PAGE 2 OF 2
The researchers still didn’t know whether they could use the virus to block gene expression in the mice. So they engineered the lentivirus to hold an RNA hairpin meant to block the gene for alpha catenin, a protein that helps cells stick together, and injected it into mouse embryo amniotic sacs.
“It worked,” Livshits says. The protein was largely undetectable in cells that had been infected, while still present at normal levels in the uninfected cells. And the infected tissue looked identical to tissue taken from a mouse unable to express alpha catenin in the skin. The team reported their results in the July 2010 Nature Medicine.
The technique may work for other tissues as well. Christer Betsholtz, a biologist at the Karolinska Institute in Stockholm, Sweden, hopes to tweak the technique to study vascular development. Rather than injecting the virus into the amniotic sac, he plans to inject it into the embryo’s heart or a large blood vessel. The hope is that the virus will travel through the circulatory system and infect the cells that line the blood vessels, Betsholtz’s research interest. But the heart is a much smaller target than the embryonic sac. Delaying the injection a few days might make the injection more feasible. “This will not be easy, but it should be doable,” he says.
Since their discovery, Fuchs and colleagues have used their new technique to investigate the molecular mechanisms behind asymmetric division, a phenomenon in which a cell divides into two different kinds of progeny cells rather than two identical daughters.
Fuchs has always been intrigued by the fact that when the skin begins to stratify, it changes its angle of cell division from parallel to perpendicular relative to the embryo’s body. She and her colleagues speculated that this spindle reorientation might play a key role in asymmetric division. Evidence from fly studies shows that a specific set of genes regulates asymmetric cell division, enabling the mother cell to partition proteins to the two daughters unequally. Only one of the daughter cells receives proteins involved in a signaling pathway called “Notch.” This pathway helps determine whether the daughters become tissue or stem cells. “We wondered whether this ancient mechanism might also be used to regulate skin progenitors in the mouse,” Fuchs says.
To test this hypothesis, she and her team infected embryos with lentiviruses that reduced expression of three key proteins involved in this process. In the absence of these proteins, the perpendicular divisions were perturbed, and the skin didn’t stratify as it should. To test whether the skin lacking these proteins formed a tight seal, the researchers dropped the embryos into blue dye. Embryos with normal skin exclude the dye, but these embryos adsorbed it, turning blue. The team reported their results in the February 17, 2011, issue of Nature.
“The amount of genetics that it would have taken to do this set of experiments with conventional techniques would have been...not insurmountable, but it would have taken a very long time—not something desirable for any postdoc aiming to start their own laboratory,” Fuchs says.
The technique has “really taken off” in the Fuchs lab, Livshits says. She estimates that 10 lab members are using it to study everything from sweat glands to corneal epithelium—and, of course, the skin and its cancers. Rather than focusing on proteins one by one as they used to do, researchers can now focus on many proteins in a genetic pathway, gaining more insights in less time. “For a skin biologist like myself, it’s heaven,” Fuchs says. “Now we can put the molecular pieces into our biological puzzles at a pace not thought possible even a few years ago.”