Zebrafish Continue to Elucidate Development of Vertebrates

Zebrafish The key to understanding vertebrate development and the origins of human birth defects may lie with the fishes--that is, with the zebrafishes (Danio rerio). Using zebrafish mutants produced primarily by chemical mutagenesis, researchers have, in recent years, turned the popular aquarium pets, originally from India, into invaluable tools for understanding the finest genetic details of vertebrate embryogenesis. Zebrafishes are the only viable candidate for large-scale vertebrate mutagenesis screens akin to those done in C. Elegans and Drosophila --a task that's usually too expensive in the mouse. In mice, so-called reverse genetics is utilized; scientists perform knockout mutations on a known gene of interest and then study the resulting mutant animals. In zebrafish, researchers, using forward genetics, first randomly mutate genes and then attempt to understand the function of those genes. The procedure allows investigators to explore much more of the unknown or poorly understood parts of the genome in less time, and without any preconceived notions as to which genes are responsible for which functions. Other advantages of the zebrafish model include plentiful offspring, and externally fertilized eggs and translucent embryos that allow for quick and thorough in vivo analysis. In December 1996, a landmark issue of Development (vol. 123) reported the discovery of hundreds of zebrafish mutants and spurred dozens of subsequent papers describing the associated genes involved in everything from proper pigmentation to muscle development to swimming ability. Recent research has continued to introduce new mutants and their associated genes; scientists have gained further insight into the genes and processes involved in vertebrate central nervous system (CNS), abdominal, and trunk development. In a recent paper, a group led by Igor Dawid of the National Institutes of Health, reports the new findings on the so-called cyclops gene and its role in central nervous system development. (M.R. Rebagliati et al., Proceedings of the National Academy of Sciences, 95:9932-9937, Aug 18 1998). A normal zebrafish embryo at 120 hours old. Originally described in 1991 (K. Hatta et al., Nature, 350:339-41, 1991), cyclops mutants don't actually have a single eye, but two eyes placed much closer together than normal due to a lack of development of the ventral forebrain. "That's all very interesting because there is a lot of evidence that signals from underlying axial mesoderm are important for patterning the ventral aspect of the CNS," says Dawid, chief of the laboratory of molecular genetics at the National Institute of Child Health and Human Development (NICHD). The findings speak to, maintains Dawid, the "general question of how the CNS of the developing neural system becomes patterned and develops different types of cells." Dawid's group discovered that the cyclops gene encodes a member of the so-called nodal factor subgroup of the transforming growth factor ß (TGFß ) superfamily, a large family of signaling factors with several important functions. Says Dawid of the study: "It shows that this particular subgroup of TGFß factors is involved in patterning the CNS." Developmental biologists had previously credited genes called hedgehog, sonic hedgehog in particular, with triggering CNS development. According to Dawid, however, there is now reason to believe that both hedgehog and TGFß factors are involved. Another significant finding: the study moves the focus of the beginnings of vertebrate CNS pattern development to a much earlier stage than previously thought. According to the findings of the research team led by Penn's Mary C. Mullins, when members of the bmp pathway are overexpressed, zebrafish embryos, here at 36 hours old, exhibit various degrees of enlarged ventral and abbreviated dorsal features (A-E). Zebrafish mutants also have been useful in better understanding the dorsoventral--back to abdomen--development of vertebrate embryonic tissues. This past July, researchers at the University of Pennsylvania Medical Center used the mutants to find three genes-- swirl, somitabun, and snailhouse --crucial to vertebrate abdominal development. Although the neural crest--a tissue at the back-belly boundary that gives rise to the peripheral nervous system, the jaw structure, much of the skull, and all pigment cells--is missing in the swirl mutant, it is abnormally expanded in the somitabun mutant. The swirl and somitabun genes are therefore believed to have a strong but opposite effect on the development of the neural crest; snailhouse plays a smaller role. "The very surprising result that we found is that ... morphologically these two mutants look identical to each other. But when we looked at the neural crest, we found an opposite phenotype," explains Mary C. Mullins , an assistant professor of cell and development biology at Penn and senior author of the published findings (V.H. Nguyen et al., Developmental Biology, 199:93-110, July 11998). The Penn researchers believe that the growth of the neural crest is determined by a gradient of bone morphogenetic protein (bmp) activity from the back to the front of the embryo. Mullins suggests that bmp activity is probably significantly higher in the somitabun mutant than the swirl mutant. Although the bmp signaling pathway has already been implicated in dorsoventral patterning in frogs, a bmp activity gradient had not yet been identified as a patterning agent in vertebrate gastrulation, the formation of the early embryo. "We hypothesize that the neural crest is basically specified by low bmp activity levels," says Mullins. As with the research into the vertebrate CNS, the neural crest findings suggest that the tissue is specified earlier in development (in this case, about five hours earlier) than previously suspected. Researchers from the University of Washington and the University of Oregon have focused their attention on the development of the zebrafish trunk. Their work, published in the August issue of Development (K.J. Griffin et al., Development, 125:3379-3388, Aug 6 1998), is the latest in a series of studies on the spadetail mutation, the first patterning mutant to be found in zebrafish. In spadetail mutants, the cells that should form the musculature along the back end up in a blob at the end of the tail. According to senior author David Kimelman, an associate professor of biochemistry at the University of Washington, the team's findings, in conjunction with previous research, suggest that spadetail may be the master regulator of vertebrate trunk formation. The investigation into a revised role for spadetail first began with the study of fibroblast growth factor (FGF), shown in previous studies to be a major regulator of body development in the in the frog (Xenopus). "Everything from the neck down was missing when you blocked FGF signals [in frogs]," explains Kimelman. "So that supported the view that FGF was required for pattern formation." Researchers then wanted to know which transcription factors were activated by FGF in order to regulate trunk and tail development. Work in frogs indicated that FGF might work by activating the T-box gene brachyury since inhibition of FGF signaling blocks gene expression. But, because brachyury mutants (called no tail mutants in zebrafish) had normal trunks and no tails, investigators concluded that FGF must target another gene or set of genes in the trunk. "We set out to define these targets of FGF in the trunk, and isolated a T-box gene that is expressed throughout the trunk in the early embryo," says Kimelman of his team's recent paper. "The really exciting result is that we have shown that this gene is mutant in spadetail embryos." In short, the T-box gene they discovered, by convention now called spadetail after the mutant phenotype, is believed to regulate the development of the trunk, whereas the development of the tail is regulated by the brachyury ( no tail ) gene. However, Kimelman cautions that it's not quite that simple. As explained in the August Development paper, a third T-box gene, called tbx6 , is part of the hierarchy---and more work needs to be done. Both Kimelman and Mullins have similar plans for future research on their respective mutants. Says Kimelman, referring to the spadetail gene, "The main thing we are focusing on now is ... what regulates its expression and what are its targets."

Zebrafish Continue to Elucidate Development of Vertebrates

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