July 6, 1998
La Jolla, CA – In a developing organism, knowledge of right from left can often mean the difference between life and death. Certainly, the direction and ultimate destination in which an embryonic heart, lung, stomach or liver travels can be critical for the proper alignment of blood vessels and nerves so that normal life may proceed. Now, a team led by scientists at The Salk Institute for Biological Studies has discovered a molecular guide, in the form of a single gene, that helps youthful cells, tissues and organs decide in which direction to take their first fateful steps–whether to go left or right.
The study, published in today’s issue of the journal Nature, solves a long-standing mystery about how vertebrates including humans organize their organs and bodies–from a single fertilized cell–along a left-right axis. It also could provide insights into certain potentially fatal syndromes caused by severe birth defects in humans including, in some cases, the reversal of organs.
“What we found is a fundamental discovery of a pivotal gene that controls embryonic handedness in vertebrates,” said Juan Carlos Izpisúa Belmonte, an associate professor at Salk, and the study’s principal investigator.
“The results ultimately may help us explain severe and debilitating human malformations associated with the incorrect positioning of the viscera at birth in either normal babies or in conjoined twins.”
In their Nature article, the researchers described how the presence of the gene, called Pitx2, is essential for interpreting and executing signals needed for what’s known as left-right asymmetry commonly seen in vertebrates such as frogs, chicks, mice and humans.
The gene not only determines whether the heart and stomach ultimately reside on the left side of the body, or the liver on the right, it also contains the architectural plans for the internal matrix of individual organs.
For example, the heart needs to be structured along its left-right axis in such a way that it permits the unidirectional flow of blood through large vessels. Likewise, the right and left lungs house different numbers of lobes (the right lung has three, the left contains two) so that vessels can be properly aligned with the heart.
In humans, mutations in one copy of the Pitx2 gene have been linked to a rare genetic disorder called Rieger Syndrome that’s characterized by, among other things, irregular-shaped eyes leading to glaucoma, craniofacial deformities and cardiac anomalies. Since the heart is properly positioned, however, it suggests the remaining copy of the gene is still functioning – unlike complete organ reversals which involve mutations in both copies of the gene.
For their studies, the researchers initially were attracted by observations that Pitx2 was expressed predominantly on the left side of developing mouse, chick, frog and fish embryos. The same held true for developing organs, with the gene’s expression focused on the left side of the heart, gut or liver.
To determine if these findings were critical or just coincidence, the researchers first turned to a naturally occurring mutant mouse born with its organs in a reversed position. Using a green fluorescent protein as a marker that would target and light up Pitx2, the researchers observed that the gene was expressed on the right side of the mutant embryo that later gave rise to organs located in the opposite side of their normal relatives.
“That was the first indication that this gene is linked to the establishment of the left-right axis,” said Izpisúa Belmonte.
As further evidence, the researchers injected the gene along the right side of embryonic chicks, using a retrovirus to carry the gene inside the developing cells.
The resulting embryos began to organize themselves so their internal structure and organs appeared as a mirror image of their normal counterparts. Typically, a heart begins as a straight tube which, during its first developmental step, curls or loops to the right in an S-shaped structure. In these chicks, the tube curled in the opposite direction. Similar reversals were seen during the formation of the gut and the body’s rotation.
Before these experiments, the researchers already were aware of several genes that appeared to direct proper left-right orientations in a variety of vertebrates including chicks, frogs and mice. But in some cases these genes apparently were switched off before any visible asymmetry appeared, suggesting there must have been another gene more directly responsible for this activity. That gene turned out to be Pitx-2.
“With this one gene we were able to put an entire organ on the other side,” said Izpisúa Belmonte. “That’s a major significance of this study. You don’t need thousands of genes to do this, just one gene that activates many genes downstream.”
In future work, the researchers hope to learn more about the cascade of biological signals controlled and subsequently activated by Pitx-2. Among other things, the team is homing in on how Vitamin A, linked to a variety of developmental disorders, affects this gene.
Also participating in the study were Bruce Blumberg, Conceptión Rodrigues-Esteban, Sayuri Yonei-Tamura, Koji Tamura, Jennifer de la Peña, Wahlid Sabbagh, Jason Greenwald, Senyon Choe and Ronald Evans, of The Salk Institute; Aimee K. Ryan and Michael G. Rosenfeld, of UCSD; and Dominic P. Norris and Elizabeth J. Robertson, of Harvard University.
Funding for the study was provided by the National Institutes of Health and a grant from the G. Harold and Leila Y. Mathers Charitable Foundation.
The Salk Institute for Biological Studies, located in La Jolla, Calif., is an independent nonprofit institution conducting basic science research dedicated to the improvement of human health and improving the quantity and quality of the world’s food supply.
Its two main fields of concentration are neuroscience and molecular-cellular biology and genetics.