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Chin Chiang and Ying Litingtung are studying the genes that regulate limb development and control digit pattern and identity. (photo by Dana Johnson)

VUMC team pinpoints genes that dictate five fingers

BY: LEIGH MACMILLAN

The skeletal structure of the forelimb (arm) in normal and genetically modified mice. The limb and hand — with digits 1 through 5 — of a normal mouse are shown in panel a. When Sonic hedgehog is absent (panel b), mice have stunted limbs and only one digit. When Gli3 is missing (panel c) or both Sonic hedgehog and Gli3 are missing (panel d), mice have full limb skeletons and more than five digits.  Mice with one copy of Gli3 in the absence of Sonic hedgehog (panel e) have an intermediate number of digits. (Courtesy of Chin Chiang and Nature)

8/30/2002 - The skeletal structure of the forelimb (arm) in normal and genetically modified mice. The limb and hand — with digits 1 through 5 — of a normal mouse are shown in panel a. When Sonic hedgehog is absent (panel b), mice have stunted limbs and only one digit. When Gli3 is missing (panel c) or both Sonic hedgehog and Gli3 are missing (panel d), mice have full limb skeletons and more than five digits. Mice with one copy of Gli3 in the absence of Sonic hedgehog (panel e) have an intermediate number of digits. (Courtesy of Chin Chiang and Nature)

As a little girl, Ying Litingtung was fascinated by the animals at the zoo — in particular by their fingers and toes. “I can remember being struck by the fact that even though the animals are very different from us, many of them have five fingers or five toes, just like we do,” said Litingtung, Ph.D., a postdoctoral fellow in the laboratory of Chin Chiang, Ph.D., associate professor of Cell & Developmental Biology.

She never imagined that she would end up studying the very genes that dictate the five-fingered hand.

Litingtung, Chiang, and colleagues from the University of Wisconsin reported this week in the journal Nature that two genes called Sonic hedgehog and Gli3 work together to regulate the number of digits and their identity. Mice that are missing both of these genes end up with between six and 11 digits per limb, and all of them are the same type of digit.

The results were a surprise, Chiang said, because previous studies had suggested that the gene Sonic hedgehog was important for both the formation of the limb skeleton and for its patterning. When Sonic hedgehog alone is missing, mice have stunted limbs with incomplete skeletal elements and only a single digit.

But when the investigators deleted another gene, Gli3, along with Sonic hedgehog, instead of having stunted limbs, the mice had complete limb skeletal elements and extra fingers and toes.

“It was assumed that Sonic hedgehog was important for skeletal formation and digit patterning, but its precise role was not clear,” Chiang said. “We show that it’s not important for skeletal formation; where it’s really important is in determining digit number and identity.”

Sonic hedgehog and Gli3 work together to pattern the pentadactyl — five-fingered — limb. Gli3 is a protein that turns other genes on or off, depending on whether it is present as an activator (the “on” form) or a repressor (the “off” form). The investigators demonstrate that Sonic hedgehog blocks the conversion of Gli3 activator forms to Gli3 repressor forms. When there is a lot of Sonic hedgehog protein around, Gli3 is present mostly in its activator forms. When Sonic hedgehog is absent, almost all of the Gli3 is converted to the repressor forms.

It is the balance of Gli3 activator and repressor forms that determines digit identity, the investigators said. Higher levels of repressor forms result in digit one, the thumb, and lower levels of repressor forms result in digit five, the pinkie. And Sonic hedgehog is the molecule that regulates the Gli3 forms. “So from the thumb to the pinkie, the master molecules that are dictating this pattern are Sonic hedgehog and Gli3,” Litingtung said.

The constraint of five digits is an evolutionary development, Chiang said. The earliest four-legged animals in the fossil record — more than 400 million years ago — were polydactyl, that is, they had many digits. In a sense, Chiang said, “our double knockout of Sonic hedgehog and Gli3 created what ancestral limbs looked like, the polydactyl potential of the primitive limb.”

Previous studies have shown that Sonic hedgehog is important to cell growth in the developing limb bud. When it is absent, there is massive cell death, Chiang said, resulting in stunted skeletal development. The current study demonstrates that Sonic hedgehog works through Gli3 to regulate expression of fibroblast growth factor (FGF), a molecule that is critical for cell proliferation. “Our study provides the molecular mechanism to explain how Sonic hedgehog regulates cell proliferation in limb precursor cells,” Chiang said.

Chiang has been tracking the role of Sonic hedgehog — a gene named after the Sega game character — for nearly eight years now. It is a molecule that plays critical roles in both cell proliferation and patterning during development. Mutations in the human version of Sonic hedgehog are associated with holoprosencephaly, a congenital malformation of the forebrain.

Gli3 mutations also lead to congenital defects in human beings; they are linked to Greig cephalopolysyndactyly and Pallister-Hall, two syndromes that share the feature of more than five digits. In fact, Chiang said, mutations affecting the function of Sonic hedgehog and Gli3 are the likely culprits in most cases of polydactyly, one of the most common birth defects in human beings.

Inappropriate Sonic hedgehog signaling also is linked to cancer. Mutation of molecules in the Sonic hedgehog signaling pathway are associated with basal cell carcinoma and medulloblastoma.

“These are key molecules with widespread roles in embryonic development and disease,” Litingtung said.

Litingtung and Chiang’s co-authors on the Nature paper are Randall D. Dahn, Yina Li, and John F. Fallon. The research was supported by the National Institutes of Health.

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