Microchimeras and Serotonin

While many of the claims at improving cognition are dubious (eg, the "Mozart effect"), there is now ample reason to suspect that parental involvement in children's brain development occurs much earlier than the first 3 years. Data now suggests that maternal cues are critical to proper brain development long before birth. In this article, I will explore this almost ridiculous example of early parenting, including the strange world of microchimeras and the role maternal serotonin plays in fetal brain development. I will begin with some information about chimeric biology and then move to data involving brain development in laboratory animals.

The interesting biology of microchimeras

In Greek mythology, Chimera was the name of a fantastic fire-breathing female monster who looked as if she were created by a committee. Her front part was usually depicted as a lion, her back end was a dragon, and her middle was a goat. She was a mythological creature of great destructive power.

As genetic and tissue engineering techniques became regular practice, the mixed-up characteristics of the Chimera were co-opted by the research community; they used the name to describe a very valuable—and equally mixed-up—biological phenomenon. Chimeras are now formally defined as any organism, organ, or body part made up of 2 or more tissues of differing genetic composition. This can result from grafting, genetic engineering, or even organ transplantation. The word is even used to describe molecules. Antibodies made from the proteins (or genes) of 2 or more different species, for example, are formally termed "chimeras."

It has become clear that chimeric characteristics are not just epiphenomena from research laboratories and clinics. A number of years ago it was shown that during gestation the human fetus acquires chimeric characteristics. Maternal cells get into the fetus and become an active part of its biology, extending into adulthood. Fetal tissues return the favor, entering the mother before birth and setting up shop. Such trafficking has potentially powerful consequences, from embryonic development to immune system reactivity.

A brief history of chimerism

More than a decade ago, a strange observation was made in umbilical cord blood samples from male infants. About 20% of the samples contained fetal cells. These results called into question the long-held assumption that maternal cells rarely, if ever, passed into fetal circulation. More refined techniques using DNA showed the incidence to be quite common: 40% to 100% of the samples showed the exchange.

Critics argued that the data could be explained by maternal-fetal exchanges during labor. Their arguments were mostly silenced when fetal blood from second- and third-trimester pregnancy terminations revealed the same chi- meric phenomenon. Indeed, exchanges were occurring throughout pregnancy and appeared to be a normal part of gestation.

Beyond that, the phenomenon was shown to be stable, with maternal cells persisting into adulthood. The idea generated a great deal of excitement. As researchers bore down on the phenomenon, it was shown that the exchange could be 2-way—fetal cells were also found to persist in the mother. Microchimerism appeared to be a normal part of pregnancy.

There are many powerful consequences of microchimerism. One is the generation of autoimmune diseases, demonstrated most notably in systemic sclerosis (scleroderma). The phenomenon has also been linked to autism, phenylketonuria, and irritable bowel syndrome.

One of the most intriguing hypotheses, however, has to do with normal development. Why would the fetal-maternal interactions, previously thought so impossible, turn out to be so prevalent? Why doesn't everybody suffer from some kind of immunological meltdown, especially women who bear more than one child? The answer is a bit of a shocker. It is very possible that, immune issues aside, the exchange is critical for the successful transit of a baby through gestation.

Just how consequential this exchange can be to normal growth has only been recently appreciated. There may even be psychiatric consequences. Two examples—one from humans, one from mice—give a strong glimpse into the powerful role cellular exchanges play in normal fetal growth. At least in mice, this may include brain development.

Inherited diabetes

The first example involves human tissues and the investigation of type 1 diabetes mellitus (T1D). T1D is the archetypal pediatric autoimmune disease, and its origins have been under intense investigation for decades.

Some researchers have suspected that the origins of T1D involve microchimeric activity (maternal-to-fetal exchanges), and a group in Seattle decided to investigate.¹ There was ample precedent—maternal cells have been shown to persist in immune-competent children in a wide variety of tissues, including the thymus, skin, thyroid, liver, and heart. They even survive in the peripheral blood of adults.

The researchers first had to develop a bulletproof assay for the detection of maternal cells in the infant. Finding fetal cells in the mother is fairly easy, especially if the fetus is a male. One simply looks for the presence of a Y chromosome. Discriminating maternal cells from fetal cells in the fetus, however, is a harder task. For that you must use quantitative polymerase chain reaction (PCR), assaying for genes that are nontransmissable and maternal-specific.

Although the details of quantitative PCR are beyond the scope of this article, the gist is easy to explain. A person makes snippets of DNA (called "primers") that flank a "target"—a region of DNA the researcher is interested in examining. Then, using an enzyme that works like a tiny movable Xerox machine, millions of copies of the target gene can be created. Under precise conditions, quantitative assessments of the target can be ascertained. If the target is not present in the population of cells being examined, no copies will be made.

The target the researchers used was genes within the human leukocyte antigen (HLA) complex. These proteins are involved in immune recognition and help the body identify self from nonself. The genes for the HLA complex reside on chromosome 6. Because there are nontransmitted, nonshared HLA genes in the complex, it is possible to use PCR to discriminate fetal cells from mater-nal cells in tissues containing mixed populations.

The researchers in the investigation used quantitative PCR with HLA as the target in their investigations of T1D; the pancreas was an obvious first tissue choice. When the researchers examined the pancreas of both affected and unaffected persons, they found evidence of microchimerism.

It was hardly new that maternal cells could penetrate the womb, but what happened once they established themselves in the fetus was a stunner. Subset populations set up shop in the pancreas, turned into islet beta cells, and began pumping out insulin. These cell types even survived into adulthood, composing anywhere from 0.4% to 1% of the total pancreatic population. For reasons not fully explained, this microchimerism occurred more often in patients with T1D than in those without.

This finding was extraordinary because it showed that microchimerism might actually provide a functional benefit. In fact, it has changed the way many of us look at heritability of cellular function. Just how deeply into brain development that involvement might be was discovered next, when a group of French researchers decided to target serotonin.

Serotonin

As you know, mammalian serotonin has many functions. In adults, it participates in the control of GI motility and secretion, appetite, sexual behavior, the perception of pain, and cardiovascular regulation—the list is long and varied. There is growing evidence that serotonin participates in the developmental biology of a wide variety of systems, exerting its effects before the onset of neurogenesis. It has been shown to affect GI and cardiovascular morphogenesis in mice, rats, and chickens, mediating its effects via the 5-HT2B receptor. Serotonin plays an important role in establishing the left-right body axis, placing internal organs in the proper orientation during development. Serotonin even affects eye morphogenesis, mediating its effects once again through the 5-HT2B receptor. Serotonin, perhaps surprisingly, is a powerful developmental morphogen before it is a neurotransmitter.

This role in development came under investigation when the FDA was reviewing new drug applications for fluoxetine (Prozac, Sarafem). A report issued in 1988 demonstrated that the drug affected the embryos of pregnant laboratory animals, creating head, heart, and facial malformations. The report delayed approval of fluoxetine until it could be shown that the medication did not produce such deformities in the human fetus.These facts demonstrated unequivocally the role of serotonin in fetal development, at least in laboratory animals.

The puzzling part in all this is that serotonin biosynthesis has never been detected in either the embryonic or extraembryonic tissues of the developing animal. From where does it get this important signaling molecule? A group of researchers headed by Jacques Mallet decided to find out.

A knockout from knockouts

Jacques Mallet is most famous for his isolation and characterization of tryptophan hydroxylase-1, an enzyme critical for the biosynthesis of serotonin. Serotonin is actually synthesized via a 2-step mechanism involving the amino acid tryptophan. Tryptophan hydroxylase mediates a tetrahydrobiopterin-dependent hydroxylation reaction (quickly followed by a decarboxylation step catalyzed by aromatic l-amino acid decarboxylase, for all interested biochemists). Tryptophan hydroxylase-1 produces the type of serotonin found in blood.

The experiment showing the maternal serotonin involvement in fetal development came from so-called knockout mice (Figure), which have been genetically engineered in such fashion that they develop without a particular genetic sequence, presenting the researchers with a site-specific loss of function. Hints about the functioning of the "knocked out" gene sequence can be obtained simply by noting any observable abnormalities in the functioning or behavior of the animal.

The researchers in Mallet's laboratory used knockout technology to investigate a fundamental mystery in mouse development. They knew the pup did not make its own serotonin until around the third trimester. Since most mouse pups develop normally, where was the serotonin? Was it not needed until then, or did the mother supply the important monoamine until the pup could make its own?

They decided to knock out the tryptophan hydroxylase-1 gene in a female mouse, and allow her to grow to sexual maturity and become pregnant. Any abnormalities associated with the development of her pups would reveal the role maternal serotonin plays in normal development.

Abnormality was exactly what the researchers found. Of the 43 mouse embryos tested, 37 displayed striking abnormalities in systems known to need serotonin. Most dramatic were the abnormalities in the brain. The rhombencephalic regions, the neopallial cortex (which is the future cerebral cortex), alterations in the ventricular tissues, and an overall loss of mitotic activity in wide regions throughout the brain were observed.

For the first time, embryonic development was unambiguously shown to need maternal serotonin to proceed normally. This is quite an achievement.

Conclusions

There are many implications of the work described above. Direct biochemical involvement of a maternal neurotransmitter in fetal development expands the notion of heritability, at the very least. Serotonin is a very small molecule, and it may be that other molecules make it across the placenta as well, perhaps affecting other tissues within the brain, or other tissues outside it. Researchers are investigating how the maternal serotonin gets there in the first place. Others are looking at how it exerts its effects once it arrives.

It must be remembered that these experiments were done only in laboratory animals. The largest questions revolve around whether humans follow a similar mechanism. Mallet certainly thinks so and was quoted in an interview saying, "There is no reason to think that this should not happen in humans. It has to be tested now."² If found to work similarly, it will change the way we look at human fetal development. It will certainly expand the whole idea of parental involvement in early childhood brain development, turning it, if you will forgive me, on its head.

References:

References

1.

Nelson JL, Gillespie KM, Lambert NC, et al. Maternal microchimerism in peripheral blood in type 1 diabetes and pancreatic islet beta-cell microchimerism.

Proc Natl Acad Sci U S A

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2007;104:1637-1642.

2.

Stix G. Selfless giving: mom's brain chemical affects embryonic development.

Sci Am.

2007;296:20-22.