Developmental Biology

Edited by: Paul Smaglik P. Carmeliet, V. Ferreira, G. Breier, S. Pollefeyt, L. Kieckens, M. Gertsenstein, M. Fahrig, A. Vandenhoeck, K. Harpal, C. Eberhardt, C. Declercq, J. Pawling, L. Moons, D. Collen, W. Risau, A. Nagy, "Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele," Nature, 380:435-9, 1996. (Cited in more than 235 papers since publication) Comments by Andras Nagy, senior staff scientist, Mount Sinai Hospital, Samuel Lunenfeld Research Institute, Toronto N. Ferrara, K. Carver-Moore, H. Chen, M. Dowd, L. Lu, K.S. O'Shea, L. Powell-Braxton, K.J. Hillan, M.W. Moore, "Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene," Nature, 380:439-42, 1996. (Cited in more than 215 papers since publication) Comments by Napoleone Ferrara, staff scientist at the Department of Cardiovascular Research, Molecular Biology, and Bioanalytical Technology, Genentech Inc., South San Francisco, Calif., and Mark W. Moore, founder of Deltagen, San Carlos, Calif. Two groups. Two techniques. One surprise result. Scientists in both the Genentech team and a coalition of Canadian and Belgian researchers racing for the vascular endothelial growth factor (VEGF) knockout phenotypes suspected that VEGF was essential for embryonic development. But neither group realized just how key. Napoleone Ferrara Napoleone Ferrara and Genentech colleagues had a hunch after they initially cloned the gene and purified its protein product in 1989.1 "We proposed at the time that this could be a very important molecule in angiogenesis, because it's potent, specific for endothelial cells, and diffusible," Ferrara recalls. "But of course, you need to get some proof of that." They began accumulating that proof: In 1993 they demonstrated that tumors needed VEGF for angiogenesis and growth.2 In 1994 they showed that VEGF was required for other forms of pathological angiogenesis, such as that associated with diabetic retinopathy.3 Still, until this paper, questions remained about VEGF's role in physiological processes. "It was very critical to show that VEGF was important for embryonic development." Andras Nagy and colleagues at the Mount Sinai Hospital, Samuel Lunenfeld Research Institute, had also had a long-standing interest in angiogenesis. "We cloned ... quite a few genes that were specific to the endothelial cells and had a role in endothelial development," Nagy recollects. "But all these genes were for membrane receptors on the endothelial cells. Obviously we were interested in knowing what was the function of the ligands for those receptors." Nagy remembers being excited when Peter Carmeliet from the Center for Transgene Technology and Gene Therapy in Leuven, Belgium, called to ask if he was interested in collaboration to find a knockout of VEGF, since VEGF serves as a ligand for two endothelial-specific receptors--Flt-14,5 and Flk-16,7--both of which had been earlier functionally characterized by Nagy's colleagues Alan Bernstein, Martin Breitman, and Janet Rossant, and implicated in embryonic angiogenesis. Ferrara's group and Nagy's team used drastically different approaches to turn off the gene. But both scientists use the same word to describe the lethality that resulted when a single copy of VEGF was inactivated: unprecedented. "This would be the first example of a gene that is so critical that even a partial inactivation is incompatible with development," recalls Ferrara. "Unexpected" would also be an apt word to describe the finding. "The hypothesis was that this would be an important molecule in embryonic development--and it was. But if I would say that we expected this to be a heterozygous lethality, that would be a big lie." Both groups anticipated that mice with both copies of the gene missing would die. Earlier knockout experiments supported this hypothesis, since mice with one mutant allele for receptors lived, while mice missing both alleles did not. "The results are particularly surprising because usually it's the receptor that is the rate-limiting step," Ferrara explains. "There are multiple ligands for these receptors. So it would not have been an unreasonable hypothesis that the other ligands could compensate for VEGF deficit. The fact that even partial inactivation of just one ligand could have such an effect was entirely unexpected." Nagy, too, recalls being surprised with the result. He was also surprised, in the early stages of the experiment, that the Genentech group was working on the same project. "We were under severe pressure of time, because we knew that they had the knockout already and we were just starting." That knowledge dictated that they use a different technique than the standard--but time-consuming--way that the Genentech scientists were using: making chimeras, getting germline transmission, and breeding the germline-transmitted chimeras into the F1 generation, then crossing them into the F2 generation. "It's usually at least a nine-month to a year process," Nagy notes. Since Genentech had a head start, Nagy and colleagues knew that they didn't have that kind of time. "In my laboratory, there was a technology to speed up the phenotyping of the knockout and get in vivo phenotype information directly out of embryonic stem (ES) cells." The only problem was, that technique was relatively untested. It involved deriving a double knockout ES cell, then making completely ES cell-derived embryos rather than chimeras. "With this little trick you are able to generate embryos from this mutant ES cell, then you see the phenotype two or three weeks after the cell line is derived," Nagy comments. "So, we cut back time significantly." The technique, which had never been used for a knockout before, yielded the heterozygous lethality in two months. "To be honest, we were very lucky," Nagy says. Mark W. Moore The unexpected result slowed the other group, since they couldn't introduce the allele into a live mouse. "We spent several months sorting out why we couldn't transmit the allele," recalls Mark W. Moore, then a Genentech scientist. The team spent those months searching for alternative explanations and, perhaps, experimental errors that could explain their results. "That slowed them down," Nagy admits. "And we could catch up." The Genentech team finally realized that they had not been mistaken when they dissected a developing embryo produced with one missing allele of the VEGF gene. "When we opened up the embryo, there were no normally developed blood vessels," Moore recalls. Nagy and Ferrara agree that the surprise heterozygosity accounts for much of the citation interest. Nagy also suspects that the new approach his group used yielded some attention. "It's a novel technology that we applied. It's a very important gene, as far as biology is concerned." Ferrara adds that the increased interest in angiogenesis and angiogenesis inhibitors--especially their role in cancer--has fueled fascination in the studies. Since the studies, Genentech has launched two related clinical trials. First, "We're using VEGF itself as a therapeutic for myocardial ischemia, to essentially grow new blood vessels in the heart." The other technique harnesses an anti-VEGF humanized monoclonal antibody to block tumor growth. Moore is considering using Nagy's faster phenotyping techniques to derive other knockouts more quickly. And both Ferrara and Nagy continue to ponder the basic science behind angiogenesis. "We still need to learn a lot about the precise mechanism of action of VEGF, as well as other mediators of angiogenesis," Ferrara comments. "We know a great deal about the biology, but at the mechanistic level, there is still quite a bit that needs to be done--for example, sorting out which receptor mediates which [process]." Nagy comments that the vessel developments both scientists hope to plumb seem increasingly complicated. "There are more and more genes that are coming up that have an effect on vessels and have key roles," Nagy comments. "We're learning more and more about the different ways of developing vessels, how you differentiate between vascular genesis and angiogenesis. The players are partially overlapping, but also there's a big set of different genes that have a unique role in one or another. It's not a single actor play. We can't say: 'This is the gene that is really responsible for that,'" concludes Nagy. "There's a constellation of genes that are responsible." D.W. Leung et al., "Vascular endothelial growth factor is a secreted angiogenic mitogen," Science, 246:1306-9, 1989. K.J. Kim et al., "Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo," Nature, 362:841-4, 1993. L.P. Aiello et al., "Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders," New England Journal of Medicine, 331:1480-7, 1994. M. Shibuya et al., "Nucleotide-sequence and expression of a novel human receptor-type tyrosine kinase gene (Flt) closely related to the Fms family," Oncogene, 5:519-24, 1990. C. Devries et al., "The Fms-like tyrosine kinase, a receptor for vascular endothelial growth-factor," Science, 255:989-91, 1992. B. Millauer et al., "High-affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis," Cell, 72:835-46, 1993. T. Mustonen and K. Alitalo, "Endothelial receptor tyrosine kinases involved in angiogenesis," Journal of Cell Biology, 129:895-8, 1995.

Developmental Biology

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