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Article from BioMedNet

From Arabidopsis to Zebra Fish New Techniques for Developmental Biology

by Amy Fluet
posted July 21,2000 --- Issue 33


The recent Society for Developmental Biology meeting offered a smorgasbord of topics from model organisms to science education, careers, and genome initiatives. This article highlights methodology and new technologies such as in vivo electroporation, double-stranded RNA-mediated interference, and morpholinos. As the author stresses, however, new technology alone does not make good science.

The 59th annual meeting of the Society for Developmental Biology (SDB), held June 7-11, 2000 at the University of Colorado at Boulder, covered development - from embryo to organism - in three days. This meeting was truly a smorgasbord, with something for everyone. Scattered among the talks about such model creatures as Arabidopsis, Drosophila, and zebra fish, were discussions on science education, career choices, and genome initiatives. Like any good buffet, it's hard to stay focused when there are so many tempting dishes. This summary, however, will stick to just one course. One highlight of the meeting was the frequent discussion of methodology and new technologies. Developmental biologists, while rooted in a field with a healthy historical perspective, make good use of new tools.

New techniques included electroporation of the chick embryo.

The Advanced Technologies in Biology workshop drew a crowd of around 250 researchers. The session's organizer, Lee Niswander of the Memorial Sloan-Kettering Cancer Center, began by describing the relatively new technique of in vivo electroporation in the chick embryo, which her lab is using "as a very rapid and inexpensive way to do promoter-enhancer analysis."

This technique relies on the same principle as DNA gel electrophoresis to move a DNA construct into a population of cells in the embryo. The orientation of the electrodes determines which population of cells will receive the DNA. Niswander showed pictures of neural tube sections in which one side of the tube glowed green from an electroporated GFP-reporter construct. With this technique, researchers can study both experimental and control cell populations in a single embryo. It also enables a rapid expression of the protein of interest (high levels in 4-6 h) and works with large DNA constructs (~10 kb), she reported.

Electrode placement refines precise targeting of gene expression.

Catherine Krull of the University of Missouri at Columbia spoke excitedly about this technique later in the week. Her lab has used the method to study the receptor tyrosine kinase EphA4 and its role in axon guidance. "But the cool thing is," she said, "by fooling around with the types of the electrodes you use and the placement, you can really target things more precisely." Along with the left-right restriction that Niswander showed, Krull further restricted the electroporation to target either the dorsal or ventral half of the tube.

A Cautionary Tale

Robert Ho of Princeton University rounded out the technology workshop with a more sobering story. Double-stranded RNA-mediated interference (RNAi) has long been known by plant biologists as a phenomenon that blocks gene expression. Researchers now use it routinely to knock out specific genes in Caenorhabditis elegans. The possibilities of such a method have led scientists to try it in other organisms. As Ho said, "We really wanted this double-stranded technique to work."

Cautionary tale: RNAi interfered with everything in zebra fish.

Ho described experiments that Andrew Oates and Ashley Bruce did in his lab using RNAi to knock out expression of the zebra fish spadetail gene. It looked like the method had worked - at first. What began as an expected knockout result became, as more controls were done, a disappointment: dsRNA in zebra fish embryos appears to decrease nonspecifically the expression of many transcripts. The kicker came when Ho's group injected dsRNA for the bacterial lacZ gene. They saw the same range of abnormalities they had seen with spadetail RNAi. "As far as we know, zebra fish don't have a lacZ gene," said Ho.

Make Way for Morpholinos

Ho's talk ended on a happier note as he described preliminary results from Stephen Ekker's laboratory, and his own, using morpholinos in zebra fish embryos. The morpholino is a short antisense oligo built on a backbone of 6-membered morpholine rings. The morpholino sequence is designed against the region surrounding the start codon of a specific gene and works by blocking translation of that gene. Its small size should allow increased specificity when knocking out individual members of a gene family. Its resistance to nucleases should make experiments easier to carry out.

The hero of the evening's talk was the morpholino.

In the open-microphone presentation that concluded the workshop, researchers described their own experiences with gene knockouts. Eddy De Robertis (University of California at Los Angeles), Eduardo Macagno (Columbia University), and Magdalena Zernicka-Goetz (Wellcome/CRC Institute, University of Cambridge) all had successes with RNAi in Xenopus, the leech, and the mouse, respectively. But it soon became apparent that the hero of the evening's discussion was the morpholino.

Lynne Angerer of the University of Rochester Medical Center's Strong Children's Research Center walked down to the stage and accepted the microphone to describe her positive experiences with this new technology. Her lab's four weeks of experimentation with morpholinos in the sea urchin embryo capped off what Angerer later described as "the most exciting six months of my scientific life."

Morpholinos aren't cheap.

Morpholinos may soon become a common tool for developmental biologists. For the time being, their cost may be the limiting factor. As Ho said, "They are not cheap. You've really got to want to knock out the gene." Based on the morpholino-buzz heard during the remainder of the meeting, quite a few researchers may have just such a desire.

"And Now for Something Completely Different"

Kathy Barton of the University of Wisconsin used the words of Monty Python to begin her plenary-session talk to the mainly animal-kingdom-oriented audience. Although in the minority, the plant biologists had new tools to talk about as well.

Promoterless GUS gene trap stains plant proteins blue.

Andrew Groover from Rob Martienssen's Cold Spring Harbor Laboratory group described a gene trap screen he has used to study the developing vascular system in plants. This gene trap relies on a promoterless construct that contains GUS (beta-glucuronidase), the plant biologist's lacZ. When this DNA element is inserted into a plant gene, that gene's promoter drives expression of GUS, leading to a blue staining product in the characteristic pattern of the endogenous gene.

In plants, said Groover, "cell-cell communication is key during patterning and other developmental events." But many of these cell-signaling proteins are underrepresented in the current sets of available developmental mutants. "So we decided to design a screen that identifies classes of proteins based not on a mutant phenotype, but instead on a functional property of these proteins."

Tunicamycin reveals secreted GUS-tagged gene products.

This screen relies on the fact that GUS loses its enzymatic activity when it is glycosylated. So whenever a GUS-tagged gene product is routed through the secretory pathway (i.e., that protein is destined to be either secreted or membrane-spanning), the blue staining disappears. If, however, Groover treats the plants with tunicamycin, an inhibitor of glycosylation, the blue color returns. When Groover sees that increase in staining, he knows that he's probably looking at an insertion in a secreted or membrane-spanning protein. And as he showed in his presentation, these insertions have already given him several candidate cell-signaling molecules to study.

A New Model Organism?

New methods are also allowing scientists to revisit old questions. The planarian, a flatworm with amazing regenerative abilities, has interested biologists for more than 200 years. Alejandro Sánchez Alvarado of the Carnegie Institute of Washington, Department of Embryology and Phillip Newmark, a postdoc in the Sánchez lab, reported on their recent studies using planarians in combination with modern techniques, such as bromodeoxyuridine labeling and RNAi. These tools will help them to understand how the tail end of a planarian can regenerate a head, and how these creatures can shrink more than eightfold during times of starvation.

It Takes More than Technology

A Hamburger casts a long shadow.

With a respectful nod to the history of developmental biology, the SDB awarded its first lifetime achievement award to Viktor Hamburger. Hamburger is known for his many contributions to developmental biology, including studies of neural development in the chick and his role in the discovery of nerve growth factor. Chris Wylie of the University of Minnesota, the society's outgoing president, showed a hand-written letter from the almost 100-year-old scientist, expressing his regrets that he was not able to attend the meeting.

When Drew Noden of Cornell University accepted the award for Hamburger, it became apparent that Hamburger had given much to the scientists he trained. During his career, said Noden, Hamburger always considered both the philosophy of the science and the scientist as well as the history behind the research. Noden stressed that these traits are just as important for today's scientists. It's important to consider the history and context of developmental biology, even among, as he put it, "the crescendo of technology and the rush to generate experiments and meet the credentials of the top journals." Good science does not come from methodology alone.