A frame-by-frame view of animal development
Today's video technology allows you to advance a film frame-by-frame, breaking down the action into snapshots that normally go by too fast to be seen. Researchers would like a similar view of embryonic growth, as a fertilized egg divides billions or trillions of times to build a complete organism. The best way to do this would be to analyze thousands of embryos at the same stage of development. This is a huge challenge because eggs are not fertilized at exactly the same instant and they grow at slightly different rates; in any traditional method of collection, they get out of synch. Now Nikolaus Rajewsky's lab at the MDC, collaborating with Fabio Piano at New York University, has found a way to capture thousands of embryos of the worm C. elegans, one of biology's most important model organisms, at precise stages of development. The method, which is described in the Sept. 6 on-line issue of Nature Methods, will allow scientists to "microdissect" molecular events in the growth of this tiny roundworm. Since C. elegans and humans are related through evolution, this work has led to important insights into the function of genes in our own species as well.
eFACS can sort embryos into groups that share a precise developmental stage based on the appearance of proteins that have been tagged with fluorescent markers.
In the past, two main methods have been used to study the role of genes in C. elegans development. One involves deactivating single molecules and observing how this affects the developing embryo. Another approach has been to track specific cells as they differentiate and help build body structures. Such studies have revealed specific molecules that play a key role at various timepoints as the worm's body structures develop.
But the picture remains far from complete. Individual molecules carry out their jobs in complex networks that may involve hundreds or thousands of genes. Researchers would like a holistic overview of how networks of genes, RNAs, and proteins change as the embryo develops. The genome era has produced high-throughput technologies capable of carrying out a "census" of all of a cell's molecules, and Rajewsky and his colleagues are experts and doing so. But these methods require obtaining enough material for analysis, meaning that thousands of "synchronized" embryos have to be studied. Because they grow quickly, researchers need a way to collect them automatically and very rapidly.
PhD student Marlon Stoeckius and other members of Rajewsky's lab have developed a new method called embryo Fluorescent Activated Cell Sorting (eFACS) to obtain them. Stoeckius is the first PhD student in a new graduate school jointly established by the MDC's newly established "Berlin Institute for Medical Systems Biology" and the new Center for Genomics and Systems Biology at New York University. The emphasis of the program is to integrate high-throughput technology, innovative computational methods, and many animal model systems to answer complex biological questions like those addressed in the current study.
In the FACS method, which was invented in the 1970s, cells in a liquid are passed through an apparatus that detects differences and separates them into groups. In the 1990s scientists developed genetic engineering techniques that allowed them to attach fluorescent tags to specific molecules. When a cell activates the gene and makes a protein, it gives off a fluorescent signal that can be used to sort the cells. But the method had not yet been used to sort larger collections of cells such as embryos.
"The idea was to add a fluorescent tag to a marker protein that appears only at a precise stage of development," Rajewsky says. "At that point FACS could theoretically be used to identify and collect embryos that have reached that stage. But doing so required solving some technical difficulties."
One problem has been that large objects like embryos have to be passed through the FACS instrument more slowly than single cells. By the time thousands of embryos are sorted, many of them have taken further steps of development and are no longer synchronized. With the next generation of cell sorter instruments, Rajewsky says, the time factor may not be as much of an issue. But even with a more traditional FACS system, Stoeckius and his colleagues found a way to get around the problem by cooling the embryos – slowing their development – and then treating them with methanol, a chemical that arrests further growth. This produced a very uniform population of tens of thousands of embryos.
Next Stoeckius and his colleagues carried out a complete "census" of small RNA molecules that do not encode proteins, such as microRNAs, which cells use to control the activity of genes involved in development and other processes. By analyzing embryos captured at six different stages, they obtained a frame-by-frame view of the complex, shifting activation of molecules that help guide embryonic growth. They discovered nearly 400 new molecules that had never been detected in previous experiments. A very important finding was that embryos make different types of small RNAs at different stages of development, and that within each type, there are shifts in which molecules are produced.
The group used high-throughput sequencing methods to collect huge amounts of data about the populations of molecules in cells. Identifying and quantifying small RNAs, Rajewsky says, is tremendously difficult – lab member Jonas Maaskola had to develop innovative computer methods to complete the job.
Alongside the concrete results produced by the study, Rajewsky says the work establishes a proof of principle: the method can be widely applied to embryos of many species at any desired stage. "The only limitation is that you need to identify a particular protein that is produced only at that stage and mark it with a fluorescent tag. But you can also tag and track several molecules and sort them based on the combined profile of their activity. This opens the door to carrying out a complete, step-by-step census of the molecules active in an embryo throughout the course of its early development."
- Russ Hodge
Highlight Reference:
Stoeckius M; Maaskola J; Colombo T; Rahn HP; Friedlaender MR; Li N; Chen W; Piano F; Rajewsky N. Large-scale sorting of C. elegans embryos reveals the dynamics of small RNA expression Nature Methods (2009-09-06).
Full text of the article
Homepage of the MDC's BIMSB project
Wikipedia entry on microRNA
A list of RNA types
