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The group probing a gene associated with organ placement includes, from left, Andrew Latimer, Bhaskarjyoti Sarmah, Ph.D., Susan Wente, Ph.D. and Bruce Appel, Ph.D. photo by Dana Johnson

Gene that helps determine organ location identified

BY: LEIGH MACMILLAN

7/15/2005 - From the outside, human beings look symmetric — the left and right sides mirror each other. But the insides are another story. The heart is on the left, as are the stomach, pancreas and spleen. The liver and gall bladder are on the right. Even the left and right hemispheres of the brain have subtle but important physical differences.

This “left-right asymmetry” results from a carefully choreographed developmental program that scientists are just beginning to decipher at the molecular level. Now, a multi-disciplinary team of Vanderbilt investigators has discovered a new player in this developmental routine: one of the genes involved in regulating left-right organ placement in zebrafish. The researchers predict that the gene, reported in the July issue of Developmental Cell, will play a similar role in all vertebrates, including humans.

The investigators didn't set out to find a left-right asymmetry signaling pathway, said leaders Susan R. Wente, Ph.D., professor and chair of Cell & Developmental Biology, and Bruce H. Appel, Ph.D., associate professor of Biological Sciences. Wente and Appel may have never even worked together had it not been for their common roots in rural Iowa, which came to their attention only after Wente joined the Vanderbilt faculty in 2002.

“We grew up 12 miles from each other, but we went to different schools and never met,” Wente recalled. “I even had sleepovers with a friend who lived right across the street from Bruce.”

When Wente's group attended its first Vanderbilt Program in Developmental Biology scientific retreat, Appel, aware of the Iowa connection, made it a priority to speak with Wente's postdoctoral fellows and students. A collaboration was born.

One of the areas of interest in the Wente laboratory is a family of signaling molecules called inositol polyphosphates. This series of small molecules are generated by actions of inositol kinases and phosphatases. Perturbations of inositol signaling can result in diseases including cancer of the brain, prostate and skin, and neurological disorders, Wente said.

Wente and collaborators, working in yeast, a simple single-celled organism, discovered several inositol kinases in 1999 and 2000. They were interested in the roles these molecules might play in disease and development and intended to move to mammalian cell culture and mouse model systems to find out.

Appel urged them to consider an alternative: the zebrafish, a vertebrate model system prized for genetic and developmental biology studies. Zebrafish develop much more quickly than mice, the embryos are transparent and genetic techniques to knock out genes and to reduce gene expression transiently are available.

Bhaskarjyoti Sarmah, Ph.D., a postdoctoral fellow in Wente's group, and Andrew J. Latimer, then a student in Appel's laboratory, joined forces to explore the role of the inositol kinase, Ipk1, in zebrafish development. Using an antisense strategy, they “knocked down” Ipk1 expression and evaluated the outcome. The first experiments looked disappointing.

“We knew the phenotype might be uninformative, which could result from any number of things,” Appel said. “They weren't really seeing anything wrong, and then Drew turned the embryos over and started looking at the heart.”

That turn proved key. In half of the embryos, the heart looped the wrong way.

The reversal of heart looping pointed to a defect in left-right asymmetry, prompting the investigators to examine the placement of other organs. They evaluated genes normally expressed in asymmetric patterns in the heart, pancreas, liver, and brain and found that in all cases, the normal asymmetry was perturbed in the altered embryos.

But knowing that Ipk1 expression was participating in left-right asymmetry wasn't enough — the investigators wanted to know how it was having this effect.

They looked to calcium. Inositol polyphosphates are known to affect calcium signaling, and calcium signaling has recently been implicated in the establishment of left-right asymmetry.

“We had to develop all of the technology to study calcium signaling in the zebrafish embryo, and that was a huge hurdle,” Wente said. But it was a hurdle that the researchers successfully leaped. They used calcium imaging to demonstrate a left-sided burst of calcium in normal embryos, but not in those with reduced Ipk1 expression.

The findings add to the debate over the “symmetry breaking event” that precedes left-right asymmetric development. Up until this event, vertebrate embryonic development is bilaterally symmetric.

“Nobody really knows what the symmetry breaking event is,” Appel said. Several lines of evidence have focused on a special portion of the vertebrate embryo called the node, a compartment of cells with finger-like cilia whose movement seems to be involved in signaling left-right asymmetry.

“A huge question is how is the information initiated at this focal point and how is it communicated to the surrounding cells,” Appel said. “That could be where these inositol signaling molecules are playing a role: in communicating the left-right asymmetry breaking event to the surrounding cells.”

There are individuals — about one in 10,000 in the United States — who suffer from laterality defects: their organs are not in the normal positions. And the heart, which depends upon coordinated left-right signaling for its complex looping and connection to the vasculature during development may be particularly prone to defects in left-right asymmetry. Other investigators have even speculated that some congenital heart defects may result from mutations in left-right patterning genes, Wente said.

Appel and Wente do not expect their findings to lead to therapies for correcting laterality defects in human beings. But since the inositol polyphospate signaling cascade plays multiple roles in human disease and could offer targets for therapeutic intervention, it's important to understand all of its many roles, they said.

Wente said it is especially gratifying to her that a discovery her laboratory made in a very simple model organism — budding yeast — is now offering insights to development in a multicellular vertebrate organism.

Wente and Appel credit the Zebrafish initiative funded by the Academic Venture Capital Fund with making their collaborative studies possible.

“That initiative put us into the position to do these studies and made the very high-end imaging equipment and software available for the calcium imaging studies,” Appel said.

The investigators are also grateful to Christopher V. E. Wright, D.Phil., professor of Cell & Developmental Biology and director of the Program in Developmental Biology, and David W. Piston, Ph.D., professor of Molecular Physiology & Biophysics and director of the W. M. Keck Free-Electron Laser Center, for their insight and technical assistance over the course of the project.

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