BETHESDA, Md. _ For Philip Leder, the recognition of a genecrucial to limb and kidney development began, as so many discoveriesdo, with "an accident of science."On this occasion, said Leder, chairman of genetics at Harvard MedicalSchool in Boston, "chance favored the prepared animal technician"who noted that "something really strange is happening to the mice."Leder, who described the incident to an audience gathered Monday atthe National Institutes of Health (NIH) to name him NIH'sdistinguished alumnus of 1994, said he was intrigued by what he sawwhen he stepped into Harvard's animal room and peered into therodents' cages.For reasons that he could not begin to fathom, the creatures' limbs hadfailed to develop normally. They had what looked like sticks instead ofpaws. Their digits had fused. So had their radius and ulna, the pairedbones running from wrist to elbow.Otherwise, they appeared normal. Only after the mice were sacrificed,and dissected, did the researchers notice another anomaly. The micehad "diminished or absent" kidneys.And no one knew why.For Leder, the course of action was apparent. The geneticist, whosepioneering effort to synthesize genes and other accomplishments, havebrought him numerous accolades _ including the prestigious AlbertLasker award, which is widely regarded as a step toward the NobelPrize _ does not make a practice of leaving interesting questionsunanswered.Serendipity has led to some of the greatest discoveries in science. Inthis case, success could lead to gene therapy for developmentalabnormalities.Leder had developed a theory to explain the anomalies even as heobserved the mice."These were transgenic mice, developed to help study breast cancer,"Leder said. "We believed that a transgene had migrated into thegenome in a place where it disrupted the development of the limbs andkidneys."He also had a clue. Both the limbs and the kidneys developsymmetrically, and at approximately the same point duringembryogenesis.Leder's team mapped the gene to chromosome two. They found twomutations that were "particularly intriguing."One produced multiple fingers and toes, which was essentially "themirror image of what was happening to the mice." The second,according to medical literature, produced "exactly the mutation we hadobserved, and with variable degrees of penetrance you get renalaplasia."It appeared that the transgene was not directly responsible, but it hadsomehow interrupted the expression of a gene that was essential fornormal development. Leder and his coworkers obtained alleles of thegene from reference laboratories."It turned out to be a very complex locus, 200 kilobases in length,"Leder said. The gene produces formins, complex proteins composed of1,400 to 1,600 amino acids, which are involved in the development ofthe limbs and organ systems.But the researchers wanted more detail on precisely what went wrongin the hobbled mice, so they began looking more closely at the gene'sactivity.They found that when the breast cancer transgene integrated into thegenome it knocked off about 10 percent of the genetic material, fromthe carboxy locus on. Perhaps, Leder mused, that somehow inhibitedthe expression of formins in the mice, and accounted for their retardeddevelopment.Examination of the mice showed that they had poorly developedstructures, called the apical ectodermic ridge, which emerges onbudding mouse limbs about nine days into gestation and produces "afactor that promotes development." At 15 days, the gene expresses aprotein that kick-starts development of kidney tubules.To get a clearer picture of how the gene functions, Leder's lab teambegan "knocking out" elements of the gene. They found that when theyknocked out one exon, a sequence of DNA that codes for proteinsynthesis, mice developed with perfectly normal limbs, but 10 percentof them had abnormal kidneys. The exon coded for tyrosine kinase in aproline-rich domain, a protein found in the nucleus of cells.To study the limb deformities, the researchers turned their attention totwo structures that are necessary for normal limb development: theapical ectodermal ridge and the zone of polarizing activity, which lieswithin the ridge.Leder said it seemed clear that the "action of the gene must precedeformation of these structures or be essential for the maintenance ofthese structures."The next step was relatively simple: mate mice who have the "limbdeformity mutation" with those who have the mirror imageabnormality, called Luxoid Strong's mutation.Not surprisingly, the mutations canceled one another out. The miceproduced 84 offspring, all of whom were normal. But when those micewere mated with "wild-type" mice, those whose genomes have notbeen tampered with, 13 of 58 offspring were born with multiple fingersand toes.Leder said that further research showed that mutant mice developedtwo zones of polarizing activity, and thus excess digits. He theorizedthat causative gene is one of many "involved in controls that act at thelevel of the genes to promote normal limb and organ development."The gene has not yet been isolated, and little of the work has so farbeen published in the medical literature, he said. That work continues."Right now," Leder says, "we're at a point at which we can approachthese questions by using a reductionist system," and knocking outelements of the genome to examine what precise effects each segmentof a gene may have."In this case," he said, "an accident of science favored a preparedanimal technician _ and disclosed a complicated locus in thedevelopment of two organ systems." n

-- Steve Sternberg Special To BioWorld Today

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