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I Want to Help Patients of Bone and Joint Diseases
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Ikegawa had served as a surgeon at an orthopedic hospital specializing in bone and joint diseases for 13 years. "Many patients who were at the end of their tether visited my hospital after visiting a number of other hospitals, thinking that they must resign themselves to the inevitable. Sadly, there are many diseases for which no effective therapy is available, and I was unable to respond to their urgent requests for me to relieve their suffering. That's heartbreaking for me as a physician and as a human being." Some ten years ago, Ikegawa relinquished his clinical practice and moved into basic research with the aim of developing a radical therapy for OA. "The reason why no effective therapy is available is that the genes that are the root cause of the disease remain unidentified. In the belief that discovering such a susceptible gene would represent the first step toward treating the disease, I started my research into the present theme." |
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| The world's first identification of an osteoarthritis-susceptible gene | |||
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OA is a disease in which cartilage tissue, which serves as a cushion between bones at joints, degenerates and/or is lost, resulting in pain and functional impairment due mainly to direct contact between otherwise isolated bones (upper panel on cover page). Although OA is one of the most prevalent bone and joint diseases, no radical therapy is available. OA can also be described as a lifestyle-related disease or multifactorial hereditary disease that develops due to the interaction of genetic factors and environmental/lifestyle-related factors such as body weight, diet, and exercise. Generally, the term hereditary disease refers to a monogenic disease. In this case, whether the relevant disease develops actually depends with a nearly 100% probability on the status of variation of a single particular gene. The correspondence between the gene and the disease is quite simple and clear. On the other hand, a number of genes are associated with lifestyle-related diseases, and each of these individual genes does not have a significant effect. For this reason, it is not always possible to explain these diseases clearly from the viewpoint of a single gene. Additionally, the mode of inheritance is quite complex; having the gene in question does not always lead to development of the disease. This is the reason why genes associated with lifestyle-related diseases are more difficult to find than the causative genes of hereditary disease. Hence, absolutely no OA susceptibility genes had been identified thus far. Genetic information is written in the form of arrangements of the four kinds of bases that constitute DNA. The human genome (entire genetic information) consists of approximately three billion base pairs, and its base sequences differ slightly among individuals. This explains the fact that individuals have different facial shapes, heights, predispositions, and susceptibilities to lifestyle-related diseases. This kind of individual difference in the sequence of genome is called polymorphism. Ikegawa and others thought that susceptibility to OA must also be associated with polymorphism of some genes. "As a result of the Human Genome Project, all base sequences were clarified and 20,000 to 30,000 genes were found. It is certain that OA susceptibility genes are included in these 20,000 to 30,000. The first step to take in search of OA susceptibility genes is to compile a large amount of data on the disease and collect a large number of gene samples. This task cannot be accomplished without the cooperation of both patients of the disease and clinicians involved in their care. Then, we proceed to search for differences in the frequency of gene polymorphism between a population with a particular disease and another without, using a statistical method called association analysis. If a polymorphism is involved in the disease, the polymorphism must be more prevalent in the diseased population." This means that a type of polymorphism prevalent in a population of individuals with a particular disease is searched for using a statistical approach. The SNP Research Center has built the world's most advanced technology for efficiently screening a number of types of polymorphism and statistically analyzing the data obtained. "There are two approaches to detecting genetic polymorphism. One is what is known as 'whole genome screen,' in which all genes are examined one after another. The other is a method of examining selected genes that are likely to be associated with the target disease based on already available knowledge and data." The first step taken by Ikegawa and others was to examine genes suspected of being associated with OA based on the then-existing relevant knowledge. Past studies had shown that many members of the SLRP family of genes play a key role in cartilage. Other reports were available on the development of OA in knockout mice lacking the functions of the SLRP family of genes. It should be noted, however, that there are 40 to 50 different genes in the SLRP family. Among those genes, Ikegawa and others attempted to examine the asporin gene, which was considered to be suspicious as it exhibits noticeable function in cartilage. First, they compared the expression level of asporin between the cartilage of OA patients and that of non-OA individuals. "Our speculation proved to be true. The expression level was significantly higher in the cartilage of OA patients than in non-OA individuals." The asporin gene includes 12 polymorphism. A comparison of these 12 between an OA population and a non-OA population revealed a close association of the polymorphism to the form of OA that affects the knee and hip joints. This polymorphism is located at a position where the sequence for aspartic acid (D), a kind of amino acid that is a constituent of protein, is present in repeats (Figure 1). The number of repeats varies from 10 to 19 among individuals. For the OA population, the 14-repeat type (D14) was the most prevalent, whereas for the non-OA population the 13-repeat type (D13) was the most prevalent. D14 accounted for slightly less than 10% in the OA population and slightly less than 5% in the non-OA population. This means that individuals with D14 are at an increased risk of developing OA in the knee and hip joints, about double the risk for those with any other repeat types. Cartilage tissue comprises chondrocytes and a substrate that fills the intercellular spaces. This substrate comprises proteins produced and secreted from chondrocytes, such as collagen and proteoglycan, and functions importantly as a cushion for cartilage tissue. Asporin is also a protein found in the substrate, and is produced and secreted from chondrocytes, but its function had remained unknown. Ikegawa and others allowed cultured chondrocytes to express asporin in large amounts in vitro. Interestingly, chondrocyte differentiation and substrate production were suppressed and, in addition, the magnitude of this suppression was the highest for the asporin of type D14. "Cartilage is constantly undergoing damage due to trauma and mechanical stress, losing its chondrocytes and substrate. If cartilage is left uncompensated for, the cushion quality of the cartilage tissue worsens and its mass decreases limitlessly. This reduction must be replenished. Accordingly, it is considered that growth factors such as TGF-  induce chondrocytes to differentiate to increase their potential for substrate production, in compensation for the reduction of chondrocytes and substrate."However, uncontrolled action of growth factors leads to other problems, including abnormal hyperplasia, ossification, and tumor development in cartilage. Consequently, a mechanism for limiting the action of growth factors and regulation to given levels is required. "We think asporin is responsible for the regulation. However, individuals with type D14 asporin are more susceptible to OA because their potential for cartilage regeneration is weakened due to excess suppression of TGF- as shown in Figure 2." Then, why does type D14 asporin potently suppress the action of TGF-? The possible mechanism remains to be clarified in detail. "Without its elucidation, we cannot produce a drug that acts directly on the mechanism." Ikegawa and others have been maintaining a joint research system with a pharmaceutical company since early times, which enables quick bridging of their achievements to the treatment of the disease. "I hope our work will open the way for the development of a radical therapy drug for OA in five years."
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| From SNP to disease-susceptible genes | |||
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"The pathogenesis of OA cannot be explained based solely on asporin. Individuals with type D14 only account for slightly less than 10% of OA patients. Additionally, D14 is only weakly correlated with the form of OA that affect the joints of the hand and the like. This finding provides evidence for different modes of pathogenesis of OA among different parts of the body. Hence, to elucidate all aspects of OA, we must detect more susceptibility genes."
Ikegawa and others are also involved in a program to search for all genes one after another. This approach is also characterized by a comparison of genes between a population of patients with a particular disease and another without. To efficiently screen as many as 20,000 to 30,000 genes for genes associated with lifestyle-related diseases, good markers are essential. It is Yusuke Nakamura, Director of the SNP Research Center, who for the first time in the world took note of single nucleotide polymorphism (SNP) as a marker for this purpose. SNP is defined as a type of genetic polymorphism in which only one kind of base at a particular position differs among individuals. Scientists consider that there are three to five million SNPs in the entire genome. Absolutely all genes are examined by means of SNP markers dispersed in the entire genome in search for genes associated with lifestyle-related diseases. That's the SNP Research Center's basic strategy.
Ikegawa and his colleagues examined 100,000 SNPs in various gene regions for difference between a population of OA patients and another of non-OA individuals. They demonstrated that one of the calmodulin genes, CALM1, is associated with susceptibility to OA of the hip joint. Calmodulin is a protein that plays a key role in signal transmission by calcium in cells. In individuals with a particular type of CALM1 SNP, the risk of OA pathogenesis is about doubled. Furthermore, individuals having both this type of CALM1 SNP and the aforementioned type D14 asporin were found to be at an increased risk about 12 times as high as that for individuals having neither type. Many of the genes associated with lifestyle-related diseases have such combined effects. This is the reason why these diseases could not been explained well on the basis of individual single genes, as described above. "The genetic risk of pathogenesis of OA cannot be quantified unless OA susceptibility genes are identified systematically. Otherwise, any diagnosis does not deserve to be referred to as 'genetic diagnosis' in the true sense, although the risk of pathogenesis does not depend solely on genes." For example, even individuals predisposed genetically to an increased risk of lung cancer from smoking can reduce the risk by refraining from smoking. "To date, the only approach available has been to examine individuals with different types of genes all together for risks associated with lifestyle factors such as body weight, diet and exercise. Provided that susceptibility genes are identified, it is possible to determine the risk of a given lifestyle for a group of individuals with the same type of genes. Although the genes themselves cannot be modified, the lifestyle can be changed. Humans can change their future by having an accurate awareness of their own risks." |
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| Basis for quest of susceptibility genes formed | |||
The International HapMap Project, a project for identifying and mapping the numerous polymorphic patterns on the human genome, to which the SNP Research Center is contributing significantly, has detected particularly useful 250,000 SNP markers. "Exploring these 250,000 SNPs would be tantamount to examining all genes on the human genome. The conventional genome-wide approach must have missed some susceptibility genes due to the sparsity of the marker meshes. From now we will be able to find absolutely all disease susceptibility genes. It's really a fantastic thought."
In addition to OA, Ikegawa and others are working on four other kinds of bone and joint diseases. "We can find genes associated with other diseases using the same approach as with OA. Last May, we published the results of our study of intervertebral disc herniation susceptibility genes* which we believe will lead to the treatment of lumbago and sciatica. Over the past five years, we have formed a basis for research in the quest for susceptibility genes associated with bone and joint diseases. I think that in the near future, more and more disease susceptibility genes will be discovered one after another. Based on the findings, I want to develop effective methods of diagnosis and treatment of the disease, and to see overjoyed patients."
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"If you see an aged person walking along the street painfully with the aid of a stick, you can be pretty sure that he or she is suffering from this disease," explains Shiro Ikegawa, Laboratory Head of the Laboratory for Bone and Joint Diseases at the SNP Research Center of RIKEN's Yokohama Institute, in talking about osteoarthritis (OA). OA is a disease characterized by erosion of the cartilage in various joints, resulting in pain, functional impairment, and disturbance of gait. Prevalence for OA in Japan is estimated at more than seven million patients. About 30% of individuals over 70 years are suffering from the disease. Hence, OA represents a major cause of the deterioration of the quality of life for the middle-aged and the elderly. However, the mechanism of its pathogenesis remains unknown, and no radical therapy is available. The Laboratory for Bone and Joint Diseases is aimed at discovering OA susceptibility genes, and bridging them to the development of an innovative diagnostic/prophylactic method and an effective radical therapy.
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Exploring the Mechanism of Dorso-ventral Patterning
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| Why was the zebrafish chosen? | |||
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"Look here!" says Hibi, guiding me to the aquatic animal building at the Center for Developmental Biology in RIKEN's Kobe Institute. The facility can accommodate 2,300 water chambers, each with a 4-liter capacity, enabling us to breed more than 20,000 zebrafish at one time. The zebrafish, a tropical fish with a body length of about five centimeters, has recently been attracting attention as a laboratory animal. Xenopus and other laboratory animals are also reared in this facility, which is one of the best equipped facilities for breeding experimental aquatic animals in Japan. "If a gene plays an important role in the formation of the dorso-ventral axis, a mutation of the gene leads to an abnormality in the axis formation. We are working to elucidate the mechanisms behind the formation of the dorso-ventral axis by finding zebrafish mutants that show abnormalities in the dorso-ventral axis formation, and identifying the causal gene," says Hibi. In the body of an organism, dorso-ventral, anterior-posterior, and left-right axes are present. The formation of these body axes represents a basic step for a fertilized egg, which consists of a single cell, to differentiate into various kinds of cells, to generate tissues, and to form a body. Then, why are zebrafish used in research into body axis formation? "Zebrafish offer a number of advantages," says Hibi. First of all, the embryogenesis of the zebrafish has a short time span and it nearly completes in 24 hours after fertilization. Additionally, the zebrafish embryo permits examination for changes in each cell because it is transparent until about 24 hours after fertilization. It is possible to introduce the gene for the green fluorescence protein (GFP) with a specific gene promoter into zebrafish genome, and visualize the promoter's activity in the embryo in real time (cover). Other preferred features of zebrafish for genetic research include easy rearing, prolificacy, and a short generation exchange cycle of two to three months. "Zebrafish are also advantageous in many technical aspects," adds Hibi. "There are two ways of destroying genes and examining their roles in development. One is to destroy a number of genes randomly and pick up the desired mutant from among the many resulting mutants. The other is to destroy a selected target gene and examine the results for knockdown of the gene's function. The first approach was successfully applied in experiments with Drosophila and nematodes, but in vertebrates it encountered difficulties, because too many genes are involved. It was the zebrafish that permitted the successful use of this approach for the first time in a vertebrate." Regarding the second approach, a groundbreaking technique was established in 1998, which is characterized by artificial inhibition of protein generation from a targeted gene using a chemical substance known as morpholino oligonucleotides. A fertilized egg of the zebrafish is 0.5 mm in diameter. This technique is quite simple in that morpholino oligonucleotides, which recognizes a part of a gene, is injected into the center of the egg using an injection needle. "With these technical innovations, the zebrafish has recently become the object of academic attention as a laboratory animal that facilitates genetic analysis of embryogenesis." Although research into the genetics of Drosophila has seen remarkable progress, investigations have brought results that often have no relevance to human genetics. Being a vertebrate like man, the zebrafish shares the basic mechanisms of embryogenesis and is expected to facilitate an understanding of the mechanisms of human embryogenesis. Then, to which extent has zebrafish research clarified the mechanisms behind the determination of the dorso-ventral axis? |
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| Formation of the dorsal organizer | |||
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In the fertilized egg of the zebrafish, the first cleavage occurs 45 minutes after fertilization, followed by a sequence of cleavages at 15-minute intervals. Some six hours after fertilization, gastrular invagination occurs, resulting in the formation of a thickly risen structure known as an embryonic shield (Figure 1). If the embryonic shield is isolated from the embryo and transplanted to another embryo, the resulting embryo would have two sets of a notochord and a nervous system. It can be considered, therefore, that this domain carries information for the generation of dorsal tissue. The domain is called the dorsal organizer. Since German-born Hans Spemann (the 1935 Nobel laureate in Medicine) and Hidle Mangold discovered the dorsal organizer in the newt embryo in 1924, many developmental biologists have worked to understand the mechanisms behind the formation of the dorso-ventral axis. Hibi is among those developmental biologists. "I have been engaged in research in an attempt to elucidate the mechanisms behind the formation of the dorsal organizer, and the interaction of signals from the dorsal organizer and other signals in the formation of the dorso-ventral axis." A fertilized egg has an animal pole and a vegetal pole. "It had been considered that egg yolk protein is the only ingredient of the vegetal pole. Because no dorsal tissue is formed if the vegetal pole is cut away, it is postulated, however, that a critical factor in dorsalization (dorsal determinant) is present there (left panel of Figure 2). It seems that in the very early stages of embryogenesis, a rail-like structure known as a microtubule is formed, via which an unidentified molecule is transported to the domain destined to become the dorsal side, and the dorsal organizer is formed. Because the dorsal organizer is not formed also if microtubule formation is inhibited, it is certain that a molecule is transported by the microtubule." It is known that a signal known as Wnt functions beyond the dorsal determinant (middle panel of Figure 2). The Wnt signal causes -catenin to be accumulated in the nucleus. This protein stimulates the expression of dorsal-specific genes, resulting in the induction of the dorsal organizer.Some genes are known to induce the dorsal organizer. Among them is the bozozok/dharma gene, which was discovered by Hibi, and which is considered essential to the induction of the dorsal organizer. "If this gene is overexpressed, the ventral side disappears and the dorsal side increases in size, resulting in a "coiled" appearance. I designated this gene "dharma" (a Japanese noun describing a doll-like shape) after its appearance. Regretfully, this gene is now often called "bozozok," a synonym given by the Polish scientist who discovered the mutant," says Hibi. According to the Polish scientist, who is a Japanophile, the mutant's behavior brought up his image of a "bosozoku" (motorcycle gang), but he seems not to have known the right spelling of the Japanese word.
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| Maternal genes and determination of the dorso-ventral axis | |||
The discovery of the bozozok/dharma gene represents a major achievement that bridges the Wnt signal and the dorsal organizer. Hibi points out, however, "The dorsal determinant, the point that we most wanted to elucidate, remains unclear."As a possible key to resolving this riddle, a mutant called tokkaebi (a Korean noun meaning a unicorn demon) is attracting attention (Figure 3). In this mutant, nuclear accumulation of -catenin does not occur and the gene that induces the dorsal organizer is not expressed, so that absolutely no dorsal tissue is formed. "During 3.3 hours after fertilization, the zebrafish embryogenesis proceeds only in the presence of RNA and protein stored during ovum formation in the mother. In this process, no transcription takes place. This phenomenon is called the maternal gene effect. tokkaebi is a gene that expresses itself just at this stage. We consider it likely that it plays a critical role in the very early stage of dorsal determination."At the Laboratory for Vertebrate Axis Formation, investigations have been progressing to the extent of identification of the causal genes for tokkaebi, and the number of candidates has been narrowed to the teens. However, if the cause of the variation resides in a maternal gene, the gene is extremely difficult to identify, says Hibi. "To date, there have been almost no cases where a maternal gene was identified as a causal gene for a mutant. I want to overcome this challenging issue to identify the causal gene for tokkaebi, and to obtain information that will lead to the elucidation of the dorsal determinant." |
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| Interactions of signals that determine the dorso-ventral axis | |||
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At the Laboratory for Vertebrate Axis Formation, a program is also ongoing to clarify how the signal(s) from the dorsal organizer determine(s) the dorso-ventral axis. It is already known that interaction between signals for Bmp protein from the ventral side and those for Bmp inhibitors, such as Chordin, from the dorsal organizer is important in the patterning of the dorso-ventral axis (right panel of Figure 2). "However, another factor was found also to play an important role," says Hibi. "Among the genes whose expression is regulated by Bmp signals is the ogon/sizzled gene. ogon/sizzled is suspected to exhibit a feedback function for the inhibition of Bmp signals. If ogon/sizzled collapses, dorsal tissues become small but instead ventral tissues are expanded. I postulate that normal embryogenesis does not occur without a good balance between the dorsal and ventral sides by the feedback mechanism of Ogon/Sizzled." We are planning to analyze the molecular mechanisms by which the ogon/sizzled gene influences the determination of the dorso-ventral axis. |
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| Working to elucidate the mechanisms of nerve generation | |||
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"Regarding the formation of the dorso-ventral axis, we have not yet identified the starting dorsal determinant, but I think we have just revealed an outline of its mechanisms. Of course, we will make further efforts to clarify details. On the other hand, I want to extend the frontiers of knowledge to proceed to exploring the formation of the nervous system," says Hibi in outlining future prospects. "After graduating from medical school, I began conducting basic research, rather than studying clinical medicine. Although it was my initial desire to work on the nerves at graduate school, I began studying immunology. I thought that immunology was the best thing to study in the context of molecular biology, since research into the nervous system from molecular biological perspectives was then only at the beginning. And I came to study developmental biology some seven years ago. Now, it can be said that the time has ripened." Interaction between signals from the dorsal organizer and those from the ventral side patterns the dorso-ventral axis on the one hand and, on the other hand, induces the dorsal ectoderm to differentiate into the neuroectoderm. Dorso-ventral axis patterning is also closely associated with neural induction. From the neuroectoderm are formed three nerves: motor, sensory, and interneuron. The Laboratory recently identified genes involved in the patterning of the three neuron-generating domains (proneuronal domains). Another program ongoing at the Laboratory is to analyze the mechanisms of higher nervous system generation using mice. This study has revealed that inhibiting the Fez-like gene, which is expressed specifically in the forebrain makes test mice 10 times more active than normal controls. Hibi says, "This mouse model not only helps in elucidating the mechanisms of forebrain development, but also has the potential for clinical applications, including the modeling of attention-deficit/hyperactivity disorder (ADHD)." The reason why Hibi entered medical school was that he wanted to understand human beings. "Various approaches are available to understand human beings. I will be happy to contribute to accomplishing the goal by elucidating the mechanisms behind the generation of the central nervous system, which is responsible for the most important function of man, on the basis of my experience in molecular biology and experimental work with zebrafish and mice."
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How is the dorso-ventral axis determined during early embryogenesis? This question has long remained unanswered in embryology. More than 80 years have elapsed since Spemann and Mangold discovered the dorsal organizer as the domain that induces the formation of dorsal tissue in 1924. Against this background, Masahiko Hibi, Leader of the Laboratory for Vertebrate Axis Formation at the Center for Developmental Biology in RIKEN Kobe Institute, says, "How is the dorsal organizer established? And what are the mechanisms behind the induction of dorsal tissues, such as neural tissues and muscles, at the right positions by these dorsal organizers? We have finally revealed an outline of these mechanisms." This article reports on the scenario surrounding dorso-ventral determination, which is becoming increasingly elucidated through research using zebrafish. 
