuroscience, UCLA professor of neurology Elizabeth Sowell and her colleagues used structural magnetic resonance imaging (sMRI) to show for the first time that individuals whose mothers abused methamphetamine during pregnancy, with or without alcohol abuse, had structural abnormalities in the brain that were more severe than those seen in children whose mothers abused alcohol alone.

The researchers identified the brain structures that are vulnerable in such exposure, which may help predict particular learning and behavioral problems in methamphetamine-exposed children.

"We know that alcohol exposure is toxic to the developing fetus and can result in lifelong problems," said Sowell, the study's senior author. "In this study, we show that the effects of prenatal meth exposure, or the combination of meth and alcohol exposure, may actually be worse, and our findings stress the importance of seeking drug-abuse treatment for pregnant women."

In particular, said Sowell, a structure in the brain called the caudate nucleus, which is important for learning and memory, motor control, and punishment and reward, was one of the regions that was more reduced by methamphetamine than alcohol exposure.

Of the more than 16 million Americans over the age of 12 who have used methamphetamine, about 19,000 have been pregnant women, according to 2002–04 data from the National Survey on Drug Use and Health.

"About half of women who say they used meth during pregnancy also used alcohol," Sowell said, "so isolating the effects of meth on the developing brain was difficult."

The researchers overcame this challenge, she said, by recruiting women who abused alcohol but not methamphetamine during pregnancy and compared them to the children with exposure to both drugs, and to a group that was not exposed.

The neuroscientists evaluated the specific effects of prenatal methamphetamine exposure by comparing brain scans of 61 children, whose average age was 11. Of these, 21 had prenatal methamphetamine and alcohol exposure, 13 had heavy alcohol exposure only and 27 were unexposed. Structural magnetic resonance imaging showed that the sizes and shapes of certain brain structures varied depending on prenatal drug exposure.

Previous studies have shown that certain brain structures are smaller in alcohol-exposed children. In this study, the authors found that these brain regions in methamphetamine-exposed children were similar to those in alcohol-exposed children and that in some areas they were even smaller. Some brain regions were larger than normal. For example, an abnormal volume increase was noted in methamphetamine-exposed children in a region called the cingulate cortex, which is associated with control and conflict resolution.

"Either scenario — smaller or larger growth — could be a bad thing in kids with prenatal drug exposure," Sowell noted. "There are enormous developmental changes that take place during adolescence. These drugs are likely altering the trajectory of development, and clearly not in a good way."

This brain imaging may also aid in treatment. Because the researchers were also able to predict a child's past exposure to drugs based on imaging and IQ information, detailed data about vulnerable brain structures may eventually be used to diagnose children with cognitive or behavioral problems, even when well-documented histories of drug exposure are not available.

"The tragedy here is that all these developmental problems are 100 percent avoidable," Sowell said. "The important message is to urge drug abusing women to seek treatment during pregnancy."

Other authors on the paper included Alex D. Leow, Susan Y. Bookheimer, Lynne M. Smith, Mary J. O'Connor, Eric Kan, Carly Rosso, Suzanne Houston, Ivo D. Dinov and Paul M. Thompson, all of UCLA.

The research was supported by the National Institute of Drug Abuse, the National Institute on Alcohol Abuse and Alcoholism, and the March of Dimes. Additional support was provided by the National Center for Research Resources, the General Clinical Research Center, and the National Institutes of Health.

Female Sex Chromosomes Regulate Blood Pressure

Male mice born with female sex chromosomes experience hypertension seen in postmenopausal women

Researchers at Georgetown University Medical Center (GUMC) have determined that something in female sex chromosomes appears to trigger a rise in blood pressure after the onset of menopause. This finding challenges the current belief that sex hormones are largely responsible for regulating blood pressure.

Reported online Monday in Hypertension, this work is the first of its kind and involves male mice engineered to have female (XX) sex chromosomes, and female mice with male (XY) chromosomes. The findings suggest that sex chromosomes regulate blood pressure. Most researchers have thought that sex hormones (estrogen and testosterone) play key roles in controlling blood pressure and that women develop hypertension after reaching menopause because of loss of estrogen.

"Up until now, it has been impossible to separate the influence of sex chromosomes from the effects of sex hormones, and in this paper, we have shown for the first time that sex chromosomes are impacting blood pressure - independent of sex hormones," says the study's lead investigator, Kathryn Sandberg, PhD, director of the GUMC Center for the Study of Sex Differences in Health, Aging, and Disease.

"That is not to say sex hormones don't matter in blood pressure regulation, because they do, but we now know they aren't the only players," she says. "Estrogen likely works to protect against hypertension, but once the hormone is depleted, something is unmasked on female XX chromosomes that allows blood pressure to rise."

Men have two different sex chromosomes (an X from their mother and a Y from their father) while women have just X (two XX chromosomes, one from each parent).

But only one gene – the all-powerful Sry gene – on the Y chromosome makes a man a male because it dictates development of male testes.

Women are the result when an Sry gene is absent resulting in the development of ovaries, Sandberg says.

In utero, males and females are bathed in a soup of associated sex hormones, also dictated by the presence or absence of the Sry gene, which leads to development of the male and female hair, breasts, and genitalia.

In this study, Sandberg's team studied mice in which the Sry gene was deleted from the Y chromosome resulting in XY females. These mice were females because they were born with ovaries (which is what occurs when the Sry gene is missing) and exposed to estrogen in utero. They also studied XX males in which the Sry gene was put on one of the other 22 non sex chromosomes in the genome, which then dictated development of testes and testosterone. "The Sry gene just needs to be present in the genome. It does not have to be on the Y chromosome to create testes," Sandberg says.

The researchers could now compare between the different mice. For example, the only difference between the XX and XY females, who developed in utero with the same hormonal environment, is the difference in the sex chromosomes. The same is true for XX and XY males. Researchers could also compare the role that hormones played in blood pressure regulation between XX females and XX males, and XY females and XY males.

"Its a two-by-two analysis," Sandberg says. "If we find a difference between XX females vs. XY females that is also found in XX males vs. XY-males then we can ascribe it to a sex chromosome effect and not a sex hormone effect."

And that is exactly what they did find in regard to blood pressure. "XX mice have a greater magnitude of hypertension than XY mice regardless of whether they are male or female," she says.

"That means there is something encoded in the sex chromosomes that is separate from hormonal influence that is impacting blood pressure in a significant manner," Sandberg says. "Researchers have found sex chromosome effects in some areas of brain and immune system function, but no one before has looked at this in the cardiovascular system."

Sandberg says there could be three possible explanations for why this is occurring.

One is known as "escape from X-inactivation." In each XX cell, only one X chromosome is functioning, but it is known that some genes from the "silent" second X chromosome are not actually silenced. "These genes are known, and it is the same set of genes that escape X-inactivation, including in the XX males," Sandberg says. "We think there may be something special about these genes that escape X-inactivation and which are only expressed in XX cells."

Another possible explanation is conflict between cells that are expressed from different parental X chromosomes - one from the father and one from the mother. While each cell in a female expresses only one X, that X could come from either the male or female parent. Conflict can arise when a cell expressing a maternal X is adjacent to a cell expressing a paternal X. "That can drive an immune response because the two X's may recognize their own molecular self differently," she says.

The third theory is that the Y chromosome contains a gene or genes that protects against hypertension, and that "since postmenopausal women don't have that added benefit from those Y genes, once the beneficial effects of estrogen are gone, blood pressure rises."

"These very exciting findings deserve much more research into what is encoded within the sex chromosomes that affects blood pressure control," Sandberg says.

If researchers can zero in on what that particular mechanism is, it may be possible to design a therapy to stop postmenopausal rise in blood pressure, Sandberg says.

"There is a real jump in blood pressure and incidence of hypertension in menopausal women, and while the condition is treatable, blood pressure in many of these women is not fully under control making them far more susceptible to cardiovascular and kidney disease and stroke. Therefore, it would be wonderful to have specific therapies that target the root cause of this hypertension."

One Gene Lost = One Limb Regained?

Mammals appear to be able to regenerate limbs through a single gene deletion.

A quest that began over a decade ago with a chance observation has reached a milestone: the identification of a gene that may regulate regeneration in mammals.

The absence of this single gene, called p21, confers a healing potential in mice long thought to have been lost through evolution and reserved for creatures like flatworms, sponges, and some species of salamander. In a report published today in the Proceedings of the National Academy of Sciences, researchers from The Wistar Institute demonstrate that mice that lack the p21 gene gain the ability to regenerate lost or damaged tissue.

Unlike typical mammals, which heal wounds by forming a scar, these mice begin by forming a blastema, a structure associated with rapid cell growth and de-differentiation as seen in amphibians. According to the Wistar researchers, the loss of p21 causes the cells of these mice to behave more like embryonic stem cells than adult mammalian cells, and their findings provide solid evidence to link tissue regeneration to the control of cell division.

“Much like a newt that has lost a limb, these mice will replace missing or damaged tissue with healthy tissue that lacks any sign of scarring,” said the project’s lead scientist Ellen Heber-Katz, Ph.D., a professor in Wistar’s Molecular and Cellular Oncogenesis program. “While we are just beginning to understand the repercussions of these findings, perhaps, one day we’ll be able to accelerate healing in humans by temporarily inactivating the p21 gene.”

Heber-Katz and her colleagues used a p21 knockout mouse to help solve a mystery first encountered in 1996 regarding another mouse strain in her laboratory.

MRL mice, which were being tested in an autoimmunity experiment, had holes pierced in their ears to create a commonly used life-long identification marker. A few weeks later, investigators discovered that the earholes had closed without a trace. While the experiment was ruined, it left the researchers with a new question: Was the MRL mouse a window into mammalian regeneration?

The discovery set the Heber-Katz laboratory off on two parallel paths. Working with geneticists Elizabeth Blankenhorn, Ph.D., at Drexel University, and James Cheverud, Ph.D., at Washington University, the laboratory focused on mapping the critical genes that turn MRL mice into healers.

Meanwhile, cellular studies ongoing at Wistar revealed that MRL cells behaved very differently than cells from “non-healer” mouse strains in culture.

Khamilia Bedebaeva, M.D., Ph.D., having studied genetic effects following the Chernobyl reactor radiation accident, noticed immediately that these cells were atypical, showing profound differences in cell cycle characteristics and DNA damage. This led Andrew Snyder, Ph.D., to explore the DNA damage pathway and its effects on cell cycle control.

Snyder found that p21, a cell cycle regulator, was consistently inactive in cells from the MRL mouse ear.

P21 expression is tightly controlled by the tumor suppressor p53, another regulator of cell division and a known factor in many forms of cancer. The ultimate experiment was to show that a mouse lacking p21 would demonstrate a regenerative response similar to that seen in the MRL mouse. And this indeed was the case.

As it turned out, p21 knockout mice had already been created, were readily available, and widely used in many studies. What had not been noted was that these mice could heal their ears.

“In normal cells, p21 acts like a brake to block cell cycle progression in the event of DNA damage, preventing the cells from dividing and potentially becoming cancerous,” Heber-Katz said. “In these mice without p21, we do see the expected increase in DNA damage, but surprisingly no increase in cancer has been reported.”

In fact, the researchers saw an increase in apoptosis in MRL mice – also known as programmed cell death – the cell’s self-destruct mechanism that is often switched on when DNA has been damaged. According to Heber-Katz, this is exactly the sort of behavior seen in naturally regenerative creatures.

“The combined effects of an increase in highly regenerative cells and apoptosis may allow the cells of these organisms to divide rapidly without going out of control and becoming cancerous,” Heber-Katz said. “In fact, it is similar to what is seen in mammalian embryos, where p21 also happens to be inactive after DNA damage. The down regulation of p21 promotes the induced pluripotent state in mammalian cells, highlighting a correlation between stem cells, tissue regeneration, and the cell cycle.”

The study was supported by grants from the Harold G. and Leila Y. Mathers Foundation, the F.M. Kirby Foundation, the W.W. Smith Foundation, the National Institute for General Medical Sciences and National Cancer Institute.

You Are What Your Gut Bacteria Want to Eat

“It could be the case that bacteria are involved in obesity in a way that’s transmissible between people.”

It reads like a plot straight from the pages of a science fiction novel: Hordes of bacteria infect mice and cause the rodents to develop voracious appetites. The ill-fated mice grow fat, their troubles compounded by insulin resistance, high cholesterol, and high blood pressure. To make matters worse, the microscopic troublemakers can move from mouse to mouse, spreading poor health habits to any rodent unlucky enough to play unwitting host.

A study published online March 4, 2010, in Science Express, revealed just such a scenario in real life, suggesting that gut microbes might one day be grouped along with inadequate exercise and overeating as a cause of obesity and metabolic syndrome, both of which shorten life expectancy.

Rob Knight, a Howard Hughes Medical Institute Early Career Scientist at the University of Colorado, Boulder, conducted some of the complex analyses that led to the discovery that gut microbes may help alter the behavior of mice. “It could be the case that bacteria are involved in obesity in a way that’s transmissible between people,” Knight said.

To study the impact of gut microbiota on obesity, researchers led by Matam Vijay-Kumar of Emory University created mice without a gene for an immune system protein called toll-like receptor 5. TLR5, normally prominent in the gut lining, recognizes the protein that forms the flagella of invading bacteria and helps target the pathogens for destruction.

Without the TLR5 gene, mice develop metabolic syndrome, a group of obesity-related abnormalities that increase the risk for type 2 diabetes and heart disease. Metabolic syndrome is on the rise in the United States, and some estimates indicate that one in four Americans may be affected by it.

The mice without the TLR5 gene ate 10 percent more than other mice. Those mice also had a more severe response to a high-fat diet, and showed signs of insulin resistance and metabolic syndrome - even when their caloric intake was restricted. When researchers transferred the gut microbiota from the TLR5-deficient mice into the guts of normal mice, these mice also developed the signs of metabolic syndrome: overeating, insulin-resistance, high blood lipid levels, and elevated blood glucose.

The experiments showed that the behavior and metabolic transformation in these animals came from within - driven by shifts in the bacteria population in their guts. When the mice were administered a broad-spectrum antibiotic, which wiped out much of their gut bacteria, their behavior and other symptoms returned to normal.

Knight’s lab has developed methods to analyze microbial diversity and its impact on health, characterized by the changing microbe populations in the mice, in collaboration with Ruth Ley at Cornell University.

An individual animal’s gut can contain hundreds of species of bacteria, and many of these refuse to grow when removed from their favorite environment. Rather than attempt to culture and characterize the thousands of bacteria, Knight and colleagues focused on their DNA. Specificly, the 16S-rRNA genes in the sample.

After amplifying the 16S rRNA, Knight used a sequencing technology called pyrosequencing to identify each genetic sequence, and tagged it with a “barcode” created by Micah Hamady, a graduate student in his lab. Using a software program called UniFrac, developed in Knight’s lab, the researchers examined the differences between many versions of the 16S rRNA genes.

“This allows you to use the universal tree of life as a ruler and measure the differences between each community,” Knight said. Their analysis indicated that the relative abundance of 116 different groups of closely related bacteria was altered significantly in the mice that overate compared to normal mice.

The next step in this research will be to determine whether gut microbes affect humans in a similar manner. Lay published work in Nature in 2006 showing a change in gut microbes accompanies obesity in humans - but that research did not show a cause-and-effect relationship.

The scientists hope to determine how often microbe activity manipulates behavior, as it did in the overweight mice. The phenomenon is already a well-documented side effect of infection with Toxoplasma gondii, the parasite that causes toxoplasmosis.

Rats and mice infected with T. gondii show no sign of fear when they smell the urine of cats –a deadly predator. When the rats’ behavior turns them into cat food, the parasite has the carnivorous host it requires.

The diversity of the microbes in humans may also hold secrets about human variation. “We’re 99 percent the same on a DNA level, but our microbiota are 80 to 90 percent different,” Knight said.

Knight points to weight reduction studies that consistently show only modest average improvements, even though a few study members respond dramatically. Maybe microbes can help explain the difference, he said.

Molecule Tells Brain Cell: Grow Up and Get To Work!

Stanford University School of Medicine scientists have now identified a molecular master switch that catalyzes oligodendrocytes brain cells to transition into mature, myelin-making cells. This may have implications for medical treatment, as defects in the myelin maturation process have been observed in multiple sclerosis and the most common adult brain cancer, gliomas.

About four out of every 10 cells in the brain are oligodendrocytes. These cells produce the all-important myelin that coats nerve tracts, ensuring fast, energy-efficient transmission of nerve impulses. Mixed among them are precursor cells that are destined to become oligodendrocytes when needed but remain immature, and in an undifferentiated state between stem cell and adult oligodendrocyte.

In a study published March 10 in Neuron, investigators found the molecule known as miR-219, a microRNA, at high levels only in oligodendrocytes, and that it is needed to induce undifferentiated precursor cells to become functioning adult cells.

“The mechanism responsible for this shifting of anatomical and behavioral gears from precursor to fully functioning oligodendrocyte was a mystery,” said Ben Barres, MD, PhD, professor and chair of neurobiology and the study’s senior author. “Finding this switch has allowed us to ferret out several of the molecules it acts on inside cells. And that in turn could open the door to new approaches to treating diseases where oligodendrocyte precursors’ failure to mature appropriately plays a role.”

Biologists have puzzled over how cells in one state switch seamlessly into another state. Shifts imply switching on and off entire banks of genes whose protein products determine a cell’s shape, activity and contribution - beneficial or otherwise - to our overall health.

RNA molecules are normally thought of as messengers that convey instructions from DNA in the nucleus of animal and plant cells to the surrounding watery zone inside the cell where molecular machines “read” the messenger RNA’s nucleic-acid sequences assembling proteins accordingly.

Unlike messenger RNA, microRNA molecules are very short strings of RNA that don’t contain instructions for making proteins. While a messenger RNA molecule has to be fairly lengthy to hold all the information necessary for generating a complete protein, a microRNA molecule plays a different role entirely.

In the same way that two DNA strands form a coordinated double strand when their nucleic-acid sequences are complementary, a microRNA molecule can bind to messenger RNAs when those messenger RNAs’ sequences complement its own. The result is that the messenger RNA’s sequence can no longer be read by the cell’s protein-manufacturing apparatus. The binding of microRNA to messenger RNA can even trigger the destruction of the bound complex.

However, not every bit of a microRNA molecule’s sequence has to match its opposite number on a messenger RNA molecule in order for the two to bind. Pairing relies on the recognition of an ultra-short “seed” sequence within the microRNA molecule.

“So a single microRNA can affect the activity levels of hundreds of different messenger RNAs,” said the study’ first author, Jason Dugas, PhD, a research associate in Barres’ laboratory.

In these experiments, Dugas, Barres and their colleagues showed that impairing all microRNA production in cells fated to become oligodendrocytes produced both behavior defects in live laboratory mice and clear anatomical defects (lack of proper myelination) in brain slices from these mice.

When the scientists induced oligodendrocyte precursor-to-adult transition in normal cells using the McManus lab’s technology, and checked for microRNAs levels - the amount of one, miR-219, increased by 100-fold. That finding has been confirmed in another laboratory that will also publish soon on the subject.

Stained brain sections revealed that miR-219 is largely restricted to the brain’s white matter - its myelinated regions - making it an excellent biomarker for oligodendrocytes. Researchers also found that delivering a synthetic copy of miR-219 to oligodendrocyte precursor cells deficient in all microRNA generation - and therefore incapable of maturing to myelin-producing oligodendrocytes - at least partially rescued those cells’ ability to mature.

Moreover, knocking out miR-219 in the oligodendrocyte precursor cells prevented them from maturing normally. Adding miR-219 to normal oligodendrocyte precursors in culture, without inducing their differentiation by standard growth-factor withdrawal, increased up to fourfold their likelihood of converting to adulthood.

Finally, the investigators were able to identify several distinct messenger RNAs that were inhibited by miR-219. These messenger RNAs encode proteins that both maintain precursors’ proliferative potential and prevent them from becoming full-fledged oligodendrocytes.

“In addition to potential importance for stimulating remyelination in multiple sclerosis, we’re especially excited about our findings’ potential significance for glioma, the most common adult brain tumor,” Barres said. “There hasn’t been any really good treatment for these tumors, in which precursor cells start dividing and dividing and don’t differentiate. Why are these cells behaving so abnormally? Maybe this microRNA switch has been downregulated or shut down entirely. Perhaps by introducing miR-219 back into glioma cells, we may actually be able to stop them from behaving like tumor cells.” Barres said his lab has entered into a collaboration with another group to test this idea.

The study was funded by the Myelin Repair Foundation. Other Stanford co-authors were Anja Scholze; Adiljan Ibrahim; Ben Emery, PhD; Jennifer Zamanian, PhD; and Lynette Foo, all of the medical school’s Department of Neurobiology.