Sizing up the brain: mutations that produce small brains may reveal how human intelligence evolved.

In the 1960s, Pakistan built a mammoth dam on the river Jhelum to generate electric power and store water for irrigation. Known as Mangla, the dam created an upstream lake that displaced about 20,000 families from the district of Mirpur. Around the same time, England's textile industry was facing a major shortage of skilled laborers, especially in the county of Yorkshire. Many of the people from Mirpur who were displaced by Mangla traveled to Bradford and other Yorkshire districts.

The coincidental timing of the dam's construction and Yorkshire's need for workers has, nearly 4 decades later, provided scientists with insight into how the human brain develops and, possibly, into how it evolved from the smaller brains of our hominid ancestors. A few years ago, a physician from St. James' University Hospital in Leeds, England, noticed something unusual among the Pakistani families he examined at a Bradford clinic. "I was seeing a lot of children who had microcephaly with moderate mental retardation but no other disease features," recalls clinical geneticist C. Geoffrey Woods.

Microcephaly is a rare condition characterized by an abnormally small head, the result of an undersized brain. In particular, the cerebral cortex--the layers of nerve cells that cover the brain's surface and are the seat of higher reasoning--is shrunken. "The cerebral cortex is the part of the brain that, for better or worse, makes us human," notes Christopher A. Walsh, a Howard Hughes Medical Institute (HHMI) investigator at Harvard Medical School in Boston. "Children who have abnormal development of the cerebral cortex fail to achieve the kind of talents we pride ourselves on, such as language."

Intrigued by his patients with microcephaly, Woods began studying the DNA of Pakistanis with the condition and their unaffected relatives. He and his colleagues gradually realized that there isn't just one gene that causes microcephaly when mutated; there are at least six genes. This year, the researchers identified two of the genes, including one responsible for microcephaly in about half of the nearly 60 Pakistani families studied to date.

These two genes, as well as another one studied by Walsh's group (see box, page 314), are shedding light on how the cerebral cortex forms. Moreover, by pinpointing genes that seem to regulate the size of the cerebral cortex, the scientists have set the stage for studies into what genetic changes produced the rapid expansion of the cerebral cortex as primates, including humans, evolved. Indeed, one of the human-microcephaly genes is dramatically different from its counterparts in much simpler animals such as worms and mice.

"I suspect there may be many things that contribute to the increased size of the primate cortex, but the microcephaly genes are a fascinating beginning," says Martin Raff of University College London, who studies regulation of nerve cell division.

BRAIN'S BLACK BOX Containing billions of nerve cells, the cerebral cortex is the largest structure of the human brain. Essentially a flat sheet not much thicker than an orange peel, the cortex folds and refolds into the familiar deep creases of the brain's surface. The cerebral cortex varies in size dramatically among species. It "mostly grows by becoming a larger sheet rather than a thicker sheet," says Walsh.

The human cortical surface area is about 1,000 times greater than that of the mouse, for example. And compared with the cortex of the chimpanzee, our closest living relative, the human cerebral cortex has three to four times more surface area. Some scientists attribute the greater intelligence of modern humans to the rapid expansion of the cerebral cortex as hominids evolved.

In microcephaly, the cerebral cortex grows unusually slowly and reaches a size no bigger than that of early hominids. Various circumstances, such as prenatal infections or a mother's alcohol abuse, can produce microcephaly, but they usually also generate other physical abnormalities. In so-called primary microcephaly, the small brain and head are the only obvious physical defects. The brain's basic architecture is preserved, albeit in a smaller form. In such cases, the child or adult is mentally retarded but has no other apparent neurological problems, such as seizures.

Primary microcephaly typically occurs when a mother and a father each pass on a mutated copy of a gene that controls brain size. Since among the families that migrated to England from Mirpur, cousins often married each other, so the chances that a baby would have two mutated copies of a gene increased. That explains the abundance of microcephaly cases that Woods saw.

In work led by Andrew P. Jackson of St. James' University Hospital in Leeds, the investigators tracked down one microcephaly gene by focusing on two Pakistani families that had an unusually large number, seven in total, of members with microcephaly. In the July American Journal of Human Genetics, Jackson, Woods, and their colleagues report finding mutations in a novel gene in affected family members but not in unaffected ones. They named the gene microcephalin and, by studying human and mouse fetal tissue, showed that the gene is active in the cerebral cortex as it develops before birth.

The protein encoded by microcephalin has several features seen in other proteins, but how the molecule regulates size of the cerebral cortex remains unclear. "Unfortunately, the gene doesn't have a specified function yet. It's been a bit of a black box," says Woods.

Also murky is whether the gene played an evolutionary role in the expansion of the cortex. The DNA sequences of the mouse and human gene do differ, but not in a way that offers an obvious explanation for the brain sizes of the two species. "We need to sequence the primate versions of microcephalin to get a better handle" on the gene's evolution, says Woods.

IQ TEST The gene microcephalin so far accounts for the small-brain disorder in only a few of the Pakistani families. A much more provocative story has emerged from the second reported identification of a gene for primary microcephaly. It's the human version of a gene called asp, which stands for abnormal spindle. Originally studied in fruit flies, this gene encodes a protein associated with cell division. When a cell divides, two networks of fibers form, each one pulling a set of chromosomes into one of the two daughter cells. In flies with asp mutations, these networks, called spindles, don't work as well as normal, and the overall rate of cell division is lowered.

In the October Nature Genetics, Woods and his colleagues report that in about half of the Pakistani families studied, the members with microcephaly had mutations in both copies of the human version of asp. While the fly gene is active throughout the insect body, the human and mouse version turns on just in the fetal brain, specifically in progenitor cells of the cerebral cortex. Woods speculates that the gene's protein somehow keeps these cells dividing. If the protein is missing or mutated, the progenitor cells may not divide as often as normal, leading to an undersized cortex.

When comparing the fly, worm, mouse, and human versions of asp, the researchers noticed something remarkable. The proteins encoded by each gene have multiple copies of a stretch of amino acids called an IQ domain--the name derives from the scientific notation for two amino acids, isoleucine (I) and glutamine (Q), present in the domain. The number of IQ domains in the protein differs considerably from one species to the next. The worm, fly, mouse, and human proteins have 2, 24, 61, and 74 IQ domains, respectively.

"The protein is physically larger in species with larger brains," he says. "As your IQ gets higher, you have more and more of these IQ repeats." This is particularly odd, adds Walsh, given that no other known protein has more than five or six IQ domains.

Although this discovery suggests that as asp encoded more and more IQ domains species evolved to have larger and larger brains, the scenario puzzles Woods. "It's difficult at the moment to make a model of why the number of IQ domains should affect the size of the brain," he admits.

To examine the evolutionary questions surrounding this microcephaly gene, the investigators intend to deactivate the mouse version and see whether that produces animals with small brains. They may also replace the mouse version with the human gene and observe whether big-brained rodents result.

Furthermore, Woods and Walsh have started to sequence the asp gene in chimpanzees. "If they have exactly the same gene as us, then, while it's clearly important for brain development, it isn't the step that has made our brain three times bigger than higher primates," says Woods.

Bruce Lahn, an HHMI investigator at the University of Chicago, is looking for genes that drove human-brain evolution. He agrees that the newly identified microcephaly genes cry out for further study. Still, Lahn cautions against prematurely crediting the genes for the human brain's impressive cerebral cortex.

"Very little, if anything, is known about the genetic basis of brain evolution. It's a complete blank slate,"he says. "It's not too far out to speculate that evolution may have played on these genes to select for a larger brain. The caveat is that there are many such genes. It takes thousands, if not tens of thousands, for the brain to develop properly."

RELATED ARTICLE: Wrinkle in mind: genetically engineered mice develop enlarged, humanlike brains.

If a human brain and a mouse brain were sitting next to each other, the first thing you would notice is the difference in size. The second most obvious difference is that folds and furrows mark the surface of the human brain, while the surface of the mouse brain is smooth. The wrinkled portion of the human brain is the cerebral cortex, the multilayer region responsible for making sense of all the information streaming into a person's head. In people, nonhuman primates, and other mammals with relatively large brains, the cerebral cortex's convolutions permit its large surface area to cram inside the skull.

Christopher A. Walsh of Beth Israel Hospital in Boston compares the process to crumpling up a large sheet of paper so that it fits inside a small container. "As the cortex gets to a certain size, [it] starts developing these wrinkles. The bigger the cortex gets, the more wrinkles it gets," he says.

In the July 19 Science, Walsh and his colleague Anjen Chenn, now at Northwestern University School of Medicine in Chicago, described genetically engineered mice that develop cerebral cortexes with greatly increased surface area, so much so that the mouse brains have a more humanlike, wrinkled appearance. "It looks as if these wrinkles don't require any special genetic tricks. It seems to be a passive response to having a brain that's bigger than your head," says Walsh.

To create the mice, the two researchers modified a gene encoding a protein called beta-catenin. This protein had drawn their interest because it regulates cell division in many tissues. In some cases of brain cancer, for example, beta-catenin drives unchecked cell proliferation. Adding to Chenn and Walsh's interest, the beta-catenin gene is active in the pool of brain-progenitor cells in which the nerve cells of the cerebral cortex originate.

In normal circumstances, beta-catenin breaks down quickly in cells once enzymes attach phosphate groups to the protein. Chenn and Walsh altered the protein's gene so that it encodes a version of beta-catenin lacking some sites to which the phosphate groups stick. "It's broken down much more slowly," says Walsh.

The resulting beta-catenin accumulation seems to enable the cerebral cortex's progenitor cells to divide extra times before maturing into nerve cells, ultimately increasing the size of the cortex. The mutant mice had brains two to three times as big as normal. "It shows how you can regulate the size of the cortex fairly dramatically by controlling one simple [genetic] switch," notes Walsh.

The large-brained mice didn't survive past birth, but the researchers don't know why. More recently, Walsh's research group tweaked the beta-catenin gene so that the mouse brains enlarge to just about 40 percent greater than normal. These rodents survive and seem healthy, although they're a bit more aggressive than normal. The researchers haven't yet evaluated whether these big-brained mice are smarter than the average mouse.

Walsh now plans to investigate whether beta-catenin plays a role in human conditions marked by abnormally large or small brains. He's also curious about whether the cerebral cortex-size differences among species may stem, at least in part, from varying activity of the beta-catenin gene. "That's a much harder question" to answer, he says.--J.T.