How do you make a mountain out of a mole hill?

That's what Harvard Medical School (HMS) Assistant Professor of Pathology Li-Huei Tsai has been trying to find out in her studies of brain development.

To date, it remains a mystery to scientists how a few cells differentiate to form the billions of cells and connections which make up the brain.

"This area is entirely a black box," Tsai says.

But a new form of mutant mouse engineered by Tsai and her colleagues at HMS is starting to unravel what is considered to be one of the greatest enigmas in biology.

Tsai and her team have recently isolated a gene in mice, called p35, which appears to play an integral part in the formation of the cortex.

To elucidate the gene's effects, Tsai used a recently-developed technique which allows researchers to selectively create "knockout mice"--mutant mice who are lacking in a specific gene. The researchers can then study the effect the loss has on the mutants by recovering the excited gene for study.

The technique is said to be invaluable in studying genetics.

The p35 mutants, which were seemingly normal at first, became consistently abnormal when grown.

Tsai says the mice lacking the gene were "not as active," were uncoordinated and walked unsteadily. Although they were still fertile, the mothers "displayed nurturing defects."

But most importantly, Tais says, the mice "were especially sensitive to suffering fatal seizures."

When the mutants' brains were sectioned and inspected, Tsai found that "the cortex [was] pretty much messed up."

Instead of the cells' elegantly moving into their correct spots, the process seemed to proceed in the dark, with cells going to the wrong places and inadvertently bumping into each other.

When finished, the normal six layers which should be present in the cortex were absent and "the distribution of cells seemed random at first," Tsai says.

The precise nature of the disruption gradually came to light. The mutation seemingly altered the genetic "sign posts" which allow the cells to orient themselves and travel to the correct places.

The mutation caused younger cells to stop and not pass the older neurons. A gradient of increasingly older neurons toward the outside of the cortex resulted, which, according to Tsai, "effectively inverted the pecking order of the cortex."

Another pattern of disruption occured in the wiring patterns that neurons form with each other.

Cracking the Mystery

The cerebral cortex, an intricately folded layer of neural tissue which is much larger and more complex in humans than in any other animal, is thought to be responsible for many of the so-called "higher brain functions"--such as the reasoning and logic capabilities which are unique to humans.

The cortex is arguably the greatest piece of engineering known. Scientists from all fields--ranging from embryology to computer science--strive to learn how its structure participates in the developmental program of our genes.

During embryonic development, millions of neurons mature over a period of a few days. Through a process which is not well-understood by scientists, these neurons grow to form the intricate connections that will become the cortex--which is organized into a pattern of six distinct layers.

"This process amazes me, but, in terms of molecular control, no one has the faintest idea what is going on," Tsai says in a newsletter.

Research Implications

In the mutant mice, the corpus callosum--the bundle of fibers which connects the two hemispheres of the brain--was almost non-existent. Additionally, the general pattern of connections in the rest of the cortex seemed to be highly altered.

The results of the finding indicate that p35 is an essential part of a proposed signaling pathway that has been thought to guide neural development.

"This mutant provides the first evidence that such a signaling pathway exists," Tsai says.

Other findings can now be put into the context of this discovery, painting a picture of how neurons orient themselves according to molecular signposts--using the molecules as signals to either turn or stop.

Although Tsai's research has focused solely on the brains of mice, she says the results are not unique to that species.

Tsai has found that the deficiencies in p35 mice closely mimic those found in several human neural disorders, such as lissencephaly, which produces the same type of seizures found in the mice and periventricular heterotopia and double cortex--which are caused by incorrect migration of cortical neurons.

Although all the evidence is not in yet, Tsai hints that her current work is showing that there are strong ties between p35 and these degenerative diseases, raising hopes that more effective treatments for them might be found in the future.