As the body’s source of a variety of cell types, stem cells must split their energy between creating copies of themselves to ensure a steady supply, and crafting new cells specialized for particular functions. Using a rapid new method of blocking gene function, Howard Hughes Medical Institute (HHMI) researchers are dissecting how stem cell progenitors in the skin perform this balancing act.
As a mammalian embryo matures, the developing skin initially forms as a single layer of cells. As skin progenitors divide, they fall into place next to one another to maintain the single layer and keep the growing embryo covered. Suddenly, many of the cells begin to pivot as they divide, leading to multiple cell layers, a process known as stratification. Each of these asymmetric divisions leaves one daughter stem cell behind in the inner layer and produces a second daughter cell that starts becoming a specialty skin cell in the layer above. As more and more specialized cells are generated from the single inner layer of stem cells, and pushed outward, a resilient skin barrier forms that protects the animal from infections and dehydration once it is born.
“The study is a proof of principle that you can conduct complicated genetics in mice in a short period of time.”
A standard cell division splits a cell into two equivalent daughter cells, parsing out the contents of the mother cell evenly. But many biological processes, including the transformation of a fertilized egg into a functioning organism, require the kind of asymmetric cell divisions that take place in the developing skin and give rise to two unequal daughter cells.
“In the stem cell field, there’s a great deal of interest in asymmetric cell division,” says HHMI investigator Elaine Fuchs of the Rockefeller University in New York City. “But we draw much of what we know from lower animals like fruit flies.”
In flies, a handful of proteins help rotate the axis along which cells divide; they also parcel out other proteins unevenly between the two cells that result from division. This asymmetry appears to direct one of the offspring cells to seek an alternative fate – that is, it should not be a stem cell like its parent. Fuchs and her postdoctoral fellow Scott Williams wondered whether the signaling proteins that had been identified in the considerably simpler fruit fly might serve a similar function in mice.
To find out, they investigated whether the mouse versions of the asymmetry proteins—called LGN, NuMA, and DCTN1--influence division in developing skin. The standard way to shut off genes to study their function is to mutate them, but making knockout mice can take years of work. “Genetics is a very slow process in mice in contrast to flies and worms,” says Fuchs. Her lab wanted faster results, so they chose a different approach.
Last year, Slobodan Beronja and Geulah Livshits in Fuchs’ laboratory devised a fast method to shut off genes specifically in the skin cells of mouse embryos. They built their technique around RNA interference, a biological process whereby short RNA molecules trigger the destruction of specific messenger RNA molecules. When messenger RNA from a specific gene is destroyed, that gene cannot produce any protein. Fuchs' team constructed viruses loaded with genetic material that makes short RNAs. Then, using ultrasound to locate the embryos in a living pregnant mouse, they injected the viruses into the amniotic fluid that cushions the developing mouse fetus. The viruses invaded the first layer of cells they found—in this case, skin cells. Unlike the genetic manipulations used to breed a mouse that lacks a gene, the invading viruses introduce the interfering RNA and shut off gene function almost immediately, and specifically in the single layer of skin stem cells.
In the new study, which was published in the February 17, 2011, issue of the journal Nature, Fuchs’ team injected viruses to block the expression of LGN, NuMA, and DCTN1. In the injected embryos, the skin cells failed to orient their divisions perpendicularly. As a result, the skin failed to stratify and produce the specialized skin cells of the barrier. It wasn’t immediately clear how a change in the orientation of cell divisions might affect cell specialization. “Is it just positioning, or is there a molecular program that’s important?” asks lead author Scott Williams, a postdoctoral researcher in Fuchs’ lab.
In many tissues, including skin, a protein called Notch sends molecular signals that control cell specialization. Fuchs and her colleagues investigated whether LGN, NuMA, and DCTN1 influenced Notch. When asymmetry genes were shut down, they found that Notch did not send its signal. Additional studies mixing and matching defects in LGN and Notch revealed that LGN, NuMA, and DCTN1 act in a common signaling network with Notch. “Our studies reveal that setting up Notch signaling properly is one of the important consequences of partitioning cells asymmetrically,” says Fuchs.
“The study is a proof of principle that you can conduct complicated genetics in mice in a short period of time,” says Fuchs. The findings reveal new details about how stem cells set up healthy tissues as an organism develops. The team plans to further dissect the biochemical pathways that link the direction a cell divides to whether it specializes.
The results might also shed light on the role of stem cells in cancer, says Williams. Tumors might develop from out-of-control stem cells, an idea that has “taken the cancer field by storm,” he says. Glitches in the asymmetry pathway could send a stem cell down a path toward cancer, a question the team is eager to address with their new technique.