Zebrafish research may help determine what causes certain birth defects

Jun 24 2004

After five years and thousands of zebrafish breeding experiments, Mary C. Mullins, PhD, associate professor of cell and developmental biology at the University of Pennsylvania School of Medicine and colleagues have published a description of dozens of mutations that will help determine the earliest steps in vertebrate development, which take the spherical embryo to a complex creature.

These discoveries are described in a pair of papers in the June Developmental Cell and are featured on the cover of that issue. In time, these discoveries may help researchers understand human sterility and fertility problems, as well as what causes certain birth defects.

Molecular control of the step-by-step process of how the zebrafish body unfolds relies to some extent on maternally driven processes. These depend on proteins derived only from the egg, which are critical to embryonic development prior to the activation of the new embryo’s own genome. In vertebrates, maternal gene products direct such essential processes as fertilization; the first cellular divisions of the embryo; and the head-to-tail arrangement of developing cells. The genes also control morphogenetic movement, the migration of cells to form the three-dimensional structure of the embryo.

In 1998 the Mullins lab embarked on a large-scale maternal-effect mutant screen, not previously performed in a vertebrate animal model. In addition to the 68 maternal mutations, through inbreeding studies, they also discovered five paternally derived mutations. Daniel S. Wagner, PhD, and Roland Dosch, PhD, both postdoctoral fellows in the Mullins lab, spent five-plus years each on this intensive research project.

“These maternal processes are well-studied in invertebrates, but not vertebrates,” says Mullins. “Genetic screens have been extremely powerful in identifying key genes and understanding the processes involved.” This collection of mutants provides the first molecular analysis of maternal control of embryonic development in vertebrates.

The process is time-consuming, yet has been worth the wait. Mullins uses a process in which genes are mutated at random with a chemical mutagen to determine what genes are required for a particular process of interest. This is opposed to reverse genetics, where there’s a particular gene of interest, which is then knocked out to determine its function. The mother—who is the mutant—is bred with a male non-mutant. “All of her progeny are affected because the father doesn’t provide any mutant genes,” explains Mullins. “It’s the mother who’s providing what’s in the egg.” The Mullins group can ultimately identify the genes because the almost-completed zebrafish genome sequence is available in public databases.

The first paper describes 21 maternal-effect mutants from the earliest stages of embryonic development, when it is literally only hours old. These mutants – some dubbed over easy, soufflé, sunny side up, jumpstart, and buckyball – include such basic processes as the first embryonic cellular divisions and the initial head-to-tail arrangement of cells.

In addition to mutants that affect basic patterning, or assembly, of embryonic tissue the researchers describe in the second paper some “totally unexpected” mutants from one-day-old embryos, explains Mullins.One was a morphogenesis mutant called pollywog. (Top; click on thumbnail image to view full-size photos). This gene is involved in orchestrating the ultimately three-dimensional character of the head. “We end up with flat-headed fish,” says Mullins. “The head is just spread out on the yolk and it doesn’t elevate to look like the normal three-dimensional structure.” The group surmises that this is a cell movement defect. Another mutant, dubbed pug, affected the embryonic body plan. (Bottom images). Embryos from pug mutant mothers have no pectoral fins – the first limbs of zebrafish that form – and narrow-set eyes, as well as defects in the midbrain and cerebellum.

Because the process that Mullins used to obtain her mutants involved three generations before she could test for a mutant mother, she didn’t know how frequently such mutant moms would be produced or be able to breed. “That was the big risk of doing the screen, a priori, you don’t know what you’re going to get,” says Mullins. “Now we feel we have this little gold mine.”

Penn researchers Keith A. Mintzer, Greg Runke, and Anthony P. Wiemelt were also co-authors on the papers. This work was funded in part by the National Institutes of Health, the March of Dimes Birth Defects Foundation, and the American Cancer Society.