Cholesterol Gets 'Thumbs Up' For Role In Digit Development

Although cholesterol has a bad rap as the sticky, fatty substance responsible for clogging arteries, Vanderbilt University Medical Center researchers recently found that the attachment of cholesterol to an important developmental protein controls the development of fingers and toes in mice. Without cholesterol, mice developed extra digits, as well as digits in the wrong places.

The new study published online in the Proceedings of the National Academy of Sciences (PNAS) last week helps to clear up some of the conflicting data about cholesterol's controversial role in limb development, said senior author on the study, Chin Chiang, Ph.D., associate professor of Cell and Developmental Biology.

The developmental protein at work here, named Sonic hedgehog after the video game character, was discovered in the early 1990s and shown to have important roles in patterning the developing embryo, including proper digit patterning.

Chiang led the early studies showing that mice without Sonic hedgehog developed only a single digit — a thumb on the front paw (or a “big toe” on the back paw).

The Sonic hedgehog protein is produced by a specialized group of cells located at the posterior part of the developing limb bud, which eventually develops into the pinkie finger or toe. At the site of its synthesis, Sonic hedgehog concentrations are high. It then diffuses out across the developing limb bud, and the declining concentrations (or gradient) of the protein dictate the identity of the other digits.

“Questions have remained about what regulates the Sonic hedgehog gradient,” said Chiang. “And we've been working on that for a number of years.”

One clue about this regulation came when other researchers discovered Sonic hedgehog's rather unusual requirement — the protein had to have a cholesterol molecule attached to work properly.

“In fact, Sonic hedgehog is the only protein known to be modified by cholesterol,” Chiang said.

Because cholesterol is typically found in cell membranes and thought to 'tether' proteins to cells, scientists speculated that cholesterol might inhibit the movement of Sonic hedgehog through the developing tissue. This unique modification might explain why concentrations of the protein were high at the site of its production and then tapered off with increasing distance from the synthesis site.

But previous studies in mice suggested that cholesterol promoted the movement of Sonic hedgehog, a counterintuitive proposal given cholesterol's supposed tethering ability.

To try and clear up cholesterol's role in digit patterning and the Sonic hedgehog gradient, Chiang and colleagues created mice with an altered form of the Sonic hedgehog protein to which cholesterol cannot attach.

They found that mice lacking cholesterol-modified Sonic hedgehog developed with malformed and ectopic, or out of place, digits. The second, or “index,” digits were stunted and misshapen, appearing more similar to a thumb than a normal second digit.

The researchers also examined mice in which only half of their Sonic hedgehog proteins could attach to cholesterol. Those mice developed normal digits 2 through 5 (index through pinkie), but had duplication of these digits anteriorly.

The findings suggested that Sonic hedgehog without cholesterol traveled further than normal, triggering the anomalous digit duplications.

Chiang and colleagues confirmed this microscopically, showing that the protein spread out more evenly across the limb bud (a weaker gradient) in mice lacking cholesterol-modified Sonic hedgehog compared to normal animals.

“We found that, without cholesterol, Sonic hedgehog moves more readily, far from its site of synthesis, all the way to the anterior part of the limb bud where it is normally never detected,” Chiang explained.

When Sonic hedgehog travels to tissue where it normally would be absent (as it does when cholesterol is missing), extra digits may form — a condition known as polydactyly.

Although the causes of polydactyly in humans are not fully understood, mutations in some part of the Sonic hedgehog signaling pathway are high on the list of suspects.

In addition to limb deformities, errors in Sonic hedgehog signaling are involved in a number of other human conditions including cancer and a condition known as holoprosencephaly, a congenital malformation of the forebrain.

Chiang is currently examining the role of cholesterol-modified Sonic hedgehog in the developing brain and spinal cord.

“We are finding some surprises,” Chiang said, “suggesting that the function of cholesterol is different in these different tissues.”

The continued study of the wide-ranging actions of Sonic hedgehog promises to expose the incredible secrets of the developing embryo and could provide clues for preventing devastating birth defects.

Yina Li, Huimin Zhang and Ying Litingtung, Ph.D., were co-authors on the paper. The research was supported by grants from the National Institutes of Health and the March of Dimes Foundation.